ZEBRAFISH GENETICS AND DEVELOPMENT

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ZEBRAFISH GENETICS AND DEVELOPMENT LECTURE
4 Patterning the Anteroposterior Axis of the
Vertebrate CNS October 31, 2005 Bruce
Appel Department of Biological Sciences
Required reading Repressor activity of
Headless/Tcf3 is essential for vertebrate head
formation (2000) Kim et al., Nature 407,
913-916. A small population of anterior cells
patterns the forebrain during zebrafish
gastrulation (1998) Houart et al., Nature 391,
788-792. Recommended reading A mutation in the
Gsk3-binding domain of zebrafish
Masterblind/Axin1 leads to a fate
transformation of telencephelon and eyes to
diencephalon (2001) Heisenberg et al., Genes
Dev. 15, 1427-1434. Establishment of the
Telencephalon during Gastrulation by Local
Antagonism of Wnt Signaling (2002) Houart et
al., Neuron 35, 255-265.
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• Lecture Overview
• A brief intro to vertebrate neural induction
• A brief intro to anteroposterior patterning of
  vertebrate CNS
• Vertebrate CNS AP patterning and Wnt signaling
• Mutations of canonical Wnt members disrupt AP
  patterning
• Embryological experiments reveal a source of
  signals

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How is a uniform pile of cells transformed into
the central nervous system?

• There are four basic (and oversimplified) steps
• Neural induction
• Anteroposterior patterning
• Dorsoventral patterning
• Neuronal and glia specification

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Remember the Spemann-Mangold organizer can
induce formation of an entire embryo, including
CNS
Works for amphibians
Cross section showing donor cells (brown) and
host cells organized into CNS (bracket)
Same goes for zebrafish
Saude et al., 00
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Hypothesis the organizer is the source of neural
inducing signals
neural
epidermal
epidermal
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TGF-? Signaling and Neural Induction
Dominant negative receptors
II
I
Inhibits formation of neural tissue
Causes formation of neural tissue from ectoderm
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The organizer expresses factors that induce
neural tissue by blocking Bmp signaling activity
neural
epidermal
epidermal
epidermal
Bmp
neural
Bmp
Chordin Noggin
Streit and Stern 99
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Once neural tissue is induced, how is it
subdivided?
Spinal cord
hindbain
midbrain
forebrain
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Anteroposterior CNS Patterning Nieuwkoop and
Nieuw Stuff
Two step model
1. All CNS is induced to have anterior
character 2. Posterior CNS is then transformed
What is the source of transformer? What is the
idendity of transformer?
Candidates retinoic acid FGFs Wnts
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Fate maps are needed to understand AP patterning
Summary of work from K. Woo and S. Fraser (1995)
T, telencephelon R, retina D, diencephelon H,
hindbrain Upper row, beginning of
gastrulation Bottom, end of gastrulation
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Transplantations reveal lateral margin, but not
dorsal margin, as a source of posteriorizing
signal
180
Ectopic Krox20 (hindbrain) expression
90
45
0
Woo and Fraser 97
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A signal from the organizer may limit the action
of a transformer
boz mutant embryos have reduced forebrain and
hindbrain appears to be moved anteriorly. These
mutants have reduced organizer activity.
wt
boz
Fekany et al 00
Interpretation
Posteriorizing activity resides in lateral
margin. boz excludes it from the dorsal midline.
Whats the signal? wnt8 is expressed in lateral
but not dorsal margin of wt embryos. Expression
is more dorsal in boz mutants (Fekany et al).
wnt8 activity is necessary for posteriorization (E
rter et al, 01).
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Zebrafish mutants reveal that Wnt signaling is
important for neural AP patterning
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How did they produce embryos that lack both
maternal and zygotic hdl function? What do the
gene expression patterns tell you?
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hdl mutation disrupts the canonical Wnt pathway
What is the evidence that hdl encodes Tcf3? What
is the evidence that Hdl/Tcf3 functions as a
repressor?
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Summary Tcf3 promotes forebrain development by
inhibiting Wnt target genes
Anterior brain
Posterior brain
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Posterior brain
Posterior brain
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Df/Df is homozygous deletion of wnt8
otx2 is a forebrain marker
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What do we learn from these three mutations about
vertebrate CNS AP patterning?
Wnt
?
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Clever embryological experiments reveal sources
of pattering information
Whats the hypothesis?
What do these experiments establish?
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How did they assay patterning defects?
emx telencephelon
dlx2 telencephelon anterior diencephelon
shh ventral forebrain
normal
Row 1 ablated
Differentiated neurons
zash1a Putative neuronal marker
normal
Row 1 ablated
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Are there brain defects because row1 cells
contribute to a big chunk of brain?
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When does signaling occur?
Labeling emx1 krox20
Early ablation
Late ablation
So, whats the row 1 signal?
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A PCR-based subtractive hybridization screen for
genes expressed by Row 1 cells produced tlc,
which is related to frizzled genes.
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Tlc is required for telencephelon development
Tlc antagonizes Wnt signaling in
concentration-dependent manner
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Inhibition of Wnt signaling from diencephalon
promotes telencephalon development.
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Tlc
Wnt
Wnt targets
Wnt targets
Anterior brain
Posterior brain
Wnt
Tlc