The activity of the Frizzled signaling pathway is required during the six hours prior to hair formation for normal planar polarization. To better understand the cell biological function of Diego protein during this time, antibodies were generated to a region COOH-terminal to the ankyrin repeats. This antibody recognizes a band of the expected size on Western blots of wild-type pupal wings that is of similar mobility to the protein produced by GAL4-driven overexpression and is undetectable in the Dgo380 mutant (Feiguin, 2001).
Immunofluorescence analysis of pupal wings at 18 hr shows that Diego localizes in a spotty pattern all around apico-lateral junctions, with no apparent polarity. By 24 hr, however, Diego has become localized to proximal and/or distal cell boundaries; whether Diego is present on one or both sides of the boundary is impossible to resolve by light microscopy. The localization persists during hair formation (which occurs at ~30 hr). These experiments show that Diego becomes localized to the proximal-distal junctional region when the Frizzled signaling pathway is actively generating planar polarity (Feiguin, 2001).
The localization of Diego to proximal-distal boundaries resembles that of Flamingo, a homophilic adhesion molecule with seven-transmembrane domains. To investigate the extent of colocalization between Diego and Flamingo, pupal wings were stained with antibodies to both proteins. Neither protein is smoothly localized at proximal-distal boundaries; instead, the proteins are more abundant at specific spots along the membrane. Diego and Flamingo show a strong tendency to be especially concentrated at the same places. This suggests that the two proteins are present in the same specialized regions of the membrane (Feiguin, 2001).
Planar cell polarity is established in the Drosophila eye through distinct fate specification of photoreceptors R3 and R4 by a two-tiered mechanism employing Fz and Notch signaling: Fz signaling specifies R3 and induces Dl to activate Notch in R4. The atypical cadherin Flamingo (Fmi) plays critical, but distinct, roles in both R3 and R4. Fmi is first enriched at equatorial cell borders of R3/R4, positively interacting with Fz/Dsh. Subsequently, Fmi is upregulated in R4 by Notch and functions to downregulate Dl expression by antagonizing Fz signaling. This in turn amplifies and enforces the initial Fz-signaling bias in the R3/R4 pair. These results reveal differences in the planar cell polarity genetic circuitry between the eye and the wing (Das, 2002).
The initial asymmetrical enrichment of Fmi in both R3 and R4, and the subsequent enrichment in R4 only, raised the question of in which cell(s) of the precluster is fmi required for PCP establishment. Interestingly, the analysis of mosaic clusters revealed a requirement for fmi in both R3 and R4. An ommatidium always adopts the correct orientation when both R3 and R4 are fmi+. When either R3 or R4 (or both) are fmi-, the ommatidium selects chirality randomly or stays symmetrical. Significantly, all ommatidia with wrong or no chirality had fmi- R3 and/or R4 cells. Loss of fmi function in any other R cells in any combination has no effect on ommatidial polarity. These data indicate that fmi is necessary and sufficient in both the R3 and R4 photoreceptor precursors for normal polarity establishment (Das, 2002).
The genetic requirement of fmi in both R3 and R4 is unique, since other PCP genes are required only in either cell (fz and dsh in R3 and stbm and N in R4), and raised the question of how Fmi relates to these genes in function and expression. Thus, the expression patterns of other PCP proteins were examined in the eye (Das, 2002).
Since Fmi is initially expressed in both cells of the pair, its inhibitory role on Dl can only be allowed in R4 and thus needs to be regulated. How is this achieved? Diego (Dgo) is a good candidate for this role. The cytoplasmic Dgo protein depends on Fmi for membrane association and generally colocalizes with Fmi at all membranes in the eye disc. The genetic interactions with sev --> Fmi (resulting in Fmi overexpression in the R3/R4 pair) identify dgo as a strong enhancer, suggesting that it is suppressing Fmi function in this context. Mosaic analysis of dgo shows that it is required in R3 and thus might keep the inhibitory function of Fmi off in R3. Since Fmi is necessary, but not sufficient, for Dgo membrane recruitment, other factor(s) are also required. Since Dgo and Fmi colocalize also in R4, a factor is needed there to antagonize Dgo function. Strabismus (Stbm), since it is required in R4, is a candidate. Since fmi mutants are enhancers of an Stbm overexpression phenotype, Stbm could serve this function in R4 (Das, 2002).
How could this be achieved? (1) There are the distinct requirements for dgo in R3 and stbm in R4; (2) the differences in Fmi levels in early R3/R4 versus late R4 could account for its individual functions. High levels of Fmi in R4 could lead to a formation of a different complex than that formed in R3. For example, an Fz/Dsh/Fmi/Dgo complex would promote Fz signaling, whereas, in R4, since there is significantly more Fmi, a different Fmi complex would inhibit Fz signaling by possibly sequestering Dsh from the Fz complex (Das, 2002).
In order to determine whether loss of diego function causes tissue polarity defects, diego mutants were generated by imprecise excision of EP 2619. Six mutants were identified harboring deletions that removed parts of the diego transcript, but left sequences upstream of the P element intact. These diego mutants are homozygous viable with wing hair polarity defects that resemble those of the strongest viable flamingo/starry night allelic combinations. Bristle polarity is not strongly affected by dgo mutation (Feiguin, 2001).
Diego mutant eyes also display typical planar polarity defects; some ommatidia have inverted chirality, others are symmetrical, whereas others are misrotated. The diego mutant phenotype is reminiscent of that observed for other 'core' polarity genes like fz. Of the diego mutant alleles, dgo 380 (which harbors the largest deletion) produces the strongest eye phenotypes, and its phenotype is not stronger over a deficiency, indicating that it is probably a null allele. Consistent with this, no Diego protein is detected on Western blots of dgo 380 homozygotes. All dgo alleles appear to have similar wing phenotypes that are not stronger when hemizygous. This suggests that whereas all the diego alleles are at least strong hypomorphs for wing-specific functions, the shorter deletions may retain some ability to function in the eye. The fact that Diego is required for planar polarization of both eye and wing epithelia places it in the 'core' group of tissue polarity genes and suggests that it forms an integral part of the polarization machinery (Feiguin, 2001).
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Das, G., et al. (2004). Diego interacts with Prickle and Strabismus/Van Gogh to localize planar cell polarity complexes. Development 131: 4467-4476. 15306567
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date revised: 30 May 2008
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