Sea Urchin Development Flashcards
1
Q
what two mechanisms of cell patterning are used in the sea urchin?
A
- autonomous specification
- conditional specification
2
Q
why are sea urchins good model organisms?
A
- they release of large number of gametes, all embryos develop synchronously, embryos are transparent, easy to manipulate genetically, has many gene regulatory networks
3
Q
what is the sea urchin life cycle?
A
- first two cell dividisions divde the embryo into 4 equal cells- first two are meridional cleavage, the third in the equatorial.
- then form 8 cell stage
- the fourth cleavage is different: the next division forms 2 smaller micrometers in the vegetal pole and 4 micromeres in the vegetal half. the top 8 cells are the mesmerise.
- the embryo is eventually divided into animal 1,2 and vegetal 1,2
- after cleavage the blastula forms which has a blostoceol
- the micrometers become the PMC and ingress within the cell, triggering gastrulation
- the cells at the vegetal pole form a blastopore
- the secondary mesoderm cells then ingress and protrude filapodia which pull the Archenteron towards the mouth end.
- the PMC form the skeletal extensions
- then the prism stage occurs
- then 6-8 weeks undergoes metamorphosis
4
Q
how long can sea urchins live?
A
200 years
5
Q
what forms around the embryo after fertilisation?
A
the fertilisation membrane
6
Q
what are the micromeres?
A
4 small cells that form at the bottom of the vegetal pole- become the PMC
7
Q
where are the mesomeres, macromeres and micromeres?
A
the mesmerise are found in the animal pole
- the micromeres are in the vegetal pole at the bottom
- the macromeres are found in the vegetal pole
8
Q
how do the micromeres ingress?
A
they undergo an epithelial to mesenhymal change- the cells lose affinity for their hyaline layer and produce proteins which allows them to migrate into the blastoceol and lose their cilium.- they first pop their nuclei into the blastocoel then fully enter
9
Q
on what side of the embryo does the mouth open?
A
oral not aboral
10
Q
what will the animal 1 area of the embryo go on to form?
A
the apical organ, oral aboral ectoderm and ciliated bands
11
Q
what will the animal 2 area of the embryo go on to form?
A
the oral aboral ectoderm and the ciliated bands
12
Q
what will the veg 1 area form?
A
hindgut and endoderm
13
Q
what will the veg 2 area form?
A
the endoderm and non-skeletal mesoderm
14
Q
what do the micromeres form?
A
the PMC and coleomic pouches
15
Q
how are the micromeres specified and what does this mean?
A
they are autonomously specified, this means that they don’t need signals from surrounding cells for the specification and instead depend on cytoplasmic, internal signals
16
Q
what do the micromeres specify?
A
the endomesoderm
17
Q
what is the key component of autonomous specification in the vegetal cells?
A
the nuclearisation of beta catenin
18
Q
what studies have shown that the nuclearisation of beta catenin causes the formation of vegetal cells?
A
if you induce nuclearisation in all cells of the embryo then all the cells become vegitalised
19
Q
what is the pathway by which beta catenin is vegetalised?
A
it requires WNT, when WNT binds to a dishevelled protein it activates frizzled protein, this prevents the degredation of beta catenin and allows it to enter the nucleus- without wnt, b-cat is degraded
20
Q
how are the micromeres autonomously specified?
A
the egg contains localised factors that are essential for triggering the specification- this is the dishevelled protein which becomes localised at the vegetal pole during oogenesis
21
Q
what is the role of disheveled in the vegetal pole during cleavage ?
A
it prevents the degradation of b- catenin that occurs without the wnt pathway- it can go into the nucleus
22
Q
where does beta catenin occur in the nucleus in the developing embryo-
A
predominately in the micromeres but also in the veg 2 regions
23
Q
what shows that micromeres are autonomously specified?
A
when grown alone, they form th same skeletal mesodermal fate
24
Q
what experiments show the role of beta catenin in the embryo in determining cell fate?
A
treating a cell with lithium chloride causes the accumulation of beta- catenin in every cell and transforms the presumptive ectoderm into endoderm.
