A mutant allele of cad99C, termed cad99C^57A, was generated in order to test whether Cad99C is required to segregate anterior and posterior cells. cad99C^57A appears to be a null allele of cad99C based on four criteria: (1) sequencing of the genomic DNA revealed that the predicted promoter region, the transcriptional start site, and the coding sequence for the first 101 amino acid residues were deleted; (2) an RNA probe recognizing the 3′ region of the cad99C transcript, outside of the deletion present in cad99C^57A, does not show detectable staining in wing imaginal discs from homozygous cad99C^57A mutant larvae, indicating that the cad99C transcript levels are highly reduced; (3) an antibody directed to the C-terminus of Cad99C does not recognize a protein of the predicted size for Cad99C in extracts from wing imaginal discs of homozygous cad99C^57A mutant larvae; (4) Cad99C immunoreactivity is highly reduced in homozygous cad99C^57A mutant clones within wing imaginal discs (Schlichting, 2005).

The requirement for Cad99C to maintain the segregation of anterior and posterior wing imaginal disc cells was tested by clonal analysis. Using this assay, Cad99C was found not to be essential for maintaining the normal segregation of anterior and posterior cells. This result can be explained in several ways. (1) Cad99C does not play any role in the segregation of anterior and posterior cells. The Hh-dependent increase in expression of Cad99C may either be irrelevant for the function of Cad99C or might reflect an unrelated function. For example, Hh signaling is required for the patterning of the longitudinal wing veins L3 and L4. It is thus conceivable that Cad99C may play a role in this aspect of Hh signaling. However, vein patterning appeared to be normal in wings from homozygous cad99C^57A mutants. (2) The activity of Cad99C in mediating the segregation of anterior and posterior cells is redundant with the activity of one or several of the remaining 16 cadherins in Drosophila. Different, partially redundant mechanisms contribute to the segregation of anterior and posterior cells. For example, one mechanism might be Cad99C-dependent whereas additional mechanisms may rely on cell surface proteins unrelated to cadherins or on cytoskeletal components (Schlichting, 2005).

The chromosomal interval 99C is poorly characterized, and available deletions are haplolethal. To determine the function of Cad99C, mutations were generated by imprecise excision of two P elements. GE21034 is inserted upstream of the sequences that have been reported to represent exon 1 of Cad99C, and GE23478 is located in intron 1 (Schlichting, 2005). Both P element insertions are homozygous viable. However, whereas GE21034 appears fully fertile, GE23478 females show reduced fertility that is fully restored by excision of GE23478, indicating that the P element insertion is responsible for this defect. Several excision lines were female sterile (but male fertile) and had subtle bristle and eye defects. These lines failed to complement each other, and molecular analysis showed that they represent genomic deletions within Cad99C. In Cad99C^21-6, a small deletion was detected removing exon 1, which is noncoding. The largest deletions are Cad99C^21-8 and Cad99C^21-5, with the latter removing most of the coding sequence for the extracellular domain (CDs 1-8 and part of CD 9) (D'Alterio, 2005).

Cad99C antibodies, raised against a portion of the extracellular (CDs 10 and 11) and cytoplasmic domains, were used to probe Cad99C expression in ovaries of wild-type and homozygous Cad99C mutant females, respectively. Immunoblots identified three protein bands in wild type that were absent in mutants that lack exon 1 -- Cad99C^21-5, Cad99C^21-6, and Cad99C^21-8. Cad99C protein was also strongly reduced in ovaries that expressed Cad99C double-stranded RNA (dsRNA) causing RNA interference (RNAi). The major band corresponds to ~217 kD, which is 30 kD larger than the predicted size of Cad99C, a difference that is partly attributable to N-linked glycosylation. Two weak additional bands (195 and 172 kD) varied in intensity between preparations (inverse proportional to the intensity of the major band) and may represent degraded or otherwise modified Cad99C protein. Together, these results suggest that null mutations for Cad99C has been isolated and that this gene is not essential for viability but required for female fertility (D'Alterio, 2005).

The female-sterile phenotype of Cad99C mutants is caused by defects in eggshell formation as revealed by the following analysis. Cad99C^21-5, Cad99C^21-8, or Cad99C^21-9 mutant females laid ~50% fewer eggs than wild-type females, and <2% of those eggs produced larvae. These larvae developed into flies that displayed defects only if they were homozygous mutants, and those defects are the same as in homozygous mutants derived from heterozygous mothers, suggesting that maternal loss of Cad99C has no effect on postembryonic development. The majority of eggs from Cad99C mutant females collapse soon after deposition, and even noncollapsed eggs are penetrable to the vital dyes neutral red and trypan blue, which do not stain wild-type eggs. These defects suggest that eggshells, which normally restrict permeability and prevent desiccation, are compromised. Eggs from mutant females were also highly intolerant to sodium hydrochlorite. Hydrochlorite treatment of wild-type eggs removes the outer eggshell, the chorion, but leaves the inner eggshell, the vitelline membrane (VM), intact. Disintegration of eggs from Cad99C mutant females in hydrochlorite suggests that the VM is not functional. The VM normally forms a continuous layer of homogeneous thickness in late follicles, whereas the VM of Cad99C mutant follicles varies in thickness and contains numerous holes. These holes are likely the cause for the observed desiccation of eggs. The variability in eggshell defects may explain why mutant females are not fully sterile, allowing a few embryos to develop. The importance of Cad99C for proper eggshell formation is consistent with its expression in follicle cells that secrete the eggshell material (D'Alterio, 2005).

