3 kb. The putative Msi1 gene from F. rubripes also has a similar structure with 15 exons. Interestingly, the Fugu genome size is quite small for a vertebrate, being approximately one-eighth the size of the human genome. However, the putative

Fugu Msi1 gene is one of the largest among all genes (~ 176 kb), being approximately six-times larger than the human MSI1 in size ( Aparicio et al., 2002 and Brenner et al., 1993). The compactness Ceritinib ic50 of the Fugu genome is thought to be the result of the deletion of unnecessary repeated sequences and minimization of intronic regions. However, for the Fugu Msi1 homolog this is apparently not the case, and is contrary to current hypotheses regarding exon-intron structure evolution. We next focused on the intronic MAPK Inhibitor high throughput screening enhancer region designated D5E2, which, in mouse, is responsible for transcriptional regulation of Msi1 (Kawase et al., 2011). The intronic D5E2 enhancer element is highly conserved among several mammalian species; however, this conserved element was not detected in chick, zebrafish or Fugu. The conservation of the D5E2 intronic enhancer in a few species suggests the acquisition of different mechanisms of gene regulation in Msi1 evolution. To explore the temporal distribution patterns of zMsi1 mRNA, total RNA was prepared from

animals at several developmental stages, from just after fertilization to the adult stage. A semi-quantitative RT-PCR analysis was performed with primer sets to detect total zMsi1 mRNA levels or expression of an internal control (β-Actin). Only a small amount of total zMsi1 expression was detected at 0 and 6 hours post-fertilization (hpf). The zMsi1 mRNA level increased dramatically from the shield stage, 12 hpf, and its expression was maintained through to 12-month-old

zebrafish in the brain ( Fig. 4A), suggesting that zMsi1 expression is initiated after the maternal-zygotic transition. To distinguish the splicing variants of zMsi1, we designed new primers for sequences in exon 10 and 12 that distinguish between the zMsi1L and zMsi1S transcripts. The long form of zMsi1 is the major form expressed in all examined developmental stages. However, a small amount of mRNA for the short form of zMsi1 was detected in 48-hpf and older embryos ( Fig. 4B). Flucloronide These results indicated that total zMsi1 expression was mainly zygotic and similar to the type of genes expressed in the pharyngula stage ( Mathavan et al., 2005). Another primer set for sequences in exon 3 and 5 was used to distinguish transcripts containing the 35 base pairs of exon 4 (Fig. 4C). Small amounts of zMsi1 mRNA with the 35 bp alternative exon were specifically detected after 24 hpf. This observed minor population of zMsi1L + 35 bp mRNA may be the result of RNA degradation by the nonsense-mediated mRNA decay (NMD) pathway. These RT-PCR results demonstrated the predominance of zMsi1L mRNA. However, the functional differences between the three variants still remain unclear.