- inhibition of accumulation of beta catenin in the vegetal cells prevents the formation of endoderm and mesoderm
25
what does treating an embryo with lithium chloride do
cause the accumulation of beta- catenin in every cell and transforms the ectoderm into endoderm
26
when does otx become localised in the micromeres?
during the fourth cleavage
27
what is otx?
a maternal transcription factor that becomes localised in the fourth cleavage
28
what is the role of otx?
it interacts with the b-catenin/TCF transcription factor binding and interacts with the enhancer of Pmar1 gene to activate the transcription of this gene in the micromeres
29
what is Pmar1?
a transcriptional repressor of HesC, a gene that encodes another repressive transcription factor
30
what is HesC?
a gene that encodes for a repressive transcription factor that represses the expression of delta
31
what is the action of Pmar1 repressing the pressing function of HesC?
a double negative gate
32
what is the result of a double negative gate?
inhibits the expression of a gene in every cell other than where its repressor is
33
what are the genes repressed by HesC involved in?
micromere specification- Alx1, Ets1, Tbr, Tel and SoxC
34
what are the genes that are involved in micrometer patterning but are repressed by HesC?
Alx1, Ets1, Tbr, Tel and SoxC
35
the genes that encode for micromere regulation are Alx1, Ets1, Tbr, Tel and SoxC. how can they be expressed normally and what occurs in the presence of HesC?
these genes can normally be expressed by ubiquitous transcription factors, however when HesC is being expressed, they can't be. thus when Pmar1 represses HesC, these ubiquitous transcription factors can have an effect
36
what signalling proteins are under the control of HesC and why is this significant?
HesC represses delta which is involved in the specification of non skeletal mesoderm cells. In the micromeres, HesC is repressed so delta can be expressed and interact with the surrounding cells that express delta- patterning them as NSM- veg 2 cells.
37
what is the feedforward mechanism seen in the micromere regulatory genes and why is it needed?
it is needed because the expression of Pmar1 is transient, but the genes that it regulates need to be able to promote their own expression.
38
describe a positive feedback loop seen in the micromere regulatory genes? what is the step after this?
eats activates Erg, Hex and Tgif erg activates Hex and Tgif
Tgif activates hez and itself
Hex activates Erg ... these activate differentiation genes
39
what is important about activation transcription factors in micromeres in terms of only expression genes when needed?
each differentiation gene must be activated by multiple differentiation genes
40
Considering that the micromeres require beta catenin for their regulatory pathway, how do they ensure that there is a constant supply of WNT8?
as soon as the micromeres form. b-catenin and otx activate Blimp1 genes, this activates the gene encoding WNT8. this means that WNT8 can continue to facilitate beta catenin in the nucleus and therefore its own expression
41
what is Blimp1?
a gene expressed following the maternal beta-catenin and otx have bound to transcription factors- it activates WNT8 expression
42
where does the initial beta catenin come from in the embryo?
the oocyte- maternal factor
43
how thick is the blastomere?
1 cell thick- achieve by adhesion to the hyaline layer and the blastoceol
44
at what stage are the cells on the embryo committed?
60 cell stage (however not irreversible)
45
what is the role of the micromeres?
- large micromeres become autonomously specified. They inherit maternal determinants that had been deposited at the vegetal pole of the egg and which become incorporated into the four micromeres at fourth cleavage. These cells are thus determined to become skeletogenic mesoderm cells that will leave the blastu- la epithelium to enter the blastocoel, migrate to particular positions along the blastocoel wall, and then differentiate into the larval skeleton.
- Second, the large micromeres produce paracrine and juxtacrine factors that specify the fates of their neighbors. The micromeres are able to produce a signal that tells the cells above them to become endoderm and induces them to invaginate into the embryo.
46
what is the difference between the large and small micromeres?
the large contribute to the skeletal components but the small contribute nothing to the embryo- form the coelom
47
what will happen if large micromeres are transplanted into the animal region of the blastomere?
f skeletogenic micromeres are transplanted into the animal region of the blastula, not only will their descendants form skeletal spicules, but the trans- planted micromeres will alter the fates of nearby cells by inducing a secondary site for gastrulation. Cells that would normally have produced ectodermal skin cells will be respecified as endoderm and will produce a secondary gut
48
what is the role of nodal in embryo development?
