Marysias Lectures 381

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  • Created by: Sarah
  • Created on: 06-01-19 11:14
how did the early NS develop?
arose with multicellularity. Evolved from surface layers from ectoderm- specialisedskin cells began to have mechanotransducer that allowed them to sense pressure, that cell split and gave a skin cell and a neuron- cells became more specialised
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as you go higher up the evolutionary scale what happens to the neural cells?
neurons scattered throughout in primitive animals but higher animals show more complex neurons and axons become aggregated. Everything becomes integrated in a hierarchy
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in vertebrates what do early neural cells show?
increased cohesion to their neighbours. They maintain cell-cell contacts forming the neural plate neuroepithelium
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how does the neural plate cells form?
differentation
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what is differentation?
cells become different form each other and acqurie speclaised propertied. Governed by changes in gene expression which dictates what proteins are synthesised. as specialisation increases, pluripotency decreases.
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what can gene expression in a cell be governed by?
extrinsic factors- morphogens intrinsic factors- TFs
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how do you get neural and ectoderm cells to be different?
different molecular pathways. Organiser secretes BMP antagonists that causes ectoderm cells to become neural cells as they don't see BMp signalling
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what's the pathway for epidermal differentation?
high smad1, Msx1, GATA1, Vent TFs -> LEF1 -> epidermal cell fate
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how do you get neural differentation?
low samd 1 -> Xlpou2, Soxd TFs -> neurogenin -> NeuroD
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what TF is needed to make the organiser secrete BMP antagonists?
goosecoid
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what else does goosecoid do?
tells the organiser to change its properties, controls cell polarity, cell adhesion genes and migration genes for intercalation and CE
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what transcitps does goosecoid turn on?
BMP antagonists- chordin, follistatin, noggin
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where are BMP antagonists secreted to? why?
the adjacent ectoderm around the organiser to induce neural tissue
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what does the organiser self differentiate itno?
anterior endoderm, PCM + notochord (axial mesoderm)
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what underlies the self differentation of the organiser?
the genes turned on by goosecoid- migration, cell adhesion + polarity. Gastrulation makes the axial mesoderm involute, intercalate and undergo hypothalamic development.
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what are the opposing gradients that set up the AP axis?
anterior- Wnt +BMP antagonists in the PCM posterior- FGF, Wnts, RA by the late organiser(growth and posteriosation)
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what is the core concept in development to set up pattern?
have 2 opposing gradients. 2 antagonist signalling molecules at either end to pattern a sheet of cells to impose different identities
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what is the disadvantage that none of the models of how the early NS is set up?
ooks at it like its a fixed neuroepithelium of 10 cells and you get this patterning with no growth or proliferation. Doesn't take into account that over time there is proliferation in development
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how do you get neural plate border cells?
over time the receptiveness of cells to signals changes. So ectoderm doesn't see BMPs -> becomes neural plate cell -> sees BMPs -> becomes NPBC. BMPs from the ectoderm act on the edges of the neural plate and upreg a num of TFs that induce other sign
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what do neural plate border cells become?
neural crest cells and roof plate cells
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what do neural crest cells give rise to?
the entire peripheral NS (enteric, sympa+ parasympa), melanocytes, cartilage of the head and neck. Vast structures all over the body arise from them- highly proliferative and potent
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how do neural crest cells form?
BMPs working with wnts + FGFs initiate a cascade of events that give rise to highly potent (many cell types) + proliferative (divides lots) neural crest cells
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what does these signals BMPs, wnts and FGFs in an early cell indicate?
having stem cell capacity- usually highly proliferative and potent
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How are most regions of the CNS neurons found?
in columns and layers. This reflexts development along the AP, DV axis.
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what is different about the hypothalamus to the other parts of the CNS?
neurons aren't found in neurons or columns completely unorganised chaotic structure was surprising. Neurons are found in clusters called nuclei
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what manner are nuclei that arranged in the hypothalamus found?
a patchwork manner
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what core body functions does the hypothalamus control?
temperature, growth, sleep, circadian cycle, mood, stress, metabolism, reproduction, electrolyte balance
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what do the interconnected nuclei in the hypothalamus control?
body homeostasis- regulation of physiological processes
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what is physical homeostasis?
maintaining reproduction, metabolism, temp (warm-blooded animals), electryolte balance, stress pathway, food intake
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what does the notochord and prechordal mesoderm underlie?
the cells of the ventral midline
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what does the layers in the CNS like cerebellum, retina, spinal cord (ventral and dorsal interneurons)?
reflects either spatial morphogens (Shh, BMPs) on opposite poles of the SC setting up opposing gradients and progenitor cells differentiating according to the gradient or time waves of neurogenesis (retina, rods, cones).Generated distinctly over time
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what paradigm did most development follow and where was this different?
the nervous system is set up through columns or layers by spatial morphogens or because things are generated distinctly over time- hypothalamus = DIFFERENT
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normally in the NS where neurons are dictates what?
what they're doing eg motor neurons are at the bottom. Spatial organization dictates cell type + function but not true in the hypothalamus
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how does the hypothalamus govern the physical stress pathway?
the levels of circulating cortisol
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why is physical homeostasis important?
they nuture and maintain a healthy individual and the species. If you don't grow to adolescence or go through puberty you won't reproduce the species will die out = hypothalamus highly conserved to prevent the species dying out
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what is the stress pathway intimately evolutionary linked to?
food intake, growth, reproduction and core homeostasis
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most studies into mental and behavioural homeostasis used mice, what is this?
desires, mood, anxiety, trust, anger, reproductive/sexual drives, aggression, mood and motivation
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why do we know so little about the hypothalamus development?
it doesn't follow the same principles as other CNS regions (no columns/layers). It's tiny so it's hard to know where it originates in the NT and how it develops
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where did the first clue about how the hypothalamus develops come from?
LOF shh studies + deletion of shh leads to failure of hypothalamic development/ventral forebrian (and cyclopia/holoprosencephaly)
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how does the loss of shh show in the mouse?
cyclopia- absence of hypothalamus + eye fields and lateral mesenchyme fail to resolve bilaterally
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how many live births had shh mutations?
1/16,000
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in 2000 genetic mutations in shh or shh signalling pathway components were found to underlie what?
cyclopia and holoprosencephaly
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what underlies the anterior forebrain/ anterior-most NT?
the prechordal mesoderm (part of the axial mesoderm)
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what does the prechordal mesoderm express?
shh
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what does the shh from the PCM do?
induces a fan-shaped set of cells in the neural plate immediately above it
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how do they know the PCM secretes shh?
shh mRNA and antibody
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how do they show that the PCM shh induces a dan shaped set of shh-expressing cells in the neural plate above it?
1) surgically ablate the PCM (no shh induced) 2) Graft extra PCM (Ectopic shh) 3) combine a piece of PCM with naive NP (shh induced) 4) same as 3 but treat PCM to block shh (no shh cells induced) 5) conditional knock down of shh in PCM in vivo (no sh
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what did they look at for hypothalamus development?
a floorplate like structure in the ventral midline of the forebrain
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around the end of this floor plate structure of shh-expressing cells what could explain the pattern of hypothalamic development?
the formation of arcs of progenitor territories around the end of this floor plate
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why did the formation of arcs of progenitors not explain it?
floorplate hen arcs of progenitors expressing different TFs eg basal plate-Nkx2.2 and Alar plate eg Pax6 but then it all becomes obscured
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what did analysis of known progenitor markers by anti sense labelling show?
chaotic clustering around the edge of the floor plate-basal plate territory. These patterns could not be explained by a simple shh morphogen gradient/arcs of progenitors
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how does it go from bands of progenitors to unorganised chaotic clustering around the edge of the ploor plate territory?
the PCM changes its signalling properties. 0.5 day after inducing shh the PCM turns on Wnt, BMP + FGF10 in the fan shaped neural cells that lie above it
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what do the fan shaped neural cells that lie above the PCM turn on?
Wnt, BMP and FGF10
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double in situ shows the population of the cells above the PCM co-expressing what?
they co-express shh, BMP7, wnt and FGF as well as their signalling pathways
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why is this co-expression unusual?
shh and BMP7 usually repress each other transcription
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where else has BMP, wnt and BMP worked together to induce a specialised population?
neural crest cell, neural border cell
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what was the hypothesis that the PCM induces?
a hypothalamic stem cell that co-expresses shh, BMP7 and FGF10
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what must stem cells be able to do?
a single cell that can divide many times and give rise to multiple cell types
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what experiment was done to find if shh, FGF + BMP7 expressing cells give rise to other hypothalamic cells?
they did fate mapping experiments by injecting DiI by a slit in the NT and this injects the dye into the neural tissue above the PCM&develop embryo in the egg until distinct forebrain regions can be identified. 160 injections into cells.
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what did this experiment show?
BMP7, shh + FGF10 - gave rise to huge amounts of basal hypothalamus
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some neurons in the hypothalamus are derivatives of what?
a mix of the stem/progenitor cell and fpartly from the patterned territory
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what does a huge amount of the hypothalamjus come from?
FGF10/Shh population
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how is hypothalamus development overwritten?
spatial patterning by shh induced which you then overwrite with proliferation of stem population thats induced early on in the embryo by the change in signalling properites of the PCM
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what is the difference in signals induced in neural crest cells and the the signals in the hypothalamic stem cell?
this region the cells expresses shh which is not the case in the neural crest cells
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what is most of the basal hypothalamus derived from?
shh+BMP7+FGF10 cells that lie above the PCM
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how are populations mixed?
early patterning of shh is obscured by the growth of the FGF10 progenitor cell
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what is always retained in the development of the hypothalamus?
a shh-FGF daughter cell
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what does the basal hypothaalmus develop from?
a shh+ FGF10+ progenitor population induced by tge prechordal mesoderm
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where are progenitor populations of FGF10+Shh maintained?
in the ventro-tuberal region
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how long is a pHypothalamus cell retained for?
throughout life- it's potentially a stem cell
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where is the highest amount of proliferation in hypothalamic development?
in cells that are downregulating FGF10 and upregulating/maintaining shh
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what analysis did they to show mirror-image gradients of proliferation-differentation in the anterior and then mamillary hypothalamus from the edges of centrally retained FGF10+ pHyp cells?
Edu and p57 analysis
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where is growth driven from?
the egdes of FGF10 pHypothalamic cell
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what showed that anterior and mamillary regions grow sequentially from centrally-retained FGF10+ pHyp cells?
fate mapping/marker analysis
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how does shh diffuse out from the PCM?
diffuses in an arc around the anterior end . It is secreted- it diffuses in many directions
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they found that early on shh induced different genes one in one arc, a different gene in the next arc but why wasn't that right?
when they looked with markers (by anti-sense in situ hybridisation) from the spinal cord to see whether you get arc like rings of TFs you do for 3 days but then obscures. 5 days later by chaotic clustering of pogenitor markers around the edges
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how do you upregulate shh in the neural plate where the hypothalamus will be?
by shh inducing it from the PCM
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how does the expression in the PCM change over time?
first upregulates shh + BMP antagonists why it maintains anterior identity. Switch lose expression of BMP antagonists half a day later then upregulate BMPS
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what do the BMPs from the PCM then do?
diffuse into the fan floorplate territory (that was expressing shh) and turn on a whole new set of TFs and gene expression
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what do fan shaped cells in the neural plate upregulate (in response to BMPS?)?
upregulate Tbx2 and upregulates BMPs themselves BMP2+7, Wnts + FGF10.
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what do neural plate border cells usually express?
wnt, FGF and BMP
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what does wnt, FGF and BMP usually mean?
has stem cell like properties
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describe how the hypothalamus cells form?
induced shh by shh from the PCM along the axis in the floorplate like structure- sets up arcs of gene expression acts as a morphogen. Later properties of the PCM change to express BMPs which causes them to upregulate FGF10, wnts, and responsive gene.
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what are we trying to work out about which bits come from where in the hypo?
which bits comes directly from proliferation verses which come from the cells that you've patterned early on
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where did the cells come from?
the cells that are patterned are intermingled with the ones growing out. An early pattern from shh arcs is obscured by massive proliferation of FGF+BMP cells
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when do you inject a diI to fate marker to see what the Shh/FGF/BMP7 dcells give rise to?
just after they upregulate FGF10
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Why do we want to know what the neural stem cells express?
we can make and maintain neural stem cells for differentation- gives us a supply of neurons
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what does the fate mapping study tell us? what does it not tell us?
cells can differentiate and give rise to lots of neurons that make up the basal hypothalamus but doesn't tell us it's a stem cell because one daughter should stay as a stem cell for it to be self-renewing
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are some cells retained as stem cells?
yes always maintain a FGF-shh progenitor all the way through life
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pathway from shh form PCM?
shh from PCM-> induced shh in a hypothalamic FP-like cell-> shh from this cell sets up arcs -> sets up patterns of progenitors -> signal of PCM change, upreg BMPS2+7-> upregulate genes convert quinsencent shh+ cells to activated hypothalamic progenit
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what does the hypothalamic progenitor express?
