Abstract
SummaryDevelopment of epithelial cell polarity is a highly dynamic process, and often established by the sequential recruitment of conserved protein complexes, such as the Par or the Crumbs (Crb) complex. However, detailed insights into the refinement of polarity and the formation of the complexes are still lacking. Here, we established fluorescently tagged Lin7c, a core member of the Crb complex, as an ideal tool to follow development of polarity in zebrafish epithelia. We find that in gastrula stages, RFP-Lin7c is found in the cytosol of the enveloping layer, while Pard3-GFP is already polarized at this stage. During development of the retinal epithelium, RFP-Lin7c localization is refined from being cytosolic at 14 hours post fertilization (hpf) to almost entirely apical in cells of the eye cup at 28 hpf. This apical Lin7c localization depends on the Crb complex members Oko meduzy and Nagie oko. Thus, fluorescently tagged Lin7c can be used in a broad range of epithelia to follow polarity maturation in vivo and specifically to elucidate the sequence of events determining Crb complex-mediated polarity.
Highlights
Epithelial tissues are built from cells with marked polarity, manifested by the polarised cell shape and the uneven distribution of organelles and molecules
During development of the retinal epithelium, RFP-Lin7c localization is refined from being cytosolic at 14 hours post fertilization to almost entirely apical in cells of the eye cup at 28 hpf
Fluorescently tagged Lin7c can be used in a broad range of epithelia to follow polarity maturation in vivo and to elucidate the sequence of events determining Crb complex-mediated polarity
Summary
Epithelial tissues are built from cells with marked polarity, manifested by the polarised cell shape and the uneven distribution of organelles and molecules. Several evolutionarily conserved protein complexes regulate establishment and maintenance of epithelial cell polarity, with the Par-3 (Pard in zebrafish) and the Crumbs (Crb) protein complexes controlling polarity from the apical pole (Goldstein and Macara, 2007; Assemat et al, 2008; Nance and Zallen, 2011; Tepass, 2012; Roignot et al, 2013). Crb is a conserved transmembrane protein, which forms a membrane-associated protein complex at the apical pole by recruiting the MAGUK (membrane-associated guanylate kinase) protein Mpp5/Pals (zebrafish Nagie Oko (Nok), Drosophila Stardust (Sdt)), the multi-PDZ (PSD-95/Dlg/ZO-1)-domain protein Patj, and Lin, which, together with Crb, are conserved from nematodes to mammals and form the core components of the Crb complex (Bulgakova and Knust, 2009; Tepass, 2012). All three are expressed in the eye, Lin7c is the predominant form expressed at early stages in the eye and the developing neural tube (Wei et al, 2006; Yang et al, 2009), we focused our attention to this isoform
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