Developmental Biology

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  • Created by: Sarah
  • Created on: 11-04-17 07:51
what was the critisms of Gurdons first nuclear transfer in xenopus?
tadpole gut cell not fully differentiated- stem cell like turnover and low frequency of success (no adults)
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what was Briggs and Kings experiment for nuclear potency?
used Rana(frog), needle to extract chrs out of egg cell, egg cell with no GM, replaced GM with nucleus from cell in blastula stage embryon--> normal tadpole, blastula nucleus pluripotent
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what are the 2 hypotheses to explain why cells lose potency?
1) gene loss 2) differential gene activity
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what experiments show that genes are not lost when cells differentiate, expression controlled by cytoplasmic factors and gene expression of differentiated can be changed?
lens regeneration in newt, human liver cell fuses with rat muscle cell expresses human muscle proteins, xenopus kidney cells in pleuroedeles oocyte produces xenopus oocyte proteins
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what happens in lens regeneration in newts?
lens usually produced from placodes, when lens destroyed iris dorsal cells proliferagion, de-differentiation and re-differentiation, iris cells make crystallin proteins for lens
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exceptions to gene loss rule?
1) gene arrangments in b lymphocytes 2) chromosome dimuntion fragmentation in unwanted chrms in parascaris except in germ plasm which become germ cells
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what can now be done to show that muscle protein genes are in white blood cell DNA?
genomic sequencing
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what marker did they use in dolly the sheep?
surrogate had black face, cell from the mammary epithelium were white
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when were the cells best for the mammary epithelial cells?
in low serum, exit mitosis and enter gap 0 phase
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how did they check the authenticity of dolly the sheep?
DNA fingerprinting (microsatellite analysis), DNA sequencing
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what is dorsal?
transcription factor
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what does dorsal activate?
mesoderm ventral side of embryo zygotic genes transcription (twist and snail)
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what genes are on the ventral side of the embryo?
twist and snail
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what are dorsalized class of mutant required for ventral fates?
STD, Spatzle, Toll and Dorsal
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what is spatzle in the toll pathway?
the ligand
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what is toll?
transmembrane receptor
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on what side does dorsal enter the nuclei?
ventral side (where toll signalling is active)
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why is the toll pathway only active on the ventral side?
spatzle ligand only present on the ventral side in the perivitelline space, reflects DV patterning, set up signalling between follicle and oocyte cells
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how did they show myod is sufficent for muscle development?
transfect fibroblats (with no Myod) with Myod -> differentiates into muscle therefore sufficient
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why is myod not absolutely required?
when no Myod muscle still develops because of redudancy another TF- Myf5
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what is redudancy?
having several genes that perform the same role to some extent, if ones not present the other jumps in
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what are stem cells?
undifferentated, they renew cells that are continually lost or repair tissue
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what are stem cells in muscle?
satellite cells
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where do satellite cells lie in muscles? what do they express?
between basal and cell membrane, expresses marker proteins pax7
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how is the Myod expression maintained?
it maintains its own expression by positive feedback
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what are cell-type specific proteins in muscle?
muscle specific actin, myosin 2, tropomyosin and enzymes (phosphate kinase)
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what does ultrabithorax act as a selector gene for?
haltere identity, T3 metathoracic pathway
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what is the ultrabithrax mutation what does it cause?
recessive mutation, causes transformation of T2 to T3
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What is a homeotic mutation?
one structure is replaced by another, does not alter the number of segments
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what is an example of non-autonomous function
signalling molecules (affects other cells)
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what are the 2 hypotheses to restriction in potency?
1) differential gene activity 2) gene loss
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what happened in the experiment with lens regeneration in the newt?
lens removed -> proliferation of cells in dorsal iris -> synthesis of crystallin proteins (found in lens) -> iris cells have genes for crystallin can make lens
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what does the experiment with human liver cell transplated into s rat muscle cell (heterokaryon) tell us?
human liver gene off, human muscle gene on, cytoplasm induces change therefore genes not lost as can change cell types
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what is holoprosencephaly caused by?
mutations of hh and loss of signalling
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why does a steroid alkaloid casuse cyclopoedia?
blocks hh pathway and inhibits action of smo pathway
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why do we need animal models of human disease?
development of diagnostic tools, dev of therapeutic (stem cells, pro structure for drug design)
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where is shh signalling expressedin the limb bud?
in the limb bud, at the midline (notochord+floorplate)
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what is the transcription factor in the shh pathway?
gli
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what is the TF in the hh pathway (invertebrates)?
ci
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what happens in prexial polydactylyl?
extra digits