FGF, Wnts, BMPS + Shh
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what mechanism are we trying to understand in every stem cell niche?
how you get one daughter stem cell and one differentiated cell
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if you combine fatre mapping and markers how does development proceed?
first cells grow out of the stem progenitor domain in one direction then they grow out from the opposite dorectoon posteriorly- cells growing in 2 directions from a proliferation front
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where is an FGF population retained?
cells grow out one way (anterior) and then cells grow out the other way *posterior) retain a central population all the way through of FGF10 cells
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what cells do you get first?
anterior cells and then posterior cells
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what does a mirror gradient with markers of differentation show?
there seems to be a proliferation front- the driving force for the generation of cells is at the edges of the domain
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where is the highest amount of proliferation?
in cells at the edges of the domain
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how do you get a shh/FGF cell and still get a shh/FGF cell maintained?
as the cell divides one expresses FGF10 and the other express shh. but you maintain ashh+FGF expressing cell because the shh cell induces upreg of shh in the FGF10 cell to maintain a Shh/FGF cell
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what important concept underlies this?
cells are in a state of flux as they divide their state can vary. They can fluctate as they divide
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what kind of population is this caused?
a transient amplifier population
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what is a transient amplifying population?
a population that can divide very rapidly to generate massive growth- this characterises the growth of the basal hypothalamus
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describe how you maintain a shh/FGF stem cell?
it divides to give 2 daughters, netheir of which look like the mother rapidly the shh cell induces the FGF cell to express shh so it looks like the mother again
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how do you get differentiated cells if it just keeps going back to making FGF/shh cells?
the cell that expresses shh before it signals badck to the other daughter to restablish the mother cell, the shh daughter can divide very very rapidly = transient amplifying progenitor population
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what is really important to what genes a cell is expressing?
the circadian cycle- express different genes at different times of the day. 35% of genes are differentially regulated over time
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why do genes vary over time?
evolutionary advantage get maximal fitness and coordinated efficiency
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do stem cells express the same genes all the way through life?
no they fluctuate- cells cycle and go through waves of oscillations of genes they're expressing
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what is the working hypothesis?
have a cellular homeostatic mechanism involving FGF and shh signalling which ensures the right balance of FGF10+ and shh+ progenitors and differentiating cells which orchestrates appropriate developent of the hypothalamus
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what does the suprachiasmatic nucleus do?
circadian cycle control
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what does the arcuate nucleus do?
eating and reproduction control
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what neurons do the arcutae nucleus contain? what do these regulate?
neuropeptide Y + POMC neurons - regulate energy homeostasis
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what does the tammarian nucleus control?
sleep-wake cycle (together with neurons born in a more lateral region)- neurons that decide if you're awake or asleep
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what are you left with as a remnants of the FGF/Shh cells?
tanycytes (Radial glia like cells)
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what do the tancytes line?
the third ventricle, sit in close proximity to the blood
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why is it good for the tanycytes to sit close to the bloo?
crucial for feedback loops- sample blood for information
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where did the idea that tancyctes might be neural stem cells come from?
mouse/rat where people challenged them eg put them on a high fat diet or stressed them to look for neurogenesis and found neurogenesis in the hypothalamus
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why would tanycytes be needed?
to set a new physiological norm for body homeostasis
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how can you understand whether an adult cell gives rise to other cells?
lineage tracing
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why would you do lineage tracing instead of using dyes?
the dyes dilute out as the cells divide so there ends up being none left whereas lineage tracing eg GFP is maintained
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how do you do genetic lineage tracing?
like a conditional KO with cre recombinase, get gene specifically expr in cell population and need promoter of that gene- tissue specific + temporal marker flox sequence with loxP sites GE. put cre recombinase under a promoter of that cell type
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how would you KO a gene in the heart?
GE BMP7 flox sequence it in one animal cross with another animal with cre cloned downstream of a heart specific gene (cardomyocin). cross mice when turn on cardomyocin in heart you turn on cre
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why couldn't you just KO the whole of BMP7?
BMP plays a role in embryogenesis so embryos wouldn't develop far
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what's a floxed sequence?
a sequence of DNA that's recognised by cre reocmbianse
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how do you alter conditional KO to do lineage tracing?
clone cre downstream of wnt1 promoter in 1 then 2nd mouse with constitutive promoter driving expression of reporter gene (lacZ/GFP) so see when cre expr. put seq of nonsense DNA, RNA pol falls off cre acts on loxP sites removes nonsense Lacz made
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whenever you have wnt 1 expressed what will you have?
cre will be expressed so it will cut out the nonsense and transcribe Lacz so the cells will turn blue. Don'tlose GFP/Lacz when a cell divides it endures all descendants will be blue
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what do you need to vary lineage tracing for stem cells?
need a gene expressed in that population + know the promoter that regulates that gene to clone CR downstream of the promoter. Then 2nd transgenic mouse which has a constitutive promoter with a reporter gene downstream- cre turned on marks SC+progeny
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SC promoter will work all the way through life so how do we choose which point in time we do it?
temporal-conditioning tracind- cre-recombinase is fused to a mutant estrogen ligand binding domain. cre only works in the presence of tamoxifen
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what is cre fused to what?
mutant estrogen ligand binding domain
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what activated cre in temporal conditioning tracing?
inject in tamoxifen
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what 3 things do you need to lineage trace from adult SCs?
1) transgenic animal with cre downstream of a SC promoter 2) cre ERT2 + inject tamoxifen 3) cross animal into a reporter allele so you mark SC+descendants
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what is a gene that is only expressed in tanycytes?
GLAST
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how would we show if tanycytes give rise to cells in the adults?
lineage tracing- put cre under a GLAST promoter
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where are tanycytes?
line the base of the 3rd ventricle
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are all tanycytes the same?
no there are molecularly distinct subset of tanycytes
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what tanycytes were they interested in?
the subset that express FGF10
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lineage trace tanycytes?
clone cre-ERT2 downstream of the GLAST promoter and cross with reporter mice (GFP/Lacz mice), inject tamoxifen for 5 days and recombines out loxP sites to turn on GFP
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what is a reporter gene?
a gene used to monitor expression of a gene
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What were tanycytes differentiating into?
found tanycytes were neurogenic and gliogenic
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is neurogenesis usually high, medium or low normally? what gives new neurons?
neurogenesis is low normally, will only get new neurons if there's a sudden change in diet or stress- change gives new neurons
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why doesn't lineage tracing from 500 tanycytes not prove it's a SC?
need to show its self-renewing- SC definition is that a single cell can give rise to multiple cell types
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what would be the killer experiment to show tanycytes are multipotent and neurogenic but has not been done yet?
kill all tancytes- put one back in and see if it can restore everything
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what happens if you disrupt FGF10+ cells?
the hypothalamic-pituitary axis fails to develop
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what does the FGF10+ shh population give rise to?
anterior and mamillary progenitors. Ultimately these differentiate into neurons that refulate homeostasis including circadian cycle, eating, reproduction and sleep-wake stares
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what ensures there is a constant supply of FGF10+ progenitor cells?
the cellular homeostatic mechanism involving FGF and shh signalling
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what gives rise to the infundibulum (ventral outpocketing)?
the FGF10+ cells
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what are the 2 roles of the infundibulum?
1) to self differentiate into the posterior pituitary (neurohyphysis) and the median eminence (ME) 2) To induce an underlying pouch of ectoderm into Rathke's pich (RP) which will self-differentiate into the anterior pitutary (adenohypophysis)
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what turns into the anterior pituitary?
Rathke's pouch
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what induces the ectoderm into Rathke's pouch?
the infundibulum
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how do you get the infundibulum?
from the FGF10+ cells
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what does the infundibulum turn into?
self-differentiates into the posterior pituitary (neurohyphysis)
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what happens if FGF10+ cells are disrupted?
the infundibulum does not form and RP does not form properly, the hypothalamic-pituitary neuraxis fails to form
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what is needed for establishment of the hypothalmo-pituitary neuraxis?
interactions between infundibulum and Rathke's Pouch
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why is it important to maintain FGF/Shh progenitors?
what you set up early in development are a group of FGF-expr progenitor cells that divide those give rise to cells that will build the hypothalamus. set up early in embryogenesis has implications all thro life for function
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why are these cells so important for later function?
they underlie steps in later dev. The population induced by the PCM above it of shh/FGF cells gives rise to progenitors that differentiate into neurons that regulate homeostasis
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what do FGF10+ cells give rise to?
differentiate into neurons that regulate homeostatic + specific behaviours
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what is optogenetics?
you can literally stimulate a specific neuron and get a behaviour.
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what does optogenetics show with sleep neurons?
there are sleep awakening and sleep promoting neurons- you can stimulate a neuron in a mouse and make it wake up or fall asleep
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what do the FGF10+shh+ populatiuon gives rise to?
anterior and mamillary progenitors- which differentiate into neurons that regulate homeostasis (Circadian cycle, eating, stress, reproduction and sleep-wake states)
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why is it important to maintain an FGF10 shh population?
so you can build a plentiful supply of neurons which can respond to homeostasis and build new neurons throughout life
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why can tou build neurons throughout life in the hypothalamus?
because you have maintained a stem progenitor population
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where do FGF10+ cells sit?
in the really ventral region of the hypothalamus
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what is the aural ectoderm?
PCM induce FGF10 cells then moves away and in its place you get an ingrowth of aural ectoderm (a sheet) that grows in and underneath dev forebrain and establishes a pouch structure is underneath FGF10 cells
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what does the FGF10 cells (together with shh signals) to the pouch tell the pouch to do?
pouch upregulates TF LHX3
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what does the LHX3 TF do?
an ectoderm cell expressing LHX3 becomes a Rathke's pouch cell LHX3 sits at the top of a hierarchy to differentiate into anterior pituitary endocrine cells
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what does Rtahke's pouch develop into?
develops into the anterior pituitary gland and that's what LHX3 TF does
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what do endocrine cells secrete?
hormones
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what does the anterior pituitary gland make?
menstrual cycle hormones- FSH, LH and MCTH which governs the stress axis. Growth hormone and gonadotropin hormone
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what does ACTH from the anterior pituitary do?
ACTH regulates release of cortisol that's made in the the adrenal gland
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what causes the pouch-like structure (RP) to change shape and differentiate into the anterior pituitary (adenohypophysis)?
LHX3
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what is the anterior pituitary made of?
6 major cell types in it which all make a particular major hormone or hormones
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what is crucial to the maintenance of the ongoing differentation from the early RP to the anteripr pituitary?
signalling from the SHH/FGF cells that lie above it
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why are FGF+ cells and shh progenitors important to maintain for the anterior pituitary?
they induce ectoderm into RP which differentiates into the anterior pituitary
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how is the infundibulum similar to the organiser?
induces adjacent cells and self differentiates
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what does the infundibulum (FGF10+ cells) self differentiate into?
the posterior pituitary (nerophyohysis) and the median eminence
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cells at the most distal tip of the infundibulum protrusion form what?
posterior pituitary
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most proximal outgrowth of the infundibulum differentiates into what?
median eminence
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why do we collect the ant + post pituitary when they are of very mixed embryonic origin?
part of its neural derived from hypo (post pituitary) + part of it's induced from the hypo (ant hypo)
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what is the infundibuum?
FGF10+ progenitors and RPs. Its absolutely vital to the proper functioning
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what underlies the correct development?
that RO forms properly, upregulates Lhx and differentiates into the anterior pituitary, the close proximity of these tissues is what underlies correct dev
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if we disrupt the FGF or FGF shh cells as they're forming by inteferring with shh signallying with cyclopamine what happens?
RP doesn't doesn't form, hypothalamic-pituitary aixs, doesn't express Lhx3, infundibulum doesn't form
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why don't you build the hypothalamus-pituitary axis when you disrupt shh signalling with cyclopamine?
1) you don't get an infundibulum 2) you don't get RP 3) they don't come into contact. Disruption to shh signalling early on could lead to a non-viable embryo
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where does the anterior pituitary come from?
RP (induced by the infundibulum) and the aural ectoderm
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what does the ant + post pituitary share?
they share the ability to to secrete hromones but the hromones are released in a very different way and in different cell types
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what hormones does the posterior pituitary release?
arginine vasopressin (ADH) and oxytocin are released into the blood
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what is oxytocin for?
the trust hormone and is released in labours. gives the ability to trust others.
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what guides axons from the hypothalamus that project to the posterior pitituary?
neurons set up early under shh signalling. FGF10 signalling guides these axons- importance of maintaining FGF10 is because it's going to guide that subset of axons to their target cells
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the neurons that are born in the hypothalamus under the influence of shh make what neurohormones and they project to the post pituitary? what do they regulate?
oxytocin and vasopressin which are made by neurons that are transported to the posterior pituitary where they've been released into the bloodstream to regulate water level and trust. They are likely to play a role in general emotion and aggressive
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neurons that project from the hypothalamus to the median eminence?
shh FGF progenitors and some are induced by shh in its patterning mode
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what do neurons that project from the hypothalamus to the median eminence do?
they're neurosecretory- they also make neurohormones. The neurohoromones are released into the portal capillary supply travel to the anterior pituitary gland where each hormone affects the release of a real hormone
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where do hormone releasing hormones go after the median eminence?
neurons project to the median eminence where they secrete hormone releasing neurohormones. Travel through the portal blood capillary to govern the release of hormones from the endocrine cells of the anterior pituitary.
