Final 2 Flashcards
Vitelline envelope
An extracellular matrix envelope all around the Drosophila embryo. Vitellin proteins DO NOT get sulfated on the dorsal side
The proteins do get sulfated on the ventral side by Pipe *enzyme
Pipe is active ventrally.
What is the gastrulation defective (GD) protein and what does it bind to?
It is a protease in Drosophila embryos. It binds to sulfated vitellin proteins on the ventral side of the embryo.
Pipe enzyme sulfated the the vitellin proteins - these serve as docking sites for GD
What are the three proteases in the dorsal-ventral axis initiation pathway?
Gastrulation Defective (GD), Snake, and Easter
What protein cleaves the Snake protein?
GD will cleave the Snake protease to make it active. the cleavage will only happen when GD is bound to the sulfated vitellin proteins
What protein cleaves the Easter protein?
The activate Snake protease cleaves the Easter protease to make it active
- this is only happening in the ventral area because GD is bound to sulfated vitellin proteins here.
What does the Easter protein cleave?
When cleaved and activated, Easter will cleave the Spatzel protein, which can then function as a ligand for the Toll receptor on the embryo’s cell membrane (syncytium)
What ligand binds to the toll receptor?
the cleaved spatzel ligand. Easter cleaved the spatzel
The Toll receptor is present on the cell membrane of the embryo (syncytium)
What happens when the Toll receptor is activated?
The cleaved Spatzel ligand will bind the Toll receptor (which is present on the cell membrane of the embryo) and this will activate the protein kinase called Pelle within the embryo’s cytopasm
What is the Pelle protein?
A protein within the cytoplasm of the embryo’s syncytium in flies. It is a protein kinase
When the cleaved Spatzel ligand binds and activates Toll this will activate the Pelle Ser-Thr kinase which will then Phosphorylate Cactus - leading to the degradation of cactus.
What causes the degradation of Cactus? Where does this occur in the embryo?
The activation of Pelle protein kinase (initiated by Spatzel binding Toll receptor) which phosphorylated Cactus, resulting in its degradation.
Cactus is generally only degraded in the ventral part of the embryo (allows Dorsal to enter the nucleus)
What do mutations in proteins of the dorsal-ventral axis initiation pathway result in?
Mutation of: GD, Snake, Easter, Spatzel, Toll, Pelle, or Dorsal will all result in a dorsalized embryo because dorsal is not able to enter the nucleus and act as a TF
No cells will have dorsal entering into the nucleus
Mutation of Cactus: ventralized embryo because dorsal will always be allowed to enter the nucleus (anywhere in the embryo)
What causes the embryo to be dorsalized? ventralized?
Dorsalized - no dorsal entering the nucleus anywhere to act as a TF - This can result from mutation of numerus proteins (not including cactus)
Ventralized - dorsal entering the nucleus everywhere - This can result from mutation of cactus, which normally functions to keep dorsal out of the nucleus on the dorsal side of the embryo
What is IRAK protein in humans homologous to in flies?
Homologue to Pelle and Tube (adaptor that helps Pelle bind to the Toll receptor)
What is the vertebrate homolog of Dpp (decapentaplegic) ?
BMPs
Dpp is a BMP ligand for Ser-Thr kinase receptors
EXPRESSED DORSALLY
What is the vertebrate homolog of Sog (BMP inhibitor) found in Drosophila?
Chordin (a BMP inhibitor) in vertebrates
Sog is an inhibitor of Dpp in flies - keeps Dpp levels low ventrally by binding to them
What is the vertebrate homolog of MAD found in Drosophila?
SMAD1,5
How do you get induction of the NS?
Inhibition of BMPs
Using Sog in flies, Chordin in vertebrates
Tolloid
A dorsally expressed protease that degrades Sog dorsally (prevents it from being active dorsally). Get more Dpp active in dorsal area because its inhibitor is being degraded)
Degrades inhibitor and thus increases Dpp expression
Is Sog active dorsally or ventrally (flies)?
Sog is active VENTRALLY - Need to inhibit Dpp ventrally for formation of the NS here. (want low Dpp ventrally)
Tolloid degrades Sog dorsally (so you get higher Dpp dorsally)
What is chordin?
A BMP inhibitor expressed in vertebrates. It is present dorsally to inhibit BMPs dorsally–> get low BMP dorsally which allows for NS induction.
Get the most phosphorylated SMAD1/5 where BMP signalling is the highest (ventrally)
BMP inhibitors in vertebrates?
Chordin, Noggin, Follistatin
Cells gain expression of these as they travel through the organizer during gastrulation
What do the cells that go through the organizer during gastrulation become?
pharyngeal endoderm, mesoderm (prechordal plate, notochord)
These cells will gain expression of BMP inhibitors as they travel through - expression of these molecules will induce the NS structures to form in the overlying ectoderm.
