Transcript HDAC4 and Cranial Development

Vishesh Khanna
Kimmel Lab
Neurocranium
Images from Kimmel et al. 2001
Image from Wada et al. 2005
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Similar mutations or genetic characteristics
responsible for neurocranial defects in
zebrafish may help explain the genetic
mechanism underlying palate defects in
humans.
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“Histone Deacetylases”
Repress transcription indirectly by binding to
transcription factors
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Morpholino knockdowns of HDAC4 mRNA
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Severe neurocranial defects
WT
6dpf
hdac4 MO
Photos courtesy of Dr. April
DeLaurier
6dpf
HDAC4
Genes Controlling Proper Neurocranium Formation
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HDAC4
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Genes Controlling Proper Neurocranium Formation
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Determine the downstream target genes of
HDAC4
Analyze the effects of HDAC4 knockdown on
these target genes
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“Within the tissue”
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Hybridization: Joining of nucleic acid strands
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Performed Double Fluorescent In-Situ
Hybridization
Step 1: Synthesis of antisense RNA probes from
template DNA
Shh
Restriction Digest
Linearized DNA
Plasmid with
gene insert
RNA probe complementary
to cellular mRNA
RNA Polymerase
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Step 2: Hybridization of probe to the target
mRNA sequence
The RNA probe is recognized by an antibody
that contains a peroxidase, which when
exposed to another substrate, creates a
fluorescent signal
POD
Label
POD
Label
Probe
Probe
mRNA
mRNA
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Surveyed literature (i.e. Pubmed, ZFIN) on
potential genes involved in neurocranial
development
Candidate genes found were:
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wnt11
shh
fgfr1b
pdgfaa
pdgfra
mirn140
sox9
Performed the In situ Hybridizations
wnt11
sox9
30 hpf
WT
pdgfra
sox9
36 hpf
WT
pdgfaa
sox9
36 hpf
WT
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Identify best methods of visualizing and
staging embryos for ideal expression
patterns
Simultaneous in-situs of wild type and
morpholino-injected zebrafish embryos
Identify genes affected by HDAC4
knockdown
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Dr. Chuck Kimmel
Dr. April DeLaurier
The Kimmel Lab:
◦ Dr. Brian Eames, Dr. Mark Sasaki, Jared Talbot, Dr.
Jamie Nichols, Bonnie Ullmann, Tyler Huycke
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Dr. Peter O’Day
Chelsie Fish
The SPUR participants