InteractiveFly: GeneBrief

Cadherin 99C: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Cadherin 99C

Synonyms -

Cytological map position - 99C4

Function - receptor

Keywords - oogenesis, microvillus formation, cell adhesion

Symbol - Cad99C

FlyBase ID: FBgn0039709

Genetic map position - 3R

Classification - cadherin superfamily

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

Actin-based protrusions can form prominent structures on the apical surface of epithelial cells, such as microvilli. Several cytoplasmic factors have been identified that control the dynamics of actin filaments in microvilli. However, it remains unclear whether the plasma membrane participates actively in microvillus formation. The function of Drosophila melanogaster cadherin Cad99C in the microvilli of ovarian follicle cells has been analyzed. Cad99C contributes to eggshell formation and female fertility and is expressed in follicle cells, which produce the eggshells. Cad99C specifically localizes to apical microvilli. Loss of Cad99C function results in shortened and disorganized microvilli, whereas overexpression of Cad99C leads to a dramatic increase of microvillus length and results in large bundles of microvilli. 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. Cad99C that lacks most of the cytoplasmic domain, including potential PDZ domain-binding sites, still promotes excessive microvillus outgrowth, suggesting that the amount of the extracellular domain determines microvillus length. Cad99C is thus a critical regulator of microvillus length, the first example of a transmembrane protein that is involved in this process (D'Alterio, 2005; Schlichting, 2006).

Microvilli are fingerlike protrusions on the apical surface of epithelial cells, where they can form a dense brush border. Microvilli are also used by several sensory cells as a basic module to form specialized structures that engage in the transduction of light and mechanosensory stimuli. Stereocilia of vertebrate inner ear hair cells are a prominent example. The core of a microvillus consists of a bundle of cross-linked parallel actin filaments, which have their barbed (+) ends inserted at the microvillus tip and their pointed (–) ends anchored in a terminal web of actin filaments. The actin filament bundle undergoes constant turnover through treadmilling. Growth of microfilaments at barbed ends is thought to push the membrane envelope forward, lengthening the protrusion (D'Alterio. 2005).

Among the molecules that serve a critical function in the formation and regulation of microvillus growth are several F-actin cross-linking proteins, including villin, epsin, fimbrin, and fascin. Epsin can induce microvillus elongation in vitro probably by affecting the actin treadmilling process, and epsin mutant deaf jerker mice have shortened hair cell stereocilia. Additional actin-binding factors that control microvillus size colocalize with the tip complex, which is thought to nucleate the microfilament bundle and to regulate actin polymerization at barbed ends. They comprise myosin XVa and its binding partner whirlin, which promote concentration-dependent elongation of hair cell stereocilia. EPS-8, another actin-binding protein located at a microvillus tip, regulates microvillus length through its barbed-end capping function in the intestine of Caenorhabditis elegans. In the early Drosophila melanogaster embryo, absence of the Abelson kinase causes abnormally long microvilli. This correlates with an ectopic accumulation of F-actin and its growth promoting factor Enabled in the apical cell cortex (D'Alterio, 2005 and references therein).

Observations like these suggested that microfilaments and their binding factors may be sufficient for microvillus formation and elongation and that the plasma membrane may serve only as an anchor for the core bundle. That coupling to the plasma membrane is important is suggested by the finding that ezrin, an ERM (ezrin-radixin-Moesin) protein that likely forms a link between actin filaments and the plasma membrane, induces microvilli in cell culture. Deficiency of ezrin, which appears to organize the terminal web of microvilli, causes shortened, irregular intestinal microvilli in mice. Similarly, the terminal web-associated ERM protein Moesin in Drosophila is required for normal organization of microvilli in rhabdomeres, and an excessive formation of irregular microvilli results from expressing constitutively active Moesin. However, there has been no evidence so far that would implicate specific integral membrane proteins as regulators of microvillus growth (D'Alterio, 2005 and references therein).

