The MHC phenotype of fz mutants. This portion of the wing contains the region of the anterior ventral surface used to quantify the MHCs of the fz alleles (see materials and methods). (A) Cells in a wild-type wing each secrete a single hair. (B) In a weak fz mutant, fz^SY, some cells may secrete more than one hair (red arrows). The number of MHCs increases in moderate (fz^MP) (C) and strong (fz^JB) (D) fz mutants. The number of MHCs observed in this region of the wing is a strong predictor of the severity of the phenotype observed in other tissues, such as the bristles on the dorsal thorax.

Arm signaling strength of fz alleles. (A) Cross to test the Arm signaling strength of fz alleles where fz* denotes the allele tested. (B) The fz alleles exhibit a spectrum of Arm signaling phenotypes in embryos. Representative mutant cuticles from a cross of fz*/fz^GL31 fz2^e2 mothers and fz^GL31 fz2^e2/TM3 fathers are shown. Twenty-five percent of the progeny are homozygous for the fz^GL31 fz2^e2 chromosome and receive maternal fz* and fz^GL31 transcripts. The order from upper left to lower right was determined by PCP signaling strength (Figure 3). Approximately 25% of the progeny from strong and moderate alleles display a cuticle similar to that shown. For the weak class of mutants, <25% of the progeny displayed a phenotype.

Model of Fz activation in PCP and Arm signaling. In the absence of a Wnt ligand, Fz has an intrinsic ability to signal to the PCP pathway. This may occur via a direct interaction with Dsh. When Wg is present, the coreceptor Arr is brought into proximity to Fz, which results in the close apposition of Dsh and Axn. This combination of intracellular components switches the output of Fz from PCP to Arm signaling.

Schematic of fz alleles. Mutations found in fz alleles isolated in PCP screens are located throughout the transmembrane portion of the receptor. There are no missense mutations located in the CRD, shaded in gray. The cold-sensitive mutation encoded by the fz^HD21 allele is green. With the exception of fz^HD21, all other missense mutations (yellow) affect amino acids that are conserved in the frizzled family. Nonsense mutations are red. Mutations that insert amino acids are indicated with open triangles. All insertion mutations encounter a stop codon that results in the synthesis of a truncated protein.

PCP signaling strength of fz alleles. (A) The number of MHCs was counted in a section of the ventral wing between the first and second wing veins in a region defined by the first 15 stout margin bristles (inset). All alleles (fz*) were assayed in trans with fz^K21, a null allele. The number of MHCs was plotted for each of 10 wings (solid circle) along with their average (plus symbol). Some alleles appear to have <10 data points because the dots overlap due to identical values. The number of MHCs correlates with the severity of the polarity disruptions in other tissues, such as the dorsal thorax. Alleles that display the greatest numbers of MHCs have the strongest polarity phenotypes in other tissues. fz^D21 is an amorphic deletion allele (Jones et al. 1996). (B) Photomicrograph of the dorsal thorax of a wild-type fly. Note the parallel arrangement and uniform posterior polarity of the bristles. (C) Alleles with the fewest number of MHCs have near wild-type bristle polarity. Only slight deviations from the normal posterior alignment are observed in fz^SY/fz^K21. (D) Alleles with an intermediate number of MHCs also exhibit a mild bristle phenotype on the thorax. The majority of the lateral bristles in fz^MP/fz^K21 deviate from wild type by 45° or less. (E) Lateral bristles in fz^JB/fz^K21 that normally point posteriorly frequently deviate from their normal orientation up to 90°, pointing straight toward the middle of the thorax. When all the alleles were assayed in trans with the hypomorphic allele fz^GL31, the ranking was similar with only minor differences (data not shown). In addition, when fz^GL31 was used as the reference allele, the correlation remained between the number of MHCs and the polarity phenotypes observed in other tissues, such as the thorax (data not shown).

Western blot of fz alleles. Characterization of protein abundance and mobility of Fz isolated from embryos produced by a sibling cross of fz*/fz^P21 Df(3L)fz2, where fz* denotes the allele tested. The only fz contribution in the resulting embryos is fz* or fz^P21. Since fz^P21 encodes a protein null allele (Jones et al. 1996), the only detectable Fz protein is translated from fz* transcripts. Fz protein cannot be observed in whole-embryo extracts (data not shown). Therefore, Fz protein was concentrated from total embryo extract by immunoprecipitation using an antibody that recognizes the hinge portion of the receptor, which is located between the end of the CRD and the first transmembrane domain. An equal amount of protein (300 μg) was used for each immunoprecipitation. Fz was detected in the immunoprecipitated material by probing Western blots with a monoclonal antibody that recognizes an epitope in the CRD (Park et al. 1994). Drosophila S2 cells do not express fz and were used as a negative control. Probing an extract prepared from S2 cells stably transformed with a fz transgene (pMKfz) marks the position of Fz with a relative molecular mass slightly >50 kDa. Fz protein isolated from embryos having a single copy of wild-type fz comigrates with Fz produced in S2/pMKfz cells. The fz^MG and fz^JB truncation alleles do not express detectable levels of protein. The nonsense mutations fz^RN and fz^CK produce both the predicted size truncated protein and an additional product that migrates near the position of wild-type Fz. The fz^JW also has a larger protein product. However, it migrates more slowly than wild-type Fz and the comparable bands in fz^RN and fz^CK. Slower-mobility products result from improper processing in the endoplasmic reticulum, which is likely the result of misfolding. The missense alleles fz^J22, fz^F31, fz^R53, fz^TT, and fz^ED all have an abundance and mobility similar to that of wild-type Fz. The remaining alleles all display a marked decrease in protein level with fz^R54, fz^HD21, and fz^MP also having a reduced mobility.