Download Delineation of ESDN-Dependent Signaling Mechanisms Required for Zebrafish Eye Development

Delineation of ESDN-Dependent Signaling Mechanisms Required for Zebrafish Eye Development
Ryan M. Joy, Erin E. Wysolmerski, Bryan A. Ballif and Alicia M. Ebert
Department of Biology, University of Vermont, Burlington, VT, 05405, USA.
Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is an orphan
transmembrane receptor, originally found in a screen to identify proteins with signal peptides
expressed in vascular endothelial cells. We identified ESDN in two large-scale
phosphoproteomic analyses. The first found ESDN tyrosine phosphorylated from murine
embryonic brain. The second was a functional proteomics screen to identify substrates of Src
family tyrosine kinases that when phosphorylated would lead to their interaction with the
signaling adapter Crk-Like (CrkL). Given ESDN’s extracellular domain is reminiscent of
neuropilins, which play important roles in the migration of both vascular endothelial cells and
neurons, we examined the expression pattern of Esdn across zebrafish development by in situ
hybridization. Esdn expression is strongly neuronal, including in the retina, and greatly overlaps
with Crkl expression. Morpholino-based knockdown of Esdn in zebrafish resulted in incomplete
development of the retina and reduced numbers of retinal ganglion cells (RGCs). The morphant
phenotype was rescued RNA encoding full-length ESDN in a dose-dependent fashion. To assess
phenotypes, we scored brain innervation by RGCs in whole-mount embryos. We also examined
RGC cell counts in sectioned embryos during the first three days of development. These data
support a role for ESDN in neuronal development, specifically in RGCs.