Schematic diagrams of the ORF of trim46a and protein structures of Trim46a. (A) Zebrafish trim46a is present on chromosome 16 and spans 27,494 bp on the genome. trim46a includes 2322 bp of open reading frame (ORF) encoding 773 amino acids long Trim46a. (B) Trim46a contains RING_Ubox superfamily domain (amino acids 26–60), zf-B box domain (amino acids 221–263), DUF745 superfamily domain (amino acids 289–360), FN3 domain (amino acids 430–540), and SPRY superfamily domain (amino acids 548–748).

Spatiotemporal expression patterns of trim46a in zebrafish embryos at 1 cell, sphere, shield, bud, 18 hpf and 24 hpf. trim46a transcripts were distributed in the precursor region of brain along the central nervous system. (A) Maternal messages of trim46a were present at 1-cell stage. (B) trim46a transcripts were abundant in the deep cell layer (DEL), enveloping layer (EVL) and I-YSL (yolk syncytial layer) at sphere stage. (C) trim46a transcripts were localized to the ventral & dorsal region at shield stage. (D) trim46a transcripts were abundant in the central nervous system at bud stage. (E and F) trim46a transcripts were evenly distributed in the brain region. (G and H) Lateral (G) and anterior (H) view of zebrafish embryos at 24 hpf; trim46a messages were restricted to the forebrain through the telencephalon, diencephalon, midbrain, MHB, cerebellum and rhombomere. All embryos were collected synchronously from WT zebrafish for WISH analysis at the corresponding stages. MHB, midbrain-hindbrain boundary; DMB, diencephalic-mesencephalic boundary; c, cerebellum; rh, rhombomere; t, telencephalon; m, midbrain; d, diencephalon; f, forebrain. (A–H) Scale bars: 50 μm.

Putative cis-acting elements in the promoter region (3899 bp) of trim46a. Putative cis-acting elements defined by the Transfac database (Wingender et al. 1996). (A) Schematic representation of zebrafish trim46a genomic region. Twelve exons (E1 to E12) and eleven introns are depicted; the translation initiation site is indicated with an arrow. (B) List of the sixteen transcription factors which might bind to their corresponding response elements within the trim46a promoter (3899 bp). (C) The putative transcription factors are highlighted in bold and blue color represents exon1 (Cut-off p-value = 0).

Inhibition of SHH signaling with cyclopamine resulted in developmental defects in the midbrain of zebrafish embryos at 24 hpf. (A–D) Zebrafish embryos at 24 hpf which were treated 0.2% and 0.6% EtOH from 4 hpf. 0.6% EtOH-exposed embryos showed similar phenotypes of midbrain and MHB in comparison to those of 0.2% EtOH-exposed embryos. (E and F) Cyclopamine-treated embryos (20 μM) showed reduction in width of the midbrain and dorsally shrunken midbrain in comparison to those of EtOH controls. (G and H) 60 μM cyclopamine-treated embryos exhibited severe defects in the midbrain. (I–L) Spatiotemporal expression patterns of otx2b in the midbrain of cyclopamine-treated embryos at 24 hpf. CCP, cyclopamine. (A–L) Scale bars: 50 μm.

Inhibition of SHH signaling by cyclopamine decreased the level of trim46a transcripts in dose-dependent manners. (A–D) WISH analysis using foxa2 specific probe detected foxa2 transcripts in the midbrain at 24 hpf. Level of foxa2 transcripts decreased in the embryos treated with cyclopamine versus the control. (E–H) WISH analysis of cyclopamine-treated embryos using trim46a as a probe. Cyclopamine caused reduction in the level of trim46a transcripts in comparison to those of EtOH control in dose-dependent manners. (I) Quantification of foxa2 transcripts in the ZLI, and trim46a transcripts in the forebrain, midbrain and cerebellum at 24 hpf using ImageJ software. CCP, cyclopamine. (A–H) Scale bars: 50 μm.