An in vivo screen to identify candidate neurogenic genes in the developing Xenopus visual system

Neurogenesis in the brain of Xenopus laevis continues throughout larval stages of development. We developed a 2-tier screen to identify candidate genes controlling neurogenesis in Xenopus optic tectum in vivo. First, microarray and NanoString analyses were used to identify candidate genes that were differentially expressed in Sox2-expressing neural progenitor cells or their neuronal progeny. Then an in vivo, time-lapse imaging-based screen was used to test whether morpholinos against 34 candidate genes altered neural progenitor cell proliferation or neuronal differentiation over 3 days in the optic tectum of intact Xenopus tadpoles. We co-electroporated antisense morpholino oligonucleotides against each of the candidate genes with a plasmid that drives GFP expression in Sox2-expressing neural progenitor cells and quantified the effects of morpholinos on neurogenesis. Of the 34 morpholinos tested, 24 altered neural progenitor cell proliferation or neuronal differentiation. The candidates which were tagged as differentially expressed and validated by the in vivo imaging screen include actn1, arl9, eif3a, elk4, ephb1, fmr1-a, fxr1-1, fbxw7, fgf2, gstp1, hat1, hspa5, lsm6, mecp2, mmp9, and prkaca. Several of these candidates, including fgf2 and elk4, have known or proposed neurogenic functions, thereby validating our strategy to identify candidates. Several candidates that we tested in the morpholino screen had no previously demonstrated neurogenic functions, including gstp1, hspa5, and lsm6, suggesting that our screen effectively identified unknown neurogenic candidates. Several of the candidate neurogenic genes have been previously implicated as human disease genes, such as mecp2 and fmr1-a, or in pathways related to disease genes, providing the groundwork to use Xenopus as an experimental system to probe conserved disease mechanisms.Together the data identify candidate neurogenic regulatory genes and demonstrate that Xenopus is an effective experimental animal to identify and characterize genes that regulate neural progenitor cell proliferation and differentiation in vivo.