Regulation of Danio Hatching by Tetraspanin cd63 Figure 7. Morphant hatching gland. A. DIC microscopy images of cells of the hatching gland in a dechorinated control embryo (I and III) and morphant (II and IV) at 32 hours post fertilisation. III and IV are areas corresponding to the area of the red box in I and II respectively. Blue arrows denote intracellular granules. B and C. Analysis of hatching gland cell characteristics. Images of 3 wild type hatching glands and 3 morphant hatching glands were used for analysis with ImageJ. Distribution of data was tested to determine the relevant statistical analysis for each parameter measured. B: From each image five cells were randomly selected. Granule number was calculated and five granules randomly selected and measured. 2 tailed t- test of morphant vs. wt no. of granules per cell- no significant difference; Wilcoxon matched pairs test of morphant vs. wt granule size2 p = ,0.0001 *** significantly different. C: From each image five cells were randomly selected and measured at the widest diameter. 2 tailed t-test of morphant vs. wt cell size- no significant difference. doi:10.1371/journal.pone.0019683.g007 Whole mount in situ hybridisation IMAGE clones (Geneservice Ltd.) of cd63 (BC056698), cd82 (BC054586) and catl (BC108031) were used as template to generate digoxigenin labelled in situ RNA probes using DIG RNA labelling mix (Roche). Whole mount in situ hybridisation was performed as described previously [49] with the following modifications: hybridisation was conducted at 65uC, PBT (1X PBS 0.1% Tween-20), 2% FCS (foetal calf serum) was used to block Figure 8. Assay for hatching enzymes. Media from the wells containing each embryo was harvested on a four hourly basis and tested for substrate cleavage ability determined by measuring release of fluorescent substrate. Doses of 10 pg of MO2 or 4 were injected, 0.8 pg MO1. A. Single control embryos. LWT are uninjected embryos. 2 way ANOVA of effect of MO3(mismatch) vs. uninjected embryos- % total variation 1.35, p = ,0.46 ns. n = 2; total of 27 embryos. B. Assay for hatching enzymes in single MO1, MO2 and MO4 injected embryos. Control embryos were uninjected or MO3 injected. 2 way ANOVA of effect of MO vs. Control- % total variation 5.68, p = ,0.0001 *** significant. n = 2; using a total of 60 embryos. In a hatching assay conducted in tandem 100% of control embryos hatched, 92% between 56 and 72 hours, n = 12 (6 MO3 injected, 6 uninjected). In the same assay only 6% of MO injected embryos hatched, n = 18 (6 MO1, 6 MO2 and 6 MO4 injected). C: Substrate cleavage for single embryos. MO = MO4 injected, Control = MO3 injected. Data represents a typical release profile for a morphant and a control embryo. doi:10.1371/journal.pone.0019683.g008 PLoS ONE | www.plosone.org 11 May 2011 | Volume 6 | Issue 5 | e19683