Dorsal and Ventral Gene Expression Associated with Neural Induction

in Xenopus embryos Notes from review paper by De Robertis and Kuroda, 2004

Jennifer Slade

B.Sc (Hon), M.Sc Candidate

Overview

• Introduction to Xenopus– Life cycle and stages of

development– Fate map of germ layers

• Discovery of Induction– Possible genes involved in

induction

• Formation of signaling centers– Dorsal

• Nieuwkoop and BCNE• Spemann

– Ventral

• Dorsal Genes– BMP antagonists:

• Chordin, Noggin and Xnr3

– Wnt antagonists:• Frzb-1, Crescent, sFRP-2 and

Dickkopf

– Cerberus

• Ventral Genes– BMP synexpression group:

• Crossveinless-2, Twisted Gastrulation, Xolloid-Related and Bambi

– Sizzled

• Summary

Dorsal/Ventral (D/V) Axis

• Formation:– Sperm fertilizes egg at animal pole– Cortical rotation shifts vegetal pole 30º– Point of sperm entry becomes ventral– Point opposite of sperm entry becomes dorsal

• D/V axis gives rise to backbone and belly• Dorsal ectoderm: Neural Plate and CNS (therefore want neural

induction)• Ventral ectoderm: Epidermis and its derivatives (therefore do NOT

want neural induction)• Mesoderm: Dorsal Ventral

» Prechordal Plate, Notochord, Somites, Kidney, Lateral plate and ventral blood islands

» Neural Induction Less Neural Induction

Spermentry

Ventral

Dorsal

Fate Map of Germ Layers in Blastula

Animal

Vegetal

Ventral DorsalVM DM

Endoderm

MesodermEctoderm

DEcVEc

VEnd DEnd

Discovery of Induction in Mesoderm

• Spemann and Mangold, 1924

• Transplanted small piece of dorsal mesoderm from one embryo into ventral mesoderm of another

• Changed differentiation of neighbouring ventral cells– dorsalized – Became CNS, somites

rather than epidermis

• Twinned embryo

What genes are involved in induction?

• Transplant was from dorsal to ventral– Neural induction occurs at dorsal, and not at ventral

• Therefore genes that affect dorsal and ventral axis formation may be involved…

• Possible genes discovered through mutations observed in other model organisms– Seven genes in zebrafish – Seven genes in Drosophila– All associated with members from bone morphogenetic

protein (BMP) signaling pathway

Another Gene Present After D/V Formation

• β-catenin accumulates in dorsal blastula after cortical rotation

• Moves from cytoplasm into nuclei to induce transcription of various genes– Siamois – dorsalizing gene

– Nodal-related genes – specify Nieuwkoop center

• Partially regulated through Wnt signaling• Important in the formation of signaling centers

Formation of Dorsal Signaling Centers

• Two signaling centers:– Nieuwkoop center (dorsal vegetal)

• Induces dorsal mesoderm in overlying cells– Nodal-related factors (Xnr1, 2, 4, 5 and 6)

• Nieuwkoop center cells themselves form dorsal anterior endoderm

– Blastula Chordin and Noggin expression center (BCNE) (dorsal animal)

• Eventually gives rise to anterior neural tissue– Chordin, noggin and Xnr3

• Both form simultaneously when zygotic transcription start

• Both require β-catenin signaling

“Combination” of Nieuwkoop and BCNE: Spemann Organizer

• Once blastula becomes gastrula, signals from Nieuwkoop center lead to the generation of the Spemann organizing center at the dorsal lip

• In addition, a ventral signaling center is established

Dorsal Gastrula Genes

• Expressed from Spemann Organizer

• Include:– BMP signaling antagonists:

• Chordin, noggin and Xnr3

– Wnt signaling antagonists• Secreted Frizzled-related

proteins (sFrps), and Dickkopf

– Cerberus

BMP Signaling

• BMP’s are growth factors

• Binds to BMP receptors (BMPR)

• Interacts and phosphorylates SMAD1 leading to interactions with other SMAD proteins from TGF-β signaling pathway

• Complex enters nucleus to repress transcription of target neural genes

• Overall: BMP signaling reduces neural induction

SMAD1

SMAD1

P

SMAD1P

SMAD4

Neural Genes

BMP

BMP Antagonist: Chordin

• Large protein, about 1000 amino acids• Contains four cysteine-rich domains (CR1 – CR4) of about 70

residues each– Constitutes BMP binding

• Binding to BMP proteins prevents them from binding to their receptor – Reduces BMP signaling– Allows neural induction

• High concentrations of Chordin dorsally– Enough to block BMP signaling by itself

