Chapter 11: Determination of Amphibian Axes Flashcards
T/F polarity in an amphibian egg is already established, even before the sperm enters
true. There is already an animal-vegetal axis, and the germ layers can be mapped onto the oocyte even before fertilziation.
animal hemisphere blastomeres will become ___ cells
vegetalhemisphere blastomers will become ___
equitorial cells (marginal zone/grey crescent area) will become ____
animal hemisphere blastomeres will become ECTODERMAL cells (SKIN AND NERVES)
vegetalhemisphere blastomers will become ENDODERMAL ORGANS (GUT)
equitorial cells (marginal zone/grey crescent area) will become MESODERM (BONE, MUSCLE, HEART)
What is bottom up processing specification?
the act of having a general rate map imposed on the embryo BY THE VEGETAL CELLS. The vegetal cells in the mebryo induce the cells immediately above them to become mesoderm.
How does bottom up specification occur? Outline the two pathways. Explain the positive feedback loop seen in this process.
mechanism is due to mRNAs tethered to the vegetal cortex.
VegT mRNA is translated in the vegetal cells shortly after fertilization. This allows for vegetal cells to become endoderm, and for cells above them in the margianl/equitorial region to become mesoderm. VegT mRNA does this through:
1) Sox17 gene activation: Sox17 is a transcription factor which activates genes for endoderm specification, guiding the vegetal cells to endodermal fates.
2) Nodal activation (paracrine factor from TGFbeta family): instructs the cells above them into mesoderm. Nodal secreted from vegetal cells due to VegT promotes the cells above them to accumulate phosphoryalted SMAD2. Phosphorylated Smad2 in the cells above the vegetal cells activates EOMESODERMIN AND BRACHYURY (XBRA) GENES, CAUSING MESODERMAL SPECIFICATION.
- eomesodermin and Smad2 proteins work together to activate zygotic genes for VegT protein, creating a positive feedback loop (vegT —> Nodal –> Smad2 in above cell -_> eomesodermin –> more veg T) This HELPS SUSTAIN THE MESODERM.
in addition to vegT (involved in bottom up processing through Sox17 and Nodal-Smad2-eomesodermin genes-brachyury), What other mRNA is expressed in the vegetal cells in order to induce germ layer specification? before mid-blastula transition)
VG1. Vg1 mRNA is also expressed and stored in vegetal cytoplasm. Vg1 is a nodal like protein which activates WNT ANTAGONIST genes in the dorsal mesoderm TO ALLOW FOR PROPER HEAD FORMATION LATER ON. If either nodal or Vg1 is blocked, there is little or no mesoderm induction.
IN SUMMARY: by late blastula stage, vegetal cells are speciied to endoderm through Sox17. Equitorial cells are specified to mesoderm through eomesodermin and brachyury. Animal cap has not yet received signals, but it becomes ectoderm.
- if the animal cap was not separated by the blastocoele cavity, it would be induced to form mesoderm or something because it would be exposed to eomesodermin/sox17/brachyury/vegetal signals.
Axes (both dorsal-ventral and anterior-posterior) are determined at fertilization, however, they are not physically visible until:
gastrulation
______ (TF) becomes localized in the region of the egg opposite to sperm entry in order to specify the __ aspect of the animal.
BETA CATENIN (TF) becomes localized in the region of the egg opposite to sperm entry in order to specify the DORSAL aspect of the animal.
Beta catenin in the dorsal region of the egg induces:
the expression of genes and thus initiate the movement of involuting mesoderm. This movement establishes the anterior-posterior axis of the embryo. The first mesoDermal (PRECORDAL PLATE MESODERM CELLS) cells to migrate over the dorsal blastopore lip will induce the ECTODERM ABOVE THEM TO PRODUCE ANTERIOR STRUCTURES SUCH AS THE FOREBRAIN.