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what are the neurons that project as far as the median eminence?
dopaminergic inhibitory neurons, GHRH, thryotropin RH, gonadotrophin RH located in the arcuate nucleus project to hypophyseal portal system in the anterior pituitary
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what neurons from the paraventricular nucleus project to the posterior pituitary?
vasopressin, oxytocin- releasing peptidergic neurons
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what are the 6 different cell types in the anterior pituitary?
corticotrophs, thyrotrophs, gonadotrophs, somatotrophs, lactotrophs, melanotrophs
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what do corticotrophs make?
make ACTH which regulates your stress axis (Cortisol). ACTH released from ant pituitary and release cortisol from adrenal gland
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what do gonadotrophs make?
LH + FSH
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What do somatotrophs make?
growth hormone
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what do lactotrophs make?
prolactin releasing
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what do them 6 cell types made?
regulate growth, reproductive status and your stress axis. The hormones made by each cell type is under the regulation of an equivalent neuron in the hypothalamus
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How do your brain-body communication regulate growth homeostasis?
Somatrophs detect GHRH from a hypothalamic neuron and then you secrete GH which makes you grow
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why is FGF10+ cells vital?
none of this would happen- no hypothalamic-pituitary axis would be built- what happens in early embryogenesis is vital for everything
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what's the infundibulum?
Early FGF10+ outgrowth that will give rise to the posterior pituitary at the distal end and at the proximal part you maintain FGF10_ cels through adulthood which line the 3rd ventricle
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where do the GHRH project to?
median eminence
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what kind of hormones project to the anterior pituitary?
releasing-hormones
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what were the FGF10+ cells?
neuroepithelial cells
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where are neural epithelial cells maintained?
at the lumen of ventricular cells- remnant of the early neural epithelial FGF10 population
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where are neurons that regulate energy homeostasis?
the arcuate nucleus
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what are tanycytes?
a remnant of the early neural epithelial FGF10+ expressing cells radial glia like cells they are maintained at the limen/SBZ
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where do tanycytes lie?
the 3rd ventricle and some line the ME
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What do tanycytes that line the ME involved in?
acute homeostasis eg you only grow at night. GH made by ant pituitary controlled by GHGR made by hypothalamic neurons in the day but these tanycytes end foot meet the end foot of GHRH neurons. tanycytes wrap around the bottom so GH not released day
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how come at night GH is released?
the end foot of GHRH neuron , at nightime the tanycytes retract and the GHRH goes into the portal capillaries to the anterior pituitary so you release GH at night- control timescale and circadian cycle
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the hypothalamus is one of the places in the brain where you maintain a neurogenic neural stem progenitor population and it derives from what population?
tanycytes that line the ME + 3rd ventricle- they are FGF responsive multipotent neurogenic stem progenic cells.
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why is the hypothalamus easy to study in animal models?
because it is a basal part of the brain behavuoru can be studied as its evolutionary conserved
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what is the difference between the NS or a single celled organism and a human?
much more integration- hierarchial levels of organisation- code organised in triplets, integrate multiple neurons receiveing sensory info to integrated centres in the brain to get a response
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what is neuroepitheliunm?
one cell thick sheet but the cells know it's going to be neural
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how thick is the NP?
one cell thick
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what kind of is neurepithelial cells?
stem cells
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what are stem cells?
can self renew, divide or give more of itself or renew to give other fates
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what happens as cells divide in the NT?
some cells divide some are retained as SCs in the neuralepithelial sheet others differentiate and move laterally
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in differentation as you increase in specialisation what decreases?
pluripotency
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what is differentiation?
process where cells become different from each other and acquire specialised protperties governed by changes in gene expression which dictate the repertoire of proteins synthesixzed
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how may a cell become committed?
start expressing a TF in that cell might be a master TF which is at the top of a hierarchy that tuns on genes that make proteins that define a cell type
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why do we look at the developmental approach?
might be easier to look at what happens in the beginning as adult brain is too complex
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How does a cell decide whether its going to be a neural plate cell or skin fate?
need to deplete or prevent a cell from being exposed or transducing the BMP signalling pathway to elicit TFs which ultimately tell that cell to become a neural cell
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what TFs are activated if sees BMP antagonists so ectoderm cell prevented from seeing BMPs?
Xlpou2, SoxD, NeuroD Neurogenin
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how does it fit in space and time?
the organiser/node- region of the embryo that becomes specialised because of signals it sees and because it expresses Gsc. Gsc acts within to turn on BMP antagonists so prevent neighbouring ectoderm cells from seeing BMP -> become neural
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what is under the trasncriptional control of goosecoid?
chordin, noggin and follistatin
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what is GSC?
a TF
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What are chordin, noggin nad follistatin?
secreted molecules go into adjeacent ectoderm cells expose to them BMPa ntagonists
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what do neural plate cells start expressing?
XLpou2, SoxD, NeuroD, neurogenenin
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where you build NP?
in the ectoderm where BMP signalling was inhibited
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when the organiser induce the neural plate at the same time what does it do?
begins to differentiate into axial mesoderm
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what other things does Gsc do?
1) turn on BMP antagonists, tells the organiser to change its properties by: 2) changes in cell adhesion molecules 3) cell polarity molecules 4) molecules that let cells respond to migration signals
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what does the organiser give rise to? how?
the axial mesoderm- self differentiates. As gastrulation proceeds, the axial mesoderm involutes, intercalates and undergoes CE giving rise to a rod of axial mesoderm that underlies the midline of the NP/NP
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what explains the formation of the AP axis?
the differentation and movements of the axial mesoderm- movements of the organiser CE and involution gives AP axis
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why do we know so much about the SC?
it was easier to study because there was more of it to play with and make mistakes on- forebrain small in dev, lots of it to study + composed of easy reflex arcs
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what is there massive amounts of in the forebrain?
proliferation
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as the organiser begins to differentiate into axial mesoderm what movements does it undergo?
undergoes CE + involution- extends under the midline of the newly-induced NP (Single celled neural epithelium)
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if we experimentally stop dev at this point in time and look with molecular markers what is the NP expressing?
the early NP expressed markers that are later confined to the forebrain so we say the early NP is anterior in character
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where does the PCM underlie?
the top part of the neural tube (anterior-forebrain)
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why is the early NP anterior in character?
because BMP antagonists induce neural tissue that has anterior identity. BMP antagonists are made in the early organiser/node and in the PCM
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how does the late node and notochord have different characterstics?
change signals to ones that promote proliferation and growth of neural cells and their posteriosation
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how do we know the organiser self assembles?
dissect out hensons node and culture overnight gives rise to notochord + PCM
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what does this tell us?
all the instructuons for making PCM + notochord and in the right space info contained in hensons node
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why do we want to understand what the instructions are?
want to take human cells and push them towards defined fates so we can grow lungs or bits of MN or spinal cord
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why does the PCM maintain anterior like tissue (for 1st couple of days in embryogenesis)?
BMP antagonists is only retained in PCM
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how is forebrain maintained?
by BMP and wnt antagonists are maintained anteriorly in the PCM
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what molecules are from the late organiser/node posteriorly?
FGF wnts RA- promote growth and posteriose
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if you have a sheet of epithelial cells and you want to impose identities on that what is the easiest way to do that?
to put 2 antagonist molecules at opposing sides if they are secreted they both set up gradients so can get opposing gradients that establish a regional pattern
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why is it a useless model of opposing gradient?
it doesn't take into account that over time cells proliferate- vast proliferation early models all problems early NS set up looked like fixed neuroepitheleium of fixed cells- no growth or division
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what can time add?
complexity
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what do the NCC give rise to?
entire peripheral NS- sympa, para + enteric, melanocytes, and cartilage of head and neck
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what do neural plate border cells give rise to?
1) NCC 2) roof plate cells
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what are the properties of NCC?
1) highly proliferative to give populations all around the body
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what induces NPBCs?
BMPs induce the NPBCs, NCCs and roof plate cells- BMPs come from the surrounding ectoderm
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how do you go from a NP cell to NPBC?
first ectoderm -> BMP antagonists -> neural plate cell -> sees BMPs -> NPBC so over time the signals cells see and can respond to change
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what is the goal? what do we need to understand for this?
to make organoids in a dish- need to understand how cells change over time to do this (eg their resceptiveness to sinals)
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what do BMPs work with to initate NCC?
BMPs work with wnt + FGF signalling to give highly potent, highly proliferative NCCs
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what is meant by a highly proliferative population?
can divide many many times
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what is a highly potent population?
cells that can give rise to many many types of differentiated cells
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how did people know that there are NCCs?
found a few TF identified in drosophila studies (cloning vert homologues how we found things) 5 genes only expressed in these cells
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what genes are expressed in NCCs?
c-myc (Cancer), snail (gastrulartion) + soxD (sex determination)
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how is the NT ventralised?
as the axial mesoderm differentiates into the notchord + the PCM underlies the cells of the ventral midline of the ventral tube, notochord upregulates shh (morphogen) then high shh upregs shh in FP
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where is shh mRNA expressed?
in notochord and the FP
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how do you see mRNA (in old days)?
took antisense probe and then a dye- know what the gene looks like, know mRNA make the converse probe
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where you cells see highest levels of shh what do you induce?
shh- so this means notchord upregs shh in the floorplate
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what does shh establish?
a morphogen gradient- acts as a morphogen to induce different progenitor cells along the DV axis of the SC. has a source and sink. High ventral low dorsal. cells respond to distinct thresholds of shh and respond by different cell fates pattern
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what happens when cells see shh?
you activate the Gli pathway, some glisa re activators and some are repressors because of that you get fine thresholds whether a cell is activated at high or low level bcos of the balance of activator and repressor you have in a particular cell
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what happens in response to how much gli activator and repressor of a cell?
that will act in the nucleus of that cell to turn on other TFs
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what did susan morton do?
take chick SC, mash them up inject into mice and make chick specific antibodies made monoclonal antibodies against TFs
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what did they show in the SC?
6 antibodies each of which detected a TF that was turned on in response to a particular level of gli activator/repressor- diff TFs induced in bands of progenitors along the DV axis these are readouts of shh morphogen grad along DV axis
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what are these TFs?
homeodomain TFs- master regulators of identities
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why doesn't the neuroepithelium look like its a single celled thick anymore?
interkinetic movement of nuclei- pseudostratified neuroepithelium
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what happens to radial glia cells?
daughter cells will choose to be retained close to the lumen or give rise to daughters that move away and build up the layers of differentiated nerves that form the SC
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what is the differences between early progenitors at the lumen and daughters that move laterally and differentiate?
progenitors still express TF, as daughter cells differentiate they downregulate these TFs
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what is the remnant of the neuropepithelium?
bit around the lumen where you have stem/progenitor cells
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how does shh confer patterning?
shh acts at early stages to confer DV pattern and establish these progenitor domains expressing distinct TFs that are the upstream master regulators of particular neuronal fates
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shh from where patterns?
ventrally- notochord + the FP
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why did we need background info from 236?
need to bring it together to understand whats going on in the hypothalamus
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most of of our understanding of how the NT was patterened came from where?
studies in the SC
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where does the PCM underlie?
the developing forebrain
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what does the PCM induce?
the hypothalamus- causes the hypothalamus to form
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what has multiple roles in development of the hypothalamus?
shh (a lot more complicated than just a spatial morphogen)
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what type of cell in the hypothalamus does shh control?
a hypothalamic stem cell
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what cell does the hypothalamus derive from?
a specialised hypothalamic stem cell
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what is really important for improving our understanding?
technology
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what did Marriane Bronner work on?
identifying this handful of genes that were expressed in the neural crest- molecularly some genes just expressed in the neural crest
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what genes expressed in the neural crest?
BMPs working with wnt and FGF signalling
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what is the neural crest?
an embryonic population that is multipotent
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how do we know NCC are multipotent?
if you inject dye or lijneage tracer later on it gives rise to cells of the body of all different cell types- it forms many structures
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why is it really difficult to do intact transcriptional profiling in in tact tissues?
the skill involved in getting a good section and doing an in situ hybridisation is hard for one gene yet alone lots hard to get resolution between diff genes
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why is it important whether the NC is a heterogenous or homogenous population?
homogeneity = implies a single stem cell heterogeneity = you've already got restriction
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what does the Bronner paper do?
paper describes a novel technique that combine in situ hybridisation with machine learning to examine complex gene expression in cells in the developing NT at single cell resolution
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what was the best they had before that paper?
taken early chick embryo got out of egg, nice section and done a double fluorescent in situ hybridisation
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what is a double fluorescent in situ hybridisation?
you've made complementary mRNA against 2 mRNA and instead of blue dye you've attached diff fluroscent labels
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what is the problem with double in situ hybridisation?
its really hard, really expensive, it takes a long time to do and its hard to resolve single cell
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how did this paper move the field forward?
worked with someone at Caltech to develop a technique that lets you peform multiplex in situ analysis, 2 to 35 genes analysed at a time much more cheaply 1) made in situ probes against 35 mRNA exp in dev NC 2)way of re-hybridising multiple probes
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why was multiplex in situ analysis good?