Some proteins that have received considerable attention in recent years as important organizers of hair cell stereocilia are protocadherin 15 (PCDH15), cadherin 23 (CDH23), myosin VIIa, harmonin, and SANS. Mutations in these genes are responsible for Usher syndrome type 1 (USH1), a genetic disorder that combines congenital deafness, vestibular dysfunction, and retinitis pigmentosa in humans. The phenotype of mice mutant for any USH1 gene is characterized by splayed and disorganized stereocilia and consequently it was proposed that the two USH1 cadherins may contribute to the links that visibly connect neighboring stereocilia. For CDH23, this model is supported by the observations that it colocalizes with lateral and tip links, is needed for tip link integrity, and can mediate homophilic adhesion. The molecular function of PCDH15, however, has not been elucidated. Cad99C, the fly orthologue of PCDH15, is a component of microvilli; loss- and gain-of-function analyses show that Cad99C promotes microvillus elongation in a concentration-dependent manner. The data also suggest that Cad99C acts through a mechanism that does not involve adhesion between microvilli (D'Alterio, 2005).

Loss of Cad99C results in shorter microvilli and overexpression in longer microvilli than in wild type, indicating that the concentration of Cad99C is positively correlated with the length of microvilli in follicle cells. Interestingly, modifications in the Cad99C mRNA expression level during oogenesis appear to be good indicators for changes in microvilli. During mid-oogenesis, prominent expression of Cad99C is seen in follicle cells that show forming and growing apical microvilli, and Cad99C expression culminates when microvilli reach their maximum extension. The following drop of mRNA levels in most follicle cells coincides with a regression of microvillus size, whereas centripetal cells express Cad99C strongly, consistent with the delayed formation of a microvillus brush border by these follicle cells. It is therefore proposed that transcriptionally regulated changes in the concentration of Cad99C are critically involved in the dynamic remodeling of follicle cell microvilli (D'Alterio, 2005).

The loss of Cad99C, which results in microvilli defects, also leads to defects in eggshell formation, suggesting that normal microvilli have an important function in eggshell development. Interestingly, the dynamic regulation of Cad99C expression correlates well temporally and spatially with described phases of eggshell secretion and with morphogenetic movements of follicle cells; this raises the question of how these processes are interrelated. Follicle cells undergo multiple morphogenetic movements to reach positions from which they secrete eggshell material, either while the movement is being completed or immediately afterwards. Therefore, the striking correlation between the Cad99C expression profile and morphogenetic movements likely reflects the close association between those movements and eggshell secretion (D'Alterio, 2005).

Cad99C specifically localizes to the plasma membrane of microvilli and is distributed throughout their entire length. PCDH15 shows a similar subcellular distribution in stereocilia on the surface of hair cells in the cochlea (Ahmed, 2003). In PCDH15 mutant mice (Ames waltzer) and zebrafish (orbiter), stereocilia are splayed and their arrangement is severely disturbed, causing deafness (Alagramam, 2001a; Raphael, 2001; Seiler, 2005). The function of PCDH15 in stereocilia, however, has remained unclear. This study indicates that its Drosophila orthologue Cad99C is a potent regulator of microvillus size. Interestingly, PCDH15 is found at a higher concentration in the longer stereocilia of a staircase-like bundle of hair cell stereocilia (Ahmed, 2003), and an irregular shortening of stereocilia in Ames waltzer mice has been described (Alagramam, 2001a; Raphael, 2001). This raises the possibility that PCDH15 regulates the length of stereocilia similar to Cad99C. In addition to being required for size regulation, Cad99C is also important for the normal shape and arrangement of microvilli. It will be interesting to determine in future studies whether the effect on size and on shape and arrangement of microvilli/stereocilia reflects two distinct functions of Cad99C/PCDH15 or whether they are two consequences of the same molecular function. Together, it is proposed that Cad99C/PCDH15-type cadherins have an evolutionarily conserved role in microvillus biogenesis (D'Alterio, 2005).

