Human Molecular Genetics, 2001, Vol. 10, No. 26 3049-3062
© 2001 Oxford University Press
The Caenorhabditis elegans homolog of FGD1, the human Cdc42 GEF gene responsible for faciogenital dysplasia, is critical for excretory cell morphogenesis
1Department of Pediatrics and Communicable Diseases and 2Department of Human Genetics, University of Michigan School of Medicine and 3Department of Biology, College of Literature, Science and the Arts, University of Michigan, Ann Arbor, MI 48109, USA
Received August 26, 2001; Revised and Accepted October 29, 2001.
DDBJ/EMBL/GenBank accession nos AF326352 and AF339059.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
FGD1 mutations result in faciogenital dysplasia, an X-linked human disease that affects skeletogenesis. FGD1 encodes a guanine nucleotide exchange factor (GEF) that specifically activates the Rho GTPase Cdc42. To gain insight into the function of FGD1, we have isolated and characterized fgd-1, the Caenorhabditis elegans homolog of the human FGD1 gene. Comparative sequence analyses show that fgd-1 and FGD1 share a similar structural organization and a high degree of sequence identity throughout shared signaling domains. In nematodes, interference with fgd-1 expression results in excretory cell abnormalities and cystic dilation of the excretory cell canals. Molecular lesions associated with two exc-5 alleles affect the fgd-1 gene, and fgd-1 transgenic expression rescues the Exc-5 phenotype. Together, these data confirm that the fgd-1 transcript corresponds to the exc-5 gene. Transgenic expression studies show that fgd-1 has a limited pattern of expression that is confined to the excretory cell during development, a finding that suggests that the C.elegans FGD-1 protein might function in a cell autonomous manner. Serial observations indicate that fgd-1 mutations lead to developmental excretory cell abnormalities that cause cystic dilation and interfere with canal process extension. Based on these data, we conclude that fgd-1 is the C.elegans homolog of the human FGD1 gene, a new member of the FGD1-related family of RhoGEF genes, and that fgd-1 plays a critical role in excretory cell morphogenesis and cellular organization.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
FGD1 is implicated as an important participant in mammalian skeletal formation because mutations in this gene result in the disease faciogenital dysplasia (FGDY; Aarskog syndrome), an X-linked skeletal dysplasia that adversely affects skeletal development (1,2). Most FGD1 mutations in faciogenital dysplasia patients are predicted to function as null alleles; thus the X-linked recessive phenotype appears to be due to the absence of the gene product in affected males (1,3). FGD1 mutations affect the developing skeleton and skeletal abnormalities dominate the clinical manifestations of the disease (2,4). Clinical findings typically include a unique pattern of developmental skeletal abnormalities including disproportionate acromelic short stature, widely spaced eyes (hypertelorism), maxillary and mandibular hypoplasia, brachydactyly with hypoplastic phalanges and delayed bone maturation. Data shows that the FGD1 mouse ortholog, Fgd1 (5), has a restricted spatiotemporal pattern of expression that is predominantly limited to osteoblasts and preosteoblasts within regions of incipient and active endochondral and intramembranous ossification (2). These studies also show that Fgd1 is expressed in skeletal components derived from the periaxial and lateral mesoderm and the neural crest, elements that are adversely affected by the disease. Fgd1 protein is expressed in mouse osteoblasts, osteosarcoma cell lines and permanent osteoblast-like cell lines (2). Thus, the accumulated data indicates that FGD1 signaling plays a critical role in skeletal formation.
FGD1 encodes a guanine nucleotide exchange factor (GEF) for the p21 GTPase Cdc42, a member of the Rho (Ras homology) family of GTPase proteins (6,7). RhoGEFs activate Rho GTPases by catalyzing the exchange of bound GDP for GTP, thus inducing a conformational change in the GTP-bound GTPase that allows it to interact with downstream effector proteins (8). The mammalian Rho protein family consists of at least 10 distinct proteins and three major subfamilies: Cdc42, Rac and Rho; all of the Rho proteins share at least 50% identity with each other and 30% identity with Ras. Together, these Rho proteins regulate several vital cellular signaling activities including organization of the actin cytoskeleton, vesicular transport, the extension of cellular processes and the transcriptional regulation of gene expression (9). Rho GTPases play a critical role in the regulation of the actin cytoskeleton in a wide variety of eukaryotic cells. Rho regulates the assembly of contractile actin-myosin filaments (stress fibers) and focal adhesion complexes; Rac controls the assembly of actin filaments at the cell periphery to produce lamellipodia and membrane ruffles. In contrast, Cdc42 regulates cellular polarity and the assembly of actin-rich surface protrusions called filopodia (10). By regulating these cellular signaling pathways, Rho proteins (and their RhoGEF activators) regulate cell shape, motility and differentiation, properties critical to cell and tissue morphogenesis (10).
Microinjection and biochemical studies show that FGD1 specifically activates the p21 GTPase Cdc42 (6,7). By activating Cdc42, FGD1 stimulates fibroblasts to form filopodia, cytoskeletal elements involved in cellular signaling and adhesion (6). Through Cdc42, FGD1 also activates the c-Jun N-terminal kinase (JNK) signaling cascade (7), stimulates the passage of fibroblasts through the G1 phase of the cell cycle (11), and causes the tumorigenic transformation of NIH 3T3 fibroblasts (12). Subcellular fractionation and immunocytochemical studies localize endogenous and ectopically expressed mouse Fgd1 protein to the subcortical actin cytoskeleton and Golgi complex (13), and imply that FGD1 is involved in the regulation of Cdc42 in these subcellular regions. Thus, amassed data indicates that FGD1 is a specific Cdc42GEF involved in cellular signaling; these studies further suggest that FGD1 is involved in the regulation of the osteoblast actin cytoskeleton. Together, these results indicate that faciogenital dysplasia is a developmental disorder of dysregulated FGD1/Cdc42 signaling. However, these results do not provide a rapid means for genetically analyzing the FGD1 signaling pathway.
