Neuron-specific expression of scratch genes during early zebrafish development

Abstract

Scratch (scrt) genes are neural-specific in mammals, but their homologues have not been well studied in non-mammalian vertebrates. In this report, we isolated three zebrafish scrt genes, scratch1a (scrt1a), scratch1b (scrt1b), and scratch2 (scrt2), which belong to the Snail superfamily of zinc finger transcription factors. Spatiotemporal expression analysis revealed that scrt1a and scrt2 were initially detected in the central nervous system (CNS) during early somitogenesis while scrt1b was first detectable in neuronal clusters in the brain during late somitogenesis. Interestingly, scrt-expressing cells largely overlapped with huC-positive differentiating neurons and partially with neurogenin1-positive neuronal precursor cells. In addition, scrt-expressing cells were dramatically increased in mind bomb, a neurogenic mutant. Taken together, these results suggest that each zebrafish scrt gene is specifically expressed in neuronal cells and may be involved in differentiation of distinct neuronal populations in the vertebrate nervous system.

Fig. 1.. Comparison of the amino acid sequences of Scratch homologues. (A) Multiple sequence alignments were performed using Clustal X. Scratch homologues contain one SNAG domain in the N-terminus, one Scratch domain in the central region, and five C2H2 zinc finger motifs in the C-terminus. Identical residues are shown in black. (B) The phylogenetic tree of zebrafish Scrts and their putative orthologues from other species. Human (Hs) SCRT1 (Genbank Acc. No. NP_112599), human SCRT2 (NP_149120), mouse (Mm) SCRT1 (NP_570963), mouse SCRT2 (NP_001153882), zebrafish (Dr) Scrt1a (NP_001107073), zebrafish Scrt1b (NP_001014369), zebrafish Scrt2 (NP_998802), Drosophila (Dm) scrt (AAA91035), and C. elegans (Ce) CES-1 (AAF01678).

Fig. 2.. Expression profile of scratch genes during early development. (A) The three scratch genes are expressed from somite-stages to the early larval stage but not the cleavage- and gastrula-stages. Actin was used as a loading control. (B) At the 12-somite stage (15 hpf), scrt1a expression was detected in subsets of neurons in forebrain, hindbrain, and spinal cord. (C) At the 22-somite stage (20 hpf), scrt1b was expressed in subsets neurons in the forebrain, hindbrain and spinal cord. (D) Expression of scrt2 was first detectable in the trigeminal ganglia at the 3-somite stage (11 hpf). (E) At 24 hpf, scrt1a was expressed in the dorsal telencephalon, diencephalon, tegmentum, hindbrain and spinal cord neurons. (F) At 24 hpf, expression of scrt1b was detected in the dorsal telelecephalon, tegmentum, and hindbrain. (G) At 24 hpf, scrt2 was specifically expressed in the dorsal telencephalon, diencephalon, tegmentum, hindbrain and spinal cord neurons. (H) At 72 hpf, scrt1a was detected in the ganglion cell layer in the retina, and the tectum, midbrain-hindbrain boundary, hindbrain, and spinal cord neurons. (I) At 72 hpf, scrt1b was expressed in the tectum and hindbrain. (J) At 72 hpf, expression of scrt2 was strongly detected in the retina, forebrain, midbrain, midbrain-hindbrain boundary, and hindbrain. Abbreviations: d, diencephalon; dt, dorsal telencephalon; f, forebrain; gcl, ganglion cell layer; h, hindbrain; m, midbrain; mhb, mid-hindbrain boundary; ret, retina; sp, spinal cord; tc, tectum; teg, tegmentum; tg, trigeminal ganglia. Scale bars = 200 μm.

Fig. 3.. Expression of scratch genes during early neurogenesis. Images are dorsal view with anterior to the left for all panels. (A-C) 36 hpf, (D-I) 24 hpf. (A, B) Expression of scrt1a partially overlapped with that of scrt1b (A) and scrt2 (B) in the CNS at 36 hpf. (C) Expression of scrt1b overlapped predominantly with that of scrt2 at 36 hpf. (D-F) scrt1a- (D), scrt1b- (E), and scrt2-expressing cells (F) partially overlapped with ngn1- positive neuronal precursor cells at 24 hpf. (G-I) scrt1a- (G), scrt1b- (H), and scrt2-expressing cells (I) are all huC-positive, differentiating neurons in the CNS at 24 hpf. Abbreviations: f, forebrain; h, hindbrain; m, midbrain; ret, retina. Scale bars = 100 μm.

Fig. 4.. Increase in scratch-expressing cells in mind bomb/mib, a neurogenic mutant. (A-C′) Lateral view with anterior to the left. (E-G′) Dorsal view with anterior to the left. The numbers of scrt1a (A, A′, E, and E′), scrt1b (B, B′, F, and F′), and scrt2 (C, C′, G, and G′)-expressing cells increased markedly in mib mutant embryos at 24 hpf. In mib mutant embryos, ectopic scrt-positive cells in the midbrain were detected compared to wild-type embryos (arrows in E′, F′, and G′). Scale bars = 200 μm.