Drosophila APC2 is a cytoskeletally-associated protein that regulates wingless signaling in the embryonic epidermis.

The tumor suppressor adenomatous polyposis coli (APC) negatively regulates Wingless (Wg)/Wnt signal transduction by helping target the Wnt effector beta-catenin or its Drosophila homologue Armadillo (Arm) for destruction. In cultured mammalian cells, APC localizes to the cell cortex near the ends of microtubules. Drosophila APC (dAPC) negatively regulates Arm signaling, but only in a limited set of tissues. We describe a second fly APC, dAPC2, which binds Arm and is expressed in a broad spectrum of tissues. dAPC2's subcellular localization revealed colocalization with actin in many but not all cellular contexts, and also suggested a possible interaction with astral microtubules. For example, dAPC2 has a striking asymmetric distribution in neuroblasts, and dAPC2 colocalizes with assembling actin filaments at the base of developing larval denticles. We identified a dAPC2 mutation, revealing that dAPC2 is a negative regulator of Wg signaling in the embryonic epidermis. This allele acts genetically downstream of wg, and upstream of arm, dTCF, and, surprisingly, dishevelled. We discuss the implications of our results for Wg signaling, and suggest a role for dAPC2 as a mediator of Wg effects on the cytoskeleton. We also speculate on more general roles that APCs may play in cytoskeletal dynamics.

Non-equivalent roles of Drosophila Frizzled and Dfrizzled2 in embryonic wingless signal transduction.

The highly conserved Wnt family of growth factors is essential for generating embryonic pattern in many animal species [1]. In the fruit fly Drosophila, most Wnt-mediated patterning is performed by a single family member, Wingless (Wg), acting through its receptors Frizzled (Fz) and DFrizzled2 (Dfz2). In the ventral embryonic epidermis, Wg signaling generates two different cell-fate decisions: the production of diverse denticle types and the specification of naked cuticle separating the denticle belts. Mutant alleles of wg disrupt these cellular decisions separately [2], suggesting that some aspect of ligand-receptor affinity influences cell-fate decisions, or that different receptor complexes mediate the distinct cellular responses. Here, we report that overexpression of Dfz2, but not Fz, rescues the mutant phenotype of wgPE2, an allele that produces denticle diversity but no naked cuticle. Fz was able to substitute for Dfz2 only under conditions where the Wg ligand was present in excess. The wgPE2 mutant phenotype was also sensitive to the dosage of glycosaminoglycans, suggesting that the mutant ligand is excluded from the receptor complex when proteoglycans are present. We conclude that wild-type Wg signaling requires efficient interaction between ligand and the Dfz2-proteoglycan receptor complex to promote the naked cuticle cell fate.

The murine Fhit gene is highly similar to its human orthologue and maps to a common fragile site region.

The human FHIT gene is a putative tumor suppressor gene that maps to human chromosome band 3p14.2 in a region that is frequently deleted in cancers. It exhibits both genomic deletions and aberrant transcripts in a variety of tumors and spans the common fragile site FRA3B. This fragile site extends over a broad region of several hundred kb within the FHIT gene and may account for its instability in tumors. As one test of this hypothesis, we isolated the murine Fhit gene and asked whether it also contains a common fragile site and if it is unstable in mouse tumors or tumor cell lines. The Fhit gene was isolated, and the sequence was found to be 87.5% identical to that of the human FHIT gene in the open reading frame. Using fluorescence in situ hybridization, Fhit was assigned to mouse chromosome band 14A2, in a region that was previously shown to contain an aphidicolin-inducible mouse fragile site. Fluorescence in situ hybridization with genomic clones containing Fhit and flanking sequences demonstrated that gaps and breaks in the fragile site occur over a broad region within and proximal to the Fhit locus. Thus, the physical relationship of Fhit to a common fragile site is similar to that observed with the orthologous human FHIT gene and FRA3B.

A phosphoglycerate mutase brain isoform (PGAM 1) pseudogene is localized within the human Menkes disease gene (ATP7 A).

We have identified a phosphoglycerate mutase brain isoform (PGAM 1, PGAM B) cDNA that is localized between exons 1 and 2 of the Menkes disease gene (ATP7 A, MNK) at Xq13.3. The cDNA shows 98% identity to the previously identified PGAM 1 cDNA (Sakoda et al., J. Biol. Chem. 263 (1988) 16899-16905) and probably represents a recent retroposition of this parent PGAM 1 mRNA. Although the typical features of a processed pseudogene are present, the open reading frame (ORF) of this PGAM cDNA is potentially expressed. There are 11 bp changes in the 765 bp ORF, none of which are nonsense mutations or deletions. The region upstream from the ORF shows some features of a possible promoter region, although it lacks a CpG island often associated with functional promoters. We analyzed the expression of this PGAM 1 cDNA using RT-PCR followed by restriction enzyme digestion based on a 1 bp missmatch in this cDNA to distinguish it from normal PGAM 1 gene expression. With this sensitive method, we could not find expression in any of the tissues examined. Taken together, we conclude that the PGAM 1 cDNA upstream from exon 2 of the Menkes gene is likely to be a processed pseudogene originating from a very recent retroposition of a PGAM 1 transcript. To our knowledge this is the first report of a pseudogene located within a gene.

