Regulation of Danio Hatching by Tetraspanin cd63 Figure 6. Determination of mesodermal markers by ISH. Morphant embryos were injected with 10 pg MO4. A. Location of pre-chordal plate marker gsc in LWT embryos at 80% epiboly. I and II give lateral view, III and IV looking down on gsc positive staining. II and IV were injected with MO4, I and III are mis-match injected siblings. Arrows indicate gsc specific staining. B. Hatching gland marker cat L in 24 hour post fertilisation LWT embryos. I and II are morphant embryos, ventral and lateral views respectively. III and IV are mismatch injected LWT embryos, ventral and lateral views respectively. Arrows indicate hatching gland specific cat L staining. doi:10.1371/journal.pone.0019683.g006 causing toxicity, or that the enzymes themselves are essential for correct migration. The intracellular granules within the hatching gland cells were also seen to change from being bulbous and well organised in clusters, to small, misshapen and loosely clustered. Morphant granules rapidly moved in a seemingly random fashion, whereas movement in control cells was less obvious, slower and restricted to the immediate area local to the granule. The reason for these changes in granule movement is unknown but a similar effect occurs in mast cells from rab27b deficient mice [46], suggested to be due to a defect in the peripheral actin-mediated stationing of granules adjacent to the plasma membrane. Alternatively, this may be a consequence of reduced granule size allowing more room for movement in the actin network. In Hermansky-Pudlak syndrome, mutation of the b subunit of AP-3 results in mislocalisation of CD63, increased lysosome granule size and a failed release of lytic factors due to defective migration of intracellular granules [47]. In cytotoxic T lymphocytes from a Hermansky-Pudlak sufferer, lytic granules were unable to move along microtubules and dock with relevant secretory domains [47]. Further to this, siRNA mediated knockout of CD63 in a neutrophil model lead to depletion of granules [16], due to a failure in CD63 mediated trafficking of factors to populate the granules, which was accompanied by a defective granule morphology similar to that seen in the hatching gland cells of morphant embryos. Finally, in RBL-2H3 cells, CD63 specific antibodies block full degranulation of lysosomes [18]. It is possible the CD63-mediated mechanism of granule population with enzymatic cargo is conserved in the zebrafish hatching gland and therefore the defects seen as a result of cd63 knockdown are similar. It seems likely that cd63 is important for transport, and could be involved in transportation of factors both to and from the intracellular stores, which in turn may involve interaction with microtubules. A final possibility is that cd63 may be required for correct production of enzymes, a role previously unassociated with CD63. The phenotype of the CD63 knockout mouse (in the C57BL/6J background) was surprisingly subtle, with mild kidney and colon water retention defects [48]. Litter sizes were not reported to be smaller than expected, suggesting that blastocyst hatching, a process known to involve proteolytic enzymes, was not affected to any great extent. It is not clear if mammals have alternative mechanisms for the secretion of these enzymes or if other tetraspanins have been selected to compensate for CD63 deficiency during the knockout procedure. The mechanism of the water retention defects is also currently unclear. In summary, cd63 morphants have multiple hatching gland defects manifested as incorrect localisation, abnormal intracel- lular granules and excessive granule movement. cd63 is a Nodally-regulated gene involved in hatching gland differentia- tion rather than specification. Disrupted hatching gland is accompanied by hatching defects. Zebrafish hatching is mediated by an enzyme release event, absent in morphants. The consequence of cd63 knockdown is likely mediated through partner proteins of which there are many potential candidates for further investigation. Materials and Methods Ethics Statement All experimentation in the CDBG conforms to the UK and EU guidelines on research practice (http://cdbg.shef.ac.uk/about/ policy/index.html). No specific ethics approval under UK and EU guidelines was required for this project, as all zebrafish used in this study were between 0 and 5 days old. Fish All experiments used 0–5 day old London wild type embryos (maintained by the MRC Centre for Developmental and Biomedical Genetics, University of Sheffield) unless otherwise stated, maintained in E3 buffer (0.5 mM NaCl, 0.017 mM KCl, 0.033 mM CaCl2, 0.033 mM MgSO4, 10–6% methylene blue). Morpholinos, plasmid DNA and RNA were injected into the yolk sac of embryos at the 1 cell stage. Transfection of mammalian cells Cd63GFP was generated by insertion of the IMAGE cd63 cDNA into the EGFP-N1 plasmid (Clonetch). Primers were designed to add a Sac II restriction site before the ATG of cd63 and to mutate the G to a C in the stop codon, add two extra bases and an Age I restriction site. Sequential restriction digest of insert and vector was followed by clean up and ligation using T4 DNA Ligase (Promega). The Cd63GFP construct was transfected into mammalian cells by electroporation using the Gene Pulser II (Bio-Rad) or injected into the yolk of embryos. CHO or RBL- 2H3 cells stably expressing Cd63GFP were selected by growth in DMEM-10% FCS containing G418 (Invitrogen), followed by florescence activated cell sorting using a FACS Aria (Beckman and Dickinson). Protein extraction and analysis Lysates from mammalian cells or embryos were prepared by resuspension in 50 mM Tris (pH 8.0), 150 mM NaCl, 0.02% NaN3 with 1% CHAPS with 10% protease inhibitors (Sigma). After an incubation on ice for 30 minutes lysates were centrifuged at 13000 g for 30 minutes at 4uC to pellet nuclei. SDS-PAGE and Western blotting were carried out using standard procedures using and Bio-rad mini Protean and mini transblot transfer cell apparatus. Anti GFP antibody was supplied by Biosera. Microscopy and image analysis Embryos were incubated in E3+4.2% tricaine before dechor- ination and mounting in E3-containing 3% methylcellulose. Imaging of cells by fluorescence microscopy used a Nikon Eclipse E400, and DIC microscopy (Axioscope, Zeiss; Jena, Jenoptik) was used for all other images and time lapse. Images were viewed using Adobe Photoshop CS2 and analysed using ImageJ (http://rsb. info.nih.gov.ij/). The brightness and contrast of some images were adjusted for clarity. All statistical analysis was performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, USA, www.graphpad.com. PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e19683