V. Organizing Power and Axis Formation

A. Background Information

B. Invertebrates1. Sea Urchins

2. Snails

3. Tunicates

4. C. Elegans

5. Drosophila melanogaster

C. Vertebrates1. The Frog

2. Zebrafish

3. The Chick Embryo

4. Mammals

• Part of these processes is the determination of axes in the organism

– The first few cleavages may produce little or no directionality to the embryo

– It starts at varying stages in various animals and can result from different mechanisms

Remember our primary axes....

Figure 5.8 Fate map and cell lineage of the sea urchin Strongylocentrotus purpuratus

Step 1: Specification of Micromeres

disheveled expression blocks B-catenin degradation

egg

Two Big Changes: Specified to become skeletogenic mesenchyme Specified to become “Organizer” for other cells

-catenin’s job

NML

ALL

Allendoand meso

NONEAllecto

Step 2: “Organizing Power”

• Secrete Wnt-8 into autocrine loop• Wnt-8Blimp-1B-cateninWnt-8

• Paracrine “early signal” induces macromeres and vegetal cells to differentiate to vegetal endoderm– Unknown signal as of yet

• Delta-Notch juxtacrine signal induces non-skeletogenic mesenchyme

• Wnt-8 makes a come-back to induce invagination

Axis Determination

• Anterior-Posterior: Cytoplasmic determinants in the egg cytosol, such as disheveled and B-catenin

• Left-Right: Nodal expression (TGF-B family member)

• Dorsal-Ventral: unclear

Spiral cleavage in molluscs

The spirally cleaving mollusks havea strong autonomous specificationfrom cytoplasmic determinants in egg.

Step 1: Polar lobe formation

The polar lobeis a cytoplasmoutpouching from the egg prior to cleavage

It isolates criticaldeterminants intoonly one of thefirst cell pair.

TF’s associatedwith the lobeturn CD into“The Organizer”

Figure 5.27 Association of decapentaplegic (dpp) mRNA with specific centrosomes of Ilyanassa

Decapentaplegic is TGF-B family member used toinduce specific cell fates secreted by the Organizer

The Organizer induces mesodermal and endodermalfates in cells that would otherwise remain ectodermal

MAP kinase activity activated by D-quadrant snail blastomeres

Figure 5.30 MAP kinase activity activated by D-quadrant snail blastomeres (Part 2)

Normal MAPK Blocked

Axis Determination

• Anterior-Posterior: Cytoplasmic determinants in the lobe

• Left-Right: Nodal expression (TGF-B family member)

• Dorsal-Ventral: Cytoplasmic determinants in the lobe

Bilateral, Holoblastic Cleavage of the Tunicate

The 8-cell embryo isalready autonomouslyspecified for cell fates

Figure 5.35 Cytoplasmic rearrangement in the fertilized egg of Styela partita

Fertilization rearranges cytoplasmic determinants

1. Animal pole cytosol determines ectoderm2. B-catenin presence determines endoderm (like urchins)3. Macho-1 in yellow crescent determines muscle cells

Figure 5.38 Antibody staining of -catenin protein shows its involvement with endoderm formation

Wherever B-catenin shows up, endoderm is formed

Figure 5.37 Autonomous specification by a morphogenetic factor

Where Macho-1 shows up tail muscle will form

Zinc-finger TF for muscle actin, myosin, TBX-6Also TF for Snail TF which blocks notochord induction

Conditional Specification also plays a role

Integrates with the autonomous specification patterns

Axis Formation accomplished prior to cleavage!

Fertilization rearranges cytoplasmic determinants

determines dorsal-ventral

determinesanterior-posterior

Left-right: unclearbut nodal shows it later

Rotational, Holoblastic Cleavage in the nematode Caenorhabditis elegans

hermaphrodite

Figure 5.42 The nematode Caenorhabditis elegans (Part 2)

Both autonomous and conditional specification at work early on.