To analyze microvillus formation in the absence of Cad99C, the distribution of F-actin and the membrane marker CD8-GFP was examined. In wild type, F-actin is seen in microvilli of follicle cells and is enriched in the cortex of follicle cells and oocyte. In Cad99C mutant follicles, F-actin staining is strongly reduced in the space between follicle cells and oocyte, suggesting defective microvilli. CD8::GFP labeling revealed a regular microvillus brush border in wild-type follicle cells, whereas Cad99C mutant follicle cells display a range of defects of microvilli. In all mutant follicles, microvilli appear substantially shorter than in wild type. In many cases, apical protrusions appeared reduced in number, and they formed an irregular spiky pattern, suggesting that microvilli are abnormally shaped and may be clumped. In other cases, no obvious protrusions were detected or only few microvilli with an abnormal, wavy shape protruded from the apical surface. Expression of Cad99C dsRNA caused the same type of microvilli defects as Cad99C mutations. The persistent strong CD8::GFP labeling of the apical membrane domain even in cases where no protrusions were detected indicates that a substantial amount of membrane material is retained apically. These findings argue against a complete loss of microvilli and suggest that microvilli are strongly shortened and may also be collapsed against the apical cell surface. Together, these data show that Cad99C is essential to form microvilli of normal length and organization in follicle cells (D'Alterio, 2005).

Egg chambers of Cad99C mutants have vitelline bodies that are irregular in size, shape, and distribution. This suggests that the defective microvilli may cause an abnormal secretion or distribution of extracellular proteins. To address this problem, the distribution of Nudel, a secreted protein that is involved in proper formation of eggshells and the dorsal-ventral axis, was examined. No difference was noted between Cad99C mutant and wild-type follicles in the distribution or amounts of Nudel, which was deposited to its proper location between oocyte membrane and VM material. This finding argues against a general defect in protein secretion in Cad99C mutants (D'Alterio, 2005).

To determine whether Cad99C is a limiting factor for microvillus outgrowth, full-length Cad99C (Cad99C-FL) was overexpressed in follicle cells. Cad99C-FL expression induces a striking increase in the length of apical microvilli. These long cellular protrusions, which sometimes exceeded the apical-basal length of the follicle cells, projected into the narrow space between follicle cells and oocyte. Very long extensions showed small knoblike dilatations. Interestingly, the vitelline bodies deposited between the overlong microvilli also appeared enlarged. Furthermore, expression of Cad99C-FL rescued microvillus formation in a Cad99C mutant background. To investigate the importance of cytoplasmic interactions for Cad99C function, a transgenic protein (Cad99CDeltacyt::GFP) in which GFP replaces a large portion of the cytoplasmic tail, including the PDZ-binding sites, was overexpressed. Strikingly, Cad99CDeltacyt::GFP induced abnormally tall microvilli that fanned out from the apical surface and enlarged vitelline bodies similar to Cad99C-FL. This effect was observed in a wild-type and in a Cad99C mutant background. These data suggest that Cad99C levels determine the length of microvilli and show that the majority of the cytoplasmic domain (256 out of 287 amino acids) can be deleted without affecting the potential of Cad99C to promote microvillus outgrowth (D'Alterio, 2005).

Microvilli are actin-filled membranous extensions common to epithelial cells. Several proteins have been identified that localize to microvilli. However, most of these proteins are dispensable for the normal morphogenesis of microvilli. The non-classical cadherin Cad99C localizes to microvilli of Drosophila ovarian follicle cells. Loss of Cad99C function leads to disorganized and abnormal follicle cell microvilli. Conversely, overexpression of Cad99C in follicle cells results in large bundles of microvilli. Furthermore, altered microvilli morphology correlates with defects in the assembly of the vitelline membrane, an extracellular layer secreted by follicle cells that is part of the eggshell. Evidence is provided that Cad99C is the homolog of vertebrate protocadherin 15. Mutations in the gene encoding protocadherin 15 lead to the disorganization of stereocilia, which are microvilli-derived extensions of cochlear hair cells, and deafness (Usher syndrome type 1F). These data suggest an essential role for Cad99C in microvilli morphogenesis that is important for follicle cell function. Furthermore, these results indicate that insects and vertebrates use related cadherins to organize microvilli-like cellular extensions (Schlichting, 2006).