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what is preaxial polydactylyl caused by?
overactivity of hh signalling (mutation in a shh enhancer region)
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what can result with problems in shh signalling?
preaxial polydactylyl, holoprosencephaly, basal cell carcinoma (cancer)
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what happens in holoprosencephaly?
cyclopia-eyes fuse together, range of midline malformations (undivided midbrain)
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what is holoprosencephaly caused by?
loss of function of hh signalling (dominat mutations in the shh gene)
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example of gain of function hh signalling?
preaxial polydactyl
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example of gain of function hh signalling (dominant mutation in shh)
holoprosencephaly
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what is cyclopamine?
steroidal alkaloid
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where is cyclopamine from?
californian corn lily- plant
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what is an example of a compound that disrupts shh signalling?
cyclopamine
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what is a teratogenic effect?
compound that disrupts shh signalling
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what does cyclopamine cause in the early embryo?
cyclopia, mimics genetic loss of shh function
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how does cyclopamine prevent shh signalling?
cyclopamine binds to the smoothened protein
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why do developmntal signalling pathways (like hh) need to be kept ast a low level in adults?
inappropriate activaton can cause cancer, act like stem cells, cells divide
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what is basal cell carcinoma caused by?
1) too much sun 2) hereditary-disruption in genes in hh pathway
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an amorphic loss of function of what gene causes basal cell carcinoma
PTC
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a gain of function (hypermorphic) mutation in what causes cancer?
SMO
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what happens in the hereditary form of basal cell carcinoma?
stem cells in stratum basale layer inappropriately inactivated
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what does inappropriate activation of the wnt pathway cause?
APC mutation and colon cancer
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how are cancer cells like embryonic cells?
rapidly dividing, undifferentiated, epithelial to mesechymal transition (metastasis)
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how are tumour cells different from embryonic cells?
escape normal controls on: proliferation, growth and differentiation
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why do most cancers occur in epithelia?
they contain many stem cells that continue to proliferate and fail to differentiate
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what is SMO?
proto-oncogene
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what kind of mutation does SMO cause that causes cancer?
gain of function (hypermorphic)
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what are tumour supressor genes in cancer? hh example?
loss of function genes eg PTC (amorphic, hypomorphic and antimorphic)
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what is an antimorphic mutation?
a dominant negative mutation, mutant copy produces a product that inteferes with WT function
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what is a nonsense mutation?
changes amino acid to stop codon
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what is an amorphic/null mutation?
one that gives complete loss of gene function
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how would you treat a cancer caused by overactivation of Hh signalling?
treat with GDC-449 (similar to cylopamine)
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what is the problem with treating cancer with drugs like GDC-449 which disturb the Hh pathway?
treatment is short lived
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what is the name used to decribe development where cells are determined late/not early in dev?
regulative
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what is mosaci development?
cells are determined early in development
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what are the features of mosaic development?
autonomous fashion, isolated cells develop as normal, a defect in part of embryo doesnt affect the other part
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what experiment was used to show mosaic development?
roux, inserted a hot needle into 1 cells of 2 cell embryo and one side developed normal embryo but other half dead cell
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what experiment shows regulative development?
driesch, took sea urchin embryos at 2 cell stage used a fine babys hair to separate 2 cells and got smaller fully formed larva
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what kind of development is unusaul?
mosaic
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why is regulative development more common?
cells signal to each other
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is patterning in development autonomous or non-autonomous?
mixture of both!
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what is autonomous mechanisms?
segregation of cytoplasmic components
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what are non-autonomous mechanisms?
cells signalling to each other, uses receptors usually
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why do we label cells?
to follow cell lineage and identify cell fates
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what do cells pass through as they differentiate?
different states of commitment- specification, determination
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what is an example of something that signalling between cells using receptors?
growth hormone
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3 types of signalling?
1) diffusible signals with rec 2) juxtacrine signalling both on m 3) diffusible signals with intracellular recs (steroid hormones)
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what is a morphogen?
chemical that gives rise to correct morphology, chemical (usally diffusible signal) forms a concentration gradient, produced from a localised source, usually sensed thro recs, cells respond diff to diff concentrations of morphogens
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what is the french flag model?
colours-pattern of cell types you want to achieve, have a graded concentration of morphogen, concentration of morphogen depends on position of embryo
