Expression analysis of GFAP like gene in Zebrafish embryogenesis

Abstract

The present study focuses on a cDNA fragment of 2.6 kb coding for glial fibrillary acidic protein (GFAP) related gene in zebrafish. Earlier work in the lab revealed that this gene showed 72% homology, at the amino acid level, with the mammalian gene coding for GFAP. Due to variations observed in sequence of head domain of GFAP and in expression pattern compared to that of rodent GFAP, this gene was named as zebrafish GFAP like gene (zfgfap-l)( Gene Bank Accession No: AY 397679).In this project, the expression pattern of zfgfap-l was studied by RT-PCR and in situ hybridization. RT-PCR results showed that the expression of zfgfap-l started as early as 1.5 hours post-fertilization (hpf) and increased steadily up to 10 hpf, then became a constant level of expression till 30 hpf. In embryos at sphere stage, the expression was detected in the cells of the superficial layer by in situ hybridization. After gastrulation, the expression became restricted to the neural tube, particularly in the presumptive forebrain, midbrain and posterior neural tube. This expression pattern continued to be similar at least until 24 hpf. At 48 hpf, the expression was mainly detected in the subventricular zone of the forebrain, midbrain, and hindbrain and the dorsoventral axis of the presumptive spinal cord. This expression pattern of zfgfap-l in neural progenitor cells prompted the present author to speculate that this gene could be one of the key molecules involved in zebrafish neurogenesis. In accord with this, zfgfap-l was found to be absent in several distinct locations of mindbomb mutants which have a defective Delta-Notch signaling pathway, resulting in excessive differentiation of progenitors into the neuronal phenotype. These results suggest that maternally transferred zfgfap-l transcripts continue to be present in the precursors of neuronal and non-neuronal cell populations during early embryogenesis and subsequently become restricted to the cellular populations in the subventricular zone where the neural stem cells lie.To confirm if zfgfap-l was indeed associated with progenitors, zfgfap-l morpholino was microinjected into embryos and the expression of two proneural markers- neuroD and neurogenin1 (ngn1) was examined. If indeed zfgfap-l was associated with progenitors, there would be significant changes in the expression of neuroD and ngn1. The expression was found to be similar in both wild type and morpholino injected embryos. However, more work needs to be done since there are a number of other factors (like redundancy of zfgfap-l) that may be involved.