Fig. 6S1

Zebrafish tmem5 temporal gene expression and MO specificity.

(A) A schematic representation of the zebrafish tmem5 transcript and protein domains. White boxes indicate the untranslated region (UTR). Black boxes indicate coding exons. The translational blocking MO target site and PCR primer binding sites are indicated. The amino acid sequence of zebrafish Tmem5 is ~73% similar and ~55% identical to that of human orthologue. Conserved protein domains are boxed: TM, transmembrane domain (7-29 aa); Exostosin (GT-47 homology) domain (254-349 aa). (B) The expression of tmem5 transcript was detected by RT-PCR using primer tmem5_1F and tmem5_3R. Zebrafish tmem5 is expressed maternally at the 2-cell stage, during the maternal-zygotic transition (6 h.p.f) and zygotically at later stages (24, 48, 72, 96 and 120 h.p.f.). (C) To assess tmem5 MO specificity, tmem5-egfp mRNA (200 pg) was injected into single cell, followed by injection of tmem5 MO (1 ng or 4 ng) into the yolk flow. Embryos that received only tmem5-egfp mRNA showed a strong Tmem5-EGFP expression at 30 h.p.f., whereas embryos received a second injection of 1 ng or 4 ng tmem5 MO showed a moderate and strong reduction in Tmem5-EGFP expression, respectively. These results confirm the specificity of tmem5 MO. n = 20 embryos in each group. Scale bar, 100 µm.