During the course of evolution, Cad99C/PCDH15 cadherins have adapted to act in apparently very different apical actin-based protrusions, such as the follicle cell microvilli of fly ovaries and the complex stereocilia of the vertebrate cochlea. Moreover, loss of Cad99C causes subtle defects in eye rhabdomeres and mechanosensory bristles, indicating that Cad99C is also required for other actin-based protrusions. Similar to PCDH15, which is more widely expressed in epithelial tissues (Murcia, 2001; Ahmed, 2003), Cad99C is found on the apical surface of several ectodermal epithelia during D. melanogaster development, including the imaginal discs (Schlichting, 2005). In wing imaginal discs as in the follicular epithelium, overexpression of Cad99C induces the formation of very large apical protrusions. However, there are epithelial tissues that possess a microvillus brush border but do not express Cad99C at detectable levels, including the midgut. Cad99C is therefore not a general component of microvilli and may serve a biological function that is specifically required in a subset of microvilli. Alternatively, another member of the cadherin superfamily or other membrane protein might take the place of Cad99C in the microvilli of tissues that lack this cadherin (D'Alterio, 2005).

It is speculated that, as a cadherin, Cad99C may mediate homophilic adhesion between microvilli. However, follicle cell microvilli in wild type and after overexpression of Cad99C are clearly separated from each other, implying that Cad99C mediates neither homophilic nor heterophilic interactions between microvilli while promoting their outgrowth. This conclusion is corroborated by the behavior of cell clones that either lack or overexpress Cad99C. In imaginal discs where Cad99C is concentrated at the apical interface between cells, cell clones with reduced or increased levels of Cad99C expression have wiggly boundaries, indicating that they do not sort out from wild-type cells (Schlichting, 2005). Furthermore, Cad99C located ectopically in the lateral membrane of follicle cells when overexpressed is not enriched at the border between Cad99C overexpressing cells compared with borders between wild-type and overexpressing cells as would be expected from a homophilic adhesion molecule. Similarly, in imaginal discs, the distribution of Cad99C along cell boundaries is uniform and independent of the concentration of Cad99C in neighboring cells. Hence, these findings argue against a function of Cad99C in adhesion between adjacent plasma membranes (D'Alterio, 2005).

A parallel bundle of actin filaments and associated factors that control their cross-linking and turnover are instrumental for the formation and stability of microvilli. To what extent the plasma membrane of microvilli contributes to microvillus morphogenesis either by linking to the actin core or independent of it remains largely unexplored. PCDH15 appears to influence the actin cytoskeleton of stereocilia as the amount of F-actin in stereocilia of PCDH15 mutant hair cells was reduced (Raphael, 2001). This effect is possibly mediated through its proposed interactions with the PDZ domain protein harmonin (Adato, 2005; Reiners, 2005). Among the USH1-associated proteins, harmonin has a central function, as it can also bind to CDH23 (Siemens, 2002), myosin VIIa, and SANS, and is able to interact with actin filaments promoting their bundling in cell culture, thereby potentially linking cadherins on the cell surface to the actin cytoskeleton (D'Alterio, 2005).

Unexpectedly, it was found that a truncated form of Cad99C that lacks most of the cytoplasmic tail, including the putative PDZ domain-binding sites, causes the same excessive lengthening of microvilli as the full-length protein, even in a Cad99C mutant background. This shows that the PDZ domain-binding sites do not have an essential positive regulatory function in microvillus outgrowth. It cannot be rule out that the remaining 31 juxtamembrane cytoplasmic amino acids interact with a cytoplasmic factor, but this short sequence contains no known motifs and is not conserved. Therefore, it seems likely that the membrane-bound extracellular domain of Cad99C is sufficient to promote microvillus extension independent of endogenous Cad99C. How does the extracellular domain of Cad99C control microvillus size? The data are consistent with two attractive models for Cad99C activity. The cadherin domains of Cad99C could influence the microvillus actin core by binding to an extracellular ligand, which directly or indirectly connects to the actin bundle promoting polymerization. Alternatively, the Cad99C extracellular domain might stabilize the plasma membrane of a microvillus. This may be important because to lengthen a cellular protrusion, the force created by actin polymerization has to overcome the counteracting force caused by tension in the plasma membrane envelope while it is pushed outward. Stabilization of the plasma membrane might alleviate the tension, allowing actin polymerization to proceed. The cadherin domains of Cad99C could stabilize the plasma membrane either by binding to the extracellular matrix or by self-assembling into an extracellular meshwork that forms a supporting scaffold surrounding the microvillus. The striking conservation of the Cad99C/PCDH15 extracellular domains may reflect the geometric constraints imposed by such a scaffold. The latter model in particular would be consistent with the concentration dependency of Cad99C activity and with its effect on the size and shape of microvilli (D'Alterio, 2005).