- nodal is expressed in the oral ectoderm at the 60 cell stage.
- it then becomes prominent on the presumptive oral side of the blastula
- when nodal translation was prevented, the larvae wenevr developed bilateral symmetry
- the archenteron did not bend to one side to form the mouth
- genes normally expressed in the oral ectoderm were not expressed
49
what happens when nodal is removed?
when nodal translation was prevented, the larvae wenevr developed bilateral symmetry
- the archenteron did not bend to one side to form the mouth
- genes normally expressed in the oral ectoderm were not expressed
50
at what time point do the large micromeres normally start to ingress?
9-10 hours
51
when the large micromeres ingress, what is their cell type now?
they ingress as skeletogenic mesoderm
52
what do the skeletogenic mesoderm once they have entered the blastocoel?
start extended filopodia into the ECM of the blastoceol membrane and become settled in the ventrolateral region on the embryo
53
where so skeletogenic mesoderm become settled once they have entered the embryo?
the ventrolateral axis
54
what do skeletogenic mesoderm do once they have settled in the ventrolateral region?
they fuse into syncytial cables which will form the axis of calcium carbonate spicules of the larval skeletal rods
55
what is the importance of the intracellular lamina inside the blastocoel?
initially all the blastopore cells have a high affinity for the hyaline layer and a low affinity for the basal lamina within the blastoceol
- the skeletogenic mesoderm loose their affinity by 98% while the affinity for the basal lamina increases a hundredfold
- surface proteins such as fibronectins, integral, laminin and cadherins have been implicated in this
56
what molecules have been implicated in ingression?
fibronectins, integral, laminin and cadherin
57
what provides the right positional information to the skeletogenic mesoderm cells once they have ingress?
prospective ectoderm cells and their basal lamina
58
what two factors are used to regulate the migration of the skeletogenic mesoderm cells?
VEGF paracrine factors are emit- ted from two small regions of the ectoderm where the skeletogenic mesoderm cells will congregate, and a fibroblast growth factor (FGF) paracrine factor is made in the equatorial belt between endoderm and ectoderm and then becomes defined into the lateral domains where the skeletogenic mesoderm cells collect. The skeletogenic mesenchyme cells migrate to these points via reception via VEGFR and FGFR expression
59
how does the epithelial to mesenchymal transition occur in the skeletogenic mesoderm cells?
- the cell membrane of the micrometer changes: the original membrane is endocytosed and replaced. the new membrane alcks the cadherin needed for cell cell adhesion and the receptors form the hyaline vs basal lamina membranes are altered.
- the transcription factor snail regulated the changing of the membrane
60
what does snail do in micromeres?
the cell membrane of the micrometer changes: the original membrane is endocytosed and replaced. the new membrane alcks the cadherin needed for cell cell adhesion and the receptors form the hyaline vs basal lamina membranes are altered.
61
once the skeletogenic mesoderm have ingress, what happens to the cells that remain on the outside?
- the vegetal plate cells remain bound to each other and the hyaline layer of egg and fill the gaps caused by the ingression.
- actin microfilaments collect in the apical ends of the vegetal cells, causing the end to constrict- bottle necked cells
62
what is the first group of cells to invaginate following the PMC?
the non skeletogenic mesoderm form the tip of the archenteron. these will form the pigment cells of the musculature and the gut
63
what is the order of invagination following the PMC?
the NSM invaginate first, then the endoderm adjacent (form the foregut), then the midgut endoderm and the last is the endoderm which form the handgun and anus - the blastopore is formed
64
what occurs following the first stage of gastulation?
then the archenteron formation begins via convergent extension mechanism which allows the archenteron to extend
65
how does the last stage of gastrulation occurr?
the NSM extend filopodia to contact the inner blastocoel wall and shorten to pull the archenteron
66
what is the nature of the filopodia that extend from the NSM when fusing with the blastoceol wall?
- there is a particular target site on what appears to the be the ventral side of the organism- the mouth then forms
67
how are non skeletogenic mesodermal gene repressed in the micomeres?