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what development does the french flag model account for?
regulative dev
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what happens if the line is cut in half in the french flag model?
system will regenerate
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what is the bicoid mutant phenotype?
lacking head structures at anterior end
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what did researchers hypothesize about the bicoid mutant?
there must be something at the anterior head that produces head structures
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how did they test that hypothesis?
took cytoplasm from WT and transplanted it into anterior of bicoid mutant egg then some anterior structures develop
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where is bicoid protein concentrated in the egg?
at the anterior end steep gradient towards posterior
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whats more tightly held in the anterior part maternal bicoid mRNA or bicoid protein?
maternal bicoid mRNA
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what is bicoid? what does it turn on?
a transcription factor, turns on hunchback but only above a certain conc
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how do we know hunchback is dependent on bicoid?
if we look in a bicoid mutant theres hardly any hunchback
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what kind of gene is a hunchback gene?
gap gene
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where are maternal genes made?
in the nurse cells in the ovary
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what is an example of a maternal gene?
bicoid
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whats the rle of bicoid?
activates zygotic genes, start dev of gap genes hunback, segmentation cascade
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where does bicoid form a gradient?
in the synctial blastoderm just lots of nuclei in one cytoplasm, no cells yet
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what is induction?
signalling from one cell type to another getting a change in responding cell (often specification)
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what is the amphibian organiser?
cut out small piece of tissue from blastopore lip on dorsal side and transplant it to ventral side of another embryo
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what result did the amphibian organiser give?
produced a second axis in the embryo
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where was the secondary axis derived from?
the host tissue
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what tissue was induced to form the axis?
the host tissue-responds to sigs
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what effect does the timing of induction in the amphibian organiser have?
early gastrula- clear 2nd axis, late gastrula- less affected, longer you leave it less competent to respond
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why is the tissue that induces a secondary axis called the speman organiser?
it organises the tissue around it
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what is competence?
the ability of tissue to respond to a signal
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what may competence involve?
expression of appropriate rec molecules
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what is the xenopus animal cap assay?
cut out middle cells, put animal cap directly on vegetal cells
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what happened in the xenopus animal cap cell?
cells like notochord and muscle were produced mesodermal cell fates that were usually made by the middle cells that were removed
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what is the inducing tissue?
vegetal cells induce animal cap cells
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what tissue is competent to respond in the animal cap assay?
animal cap cells
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what are animal cap cells fated to be?
neural tissie
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what are animal cap cells specified to be?
epidermis
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what can animal cap be induced to become?
muscle tissue
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is FGF signalling necessary or suficient for limb bud formation?
necessary and sufficient
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what kindof tissue is in thelimb bu?
meschenymal withetoderm on the outside
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what isthe apical ectodermal ridge?
a ridge of thick epithelium around the limbud
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what lost limbs in evolution
snakes
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what is the role of the apical ectodermal ridge?
1) keeps limb bud flattened, 2) proliferation in the progress zone
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what forms first in the chick the limb bud or axes?
axes
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what are limb buds?
appendages to the basic body plan
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how many limbs do vertebrates usually have?
4
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What happens if you dissect the apical ectodermal ridge off?
Limb dev outgrowth stops, only get proximal structures
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What happens if you dissect the apical ectoderm ridge off at later stages?
More distal structures form, more outgrowth but no digits
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what happens if you remove apical ectodermal ridge and replace it with FGF4 protein bead?
FGFs substitute for AER and allow outgrowth of limb
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what is the fgfs in the AER signalling to?
the progress zone
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what is the progess zone
a distal region of mesenchyme tissue
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what is the progress zone derived from?
mesoderm
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AER secretes FGF to progress zone what does this do in the progress zone?
undifferentiated cells rapidly divide
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what does the length of time in the progress zone affect?
length of time determines cell fate
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how did they test the role of the progress zone?
grafting experiments of old and young limb buds
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what happens with an old stump with young tip?
duplicated some of the structures in middle of limb
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what happens if you have a young tip with old stump?
distal elements attached to humerus