Exclusion of BMP6 as a candidate gene for cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant, generalized skeletal dysplasia in humans that has been mapped to the short arm of chromosome 6. We report linkage of a CCD mutation to 6p21 in a large family and exclude the bone morphogenetic protein 6 gene (BMP6) as a candidate for the disease by cytogenetic localization and genetic recombination. CCD was linked with a maximal two-point LOD score of 7.22 with marker D6S452 at theta = 0. One relative with a recombination between D6S451 and D6S459 and another individual with a recombination between D6S465 and CCD places the mutation within a 7 cM region between D6S451 and D6S465 at 6p21. A phage P1 genomic clone spanning most of the BMP6 gene hybridized to chromosome 6 in band region p23-p24 using FISH analysis, placing this gene cytogenetically more distal than the region of linkage for CCD. We derived a new polymorphic marker from this same P1 clone and found recombinations between the marker and CCD in this family. The results confirm the map position of CCD on 6p21, further refine the CCD genetic interval by identifying a recombination between D6S451 and D6S459, and exclude BMP6 as a candidate gene.

Functional analysis of Wingless reveals a link between intercellular ligand transport and dorsal-cell-specific signaling.

The Drosophila segment polarity gene wingless (wg) is essential for cell fate decisions in the developing embryonic epidermis. Wg protein is produced in one row of cells near the posterior of every segment and is secreted and distributed throughout the segment to generate wild-type pattern elements. Ventrally, epidermal cells secrete a diverse array of anterior denticle types and a posterior expanse of naked cuticle; dorsally, a stereotyped pattern of fine hairs is secreted. We describe three new wg alleles that promote naked cuticle cell fate but show reduced denticle diversity and dorsal patterning. These mutations cause single amino acid substitutions in a cluster of residues that are highly conserved throughout the Wnt family. By manipulating expression of transgenic proteins, we demonstrate that all three mutant molecules retain the intrinsic capacity to signal ventrally but fail to be distributed across the segment. Thus, movement of Wg protein through the epidermal epithelium is essential for proper ventral denticle specification and this planar movement is distinct from the apical-basal transcytosis previously described in polarized epithelia. Furthermore, ectopic overexpression of the mutant proteins fails to rescue dorsal pattern elements. Thus we have identified a region of Wingless that is required for both the transcytotic process and signal transduction in dorsal cell populations, revealing an unexpected link between these two aspects of Wg function.

Two highly polymorphic CA repeats in the Menkes gene (ATP7A).

Two highly polymorphic CA repeats have been identified in the Menkes gene (ATP7A). These repeats should be useful for prenatal diagnosis and carrier detection in families with Menkes disease and X-linked cutis laxa. The observed heterozygosity for these two repeats was 0.778 and 0.60 in Centre d'Etude du Polymorphisme Humaine (CEPH) families.

Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network.

We have generated polyclonal antibodies against the amino-terminal third of the Menkes protein (ATP7A; MNK) by immunizing rabbits with a histidine-tagged MNK fusion construct containing metal-binding domains 1-4. The purified antibodies were used in Western analysis of cell lysates and in indirect immunofluorescence experiments on cultured cells. On Western blots, the antibodies recognized the approximately 165 kDa MNK protein in CHO cells and human fibroblasts. No MNK signal could be detected in fibroblasts from a patient with Menkes disease or in Hep3B hepatocellular carcinoma cells, confirming the specificity of the antibodies. Immunocytochemical analysis of CHO cells and human fibroblasts showed a distinct perinuclear signal corresponding to the pattern of the Golgi complex. This staining pattern was similar to that of alpha-mannosidase II which is a known resident enzyme of the Golgi complex. Using brefeldin A, a fungal inhibitor of protein secretion, we further demonstrated that the MNK protein is localized to the trans-Golgi network. This data provides direct evidence for a subcellular localization of the MNK protein which is similar to the proposed vacuolar localization of Ccc2p, the yeast homolog of MNK and WND (ATP7B), the Wilson disease gene product. In light of the proposed role of MNK both in subcellular copper trafficking and in copper efflux, these data suggest a model for how these two processes are linked and represent an important step in the functional analysis of the MNK protein.

Molecular structure of the Menkes disease gene (ATP7A).

We report a detailed molecular analysis of the genomic structure of the Menkes disease gene (MNK; ATP7A). There are 23 exons in ATP7A covering a genomic region of approximately 140 kb. The size of the individual coding exons varies between 77 and 726 bp, and introns vary in size between 196 bp and approximately 60 kb. All of the splice sites obey the consensus GT-AG rule except the splice donor of intron 9, which is GC instead of GT. The exon following this rare splice donor variant is alternatively spliced. A PGAM pseudogene and two highly polymorphic CA repeats map to introns within the gene. The structure is very similar to that of the closely related Wilson disease gene (WND; ATP7B). From exon 5 (exon 3 in ATP7B) to the end, all of the splice sites occur at exactly the same nucleotide positions as in the WND gene, except for the boundary between exons 17 and 18 (exons 15 and 16 in ATP7B) and a single codon difference at the boundary between exons 4 and 5 of the MNK gene (exons 2 and 3 in ATP7B). In contrast to the WND gene, in which the first four of six metal binding domains are contained in 1 exon, metal binding domains 1 to 4 are divided over 3 exons. The striking similarity of the MNK and WND genes at the genomic level is consistent with their relatively recent divergence from a common ancestral gene.

Molecular cloning of a novel receptor (CMKLR1) with homology to the chemotactic factor receptors.

We report the cloning of a novel human gene, CMKLR1, which encodes a protein that has notable sequence and structural homology to the seven transmembrane G-protein linked chemokine receptors. This gene has 55% nucleotide sequence homology to the IL-8 type 1 receptor and 53% to the N-formyl peptide related receptor 1 genes. The mRNA of this receptors is expressed in a broad array of tissues associated with hematopoietic and immune function including, spleen, thymus, appendix, lymph node, bone marrow, and fetal liver. Using fluorescence in situ hybridization the gene encoding CMLKR1 (chemokine-like receptor 1) was localized to human chromosome 12q24.1.