BIOL 303 Lecture Notes - Lecture 5: Wnt Signaling Pathway, Polarity In Embryogenesis, Transforming Growth Factor Beta
14 May 2018
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Review from Tuesday
• Looking at amphibian axis specification, gastrulation, and formation of three germ layers
• Upon fertilization there is a cortical rotation that is important for anterior-posterior
polarity
• Dorsal mesoderm = notochord + somites
• Ectoderm at the anterior pole
• Endoderm at the vegetal pole
• At the meeting between the two ^: mesoderm
• The interaction between the endoderm and
the animal cap that give rise to the mesoderm
• VegT and Vg1 from vegetal cells signal and
convert the animal cap into mesoderm
• Transplantation experiments showed us how
the ventral and dorsal vegetal cells give rise
to ventral and dorsal mesoderm (figure 1)
• Beta-catenin presence was the differing factor
between dorsal and ventral mesoderm fate →
the dorsal mesoderm has nuclear beta-catenin
and the ventral mesoderm does not
• Experiment: inject Wnt signalling effectors
(Beta-catenin) into the ventral vegetal cells,
this is enough to cause axis duplication
• The events that occur at fertilization (cortical
rotation) determine where Beta-catenin will
be located
• The Wnt pathway occurs where Dsh is located in the cortical membrane
• Disheveled inactivates GSK-3 kinase (which normally degrades Beta-catenin) in the
dorsal mesoderm nuclei
• Purple region = Neiuwkoop centre = overlap of Beta-catenin and TGF-Beta signal
• Neiuwkoop centre is not an organizing cell because its fate is not dorsal mesoderm → it
goes on to form endoderm (only fits ¾ criteria)
• Why is this region so special? → this initiates a signalling cascade where Beta-catenin
activates the first zygotic gene to be expressed, Siamois
• Within the promoter region of the Siamois gene, there are three Beta-catenin binding sites
• Siamois is a transcription factor (like Beta-catenin), that goes and binds to organizer-
specific genes AKA within the dorsal mesoderm
• Siamois alone will not activate these genes, you need two activators
• Smad phosphorylation and accumulation in the nuclei → binding to the promoter element
and transcription of dorsal mesoderm genes Ex. Goosecoid
• Nodal-related protein (a member of the TGF-beta), is higher in the Dorsal mesoderm
region but is also present in a ventral mesoderm (it is in a gradient)
• High Nodal activity = activation of organizer genes (dorsal mesoderm)
• Low Nodal activity = ventral mesoderm specification
Figure 1
find more resources at oneclass.com
find more resources at oneclass.com
Biol 303 Feb 22nd
2
Today – 1) Mesoderm fate
• If you transplant the dorsal mesoderm to another tissue you get axis duplication
• What does the dorsal mesoderm tissue do to induce axis duplication?
• A simple mechanism
• How to figure out how it works: screen for gene + protein expression
Goosecoid induces a second axis
• Take a blastula and inject cDNA libraries within the vegetal region, let these develop, and
see if any of the pools of cDNA cause axis duplication
• They found one gene, a transcription factor: Goosecoid, injected in the ventral region
caused dorsal mesoderm formation and axis duplication
Noggin Expression
• UV irradiated Xenopus embryo – becomes a mass of ventral mesoderm tissue
• If you inject Noggin into this, you start to rescue some of the Dorsal structures of the
embryo → if you increase the amount that you inject, you can totally rescue the
formation of the tadpole → injection of Noggin alone was able to fully save axis
formation
• If you add too much Noggin, you start to degrade ventral tissue and you get a big dorsal
blog
Expression of genes encoding secreted proteins (figure 2)
• Chordin, noggin, frzb, follistatin, Cerberus → organizer
region
• Bmp4 → endoderm and ectoderm
• Wnt8 → mesoderm except organizer region
What is the function of Bmp4?
• BMP4 = Bone Morphogenic Protein 4
• Bmp4 is expressed throughout the blastula but not in the
dorsal mesoderm
• Experiment: Inject BMP4 into the Xenopus egg → all the
mesoderm of the embryo became ventrolateral mesoderm
and no dorsal mesoderm was formed
• Experiment: Prevent bmp4 signalling via overexpression of a dominant negative Bmp4
receptor → formation of two dorsal axes
• Normally the receptor is encoded with a receptor domain → Create a cleaved version
where it has no intracellular signalling
• If you overexpress the BMP4 receptor, you get axis duplication
• Prevent Bmp4 signalling → Dorsal mesoderm
• Allow Bmp4 signalling → Ventral mesoderm
• Therefore, Bmp4 is involved in ventral mesoderm specification
Figure 2
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Review from tuesday: looking at amphibian axis specification, gastrulation, and formation of three germ layers, upon fertilization there is a cortical rotation that is important for anterior-posterior. Noggin expression: uv irradiated xenopus embryo becomes a mass of ventral mesoderm tissue. If you add too much noggin, you start to degrade ventral tissue and you get a big dorsal blog. Expression of genes encoding secreted proteins (figure 2: chordin, noggin, frzb, follistatin, cerberus organizer region, bmp4 endoderm and ectoderm, wnt8 mesoderm except organizer region. If you overexpress the bmp4 receptor, you get axis duplication: prevent bmp4 signalling dorsal mesoderm, allow bmp4 signalling ventral mesoderm, therefore, bmp4 is involved in ventral mesoderm specification. Today 2) neural induction: epidermis = highest levels of bmp4, several incorrect experiments before the correct function was found, you need bmp4 to cause epidermal formation bmp is an epidermal inducer. It was found that neural tissue formation was the default for isolated ectodermal cells.