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what is length of time in the progress zone setting?
the proximo-distal axis cell fate
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short time in the progress zone produces what cell fate?
proximal cell fates
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longest time in the progress zone gives what strucures
distal cell fates
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how did they show FGFs were sufficient for limb bud formation?
FGF4 protein bead induced ectopic limb buds
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whats the zone of polarizing activity
region of posterior limb bud mesenchyme which when transplanted under the AER induces symmetrical duplications of normal limbs along the AP axis
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is the back of your hand ventral or dorsal?
dorsal
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what does transplanting the zone of polarising activity from posterior to anterior do?
gives mirror image
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what axis does the zpa affect?
AP axis
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what did they think might be in the zpa giving the mirror image?
morphogen?
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what is being expressed in the zone of polarising activity?
shh
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how did they prove shh was in the zpa>
bead of shh to anterior did the same mirror duplication as the zpa transplant
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what hox genes are expressed in nested domains in the limb bud?
hoxa and hoxd
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what did deletion of hoxa and hodd do?
absence of shh expression and truncation of the limb
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what is it thought that hoxa and hoxd do?
specify the zpa,where shh expression is going to be
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a zpa graft sets up new expression of what
hox genes
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what is therre a feedback loop of in the zpa
hox genes and shh expression
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2 ways digits are formed?
1) differential growth (amphibians) 2) cell death mammals+birds
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what controls the pattern of cell death?
mesoderm
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transplantation exps of limb buds of chickens and ducks showed what?
the mesoderm of the limb determines pattern of cell death
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if you take mesoderm from chick limb bud and ectoderm from duck what do you get?
a chick foot
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if you put duck mesoderm with chick ectoderm the foot is what?
duck foot
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what is cell death for digits regulated by?
hox genes
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what is the wing of a bat?
its fingers!
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why has snakes lost limbs?
expansion in domains of hox expression prevents FGF being expressed
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how do we know FGF is necessary for limb bud formation?
KO of Fgf10 (ligand) or FGFR (rec) genes result in embryos lacking limb buds
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what do hox genes do in limb bud formation?
restricts expressionof FGFs to limb forming regions
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what does the hoxb5 KO mutant give?
limbs develop at a more anterior level
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what is forward genetics?
mutant phenotype -> cloned gene
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what is an example of reverse genetics?
mouse KOs
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what is reversed genetics?
cloned gene -> mutant phenotype
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what genes do mesoderm express?
twist and snail
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what group of cells are set aside in the neuroectoderm (on ventral side)
proneural cluster
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what genes do the proneural cluster express?
achaete and scute
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AP axis triggered by what?
bicoid at anterior of embryo
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what is the primary fate of he proneural cluster?
neuroblast
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what is the secondary fate of the proneural cluster?
the epidermis
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where is the neuroectoderm in the embryo?
ventral side
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how does neuroectderm turn into a neuroblast?
lateral inhibition- inhibits the cells around it
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what happens in homozygous loss of function mutants for notch and delta?
all cells in the proneural cluster become neuroblasts
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what does it mean that the proneural cluster is an equivalence group?
all cells have an equivalent competence to form the neuroblast fate
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what s the receptor notch or delta?
notch rec, delta-ligand
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what kind of signalling is notch and delta?
juxtacrine signalling
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what does the notch delta oathway do?
expresses genes by disocciating repressor
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what is the notch delta pathway for lateral inhibition?
cell 2 expresses delta (ligand) more strongly, activates notch rec in cell 1 and notch pathway > down regulates delta in same cell as notch (cell 1) -> notch on cell 2 weaker, delta on cell 2 stronger
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stronger delta signalling gives what cell?
neuroblast
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cell with stronger notch activity gives what cell?
epidermal cell
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what happens when cells are specified to become neuroblasts?
undergo a dramatic change in behaviour, lose junctions with epithelial neighbours, delamination, move into embryo, localising determinats
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what determinants are localised to a neuroblast after specification?
segregation of cytoplasmic components- numb (before cell division) + prospero
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how is the spindle in the neuroblast determined for cell division?
spindle forms perpindicular to where numb is placed
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the cell with the numb segregation becomes what?