REGULATION

Transcriptional Regulation

The subdivision of the Drosophila wing imaginal disc into anterior and posterior compartments requires a transcriptional response to Hedgehog signaling. However, the genes regulated by Hedgehog signal transduction that mediate the segregation of anterior and posterior cells have not been identified. The previously predicted gene Cad99C has been molecularly characterized and shown to be regulated by Hedgehog signaling. Cad99C encodes a transmembrane protein with a molecular weight of approximately 184 kDa that contains 11 cadherin repeats in its extracellular domain and a conserved type I PDZ-binding site at its C-terminus. The levels of cad99C RNA and protein are low throughout the wing imaginal disc. However, in the pouch region, these levels are elevated in a strip of anterior cells along the A/P boundary where the Hedgehog signal is transduced. Ectopic expression of Hedgehog, or the Hedgehog-regulated transcription factor Cubitus interruptus, induces high-level expression of Cad99C. Conversely, blocking Hedgehog signal transduction by either inactivating Smoothened or Cubitus interruptus reduces high-level Cad99C expression. Finally, by analyzing mutant clones of cells, it was shown that Cad99C is not essential for cell segregation at the A/P boundary. It is concluded that cad99C is a novel Hedgehog-regulated gene encoding a member of the cadherin superfamily in Drosophila (Schlichting, 2005).

Cad99C is expressed at low levels throughout the wing, haltere, and leg imaginal discs, whereas elevated levels of Cad99C expression are confined to a strip of cells along the A/P boundary of the wing imaginal disc pouch that is known to respond to the Hh signal. Even though anterior cells along the A/P boundary of haltere and leg imaginal discs as well as cells outside the pouch region of wing imaginal discs also respond to the Hh signal, no elevated level of Cad99C was observed in these cells, indicating that cad99C is a region-specific Hh target gene. The Cad99C protein profile resembles cad99C RNA levels, indicating that the elevated expression of Cad99C is mainly due to transcriptional and not translational or posttranslational regulation. High-level Cad99C expression was reduced to the low level present in cells far away from the A/P boundary in clones of cells lacking Hh signal transduction due to mutations in either smo or ci. Conversely, ectopic expression of either Hh or Ci was sufficient to increase Cad99C expression in the wing imaginal disc pouch, indicating that high-level cad99C expression is controlled by Ci-mediated Hh signaling (Schlichting, 2005).

Different Hh-regulated genes respond differently to Ci[act] and Ci[rep]. For example, the expression of dpp is regulated both by Ci[act] and Ci[rep], whereas hh and ptc only respond to one form of Ci, Ci[rep] or Ci[act], respectively. Like ptc, cad99c appears to respond exclusively to Ci[act]. This is inferred from five observations: (1) ectopic expression in posterior cells of Ci, which under the influence of Hh is converted to Ci[act], induces high-level cad99C expression; (2) misexpression of a constitutively active form of Ci, CiPKA4, also induces high levels of cad99C expression; (3) ci null mutant clones in the anterior compartment close to the A/P boundary, where Ci[act] is the predominant form of Ci, fail to upregulate cad99C expression; (4) expression of a constitutive repressor form of Ci, CiCell, does not reduce the low-level expression of cad99C; (5) ci null mutant clones in the anterior compartment away from the A/P boundary, where Ci[rep] is the prevailing form of Ci, show no increase in the expression of cad99C. Taken together, it is concluded that cad99C expression is regulated by Ci[act] and not Ci[rep] (Schlichting, 2005).

The segregation of cells at compartment boundaries is thought to depend on the differential adhesiveness (affinity) of cells on both sides of the compartment boundaries. Based on thermodynamic considerations, it has been proposed that cells will maximize the total strength of their adhesive interactions with neighboring cells by replacing weak cell–cell interactions with stronger ones. Cells with strong adhesive interactions will thus associate preferentially with one another and will segregate from less avidly adhering cells. As predicted by this model, cells expressing different levels of the same adhesion molecule segregate from one another. However, few adhesion molecules have been identified that can promote the differential adhesiveness of cells at compartment boundaries (Schlichting, 2005).

