E encoding the full-length Crb protein was unable to rescue embryonic

2009; Klebes and Knust 2000; Muschalik and Knust 2011; Wodarz et al. 1995). Therefore, we analyzed the levels of Crb protein in flies carrying distinct copy numbers with the D-3263 site endogenous and/or fosmid-encoded crb gene. Surprisingly, the presence of four copies of crb did not enhance the overall Crb protein levels. In the absence of endogenous crb, each and every from the 3 fosmids that rescued embryonic lethality expressed comparable amounts of Crb protein when present in two copies (SB-269970 supplier Figure 1, C and D). foscrb and foscrbEGFP show wild-type expression pattern and subcellular localization in the Crb protein To analyze the expression pattern of foscrb- and foscrbEGFP-encoded Crb protein in embryos within the absence of endogenous crb, we stained foscrb; crbGX24, foscrbEGFP; crbGX24 and foscrbY10F; crbGX24 embryos with distinctive markers at distinct developmental stages. At all developmental stages, fosCrb protein in these embryos was expressed in epithelia of ectodermal origin as in wild-type, i.e., within the epidermis, the amnioserosa, the tracheae, the salivary gland, the hindgut as well as the Malpighian tubules (Figure two and information not shown). As revealed by the apical marker Sas, epithelia in embryos carrying one of these fosmids keep proper apicobasal polarity (Figure 2, C92E9). In embryos with these genotypes, fosCrb is localized apical for the basolateral marker Dlg (Figure 1E and data not shown).Figure 2 foscrb variants fully rescuing crb mutant embryos. Lateral view of stage 13/14 whole-mount embryos stained for Crb and Sas (correct). (A) Wild-type. (B) crbGX24. (C) foscrb; crbGX24. (D) foscrbEGFP; crbGX24. (E) foscrbY10F; crbGX24. Arrows in A9, C9, D9, and E9 indicate salivary glands, and arrowheads within a, C, D, E the tracheae. In B, the embryo negative for Crb staining is outlined with all the dotted line. Inset in B9 is actually a maximal projection of a stack through the remnant tracheal system. The arrowheads inside the inset in B9 indicate discontinuities in the transverse connective (TC) and dorsal trunk (DT) branches. Anterior would be to the left, dorsal up. Scale bar, 100 mm.Volume 3 February 2013 |Crb and Epithelial Morphogenesis |foscrbY10A, foscrbDERLI, and foscrbY10A, DERLI usually do not rescue crb-induced embryonic lethality crb mutant embryos lack a continuous cuticle, and only grains of cuticle is usually detected (examine Figure three, A and B) (J gens et al. 1984; Tepass and Knust 1990). crb mutant embryos with transgenes that either carried a mutated FERM-binding domain (foscrbY10A; crbGX24), lacked the PDZ-binding motif (foscrbDERLI; crbGX24), or carried both mutations (fosc.E encoding the full-length Crb protein was unable to rescue embryonic lethality and could only suppress some elements from the crb mutant embryonic phenotype (Wodarz et al. 1995). Surprisingly, 82 in the embryos with all the genotype foscrbY10F; crbGX24 hatched and gave rise to adult viable and fertile flies. This result is striking mainly because expression of UAS-crbintraY10F, which encodes a protein consisting of the transmembrane and also the cytoplasmic domain of Crb, in which Tyr10 was mutated to Phenylalanine, did not rescue embryonic lethality and showed only minor suppression in the crb mutant embryonic cuticle phenotype upon ubiquitous expression, in comparison with that of UAS-crbintra (C. Clemens and E. Knust, unpublished data).