Regulation of Danio Hatching by Tetraspanin cd63 Figure 1. CD63 protein properties. A. Alignment between Homo sapiens (CAG46893) and Danio rerio (NP_955837). Functional motifs are highlighted; glycosylation sites are in green, lysosomal targeting motif in red. BI. CHO (Chinese Hamster ovary) cells, and BII. RBL-2H3 (rat basophilic leaukaemia) cells, transfected with Cd63-GFP, GFP, or untransfected. C. Western blot of lysates from CHO cells probed with anti GFP, with anti actin as a loading control. Ladder positions are indicated on the left in kDa. * denotes Cd63GFP, { denotes GFP. doi:10.1371/journal.pone.0019683.g001 the three glycosylation sites in the large extracellular domain and the internalization motif at the C-terminus, are retained (Figures 1A and S1). GFP-tagged zebrafish Cd63 expressed in mammalian cell lines (CHO and RBL-2H3) and live embryos, displayed the intracellular localisation typical of CD63 in other species (Figures 1BI and II and S2A and B) [29]. Western blot analysis of lysates using anti-GFP antibodies revealed a smeared banding pattern from ,50–75 kDa in Cd63GFP transfected CHO cells, indicating heterogeneous glycosylation of Cd63 as reported for the human protein [30,31,32] (Figure 1C). Similar results were seen with RBL-2H3 and fish lysates. Investigation of cd63 in embryonic zebrafish Existing ISH data indicates that zygotic cd63 transcripts are expressed during early development [27,33]. Here, we have confirmed and extended these observations. Maternally-derived cd63 transcript (pre-zygotic transcription) is present in the early stages of embryonic development with positive staining for cd63 at the 1 cell stage (Figure 2A) and cd63 positive RT-PCR bands from the 1-cell to dome stage embryos (Figure 2C). Using ISH, zygotic cd63 expression was first detectable between 6–8 hpf in the pre-polster, located under the forebrain (Figure 2B I, black arrow; Figure 2C 30% epiboly). This structure gives rise to the polster where cd63 expression continues and increases. The polster is the precursor to the hatching gland, which develops and spreads (Figure 2B III-IV), with cells finally migrating across the yolk (Figure 2B V–VII) to form a mature hatching gland (Figure 2B VII, arrow). After hatching (a heterogeneous event within a clutch occurring between 48 and 72 hours post fertilisation [34,35]), cd63 levels in the hatching gland diminish (Figure 2B VIII, arrow). Knockdown of cd63 To investigate the role of cd63 in zebrafish development, we used morpholinos to knockdown gene function. Danio embryos were injected with morpholinos targeting the translation start site (MO1) and two intron/exon boundaries (MO2, 4) along with a MO1 mismatch control (MO3), to observe the effects of cd63 knockdown on Danio development. MO1, MO2, MO4 but not MO3 caused a failure of apparently viable embryos to hatch, even when fish embryos appeared grossly normal (Figure 3BI and CI), despite MO induced non specific defects [36]. To investigate the hatching defect seen in morphants, a titration of MO against hatching was carried out to confirm that the phenotype was dose dependent. Embryos were allowed to develop until 100 hours post fertilisation at which point the % of hatched embryos was calculated. A dose-dependent inhibition of hatching was seen in morphant embryos, manifested as failure to hatch or a delay in hatching compared to control embryos (Figure 4A), suggesting that cd63 has a specific role in the mechanism of hatching. RT-PCR investigation of transcripts from embryos injected with splice blocking MO4 provided further evidence of specific targeting: PCR products generated from mis-spliced transcript revealed a truncation of the 75 base pairs of exon 4 at the point of MO4 targeting, confirmed by sequencing of embryo derived transcript and visible on agarose gels (Figure 4B). Relation of cd63 to the Nodal signalling pathway and hatching gland specification Given the observed hatching defect as a result of cd63 knockdown, we attempted to determine if the influence of cd63 on hatching gland formation and function was due to a position in a signalling pathway responsible for hatching gland specification. Firstly, we tested cd63 expression in morphants using ISH (Figure 5A). This demonstrated that a consequence of knockdown is organisational disruption to hatching gland tissue in the absence of changes in tissue density (Figure 5A). Therefore it would appear that cd63 knockdown does not result in a reduction in hatching gland tissue and so cd63 is not required for specification of the pre- hatching gland cells fated to become hatching gland cells. cd63 is highly expressed from an early developmental stage in structures derived from the pre-chordal plate, formation of which relies on the Nodal signalling pathway. To address the possibility that cd63 might be involved in the Nodal signalling pathway, the Nodal deficient mutant oep was probed for expression of cd63. Oep mutants have a complete absence of cd63 staining (Figure 5B, bottom row), whereas WT siblings have strong hatching gland staining as well as notochord staining (Figure 5B, top row). This demonstrates that cd63 is Nodally regulated and therefore any role of cd63 in tissue differentiation occurs downstream of Nodal signalling. To test for defects in dorsal mesoderm specification as a consequence of cd63 knockdown, ISH was used to probe for the marker of dorsal mesoderm gsc. A change in gsc expression in the prechordal plate caused by cd63 knockdown could have downstream influence on tissues derived from here, including the hatching gland. No difference was seen in gsc expression patterns between morphant embryos and uninjected controls in the pre-chordal plate (Figure 6A, arrows), suggesting that cd63 operates downstream of gsc. To further explore a potential role for cd63 in the hatching gland expression of hatching gland marker cathepsin L (cat L) [37] was investigated in morphants using ISH. Morphant embryos showed no difference in expression levels of cat L compared to WT controls (Figure 6B, arrows). This indicates that cd63 is not involved in formation of the hatching gland tissue, as cat L is present at equivalent levels in the morphant and control. cd63 knock down does not prevent formation of the hatching gland or cause a reduction in hatching gland tissue density. Taken together this suggests that cd63 is not involved in specification of hatching gland tissues from precursor cells, but may be important for terminal differentiation of the secretory machinery and is required for the proper hatching gland function. Hatching gland phenotype alteration due to knockdown On closer inspection of embryos, morpholino-mediated cd63 knockdown disrupted the organisation of the hatching gland. In contrast to the typical band of hatching gland cells, which narrow centrally and span the yolk surface under the face in WT and mismatch embryos (red arrows Figure 3A and Figure 7A I, and blue arrows Figure 5A WT and Mismatch), embryos injected with MO4 or MO1, MO2 (data not shown) underwent aberrant migration (Figure 3C, blue arrows; Figure 5A, MO, blue arrows; Figure 7II) with hatching gland cells often aggregating into two balls located each side of the yolk centre (Red arrows Figure 3B I–III and 3C I–III; green arrows Figure 5A). A striking PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e19683