The maintenance of the A/P boundary in the developing Drosophila wing requires Ci-mediated Hh signal transduction in anterior cells. This suggests that Hh signaling may regulate the transcription of one or more genes that in turn affect the adhesiveness of anterior cells. Members of the cadherin superfamily are known to mediate adhesion between cells and several cadherins have been shown to be involved in cell segregation. Even though most cadherins implicated so far in cell segregation contain cytoplasmic β-catenin binding sites, which are absent in Cad99C, several cadherins lacking β-catenin binding sites have also been shown to mediate cell segregation. The discovery of a gene that is both regulated by Hh signaling and encodes for a cadherin, therefore, provides an attractive candidate for mediating the segregation of anterior and posterior cells. However, cad99C expression is not elevated in cells along the A/P boundary of haltere and leg imaginal discs or outside the pouch region of wing imaginal discs, indicating that if the elevated expression of Cad99C were important for cell segregation, this could not be a general mechanism for segregating anterior and posterior cells. However, since wing imaginal disc pouch cells differ in their expression profile from wing imaginal disc cells outside of the pouch, it is not inconceivable that different molecules could operate to segregate cells at the A/P boundary in different regions of the wing imaginal disc or in different imaginal discs (Schlichting, 2005).

A mutant allele of cad99C, termed cad99C57A, was generated in order to test whether Cad99C is required to segregate anterior and posterior cells. cad99C57A 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 cad99C57A, does not show detectable staining in wing imaginal discs from homozygous cad99C57A 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 cad99C57A mutant larvae; (4) Cad99C immunoreactivity is highly reduced in homozygous cad99C57A mutant clones within wing imaginal discs (Schlichting, 2005).

The identification of cad99C as an Hh-regulated gene provides a starting point to investigate a cell biological mechanism used by Hh signaling to control the development of the Drosophila wing. It also provides a further step towards the functional characterization of all remaining members of the cadherin superfamily present in Drosophila that have so far only been predicted based on the genomic sequence (Schlichting, 2005).


DEVELOPMENTAL BIOLOGY

Cad99C is expressed at low levels throughout the wing, haltere, and leg imaginal discs, whereas elevated levels of Cad99C expression are confined to a strip of cells along the A/P boundary of the wing imaginal disc pouch that is known to respond to the Hh signal. The Cad99C protein profile resembles cad99C RNA levels, indicating that the elevated expression of Cad99C is mainly due to transcriptional and not translational or posttranslational regulation (Schlichting, 2005).

Cad99C is expressed in follicle cells surrounding the oocyte and is component of follicle cell microvilli

To identify novel regulators of cell and tissue morphogenesis, the expression patterns of uncharacterized members of the cadherin gene superfamily were studied during D. melanogaster oogenesis, when follicle cells undergo a series of well-described morphogenetic movements. Cad99C, a cadherin gene that is named after its chromosomal map position, is transcribed in cells of the follicular epithelium, but not in the germline cyst. Changes in the Cad99C expression level largely coincide with morphogenetically active phases of follicle cells. Cad99C mRNA was found in anterior and posterior follicle cells at stages 2-5 but is restricted to posterior follicle cells at late stage 6. Follicle cells that move over the oocyte at stage 9 and form a columnar epithelium at stage 10a express very high levels of Cad99C transcript. By stage 10b, high levels of expression are retained only in centripetal follicle cells that migrate inward to envelope the oocyte anteriorly. Expression levels peak again in all follicle cells when they flatten to accommodate the growth of the oocyte. During late oogenesis, some follicle cells form tubelike structures -- the micropyle and dorsal appendages. This is a process accompanied by a local increase of Cad99C expression. This dynamic expression profile suggests that Cad99C makes important contributions to follicle cell development. Consistent with its mRNA distribution, Cad99C protein is first seen during stages 2–5 of oogenesis in anterior and posterior terminal follicle cells. Beginning with stage 6, Cad99C is detected only in follicle cells that are in contact with the oocyte, a distribution that persists for the rest of oogenesis. At all stages, Cad99C is located on the apical plasma membrane of follicle cells, which faces the oocyte (D'Alterio. 2005).