Microvilli are extensions of the apical plasma membrane that are common to epithelial cells. Intestinal epithelial cells, for example, display numerous closely spaced microvilli on their apical surface. This tightly packed array of microvilli, known as the brush border, greatly increases the cell-surface area available for nutrient absorption. Microvilli contain a bundle of crosslinked actin filaments at their center that extend from the plasma membrane to the cell cortex. Microvillar proteins, especially those of the intestinal brush border, have been identified. Among these are the actin-crosslinking proteins villin, fimbrin and espin, as well as the motor protein myosin-1A. Expression of villin and espin in tissue culture cells promotes the formation and elongation of microvilli, indicating that these proteins can play a role in the biogenesis of microvilli. Mice lacking myosin-1a display defects in the morphology and organization of brush border microvilli. However, a normal-sized microvillar brush border can be assembled in the intestine of mice with defects in villin. Thus, it seems that not all microvillar proteins are essential for microvilli morphogenesis. To understand the morphogenesis and function of microvilli better, it will be important to identify additional microvillar proteins and to test their requirement for the formation of microvilli (Schlichting, 2006).

Drosophila ovaries were used to study the morphogenesis of microvilli. The ovary is composed of approximately 16-20 ovarioles, each containing a chain of egg chambers proceeding through 14 stages from the germarium to the oviduct. Each egg chamber consists of 16 germline cells, one oocyte and 15 nurse cells, encapsulated by a monolayer of somatic, epithelial follicle cells. During stage 9, the majority of follicle cells move towards the posterior of the egg chamber, forming a columnar epithelium covering the oocyte. The remaining follicle cells stretch to cover the nurse cells. During stage 10, follicle cells covering the oocyte display on their apical surface numerous microvilli approximately 1 µm in length. These microvilli closely approach the surface of the oocyte and interdigitate with microvilli protruding from the oocyte. Gap junctions have been reported between microvilli of follicle cells and the oocyte, suggesting communication through small molecules among these cells (Schlichting, 2006).

During stages 8-10, follicle cells surrounding the oocyte secrete vitelline membrane proteins from their apical surface that are needed, in conjunction with the subsequently secreted chorion proteins, to build the protective eggshell. Initially, vitelline membrane proteins aggregate and form vitelline bodies located in between the microvilli. In stage 11 of oogenesis, follicle cell microvilli then shorten and the vitelline bodies coalesce into a continuous layer of vitelline membrane, which is important for the formation of a normal eggshell. The precise role of the follicle cell microvilli during the process of oogenesis is not known (Schlichting, 2006).

Cad99C is a non-classical member of the cadherin superfamily of Ca²⁺-dependent cell adhesion molecules and contains 11 cadherin repeats in its extracellular region and a consensus site for a class I PDZ (PSD-95, Dlg, ZO-1) domain-binding site at its cytoplasmic C-terminus (Schlichting, 2005). In general, cadherins provide molecular links between plasma membranes (and in some cases between plasma membranes and extracellular matrix) through homophilic or heterophilic binding of the extracellular cadherin repeats on adjacent plasma membranes and the binding of their intracellular region to cytoskeleton-associated proteins. In this way, cadherins mediate physical adhesion and/or cell-to-cell signaling. Cadherins are involved in various processes, including the maintenance of epithelial integrity and cell polarity, cell sorting, growth control, synaptic specificity and tissue morphogenesis. Flies mutant for Cad99C have been generated; whereas adult homozygous mutant Cad99C flies are viable, female mutant flies are sterile (Schlichting, 2005). This study analyzes the role of Cad99C during oogenesis. Cad99C is shown to be required for the normal assembly of the vitelline membrane and the integrity of the eggshell. Cad99C localizes to the microvilli of follicle cells and, in the absence of Cad99C, follicle cell microvilli are disorganized and abnormal. Conversely, overexpression of Cad99C in follicle cells results in large bundles of microvilli. Finally, evidence that Cad99C is the homolog of vertebrate protocadherin 15, a protein required for the proper organization of cochlea hair cell stereocilia, suggesting that insects and vertebrates use related cadherins to organize microvilli-like cellular extensions (Schlichting, 2006).