ganglion mother cell
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what kind of divisions do neurblasts show?
asymmetric
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what kind of fate division is assymetric division of neuroblasts?
autonomous cell fate as cytoplasmic segregation
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what does the neuroblast remain?
a stem cell
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what does the progeny of ganglion mother cell become?
neurons
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what are mechanosensory bristles for?
mechanical movement like dust
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whatis the bristle embedded in?
the socket cell
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what encapsulates the sensory neuron in the sensory bristle?
the sheath cell
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what are the parts of the sensory bristles?
sensory neuron, hair cell, socket cell, sheath cell
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where are the proneural clusters for the sensory bristles?
imaginal discs
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what are the imaginal discs for?
adult structures in the fruit fly
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what is the precursor for the sensory bristle?
sensory organ precursors
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what singles out sensory organ precursors from proneural clusters?
delta-notch signaalling
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the cell with more what becomes the neuroblast
delta
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how does the sensory bristle cells form?
Sensory organ precursors are singled out from proneural clusters by delta-notch signalling it is then undergoes further divisions
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what does 2a from SOP form?
socket and hair cell
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what does 2b give rise to?
sheath and neuron
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how does a SOP divide?
asymetric division in plane of epithelium
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the SOP cell divides, the cell with numb becomes what?
a 2b cell (sheath and neuron)
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what is the function of numb?
inhibits notch function
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what cells have high notch activity from divsion of SOP?
2a and socket and sheath
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loss of notch (high numb inhibits) gives what?
4 neurons
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too much notch (less numb) gives what?
4 socket cells
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what is each sensory bristle organ?
a clone of 4 cells
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where does the 4 cell of the sensory bristle derive from?
sensory organ precursor
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how are sensory organ precursors singled out from proneural clusters in the imaginal discs?
lateral inhibition, delta-notch signalling
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what is at the end of the axon?
the growth cone
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how many growth cones are sent out from one axon?
multiple then refined
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what happens to excess connections?
they are pruned back to get a 1:1 correspondance
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what is the growth cone made of?
actin
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how does the growth cone find the other cell?
chemoattraction and chemorepulsion- diffusible molecules long distance cues
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if a chemoattraction is about what happens to growth cone?
moves up the concentration gradient
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what are diffusible short range cues in axon guidance?
contact mediated cues like pros on the cell surface like cadherins and Eph ligands- attractive or repulsive
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are cadherins contact mediated cues attractive or repulsive?
attractive
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what is an example of contact repulsion~?
Eph ligands
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what happens to the num of neurons during dev?
generated in excess then pruned away only those with connections will survive
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what are neutrophic factors needed for?
neuronal survival
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what does neuronal neutrophic factors specifically do?
ensure num of neurons = num of targets
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what does the growth cone send out?
filopodia-exentio of cell m
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stages in zebrafish dev?
egg surrounded by chorion -> yolk-free cytoplasm+yolk -? blastoere and yolk -> morula->blastocyst
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what is dev?
a change in structure (anatomy+morphology over), how single cell devs into a fully formed organism with many cell types
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what is regional specification?
how a pattern appears in a similar population of cells
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what side is your tummy ventral or dorsal?
ventral
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what does morphogenesis requre?
cell and tissue movement
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what is morphogenesis?
shaping of organs and tissues in 3d
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what does cell diffentation require?
cell-type specific gene products, differential gene transcription
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what is the broad diffention of cell differentation?
formation of specialised cell types
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what do many embryos absorb to grow?
the yolk
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what does growth involve?
cell death and cell proliferation
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differentiated cells start off as what?
stem cells
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what model organisms are used for model organisms?
chick and frog
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why do we need model organisms?
to identify genes with roles in dev processes, understand gene function, provide models for human disease
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what model organisms are used in dev genetics?
fish, mouse, worm, fly
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why is it okay to apply some model organism stuff to humans?
conservation of body plan, conservation of genes and gene function