• When Chordin is knocked-out in mice:– Small percentage of embryos ventralized

• Therefore must be other anti-BMP genes present

BMP Antagonist: Noggin

• Binds to BMP as Chordin does– Contains cysteine-knot structural motif

– Shares conserved protein fold as BMP

• Knocked-out Noggin in mice:– Still form normal gastrula and neural plate

• Both Chordin and Noggin knocked out in mice:– Embryos exhibit loss of neural development, thus both

are required

– Redundant function – same as Chordin

BMP Antagonist: Xenopus Nodal Related – 3 (Xnr3)

• Nodal-related protein– Similar to those expressed in Nieuwkoop center– Lacks mesoderm inducing activity

• When overexpressed in animal pole:– Induces neural differentiation

• Antagonizes BMP signal through amino-terminal proregion– Mechanism still unclear

• In Xenopus, after Chordin, gene most induced by early β-catenin signal

Wnt Signaling• Wnt is a secreted glycoprotein

that binds to Frizzled receptor• Has 2 functions:

– Activates Dishevelled (Dsh) which leads to neural induction

– Deactivates a degradation complex to reduce degradation of β-catenin

• Increase β-catenin induced transcription of genes

• Can either have:– Blocked Wnt signaling– Just blocked Wnt/β-catenin

signaling

Dsh

DegradationComplex

Nucleus

Wnt

NeuralInduction

Βeta-catenin

Wnt Antagonist: sFRPs

• secreted Frizzled-Related Proteins• Secrete forms of the amino-terminal cysteine-rich

domain of Wnt receptor– Like Frizzled receptor without “bottom”– When Wnt binds, no signaling cascade is initiated– Binding of Wnt proteins lowers binding to normal

Frizzled receptor and signaling

• High level of expression in Xenopus dorsal gastrula• Include Frzb-1, Crescent and sFRP2

– Frzb-1 dorsalizes mesoderm

Wnt Antagonist: Dickkopf (Dkk-1) • Encodes cysteine-rich secreted protein • Antagonizes Wnt/β-catenin signaling only• Dkk-1 binds Wnt coreceptor LRP6

– Binding of LRP6 to Wnt and Frizzled forms a ternary receptor complex– Draws one member of the β-catenin degradation complex to cell

membrane, thereby reducing degradation of β-catenin

• Therefore Dkk-1 can selectively inhibit Wnt induced degradation of β-catenin without affecting rest of Wnt signaling

• Dkk-1 also binds transmembrane protein Kremen– Binds to LRP6 at same time– Endocytosed, depleting LRP6

• Inactivation of Dkk-1: no head or prechordal plate formation

Cerberus• Cytokine• Antagonist of:

– BMP – Wnt– Nodal signaling

• Responsible for head induction

– Can induce ectopic heads

• Knocked-out:– No head formation– Only trunk and tail

Ventral Gastrula Genes• Expressed at ventral signaling center

– Ventral mesoderm and ectoderm – 180º from Spemann Organizing center

• Ventral signaling was not viewed important as it lacked inductive activity, but now that loss of function mutations in ventral signaling centers are known, this may be an area of focus in the future

• Include:– BMP synexpression group:

• Crossveinless-2 (Cv-2)• Twisted Gastrulation (Tsg)• Xolloid-Related

– Sizzled

BMP Synexpression: Crossveinless-2 (Cv-2)

• Originally discovered in Drosophila– Forms wing cross-veins

• Contains 5 cysteine-rich domains– Similar to those of Chordin

• Normal expression is upregulated by BMP4– Represses Chordin expression– Allows BMP signaling

• When overexpressed, has a BMP antagonist activity

BMP Synexpression: Twisted Gastrulation (Tsg) and

Xolloid-Related• Tsg binds to both BMP and Chordin• Forms ternary complex to prevent binding of BMP

to its receptor• Promotes cleavage of Chordin

– Xolloid-Related acts as scissors to cut Chordin• Tolloid metalloprotease• Expression upregulated by BMP4• Plays critical role in inactivation of Chordin (in conjunction with

Tsg)

• Releases BMP• Allows BMP signaling

Sizzled

• Encodes sFRP-like molecule• Has a ventralizing function:

– Increases ventral mesoderm– Decreases neural plate formation

• Overexpression phenotype is indistinguishable from loss of Chordin phenotype

• Knock-down of Chordin leads to increase in Sizzled expression and increased BMP signaling

• Exact function still undetermined– Expected to be associated with BMP signaling

Summary

• Number of dorsal and ventral genes affect downstream neural induction

• Similarities:– Both sets of genes secrete related growth factor antagonists

• Differences:– Under opposite transcriptional control by BMP signaling

• Established communication between dorsal and ventral genes

• Now need to discover what it means and why it happens

References

• De Robertis and Kuroda, 2004. Dorsal-Ventral Patterning and Neural Induction in Xenopus Embryos. Annu. Rev. Cell. Dev. Biol. 20: 285-308

• Wolpert, L., et al. 2002. Principles of Development, 2nd Edition. Oxford University Press, New York. Chapters 2-4