Here we describe the isolation and characterization of fgd-1, the Caenorhabditis elegans homolog of the human FGD1 gene. Comparative sequence analyses show that the predicted fgd-1 and FGD1 sequences share a similar structural organization and a high degree of sequence identity throughout shared signaling domains. Interference with fgd-1 expression results in excretory cell abnormalities and cystic dilation of the excretory canals. Transgenic expression studies indicate that fgd-1 has a limited pattern of expression that is confined to the excretory cell during development, the only cell affected in fgd-1 mutant animals; these results suggest that the C.elegans FGD-1 protein might function in a cell autonomous manner. Serial observations indicate that fgd-1 mutations cause cystic dilation of the excretory cell canals and interfere with canal process extension early in larval development. These data indicate that fgd-1 is a new and novel member of the conserved FGD1-related family of RhoGEF genes and that fgd-1 is critical in C.elegans excretory cell morphogenesis.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Isolation and characterization of fgd-1, the C.elegans FGD1 homolog
A C.elegans genomic sequence (cosmid C33D9; accession no. Z68159) showing homology to the human FGD1 gene was reported previously by Bassett et al. (14). cDNA fragments corresponding to a large part of the putative mRNA were isolated by RT–PCR; the 5' and 3' ends of the transcript were subsequently isolated by using the rapid (PCR) amplification of cDNA ends (RACE). The full-length 2911 bp cDNA contained a 2478 nucleotide open reading frame (ORF) that was predicted to encode a protein of 826 amino acids. Consistent with the previous report (14), based on the similarity of this sequence to human FGD1 (see below), the gene encoding this cDNA was designated fgd-1 (GenBank accession no. AF339059). This sequence was identical to that recently reported by Suzuki et al. (16). Genomic and cDNA sequences were compared to derive the genomic organization of the fgd-1 gene; the genomic organization of the fgd-1 gene is shown in Figure 1A. In contrast to the report by Suzuki et al. (16), this analysis showed that the fgd-1 gene was composed of a total of 16 exons and that the fgd-1 transcript was alternatively spliced. Semi-quantitative RT–PCR analysis showed that fgd-1 transcripts containing exon 14 comprised a small proportion of all the transcripts, and that transcripts lacking exon 14 were present in
20-fold excess relative to transcripts containing this exon (Fig. 1B). DNA sequence analysis predicted that, relative to transcripts lacking this exon, transcripts containing exon 14 encode a protein containing 18 additional amino acids inserted between residues 727 and 728 (GenBank accession no. AF326352). Sequence analyses predicted that these 18 additional residues would be inserted into the N-terminal region of the predicted pleckstrin homology 2 (PH2) domain to yield an altered PH2 domain (Fig. 2A). Functional studies indicate that the human PH2 domain may, in part, regulate FGD1 GEF activity (13); however, the significance of the alternatively spliced fgd-1 transcripts remains to be determined.
|
|
N, 3/10 and An analysis of the fgd-1 sequence showed that the predicted C.elegans FGD-1 protein (CeFGD-1) had a structural organization strikingly similar to human FGD1 (hFGD1) and other mammalian FGD1-related gene family members (Fig. 2A). The CeFGD-1 protein was predicted to contain (in order) adjacent RhoGEF and PH domains, a FYVE domain, and a second C-terminal PH domain (PH2). Within each of these domains, CeFGD-1 was found to be markedly similar to hFGD1 and the other FGD1-related gene family members (Fig. 2A). These results indicated that, like the other FGD1-related genes, Fgd2 and Fgd3 (17,18), CeFGD-1 contained a conserved canonical structure of signaling domains (RhoGEF, PH, FYVE and PH2) spanning 540 contiguous amino acid residues. The observation that, like FGD1, other FGD1-related family members selectively activate Cdc42 (18,19) supports the hypothesis that the high degree of similarity found among FGD1-related proteins indicates that these proteins play similar functional roles.
A search of the C.elegans genome (>99.5% complete) showed that only one other sequence, the predicted ORF C28C12.10 (accession no. Q18284), contained an FGD1-like domain structure (20). However, compared to C28C12.10, sequence alignments showed that the CeFGD-1 sequence was more closely related to hFGD1, especially within the functionally significant 320 residue RhoGEF and PH domains (Fig. 2B). Compared to C28C12.10, CeFGD-1 and the other FGD1-related sequences shared a higher degree of sequence identity throughout the RhoGEF and PH domains and regions of identity extended to sequences outside of the designated conserved regions (CRs; Fig. 2B). In contrast to the 34 and 31% amino acid residue identity shared by CeFGD-1 and hFGD1 within the RhoGEF and PH domains, respectively, C28C12.10 and hFGD1 shared 13 and 10% amino acid residue identity in the same domains. Based on these data we conclude that fgd-1 is the C.elegans homolog of the human FGD1 gene.
The fgd-1 transcript is expressed throughout development
Northern blot studies were performed to measure the relative quantities of fgd-1 transcripts during development. As shown in Figure 3A, an apparently single 3.0 kb fgd-1 transcript was present in all developmental stages without significant variation. The size of the detected transcript was consistent with the size of the isolated cDNA; however, these studies were not sensitive enough to detect the two alternatively spliced fgd-1 transcripts. RT–PCR experiments were performed with stage-specific RNA to determine the relative abundance of each of the alternative transcripts during development. Consistent with our initial results, these studies showed that, throughout development, most of the fgd-1 transcripts lack exon 14 (Fig. 3B). In contrast, transcripts containing exon 14 appeared to be expressed only during the larval stages of development (L1–L4). Based on these results, we conclude that fgd-1 transcripts are alternatively spliced and that this process is developmentally regulated. These results suggest that alternative CeFGD-1 isoforms might play individualized roles during nematode development.
|
Down-regulation of fgd-1 leads to abnormalities in excretory cell morphogenesis
To begin to understand the biological function of the fgd-1 gene, RNA mediated interference (RNAi) was used to transiently down-regulate gene expression during development. These studies were facilitated by using bgIs310 nematodes; this strain carries a transgenic insertion with a construct that expresses GFP in an excretory cell-specific pattern (T.Bogaert, personal communication). In nematodes, the single H-shaped excretory cell forms a major component of the nematode renal system (21). In bgIs310 worms, fluorescent microscopy shows that, from the cell body (located ventral to the pharynx), the excretory cell extends tubular processes, called canals, anteriorly and posteriorly along the basolateral surface of the hypodermis (Fig. 4A and B). Whereas the fgd-1 dsRNA had no obvious effect on the injected individuals, F1 descendents were found to have a strikingly abnormal phenotype. As shown in Figure 4C, fgd-1(RNAi) worms developed large cysts that were easily visible under the dissection microscope. Cysts were first visible in the later larval stages (L3 and L4) and were readily visible in the adult. These changes appeared to be fully penetrant; >95% of the F1 descendents were found to develop cysts by young adulthood. Close examination showed that the cysts communicated with the excretory cell canal (Fig. 4C); these results suggested that fgd-1(RNAi) affected excretory cell formation. To confirm our initial results, additional fgd-1 RNAi experiments were performed using bgIs310 nematodes. In contrast to the smooth uniform excretory canals of the bgIs310 nematodes (Fig. 4A), the F1 descendents of bgIs310 worms injected with fgd-1 dsRNA developed multiple large and small cysts in their excretory cell canals (Fig. 4D). In these worms, confocal microscopy showed that the cysts were associated with both dilation of the excretory canal and the focal accumulation of cytosol. These results strongly suggested that fgd-1 activity was important for excretory cell formation.