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what kind of disease is aniridia?
autosomal dominant
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consequences of aniridia?
lack of iris, decreased vision and cataracts
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what is the homozygous phenotype for aniridia?
homozygous perinatal lethal
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what gene is mutated in aniridia?
PAX6
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what year was PAX6 associated ith human and mouse developmental abnormalities (aniridia, small eye)
1992
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what is the mutation in aniridia?
semi dominant- intermediate phenotype
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running order of genes being turned on?
maternal gene -> gap genes -> pair rule genes -> segmentation genes
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when were paired genes found to be a key regulator in drosophila dev?
1980
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gene in the mouse first identified simple to paired genes in dropsophila IN 1988?
PAX1
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How many PAX genes were found in the mouse by 1991?
8
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what is small eye caused by?
mutations in pax 6 gene
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what did pax 6 mutations in drosophila map to?
eyeless mutation- no eye
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in 1995 ectopic pax6 induces what in drosophila?
an ectopic eye (so does the vertebrate)
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homeodomain of pax6 structure?
3 alpha helices 1 in major groove of DNA, crystal structure of paired domain similar
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how many mutations are there in pax6 characterised?
220
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what is conserved?
gene sequence and function
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how were pax family genes first discovered?
by genetic studies in flies
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what does PAX6 gene code for?
a TF
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is PAX6 neccessary or sufficient for eye formation in the embry?
necessary and sufficient
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pax6 gene sequence are what from flies to humans?
highly conserved
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what is making a targetting construct?
disrupt gene like pax6 by introduce another gene (drug resistance)right in the middle of he genes exon
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what is homologous recombination?
done in embryonic stem cells recombination event 2 strands dna cross over , target dna and targetting gene cross over
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how into every cell of the mouse
homologous recombination in embryonic stem cells,
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what stage are the stem cells in?
blastocyst stage cells before implantation of embryo (
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what tissue becomes the embryo itself?
the epiblast
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what does the trophoblast make?
extra embroyinc tissue, placenta and membranes
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what is ES?
undifferentiated in cultured cells
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how do you replace pax6 gene with targetting construct?
treat cells, DNA into nucleus by homology seq similarity it will find similar sequences very rarely have recombination event 2 strands dna cross over
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how do we tell which cells took up the targetting construct?
selection in antibiotic/drug as targetting construct has gene resistance
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what happens with the stem cells that have the gene knocked out?
replace cells in blastocyst by microinjection (fine glass pippette) -> blastocyst into foster mother
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what is a chimera?
has some cells with mutation in cells only one allele (+/-) heterozygous for mutation
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what is the marker in chimera mice usually?
colour coat patches
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how do you get a heterozygote with all cells being mutant?
chimera x wild type
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how do you get a homozygous knockout?
breed brother and sister with all cells being mutant
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why is the mouse particularly used for knockout?
you can get embryonic stem cells from them
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what KO models of human disease are there?
cystic fibrosis, atherosclerosis, susceptibility to cancer
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what characteristic of zebrafish allows easy characterisation of mutant phenotypes and visualisation of labelled cells?
transparency
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what does early embryogenesis involve?
gametogenesis, fertilisation, cleavage, formation of a blastula, generation of cell differences, germline and somatic cells, zygotic genome activation
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what was the problem when looking at differentation?
how do chromosomes (identical in every cell) direct differences between the cytoplasmic contents of different cells
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what does pluripotent + mulipotent mean?
multiple cell fates
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what does totipotent mean?
can form embryonic and extra embryonic cells
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what does pluripotent mean specifically?
any cell in the embryo but not extra embryonic tissue
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hypotheses to explain a restriction in potency?
gene loss, diferential gene activity
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what did lens regeneration in the newt show?
iris cells retain genes to turn on cystallin, only lens express crystallin genes
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put human liver cell in rat muscle what happens?
liver cell turns on human muscle specific genes, liver genes switched off
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pleurodeles (newt) oocyte put in xenopus (frog) kidney cells nuclei in what happens?
1st) kidney specific genes switched off 2) re-expressed oocyte specific genes for xenopus
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what is the method in the last few cards called?
nuclear transfer
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what is the newer technique to see if genes are still present?