What is the organizer?
the first mesodermal cells to involute over the dorsal plastopore lip (due to beta catenin in dorsal region of embryo initiating cell movement) that induce the ECTODERM ABOVE THEM TO PRODUCE ANTERIOR STRUCTURES SUCH AS THE FOREBRAIN.
- the mesoderm that involutes later will signal the ectoderm to form structures such as the hind brain and the spinal cord.
FROM TEXTBOOK:
the organizer is the dorsal lip of the blastopore, along with the dorsal mesoderm and pharyngeal endoderm that form from it, constituted an “organizer” that was able to instruct the formation of embryonic axes, specifically the dorsal-ventral polarity.
the process of involution of initial mesoderm cells (the organizer) which specifies the ectoderm cells above them to form hindbrain and spinal cord is known as ___ ___ ___: which is involved in ANTERIOR-POSTERIOR PATTERNING.
primary embryonic induction: the central nervous system forms thorugh interactions with the underlying esoderm. When progeny or dorsal lip induces the dorsal axis and CNS.
The first (prechordal) mesoderm cells to migrate over the dorsal blastopore lip later derives the notochord and head/ventral endomesoderm. These cells are named the organizer because:
1) these cells induced the hosts ventral tissues to change their fates to form a neural tube and DORSAL mesodermal tissue (such as somites)
2) these cells induce the above dorsal ectoderm into a neural tube, and transform the flanking mersoderm into the anterior-posterior body axes.
There are two signals that allows the organizer (first mesodermal cells to involute over the blastopore lip) to form :
1) the first tell the cells that they are dorsal (nieukwoop)
2) the second signals says these cells are mesodermal cells. These signals interact to create a polarity within the mesoderm that is the basis for specifying the organizer and for creating dorsal-vetnral polarity. (beta catenin)
What are the two signals that allow for organizer formation?
1) nieukwoop center: the dorsal-most vegetal cells that are capable of inducing the organizer. These dorsal-most vegetal cells in blastula induce the animal pole cells on top of them (closest to it; recall that the most anterior animal cells become ectoderm) to become dorsal mesoderm, which form somites, notochord (chordal mesoderm), and the organizer. (Mesoderm arises from equatorial marginal cells between the top-mostanimal and bottom vegetal poles)
2) Beta catenin: the major candidate factor that forms the niewkwoop center in these vegetal cells is beta catenin, usually is synthesized in embryo from maternal mRNA. Beta catenin begins to accumulate in the dorsal aspect of the egg during cytoplasmic movements (rotation) during sperm entry and during early cleavage.
THEREFORE BETACAT IS IMPORTANT FOR FORMING ORGANIZER BECAUSE IT MAKES THE NIEUKWOOP CENTER
What three proteins allow for beta catenin to accumulate in the dorsal aspect?
The movement of Wnt11, GSK3 binding protein (GBP), and Dsh (disheveled) from vegetal cells to cells in the prospective dorsal side of the embryo following fertilization allows for beta catenin to accumulate in the DORSAL SIDE (Side opposite to sperm entry)
Explain how Wnt11, GSK3 binding protein and Disheveled allow for beta catenin accumulation in the dorsal region of the embryo prior to gastrulation
usually, GSK3 in a cell tagets beta catenin for destruction and prevents it from accumulating and acting as a transcription factor.
GSK binding protein and Dsh DEACTIVATES GSK3
- GBP and DSh release GSK3 from the degradation complex and prevent it from binding to beta catenin thus preventing GSK3 from targeting betacatenin to destruction.
- GBP and DSH get localized on the dorsal aspect of the egg (opposite to sperm entry) becuase GBP hithces a ride on Kinesin ATPASE MOTOR PROTEIN, and gets shuttled to the growing end of the microtubules. DSH also grabs onto GBP on kinesin, and is relocated from the vegetal pole cortex to the future dorsal aspect of the egg.
- Once on the opposite side of sperm entry, GBP and Dsh are released from the microtubules. They then inactivate GSK3 on the dorsal side of the embryo, allowing BetaCatenin to accumulate on the dorsal side while ventral beta catenin is degraded (there is active GSK3 there).