|
The fgd-1 gene corresponds to exc-5
At least 12 different genes are known to affect excretory cell morphogenesis in C.elegans (22). One of these genes, exc-5, genetically mapped close to the physical position of fgd-1 on chromosome IV. As shown in Figure 4E and F, exc-5 worms had excretory cell cysts that were strikingly similar to those observed in fgd-1(RNAi) nematodes. Therefore, we tested whether exc-5 mutants had molecular lesions in fgd-1. The most severely affected exc-5 mutant allele, exc-5(rh232), caused decreased viability in addition to the excretory cyst (Exc) phenotype. PCR amplification of genomic DNA showed that exc-5(rh232) DNA contained a deletion within the fgd-1 gene (Fig. 5). Although the exact limits of the deletion were not determined, at a minimum this deletion spans fgd-1 exons 8–13 (Fig. 1A). Since these exons encode the functionally significant RhoGEF and PH domains, we concluded that this mutation encodes a null fgd-1 allele. Sequence analysis showed that the second mutant allele, exc-5(n2672), contained a TGG
TAG nonsense mutation at codon 604 of fgd-1 (W604TER; Fig. 1A and Fig. 2B). These results are consistent with those recently reported by Suzuki et al. (16). Based on this mutation, the n2672 allele was predicted to encode a prematurely terminated CeFGD-1 protein that contained an abbreviated PH domain and lacked the evolutionarily conserved C-terminal FYVE and PH2 domains. Since missense mutations within the human FGD1 PH domain have been shown to disrupt hFGD1 function and result in faciogenital dysplasia (23), it seemed reasonable to conclude that the n2672 W604TER nonsense mutation would interfere with CeFGD-1 activity.
|
Transgenic fgd-1 expression rescues the exc-5 mutation
Transgenic experiments were used to determine if a wild-type copy of the fgd-1 gene could correct the phenotype caused by an exc-5 mutation. Using the marker plasmid pRF4 that confers a roller (Rol) phenotype to detect worms carrying transgenic extrachromosomal DNA, both pRF4 and fgd-1 expression construct DNA were co-injected into the gonads of exc-5(rh232);bgIs310 nematodes (24). Sixteen independently derived Rol F1 individuals were identified among the progeny of the injected worms. When inspected with a dissecting microscope, these 16 Rol F1 adult transgenic animals did not appear to contain cysts. Using fluorescent microscopy to confirm these observations, 11 of 11 Rol F1 worms were found to have normal excretory canals that were devoid of cysts; the excretory canals of two typical Rol F1 exc-5(rh232);bgIs310 worms are shown in Figure 6A and B. To verify that the rescue of the Exc phenotype was dependent on the presence of the injected DNA, non-roller F2 worms derived from the rescued Rol F1 nematodes were examined by fluorescent microscopy. As shown in Figure 6C and D, these non-roller F2 exc-5(rh232);bgIs310 worms had numerous excretory canal cysts. These results show that, in exc-5 worms, a wild-type fgd-1 gene rescues the Exc phenotype, and that in the absence of the fgd-1 gene, the progeny of these rescued worms revert to the Exc phenotype. These results confirm that the fgd-1 gene corresponds to exc-5.
|
fgd-1 is expressed in the excretory cell during development
Transgenic expression studies were performed to study the spatiotemporal pattern of fgd-1 expression during nematode development. For these studies we generated a CeFGD-1–GFP fusion expression construct that contained 2.2 kb of the fgd-1 upstream region (the putative fgd-1 promoter) and the first six exons of the fgd-1 gene fused in frame to GFP (Materials and Methods). Wild-type N2 nematodes were injected with both CeFGD-1–GFP and marker plasmid pRF4 DNA (24). Thirty-one independently derived Rol F2 individuals were identified among the progeny of the injected worms; of these, 20 different Rol F2 transgenic lines were examined in greater detail. Among these transgenic worms, the transmission frequency of the Rol phenotype ranged from 6.2 to 14.3% with an average frequency of 9.7%. For each of these transgenic lines, at least 20 different Rol worms were examined by fluorescent microscopy to determine the pattern of GFP expression. Without exception, Rol adult and larval nematodes showed a restricted pattern of GFP expression with GFP fluorescence limited to the body and canals of the excretory cell; typical F2 and F3 transgenic worm embryos and larvae are shown in Figure 7. Embryos and first stage larvae derived from Rol worms were examined by fluorescent microscopy to study the pattern of fgd-1 expression in earlier developmental stages, before the Rol phenotype was detectable. Of the 500 progeny examined, 7.5% of the embryos and larvae expressed GFP, a frequency consistent with the transgenic transmission frequency of the extrachromosomal array. Among these progeny, like the Rol larvae and adults, GFP expression was limited to the excretory cell (Fig. 7A and B). During embryogenesis, cell lineage studies show that the excretory cell is formed shortly before the embryo begins to elongate (25). We first saw GFP expression in comma-shaped embryos (Fig. 7A), the first stage of embryonic elongation (26). GFP expression was also observed in later-staged embryos. In contrast, no GFP expression was observed in pre-elongation embryos, a finding consistent with the timing of excretory cell formation. All of the examined Rol worm larvae expressed GFP in their excretory cells; in addition, no GFP expression was observed in cells other than the excretory cell (Fig. 7C–E). Adult worms exhibited the same expression pattern; in contrast, none of the non-roller worms expressed GFP (data not shown). Together, these results strongly suggest that fgd-1 has a restricted pattern of expression that is limited to the excretory cell during development.
