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Published byDora Shields Modified over 8 years ago
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Vishesh Khanna Kimmel Lab
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Neurocranium Images from Kimmel et al. 2001
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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 ⇩ Severe neurocranial defects WT 6dpf hdac4 MO 6dpf Photos courtesy of Dr. April DeLaurier
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Genes Controlling Proper Neurocranium Formation HDAC4
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X Genes Controlling Proper Neurocranium Formation X
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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” Hybridization: Joining of nucleic acid strands Performed Double Fluorescent In-Situ Hybridization
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Step 1: Synthesis of antisense RNA probes from template DNA Linearized DNA RNA Polymerase RNA probe complementary to cellular mRNA Shh Plasmid with gene insert Restriction Digest
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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 Label POD mRNA Probe Label POD mRNA Probe
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Surveyed literature (i.e. Pubmed, ZFIN) on potential genes involved in neurocranial development Candidate genes found were: ◦ wnt11 ◦ shh ◦ fgfr1b ◦ pdgfaa ◦ pdgfra ◦ mirn140 ◦ sox9 Performed the In situ Hybridizations
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30 hpf WT wnt11sox9
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36 hpf WT pdgfrasox9
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36 hpf WT pdgfaasox9
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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 Dr. Peter O’Day Chelsie Fish The SPUR participants
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