1) could make antisense probes much more cheaply 2) letting you combine 5 different colours at once 3) doing 1st hybridisation with your first 5 probes then washing and doing 2nd hybridisation 4) allowed you to analyse 35 genes at a time
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what was the technique of re-hybridising with multiple probes?
spatial genomic analysis
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what was there a big advance in? advantages of this?
microscopy- see much better resolution
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whats made the difference in the last 5 years?
advances in microscopy- have much better resolution (individual transcripts as a single dot)
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what did they use an algorithm for?
to determine what is real signal to what is background noise in diff colours- but room for human error
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how did they combine RNA transcripts and dot counting with?
combined with an antibody that defines just cell membranes on the same section then develop a computer programme to segment cells and assign dots to individual cells
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what was this doing overall?
analysing 35 mRNA at a time at single cell resolution
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what is bioinformatics then used for?
get a list of genes and you cluster them into different spatial groups- where expressed. hieracrchially cluster genes
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what did hierarchally clustering show?
2 main populations: stem/pluripotent and those in between )without pluripotent) and neural crest or neural
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what does clustering genes allow you to do?
which of the 35 are clustering with the stem genes and which of them are clustering with the differentiated genes
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how are cells expressing stem cells and neural cells different?
they are spatially distinct
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further clustering shows what?
if its a neural crest cell or neural cell
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what can you do to move forward from this analysis?
use best candidate gene (eg stem cell marker) and look at in detail go back into the NT and analyse the spatial expression of particular genes at high resolution
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what zones did you see when you go nack with markers?
zones that are characterstic of cells that either NCC stem cells, migratory crest genes = can see spatial map of what the niche looks like. Cells in that part of the NT are NCC stem cells, further away begin to differentiate
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what do you have to show?
that patterns are repoducible from embryo to embryo
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when you see overlap of the 2 genes what does this mean?
suggests a relationship between 2 genes or a common lineage between 2 cells
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broadly what did they do in this paper?
took sections of chick NT and analysed it with 35 different mRNAs then outlined individual cells with CM antibody and said what transcriptis are expressed in a cell, feed into computer, catergorised whether a cell is expr gene at high/low expression
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what does the pattern from the computer tell us?
catergorises high/low expression of gene in a cell- showed pattern where all 35 genes high expr, another some low+high -> found 4 patterns
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what did the 4 patterns of gene expression suggest?
there maybe 4 different cell types
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how did they show that there was 4 diff cell types?
made probes against genes from each different catergory (gene expression) and showed you could map 4 diff types of cell
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what is hierarchially clustering?
method where a computer analyses a large amount of data and sorts them into groups or picks up patterns. Clusters data into groups+hierarchies. feed large data set into computer -> catergorises into groups
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where in the developing CNS and mature CNS are neurons found in columns and layers?
spinal cord (MNS ventral, dorsal interneurons arranged in columns), cortex, retina, cerebellum
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why are CNS neurons found in columns or layers?
it reflects development along the AP/DV axis, reflects either spatial morphogens on opposite poles of SC setting up opposing gradients and progenitros differentiating according to gradient OR in cortex/retina = reflects time waves of neurogenesis
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what do the layers in development translate into?
the different layers found in the adult (eg found in the adult cerebral cortex)
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how are cells built up in the retina?
reflects time- you get waves of neurogenesis so amacrine cells, bipolar cells, ganglion cells etc, progressive ways
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what is our understanding of how things are set up in the NS been underlain by?
assembly of NS through how you set up columns or layers under the influence of spatial morphogens or because you set up things distinctly over time
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why did people think CNS formation all was by columns and layers?
because there was so many examples where that happened
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how is the adult hypothalamus different?
neurons are found in clusters called nuclei that are arranged in a bewildering patchwork manner
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is there columns or layers in the hypothalamus?
no found in nuclei not columns or layers
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what else is different about the cortex/retina to the hypothalamus?
where they dictates what they're doing eg motor neurons in ventral SC, retina rods+cones at back distirnct function in layers spatial organisation reflects cell type+cell function, in hypo every nucleus has whole cluster of neurons do opposite things
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what is an example of neurons doing the opposite thing to each other in hypothalamus nucleus?
one tells you to eat more, one tells you to eat less (energy metabolism)
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what does the hypothalamus regulate?
core body homeostasis- physical proccesses
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what physical aspects of homeostasis does the hypothalamus control?
temperature (warm-blooded animal), electrolyte balance (kidney reabsorption), growth, reproduction, stress, mood, circadian cycle, sleep
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how does the hypothalamus control the physical stress pathway?
levels of circulating cortisol
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what aspects of reproduction odes the hypothalamus regulate?
the onset of adolescence and puberty, pregnancy and birth, all governed by neurons of the hypo
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why is maintaining physical homeostasis (by hypo) important?
they nurture and maintain a healthy and individual and species- prevent the species from dying out
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why is hypo evolutionarly conserved?
if you don't grow to adolscence or go through puberty oyu won't reproduce and your species will die out
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what do you need to survive as an individual?
sleep, eat, but if animal need to respond to stress either predator or prey- stress pathway is evolutionarly linked to food-intake, growth, reproduction and core homeostasis
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what mental and behavioural homeostasis does the hypothalamus maintain?
studies of mouse model organisms show desires, aggression, stress, trust, motivation, anxiety all of which link into reproductive+sexual drives
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studies in mice of hypo give us insight into what?
human behaviours
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what is mental and behavioural homeostasis just important for?
nutruing and maintaining a helathy individual and the species as are the physical aspects (stress links into the stress pathway)
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why do we know little about the hypothalamus development?
it doesn't follow the same principles as other regions, its neurons are not arranged in- it's tiny, SC there's lots of it to study
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why is it bad that the hypothalamus is tiny?
its hard to know where it originates from in the NT and how it develops
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where was the first clue to how the hypothalamus develops?
identified shh (role in drosophila segmentation) similar genes in vertebrate homologues = shh, was expr along the FP of SC + forebrain, shh KO failure of hypothalamic dev and holoprosencephaly/cyclopia)
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why did nobody focus on what shh was doing in the forebrain?
in 1990s no one looked at forebrain as it was too complicated they looked at the SC
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features of a shh KO?
no development of the hypothalamus, no resolving of bilateral mesenchyme (cyclopia- eye fields fail to form) and holoprosencephaly (forebrain fails to develop into 2 halves)
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why did they initially KO shh?
to see if they still get ventral neurons in the SC
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what was it known about holoprosencephaly?
it was already known that reflected a lack of development of the midine where the hypothalamus was likely to arise
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what did this focus shh in brain development?
focused idea that shh was important in development of the ventral part of the brain including the hypothalamus
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what did genetic mutations in humans, shh or the shh signalling pathway found to underlie?
cyclopia/holoprosencephaly- same phenotype, did genetics on babies and had mutations in shh or components
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what part of the axial mesoderm did this focus attention on?
the PCM as it expresses shh and underlies the anterior-most part of the NT, it is the 1st set of cells that expr shh in anterior so KO shh what stopping is shh action from this tissue
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what shape is the PCM?
a fan shaped structure at the anterior end
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why is it important that the PCM doesn't extend all the way up to the end of the NT?
sets up arcs of progenitors around it by shh secretion
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what experiments were done to show that shh from the PCM induced a fan shaped set of cells in the NP immediately above it that themselves express shh?
labelled embryo with shh (in notchord+FP+ shh in PCM
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what can't you tell about the fan shaped shh expressing cells in the PCM ventral view?
thats also shh induced in the neural layer above- can see in side view or transverse view
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where is shh induced by PCM?
in ventral midline cells of the NP that lie directly above the PCM
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what is this similar to?
posteriorly- notchord induces the FP, anteriorly-fan shaped PCM induces fan shaped NP cells to upregulate shh
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how would you show that shh from the PCM induces a fan shaped set of shh expressing cells in the NP?
1) surgically ablate PCM (no shh induced in cells) 2) graft extra PM (ectopic shh) 3) combine a piece of PCM with naive NP (shh induced) 4) same as 3 but block shh in the PCM (no shh induced) 5) conditionla KD of shh in PCM in vivo (shh not induced)
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what GOF experiments could you do?
1) plant a bead of PCM in the NP (induces ectopic shh) 2) graft extra PCM (ectopic shh) 3) combine a piece of PCM with naive NP 4
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LOF studies?
1) surgically ablate PCM 2) conditional KD of shh in PCM ) 3) combine PCM with naive NP but block shh
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the finding that the PCM induces a fan shaped set of cells that then express shh focues attention on what?
focused attention on the ventral midline of the forebrain
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why did they think of the ventral midline of the forebrain?
it looked like a FP, it expressed the same molecular markers as FP cells in the posterior axis so shh and FoxA2
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how could PCM pattern arcs of progenitor around the end of this FP (ventral midline of the forebrain)?
PCM doesn't extend to the end of the NT- get fan shaped group of shh cells, shh diffuses in multiple directions in arc from a locus- arcs
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how would the PCM set up arcs of progenitors?
shh is a morphogen diffuses in multiple directions get gradient in arcs so get arcs of gene expression
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how long do you see arcs of progenitors for?
0.5 days in chick but they become rapidly obscured
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what did they think this arcs of progenitors?
gave understanding of DV axis but also how you get patterning along the AP axis
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how did they find out it wasn't just these arcs of progenitors that underlied hypothalamic development?
looked with markers from the SC and see if you get arc like rings and you did for 0.5 days in chick but then it obscures
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how did they see concentric rings for half a day?
examining known progenitor markers from the SC by anti sense in situ hybridisation
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days later what did arc patterns of progenitors become obscured by?
massively chaotic clustering of progenitor markers around the edges of this region called the FP/basal plate territory- whole array of TFs to see concentric rings but TFs piled up in one domain couldn't be explained thro morphogen gradient or arcs
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how does the PCM change its signalling properties?
about 0.5 day after inducing shh, Wnt, BMP and FGF10 are turned on in the fan-shaped neural cells that lie above it
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when does the signalling properties of the PCM change?
as early PCM is inducing the NP
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why does the PCM maintain anterior identity?
PCM is expressing shh + BMP antagonists
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when do you lose expression of BMP antagonists in the PCM in chick?
1/2 a day after upregulating shh expression PCM loses expression of BMP antagonists and upregulates BMP
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what makes the PCM start expressing BMPs?
anterior endoderm starts expressing BMPs and makes the PCM start expressing BMPS
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what does the switch from BMP antagonists to BMPs in the PCM do?
BMPs diffuse into the fan FP territory of the hypothalamus that was expressing shh and turn on a whole new set of TFs and whole new set of gene expression
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what do the fan shaped cells in the NP upregulate in response to BMPs?
Tbx2, BMPs 2+7, wnts + FGF10 and the signalling components that let cells respond to these signals
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why is it unusual that
shh and BMP7 usually repress each others transcription
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what did double in situ of the FP like hypothalamic territory show?
co-expression of Shh, BMP7, wnt and FGF
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what other cell is induced in response to BMP, Wnt and FGF?
NPBC= stem cell like properties
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so what happens as the PCM properties change?
shh is induced along the FP and in the FP hypothalamic structure where it sets up arcs of gene expression (as a morphogen) later PCM changes to only expressing BMPs so hypo-FP upregs FGF10, BMPs and Wnts
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what are cells expressing BMPs, Wnts + FGF associated with?
having some stem cell capacity
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how d properties of the PCM change?
shh + BMP antagonists --> only expresses BMPs --> inudces Hypo-FP cells to upreg FGFs, BMPs +wnts
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what is the definition of a stem cell?
a stem cell is a single cell that can self divide many many times (highly proliferative) + can form many different differentiated cell types (highly potent)
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how did they test whether this hypothalamic FP cell was a stem cell?
labelling FP by injecting a dye into it and see what they became
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what did they try and work out in the hypo experiement?
which bits of the hypothalamus comes directly from proliferation verses which come from cells you've patterned early on
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what did they find was it patterning or proliferation?
cells patterned early on were intermingled with proliferating cells = an early pattern from shh arcs is rapidly obscured by massive proliferation+growth of cells
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where is the massive proliferation and growth of cells coming from?
the set of cells that co-express FGF10 + BMP7 + shh
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What was the hypothesis?
the PCM induces a hypothalamic stem cell that co-expresses shh, BMP7 and FGF10
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how did they test whether the PCM induces a hypothalamci SC that co-expresses BMP7, FGF10 +Shh?
performed fate mapping studies to ask if cells co-expressing these give rise to many other hypothalmic cells (multipotent) and whether they self-renew
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what did they use to do fate mapping?
took a chick egg thats just upregulated FGF10 and inject with a Di-I and see where it ends up, comine with markers to see did that SC give rise to a particular neuron. inject dye via slit in NT into neural tissue just above PCM
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how did they show this territory gave rise to a huge amount of the basal hypothalamus?
