V. Organizing Power and Axis Formation Background Information B. Invertebrates Sea Urchins Snails Tunicates C. Elegans Drosophila melanogaster C. Vertebrates The Frog Zebrafish The Chick Embryo 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.... DevBio9e-Fig-05-05-0.jpg

Figure 5.8 Fate map and cell lineage of the sea urchin Strongylocentrotus purpuratus DevBio9e-Fig-05-08-0.jpg

Step 1: Specification of Micromeres Two Big Changes: Specified to become skeletogenic mesenchyme Specified to become “Organizer” for other cells egg DevBio9e-Fig-05-11-1R.jpg disheveled expression blocks B-catenin degradation

-catenin’s job NML All endo and meso ALL All ecto NONE DevBio9e-Fig-05-11-2R.jpg All ecto NONE

Step 2: “Organizing Power” Secrete Wnt-8 into autocrine loop Wnt-8Blimp-1B-cateninWnt-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 have a strong autonomous specification from cytoplasmic determinants in egg. DevBio9e-Fig-05-24-0.jpg

Step 1: Polar lobe formation The polar lobe is a cytoplasm outpouching from the egg prior to cleavage It isolates critical determinants into only one of the first cell pair. TF’s associated with the lobe turn CD into “The Organizer” DevBio9e-Fig-05-28-0.jpg

Decapentaplegic is TGF-B family member used to Figure 5.27 Association of decapentaplegic (dpp) mRNA with specific centrosomes of Ilyanassa Decapentaplegic is TGF-B family member used to induce specific cell fates secreted by the Organizer DevBio9e-Fig-05-27-0.jpg The Organizer induces mesodermal and endodermal fates in cells that would otherwise remain ectodermal

MAP kinase activity activated by D-quadrant snail blastomeres DevBio9e-Fig-05-30-1R.jpg

Figure 5.30 MAP kinase activity activated by D-quadrant snail blastomeres (Part 2) Normal MAPK Blocked DevBio9e-Fig-05-30-2R.jpg

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 is already autonomously specified for cell fates DevBio9e-Fig-05-34-0.jpg

Fertilization rearranges cytoplasmic determinants Figure 5.35 Cytoplasmic rearrangement in the fertilized egg of Styela partita Fertilization rearranges cytoplasmic determinants DevBio9e-Fig-05-35-0.jpg 1. Animal pole cytosol determines ectoderm 2. B-catenin presence determines endoderm (like urchins) 3. Macho-1 in yellow crescent determines muscle cells

Wherever B-catenin shows up, endoderm is formed Figure 5.38 Antibody staining of -catenin protein shows its involvement with endoderm formation Wherever B-catenin shows up, endoderm is formed DevBio9e-Fig-05-38-0.jpg

Figure 5.37 Autonomous specification by a morphogenetic factor Where Macho-1 shows up tail muscle will form DevBio9e-Fig-05-37-0.jpg Zinc-finger TF for muscle actin, myosin, TBX-6 Also TF for Snail TF which blocks notochord induction

Conditional Specification also plays a role Integrates with the autonomous specification patterns DevBio9e-Fig-05-39-0.jpg

Axis Formation accomplished prior to cleavage! Fertilization rearranges cytoplasmic determinants determines dorsal-ventral DevBio9e-Fig-05-35-0.jpg determines anterior-posterior Left-right: unclear but nodal shows it later

Rotational, Holoblastic Cleavage in the nematode Caenorhabditis elegans DevBio9e-Fig-05-42-1R.jpg hermaphrodite

Figure 5.42 The nematode Caenorhabditis elegans (Part 2) Both autonomous and conditional specification at work early on. If cells are separated: P1 will develop autonomously Stem cell divisions are meridional DevBio9e-Fig-05-42-2R.jpg Founder cell divisions are equatorial 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 axis is determined by egg shape Which end is posterior is determined by sperm (the closest end is back) DevBio9e-Fig-05-43-0.jpg Sperm CYK-4 activates egg rho, actin rearrangement causes assymetric first cleavage division

AB division leads to both dorsal ventral and left-right axes Assymetrical division of AB-MS forces AB dorsal and MS ventral DevBio9e-Fig-05-48-0.jpg Delta-notch recognition between daughters of AB and MS gives left-right

Cytoskeletal rearrangement also pushes P-granules into the germ line DevBio9e-Fig-05-44-0.jpg

The cells of the blastula have specified fates in Xenopus..... DevBio9e-Fig-07-05-0.jpg Gastrulation changes all of that, .....afterwards all cell fates are determined!

