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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, XolloidRelated and Bambi
– Sizzled
• Summary
Dorsal/Ventral (D/V) Axis
• Formation:
–
–
–
–
Sperm
entry
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
Ventral
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
Fate Map of Germ Layers in Blastula
Animal
VEc
DEc
Ventral
Dorsal
VM
DM
DEnd
VEnd
Ectoderm
Mesoderm
Endoderm
Vegetal
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
P
SMAD1
P
SMAD1
SMAD1SMAD4
Neural Genes
• 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
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
Degradation
Complex
Dsh
Neural
Induction
• 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:
Nucleus
Βeta-catenin
– Blocked Wnt signaling
– Just blocked Wnt/β-catenin
signaling
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. DorsalVentral 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