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

Abstract

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. Genes with no previously demonstrated neurogenic functions, gstp1, hspa5 and lsm6, were identified from the morpholino experiments, suggesting that our screen successfully revealed unknown candidates. Genes that are associated with human disease, such as such as mecp2 and fmr1-a, were identified by our screen, providing the groundwork for using 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.

Using Xenopus to understand human disease and developmental disorders

“…Xenopus also has greatly enhanced our understanding of DNA replication and repair (Kalb, Mallery, Larkin, Huang, & Hiom, ; Long, Joukov, Budzowska, & Walter, ; Räschle et al, ; Shintomi, Takahashi, & Hirano, ; Zhang et al, ), and the mechanisms of genome organization, transcriptional regulation, and epigenetics (Belikov, Berg, & Wrange, ; Bogdanović et al, ; Buisine et al, ; Gazdag, Jacobi, van Kruijsbergen, Weeks, & Veenstra, ; Gao et al, ; Hontelez et al, ; Owens et al, ; Tamaoki et al, ; Wang et al, ; Wen, Fu, Guo, Chen, & Shi, ). Numerous studies in Xenopus elucidate mechanisms of morphogenesis and organogenesis ( Agricola et al, ; Bestman, Huang, Lee‐Osbourne, Cheung, & Cline, ; Metikala, Neuhaus, & Hollemann, ; Mimoto, Kwon, Green, Goldman, & Christian, ; Nie & Bronner, ; Okada, Wen, Miller, Su, & Shi, ; Ossipova et al, ; Tanizaki, Ishida‐Iwata, Obuchi‐Shimoji, & Kato, ). Many of the components of the well‐known signaling pathways were discovered in Xenopus and fly, and new discoveries in this field continue to rely on Xenopus (e.g., Lee et al, ; Lee, Shi, & Zheng, ; Park et al, ; Zhang et al, ; Zhao, Shi, Winey, & Klymkowsky, ).…”

Using Xenopus laevis retinal and spinal neurons to study mechanisms of axon guidance in vivo and in vitro

“…Co-electroporation coupling DNA/DNA or DNA/MO also yields efficient uptake with this technique [24], [39]. For example, Bestman et al used this approach and co-electroporated MO along with a plasmid that drives GFP expression under the control of cell specific promoter to identify genes that contribute to neurogenesis of the optic tectum in Xenopus tadpoles [40]. …”

TRA2A-induced upregulation of LINC00662 regulates blood-brain barrier permeability by affecting ELK4 mRNA stability in Alzheimer’s microenvironment

“…ELK4 is a member of the Ets transcription factor family, which is a critical regulator of many neurodevelopmental events [51] . ELK4 can not only bind to gene promoter directly but also cooperate with other transcription factors to regulate target gene transcription [19,52] .…”