|
fgd-1 mutations result in abnormal excretory canal morphogenesis
By expressing GFP in the excretory cell alone, bgIs310 nematodes provide a unique and powerful reagent for monitoring excretory cell development. Formally, fgd-1 mutations could result in excretory cysts either by interfering with morphogenesis or by the cystic degeneration of a previously formed excretory canal. Thus, we observed exc-5(rh232);bgIs310 nematodes during embryonic and larval development to define the role that fgd-1 plays in shaping the excretory cell. The excretory cell forms its canals by extending processes from the cell body dorsally on both sides. During embryogenesis, upon reaching the lateral epidermis, these canals bifurcate and grow anteriorly and posteriorly. The longer posterior canals reach epidermoblast V3 by hatching, and normally, the posterior canals continue to actively extend their processes during the first larval stage to reach V6p by the beginning of the second larval stage (27). As shown in Figure 8, the excretory canals of exc-5(rh232);bgIs310 larva displayed at least two different abnormalities. First, numerous cysts were present within the posterior canals of L1 larvae. In some L1 worms, numerous cysts were distributed along the length of the canal (Fig. 8A); in others, either an aggregate of cysts (Fig. 8B) or a single cyst was located at the end of the extending process. Serial observations of the same mutant worm showed that the cysts persisted, and that over time, the cysts tended to gradually increase in size (Fig. 8C–F). In contrast, an examination of exc-5(rh232);bgIs310 embryos failed to identify any excretory canal abnormalities (data not shown). Although these observations do not rule out the possibility of more subtle abnormalities during embryogenesis, the accumulated data do suggest that the observed abnormalities are developmental and progressive, and not the result of a degenerative process. Secondly, compared to normal worms, the posterior canals of exc-5(rh232);bgIs310 nematodes were markedly short. This was most dramatically illustrated in mutant worms containing large cysts at the ends of their posterior canals (Fig. 8E and F). Although, the excretory processes of these worms had partially extended, without exception, the excretory canals of the mutant worms were dramatically shorter than the canals of normal L2 worms (Fig. 8G).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
In this study, we describe the isolation and characterization of fgd-1, the C.elegans homolog of the human FGD1 gene, and demonstrate that fgd-1 is critical in excretory cell morphogenesis. Comparative sequence analyses show that the predicted fgd-1 sequence and FGD1 share a similar overall structural organization; the shared signaling domains span over 540 contiguous amino acid residues that include adjacent RhoGEF and PH domains, a FYVE domain, and a second C-terminal PH domain. In addition, sequence alignments show that CeFGD-1 and FGD1 share a high degree of sequence identity throughout the shared signaling domains. Several lines of evidence indicate that fgd-1 plays an important role in the morphogenesis of the C.elegans excretory cell. Interference with fgd-1 expression results in excretory cell abnormalities and cystic dilation of the excretory canals. Molecular lesions associated with two exc-5 alleles affect the fgd-1 gene, and fgd-1 transgenic expression rescues the Exc-5 phenotype; together, these data confirm that the fgd-1 transcript corresponds to the exc-5 gene. Transgenic expression studies indicate that fgd-1 has a limited pattern of expression that is confined to the excretory cell during development, the only cell affected in fgd-1/exc-5 mutant animals; these results suggest that CeFGD-1 might function in a cell autonomous manner. Serial observations indicate that fgd-1 mutations cause cystic dilation of the excretory cell canals and interfere with canal process extension early in larval development. Based on these data, we conclude that fgd-1 is a new and novel member of the conserved FGD1-related family of RhoGEF genes and that fgd-1 is critical in C.elegans excretory cell morphogenesis.
The genetics of excretory cell morphogenesis
The nematode excretory cell provides a unique opportunity to study cellular morphogenesis. The excretory cell is the largest non-multinuclear cell in the worm; together with its associated duct and pore cells, the excretory cell forms the nematode renal system (21). Since the ablation of any of these cells results in the accumulation of fluid and subsequent death, these three cells are believed to function in osmoregulation (28). The body and nucleus of the excretory cell is located in the head of the worm, ventral to the pharynx, between the ventral epidermis (hypodermis) and the epidermal basement membrane (21). The morphogenesis of the excretory cell is complex and occurs in stages, and a variety of different mutations are known to affect each stage. During embryogenesis, the excretory cell begins its morphogenesis by extending bipolar circumferential cellular processes dorsally to the lateral hypodermis (21,27). Upon reaching the lateral sides, these processes branch to extend canals anteriorly and posteriorly along the basolateral surface of the lateral hypodermis to form the characteristic H-shaped excretory cell. Several mutations, including unc-5, unc-6 and unc-34, affect the first stage in excretory cell morphogenesis. In these mutants, rather than extending the processes laterally, the excretory canals are abnormally positioned along the ventral epidermal ridge (27); consequently, the excretory cell has an abnormal shape. In contrast, our results show that the excretory canals of fgd-1/exc-5 mutant worms have a normal H-shape and are normally positioned. Thus, compared to the unc-5, unc-6 and unc-34 gene products, fgd-1 appears to affect the excretory cell later in morphogenesis.
After the excretory canal processes reach the lateral hypodermis, the processes branch to extend canals anteriorly and posteriorly along the basolateral surface of the lateral hypodermis. During larval development, these processes grow through a combination of passive stretching and active extension (27). Several mutations affect excretory canal extension including lin-17, unc-53 and unc-73. Like fgd-1/exc-5 mutations, the posterior excretory canals of unc-53 and unc-73 mutants are shortened as a result of incomplete extension; in contrast, the posterior canals of lin-17 mutant worms are abnormally long and extended past the normal V6/T boundary (27). The lin-17 gene is predicted to encode a seven-transmembrane protein that is homologous to the Drosophila gene frizzled (29); the unc-53 gene is predicted to encode an actin-binding protein (30); and unc-73 encodes the ortholog of the mammalian RhoGEF Trio (31). Although unc-73 mutants have abnormally short excretory canals, unlike the fgd-1/exc-5 mutant worms, these canals fail to develop cysts (27,31). These data suggest that extension of the excretory canal processes involves at least two different RhoGEFs, unc-73 and fgd-1.
Each excretory canal is comprised of a fluid-filled channel that is surrounded by a thin continuous wall of cytoplasm. These canals are polarized; whereas the external/basolateral surface of the canal lies against the surface of the surrounding hypodermis and basement membrane, the internal/apical surface of the excretory cell process faces the extracellular matrix contained in the lumen of the internal channel (21,22). At least 12 different mutations are known to result in excretory canal cysts; these include exc-1 through exc-9, let-4, let-653 and sma-1 (22). Depending upon the particular genotype, canal defects range from focal cysts flanked by apparently normal canal segments (i.e. exc-1 and fgd-1/exc-5), to cystic canals with a convoluted and/or branched morphology (22). In addition to fgd-1, two other genes that cause excretory canal cysts have been molecularly cloned. The let-653 gene is predicted to encode a mucin-like protein that contains a cuticlin-related domain (32); mucins comprise a large family of glycoproteins that form the mucous barrier at the apical surface of internal epithelia. The sma-1 gene encodes a ßH spectrin homolog (33). Spectrin proteins comprise a family of filamentous actin cross-linking proteins that are associated with the plasma membrane (34); in Drosophila, ßH spectrin is localized to the apical region in internal epithelia (35). These data suggest that canal cysts result from defects in the formation of the canal actin cytoskeleton or from abnormal signaling through the excretory cell apical membrane (22). If the identified exc genes encode functionally related proteins, these data suggest that fgd-1 may be involved in the regulation of excretory cell polarity and/or the regulation of the excretory cell actin cytoskeleton.
