Amphibian Development and the Organizer

Is the vertebrate body plan identical to an invertebrate body plan?

No! The vertebrate body plan is an inversion of the invertebrate body plan, dorsoventrally

• The nervous system instead forms on the dorsal side!

• BMP inhibitors in induction of nervous system is conserved, but expressed on opposite sides

What is the organizer in amphibians?

Dorsal lip of the blastopore

What do cells that gastrulate through the organizer become?

They become the dorsal mesoderm (prechordal plate and notochord)

What do dorsal mesoderm cells (that gastrulate through the organizer) secrete? Why?

They secrete BMP inhibitors (noggin, chordin, follistatin) in order to induce dorsal structures in the ectoderm above them (most obviously the nervous system)

• Same as in Drosophila, except this is happening dorsally instead of ventrally

What does the sperm contribute to the amphibian egg during fertilization?

A centriole that helps organize the egg's microtubule network.

What happens to the microtubules following fertilization in amphibians?

Microtubules become organized into a parallel array beneath the egg cortex, helping drive cortical rotation and transport dorsal determinants

What is cortical rotation?

The rotation of the egg's outer cytoplasmic layer (cortex) approximately 30° relative to the inner cytoplasm following fertilization.

What is the purpose of cortical rotation in amphibian development?

To redistribute dorsal determinants toward the future dorsal side of the embryo, helping establish the dorsal–ventral axis.

Where does the future dorsal side of the amphibian embryo (organizer) typically develop relative to the sperm entry site?

Opposite the sperm entry site.

What is the grey crescent?

A lighter-coloured region that becomes visible on the future dorsal side of the amphibian embryo following cortical rotation.

What does the grey crescent indicate?

The future dorsal side of the amphibian embryo, where dorsal structures will develop.

What do the different colored regions indicate?

• Blue: Animal pole → Ectoderm

• Orange: Marginal zone → Mesoderm

• Yellow: Vegetal pole → Endoderm

What kind of cleavage do Xenopus undergo?

Radial holoblastic mesolecithal cleavage

• Whole cleavage

• Yolk at vegetal pole inhibits cleavage, resulting in large cells at the vegetal pole

What types of cell movements are there during gastrulation?

1. Epiboly

   1. Radial intercalation to spread outer cell out

2. Vegetal rotation

3. Invagination

   1. Bottle cells of the dorsal lip

4. Involution and migration

   1. Migration of mesenchymal bottle cells in the interior

5. Convergent extension

   1. Intercalation towards the midline

What initiates gastrulation and formation of the blastopore?

• Bottle cells undergo apical constriction on the future dorsal side of the embryo.

• Their apical surfaces narrow, causing the cells to become wedge-shaped.

• This creates an indentation that forms the dorsal lip of the blastopore.

Describe the early movements of cells during early gastrulation following blastopore formation by bottle cells

Once gastrulation begins, two major movements occur simultaneously:

• Involution: Mesodermal and endodermal cells move inward over the blastopore lip, spreading along the inner surface of the embryo.

• Epiboly: The ectoderm thins and spreads over the embryo's surface, gradually enclosing the deeper tissues.

The first cells to involute are those that will make up the _____?

Pharyngeal endoderm, then the prechordal plate (future head mesoderm) followed by the chordal mesoderm (future notochord) followed by somitic mesoderm

Does the blastopore lip always contain the same cells?

No! Cells are constantly moving through it, and so the cells that make the blastopore up are also constantly changing throughout gastrulation

What did Hans Spemann discover?

Recognized the importance of the gray crescent for proper development

• Noticed that the 1st cleavage usually goes through the gray crescent

• Applied pressure with hair to alter cleavage pattern:

  • ½ containing the gray crescent underwent normal development

  • ½ missing gray crescent → only forms ventral tissue

What model was used to discover the function of the organizer?

Two species of Triton newts

• One is darkly pigmented, other is light

What was the Mangold-Spemann experiement?

Sought to discover the function of the gray crescent - why was it so important?

• Took the dorsal lip from a lighter-coloured newt and transplanted it into a darker newt

• This caused a secondary invagination, followed by a secondary blastopore and a secondary nervous system to form

  • Ventral tissue → dorsal tissue

Is organizer function discovered in amphibians conserved across all vertebrates?

Yes

What does the organizer produce that makes it so important?

BMP inhibitors (Noggin, Chordin, Follistatin) are necessary to form dorsal structures such as the notochord and CNS

What is this image showing?

Stained for chordin expression

• Expression expands as more cells pass through the organizer

What two signals are required to induce the formation of the organizer in a specific location?

1. Nuclear Beta-catenin stabilized in dorsal cells of the blastula in cells below where the organizer will form and at the site of future organizer formation

2. Dorsal mesoderm-inducing signals (Nodal-related TGF-B family members; Smad2,3) are secreted by cells of the Nieuwkoop center (below futute organizer site)

What blocks mesoderm-inducing signals from diffusing to the animal pole from the vegetal pole?

Blastocoel cavity

Vegetal cells, which will form the ____, induce the cells _____ them to form the _____

Vegetal cells, which will form the endoderm, induce the cells above them to form the mesoderm

How do vegetal cells induce the mesoderm above them?

Maternal mRNAs → proteins that diffuse

• VegT: T-box transcription factor

• Veg1: Nodal-like TGF-B superfamily member

What is the importance of VegT?

VegT controls the expression of several Nodal-related genes (TGF-B superfamily). The protein products of these genes diffuse toward the animal pole where they are required for mesoderm induction

Without VegT, what happens?

No blastopore forms → no mesoderm formed → No development (ball of tissue)

What is the dorsalizing signal?

Beta-catenin and canonical WNT signaling

• Remember GSK3/Axin bound by disheveled via Frizzled → Free APC → Stable Beta-catenin → Nuclear beta catenin to turn target genes on

At fertilization, where is disheveled protein located?

Vegetal pole (very bottom of embryo)

What causes Disheveled to be transported from vegetal pole and up towards the marginal zone where the organizer will form?

1. Cortical rotation by 30 degrees

2. Kinesin motor proteins on the organized microtubule array

Besides disheveled, what else do the kinesins/microtubules/cortical rotation move?

Kinesins also bind GBP (GSK3 Binding Protein)

• Together, Disheveled (binds GSK3 and Axin) and GBP help keep beta-catenin active and stabilized due to the inhibition of the beta-catenin destruction complex)

How does the transport of GBP and Disheveled affect Beta-catenin localization?

Causes dorsal enrichment of beta-catenin

• No beta catenin in ventral nuclei

• Beta catenin in dorsal nuclei

In dorsal cells, what target genes does beta-catenin turn on?

Siamois and twin genes → Siamois and twin proteins

What is the role of Siamois and twin protein?

With Smad2 (from Nodal paracrine factors), they bind organizer genes → Chordin, Noggin which produce BMP inhibitor proteins