To address the function of Cad99C in the follicular epithelium, the subcellular localization of this cadherin was examined and it was found to be confined to the apical microvillus brush border. Cad99C is located apical to DE-cadherin, which marks the adherens junctions. In contrast to DE-cadherin, Cad99C is not seen at the apicolateral and lateral plasma membrane, where follicle cells are in contact with one another. Cad99C shows a spiky pattern in a side view and a carpetlike pattern in a front view of the apical cell surface that is consistent with a localization to microvilli. Several observations support the idea that Cad99C is specific for follicle cell microvilli and not found in oocyte microvilli, with which they make contact: (1) Cad99C expression was not detected in the germline; (2) the pattern of Cad99C-positive microvilli reflects the honeycomb pattern of follicle cells, whereas oocyte microvilli form a continuous lawn; (3) Cad99C does not overlap with Yolkless, a marker that labels the oocyte cortex but colocalizes with a marker specifically expressed in follicle cells (CD8-GFP). It is inferred that Cad99C does not mediate homophilic adhesion between follicle cells or between microvilli of follicle cells and those of the oocyte (D'Alterio. 2005).

As a marker for follicle cell microvilli, Cad99C revealed that the length of these apical protrusions increases from stage 7 to late stage 10a, when they reach their maximum extent of 2-3 µm. At stage 10a, longer microvilli are found in the center and shorter ones in the periphery of the apical follicle cell surface. After stage 10, microvilli regress, but short microvilli remain until the end of oogenesis. Concurrent with the development of the microvillus brush border, follicle cells secrete vitelline membrane (VM) material into the extracellular space between microvilli, producing so-called vitelline bodies, which correspond in height to the microvilli and subsequently fuse into a continuous VM layer above the microvilli. The extracellular space seen between individual microvilli at the light-microscopic level suggests a separation of >400 nm. With a predicted length of 50 nm for the Cad99C extracellular region, a Cad99C trans-dimer would not be able to bridge such a gap. It therefore appears unlikely that Cad99C promotes homophilic adhesion between adjacent microvilli along their entire length, although spotlike sites of adhesion cannot be excluded (D'Alterio. 2005).

To begin to understand how Cad99C might contribute to the formation of a proper vitelline membrane, a determination was made of which cells of the ovary Cad99C RNA and protein are present. RNA in situ hybridization using a Cad99C-specific antisense RNA probe was performed on wild-type ovaries. Little hybridization signal was detected in the germarium and stage-2 and stage-3 egg chambers. During stages 4-8, a hybridization signal is present in the follicle cells located at the anterior and posterior poles of the egg chamber. During stages 9-14, a hybridization signal was detected in follicle cells surrounding the oocyte. No signal above background was detected in nurse cells or the stretched follicle cells surrounding the nurse cells, and only background signal was detected from a control sense Cad99C RNA probe (Schlichting, 2006).

To determine the distribution of Cad99C protein in wild-type ovaries, an anti-Cad99C antiserum was used. Little immunoreactivity was detected in the germarium and stage-2 and stage-3 egg chambers. During stages 4-8, the anti-Cad99C antiserum stained the border between the follicle cells and the oocyte at the anterior and posterior poles of the egg chamber. During stages 9-14, the anti-Cad99C antiserum immunoreacted with structures at the entire border between the oocyte and the follicle cells. Follicle cells covering the nurse cells, or the nurse cells themselves, were not stained. Little immunoreactivity was detected in homozygous mutant Cad99C57A/57A egg chambers. The detection of Cad99C protein at the border between the oocyte and the follicle cells, along with the detection of Cad99C RNA only in follicle cells, indicates that Cad99C protein is most probably present only in the follicle cells but not in the oocyte. Thus, both Cad99C RNA and protein are present at the proper time and place for Cad99C to play a role in deposition of the vitelline membrane (Schlichting, 2006).