The role of fgd-1 in excretory cell morphogenesis
Like human FGD1, fgd-1 is predicted to encode a RhoGEF; thus, it is reasonable to hypothesize that the abnormalities observed in fgd-1 mutant animals are the result of disrupted Rho GTPase signaling consequential to deficient CeFGD-1 activation. Rho GTPases participate in a variety of essential functions during morphogenesis including dynamic processes that regulate cellular outgrowth, cell elongation, and cellular shape and form (9). For example, in developing Drosophila neurons, the expression of both constitutively activated and dominant-negative Rac1 mutants disrupts neuronal elongation and axonal outgrowth (36), and in transgenic mice, the expression of a constitutively active Rac perturbs Purkinje cell axon formation (37). Studies show that, in cultured neuron-like N1E-115 cells, Cdc42 acts upstream of Rac1 to promote neurite outgrowth whereas Rho inhibits neurite extension and promotes growth cone collapse (38). Mutations in mig-2, a C.elegans Rho GTPase, result in defects in cellular migration and axonal outgrowth misdirection (39). In addition to having short excretory canals, unc-73 worms exhibit cell migration and axon outgrowth defects (31). Similarly, fly studies show that the Drosophila Trio ortholog stimulates Rac to regulate axonal guidance signals during eye (40) and intersegmental nerve development (41). Like neurons, the excretory cell actively extends processes between the cell membrane and the basal lamina of the epidermis to achieve its final shape (27). Thus, our results suggest that, like unc-73, fgd-1 regulates Rho signaling during excretory cell morphogenesis to control canal elongation.
In fgd-1/exc-5 mutant worms, electron microscopic studies suggest that excretory canal cysts are associated with defects in the canal apical membrane cytoskeleton (22). Compared to normal canals, fgd-1/exc-5 canals contain amorphous deposits that resemble disorganized actin cytoskeleton and large vesicles below the apical membrane. Although these observations do not distinguish between primary and secondary effects, these findings do suggest that fgd-1 mutations might affect the excretory cell cytoskeleton. It is firmly established that the Rho GTPases (and activating RhoGEF proteins) play a critical role in the regulation of the actin cytoskeleton in a wide variety of eukaryotic cells (9,10). Rho regulates the assembly of contractile actin-myosin filaments (stress fibers) and focal adhesion complexes; Rac controls the assembly of actin filaments at the cell periphery to produce lamellipodia and membrane ruffles; and Cdc42 regulates the assembly of actin-rich surface protrusions termed filopodia (10). Extensive data indicate that Rho signaling regulates signal transduction pathways linking extracellular stimuli to the organization of the actin cytoskeleton and cell shape (9,10). Since fgd-1 is predicted to act as a RhoGEF, it is reasonable to hypothesize that fgd-1 plays a role in regulating the excretory cell actin cytoskeleton. Alternatively, fgd-1 mutations may act through Rho signaling to disrupt vesicular transport in affected excretory cells. Vesicular transport along the biosynthetic and secretory pathway is essential for the biogenesis and maintenance of subcellular organelle integrity and for the trafficking of proteins and lipids within and to the external region of the cell. The Rho GTPases are implicated in the regulation of various membrane-trafficking processes including the regulation of secretory vesicular transport (10,42). Rho proteins also play a critical role in the regulation of cellular polarity. It is firmly established that the Rho GTPases play a critical role in the regulation of cellular polarity in a wide variety of eukaryotic cells including the budding yeast Saccharomyces cerevisiae (43), and mammalian fibroblasts (44). More specifically, studies also show that Rho GTPases plays a critical role in the regulation of polarized vesicular transport and epithelial cell polarity (45). Accumulated data indicate that the excretory cell is a highly structured polarized epithelial cell. Thus, it is possible that fgd-1 mutations adversely affect the polarity of the developing excretory cell and/or result in abnormalities in directional vesicular transport.
The fgd-1 and human FGD1 homologs are expressed in markedly different cell types, and data show that both genes play critical roles in development, human FGD1 in osteoblasts and fgd-1 in nematode excretory cells. These observations suggest that, rather than playing a role in maintaining a specific cellular phenotype, the human FGD1 and CeFGD-1 proteins may play critical roles in regulating more basic cell processes, such as regulating cell polarity and/or cytoskeletal organization. Bone formation is a complex process that involves cell–cell and cell–extracellular matrix (ECM) interactions, processes that involve Rho GTPase signaling (9,10). A variety of studies have shown that osteoblasts are responsive to ECM, and that integrin-mediated signaling plays a critical role in osteoblast differentiation and function (46,47). In addition, protein transport studies show that osteoblasts are polarized (48). Thus, it is reasonable to hypothesize that FGD1 and fgd-1 may play similar roles in the regulation of Rho GTPase in mammalian osteoblasts and nematode excretory cells, respectively. Additional studies will be necessary to determine the precise role of each homolog.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Strains and general methods
The wild-type C.elegans strain N2, mutants and transgenic worms were maintained using standard methods (49). Strain UG754: bgIs310 [pGF2006] X was kindly provided by T.Bogaert, University of Ghent, Belgium. This strain carries a transgenic insertion of a GFP expression construct that is driven by the Ul-6 NruI–SphI genomic fragment; this strain has an excretory cell-specific pattern of GFP expression (T.Bogaert, personal communication). Mutant exc-5 strains (rh232 and n2672) were obtained from M.Buechner, University of Kansas (22). Double mutant exc-5(rh232) IV;bgIs310 X nematodes were constructed using standard genetic methods.
