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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 Photos courtesy of Dr. April DeLaurier 6dpf HDAC4 Genes Controlling Proper Neurocranium Formation X HDAC4 X Genes Controlling Proper Neurocranium Formation 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 Shh Restriction Digest Linearized DNA Plasmid with gene insert RNA probe complementary to cellular mRNA RNA Polymerase 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 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 wnt11 sox9 30 hpf WT pdgfra sox9 36 hpf WT pdgfaa sox9 36 hpf WT 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