The Crumbs_C isoform ofDrosophilashows tissue- and stage-specific expression and prevents light-dependent retinal degeneration

Abstract

Drosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress.

Changes in endolysosomal organization define a pre-degenerative state in thecrumbsmutantDrosophilaretina

“…2C' (arrows) and E). It is noteworthy that these patches are present in the crb P13A9 retina under non-degenerative conditions, and in the absence of any obvious loss of rhabdomeres indicative of the onset of degeneration [32]. The patches were not restricted to photoreceptors (arrow, Fig.…”

“…Fly stocks used in this study were: w* [32] (used as control), w; crb P13A9 [32], w; Rh1-Gal4 [39], w; UAS-LAMP-GFP (a gift of H. Krämer; [40]), GeneTrap CG6707 [41], w*; UAS-Rab7-GFP (a gift of M. Gonzalez-Gaitan; [42]), yw;otd 1.6 -Gal4 pWIZ8 (UAS-w RNAi ) (a gift of T. Cook; [43], yw; UAS-spinster-RFP (a gift of S. Sweeney; [44]), UAS-Atg8a-mCherry (a gift of T. Neufeld; [45]). For expression of transgenes encoding fluorescent protein (FP) in the crb background, the stock w; Rh1-Gal4; crb P13A9 was crossed to UAS-FP; crb P13A9 , and Rh1-Gal4/UAS-FP; crb P13A9 adults were selected.…”

“…In contrast, flies homozygous mutant for the hypomorphic crb P13A9 allele are viable and fertile, and their retina does not exhibit any of the developmental or polarity defects caused by loss-of-function crb alleles. Yet, the retina of homozygous mutant crb P13A9 flies undergoes degeneration under prolonged intense light conditions in a white-eyed (white, w*) background [32]. Therefore, this allele is ideally suited to further study the effects linked to degeneration, but independent from any developmental effect.…”

“…Besides genes encoding components of the trafficking machinery such as the retromer, many other fly genes are required to ensure photoreceptor survival [29]. One of these is crumbs (crb), mutations in which result in light-dependent retinal degeneration [30][31][32]. Similarly, mutations in one of the three human orthologues, CRB1, result in Retinitis pigmentosa (RP12) and Leber congenital amaurosis, two of the most severe retinal dystrophies associated with blindness [33][34][35].…”

“…In addition, lack of crb impairs MyosinVdependent rhodopsin trafficking to the rhabdomere [36]. We recently showed that the retina of a hypomorphic crb allele, crb P13A9 , degenerates upon constant exposure to light [32]. The retina of this allele lacks the morphological defects in cell shape observed in other crb alleles [31,37,38], suggesting that retinal degeneration associated with lack of crb is independent of its developmental phenotype.…”