The Role of Crb2 in Vertebrate Development

The Drosophila 2146 amino acid (aa) single-pass transmembrane protein Crumbs (Crb), is a key regulator of epithelial apico-basal polarity and adhesion. The short 37aa intracellular domain is both necessary and when combined with the transmembrane domain, sufficient for apico-basal polarity (Wodarz, 1995, Cell 82:67). In contrast, comparatively little is known about the role of the large extracellular domain. Three vertebrate homologues to Crb have been identified. Mutations in Crb1 are associated with a range of retinal dystrophies (den Hollander, 2001, 69:198), whilst Crb3 is required for cilia formation (Fan, 2004, Curr Biol 14:1451). However, the role of Crb2 is largely unknown. We have isolated three major isoforms of Crb2 that differ in their extracellular domains. Isoform 1 encodes the whole protein, containing a signal sequence, 15 EGF repeats, 3 laminin G domains, a transmembrane domain and the 37aa intracellular domain. Isoforms 2 and 3 are shorter variants. Isoform 2 encodes a secreted protein, as it lacks the transmembrane and intracellular domains, we show that it can be detected in the supernatant of transfected cells. In contrast, Isoform 3 is a transmembrane protein that lacks the first 7 EGF repeats of isoform 1 and has a different signal sequence. We demonstrate that in ovo electroporation of RNA interference constructs targeted to all three isoforms results in abnormal apico-basal polarity, cell proliferation and neural differentiation in the chick neural tube. We are now investigating the role that the different isoforms play in producing the phenotype.