Vishesh Khanna Kimmel Lab

Neurocranium Images from Kimmel et al. 2001

Image from Wada et al. 2005

 Similar mutations or genetic characteristics responsible for neurocranial defects in zebrafish may help explain the genetic mechanism underlying palate defects in humans.

 “Histone Deacetylases”  Repress transcription indirectly by binding to transcription factors

 Morpholino knockdowns of HDAC4 mRNA ⇩  Severe neurocranial defects WT 6dpf hdac4 MO 6dpf Photos courtesy of Dr. April DeLaurier

Genes Controlling Proper Neurocranium Formation HDAC4

X Genes Controlling Proper Neurocranium Formation X

 Determine the downstream target genes of HDAC4  Analyze the effects of HDAC4 knockdown on these target genes

 “Within the tissue”  Hybridization: Joining of nucleic acid strands  Performed Double Fluorescent In-Situ Hybridization

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

 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

 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

30 hpf WT wnt11sox9

36 hpf WT pdgfrasox9

36 hpf WT pdgfaasox9

 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

 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