Regulation of Zebrafish Hatching by Tetraspanin cd63 Michael Z. Trikic´ 1*, Pete Monk2, Henry Roehl3, Lynda J. Partridge1 1 Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom, 2 Department of Infection and Immunity, University of Sheffield, Sheffield, United Kingdom, 3 Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom Abstract Tetraspanins cause the clustering of membrane proteins into a level of organisation essential for cellular function. Given the importance and complicated nature of this mechanism, we attempted a novel approach to identify the function of a single component in a biologically relevant context. A morpholino knockdown strategy was used to investigate the role of cd63, a membrane protein associated with intracellular transport and a melanoma marker, in embryonic zebrafish. By using three separate morpholinos targeting cd63, we were able to identify a specific phenotype. Strikingly, morphant fish failed to hatch due to the lack of secreted proteolytic enzymes required for chorion-softening. The morphology of the hatching gland at both the cellular and intracellular levels was disorganised, suggesting a role for cd63 in the functioning of this organ. This work identifies a specific role for cd63 in the zebrafish embryo and provides evidence for the suitability of zebrafish as a model system for the investigation of tetraspanin enriched microdomains. Citation: Trikic´ MZ, Monk P, Roehl H, Partridge LJ (2011) Regulation of Zebrafish Hatching by Tetraspanin cd63. PLoS ONE 6(5): e19683. doi:10.1371/ journal.pone.0019683 Editor: Jean Kanellopoulos, University Paris Sud, France Received December 17, 2010; Accepted April 5, 2011; Published May 19, 2011 Copyright: 2011 Trikic´ et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a University of Sheffield Krebs Studentship grant, in the department of Molecular Biology and Biotechnology. The funding source no longer exists, however the link to the source of funding follows: http://www.shef.ac.uk/mbb/prospectivepg. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: m.trikic@shef.ac.uk Introduction CD63 is a member of the tetraspanin superfamily of four- span membrane proteins, distinguished by the presence of 4–8 cysteine residues in the second extracellular domain (EC2) in specific sequence motifs and conserved polar residues in the transmembrane domains [1]. Tetraspanins have the ability to cause the clustering of membrane proteins in tetraspanin- enriched microdomains (TEMs), a level of organisation essential for cellular function [2]. Human CD63 was first reported as the ME491 antigen, a tumour marker [3] and is now more commonly known as a marker of lysosomes and multivesicular bodies. The movement of CD63 from intracellular organelles to the cell surface during secretion has led to its use as an activation marker for several haematopoietic cell types including neutrophils, eosinophils, basophils, mast cells and platelets [4]. It is also present in melanosomes, cytotoxic T cell granules, Weibel-Palade bodies of endothelial cells, MHCII compart- ments of dendritic cells [5] and is enriched on exosomes [6]. All mammalian cell types so far studied express CD63, suggesting that it may be ubiquitous [5]. Many proteins are known to associate directly with CD63, including H+/K+ ATPase b subunit [7], syntenin 1 [8], MT1-MMP1 [9], PI-4-kinase [10], AP3 [11], TIMP1 [12], amelogenin [13] and PTK [14]. In many cases, the association with CD63 relates to the cellular trafficking of the partner protein: MT1-MMP1 is targeted by CD63 for lysosomal degradation, as is the b subunit of H+/K+ ATPase and amelogenin. CXCR4 surface expression [15] and the delivery of elastase to neutrophil primary granules (a form of secretory lysosome) [16] appears to be controlled by CD63. In rat basophilic leukaemia mast cells (RBL-2H3) CD63 is expressed in secretory lysosomes [17], were it is critical for full degranulation [18]. The protease cathepsin L (CatL), which has both intracellular roles (i.e. neuropeptide processing in chro- maffin cells [19]; transcription factor activation in the nucleus [20,21]) and extracellular roles (i.e. cancer cell migration [22] and extracellular matrix degradation [23]), also co-localizes with CD63, although it is not known if these molecules are co- trafficked [24]. Phylogenetic analysis has described the CD63 family as constituting one of the four major families of vertebrate tetraspanins [25]. CD63 is likely to have had a particularly ancient origin, as it is associated with a gene expansion in Drosophila and is also reported in sponges [26]. The zebrafish homologue is highly expressed from an early developmental stage in the pre-polster [27], a tissue which gives rise to the hatching gland, and has a key role in patterning of the embryo [28]. Although members of the tetraspanin superfamily are well represented in the zebrafish genome, little work has been conducted on their role in fish development. For this reason we chose to investigate CD63 in embryonic zebrafish. Expression during early development was confirmed and the role of CD63 investigated using antisense morpholino-mediated knockdown. Strikingly, morphant fish failed to hatch, due to the lack of secreted proteolytic enzymes required for chorion-softening. The morphology of the hatching gland at both the cellular and intracellular levels was disorganised, suggesting a role for CD63 in the function of this organ. Results Analysis of CD63 protein sequence The zebrafish Cd63 molecule is 45.4% identical (62.2% similar) to human CD63 and the key structural features, such as PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e19683