160 injections above the PCM into cells expr BMP7+Shh+FGF10
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what were the 2 regions in the hypothalamus?
region we though might be arising from earlier patterned domain but in light pink cell mixing going on and neurons that arise here are partly derivatives of the SC andderived from patterned territory 1) patterned shh progenitors 2) stem cell 3) mix
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what was the main finding from this?
a huge amount of hypothalamus comes from the FGF10/shh population (patterned from shh progenitors+ from stem cells mixing with patterned)
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hypothalamus development is a combination of what?
early spatial patterning by shh which then you overwrite with proliferation of a stem cell population thats induced in the early embryo by changes in signalling properties of the PCM
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what did this combination of signals (BMP, Shh, FGF10 suggest
that these ar ethe combination of signals you need for a neural stem cell
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why does this study tell us that the cells can differentiate into lots of other neurons but does not tell us that it's a stem cell?
a stem cell should be self-renewing- one daughter should stay as a stem cell
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*** did they show the extent of the territory to which FGF10+ progenitors contribute?
by combining fate mapping (Di-marker) with cell markers
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what did they find?
much of the basal hypothalamus derives from shh+BMP7+FGF10+ population that lie above the PCM
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the early patterning effect of shh is obscured by whta?
the growth of cells from the FGF10+ progenitor cell
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how if the hypothalamic BMP7+Shh+FGF10 cell divides to give a shh and FGF10 cell is one always retained as a Shh/FGF10 SC?
the shh/FGF10/BMP7 cell divides into FGF10 and a shh+ cell, the shh+ cell then acts back to reupregulate shh in the FGF10 cell after the shh cell undergoes transient amplifying progenitor divisions
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what signals from the PCM are needed to induce the hypothalamic FP cell?
shh + nodal
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what is needed to give a hypothalamic progenitor cell?
nodal + BMP7 (gives a BMP7 + FGF10 cell then shh reupregs itself?)
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how long do you maintain shh+ FGF10 expressing cells?
all the way through life- have a mechanism to generate a progenitor expressing Shh + FGF10 cell
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if you look in situ at shh + FGF10 what do you see in the mouse?
there is a small group of cells in the hypo that are still expressing shh + FGF
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go through from shh from the PCM -> hypothalamic progenitor
shh from PCM -> induces shh in a hypothalamic FP-like cell -> shh from this cell sets up arcs of progenitor populations -> signal properties of PCM change -> upregulate BMP -> upregulate genes convert quiescent shh-expr cell to activated hypo prog
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what does the activated hypothalamic progenitor express?
BMPs, FGFs, shh (+wnts?)
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what did Marysias paper find
Fgf10+ progenitors give rise to anterior (Shh+Fgf10−) and mammillary (Emx2+Fgf10−) progenitors through bi-directional (rostral and caudal) expansion, whereas a stable population of Fgf10+ cells becomes restricted to the tuberal hypothalamus
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what did shh signalling find shh to be essential for?
growth and differentiation of the hypothalamus, and for development of the infundibulum and RP
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fate mapping showed the hypothalamic cell gave rise to differentiate neurons, what q is asked with any SC niche?
need to understand the mechanism to give one identical daughter + one differentiated cell
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what did Travis find about the way cells were differentiating by combining fate mapping and markers?
first of all cells frow out of the stem progenitor domain in 1D and then grow out from the opposite direction = cells grow out in 2 directions
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which way do the cells grow out of first?
fiest grow out of the anterior and then it gives rise to cells that grow out the posterior
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what is retained whilst cells grow out of either direction?
FGF10+ population
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what cells squeeze out in 1D then the other?
first FGF10/shh progenitor cells squeeze out in 1D and then change and come out of the other directions
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when they looked with markers of differentation what did they see?
mirror image gradients- seems to be a proliferation front
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where is the driving force for the generation of cells?
at the edges
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when the shh/FGF10/BMP7 progenitor cell splits what 2 cell types do you get?
1) FGF+BMP 2) Shh+
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what grows sequentially from centrally-retained FGF10 hyptohalmic cells?
anterior and mammillary regions grow sequentially
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how did they find out that there was mirror-image gradients of proliferation-differentation?
EdU analysis and analysis of p57
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what does Edu and P57 show?
If cells are dividing/proliferating
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what did the EdU and p57 analysis show?
mirror image gradients of proliferation-differentation in 1) anterior and 2) then mamillary hypothalamus
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where was the prolfieration of anterior and then mamillary hypothalamus form?
from the edges of the centrally retained FGF10+ hypothalamic stem cell
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why was there highest amount of proliferation in cells that downreg FGF10 and upreg/maintaining shh?
shh cells are a transient amplifying progenitor population they proliferate a lot
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what was the working hypothesis?
that there is a cellular homeostatic mechanism involving FGF and shh signalling which ensures appropriate balance of FGF10 and shh progenitors and differentiating cells and orchestrates appropriate development of the hypo
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how do shh/FGF10 cells overlap?
shh/FGF10 cells resolve and overlap to give you diff progenitor domains and progenitor cells that give rise to other populations that form the basal hypo
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where do you get feedback loops?
between progenitor populations between FGF10-expr + shh-expr and in some way that maintains stable steady state progenitor populations that give appropriate dev of the hypo
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what do you set up in early development?
a group of progenitor cells that are going to divide but they're expressing FGF10 which give rise to cells that build the hypo
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how long do FGF10 progenitors last?
all the way the way through life- left from cells set up in embryogenesis
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where is an example that what you set up early in embryogenesis had profound implications throughout life?
set up FGF10 progenitor populations in embryogenesis and they important for function- underlie a number of steps in later development
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what steps in later development do FGF10 cells underlie?
gives rise to other progenitors that differentiate into neurons that regulate body homeostasis FGF10+ population gives rise to neurons in nucleus of 1) superchiasmatic nucleus- circadian cycle 2) arcuate nucelus- reproduction and eating 3) sleep wake
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what does arcuate nucleus control?
energy metabolism (eating) and reproduction
425 of 761
what does the tuberomamillary nucleus do?
sleep wake- FGF10 cells give rise to these populations
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FGF10 + Shh populations give rise to what?
anterior and mamillary progenitors
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what have we learnt in the last couple of years?
early FGF10 stem progenitors -> differentiating -> understand how they're involved in the differentation of neurons that regulate specific behavioursf
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what do FGF10+ shh+ populations give rise to?
anterior and mamillary progenitors that differentiate into neurons that regulate homeostasis- circadian cycle (suprachisamatic), eating + reproduction (arcuate) = tuberomamillary (sleep-wake)
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how you can stimulate a neuron in a mouse to get a specific behaviour?
optogenetics
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why is it important that FGF10/Shh stem progenitors are maintained?
so you can build a plentiful supply of neurons- neurons are born in waves from progenitor cells. Maintain a progenitor population so you can build new neurons all through life
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whats different about neurogenesis in hypothalamus to other parts of the body?
other parts of the body neurons born early in emrbyogenesis but in the hypo you build neurons throughout life and you do that because you have maintained stem/progenitor population
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what's the other reasons its important to maintain these pool of progenitor cells?
some of the FGF10 cells give rise to a ventral outpocketing called the infundibulum
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what is the 2 roles of the infundibulum?
1) to self differentiate into the posterior pituitary (neurohypophysis) and median eminence 2) to induce a underlying pouch of ectoderm into RP which self differentiates into the anterior pituitary
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what is the other name for the anterior pituitary?
the adenphypophysis
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what does the infundibulum self differentiate into?
the median eminence and posterior pituitary
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what does the infundibulum (FGF10+ cells) induce?
induces ectoderm underneath into Rathke's pouch
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what does RP self differentiate into?
anterior pituitary
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where does the PCM go?
PCM moves away because differential movement
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what do you get replacing the PCM?
an ingrowth of aural ectoderm- sheet of ectoderm that grows in and underneath the bottom of the developing forebrain- forms pouch structure (RP)
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what does the combination of FGF + Shh from these cells signalling to the pouch cells do?
makes them upregulate a TF = Lhx3
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what is Lhx doing?
ectoderm cell expressing Lhx3 becomes a Rathke's pouch cell
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what does RP develop into?
self differentiates into anterior pituitary
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what does Lhx3 TF do?
TF that sits at the top of a hierarchy that instructs the differentiation of all of the endocrine cells in the anterior pituitary
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what does anterior pituitary make?
LH, FSH, growth hormone, gonadotrophic hormone, ACTH- governs stress axis
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the expression of Lhx3 in RP does what?
causes the pouch to change its shape and differentiate into the anterior pituitary
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how many cell types does the anterior pituitary have? what do they do
6 major cell types all make a major hormone
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how long does the differentation from RPs to anterior pituitary take?
several days in chicks and several weeks in humans
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what is crucial to the ongoing differentation programme?
signalling from the shh + FGF10 population above it
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so reasons its important to maintain FGF + Shh progenitors is?
they will induce this pouch of ectoderm -> RP -> differentiates into the anterior pituitary
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how are these FGF10 + Shh progenitors like the organiser?
organiser induces neural tissue by secreting signals and self differentiates into axial mesoderm, these populations induce cells to become RP + Ant pit and self differentiate into post pit + ME
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How do hte FGF10 progenitors proliferate?
progenitors are proliferating and they give rise to a new set of progenitors that protrude ventrally
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cells at the most distal tip of that peostrusion five rise to what?
the posterior pituitary (neurohypophysis)
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cells at the proximal part of that outgrowth give rise to what?
the median eminence
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why are post + ant pituitary collectively grouped into pituitary gland when they have different origins?
post pituitary- derived from hypo and part of its induced by hypo (ant) so why its so important to set up those cells and then maintain them
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what happens if FGF10 cells are disrupted?
the infundibulum and RP does not form
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ventral outgrowth at distal end gives rise to what? proximal end?
distal end- posterior pituitary proximal end- median eminence
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what gives rise to the ventral outgrowh?
FGF10 expressing cells (and shh progenitors)
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what do FGF10 cells lie in close contact with?
Rathke's pouch- as FGF10-> infunidibulum -> induces RP next to it (lhx induced by FGF + shh signalling mix)
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what is the ventral outgrowth called?
the infundibulum
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interactions between what are needed to establish the hypothalomo-pitutary neuraxis?
interactions between infundibulum and Rathke's pouch- close proximity underlies correct development
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how did they disrupt FGF10/shh cells as they're forming?
with cyclopamine- a blocker of shh signalling
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what happened when they used cyclopamine inhibitor to distupt FGF10 cells?
no pouch of ectoderm, no RPs (no Lhx3), infundibulum doesn't form properly, pouch isn't close to infundibulum, disrupt early FGF--shh loop don't build hypothalamic-pituitary axis
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what does this tell us about early shh signalling?
disruption to shh at an early time point has massive consequences- no infundibulum, no RP
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what is key to homeostasis in the adult?
the hypothalamamo-pituitary axis
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what do hypothalamic neurons that project to the posterior pituitary secre?
oxytoxcin + vasopressin into the bloodstream
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where did does the posterior pituitary come from?
the distal end of the ventrally growing FGF10+ cells (the infundibulum)
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where does the anterior pituitary come from?
induced by shh+FGF10 signalling from infundibulum- induces aural ectoderm/RP -> Lhx3 transforms to anterior pituitary
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why are they called the pituitary collectively?
share the ability to secrete hormones
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whats the difference about the ant + post pituitary?
hormones released in very different ways and from very different cell types
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what do hypothalamic neurons that project to the median eminence secrete?
hormone-releasing neurohormones
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neurons secreting hormone releasing neurohormones that project to the ME where do they go next to travel to the anterior pituitary?
hypothalamic neurons project to ME -> hormone-releasing neurohormones released -> travel in portal blood capillaries -> to ant pituitary
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what does oxytocin from the post pituitary do?
trust hormone + labour contractions + tendency to become aggressive
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what does vasopressin (ADH) do?
regulates how much water is absorbed from the kidney
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amongst the neurons that are set up early early in dev by shh signalling form shh+ cells early are what?
cells that will differentiate to hypothalamic neurons that secrete posterior pituitary hormones
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what guides hypothalamic axons to the posterior pituitary?
