The Characterization of RhoGEFs and The Role of NET1 in Zebrafish Gastrulation

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The Characterization of RhoGEFs and The Role of
NET1 in Zebrafish Gastrulation

• By Julie Li Yang
• Mentor Dr. Alexander F. Schier and Dr.
  Katherine Joubin
• Skirball Institute of Biomolecular Research,
  N.Y.U. Medical School

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Introduction

• The central aspect of gastrulation is cell
  movement
• Movements are regulated by signal transduction in
  the embryo
• Many Intercellular signaling molecules have been
  identified
• Movement is driven by the actin cytoskeleton
  inside the cell
• Little is known about the details of cellular
  signaling pathways
• The Rho Guanine Nucleotide Exchange Factors
• RhoGEFs are molecular switches that activate Rho
  GTPases,
• which regulate the actin cytoskeleton
• We propose that the RhoGEFs play crucial a role
  in the regulation of actin cytoskeleton dependent
  cell movements during gastrulation.

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The Principle Gastrulation Movements in the
Zebrafish

• Epiboly (red arrows)
• Cells spread and thin over the yolk
• Internalization (green arrows)
• Leading cells internalize to form germ layers
• Convergence Extension (blue / yellow arrows)
• Embryonic axis narrows lengthens

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The Gastrulation Period of Zebrafish Embryonic
Development
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The Function of RhoGEFs in Actin Cytoskeleton
Dependent Cell Movements
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Method and Materials

• Identify Zebrafish RhoGEFs
• We obtain the cDNA and primers for our experiment
• Reverse Transcriptase PCR
• We determine presence of RhoGEFs in the fishy
• In Situ Hybridization
• We determine the localization of RhoGEFs in the
  fishy
• Morpholino-induced Knock Down
• Morpholinos Block Translation of mRNA
• Defects in the fish may reveal Gene function
• Over Expression of mRNA
• Over expression causes increased RhoGEF activity
• Similar to the morpholino experiment, we examine
  morphological defects to determine gene function

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Results

• GEF Search produced 30 primers for PCR 42 ESTs
  for ISH
• PCR revealed no GEFs that were present at shield
  stage
• ISH yielded one GEF with an expression pattern of
  interest in WT and MZoep embryos
• Zebrafish homologue of Neuro Epithelioma
  Transforming Gene 1 (NET1) in mice and humans.
• Two isoforms of NET1 in zebrafish present during
  gastrulation.
• Morpholino-induced Knock Down of NET1-long
  produced broader and diffuse axis
• Over Expression of NET1-long produced gaps of
  expression around the margin of the embryo