Transcriptomic networks of gba3 governing specification of the dopaminergic neurons in zebrafish embryos

Abstract

Background Molecular networks associated with dopaminergic (DA) neurogenesis remain undefined within mammalian models. To address this issue, the transient zebrafish model lmx1al: EGFP was generated, which expresses GFP in the DA precursor cells as well as in the DA neurons of the ventral diencephalon (VD). We found that the novel pseudogene gba3 has not been well studied in zebrafish neurogenesis. Objective Crucial networks associated with gba3 transcripts were investigated because the biological functions of these networks have not been reported in DA neurogenesis in zebrafish. Methods RNA isolation and sequencing were employed with GFP-expressing cells from 20-, 22-, and 24 h post-fertilization (hpf), while subsequent transcriptomic analysis generated differentially expressed genes with DA neurogenesis (DEG-DA) list. Hierarchical cluster analysis provided absolute guidance for the collection of gba3, an essential transcript that is strictly spatiotemporally expressed during DA neurogenesis, which was proven with whole-mount in situ hybridization (WISH) and knockdown and rescue of the gba3 transcripts in zebrafish embryos. Results The gba3 transcripts were restricted to the midbrain at 24 hpf and the midbrain and pectoral fins at 30 hpf in zebrafish embryos. Functional studies with knockdown of gba3 found a diminished state in the midbrain and midbrain-hindbrain boundary (MHB) and an underdeveloped condition in the anteroposterior and dorsolateral axis relative to the wild type (WT) at 24 hpf. However, it was recovered after forced expression of gba3 transcripts at 24 hpf. Molecular markers for the DA precursors and mature neurons lmx1al, nurr1, th, and pitx3 were analyzed in the gba3 MOs. The levels of transcripts lmx1al, nurr1, and th were significantly reduced in the midbrain ventral diencephalon (VD) and hindbrain of gba3 morphants compared to the WT at 24 hpf, while expression patterns of pitx3 transcripts showed no differences in the identical regions between gba3 MOs and the controls. Conclusions We discuss transcriptional networks in which transcripts of gba3 plausibly govern the specification of dopaminergic neurogenesis in zebrafish embryos.