Pmar1 activates axle which represses gcm which is expressed in non skeletogenic mesoderm
68
what happens in micromeres in the absence of axl1?
skeletal mesoderm cells acquire NSM features
69
what does conditional specification of the endomesoderm mean?
it means that the endomesoderm's fate is specified via interactions wit other cells
70
how can you determine when micromeres have their effect on patterning the endomesoderm?
by removing them at different stages in the cell cycle - found that they have an effect around the 5-7 cleavage stage
71
how is beta cetanin nuclearization maintained by ants signalling in veg2 endomesoderm?
the wnt that is produced via Blimp1 can diffuse into the Veg 2 layer and have an effect ***
72
what is required to induce endomesoderm?
the micromeres- transplantation
73
where is beta catenin found in the embryo?
it is found all around the embryo but only nuclearised in the micromeres and veg2 poles.
74
where is soxB found?
nuclearised in cells other than the micromeres
75
how is nuclear beta catenin maintained in the Veg2 endomesoderm?
wnt8, wnt6 and wnt1
76
in the endomesodermal cells, what do the signals from the micromeres trigger?
the expression of foxA, Blimp1b, Hox11 and bra in the veg2 cells.
77
why are the signals from the micromeres causing the expression of certain genes in the endomesoderms?
due to maternal factors that determine how the cells will respond to micromeres signals
78
what genes do endomesodermal cells express?
blimp1b, foxA, Hox11 and bra and eve (in veg2)
79
how is expression of blimp1b maintained?
activate otx which activates GataE which activates Blimp1b
80
what are the two divisions of the endomesoderm?
the NSM and the endoderm
81
what do the micromeres produce that induce NSM?
delta- released from repression by Pmar1
82
why can only the cells next to the micromeres become NSM?
delta doesn't diffuse, binds to cells next to it on notch
83
what is the notch pathway
once delta has bound, intracellular notch is cleaved and enters the nucleus and activates the expression of hairless which activates GCM which patterns the veg 2 domain which causes NSM
84
what are the stages of specification in the endomesoderm?
- micromeres form SM
- the veg1 and veg2 areas specify as endomesoderm via the expression of Foxa, Blimp1b, Hox11, Bra and eve
- only some of these genes are expressed in both: foxA, hoax and bra
- eve is only in veg2
and blimp is only be veg 2
85
what is the role of delta signal?
to differentiate between the mesoderm and the endoderm
86
where are pigment cells always found?
always towards the aboral side
87
where are the blastoceolar cels found?
always on the oral side
88
on what side of the embryo does gcm act/ get restricted to?
the aboral side
89
what represses gcm?
not
90
what does most of veg1 become?
ectoderm
91
draw the animal 1 and 2 and veg 1 and 2 and label what each will become, also the large and small micromeres
animal 1 and 2 will become the dorsal and ventral ectoderm including the oral and aboral.
veg1 will become endoderm and some ectoderm
veg2 will become mesoderm and endoderm- endomesoderm
large micromeres become the skeletal mesoderm
small become the germ cells in the adult- held in polemic pouches.
92
what are the first 2,3 and 4 cleavages?
1 and 2 are meridional and 3 is equatorial, 4th creates the asymmetric cleavages of meso, macro and micro
93
what happens to the veg2 cells after PMC ingression?
they flatten to form the veg plate. they then apically constrict and invaginate. the archenteron elongates via convergent extension.
94
what do the PMC extend after invagination?
filopodia
95
how are processes conserved or differ in the sea urchin?
- differ in that invagination occurs via individual or small clusters of cells in the sea urchin but via sheets of cells in vert and dros
- twist and snail is involved in mesodermal invagination in sea urchin and dros
96
what gene is implicated in cadherin endocytosis during PMC ingression?
snail
97
what two genes trigger ingression in PMC?
twist and snail
98
what pathway is used in gastrulation?
PCP WNT pathway
99
how is convergent and extension not regulated in secondary invagination?
via the WNT pathway
100
what is the endomesoderm?
veg2
101
how is the vegetal pole patterned?
via maternal factors which is dishevelled- allowing the nuclearisation of b-cat- this continues to the case in veg2 through WNT8/5 signalling