To begin to address the role of Cad99C during oogenesis, the eggs laid by mated Cad99C mutant flies were analyzed. Four independent Cad99C mutant alleles have been analyzed. Western blot analysis, using an anti-Cad99C antiserum, failed to detect Cad99C in extracts from ovaries of any of the four mutant fly lines. In contrast with wild-type flies, or heterozygous mutant Cad99C flies, flies homozygous for any of the four Cad99C alleles lay eggs that spontaneously collapsed after deposition and that do not develop into larvae. A similar phenotype has been reported for mutants with defective vitelline membranes. To test whether the vitelline membrane is affected in eggs laid by Cad99C mutant flies, the permeability of the eggs to the vital dye Neutral Red was assessed. Eggs in which the outer chorion layer has been experimentally removed are normally impermeable to small molecules such as Neutral Red, apparently due to the lipid wax layer covering the vitelline membrane. Defects in the vitelline membrane are thought to disrupt this lipid wax layer, thereby leading indirectly to the permeability of the eggs to Neutral Red. Fewer than 3% of eggs laid by control heterozygous Cad99C mutant flies take up Neutral Red. By contrast, more than 75% of eggs laid by homozygous mutant Cad99C flies take up Neutral Red. A similar phenotype was obtained with trans-heterozygous Cad99C allelic combinations inter se, indicating that the altered permeability to Neutral Red is due to the lesion in Cad99C (Schlichting, 2006).

To test whether the altered permeability to Neutral Red correlates with defects in the vitelline membrane, ultrathin longitudinal sections of stage-13 egg chambers were fluorescently stained with antibodies against the vitelline membrane proteins sV17 and sV23. Control egg chambers displayed a continuous line of sV17 and sV23 staining. By contrast, the sV17 and sV23 stainings are disrupted in Cad99C^57A/57A mutant egg chambers, suggesting that Cad99C is required for the formation of a continuous vitelline membrane. Stainings for the chorion proteins S18 and S36 showed continuous lines, indicating that the chorion is normal in Cad99C^57A/57A mutant egg chambers. To study the vitelline membrane defects further, the vitelline membrane of egg chambers was analyzed by electron microscopy. Wild-type egg chambers have a continuous vitelline membrane of even thickness. By contrast, the vitelline membrane of egg chambers from Cad99C^57A/57A, Cad99C^51C/51C and Cad99C^57A/51C mutants are of uneven thickness and occasionally display holes. The holes in the vitelline membrane explain the abnormal permeability of mutant eggs to the dye Neutral Red and the collapse of embryos through desiccation. Therefore, it is concluded that Cad99C is required for the assembly of a proper vitelline membrane and is important for the production of normal eggs (Schlichting, 2006).

The presence of Cad99C on follicle cell microvilli suggests that Cad99C might play a role in microvilli morphogenesis. To test this, three approaches were used. (1) CD8-GFP as a marker for microvilli was expressed in follicle cells of control and Cad99C mutant flies and the morphology of CD8-GFP-labeled protrusions was analyzed. In confocal microscope sections of control follicle cells, regularly spaced CD8-GFP-labeled protrusions of similar apparent length were detected. A few follicle cells of Cad99C^57A/51C or Cad99C^57A/120B mutant flies displayed some CD8-GFP-labeled protrusions of similar apparent length as control follicle cells, but no regularly spaced assembly of similar length protrusions was detected. Most Cad99C mutant follicle cells had CD8-GFP-labeled protrusions that appeared very short in confocal sections. Apart from the abnormal microvilli, the overall morphology of the mutant follicle cells was not overtly altered (Schlichting, 2006).

(2) Microvilli contain bundles of actin filaments at their core and can be detected by Phalloidin staining. Thus, as a second approach, marked clones of follicle cells homozygous mutant for Cad99C^57A/57A were generated using the FRT-FLP system and the microvilli were analyzed by Phalloidin staining. Regular-spaced stripes of Phalloidin staining between the follicle cells and the oocyte, presumably representing microvilli, were present in the region of control Cad99C^57A/+ follicle cells. In contrast, no regular-spaced stripes of Phalloidin staining were detected in between the oocyte and homozygous mutant Cad99C^57A/57A follicle cells (Schlichting, 2006).

(3) The morphology of follicle cell microvilli from control Cad99C^57A/+ and Cad99C^57A/57A mutant stage-10 egg chambers was compared at the ultrastructural level by electron microscopy. In the control, follicle cell microvilli are predominantly oriented towards the oocyte and are separated from one another by vitelline bodies. In contrast, microvilli of Cad99C^57A/57A mutant follicle cells are mainly oriented towards neighboring follicle cells and are often observed in the space between the vitelline bodies and the apical follicle cell surface. Taken together, these data indicate that Cad99C is required for the normal morphology and organization of follicle cell microvilli (Schlichting, 2006).

Since loss of function of Cad99C results in abnormal follicle cell microvilli, whether overexpression of Cad99C might also alter microvillar morphology was tested. To this end, a HA-tagged version of Cad99C was co-expressed with CD8-GFP in follicle cells using the Gal4 line CY2. Compared with control follicle cells expressing only CD8-GFP, the follicle cells co-expressing Cad99C-HA and CD8-GFP had more prominent CD8-GFP-labeled protrusions that formed roof-like structures. Similar results were obtained by co-expressing Cad99C-HA and CD8-GFP in clones of follicle cells using the Act5C>GAL4 driver line (Schlichting, 2006).