cDNA isolation and characterization
Primers based on the predicted C33D9.1 sequence were used to amplify portions of the fgd-1 cDNA by RT–PCR from total RNA isolated from mixed-stage N2 C.elegans. Primers fgd-1Fa (5'-GAC CAC AAT CGA ACG AGT GGA ACG ACT TTG TGC) and fgd-1Ra (5'-CTA TCC ACC AGC TCT TTT GCC GCA TAC CAA GC) were used to amplify a single 980 bp product from the 5' end of the cDNA; fgd-1Fb (5'-GGA TAA GAG ATA TCA AAA AGC TTG GTA TGC) and fgd-1Rb (dn16) (5'-TCA TCA TTC AGA TTG CTC GGA TCC AGA ATT CCG) were used to amplify a single 1525 bp product from the central portion of the cDNA. Product contents were confirmed by sequencing. The 3' end of the cDNA was isolated by RACE–PCR as described by Frohman (50), by using fgd-1-specific nested primers (5'-CTA CCA GCT AAG GGT TCA GGA G and 5'-ATG TGC CTT GGC TAT GCT GCC AC) sequentially with the QO and QI primers (50), respectively. To detect trans-spliced transcription products, RT–PCR was performed with either the SL-2 primer (5'-GGT TTT AAC CCA GTT ACT CAA G) or the SL-1 primer (5'-GGT TTA ATT ACC CAA GTT TGA G). To detect alternatively spliced forms of the fgd-1 transcript, RT–PCR was performed with the oligonucleotide dn16 (fgd-1Rb) and an oligonucleotide that was either entirely contained in exon 13 (up13b: 5'-CAG GAG TAT TGC ATT CAA GCC AGA TTA GG) or an oligonucleotide that spanned exons 13 and 14 (up13/14: 5'-TGC ATT CAA GCC AGA TTA GGC ACC TTC GCC). After electrophoresis, PCR products were purified from agarose gel by using Qiaex II gel extraction kit (Qiagen); PCR products were sequenced directly as described by Pasteris et al. (1). Oligonucleotides for sequencing and for PCR amplifications were synthesized by standard phosphoramidite chemistry. Purified PCR products were subcloned into the pCR II vector (Invitrogen). Homology searches were carried out at the National Center for Biotechnology Information by using the BLAST network service (51). Sequence alignments were performed by using CLUSTALW software (52). Protein domains and motifs were identified by performing Pfam database searches (53).
RNA analysis and mutation studies
Timed liquid cultures were used to grow nematodes as described by Sulston and Brenner (54). Stage-specific total RNA was isolated by acid guanidinium thiocyanate–phenol–chloroform extraction (55). For northern blot analysis, RNA was isolated from him-5(e1490) nematodes; these populations contain 30% males and 70% hermaphrodites (56). Total RNA was fractionated by formaldehyde–agarose gel electrophoresis, transferred to positively charged nylon membranes, and hybridized to a radiolabeled antisense fgd-1-specific RNA probe as described by Chen et al. (57).
To detect mutations, single-worm PCR amplifications were performed by using the Expand Long Template PCR System (Roche) as described by Plasterk (58). Multiple primer pairs were used to amplify partially overlapping fragments that spanned a 6.4 kb region containing the entire fgd-1 gene from both mutant and wild-type nematode DNA. PCR products were purified from agarose gel and both strands of the PCR products were sequenced directly (1). Additional genomic DNA PCR reactions were performed to detect deletions of the fgd-1 locus. PCR amplifications were performed with primer pair up5 (5'-GGA CCA ACA GAT TTC TCG TG) and dn8 (5'-GAT GAC ACA TTG GCA AGA AGT CGA G) to detect the 1430 bp product derived from the 5' end of the gene. PCR amplifications were performed with primer pair up13a (5'-TGT GTC AAA TGC AGT CGT C) and dn16 to detect a 590 bp product derived from the 3' end of the gene, and with primer pair dn10 (5'-CCT GCA TAT GAG AAG CAC TG) and up8 (5'-GCC AAT GTT GTT CGA AAA CAA GCT CCG TTC CTC) to detect a 632 bp product derived from the middle of the fgd-1 gene. To insure accuracy, at least two independent PCR amplifications were performed for each analysis; both strands of the PCR products were sequenced directly.
RNA-mediated interference
Double-stranded RNA used for the inhibition of fgd-1 was synthesized from a linearized template cloned into the pCRII vector using either SP6 or T7 RNA polymerase (Promega). The template used was a 313 bp fragment of the fgd-1 cDNA obtained by RT–PCR using oligonucleotides directed against nucleotides 1176–1210 and 1457–1489 (5'-CGC CAA TGT TGT TCG AAA ACA AGC TCC GTT CCT C and 5'-TTG CAT GTG CAG CTG CTT GTA ATA CCA ATT CC). Sense and anti-sense RNA were produced from separate transcription reactions. Reaction products were treated with DNase, combined, and annealed as described by Fire et al. (59); dsRNA formation was confirmed by formaldehyde agarose gel electrophoresis. RNA was resuspended in RNase-free water at a concentration of 5 µg/µl and injected into the syncytial gonads of young adult hermaphrodite nematodes as described by Mello and Fire (24). The progeny of 20 individuals were analyzed; the penetrance of the effect was >95%. Worms containing GFP expression constructs were examined with a MRC-600 BioRad confocal microscope and CoMOS software (BioRad) or a Zeiss Axiophot 2 epifluorescent microscope equipped with a Axiocam CCD camera and Axiovision digital imaging software (Zeiss).
Transgenic rescue and expression analyses
For rescue experiments, a 11 kb genomic DNA fragment containing the fgd-1 gene and 5.0 kb of 5' sequence was PCR amplified from wild-type nematode DNA by using the Expand Long Template PCR System and two oligonucleotides directed against the C33D9 sequence (5'-TGC TGA CGT CAT AAA ACG CAC ACT AAA ACC ACG ATG TGT GTT GCG and 5'-TGT GAA CTG GTT GTT AGT TAT TTT TAC GAA AA GCC GAG AGC ACG G). The PCR product was cloned into the pCRII vector, and the integrity of the fgd-1 sequence was verified by DNA sequence analysis. To study fgd-1 expression, a CeFGD-1–GFP fusion expression construct under the control of the fgd-1 promoter was generated by using PCR to amplify a 5.1 kb fgd-1 fragment with oligonucleotides (5'-AAG CTT GCA TGC CTG CAG GAA TTT GAA TGT CAG CCC AAA GTA AAT AGG and 5'-TAC CGG TAC CGG TAC CGC AGC GGC CGC CTT GTA CTC AAC AAA TGC ACG AAA CTTG C); this fragment contains the first six fgd-1 exons and 2.2 kb of the 5' upstream sequence. This 5.1 kb fragment was PstI–NotI digested and directionally cloned into the pPD117.01 vector [S.-Q.Xu, B.Kelly, B.Harfe, M.Montgomery, J.Ahnn, S.Getz and A.Fire (1997) Fire lab 1997 vector supplement (World Wide Web address: www.ciwemb.edu)], to generate an in-frame CeFGD-1–GFP expression construct that contained the first 238 CeFGD-1 residues under the putative control of the fgd-1 promoter. The integrity of the CeFGD-1–GFP expression construct sequence was verified by DNA sequence analysis. Transgenic worms were generated by injecting nematodes as described by Mello and Fire (24). Unless specified, worms were injected with a DNA solution containing fgd-1 DNA (5 ng/µl) and the marker plasmid pRF4 [rol-6(su1006dm)] (100 ng/µl); these relative concentrations were selected to favor a limited number of fgd-1 constructs in any given extra-chromosomal array.