FGF10 = again the importance of maintaining FGF10 in that area guides axons to target cells
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what are hypothalamic neurons that secrete neurohormones called?
neurosecretory neurons- they're secreting a hormone
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what hypothalamic neurons project to the posterior pituitary?
neurons of the paraventricular and supraoptic nuclei
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does the posterior pituitary produce hormones?
the posterior pituitary doesn't make hormones but stores + secretes hormones produced by the hypo, releases hormones by receiving signals from the neurosecretory neurons
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what does the paraventricular nuclei make?
oxytocin
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what does the supraoptic nuclei make
vasopressin
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does the anterior pituitary make hormones?
yes makes hormones but hormone releasing hormones from the hypothalamus stimulate secretion of hormones in the anterior pituitary
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what hormones does the anterior pituitary make?
prolactin, LH, FSH, TSH, GH, ACTH
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where do the neurons that project to the ME derive from?
from the FGF/shh progenitors others again are induced by shh in its patterning mode
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pathway from hypothalamic neurons to ant pituitary?
hypothalamic neurons project to the ME -> secrete hormone releasing neurohormones -> released intot he portal capillary supply -> travel to ant pituitary -> each hormone affects release of the real hormone
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what neurons project to the hypophyseal portal system in the anterior pituitary?
located in arcuate nucleus: dopaminergic inhibitory neurons, GHR hormone, thyrotropin releasing hormone, gonadotrophin releasing hormone
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vasopressin and oxytocin releasing neurons located in the supraoptic and paraventricular nucleus project to where?
posterior pituitary
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where do neurons that project to the ME project to?
endocrine cells
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6 cell types in ant pituitary?
corticotrophs, thyrotrophs, gonadotrophs, somatrophs, lactotrophs, melatrophs
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how is the stress axis regulated?
hypothalamic neurons make ACTH releasing hormone projects to ME to portal capillary supply releases ACTH from the ant pituitary travels to adrenal gland release cortisol
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what do thrytrophs make?
thyroid hormone- instrumental in health
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what cells make LH + FSH?
gonadotrophs
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these are key hormones that regulate what?
growth, reproductive status, stress axis and each one is regulated by a neruon in the hypothalamus that releases the releasing hormone
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how is GH regulated?
GH is released from somatotrophs when they detect GHRH from a hypothalamic neuron = how you get brain body communication that regs homeostasis
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what are FGF10+ cells at the proximal part of the infundibulum maintained as?
maintained as radial glial cells = tanycytes
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why do you need to set up FGF10+ cells?
what happens in early embryogenesis is vital- need to build hypothalamic neurons and to build hypothalamic-pituitary axis and to give tanycytes
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at its proximal end of the infundibulum what do you maintain?
a population of cells that continue to express FGF10 all the way through into adulthood = tanycytes
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where do tanycytes line?
the base of the 3rd ventricle + the ME
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how are tanycytes involved in acute homeostatis?
only grow at night as end foot of hypothalamic neuron that secretes GHRH is covered by end foot of tanycyte when you sleep tanycyte moves away and lets GHRH be released
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what is the median eminence full with?
end feet of hypothalamic neurons that have projected there, full of releasing hormones travelling to the anterior pituitary
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what did FGF10 cells start off as?
neuroepithelial cells (NP cells)
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what do you maintain at the lumen/SVZ?
neuroepithelial cells are maintained even when you're building at the SVZ
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what are these tanycytes?
these are a remnant of that early neuroepithelial FGF10 population
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where are FGF10+ cells?
cell bodies located at the ventricular zone but have a long basal extending process and depending on where that cell is located there processes project to diff parts of the hypothalamus
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where do FGF10+ cells (tanycytes project to) depending on where they are?
either the median eminence or the arcuate nucleus which regulates reproduction and eating
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we say tanycytes have radial glial like morphology what is a radial glial cell?
a cell that derives from a neural epithelial cell and its CB is maintained at the SVZ but has long basal extending process- they look like radial glial cells but actually called tanycytes
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tanycytes that line the ME are involved in what?
acute homeostatsis- only grow at night stop GHRH being released
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pathway of GHRH?
GHRH released from neurosecretory hypothalamic neurons in the day travels down and the end feet of those neurons project to the ME where tanycytes project to so tanycytes end foot block release of GHRH wrap around neuron in daytime
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what is acute homeostasis?
what is happening over a short timescale
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tanycytes that line the sides are thought to be what?
FGF-responsive multipotent and neurogenic stem and progenitor cell- hypo one of only places in brain that maintains a SC (derives from FGF10 population)
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in what other parts of the brain do we know that neural stem cells exist? what are these?
dentate gyrus of the hippocampus, lateral ventricle of the SVZ- neurogenic neural stem cells that give rise to new neurons through life
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where do they arise from?
neuroepithelial cells via radial glial like cells
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why is it debated how important these cells are in humans?
in mouse neural SCs of the SVZ give rise to neurons of the nasal epithelium let mouse smell through life but humans don't use smell (evolutionarly conserved)
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where is there more evidence for SCs being important in humans?
in the dentate gyrus of the hippocampus- involved in forming new memories by forming new circuits
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progression of a neural stem cell over the life course of an animal?
single cell neuroepithelium -> embryonic + fetal period -> give rise to radial glial cells (CB maintained at SVZ but basal projection) -> differentiate into diff neurons
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what is there controversy about this radial glial cells?
in other parts of the CNS where you have neural stem cells controversy whether nerual SC is mainted as a radial glial cell or ependymal cell or astrocytic cell or oligodendrocyte
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what SC do you see in the hypothalamus?
an FGF10+ population that starts off as a neruoepithelial population transits through a radial glia like phase and maintained as a tanycyte
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what made us suspect that there might be a SC in the hypothalamus?
hypo involved in core body functions- growth, rerpoduction ,stress, metabolism
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examples of why metabolism would change and need to be adapted?
metabolism in polar bear changes as they hibernate- metabolic status is different at diff times of the year, sheep- breeds only in spring, puberty- growth changes
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how does the body adapt to anticipitate and meet changing conditions in pregnancy?
pregnancy- more RBCs
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these observations made us think what about the hypo?
it would be useful if the hypo could adapt and meet changing conditions by developing new neurons at diff times to anticipate ready for reproduction etc
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what is the general idea behind this?
what you set up early on in embryogenesis in terms of cell populations and especially progenitor populations is incredibly important in dev and later on in life
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what progenitors are maintained?
FGF10 expressing progenitors maintained all the way through life
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3 ways FGF10 expr cells important?
1) involved in generation of particular classes of neurons that underlie homeostatic function 2) maintained- as from them infundibulum arises that forms hypo-pit axis 3) out of infundibulum get tanycytes that express FGF10
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in other regions of the NS where you maintain a neural SC, SCs that have a radial glial like morpholy derives from neuroepithelial cells what idea arises from this?
this FGF10 population (radial glial like) tanycytes might be derived from that earlier FGF10 neuroepithelial population
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what does the hypothalamus maintain?
homeostasis- optimal physiology- not the same throughout life
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the idea that came from changing diff states through life (like metabolism)?
maybe there is remodelling - new neurons, new circuits
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what model organism did people look at to see if hypo adapts? was it a good model?
the adult mouse- no kept breeding them selected out important survival mechanisms no predators or find food so people use hamsters where you don't breed out mechansims
528 of 761
what do you see in a slice through the median eminence is?
the hypo is arranged around the 3rd ventricle of the brain, the tanycytes extend all the way around the lumen of the 3rd ventricle, so in ventral 2/3rds cells that form 3rd ventricle are the tanycytes
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what do we catergorise tanycytes into?
diff subsets- tancytes that lie in a ventral region and project into the ME, then other project to particular nuclei of the hypo
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what do tanycytes form close appositions between that line the ME?
between the fenestrated capillaries and neuronal end feet of GHRH neurons
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what is one of the key nuclei that tancytes project to? what does it do?
arcuate nucleus- energy balance (eating) + reproduction
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why are tanycytes in a great place?
they can sample the CSF and blood
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how do they sample the blood?
they sit on fenestrated capillaries- end feet physical contact with blood, sample blood for levels of hormones. lipids, glucose
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how do they sample CSF?
they sit next to the 3rd ventricle that contains CSF, CSF is full of signals about internal body conditions
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what does homeostasis work on?
negative feedback loop- something changes then we bring it back to the norm eg stress -> cortisol up -> ACTH releasing hormone -> ant pit -> adrenal gland put back down stress so cortisol feedback to hypo to stop making ACTH
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What allows this feedback?
the fact tanycytes are there sampling the env
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why might hypothalamic tanycytes might be SCs?
look like radial glial cells, look like they're in a niche with blood capillaries, labelled tanyctes by protein selectivly translated in tanycytes
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what animal model did they use to show de novo neurogenesis in the adult hypothalamus that can be affected by long term changes eg high stress or high fat diet?
rat/mouse
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whats the first thing a cell has to do before it divides?
double up its DNA
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how do you mark S pahse?
Bromoxydeuridine- make a T analogue synthetically and label it in a way so you can detect it will introdue thymidine analgoue Brdu- then go in with an antibody to see if its dividing
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why did they use Brdu?
looking to see are there newly born neurons in the hypo- then will see divided cell will see Brdu incorporated
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what did they do with mice to see if neurons are dividing in the hypo (new neurons generated)?
anasethetize, use fine needle and inject Brdu into the third ventricle and so tiny diffuses into hypo- then say do we see any wait then section any new neurons have divided
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what did they find?
they found de novo neurogenesis- no neruons born in an adult mouse
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what did they get new neurons in response to?
acute physiological changes- eg fed mice high fat diet triggered neurogenesis or new neurons in sheep to anticipate oestrogen cycle or stress conditions
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what is the question they're trying to ask?
does an adult cell, a tanycyte give rise to a neuron? division in tanycytes doens't mean that tanycytes give rise to a neuron
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what do the tancyctes form a physical interface between?
between the brain and the body as sample the blood (body) which is critical for negative feedback loops for homeostasis
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how did they test whether new neurons came from tanycytes?
old: injected dyes now: genetic lineage tracing
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why don't we inject dye now?
dyes dilute out in dividing cells so can't mark all the progenitors so lose dyes, lose track of descendats
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what cells only express nestin?
tanycytes
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what does the promoter do?
determines whether a gene is transcribed- promoter/enhancers are activated in a cell specific manner so its the promoter thats important in regulating that gene
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how do they do genetic lineage tracing? (cre bit)
put cre under the promoter of a gene only expressed in the hypothalmus so clone this gene and GE to have cre downstream
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how do they do genetic lineage tracing? what do you cross with the cre mice?
cross with a transgenic mouse that has a reporter gene downstream of a constitutive promoter gene only expr in the hypothalamus but put in a nonsense DNA sequence that is floxed so LacZ/GFP will only be transcribed when cre is turned on
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Why do you have to do a conditional KO?
gene may play a role in early embryogenesis so dies before the animal develops
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what does cre do?
acts on lox sites and recombines the end, double stranded break and then splice together 2 cut sites
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so when cre is acting what will you see?
cells will turn blue
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what is the reporter gene under?
a constitutive promoter
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what is the advantage of GFP/LacZ?
they endure- you don't lose them as they divide- so animal now where a cell and all its descendants will now be blue/green
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how do you get a conditional KO at a specific time?
fuse the cre to ERT2 (estrogen ligand binding domain 2) that requires injection of tamoxifen to activate cre = cre only activated when tamoxifen injected into mice
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do how do you get that just in a cell that you think is a stem cell eg tanycyte?
put cre under a stem cell specific promoter, put reporter gene downstream of constitutive promoter with stop gene in reporter gene so when cre acts will cut out nonsense DNA and get lacZ expressed
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variation for lineage tracing from a SC?
Cre-ERT2 (fused), gene only expr in stem cell population put cre downstream of that gene and put reporter gene downstream of constitutive promoter- mark SC and all progeny
561 of 761
how does it mark SC and all prgeny?
as the SCs divide have the same genes and DNA so all have this LacZ and cre downstream
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what is temporal conditional tracing?
the cre recombinase is fused to ERT2 (estrogen ligand binding domain) so you inject tamoxifen when you want to activate cre
563 of 761
need 3 to lineage trace from SC in adult?
1) transgenic animal with cre downstream of promoter only expressed in that SC 2) cre-ERT2 so only activated in presence of tamoxifen 3) cross with transgenic animal with reporter downstream of consitituve promoter
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what gene did they put cre downstream of (only expressed in tanycytes)?
used GLAST (antibdoy recognises- onyl expr in tanycytes)
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normally tanycytes line all the way around the base of the 3rd ventricle but what did they find?
it was clear that there were molecularly distinct subset of tanycytes and were interested in the subsets that express FGF10 (which have GLAST promoter)
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how did they lineage trace from SC?
1) cre downstream of GLAST 2) cross with transgenic mice 3) inject tamoxifen in 5 consecutive day then recombine out lox sites 4) turns on GFP 5) do we see green cells being green neurons or green glia?