To examine the microvilli in Cad99C-overexpressing cells at an ultrastructural level, clones of follicle cells expressing a GFP-tagged Cad99C, Cad99C-GFP, were analyzed under the control of the Act5C>GAL4 driver line by immunoelectron microscopy using an anti-Cad99C antiserum. Electron micrographs of Cad99C-GFP-expressing follicle cells showed large bundles of Cad99C-labeled, parallel, sectioned microvilli between the vitelline bodies that were not observed in electron micrographs of control follicle cells. In addition, sectioned Cad99C-labeled microvilli were detected close, and aligned parallel, to the surface of the oocyte. The latter microvilli might reflect the CD8-GFP-labeled roof-like structures that are observed when Cad99C-HA and CD8-GFP were co-expressed (Schlichting, 2006).

During stages 11-12 of oogenesis, the microvilli of wild-type follicle cells shorten and the vitelline bodies coalesce into a continous vitelline membrane. However, when Cad99C-GFP was expressed in follicle cells, some inappropriately long microvilli persisted through stage 12, interrupting the vitelline membrane, indicating that overexpression of Cad99C prevents the timely shortening of microvilli. Together, these data indicate that overexpression of Cad99C is sufficient to alter the morphology of follicle cell microvilli, resulting in large bundles of microvilli and defects in the vitelline membrane (Schlichting, 2006).

The above experiments indicated that expression of Cad99C promotes the formation of abnormally large bundles of follicle cell microvilli. To gain insights into the mechanisms used by Cad99C to induce large bundles of microvilli, a structure-function analysis was undertaked. Cad99C contains extracellular domains, such as cadherin repeats, as well as intracellular motifs, such as a PDZ-binding site, that could be important for promoting the bundling of follicle cell microvilli, for example, through interactions with other proteins. To address whether the extracellular region, the intracellular region, or both regions of Cad99C are required to promote the formation of large microvilli bundles, mutant versions of Cad99C were generated lacking either the intracellular or the extracellular region, Cad99C-EXTRA-HA and Cad99C-INTRA-HA, respectively. Clones of follicle cells expressing full-length Cad99C-HA, Cad99C-EXTRA-HA and Cad99C-INTRA-HA were analyzed by immunoelectron microscopy using an anti-HA antibody. Anti-HA immunoreactivity for all three proteins was detected on follicle cell microvilli. Follicle cells expressing Cad99C-EXTRA-HA showed large bundles of HA-labeled, parallel, sectioned microvilli similar to those observed when full-length Cad99C-HA was expressed. Expression of Cad99C-INTRA did not result in large bundles of microvilli. Thus, the extracellular region of Cad99C fused to the transmembrane domain of Cad99C is sufficient to promote the formation of microvilli bundles, suggesting that the extracellular region of Cad99C is important for the function of Cad99C in organizing follicle cell microvilli (Schlichting, 2006).

Microvilli are common to epithelial cells in most organisms. Whether protein sequences similar to Cad99C were also present in other organisms was tested. The entire Drosophila Cad99C protein sequence was used to search databases for Cad99C-related proteins using the BLAST algorithm. In addition to a putative Cad99C Anopheles gambiae homolog (XP_312660; E-value: 0.0), BLAST also identified protocadherin 15 proteins from Mus musculus (AAG53891; E-value: e-105), Homo sapiens (AAK31804, E-value: e-104), Tetraodon nigroviridis (CAG12628, E value: e-100) and other species as putative Cad99C homologs. Human PCDH15 is a non-classical member of the cadherin superfamily that localizes to stereocilia, microvilli-derived extensions of inner ear cells (Ahmed, 2003). Mutations in PCDH15 are etiologically associated with non-syndromic deafness and Usher syndrome type 1F, an autosomal recessive disease characterized by hearing loss, vestibular dysfunction and retinopathy (Ahmed, 2001; Alagramam, 2001b). A back-BLAST analysis using human PCDH15 as the query sequence identified Drosophila Cad99C as the sequence from Drosophilawith the highest similarity (E-value: 4e-98) to human PCDH15, suggesting that Cad99C is the cadherin in Drosophila that is most closely related to human PCDH15, and vice versa (Schlichting, 2006).

How could Cad99C play a role in the formation or maintenance of normal follicle cell microvilli? Cadherins are known to provide molecular links between plasma cell membranes (and sometimes between cell membranes and the extracellular matrix) through homophilic or heterophilic binding of the extracellular cadherin repeats on adjacent plasma membranes and the interaction of their intracellular region with the cytoskeleton. These links are important for physical adhesion and/or cell-to-cell signaling. Cad99C may, therefore, constitute a molecular link used to stabilize or promote the assembly of follicle cell microvilli by providing physical adhesion of the microvilli to a target and/or signaling to the cytoskeleton. The finding that the extracellular region of Cad99C fused to its transmembrane domain is sufficient to promote the formation of large microvilli bundles, indicates that the intracellular region of Cad99C might be dispensable for this function of Cad99C, and that Cad99C might mainly act through its extracellular region. Therefore, a direct interaction between Cad99C and the cytoskeleton may not be important. However, Cad99C may indirectly interact with the cytoskeleton, for example by binding with its extracellular region to a cytoskeleton-linked transmembrane protein (Schlichting, 2006).

