Regulation of Danio Hatching by Tetraspanin cd63 Figure 5. Location of cd63 transcript as revealed by ISH. A. 32 h.p.f LWT dechorinated embryos injected with 10 pg MO4 as indicated. Blue arrows indicates hatching gland; green arrows denote mis-localised hatching gland cell clumps B. One Eyed Pinhead (oep) mutants and siblings. The top row are sibling embryos homozygous or heterozygous for WT oep, where as the bottom row are homozygous for the oep deletion. doi:10.1371/journal.pone.0019683.g005 insensitive to detect low levels of expression. This possibility is supported by micro array data showing that cd63 is over expressed in the zebrafish retinal pigment epithelium (RPE) relative to cells of the retina [42]. cd63, like other markers of anterior mesoderm, was not expressed in oep mutants. Further to this we observed that gsc and catl expression, as well as apparent tissue density in morphant embryos, was unaffected by cd63 knockdown. From this we conclude that cd63 is a nodally regulated gene and that expression is not required for the specification of hatching gland tissue. MO-induced defects in both hatching gland morphology and hatching demonstrate a potential role for cd63 in the organisation of hatching gland cells and the function of the secretory machinery. The phenotype seen in the hatching gland as a result of CD63 knock down is not unique to Cd63, as knockdown of xbp1, a protein reported to be involved in terminal differentiation of the secretory machinery of the hatching gland, resulted in reduced granule size and a similar morphology to that seen in this work. A further consequence of xbp1 knock down was failure to hatch [43]. This raises the possibility that xbp1 and cd63 may have overlapping functions or be involved in the same hatching gland processes, as is also the case for zKLF4. Failure to hatch as a consequence of MO mediated knockdown of zKLF4 was due to an extensive disruption to hatching gland morphology [44]. Our detailed microscopic analysis revealed that organisation of the gland was also disrupted by cd63 knockdown, evident by mislocalisation of cells and an altered shape. This mislocalisation of cells may have been due to altered CD63-dependent integrin function, thereby affecting processes of migration and cellular adhesion to the extracellular matrix [45]. An alternative is that hatching enzymes are not delivered to granules properly in morphants, PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19683