567 of 761
what did they find about tanycytes?
they're neurogenic and gliogenic- lots more tanycytes but have green neurons and green astrocytes
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what subset of tanycytes did they look at?
alpha tanycytes
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what can alpha tanycytes do?
self renew, give rise to ther tanycyte subsets, give rise to neurons and astrocytes
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in normal conditions are tanycytes dividing lots?
no in normal caged conditions the proliferation and neurogenesis/gliogenesis are very low
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when do yu get increased neurogenesis/gliogenesis?
when conditions change- to adapt, stress or change diet
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what is the problem with this assay?
cells initially labelled- 500 tanycytes lineage tracing from 500 cells so doesn't prove its a stem cell as definition of SC is that a single cell can self-renew and is multipotent can self renew to give itself or differentiates into diff cell types
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why can you not conclude that its a SC?
lineage tracing studies is done from a population of cells, definition of SC is a SINGLE cell that can do them things
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what would be the killer experiment to show tanycytes are a SC that hasn't been done yet?
kill all the tanycytes then put in 1 tanycytes see if it can restore everything and give many diff cell types
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what have they done in vitro to show this?
mechanically dissect out hypo, dissociate it down to single cells and plate 1 cell in 1 plate, then in well has 1 plate and if SC must proliferate so ask can give it rise to a neurosphere?
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what can you do to the neurospheres?
analyse neurosphere for expression of stem cell markers or you can put them under differentiation conditions and ask whether they can differentiate into diff hypothalamic neurons
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can in vitro tanycytes differentiate into diff hypothalamic cells?
yes they can - in vitro there is a cell in the hypo that looks like a neural stem cell potential- it can self renew, give rise to neurospheres, differentiate from those diff neurons formed
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what did neurospheric studies show?
that a lot of the hypothalamic neurons that you;ve got from tanycytes were neurons born in the arcuate nucleus
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where does the arcuate sit close to?
the ME
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what neurons does the arcuate nucleus contain?
neuropeptide Y and POMC neurons, dopinergic, GHRH, GABA
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what do neuropeptide Y and POMC neurons regulate?
energy homeostasis (food)
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the sorts of neurons that have been shown to differentiate from tanycytes in vivo include?
dopiminergic TH expressing neurons, neuropepitde Y neurons, GHRH neurons
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what do neuropeptide Y neurons regulating?
how much you want to eat, whether hungry or full
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if you can make new neuropeptide Y neurons in the adult what can you do?
you can reset your norm for energy homeostasis
585 of 761
what do dopimergic neurons play a role in?
play a role in reproductive behaviours- can make those, made to adapt to reproductive requirements probably
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what do GHRH neurons make?
GHRH which acts on the gonadotrophs in the anterior pituitary that secrete GH and you grow
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what are these behaviours not?
not linear through life, key times in life tanycytes make GHRH eg in puberty growth spurt- very dynamic
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where do we think these tanycyes are derived from?
FGF10+ embryonic multipotent hypothalamic population- first tanycytes express FGF10
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how do you get increased proliferative response of tanycytes (seen by BrdU staining)?
by elevating levels of FGF- so FGF that's there is under careful control and is part of the mechanism to get a quiscent tanycyte to be activated and begin to proliferate to give new neurons, FGF tilts that balance
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what makes tanycytes come out of quiscence?
FGF
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where was BrdU uptaken?
into tanycytes
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like the embryonic cell tanycytes continue to proliferate in response to what?
elevated FGF10 signalling
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why do you want a stem cell?
it gives flexibility, it lets you adapt, can change and build new neurons as you need to support changes through life
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what is the problem with a SC?
cancers- arise in proliferating tissue because things can go wrong, if you insult it one way or another eg eat too much/little then you stress system and spiral of dysregulation of homeostasis
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what is one core feature of a SC population?
you deplete SC over life so there's evidence emerging as you deplete the tanycytes over life the integrity of the ventricle has started to be disrupted
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what might tanycytes be linked to?
type 2 diabetes and alzeheimers
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what is tanycytes regulation key to?
normal homeostasis
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why are tanycytes important in the changing lifecourse?
you get new neurons involved in energu homeostasis an be generatd in adulthood from a FGF10/FGF10 responsive population
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in neural crest cell development in response to what?
BMPs + FGFs, came onto that to say interesting in hypothalamus get multipotent populations also expressing BMPs+ FGFs
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why is shh getting complicated?
involved in growth of different progenitors and get a resolution of a shh, FGF territory into 2 adjacent territories
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how do we do science?
build on what we already know, apply simpler system to complex system (the hypo)
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how does shh act in the SC?
its initially made in the notochord -> induced in the FP, protein diffuses establishes a conc gradient and cells respond to diff threshold concs and give rise to patterned progenitors that migrate out to give sets of different sets of differentiated
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how are sets of neurons arrayed in the SC?
along the DV axis
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where have 2 different mechanisms of shh been worked out? where is this relevant to?
worked out in the spinal cord but relevant to the hypothalamus
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what do the 2 mechanisms involve?
1) what happens in the producing-responding cell: how you control shh spread 2) what happens in the nucleus: how transcription of shh is governed
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what is the key receptor for shh?
ptch
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what are the obligate co-receptors?
hhip - hh interacting protein, gas, CDO/BOC
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what happens when a cell makes hh?
hh transcribed + translated -> shh out of cells -> when hh binds to ptch -> ptch stops repressing smo -> negative repression of transcr by gliR is allievated -> GliA activates transc genes
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what is one of the main transcriptional target that is turned on response to the signal?
the receptors
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what is one of the first thing shh does?
turn on the transcription of ptch itself + other co receptors- BOC/CDO and gas
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ptch working in combination of BOC/CDO and gas whats its normal function?
to repress smo
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once shh binds ptch what happens to ptch recs?
shh upregs ptch rec gene so more ptch receptors on the CS -> overall effect is non-bound ptch will suppress smo more shut it down
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what does the change in whether a cell is responding to a signal or not dependent on?
time
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what is ligand dependent antagonism?
the ligand turns on its own repressor
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if 2 cells next to each other and 1 molecule of ptch and there is one closer to the source will the one further from the source been activated by single hh?
no because it will bind to the first ptch on the first cell
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now if you make 100 hh molecules if there are more ptch recs in the first cell there's a more likelihood that what?
likelihood that hh will get bound in a non-productive way
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what situation do you get to?
depending on the number of ptch recs you've got on the cell the hh can or cannot diffuse more easily over a long distance
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if you've got more receptors then more molecules are going to what?
be bound and less are going to go further to further away cells
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what is LDA important for?
determing how far shh can diffuse, how local its effect is and how strong its act is
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what is the effect of LDA?
to steepen and restrict the gradient- to produce a smaller and more local effect
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what is a paradigm shift?
do experiments that follow whats gone before and sometimes an experiment makes you look at everything in a diff way- everything prior was not correct that's how hypo develops- things are working models
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absence of hh signalling?
ptch working with 2 other recs- Gas/CDO/BOC sitting at the M and represses Smo and if Smo not active then hh signalling pathway not active
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active hh signalling?
shh binds to recs --> repression of smo by ptch allievated --> smo activated
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what does smo do?
it activates the Gli TFs that turn on expression of a number of genes oincluding ptch1+2 and hhip
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in response to shh activation what do you begin to put onto the surface?
transcribe and translate and put onto the CS more Ptch1+2
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what is repressed in response to shh signalling?
CDO/BOC/gas
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what do CDO/BOC and gas usually do?
let ptch that sees hh operate so fewer cdo/boc/gas but more hhip + ptch1/2
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what happens when you have lots of recs that are repressors of smo and 3 molecules of hh?
all repress smo => balane is ptch thats repressing a smo so not get hh signalling acitvatd
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what is the negative feedback mechanism of shh?
downregulation of Gas/CDO and Boc and the upreg of ptch1/2
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what is in italics?
mRNA transcripts (will later be transcribed)
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hh feedback inhibition?
ptch 1+2 and hhip bind to hh ligands and compete with productive ligand-receptor interactions to alter the balance between bound and unbound ptch
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what is the key?
balance of bound versus unbound ptch- unbound ptch can modulate smo acitvity so won't get shh signalling that cell.
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what is important at imagining?
imagine there's another cell and if you have so many more hh recs on the first cell if if they're not productive they bind+mop up hh so shh captured none left for the next cell
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that effect of LDA in cell 1 has implications for what?
what happens in a distal cell in a non-autnomous manner 'adtitionally ligand sequestration by CS hh antagonists results in non-cell autonomous hh pathway inhibition in distal cells' (to source)
635 of 761
describe idea at first
2 cells, source of shh- have 3 recs of ptch on c1 and 2 recs on c2 and make 8 shh diffuse and bind to the recs on c1 but 3 more go and bind c2
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after shh binds to ptch recs on c1?
turn on shh on cell 1 upreg ptch1/2 + hhip over time they're put onto the CS now same num of shh mols but because there's more ptch on cell 1 less will reach cell 2
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but what happens to cell 1?
more shh bound in an unproductive way so the ratio of ptch:smo is tipped so smo won't be repressed in this cell
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because you've captured more molecules on the first cell what does this mean for cell 2?
there's fewer molecules gong to the second cell so there's less likelihood that the 2nd cell will be activatd
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what is the effect of LDA?
to steepen and restrict the gradient, produce a smaller (could be larger) and more local effect- distance from source reduced, alter high point of shh + restrict how far it can go
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how do you stop LDA?
stop expressing shh and shh receptors then you can stop LDA in that region
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what does shutting it down by stopping expressing shh and shh recs?
LDA is stopped in tat region which will support the continual spread of shh into neighbouring cells. Mechanism stopped then get higher shh and shh operating over more of a distance?
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how do you stop LDA?
get rid of shh expression and ptch expression then the loop stops
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if you want to go from LDA to without how do you shut down shh and shh signalling completely?
if briefly that means you restore high shh activity over a longer distance well in response to very high levels of shh it induces shh itself
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what induces shh expression?
high shh levels- shh is only induced by very high levels of shh
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by shutting down shh + shh signalling briefly induces what?
re-initiate its expression because you've restored the levels of activity which will induce shh transcription again and it starts again
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how could you shut down shh, how do you regulate transcription of a gene at mRNA level?
genes regulated by multiple TFs
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where did our understanding of this come from?
from what is going on in the spinal cord because progenitor genes expressed along the DV axis are regulated by the activity of multiple TFs that act on the enhancers of a gene
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what are upstream elements?
upstream regulatory sequences that are enhancer elements that govern whether the promoter is active or not
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BMP antagonists i(chordin, noggin, follistatin) induce which genes?
Pou and Sox2
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What are Pou and Sox?
these are TFs that bind to enhancers and turn on neural genes and these neural genes are expressed throughout the VZ- early stages when induce NP under BMP antagonists. generalised factors to activate many many downstream general neural TFs
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initially these general neural property TF will be expressed where?
throughout the neuroepithelium then as NT folds restricted to the VZ (throughout DV axis)
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what does shh do when its activated by the notchord and FP?
it activates a homeodomain TF in response to high levels of shh
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what does that Gli activated homeodomain TF do?
binds to the enhancer of the same gene but its a repressor so consequently you repress transcription of that gene so wherever homeodomain is on pax7 is off
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how are genes usually regulated?
not regulating genes by activating them but mainly be removing repression
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what is the take home message from the SC studies?
enhancer elements are really important
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what did they find out was different about the regulation of shh in different places?
in the hypothalamus the transcription of the shh gene is regulated by a different enhancer element compared to the future spinal cord/hindbrain and midbrain
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who found the enhancer for shh gene was diff in hypo?
Doug Epstein working in mice
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what is the enhancer in the hypothalamus called?
SBE2- shh brain enhancer 2
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what did Doug Epstein do?
looked at shh gene in the mouse looked at all the DNA around the shh gene, then mutated little bits of looked to be regulatory elements and made mice in which they're mutated and said what happens to shh?
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what happened when he mutated SBE2?
alterations in the regulation of shh just in the hypothalamus- special enhancer just regulating shh in the hypo
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what do you normally need to activate the shh gene?
GliA and Sox2- 2 activators
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what is sox2?
general neural gene
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why do you need GliA?
need glia activator- high levels of shh signalling to activate shh itself
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What's different about the enhancer in the hypothalamus?
in the hypo there's an enhancer that only matters in the hypo, it encodes a binding site for a TF = T box TF Tbx2
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what happens if Tbx2 protein binds to the enhancer what does it do?