If Cad99C does serve as a molecular link, it could establish, through its extracellular cadherin repeats, a molecular link to three different targets: (1) Cad99C present on follicle cell microvilli might interact with a component of the vitelline membrane; (2) Cad99C molecules present on neighboring follicle cell microvilli might interact homophilically with one another or heterophilically with a different binding partner, resulting in the crosslinking or bundling of microvilli (as shown by electron microscopic preparations of wild-type ovaries that demonstrated several follicle cell microvilli in close proximity). On the basis of this model, microvilli bundles would be less efficiently formed or maintained in Cad99C mutants, resulting in the destabilization or mis-orientation of microvilli, whereas overexpression of Cad99C would result in abnormally large bundles of microvilli. Indeed, mis-oriented follicle cell microvilli were detected in Cad99C mutant flies and abnormally large bundles of microvilli were detected upon overexpression of Cad99C in follicle cells. In this scenario, the role of Cad99C would be mechanistically similar to the proposed function of PCDH15 in bundling human stereocilia (Ahmed, 2003). (3) Follicle cell microvilli closely approach the surface of the oocyte and form gap junctions with the oocyte. Thus, as a third potential target, Cad99C might provide a molecular link between follicle cell microvilli and the oocyte by interacting with a protein located at the oocyte plasma membrane. This interaction would most probably be heterophilic, since it has not been possible to immunolocalize Cad99C to the oocyte plasma membrane. In this model, the absence of Cad99C would cause follicle cell microvilli to no longer be efficiently linked to the oocyte, leading to their destabilization and/or mis-orientation. By contrast, overexpression of Cad99C in follicle cells could further stabilize microvilli at the surface of the oocyte, resulting in the observed roof-like structures. It might be advantageous to employ heterophilic rather than homophilic interactions to link follicle cell microvilli to the oocyte as the presence of homophilic adhesion molecules on microvilli might result in an excess of bundling of microvilli. The identification of proteins interacting with Cad99C could help reveal the target(s) of Cad99C and, in addition, help further elucidate the function of Cad99C on microvilli (Schlichting, 2006).

Human chromosome 10q21-22 harbors USH1F in a region of conserved synteny to mouse chromosome 10. This region of mouse chromosome 10 contains Pcdh15, encoding a protocadherin gene that is mutated in Ames waltzer and causes deafness and vestibular dysfunction. Two mutations of protocadherin 15 (PCDH15) have been found in two families segregating Usher syndrome type 1F. A Northern blot probed with the PCDH15 cytoplasmic domain showed expression in the retina, consistent with its pathogenetic role in the retinitis pigmentosa associated with USH1F (Ahmed, 2001).

The molecular basis for Usher syndrome type 1F (USH1F) has been determined in two families segregating for this type of syndromic deafness. By fluorescence in situ hybridization, the human homolog of the mouse protocadherin Pcdh15 has been placed in the linkage interval defined by the USH1F locus. The genomic structure of this novel protocadherin was determined, and a single-base deletion was found in exon 10 in one USH1F family and a nonsense mutation in exon 2 in the second. Consistent with the phenotypes observed in these families, expression of PCDH15 was demonstrated in the retina and cochlea by RT-PCR and immunohistochemistry. This report shows that protocadherins are essential for maintenance of normal retinal and cochlear function (Alagramam, 2001b).

Recessive splice site and nonsense mutations of PCDH15, encoding protocadherin 15, are known to cause deafness and retinitis pigmentosa in Usher syndrome type 1F (USH1F). Non-syndromic recessive hearing loss (DFNB23) is caused by missense mutations of PCDH15. This suggests a genotype-phenotype correlation in which hypomorphic alleles cause non-syndromic hearing loss, while more severe mutations of this gene result in USH1F. Protocadherin 15 has been localized to inner ear hair cell stereocilia and to retinal photoreceptors by immunocytochemistry. These results further strengthen the importance of protocadherin 15 in the morphogenesis and cohesion of stereocilia bundles and retinal photoreceptor cell maintenance or function (Ahmed, 2003).

Protocadherin 15 (Pcdh15) is associated with the Ames waltzer mutation in the mouse. By situ hybridization Pcdh15 is found to be expressed in the sensory epithelium in the developing inner ear, in Rathke's pouch, and broadly throughout the brain with the highest level of expression being detected at embryonic day 16 (E16). Pcdh15 transcripts are also found in the developing eye, dorsal root ganglion, and the dorsal aspect of the neural tube, floor plate and ependymal cells adjacent to the neural canal. Additionally, expression is also detected in the developing glomeruli of the kidney, surface of the tongue, vibrissae, bronchi of the lung, and in the epithelium of the olfactory apparatus, gut and lung (Murcia, 2001).

