The spatio-temporal expression patterns of trim45 in early embryonic stages of zebrafish. Whole mount in situ hybridization (WISH) was performed using digoxygenin-labeled trim45 RNA probe at one cell, 30% epiboly, shield, 75% epiboly, bud, 12, 24, 36, 48 hpf stages. trim45 had maternal messages from one cell stage (A). From 30% epiboly stage (B) to shield stage (C), expression was restricted to the yolk syncytial layer (YSL). At 75% epiboly (D), trim45 was mainly expressed in dorsal mesoderm. At bud and 12 hpf stage (E and F), trim45 was strongly expressed in the head region of the anterior pole. At 24 hpf stage, trim45 was expressed in pharyngeal endoderm, tegmentum, optic tectum, and hindbrain (G and J). At 36 hpf stage, trim45 was expressed in hypothalamus, pharyngeal endoderm, and cranial ganglion (H and K). At 48 hpf stage, trim45 was expressed in pharyngeal endoderm, cranial ganglion, and retinal ganglion cell layer (I and L). Embryo orientations: lateral views (A–I) and dorsal views (J–L). Black lines point to various anatomical structures. pe, pharyngeal endoderm; ot, optic tectum; tg, tegmentum; te, telencephalon; mb, midbrain; hb, hindbrain; hy, hypothalamus; rgc, retinal ganglion cell layer; cg, cranial ganglion.

Phenotypes of the embryos from the overexpression or knock-down of trim45 in zebrafish embryos. Bright-field images of live embryos observed at 24 hpf stage. Un-injected embryos were used as a control (A and A’). trim45 mRNA (50 pg), trim45-MO (5 ng), and a mixture of trim45-MO (10 ng) and trim45 mRNA (10 pg) were injected into one cell stage zebrafish embryos respectively. Overexpression of trim45 showed a slight size expansion of midbrain and eyes (B and B’). trim45 morphants showed shrinkage in the size of midbrain and eyes (C and C’). About 79% of embryos, which were co-injected with trim45 mRNA, restored the wild-type phenotype (D and D’). Red dots – eye. Scale bar – 200 μm.

Spatio-temporal expression of olig2, rx1 and rx3, markers for diencephalon, retina, and optic primordia, in zebrafish embryos at 24 hpf. WISH was carried out with the following markers: olig2, rx1, and rx3 on each embryo (Figure 3 embryos were used). Overexpression of trim45 showed similar expression patterns with control for olig2 (A and B), rx1 (E and F), and rx3 (I and J). But, in knock-down, trim45 morphants showed compression of olig2 expression region and decrease in the size of thalamus (C). Also, knock-down of trim45 induced size reduction on eyes and decrease in transcript level of rx1 (G) and rx3 (K). Co-injection of trim45 mRNA with trim45-MO rescued the morphological phenotype of trim45 morphants (D, H and L). Lateral views – (A–D); anterior views – (E–L) th, thalamus; npo, neurosecretory preoptic area; pt, posterior tuberculum; ath, anterior hypothalamus. Scale bar – 200 μm (A–H), 100 μm (I–L).

Spatio-temporal expression of emx1 and fgf8, markers for telencephalon and midbrain-hindbrain boundary, in zebrafish embryos at 24 hpf. In situ hybridization of telencephalon marker emx1 was carried out on control (A and E), trim45 overexpressed (B and F), trim45 morphants (C and G), and rescued embryos (D and H) at 24 hpf. There were no significant changes on each embryo. te, telencephalon; hyp, hypothalamus; MHB, midbrain-hindbrain boundary. Scale bar – 100 μm (A–D), 200 μm (E–H).