genomic sequencing-see muscle genes in WBCs
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what do you find in the genome?
the entire genome stays in tact in the human-no gene loss
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what do nuclear transfer experiments tell us?
gene expression in nuclei from differentiated cells can be changed, gene expr can be controlled by cytoplasmic factors, genes are not lost
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what do cells lose as they differentiate?
potency
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if the nuclei retain the whole genome do they remain totipotent, who tested this?
briggs and king
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what did briggs and kinds do in their nuclear potency experiment?
took a fert egg + extracted cytoplasm (with chromosomes), replaced genetic material with nucleus taken from a blastocyst stage embryo, generated normal Rana (tadpole)
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what did briggs and kings conclude?
early blastula nuclei in rana are pluripotent, got tadpoles but not adults
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what did doing it at each stage of the embryo show?
the later in dev eg neurula the less potency the nucei had
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what happens to nuclei as we develop?
they lose potency
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hat did gurdon do to test nuclear potency?
took nuclei from cells of guts in tadpoles put it in a unfertilised egg that had its chromosomes irradiated by UV to destroy DNA,
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what genetic marker did gurdon use?
gut cell nuclei from albino tadpole, unfertilised egg from pigmented frog
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what happened to the embryo in gurdons experiment?
only half the blastocyst were dividing so did serial transfer (nuclei from dividing into new eggs) got clones of genetically identical tadpoles = allalbino (from gut cell nuclei)
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what did gurdon conclude from this?
nucleus retains entire potency to form all the different cell types
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critisims of gurdons gut cell exp?
not fully differentiated stem cell like gut regenerated, low success rate no adults from germ cells
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briggs and king nuclear potency exp?
needle take out dna in fert egg -> nucleus out of blastula stage egg -> generated normal tadpole
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why was rhe nuclei pluripotent not totipotent?
no adults so cant have all info to gen adult
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gurdon nuclea transfer in zenopus gut cell exp?
destroy dna in egg by uv irration -> replace with gut cell nucleus, albino gut cell, pigmented egg -> serial transfer into other eggs
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nuclear potency gurdons ADULt cell experiment?
keratin expressing skin cells -> take out nucleus -> transfer into irradiated egg -> serial transfer -> genetically identical clones
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what was the conclusions from gurdons 2nd experiment?
nuclei of differentiated adult skin cells can be reprogrammed to become pluripotent
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how do you clone sheep?
mammary epithelial cells grown in culture low serum (white) -> unfertilised egg chrs removed with needle (blackface ewe) -> fusion by electric current -> nucleus of mammary cell in egg -> embryo culture -> implant in foster mother
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what were the mammary cells grown in? why?
low serum -> cells exit mitosis go into G0 best for cloning
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how do you check the authentity of clones like dolly?
DNA fingerprinting like microsatellite analysis or dna sequencing
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what did gurdon receive an award for finding out?
the discovery that mature differentiated cells can be reprogrammed to become pluripotent
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what did jacob and monod understand from their bacterial genetics?
problem with embryology is why cells dont express all of the time everything they can in their genome
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conclusions of stem cells and potency?
most cells lose potency during dev, stem cells can remain multipotent, nuclei retain potency
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what happens in V J and C regions in immunoglobin genes?
lose genes in immunoglobins by gene rearrangements in b lymphocytes
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what organism has chromosome diminution (gets rid of chromosomes during development?
parascaris
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how do they germ cells in the parascaris keep all genome in the germ cells?
has germ plasm -> cells that inherit this become germ cells -> retain chromosomes but somatic cells fragment their chromosomes they dont need
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win the dorsoventral axis what is the difference between where neural tissue is in insects and vertebrates?
Insect- ventral, vertebrates: dorsal
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what axis does bicoid set up?
AP axis
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blastoderm stage is what?
surround yolky filled interior, nuclei all migrated and membranes have come down
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what is the dorsal ectoderm going to become?
epidermis
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where is mesoderm of fly?
ventral side
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what is in between the dorsal ectoderm and mesoderm?
neuroectoderm
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how is the DV axis of embryo established in the oocyte?
AP+DV pattern already, egg chambers grow-multiple chain, end is ost mature oocyte with nurse cells at anterio, nucleus always anterodorsal
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what are follicle cells for?
important signalling role, not laid with the egg
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what maternal effect mutants affect DV pattern?
dorslaised and ventralised class
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what are the dorsalised class of mutant caused by?
spatzle, toll and dorsal STD
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what are dorsalised class of genes required for?
ventral cell fates in the fruit fly
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what genes are mutated for the ventralised mutants?
cactus
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hat are ventralised mutant genes needed for?