Tbx2 comes along and its sobig it displaces sox2 so consequently you don't activate shh, no shh mRNA degrades transcription ceases
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how do i look at this in the contect of the earlier lectures?
how are hypothalamic cells induced- from signals from the PCM = shh itself early on shh from early PCM induces in NP cells GliaA and Sox2 they activate shh transcription but later PCM change
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what happens when PCM signalling change?
turn ons BMP7 and BMP7 induces Tbx2 in those cells, Tbx2 displaces sox2 so you downregulate shh in those cells and shh transcription in those cells ceases
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in early embryogenesis get a hypothalamic progenitor that briefly coexpresses BMP, shh and FGF10 but then what?
in some way the cell that cell expressing shh,FGF/BMP goes on to establish other progenitor populations so shh+ population and FGF10+ population
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what does the maintenance of that FGF10 positive population depend on?
feedback signals from that FGF10+ population- those feedback mechanisms depend on the feedback of shh to the FGF10 progenitor to transiently re-induce a FGF/shh cell
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what do we want to understand?
how you get the progenitor populations as maintaining that population of FGF10 progenitors are important and the maintenance of that population is dependent on getting a loop of generating shh/fgf cell that resolves and restablishes a shh/fgf dont un
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why do we want to understand how that happens?
think it holds the clue to how you maintain the FGF10+ cells all the way through life
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where is the working model at at the moment?
in the hypothalamus a mechanism where the expression, concentration and spread of shh are very carefully regulated could help to explain how we generate the key progenitor types that build the hypo and maintain progenitors in the adult
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understanding how these progenitors cells are established is going to be important for understanding what?
how the hypo is built in a way that's sustainable over the life course
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what is the current working model?
this is to do with the way shh is downregulated by BMP7 -> Tbx2 and how that involves a LDA mechanism
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what is the idea?
start off with shh from PCM inducing a shh+ cell in NP cells above -> PCM change -> upregulate BMP7 -> upregulates Tbx2 -> displaces Sox2
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at this point in time what do you still have going on?
LDA because you've just got shh and so you have LDA working so shh can't diffuse very far
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but as Tbx2 downregulates shh what do you also get the downregulation of?
the downregulation of ptch (as upreg in response to shh) now you prevent LDA so shh is now at higher levels and allowed to diffuse so the next cell along (a progenitor) becomes the cell induce shh so now source of shh acts back on FGF cell upreg shh
678 of 761
what induces pre-hypothalamic cells at the ventral midline?
shh and nodal (from PCM) induces expression of shh in the midline cells of the hypothalmus, shh turns on Ptch
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what happens at a later time?
BMP7 upregulated in the PCM which upregulates transcriptional repressor displaces sox2 so downregulate shh
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Tbx2 and BMP induce what?
FGF10 so now you get a transient population of shh/FGF cells but almost immediately tbx2 downregulates shh but FGF promotes proliferation so population proliferates as downreg shh prevent LDA
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what do you get?
a cell thats further out where its divided is induced to express shh so you get a spread of shh (FGF progenitors in middle and shh around it) but constant reinduction of FGF10/shh around the edge
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what do they think plays a role in establishing htis loop of progenitors?
LDA
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what do the 2 papers show?
how early patterning events like shh or how establishment of the shh/fgf progenitor domains are important for differentation of particular classes of neurons mediate different functions
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how is sleep controlled?
have neurons that control sleep- 2 catergories sleep promoting and sleep awakening neurons
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what is sleep pressure?
the different neurons respond differently to sleep pressure. A lot of sleep pressure then sleep promoting neurons become activated
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what neurons are activated when there's a lack of sleep pressure?
sleep awakening neurons
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how do we know about sleep in early studies?
through studies of people who had narcolepsy- fall asleep for no apparent reason = excessive daytime sleepiness
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what did they find with narcolepsy patients?
there's a neuropeptide called hypocretin that is made by hypothalamic neurons, found narcolepsy patients had very low levels of hypocretin
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how could you test if the correlation with low hypocretin levels and narcolepsy means hypocretin is needed to be awake?
KO hypocretin- make a KO mouse and see that mouse fall asleep all the time without hypocretin (do expers at night as mice nocturnal)
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what is this a good indication of?
this neuropeptide hypocretin is needed to wake up
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what is this research end goal?
to be able to do cell replacement therapy- take hypocretin expressing neurons generated in vitro and stick into someones brain but could work out new therapeutics if had a better source of neurons making hypocretin
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what is the starting point?
how could we make neurons that secrete hypocretin
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what could having a dish of neurons that just make hypocretin could be valuable for what?
screening purposes for new drug developments
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what kind of TF is Lhx9?
A homeobox domain TF
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what is the paper saying?
hypocretin neuron sare beingregulated in a way that's being conserved throughout evolution by this TF Lhx9
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where is hypocretin expressed?
hypothalamic neurons
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so where would you expect Lhx9 if its controlling them neurons?
in the hypothalamus
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what do you expect the paper to show?
that hypocretin neurons are in the F hypothalamjus and Lhx9 is in the hypothalamus and claiming Lhx9 regulates neurons show this by LOF + GOF
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what would you expect to see if you LOF Lhx9?
no more hypocretin
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if you overexpressed Lhx9 what would you expect to see?
more hypocretin
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loss of neurons that secrete hypocretin have been implicated in what?
narcolepsy
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what did they do using zebrafish (in abstract)
screen for factors that can specify hypocretin neurons in vivo, screen to look for factors that can specify hypocretin neurons and identify Lhx9
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what do you have to do to show somethings necessary and sufficient?
necessary- LOF sufficient- GOF
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how does Lhx9 induce hypocretin neurons?
directly induces hypocretin neurons- directly binds to the promoter of the hypocretin gene to upregulate it
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what does microarray analysis identifies transcripts enriched in hypocretin neurons mean?
if TF is specifiying a cell type you'd expect that TF to be expressed specifically in that cell type- do by transcriptomic analysis burst open cells and interrograte every single mRNA in that population
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where did they find hypocretin neurons expressed?
dioxygenin labelled probe (blue) in the ZF hypothalamus
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what did they do to make a transgenic ZF?
made a transgenic ZF in which the hypocretin promoter is controlling RFP- shine fluroscent light onto animals then see red fluroscence whether hypocretin neurons are
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why would you bother make a transgenic line to just pick out hypocretin neurons?
if you take this ZF line and dissociate all of the cells and then shine fluroscent light onto them only hypocretin expressing cells will be red so can FACs cell sort- collects hypocretin neurons get a dish of isolated hypocretin neurons
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then what do you with them hypocretin neurons?
you take them cells and interrogate them (microarray)- say what mRNA are they expressing at particularly high level (bioinformatics)
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what did they come up with?
a list of TFs that are enriched in hypocretin neurons
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how did they check that the trasncriptomics analysis (microarray) was correct and Lhx9 was expressed in hypocretin neurons?
did in situ hybridisation- expression patterns of candidate genes validate the microarray result
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where did they find hypocretin and lhx9 expressed?
hypocretin neurons are lying within a broader set of cells that express Lhx9
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what did double in situ hybridisation show?
double in situ hybridsation expression overlaps with expression of hypocretin (EFP transgenic line)
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what is this saying?
Lhx9 is expressed in vivo in hypocretin neurons (transcripts expressed in the right place)
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how did they show Lhx9 is sufficient to specify hypocretin neurons?
to determine whether any candidate genes is sufficient to induce specification of hypocretin neurons we cloned each gene downstream of a heat shock inducible promoter and vector (put into transgenic animal and turn on expression of that gene)
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what is Lhx9 coding sequence downstream of?
a heat shock promoter- a promoter that is turned on when you elevate the ZF embryo temperature
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what did they do when they ectopically expressed Lhx9 with a heat shock promoer?
saw extra hypocretin positive neurons
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you normally don't see hypocretin there why do you now?
Lhx9 is being expressed there and that's why we have more hypocretin positive cells = lhx9 is sufficent to specify hypocretin neurons
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what was wrong with this?
why were hypocretin neurons only expressed there? probably due to other competency factors we don't know about
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what do they go onto show?
they show that these hypocretin neurons can be defined by markers express the same markers as hypothalamic neurons and hypocretin enurons normally project to the locus coerulues and ectopic neurons project there to = not just expr hypocretin but is
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why did they do all this other stuff to show that these ectopic neurons were like real hypothalamic hypocretin neurons?
just because there was hypocretin mRNA expressed does that mean they are hypocretin neurons- have same signature + project to LC
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how did they show Lhx9 is necessary for hypocretin specification?
KO 2 methods: 1) morpholinos and 2) CRISPR-KAS9
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what do you see with the morphilnos and crispr-kas9?
reduced or total loss of hypocretin expression = no Lhx9 don't turn on hypocretin
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how did they show that Lhx9 TF directly binds to the hypocretin promoter to turn on transcription of that gene?
performed double fluroscence ISH against Lhx9 and hypocretin on ZF embryos fixed 1hr after heat shock
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what did they observe?
see hypocretin expression in almost every cell that;s overexpressing Lhx9 1 hour after
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what killer experiment did they do?
in a previous study where Lhx9 was reg other cell types/genes Lxh9 turns on WT but worked up enhancer that Lhx9 binds to drive gene expression looked for similar DNA BS upstream of hypocretin gene
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what was the thought behind this?
maybe Lhx9 governs all the genes it regulates by this enhancer
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what did they do?
mutate that binding site in the enhancer upstream of Lhx9 and then make a transgenic animal and see if Lhx9 is no longer able to activate transcription of hypocretin
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what do you do to show that a TF is directly regulating a gene?
show where its normally binding in the enhancer, mutate enhancer and show its not doing what it would normally do
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what was the paper saying?
know hypocretin neurons are important in controlling sleep and now know how in dev hypocretin neurons are regukated (by Lhx9)
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how is the evidence that hypocretin neurons regulate sleep indirect?
its a correlation that in humans with narcolepsy have a tendency to sleep or if you KO hypocretin in mice get more sleepy but not direct evdiece hypocretin neurons are needed to be active to stimulate wakefulness
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what does the locus coerulues seem to be crucial in establishing?
the appropirate dynamic of spontaneous awakenings
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what principle underlies optogenetics?
if you know the control elements that are specific in a cell then you can activate that cell by light
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what happens in an optogenetics experiment?
in a transgenic mouse so that just hypocretin neurons are activated by stimulating them with light, attach electrode into them, press a button and activate a specific set of neurons
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what are they stimulating neurons involved in?
aggression, dominance, sleep wake, eating- all dependent on knowing the genetic control elements that build the neuron so huge push
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what are Lhx9 neurons induced by?
Shh- early patterning in the arcs of progenitors. Lhx9 + 6 probably derive from the Pax6 population
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how do you go from a progenitor cells that specifices hypocretin neurons that when they're active they wake the animal up?
shh from PCM -> arcs of progenitors -> progenitors expressing pax6 -> turn on Lhx9 -> specifies hypocretin neurons -> sleep promoting neurons
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in they hypothalamus what are you doing all the time?
setting up sets of neurons that have opposing functions and that behaviour is regulated by who wins out
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what does the Liu paper start to say?
we've now characterised a number of wake promoting neurons eg hypocretin + other populations but there was few sleep promoting neurons identified neurons
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what do sleep promoting neurons respond to?
sleep pressure- as the day goes on your sleep pressure increases to a point where you activate neurons that are sleep promoting activate and fall asleep
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what does the Liu paper say?
there's a TF Lhx6 and they find they can idneityf a subset of neurons based on expression of Lhx6, they also express GABA a main inhibitor
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where are these GABAergic neruons?
in the miuse ventral xona incerta
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what kind of TF is Lhx6?
A lim homeodomain TF
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what did the paper show?
a subset of neurons that express GABA in the ventral zona incerta (hypothalamus) express a LIM homeodomain TF Lhx6 but they're activated by sleep pressure
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how could you show that a neurons activated by sleep pressure?
tell whether the cell is activated- cFos is an immediate early response gene
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what is the paper saying?
in response to sleep pressure you begin to activate these neurons and they project to the hypocretin neurons and they inhibit them
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what is the mechanism for falling asleep?
making sure you inhibit the neurons that are keeping you awake, a lot of 2 systems that have oppositions- constant battle
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what did they do to Lhx6?
conditionally deleted it in an adult mouse- get rid of neurons that make you fall asleep so they found mice didn't sleep
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how do you know where Lhx6 neurons normally project in the brain?
normally project to the hypocretin neurons and inhibit them- know this by making a trangenic mouse with a reporter gene downstream of the Lhx6 promoter.
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what did they show about the visual system?
that bits of the visual system usually feed into the sleep circuitry
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why is the sleep circuits set up so we're optimally awake when its light and asleep when its dark
predators etc
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what technique is important to trace neurons?
transgenic mice- can follow cells that follow Lhx
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what are Lhx9 and Lhx6 neurons probably induced by?
shh signalling
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what do people think about this?
you set up ultimate function many years before it happens eg week 20 embryogenesis
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why is this important?
more and more people are interested in how disruptions in embryogensis have an effect in later life- resillence to stress or obesity, non-resilent populaton dependent on what happened in embryology
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why is earlyness important?
set up everything early
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why is it important to undertsnad how the hypo develops?
understanding that to understnad how circadian cycle, eating, reproduction and sleep-wake come about
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what do tanycytes generate neurons that do what?
regulate feeding and reproduction
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another paper showed what about circadian cycle?
Lhx1 population (some induced early by shh, some derived by shh/FGF progenitors) but 3rd LIM homeodomain TF if you delete that in mouse you don't get neurons of the suprachiasmatic nucleus which regulates circadian cycle
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lhx1 develops when?
when shh patterns arcs of progenitors - important for early develeopmental steps for later development
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in vertebrates what do early neural cells show?

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how does the neural plate cells form?

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