Cad99C protein localizes to the border between the oocyte and surrounding follicle cells. To determine more precisely the subcellular localization of Cad99C, stage-10 egg chambers were stained with a marker for the zonula adherens, DE-cadherin, and Cad99C. Cad99C immunoreactivity was found apical to DE-cadherin, indicating that, unlike DE-cadherin, Cad99C does not localize to the zonula adherens, but rather to the apical plasma membrane. To test whether Cad99C protein localizes to the apical plasma membrane of follicle cells, the follicle-cell-specific Gal4 line CY2 was used in conjunction with UAS-mCD8-GFP to express CD8-GFP, a transmembrane protein routinely used to mark plasma membranes, in follicle cells. CD8-GFP labeled the basolateral plasma membrane as well as the apical plasma membrane. On the apical surface of follicle cells, CD8-GFP labeled protrusions that presumably represent single microvilli, or bundles containing a few microvilli. Cad99C colocalizes with the apical protrusions labeled with CD8-GFP, consistent with the notion that Cad99C localizes to microvilli. To test whether Cad99C localizes to microvilli at the ultrastructural level, immunogold electron microscopy using an anti-Cad99C antiserum was performed on control and Cad99C57A/57A egg chambers. Very little immunoreactivity was detected on the apical plasma membrane of Cad99C57A/57A mutant egg chambers. By contrast, immunoreactivity for Cad99C was observed in control Cad99C57A/+ stage-9 to stage-14 egg chambers on microvilli of follicle cells. At stage 10, 97% of the immunogold particles on the apical plasma membrane were present on microvilli, whereas 3% were detected on the apical plasma membrane outside microvilli, indicating that Cad99C is highly enriched on the microvilli of follicle cells. Cad99C was not detected on the oocyte microvilli. Thus, Cad99C specifically localizes to microvilli of follicle cells (Schlichting, 2006).


EFFECTS OF MUTATION

A mutant allele of cad99C, termed cad99C57A, was generated in order to test whether Cad99C is required to segregate anterior and posterior cells. cad99C57A 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 cad99C57A, does not show detectable staining in wing imaginal discs from homozygous cad99C57A 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 cad99C57A mutant larvae; (4) Cad99C immunoreactivity is highly reduced in homozygous cad99C57A 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 cad99C57A 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).

Cad99C mutations affect length, shape, and arrangement of follicle cell microvilli

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 Cad99C21-6, a small deletion was detected removing exon 1, which is noncoding. The largest deletions are Cad99C21-8 and Cad99C21-5, 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 -- Cad99C21-5, Cad99C21-6, and Cad99C21-8. 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. Cad99C21-5, Cad99C21-8, or Cad99C21-9 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).

Cadherin Cad99C is required for normal microvilli morphology in Drosophila follicle cells

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 Cad99C57A/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 Cad99C57A/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 Cad99C57A/57A, Cad99C51C/51C and Cad99C57A/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 Cad99C57A/51C or Cad99C57A/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 Cad99C57A/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 Cad99C57A/+ follicle cells. In contrast, no regular-spaced stripes of Phalloidin staining were detected in between the oocyte and homozygous mutant Cad99C57A/57A follicle cells (Schlichting, 2006).

(3) The morphology of follicle cell microvilli from control Cad99C57A/+ and Cad99C57A/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 Cad99C57A/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).


EVOLUTIONARY HOMOLOGS

Mutation in Protocadherin 15 cause Usher syndrome deafness

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).

Expression of mammalian Protocadherin 15

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).

Murine Ames waltzer mutation is caused by a mutation in protocadherin

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).

Zebrafish Pcdh15 genes

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).

Protein interactions of protocadherin 15

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).


