non-specific antibody binding and anti digoxigenin Fab fragments (Roche) were used to detect bound probe with detection achieved using NBT/BCIP (Sigma). Antisense morpholino oligonucleotide and RNA injection Four morpholino oligonucleotides (MO, Gene Tools) were designed to target the 59 CD63 sequence. MO1 (59-TTTCG- CTCCTCCTTCTACAGCCATG-39) was designed against the start codon to block translation; MO2 (59-ACAGCACTTGAGC- TAATGGAAAAAC-39) and MO4 (59-TGCAAACTGAGG- GAACACAAAGAT-39) were targeted against the splice donor site of exon 6 and exon 4 respectively to disrupt splicing and MO3 (59-TTTCCCTGCTGCTTATACAGCGATG-39) was a 5 base mispair control for MO1. Initially 3–8 pg MO1 and 4–12 pg of MO2 or MO4 were injected into single cell embryos to determine an optimal experimental dose. In further experiments MO2 and MO4 were used at a dose of 10 pg and MO1 at a dose of 1 pg in order to balance specific phenotype (Figure 4B and C; 5A II) against typical morpholino-induced non-specific defects (such as oedema and a crooked body plan). The data presented in this work is from MO4 injected embryos for which we obtained direct molecular evidence for its action (Figure 3B), but similar results were seen with MO1 and 2. RT-PCR Total RNA was extracted from single embryos homogenised in Tri Reagent (Ambion) following the manufacturer’s instructions and cDNA generated from subsequent extracts using SuperScript II reverse transcriptase (Invitrogen) and oligo dT (Invitrogen) following the manufacturer’s instructions. PCR was conducted on resulting cDNA using primers 59-GTTTCGCTTTGCTGA-39 and 59-AAAGAGATGAAGAAGATGACGC-39 to generate full length cd63 as well as with primers 59-GCCCCTGCCAATG- TAACCAC-39 and 59-TGCCAGGGACCATCTCAACAA-39 to generate a fragment of elongation factor EF1a as a control. PCR conditions were 95uC 5 minutes, 30x 95uC 30 seconds, 30x 55uC 30 seconds, 30x 72uC 2 minutes, 72uC 5 minutes. Resulting PCR products were electrophoresed on 1.2% agarose gels containing 0.5 mgml ethidium bromide. Hatching assays Embryos were incubated in petri dishes in E3 at 28.5uC. Media was changed once a day with minimal disturbance to embryos. A hatching event was recorded when an embryo became free of its chorion. Cathepsin L assay The cathepsin L assay was developed through adaptation of an established Calbiochem protocol supplied by Merck Chemicals Ltd, based on the capacity of z-Phe-Arg-7-amido-4-methylcour- marin to be cleaved by cathepsin L to release the fluorescent product 7-amido-4-methylcoumarin (AMC). AMC was quantified References 1. Hemler ME (2003) Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu Rev Cell Dev Biol 19: 397–422. 2. Yanez-Mo M, Barreiro O, Gordon-Alonso M, Sala-Valdes M, Sanchez- Madrid F (2009) Tetraspanin-enriched microdomains: a functional unit in cell plasma membranes. Trends Cell Biol 19: 434–446. 3. Hotta H, Ross AH, Huebner K, Isobe M, Wendeborn S, et al. (1988) Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression. Cancer Res 48: 2955–2962. 4. Lettau M, Schmidt H, Kabelitz D, Janssen O (2007) Secretory lysosomes and their cargo in T and NK cells. Immunol Lett 108: 10–19. Regulation of Danio Hatching by Tetraspanin cd63 using a spectrophotometer (WPA Lightwave; excitation of 370 nm, emission of 460 nm). The assay was carried out by placing single dechorinated embryos into wells of a v-bottomed 96 well plate in 110 ml E3 containing 5 mM HEPES. The plates were sealed and incubated at 28uC. After 4 hours a 100 ml sample of E3 was removed from each well and immediately replaced with 100 ml of preheated fresh E3. The plate was then returned to 28uC. 50 ml of assay buffer (340 mM sodium acetate, 60 mM acetic acid, 4 mM EDTA P.H 5.5, 8 mM DTT) was added to a 100 ml sample, and incubated at 30uC for 1 minute. 50 ml of 20 mM substrate was then added and incubated at 30uC for a further 10 minutes. The reaction was stopped by addition of 200 ml of stop solution (100 mM sodium monochloroacetate, 70 mM acetic acid, 30 mM sodium acetate). This process was repeated on a four hourly basis for the duration of the assay. A hatching control, consisting of individual embryos with intact chorions was carried out in spare wells of the assay plate. Control wells were treated identically to experimental wells and therefore were exposed to the same temperature fluctuations as dechor- ionated embryos. In these matched control embryos, hatching from the chorion was also recorded. Supporting Information Figure S1 Schematic representation of zebrafish Cd63 protein. Residue colour changes indicate separate exons within the reading frame of cd63. Solid black lines between residues denote disulphide bonds. (TIF) Figure S2 A. Localisation of Cd63GFP in 24 hr LWT embryos injected with buffer or plasmid DNA encoding Cd63GFP or EGFP, as indicated. Scale bars = 52 mm. B. Zoom of area of fluorescent Cd63GFP and GFP images in S2 A. Scale bars = 52 mm. (TIF) Movie S1 Typical WT Hatching Gland. The time lapse is one frame every 5 seconds for a total of 3 minutes (36 frames), using a 40x objective. Little movement of intracellular hatching gland granules is apparent. (MOV) Movie S2 Typical morphant Hatching Gland. The time lapse is one frame every 5 seconds for a total of 3 minutes (36 frames), using a 40x objective. The morphology of the intracellular granules in morphant embryos is altered and there is a large amount of granule movement when compared to WT. (MOV) Author Contributions Conceived and designed the experiments: MZT PM HR LJP. Performed the experiments: MZT. Analyzed the data: MZT PM LJP. Contributed reagents/materials/analysis tools: HR LJP. Wrote the paper: MZT PM. 5. Pols MS, Klumperman J (2009) Trafficking and function of the tetraspanin CD63. Exp Cell Res 315: 1584–1592. 6. Escola JM, Kleijmeer MJ, Stoorvogel W, Griffith JM, Yoshie O, et al. (1998) Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J Biol Chem 273: 20121–20127. 7. Duffield A, Kamsteeg EJ, Brown AN, Pagel P, Caplan MJ (2003) The tetraspanin CD63 enhances the internalization of the H,K-ATPase beta-subunit. Proc Natl Acad Sci U S A 100: 15560–15565. 8. Latysheva N, Muratov G, Rajesh S, Padgett M, Hotchin NA, et al. (2006) Syntenin-1 is a new component of tetraspanin-enriched microdomains: PLoS ONE | www.plosone.org 12 May 2011 | Volume 6 | Issue 5 | e19683