Natural killer cells are well known to play an important role in immune defense against tumor development and viral infections. To further characterize new functionally relevant structures in these cells, A series of monoclonal antibodies were studied that were raised against the NK cell line YT. One of these antibodies previously described as AY19, recognizes a 85 kD surface glycoprotein. This study reports the identification of a new secreted isoform of protocadherin 15, PCDH15C, which represents a potential associated protein for p85. Importantly, whereas protocadherins are absent from the surface of normal hematopoietic cells, this study shows that PCDH15 is expressed in cytotoxic tumor-derived T- and NK-cell lines as well as in biopsies of nasal NK/T-cell lymphomas (Rouget-Quermalet, 2006).

The neuroepithelia of the inner ear contain hair cells that function as mechanoreceptors to transduce sound and motion signals. Mutations affecting these neuroepithelia cause deafness and vestibular dysfuction in humans. Ames waltzer (av) is a recessive mutation found in mice that causes deafness and a balance disorder associated with the degeneration of inner ear neuroepithelia. As reported here, the gene that harbours the av mutation encodes a novel protocadherin. Cochlear hair cells in the av mutants show abnormal stereocilia by 10 days after birth (P10). This is the first evidence for the requirement of a protocadherin for normal function of the mammalian inner ear (Alagramam, 2001a).

The deaf-circling Ames waltzer (av) mouse harbors a mutation in the protocadherin 15 (Pcdh15) gene and is a model for inner ear defects associated with Usher syndrome type 1F. Earlier studies showed altered cochlear hair cell morphology in young av mice. In contrast, no structural abnormality consistent with significant vestibular dysfunction in young av mice was observed. Light and scanning electron microscopic studies showed that vestibular hair cells from presumptive null alleles Pcdh15(av-Tg) and Pcdh15(av-3J) are morphologically similar to vestibular sensory cells from control littermates, suggesting that the observed phenotype in these alleles might be a result of a central, rather than peripheral, defect. In the present study, a combination of physiologic and anatomic methods was used to more thoroughly investigate the source of vestibular dysfunction in Ames waltzer mice. Analysis of vestibular evoked potentials and angular vestibulo-ocular reflexes revealed a lack of physiologic response to linear and angular acceleratory stimuli in Pcdh15 mutant mice. Optokinetic reflex function was diminished but still present in the mutant animals, suggesting that the defect is primarily peripheral in nature. These findings indicate that the mutation in Pcdh15 results in either a functional abnormality in the vestibular receptor organs or that the defects are limited to the vestibular nerve. AM1-43 dye uptake has been shown to correlate with normal transduction function in hair cells. Dye uptake was found to be dramatically reduced in Pcdh15 mutants compared to control littermates, suggesting that the mutation affects hair cell function, although structural abnormalities consistent with significant vestibular dysfunction are not apparent by light and scanning electron microscopy in the vestibular neuroepithelia of young animals (Alagramam, 2005).

Mutations in genes coding for cadherin 23 and protocadherin 15 cause deafness in both mice and humans. Evidence is provided that mutations at these two cadherin loci can interact to cause hearing loss in digenic heterozygotes of both species. Using a classical genetic approach, mice were generated that were heterozygous for both Cdh23 and Pcdh15 mutations on a uniform C57BL/6J background. Significant levels of hearing loss were detected in these mice when compared to age-matched single heterozygous animals or normal controls. Cytoarchitectural defects in the cochlea of digenic heterozygotes, including degeneration of the stereocilia and a base-apex loss of hair cells and spiral ganglion cells, were consistent with the observed age-related hearing loss of these mice beginning with the high frequencies. In humans, evidence has been obtained for a digenic inheritance of a USH1 phenotype in three unrelated families with mutations in CDH23 and PCDH15. Altogether, these data indicate that CDH23 and PCDH15 play an essential long-term role in maintaining the normal organization of the stereocilia bundle (Zheng, 2005).

In the sensory receptors of both the eye and the ear, specialized apical structures have evolved to detect environmental stimuli such as light and sound. Despite the morphological divergence of these specialized structures and differing transduction mechanisms, the receptors appear to rely in part on a shared group of genes for function. For example, mutations in Usher (USH) genes cause a syndrome of visual and acoustic-vestibular deficits in humans. Several of the affected genes have been identified, including the USH1F gene, which encodes protocadherin 15 (PCDH15). Pcdh15 mutant mice also have both auditory and vestibular defects, although visual defects are not evident. Zebrafish have two closely related pcdh15 genes that are required for receptor-cell function and morphology in the eye or ear. Mutations in pcdh15a cause deafness and vestibular dysfunction, presumably because hair bundles of inner-ear receptors are splayed. Vision, however, is not affected in pcdh15a mutants. By contrast, reduction of pcdh15b activity using antisense morpholino oligonucleotides causes a visual defect. Optokinetic and electroretinogram responses are reduced in pcdh15b morpholino-injected larvae. In electron micrographs, morphant photoreceptor outer segments are improperly arranged, positioned perpendicular to the retinal pigment epithelium and are clumped together. These results suggest that both cadherins act within their respective transduction organelles: Pcdh15a is necessary for integrity of the stereociliary bundle, whereas Pcdh15b is required for alignment and interdigitation of photoreceptor outer segments with the pigment epithelium. It is concluded that after a duplication of pcdh15, one gene retains an essential function in the ear and the other in the eye (Seiler, 2005).