P1 will develop autonomously

Stem cell divisions are meridional

Founder cell divisions are equatorial

If cells are separated:

AB requires input from P lineage

• Autonomous specification in P1– SKN-1, PAL-1 and PIE-1 TFs from egg– as P1 divides these determine daughter fates– P lineage becomes “Organizer”

• Conditional specification in AB– P2 secretes Wnt family member MOM-1 to

induce endodermal specification in AB lineage– P2 use Delta-Notch signals to induce

ectodermal fates in AB lineage

Axis Determination in C. elegans

Anterior-Posterior axisis determined by egg shape

Which end is posterioris determined by sperm(the closest end is back)

Sperm CYK-4activates egg rho,actin rearrangementcauses assymetricfirst cleavage division

AB division leads to both dorsal ventral and left-right axes

Assymetrical division of AB-MS forces AB dorsal and MS ventral

Delta-notch recognition between daughters of AB and MS gives left-right

Cytoskeletal rearrangement also pushes P-granules into the germ line

The cells of the blastula have specified fates in Xenopus.....

Gastrulation changes all of that, .....afterwards all cell fates are determined!

Development of “Organizing Power” at the dorsal blastopore lip

The bottle cellsget the ball rollingbut the real poweris conferred on thefirst cells throughthe blastopore.

The dorsal mesoderm keeps the power to determine other cell’s fates throughout gastrulation: “Spemann’s Organizer”

This ability to determinecell fates is called...

“Primary Embryonic Induction”

The dorsal lip cells first have to become competent to be “Organizer”

The area of Dshaccumulation isseen as a graycrescent in someamphibian embryos

Cortical rotation shiftsdisheveled, GBP, Wnt-11to dorsal side of embryo

β-catenin starts out everywhere in the embryo but only survives GSK3 in the dorsal portion due to Dsh, GBP and Wnt-11

The dorsal vegetal cells of the Nieuwkoop Center turn on “Organizer”

Wnt and Vg-1 (TGF-B family)induce pre-dorsal lip mesoderm

FGF needed for all mesoderm

Figure 7.22 Summary of events hypothesized to bring about induction of the organizer in the dorsal mesoderm

Vg-1

Nodal

Figure 7.23 Vegetal induction of mesoderm (Part 2)

So, what can the “Organizer” do?

• Initiate gastrulation

• Become the notochord and other dorsal mesoderm

• Dorsalize ventral mesoderm into paraxial mesoderm, somites, etc.

• Dorsalize the ectoderm into the neural plate and neural tube

Figure 7.26 Localization of chordin mRNA

Dorsal blastopore lip Blastopore Dorsal mesoderm

The “Organizer” is induced prior to gastrulation

Continues to organize events throughout its own differentiation

Interestingly, the primary mechanism is by means of inhibition....

Presumably, the Wnt, FGF and RA signals arise from endoderm and ectoderm

Without the “Organizer”you get mainly skin and gut

Figure 7.31 Cerberus mRNA injected into a single D4 blastomere of a 32-cell Xenopus embryo induces head structures as well as a duplicated heart and liver

Don’t underestimate the power of the “Organizer”!

Axis Formation

• Dorsal-Ventral: sperm penetration and cortical rotation

• Anterior-Posterior: migration direction of the dorsal mesoderm

• Left-Right: nodal expression exclusively on left side of the lateral plate mesoderm

Nodal expression causes Pitx2 expression

Nodal and Pitx2 on left Injected on both sides

Relationships between the frog and chick “Organizers”

• The hypoblast = dorsal vegetal cells

• Koller’s sickle = pre-dorsal lip mesoderm

• Hensen’s node = dorsal blastopore lip and dorsal mesoderm

• Primitive streak = blastopore

Formation of Hensen’s node from Koller’s sickle

Wnt and FGF from the hypoblastinduce Koller’s sickle epiblast

Figure 8.10 Induction of a new embryo by transplantation of Hensen’s node (Part 1)

Possible contribution of inhibition of BMP signaling

Appears to be similar to the frog....

In the chick, the hypoblast plays a large role much like the frog endoderm

Figure 8.8 Specification of the chick anterior-posterior axis by gravity

Anterior-Posterior axis parallels the rotation inside the shell

Rostral-Caudal (Anterior-Posterior) axis extension in chick embryos

The combination ofpositional specification,complex signaling andTF (Hox, etc.) expressionis thought to cause axis.

Left-right asymmetry in the chick embryo

This is farther along Nodal and Pitx2 again are implicated