Developmental studies
To serially examine nematode larva during development, isolated stage 1 larval worms were transferred to a drop of M9 buffer on a microscope slide containing a 0.4 mm thick agar pad, one worm per pad. A coverslip was precoated with a thin layer of bacteria and gently lowered onto the slide to avoid injury. Worms were briefly examined by confocal microscopy. Immediately afterwards, the coverslip was gently removed and the solitary worm was transferred to a fresh plate in a drop of M9 buffer, one worm per plate. After 12 h, the now stage 2 larval worms were again transferred to an identically prepared microscope slide for re-examination.
|
ACKNOWLEDGEMENTS |
|---|
We are grateful to Dr Thierry Bogaert, Dr Matthew Buechner and the Caenorhabditis Genetics Center (which is funded by the National Center for Research Resources of the NIH) for mutant worm strains, and to Dr Andrew Fire for providing expression vectors. We thank Dr Bruce Donohoe and Dr Chris Edwards for technical advice and assistance. This work was funded, in part, by the American Cancer Society grant RPG-97-172-01 (R.E.E.), by the NIH training grant 5T32HD07505-02 (L.E.), and by grants from the March of Dimes-Birth Defects Foundation (6-FY99-425) and the National Institutes of Health grant HD34446 (J.L.G.).
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed at: Division of Pediatric Genetics, Room 3570 Medical Science Research Building II, Box 0688, University of Michigan Medical School, Ann Arbor, MI 48109-0688, USA. Tel: +1 734 647 2908; Fax: +1 734 763 9512; Email: jlgorski@med.umich.edu
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Pasteris, N.G., Cadle, A., Logie, L.J., Porteous, M.E.M., Schwartz, C.E., Stevenson, R.E., Glover, T.W., Wilroy, R.S. and Gorski, J.L. (1994) Isolation and characterization of the faciogenital dysplasia (Aarskog-Scott syndrome) gene: a putative rho/rac guanine nucleotide exchange factor. Cell, 79, 669–678.[ISI][Medline]
2 Gorski, J.L., Estrada, L., Hu, C. and Liu, Z. (2000) Skeletal-specific expression of Fgd1 during bone formation and skeletal defects in faciogenital dysplasia (FGDY; Aarskog syndrome). Dev. Dyn., 218, 573–586.[ISI][Medline]
3 Gorski, J.L. (2001) Aarskog-Scott Syndrome. In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D. (eds), The Metabolic and Molecular Basis of Inherited Disease, 8th edn. McGraw-Hill, New York, pp. 6153–6165.
4 Gorski, J.L. (1998) Aarskog-Scott syndrome. In Jameson, L. (ed.), Principles of Molecular Medicine. The Humana Press, Totowa, pp. 1039–1045.
5 Pasteris, N.G., de Gouyon, B., Cadle, A.B., Campbell, K., Herman, G.E. and Gorski, J.L. (1995) Cloning and regional localization of the mouse faciogenital dysplasia (Fgd1) gene. Mamm. Genome, 6, 658–661.[ISI][Medline]
6 Olson, M.F., Pasteris, N.G., Gorski, J.L. and Hall, A. (1996) Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development are upstream regulators of Rho GTPases. Curr. Biol., 6, 1628–1633.[ISI][Medline]
7 Zheng, Y., Fischer, D.J., Tigyi, G., Pasteris, N.G., Gorski, J.L. and Xu, Y. (1996) The faciogenital dysplasia gene product FGD1 functions as a Cdc42Hs-specific guanine-nucleotide exchange factor. J. Biol. Chem., 271, 33169–33172.
8 Cerione, R.A., and Zheng, Y. (1996) The Dbl family of oncogenes. Curr. Opin. Cell Biol., 8, 216–222.[ISI][Medline]
9 Van Aelst, L., and D’Souza-Schorey, C. (1997) Rho GTPases and signaling networks. Genes Dev., 11, 2295–2322.
10 Hall, A. (1998) Rho GTPases and the actin cytoskeleton. Science, 279, 509–514.
11 Nagata, K., Lamarche, N., Gorski, J.L. and Hall, A. (1998) Activation of G1 progression, JNK MAP kinase and actin filament assembly by the exchange factor FGD1. J. Biol. Chem., 273, 15453–15457.
12 Whitehead, I.P., Abe, K., Gorski, J.L. and Der, C.J. (1998) Cdc42 and FGD1 cause distinct signaling and transforming activities. Mol. Cell. Biol., 18, 4689–4697.
13 Estrada, L., Caron, E. and Gorski, J.L. (2001) Fgd1, the Cdc42GEF responsible for faciogenital dysplasia, is localized to the subcortical actin cytoskeleton and Golgi membrane. Hum. Mol. Genet., 10, 485–495.
14 Bassett, D.E.,Jr, Boguski, M.S., Spencer, F., Reeves, R., Kim, S., Weaver, T. and Hieter, P. (1997) Genome cross referencing and XREFdb: implications for the identification and analysis of genes mutated in human disease. Nat. Genet., 15, 339–344.[ISI][Medline]
15 Blumenthal, T. and Steward, K. (1997) RNA processing and gene structure. In Riddle, D.L. (ed.), C.elegans II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 117–145.
16 Suzuki, N., Buechner, M., Nishiwaki, K., Hall, D.H., Nakanishi, H., Takai, Y., Hisamoto, N. and Matsumoto, K. (2001) A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans. EMBO Rep., 2, 530–535.[ISI][Medline]
17 Pasteris, N.G. and Gorski, J.L. (1999) Isolation, characterization, and mapping of the mouse and human Fgd2 genes, faciogenital dysplasia (FGD1; Aarskog syndrome) gene homologues. Genomics, 60, 57–66.[ISI][Medline]
18 Pasteris, N.G., Nagata, K., Hall, A. and Gorski, J.L. (2000) Isolation, characterization, and mapping of the mouse Fgd3 gene, a new faciogenital dysplasia (FGD1; Aarskog syndrome) gene homologue. Gene, 242, 237–247.[ISI][Medline]
19 Obaishi, H., Nakanishi, H., Mandai, K., Satoh, K., Satoh, A., Takahashi, K., Miyahara, M., Nishioka, H., Takaishi, K. and Takai, Y. (1998) Frabin, a novel FGD1-related actin filament-binding protein capable of changing cell shape and activating c-Jun N-terminal kinase. J. Biol. Chem., 273, 18697–18700.