Defects in myosin VIIa, harmonin (a PDZ domain protein), cadherin 23, protocadherin 15 and sans (a putative scaffolding protein), underlie five forms of Usher syndrome type I (USH1). Mouse mutants for all these proteins exhibit disorganization of their hair bundle, which is the mechanotransduction receptive structure of the inner ear sensory cells, the cochlear and vestibular hair cells. Harmonin interacts with cadherin 23 and myosin VIIa. This study addresses the extent of interactions between the five known USH1 proteins. The previously suggested sans-harmonin interaction has beem established; sans also binds to myosin VIIa. Sans can form homomeric structures and harmonin b can interact with all harmonin isoforms. Harmonin also binds to protocadherin 15. Molecular characterization of these interactions indicates that through its binding to four of the five USH1 proteins, the first PDZ domain (PDZ1) of harmonin plays a central role in this network. Sans localizes in the apical region of cochlear and vestibular hair cell bodies underneath the cuticular plate. In contrast to the other four known USH1 proteins, no sans labeling is detected within the stereocilia. It is proposed that via its binding to myosin VIIa and/or harmonin, sans controls the hair bundle cohesion and proper development by regulating the traffic of USH1 proteins en route to the stereocilia (Adato, 2005).

The human Usher syndrome (USH) is the most common form of deaf-blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction and prepubertal onset of retinitis pigmentosa. Five corresponding genes of the seven USH1 genes have been cloned over the years. Recent studies indicated that three USH1 proteins, namely myosin VIIa (USH1B), SANS (USH1G), and cadherin 23 (USH1D) interact with the USH1C gene product harmonin. In these protein-protein complexes harmonin acts as the scaffold protein binding these USH1 molecules via its PDZ domains. The aim of the present study was to analyze whether or not the fifth identified USH1 protein protocadherin 15 (Pcdh15) also binds to harmonin and where these putative protein complexes might be localized in mammalian rod and cone photoreceptor cells. In vitro binding assays (GST pull-down, yeast two-hybrid assay) were applied. Antibodies against bacterial expressed USH1 proteins were generated. Affinity purified antibodies were used in immunoblot analyses of brain fractions and isolated retinas, in immunofluorescence studies, and in immunoelectron microscopic studies of rodent retinas. Pcdh15 (USH1F) has been shown to interact with harmonin PDZ2. Immunocytochemistry revealed that Pcdh15 is expressed in photoreceptor cells of the mammalian retina, where it is colocalized with harmonin, myosin VIIa, and cadherin 23 at the synaptic terminal. Colocalization of Pcdh15 with harmonin was found at the base of the photoreceptor outer segment, where newly synthesized disk membranes are present. These data indicate that harmonin-Pcdh15 interactions probably play a role in disk morphogenesis. Furthermore, evidence is provided that a complex composed of all USH1 molecules may assemble at the photoreceptor synapse. This USH protein complex can contribute to the cortical cytoskeletal matrices of the pre- and post-synaptic regions, which are thought to play a fundamental role in the structural and functional organization of the synaptic junction. Defects in any of the USH1-complex partners may result in photoreceptor dysfunction causing retinitis pigmentosa, the clinical phenotype in the retina of USH1 patients (Reiners, 2005).

Hair cells of the mammalian inner ear are the mechanoreceptors that convert sound-induced vibrations into electrical signals. The molecular mechanisms that regulate the development and function of the mechanically sensitive organelle of hair cells, the hair bundle, are poorly defined. Two gene products that have been associated with deafness and hair bundle defects, protocadherin 15 (PCDH15) and myosin VIIa (MYO7A), can be linked into a common pathway. PCDH15 binds to MYO7A and both proteins are expressed in an overlapping pattern in hair bundles. PCDH15 localization is perturbed in MYO7A-deficient mice, whereas MYO7A localization is perturbed in PCDH15-deficient mice. Like MYO7A, PCDH15 is critical for the development of hair bundles in cochlear and vestibular hair cells, controlling hair bundle morphogenesis and polarity. Cochlear and vestibular hair cells from PCDH15-deficient mice also show defects in mechanotransduction. Together, these findings suggest that PCDH15 and MYO7A cooperate to regulate the development and function of the mechanically sensitive hair bundle (Senften, 2006).