Where is the organizer formed in amphibians?
Dorsal lip of the blastopore
In what pole of the amphibian egg does the blastopore (and thus organizer) form?
Vegetal pole
When cells pass through the organizer, what molecules do they begin expressing?
BMP inhibitors (chordin, noggin, follistatin) as well as Wnt inhibitors that are necessary to induce the formation of anterior/head structures (such as the brain)
For amphibians, when is the location of the organizer determined?
At fertilization! The location depends on where the sperm enters the egg. The centriole brought in by the sperm is used to reorganize the disorganized arrangement of microtubules within the egg
- the organization of the microtubules then allows for cortical rotation
- The organizer forms at the area opposite of sperm entry!
Cortical rotation
Rotation generated by now organized microtubules (the sperm brought in centrioles that could be used for organization)
- 30 degree rotation at the cortex of the cytoplasm - important for establishing the axis of the embryo
- in some species the rotation will reveal a lightly pigmented cytoplasm - the gray crescent
The cortical rotation relocates factors towards where the organizer will form
- required for the stabilization of B-catenin
The organizer forms at this gray crescent area!
What kind of cleavage does a xenopus egg undergo?
Holoblastic mesocithal cleavage (moderate amount of yolk)
Are vegetal pole cells smaller or larger than animal pole cells?
larger!
Which cells are the first to head inside during frog gastrulation?
The bottle cells (ingress) – these cells are constricted at their apical end which triggers their involution through the dorsal lip of the blastopore - These cells will later become the pharyngeal endoderm.
The next cells to involute become the mesoderm (prechordal plate (future head meso) and chordal meso (becomes the notochord), followed by the somitic meso)
- anterior endoderm – followed by dorsal mesoderm
What does epiboly do during gastrulation?
Covers the entire endoderm and mesoderm with ectoderm. Spreading out of an epithelial layer.
What combination of molecules induces the amphibian organizer to form where it does?
- Stabilized B-catenin (Wnt signalling) in dorsal part of the blastula
- Cocktail of dorsal mesoderm-inducing signals - mostly NODAL-RELATED TGF-β superfamily members – which are secreted by the Nieuwkoop centre (located just below where the organizer will form). Thus, there will be phosphorylated Smad2,3 here
What signals do the Nieuwkoop centre cells secrete to help in forming the organizer at the dorsal lip of the blastopore?
Nodal-like TGF - B superfamily members which function through Smad2,3
These are dorsal mesoderm-inducing signals!
Which maternal mRNAs are necessary for specification of the mesoderm?
vegT and Vg1
VegT
Maternal mRNA necessary for mesoderm specification
Located at the vegetal pole. Translated to form a TF that will turn on Nodal-related genes
Vg1
maternal mRNA that is necessary for specification of the mesoderm.
It is a nodal-like TGF-B superfamily member as a protein and therefore signals through Smad 2/3
Translated only in the dorsal region of the embryo (where the organizer forms)
-protein induces the future dorsal mesoderm to express Wnt inhibitors
.
GSK3 Binding protein (GBP)
binds GSK3 and inhibits it so that B-catenin can remain intact
Kinesin takes GBP and Disheveled toward the dorsal end where the organizer will form - allows for B-catenin stabilization here.
Stabilized B-catenin plus what other factors required for organizer formation? *
Association of B-catenin with a coactivator will get activation of target genes (siamois and twin gene) - these genes code for TFs that activate expression of BMP inhibitors.
Siamois and Twin transcription factors must bind in association with Smad2 to get transcription of BMP inhibitors activated
SO
1. TGF-B Nodal-like members (required to get phosphorylated Smad2
2. Stabilized B-catenin
are both needed to get organizer formation
What combination of factors gives ventral mesoderm induction?
low nodal related signalling and no stabilized B-catenin
What are involuting cells that form the pharyngeal endoderm expressing to specify the brain and anterior structures to form?
Head inducers = Wnt inhibitors - cerberus, frisbee, and dickkopf
What are examples of Wnt inhibitors?
Frisbee, cerberus, dikkopf, crescent (humans)
These molecules will bind Wnt ligands and prevent them from binding to Frizzled (even though ligand is present - no signalling)
What are two molecules needed to get formation of the head?
Chordin (BMP inhibitor) and frisbee/cerberus/dikkopf (Wnt inhibitor), both in the anterior!
See expression of BMP inhibitors and Wnt inhibitors
Both the prechordal plate and the pharyngeal endoderm express these
What molecules are expressed in the posterior of the embryo?
BMP inhibitors (expressed in anterior and posterior) No Wnt inhibitors - so there is Wnt signalling occurring.
What molecules are expressed to induce the epidermis?
Get both BMP and Wnt signalling to specify the epidermis.
No BMP inhibitors or Wnt inhibitors.