Development of “Organizing Power” at the dorsal blastopore lip The bottle cells get the ball rolling but the real power is conferred on the first cells through the blastopore. DevBio9e-Fig-07-06-1R.jpg

This ability to determine cell fates is called... The dorsal mesoderm keeps the power to determine other cell’s fates throughout gastrulation: “Spemann’s Organizer” This ability to determine cell fates is called... “Primary Embryonic Induction” DevBio9e-Fig-07-06-2R.jpg

The dorsal lip cells first have to become competent to be “Organizer” Cortical rotation shifts disheveled, GBP, Wnt-11 to dorsal side of embryo DevBio9e-Fig-07-21-1R.jpg The area of Dsh accumulation is seen as a gray crescent in some amphibian embryos

β-catenin starts out everywhere in the embryo but only survives GSK3 in the dorsal portion due to Dsh, GBP and Wnt-11 DevBio9e-Fig-07-21-2R.jpg

The dorsal vegetal cells of the Nieuwkoop Center turn on “Organizer” Wnt and Vg-1 (TGF-B family) induce pre-dorsal lip mesoderm DevBio9e-Fig-07-18-2R.jpg FGF needed for all mesoderm

Figure 7.22 Summary of events hypothesized to bring about induction of the organizer in the dorsal mesoderm Nodal DevBio9e-Fig-07-22-0.jpg Vg-1

Figure 7.23 Vegetal induction of mesoderm (Part 2) DevBio9e-Fig-07-23-2R.jpg

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

DevBio9e-Table-07-02-0.jpg

Figure 7.26 Localization of chordin mRNA The “Organizer” is induced prior to gastrulation Dorsal blastopore lip Blastopore Dorsal mesoderm DevBio9e-Fig-07-26-0.jpg Continues to organize events throughout its own differentiation

Interestingly, the primary mechanism is by means of inhibition.... DevBio9e-Fig-07-30-2R.jpg

Without the “Organizer” you get mainly skin and gut Presumably, the Wnt, FGF and RA signals arise from endoderm and ectoderm Without the “Organizer” you get mainly skin and gut DevBio9e-Fig-07-35-0.jpg

Don’t underestimate the power of the “Organizer”! 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 DevBio9e-Fig-07-31-0.jpg 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 DevBio9e-Fig-07-36-0.jpg

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 DevBio9e-Fig-08-09-0.jpg Wnt and FGF from the hypoblast induce Koller’s sickle epiblast

Figure 8.10 Induction of a new embryo by transplantation of Hensen’s node (Part 1) DevBio9e-Fig-08-10-1R.jpg

Possible contribution of inhibition of BMP signaling DevBio9e-Fig-08-11-0.jpg Appears to be similar to the frog....

In the chick, the hypoblast plays a large role much like the frog endoderm DevBio9e-Fig-08-12-1R.jpg

Anterior-Posterior axis parallels the rotation inside the shell Figure 8.8 Specification of the chick anterior-posterior axis by gravity DevBio9e-Fig-08-08-0.jpg Anterior-Posterior axis parallels the rotation inside the shell

Rostral-Caudal (Anterior-Posterior) axis extension in chick embryos The combination of positional specification, complex signaling and TF (Hox, etc.) expression is thought to cause axis. DevBio9e-Fig-08-13-0.jpg

Left-right asymmetry in the chick embryo This is farther along Nodal and Pitx2 again are implicated DevBio9e-Fig-08-14-0.jpg