development of dorsal cell fates
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what does epistasis analysis do?
order genes into a pathway
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what is the toll pathway?
spatle --> toll --/ cactus --/ dorsal (STCD)
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what is the ligand in the toll signalling pathway?
spatzle
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where is spatzle?
in the perivitelline space of ventral side of embryo
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what is toll?
transmembrane recptor
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what gets broken down when toll is activated?
cactus degraded
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what does cactus do?
binds to dorsal holds it in cytoplasm
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what does toll do to catcus?
inhibits it from holding dorsal, degrades it
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what is dorsal?
a transcription factor
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where does dorsal move to when cactus gets degraded?
into the nucleus, change of gene expression
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how is dorsal distribued in the egg?
it is distributed uniformly (maternal factor)
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what side does dorsal enter the nuclei on?
ventral side
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why does it only enter on the ventral side?
spatzle is only on ventral side, reflects DV patterning of the egg chamber, set up by signalling between oocyte and follicle
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dorsal is a TF what does it turn on?
zygotic genes in the mesoderm (on ventral side) like twist and snail
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what does dorsal TF repress?
represses transcription of dorsal cell fates egg dpp
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why can dorsal act on these genes?
they have binding sites on twist+snail(mesoderm on ventral), rhomboid(middle) and dpp (dorsal)
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what does dorsal act as?
a morphogen so respond to different thresholds of dorsal pro conc\
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wherre ie twist oly activated?
ventral side of the embryo
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what is dorslal an activator for?
rhoboid, twist and snail
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what is dorsal a repressor for?
dpp
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affinity for dorsal is high or low in twist and snail?
low afinity so need lots to activate
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does rhomboid and dpp have high or low affinity for dorsal?
high affinity therfore only need a little bit
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what does neuroectoderm gene?
rhomboid
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what gene is for dorsal ectoderm?
dpp
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what for ventral mesoderm?
twist and snail
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what happens in a ventralised mutant?
cactus cant hold dorsal in cyto (mutation in cactus) dorsal pro moves into nuclei of all cells in embryo
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what genes are activated throughout the whole embryo?
twist and snail
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all cells adopt what fate in teh ventralised mutant?
ventral cell fate
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what happens in a dorsalised mutant?
dorsal doesnt enter the nuclei, dpp expressed throughout, twist and sbnails not expressed, all cells dorsal fate
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dpp is what?
signalling molecule it forms a morphogen gradient (high in dorsal regions)
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what does dpp have?
its own pathway with recs and signalling cascade
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what happens in gastrulation?
mesoderm invaginates and moves in the embryo (forms msucle) neuroectoderm covers ventral side, neuroectoderm top dorsal side
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what is the role of the toll pathway?
innate immune response in fly
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what is the toll pathway stimulated by?
bacteria and fungal infection in adult flies
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where are toll like receptors expressed on?
macrophages
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what is the toll pathway required for?
the innate immune repsonse to microbial infection (bacterial and fungi)
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what does the toll like rec bind to?
proteins on fungi, bacteria, parasites or viruses
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what happens when they bind to a toll rec?
stimulate toll pathway, changes in gene expression in nucleus, eg inflammatory cytokines
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what does the antigen and toll like rec do?
gets eaten in phaglysome
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what is the human homologue of Dorsal?
NF-KB
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what is the homologue of cactus?
I-KB
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what does I-kb do to NF-KB
I-KB sequesters and holds NF-KB in the cytoplasm
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what does NF-KB regyulate?
gene expression in B cells of the immune system
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what is the dpp homologue?
bone morphogenetic protein
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what is bone morphogenetic protein for?
formation of bone, plays a role in the DV patterning of the vertebrate
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if BMPs get out of hand and dysregulated and muscle turns into skeleton what disease do you get?
fibrodysplasia ossificans progressiva
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Other cards in this set

Card 2

Front

what was Briggs and Kings experiment for nuclear potency?

Back

used Rana(frog), needle to extract chrs out of egg cell, egg cell with no GM, replaced GM with nucleus from cell in blastula stage embryon--> normal tadpole, blastula nucleus pluripotent

Card 3

Front

what are the 2 hypotheses to explain why cells lose potency?

Back

Preview of the front of card 3

Card 4

Front

what experiments show that genes are not lost when cells differentiate, expression controlled by cytoplasmic factors and gene expression of differentiated can be changed?

Back

Preview of the front of card 4

Card 5

Front

what happens in lens regeneration in newts?

Back

Preview of the front of card 5
View more cards

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