Regulation of Danio Hatching by Tetraspanin cd63 Figure 2. cd63 transcript. A & B. Location of cd63 transcript as revealed by ISH, using a probe against cd63. Developmental stages are noted below with hpf (hours post fertilisation) in brackets. In the top panel of B (I–IV), the top row gives the lateral view, bottom row ventral. In BI, arrows indicate the first expression in the pre-polster. cd63 expression continues (BII–VII). Expression in the hatching gland is indicated in BVII and BVIII, by arrows. C. RT-PCR time course using single embryos. PCR primers are indicated on the right, ef1 is a ubiquitous transcription factor used as a control. Numbers indicate ladder position in base pairs. Developmental stages are indicated at the top of the figure, each carried out in duplicate. This gel is representative of three replicates. doi:10.1371/journal.pone.0019683.g002 characteristic of cells of the hatching gland are the large, bulbous intracellular granules which are normally seen tightly clustered together in an approximately central location (Figure 7A I and III, blue arrows). Granule motility changes as a result of cd63 knockdown The second defect seen in morphants was transformation of the granules, which were significantly smaller and showed loose clustering (Figures 7A IV, blue arrows and Figure 7B). Further to this, use of a x40 objective revealed barely perceptible granule movement in control cells (Movie S1), whilst the granules in morphant cells rapidly moved around in a seemingly chaotic manner (Movie S2). Although granule morphology was altered, no difference in granule number per cell or hatching gland cell size was seen (Figure 7 B and C). Attempted RNA-mediated rescue of this phenotype was unsuccessful, presumably due to over expression phenotypes seen as a result of cd63 RNA injection, and limited stability of RNA (data not shown). The failure to hatch is due to a loss of hatching enzyme release The hatching gland releases enzymes that break down the chorion and allow embryos to hatch [38,39], a process possibly involving cd63. Cathepsin L encodes a secreted gene product [40] and is a putative zebrafish hatching enzyme that is expressed at high levels in the hatching gland ([37] and figure 6B). The release of PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e19683