Regulation of Danio Hatching by Tetraspanin cd63 Figure 3. Morphology of cd63 knockdown. DIC microscopy images of the hatching glands of dechorinated 52 hour post fertilisation embryos. In each panel (A–C) I is x5 objective, white box indicates frame of II. II is x20 objective, white box indicates frame of III. III is x40 objective. II and III are montages of more than one focal plane. Red arrows point to the hatching gland, blue arrows indicate mislocalised hatching gland cells. A: Hatching gland typical of a LWT embryo. I is a montage of more than one focal plane. B: Moderately disrupted hatching gland typical after injection of 10 pg of MO4. C: Severely disrupted hatching gland typical after injection of 12 pg of MO4. doi:10.1371/journal.pone.0019683.g003 hatching enzymes was measured using Z-Phe-Arg-7-amido-4- methylcoumarin as a substrate for cat L, although it is also cleaved by other proteolytic enzymes. This lack of absolute specificity was desirable for this assay due to the potential presence of multiple zebrafish hatching enzymes. Dechorinated morphant embryos were assayed alongside mismatch MO and uninjected controls. No significant difference was seen in substrate cleavage between WT and mismatch MO injected embryos confirming the suitability of mismatch MO3 as a negative control (Figure 8A). Injection of experimental MO resulted in strong inhibition of substrate cleavage (Figure 8B) and the majority of MO injected embryos in a matched hatching assay did not hatch (Figure 8B, legend). In contrast, mismatch MO injected and uninjected controls had peak substrate cleavage (Figure 8B) at a time which corresponded to hatching in control embryos (Figure 8B, legend). Analysis of data from individual embryos revealed that in control embryos substrate cleavage was the result of a one-off release event, rather than a gradual release over a sustained period (Figure 8C). Taken together these results demonstrate that zebrafish have a single release event of enzymes capable of substrate cleavage that corresponds with hatching. cd63 knockdown inhibits substrate cleavage suggesting that cd63 has a specific role in the hatching mechanism. Discussion Like many zebrafish genes [41], maternal cd63 transcript is present during the blastula stage of embryogenesis. Early zygotic cd63 expression was observed in important structures during development of zebrafish and this could signify a role for cd63 during early development. Expression was apparent in the prechordal plate, which is the origin of the anterior mesoderm and gives rise to head and eye muscles as well as pharyngeal endoderm [28]. The polster is part of the prechordal plate and is the rudiment to the hatching gland. Given the widespread expression of CD63 orthologs it is surprising that in zebrafish expression is restricted in the fashion observed. It may be the case that cd63 is expressed at low levels in tissues throughout the zebrafish, as would be expected by analogy with mammalian CD63, and that the high through put cd63 ISH data published electronically [27,33], as well as the ISH presented here, is too Figure 4. Effect of knockdown. A. Effect of morpholino-induced knockdown of cd63 on hatching. Buff = buffer/mismatch injected control. Units of time are hours. Data from two experiments each using MO2 and 4 and a combined total of 671 embryos. B. MO4 mediated band shift. 1% Agarose gel of RT-PCR products from 48 hr LWT embryos using primers corresponding to full length cd63 transcript or ef1, a ubiquitous transcription factor used as a control. The 3 lanes next to the ladder are from embryos injected with 10 pg MO4, remaining lanes are from mismatch MO3 injected embryos. Similar results were seen in the 30 MO4 injected embryos tested. doi:10.1371/journal.pone.0019683.g004 PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e19683