Axis determination and early development in amphibians

We are standing and walking with parts of our body which could have been used for thinking had they developed in another part of the body

(Hans Spemann 1943)

Spemann’s initial experiments

Spemann’s organiser

• The term "Organizer" was coined to emphasize the ability of this dorsal blastopore lip tissue to direct the development of the host tissue and to give these redirected cells a coherent, unified, organization. "A piece of the upper blastoporal lip of an amphibian embryo undergoing gastrulation exerts an organizing effect on its environment in such a way that, if transplanted to an indifferent region of another embryo, it causes there the formation of a secondary embryonic anlage. Such a piece can therefore be designated as an organizer." This tissue had the ability to invaginate and differentiate autonomously, to induce the neural plate and, by assimilative induction, to organize somites from the lateral plate mesoderm of the host.

The functions of the organiser

• The ability to self-differentiate dorasl mesoderm

• The ability to dorsalise the surrounding mesoderm into paraxial (somite-forming) mesoderm (when it would otherwise form ventral mesoderm)

• The ability to dorsalise the ectoderm, inducing the formation of the neural tube

• The ability to initiate the movements of gastrulation

Molecular events forming the Organiser

sperm

Dsh

Dsh

GSK Dsh

GSK

-catenin degraded -catenin stable

No -catenin in nuclei -catenin in nucleiCortical rotation

V D

DV

WNT pathway

Transcriptional events

-catenin degraded -catenin stabilised (nuclear)

Siamois gene

V D

Tcf3 proteins

Repressed

Tcf3 proteins+-catenin

Activated

goosecoid gene

Siamois proteinInput from TGF- (Vg1, Nodal)signalling pathway

Activated

Dorsal axis

Ability of goosecoid mRNA to induce a new D-V axis

• An embryo (16-cell stage) whose ventral vegital pole was injected with goosecoid mRNA induced a second axis (with two complete sets of head structures precursors). This did not happen when embryos were injected with a mutant form of the DNA-binding homeodomain (Niehrs et al, 1993 Cell 72: 491-503)

Model for organiser induction and mesoderm formation

V D V D V D

VegT, Veg1 -catenin

Xnr

Stage 8 Stage 9 Stage 10

-catenin Nodal related high Organiser

VegT, Vg1 Nodal related low Ventral mesoderm

BMP4 gradient (ventral and lateral mesoderm)

Organiser

The Diffusible proteins of the Organiser I: BMP inhibitors

• Noggin: diffusible protein accomplishing two of the major functions of the organiser: induction of dorsal ectoderm to form neural tube and formation of dorsal mesoderm. Binds to BMP4 and BMP2 and inhibits their binding to

receptors (Zimmerman et al 1996 Cell 86, 599-606). • Chordin: diffusible protein directing central nervous system development. Binds

to and inhibits BMP4 (Sasai et al 1994 Cell 79, 779-790). • Nodal-related protein 3 (Xnr-3): diffusible protein synthesized by the superficial

cells of the organiser and is also able to block BMP4 (Smith et al 1995 Cell 82, 37-46).

• Follistatin: secreted glycoprotein that induces epidermal cell fate (skin) overriding the default neural fate (Hemmati-Brinvalou and Melton 1994 Cell 88, 273-281).

Rescue of dorsalised structures by Noggin

Noggin expression

Chordin expression

Bone Morphogenetic Factor 4 (BMP4): an Organiser antagonist

• The Xenopus BMP4 is a member of the TGF- family of ligands. It induces ventral mesoderm and (ventral) epidermal fates and suppresses neural ones.

Blood cells

kidneys

muscle

BMP4 relative concentration

The Diffusible proteins of the Organiser II: Wnt inhibitors

• Cerberus responsible for the anterior-most head structures; cytokine of the cysteine knot superfamily binds Xwnt8 (member of the Wnt family) as well as BMPs and Nodal-related (Bouwmeester et al 1996 Nature 382, 595-601).

• FRZB and Dickkopf Wnt receptor antagonists prevent signalling from Wnt receptors. Frzb is able to bind Wnt agonists and by binding to Frizzled proteins prevent the interaction between ligand and receptor (Wang et al, 1997 Cell 88, 757-766; Glinka et al, 1998 Nature 391, 357-362).

Cerberus induces head structures

Frzb expression and function

Frzb binds to Xwnt8

Cerberus Dickkopf Frzb Chordin Noggin Follistatin

BMP4Nodal-relatedXwnt8

Ventral

Dorsal

AnteriorPosterior

Notochordal mesodermPrechordal plate mesodermPharyngealendoderm

Posterior transforming proteins: Wnt signals and retinoic acid

In Xenopus embryos a gradient of Wnt signalling (Xwnt8) and -catenin is highest in the posterior and absent in the anterior. There is also a high concentration of retinoic acid (RA) at the posterior end of the neural tube. RA is especially important in patterning the hindbrain but not the forebrain (Dupé and Lumsden 2001 Development 128, 2199-2208)

The anterior transforming proteins: Insulin-like growth factors

In addition to proteins that block BMP4 and Wnt signalling in the head there is a positive signal that promotes anterior head development. Insulin-like growth factors (IGFs) are required to form the anterior-neural tube with its brain and sensory placodes. (Pera et al Dev Cell 1, 655-665)

Summary

Ventral Dorsal

Anterior

Posterior

Wnt

BMP4

RA

IGFs

ORGANISER