- Wnt11 is also required in order to activate the betacatenin protection pathway. If Wnt11 mRNA is not created, the organizer doesn’t form. Wnt 11 mRNA is localized in the vegetal cortex during oogenesis and is translocated to the future dorsal position by the CORTICAL ROTATION OF THE EGG CYTOPLASM. Wnt 11 protein signal amplifies the singal produced by GBP and DSV and stabilized their activity so they can effectively protect beta catenin from degradation.
Recall: nodal is secreted by vegetal cells beneath the mesoderm in the blastula, which results in the phosphorylatino of Smad2 in mesodermal cells.
- at the late blastula stage, there is a gradient of nodal related proteins across the (vegetal) endoderm: low conc of nodal ____< and high concentrations of nodal ___.
low conc of nodal VENTRALLY and high concentrations of nodal DORSALLY. (or Xnr)
therefore, for organizer formation, there is high levels of dorsal betacatenin, which promotes high levels of nodal/xnr/related protein
Forming the organizer (first mesodermal cells) through further additive effects:
The nodal- related gradient is produced in large part due to beta catenin of the nieukwoop center. High beta catenin levels in the dorsal region activates the nodal like protein expression. In the most dorsal blastomeres, beta catenin and ____ activate ___ 1,5,and 6 genes, which are amphibian Nodal versions. In the ventral regions, blastomeres do not have these genes, instead they have ___ and___, which signals ___ ____ formation.
in the area of the organizer (these vegetal cells will become the most anterior aspect because they migrate up during gastrulation), the ___ endoderm, there are increased levels of ___ proteins and acitvated ___ will be present.
Recall: in the pharyngeal endoderm, these vegetal cells transcribe the Hhex gene. Thus, in the organizer area, ___ will bind to Hhex gene. In CONCERT WITH TWIN AND SIAMOIS, WHICH ARE INDUCED BY BETA CATENIN, THEY ACTUVATE GENES THAT SPECIFY PHARYNGEAL ENDODERM CELLS TO BECOME:
In the most dorsal blastomeres, beta catenin and VEGT activate XNR 1,5,and 6 genes, which are amphibian Nodal versions. In the ventral regions, blastomers do not have these genes, instaed they have BMP4 and WNT8, which signals VENTRAL MESODERM formation.
in the area of the organizer (these vegetal cells will become the most anterior aspect because they migrate up during gastrulation), the PHARYNGEAL endoderm, there are increased levels of XNR proteins and acitvated SMAD2 will be present.
Recall: in the pharyngeal endoderm, these vegetal cells transcribe the Hhex gene. Thus, in the organizer area, Smad 2 will bing to Hhex gene. In CONCERT WITH TWIN AND SIAMOIS, WHICH ARE INDUCED BY BETA CATENIN, THEY ACTUVATE GENES THAT SPECIFY OHARYNGEAL ENDODERM CELLS TO BECOME:
1) foregut endoderm
2) to induce anterior brain development.
- The first cells that invaginate over the dorsal lip have high Smad 2 (because of Nodal expression) and activate HHex to form notocorn and anterior brain regions. Cells that invaginate later and later result in lower levels of Smad 2. Lowest levels of Smad2 triggers the formation of lateral and ___ mesoderm.
In summary for organizer formatino:
- formation of the dorsal mesoderm and the orgnaizer orginiates through activation of crticial trnascription factors by intersecting pathways:
1) Wnt/Betacatenin pathway in nieukwoop cetner activates genes encoding for Twin and Siamois TFs. Allows for dorsality axis formation.
2) vegetal cell pathway that activates the expression of nodal-related paracrine factors (partially due to beta catenin), which in turn activate the smad2 TF in the mesodermal cells above them. - high levels of Smad2 and siamois/twin TFs proteins work WITHIN THE DORSAL MESODERM CELLS AND activate genes that give these cells their “organizer” properties.