REFERENCES

Search PubMed for articles about Drosophila Cadherin 99C

Adato, A., et al. (2005). Interactions in the network of Usher syndrome type 1 proteins. Hum. Mol. Genet. 14(3): 347-56. 15590703

Ahmed, Z. M., Riazuddin, S., Bernstein, S. L., Ahmed, Z., Khan, S., Griffith, A. J., Morell, R. J., Friedman, T. B. and Wilcox, E. R. (2001). Mutations of the protocadherin gene PCDH15 cause Usher syndrome type 1F. Am. J. Hum. Genet. 69: 25-34. 11398101

Ahmed, Z. M., Riazuddin, S., Ahmad, J., Bernstein, S. L., Guo, Y., Sabar, M. F., Sieving, P., Griffith, A. J., Friedman, T. B., Belyantseva, I. A. et al. (2003). PCDH15 is expressed in the neurosensory epithelium of the eye and ear and mutant alleles are responsible for both USH1F and DFNB23. Hum. Mol. Genet. 12: 3215-3223. 14570705

Alagramam, K. N., Murcia, C. L., Kwon, H. Y., Pawlowski, K. S., Wright, C. G. and Woychik, R. P. (2001a). The mouse Ames waltzer hearing-loss mutant is caused by mutation of Pcdh15, a novel protocadherin gene. Nat. Genet. 27: 99-102. 11138007

Alagramam, K. N., Yuan, H., Kuehn, M. H., Murcia, C. L., Wayne, S., Srisailpathy, C. R., Lowry, R. B., Knaus, R., Van Laer, L., Bernier, F. P. et al. (2001b). Mutations in the novel protocadherin PCDH15 cause Usher syndrome type 1F. Hum. Mol. Genet. 10: 1709-1718. 11487575

Alagramam, K. N., Stahl, J. S., Jones, S. M., Pawlowski, K. S. and Wright, C. G. (2005). Characterization of vestibular dysfunction in the mouse model for Usher syndrome 1F. J. Assoc. Res. Otolaryngol. 6(2): 106-18. 15952048

D'Alterio, C., et al. (2005). Drosophila melanogaster Cad99C, the orthologue of human Usher cadherin PCDH15, regulates the length of microvilli. J. Cell Biol. 171(3): 549-58. 16260500

Murcia, C. L. and Woychik, R. P. (2001). Expression of Pcdh15 in the inner ear, nervous system and various epithelia of the developing embryo. Mech. Dev. 105(1-2): 163-6. 11429292

Raphael, Y., et al. (2001). Severe vestibular and auditory impairment in three alleles of Ames waltzer (av) mice. Hear. Res. 151: 237-249. 11124469

Reiners, J., Marker, T., Jurgens, K., Reidel, B. and Wolfrum, U. (2005). Photoreceptor expression of the Usher syndrome type 1 protein protocadherin 15 (USH1F) and its interaction with the scaffold protein harmonin (USH1C). Mol. Vis. 11: 347-55. 15928608

Rouget-Quermalet, V., et al. (2006). Protocadherin 15 (PCDH15): a new secreted isoform and a potential marker for NK/T cell lymphomas. Oncogene. 2006 May 4;25(19):2807-11. 16369489

Schlichting, K., Demontis, F. and Dahmann, C. (2005). Cadherin Cad99C is regulated by Hedgehog signaling in Drosophila. Dev. Biol. 279(1): 142-54. 15708564

Schlichting, K., Wilsch-Brauninger, M., Demontis, F. and Dahmann, C. (2006). Cadherin Cad99C is required for normal microvilli morphology in Drosophila follicle cells. J. Cell Sci. 119: 1184-95. 16507588

Seiler, C., Finger-Baier, K. C., Rinner, O., Makhankov, Y. V., Schwarz, H., Neuhauss, S. C. and Nicolson, T. (2005). Duplicated genes with split functions: independent roles of protocadherin15 orthologues in zebrafish hearing and vision. Development 132(3): 615-23. 15634702

Senften, M., et al. (2006). Physical and functional interaction between protocadherin 15 and myosin VIIa in mechanosensory hair cells. J. Neurosci. 26(7): 2060-71. 16481439

Siemens, J., et al. (2002). The Usher syndrome proteins cadherin 23 and harmonin form a complex by means of PDZ-domain interactions. Proc. Natl. Acad. Sci. 99(23): 14946-51. 12407180

Zheng, Q. Y., et al. (2005). Digenic inheritance of deafness caused by mutations in genes encoding cadherin 23 and protocadherin 15 in mice and humans. Hum. Mol. Genet. 14(1): 103-11. 15537665


date revised: 2 July 2006

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