20 The C.elegans Sequencing Consortium (1998) Genome sequence of the nematode Caenorhabditis elegans: a platform for investigating biology. Science, 282, 2012–2018.
21 Nelson, F.K., Albert, P.S. and Riddle, D.L. (1983) Fine structure of the Caenorhabditis elegans secretory–excretory system. J. Ultrastruct. Res., 82, 156–171.[ISI][Medline]
22 Buechner, M., Hall, D.H., Bhatt, H. and Hedgecock, E.M. (1999) Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell. Dev. Biol., 214, 227–241.[ISI][Medline]
23 Orrico, A., Galli, L., Falciani, M., Bracci, M., Cavaliere, M.L., Rinaldi, M.M., Musacchio, A. and Sorrentino, V. (2000) A mutation in the pleckstrin homology (PH) domain of the FGD1 gene in an Italian family with faciogenital dysplasia (Aarskog-Scott syndrome). FEBS Lett., 478, 216–220.[ISI][Medline]
24 Mello, C. and Fire, A. (1995) DNA transformation. In Epstein, H.F. and Shakes, D.C. (eds), Caenorhabditis elegans: modern biological analysis of an organism. Methods Cell Biol., 48, 452–482.
25 Sulston, J., Horvitz, H.R. and Kimble, J. (1988) Cell lineage. In Wood, W.B. (ed.), The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 457–489.
26 Wood, W.B. (1995) Embryology. In Wood, W.B. (ed.), The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 215–241.
27 Hedgecock, E.M., Culotti, J.G., Hall, D.H. and Stern, B.D. (1987) Genetics of cell and axon migrations in Caenorhabditis elegans. Development, 100, 365–382.[Abstract]
28 Nelson, F.K., and Riddle, D.L. (1984) Fuctional study of the Caenorhabditis elegans secretory–excretory system using laser microsurgery. J. Exp. Zool., 231, 45–56.[ISI][Medline]
29 Sternberg, P.W. and Horvitz, H.R. (1988) lin-17 mutations of Caenorhabditis elegans disrupt certain asymmetric cell divisions. Dev. Biol., 130, 67–73.[ISI][Medline]
30 Hekimi, S., and Kershaw, D. (1993) Axonal guidance defects in a Caenorhabditis elegans mutant reveal cell-extrinsic determinants of neuronal morphology. J. Neurosci., 13, 4254–4271.[Abstract]
31 Steven, R., Kubiseski, T.J., Zheng, H., Kulkarni, S., Mancillas, J., Ruiz Morales, A., Hogue, C.W.V., Pawson, T. and Culotti, J. (1998) UNC-73 activates the Rac GTPase and is required for cell and growth cone migrations in C.elegans. Cell, 92, 785–795.[ISI][Medline]
32 Jones, S.J.M. and Baillie, D.L. (1995) Characterization of the let-653 gene in Caenorhabditis elegans. Mol. Gene Genet., 248, 719–726.
33 McKeown, C., Praitis, V., and Austin, J. (1998) sma-1 encodes a ßH-spectrin homolog required for Caenorhabditis elegans morphogenesis. Development, 125, 2087–2098.[Abstract]
34 Drubin, D.G., and Nelson, W.J. (1996) Origins of cell polarity. Cell, 84, 335–344.[ISI][Medline]
35 Thomas, G.H., Zarnexcu, D.C., Juedes, A.E., Bales, M.A., Londergan, A., Korte, C.C. and Kiehart, D.P. (1998) Drosophila ßHeavy-spectrin is essential for development and contributes to specific cell fates in the eye. Development, 125, 2125–2134.[Abstract]
36 Luo, L., Liao, Y.J., Jan, L.Y. and Jan, Y.N. (1994) Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev., 8, 1787–1802.
37 Luo, L., Hensch, T.K., Ackerman, L., Barbel, S., Jan, L.Y. and Jan, Y.N. (1996) Differential effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines Nature, 379, 837–840.[Medline]
38 Kozma, R., Sarner, S., Ahmed, S. and Lim, L. (1997) Rho family GTPases and neuronal growth cone remodeling: relationships between increased complexity induced by Cdc42, Rac1 and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol. Cell. Biol., 17, 1201–1211.[Abstract]
39 Zipkin, I.D., Kindt, R.M. and Kenyon, C.J. (1997) Role of a new Rho family member in cell migration and axon guidance in C.elegans. Cell, 90, 883–894.[ISI][Medline]
40 Newsome, T.P., Schmidt, S., Dietzl, G., Keleman, K., Asling, B., Debant, A. and Dickson, B.J. (2000) Trio combines with Dock to regulate Pak activity during photoreceptor axon pathfinding in Drosophila. Cell, 101, 283–294.[ISI][Medline]
41 Bateman, J., Shu, H. and Van Actor, D. (2000) The guanine nucleotide exchange factor Trio mediates axonal development in the Drosophila embryo. Neuron, 26, 93–106.[ISI][Medline]
42 Norman, J.C., Price, L.S., Ridley, A.J. and Koffer, A. (1996) The small GTP-binding proteins, Rac and Rho, regulate cytoskeletal organization and exocytosis in mast cells by parallel pathways. Mol. Biol. Cell., 7, 1429–1442.[Abstract]
43 Schmidt, A. and Hall, M.N. (1998) Signaling to the actin cytoskeleton. Annu. Rev. Cell. Dev. Biol., 14, 305–338.[ISI][Medline]
44 Nobes, C.A., and Hall, A. (1999) Rho GTPases control polarity, protrusion, and adhesion during cell movement. J. Cell Biol., 144, 1235–1244.
45 Korschewski, R., Hall, A. and Mellman, I. (1999) Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells. Nat. Cell Biol., 1, 8–13.[ISI][Medline]
46 Franceschi, R.T. and Iyer, B.S. (1992) Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3-E1 cells. J. Bone Miner. Res., 7, 235–246.[ISI][Medline]
47 Xiao, G., Wang, D., Benson, M.D., Karsenty, G. and Franceschi, R.T. (1998) Role of the ß2-integrin in osteoblast-specific gene expression and activation of the Osf2 transcription factor. J. Biol. Chem., 273, 32988–32994.
48 Ilvesaro, J., Metsikko, K., Vaananen, K. and Tuukkanen, J. (1999) Polarity of osteoblasts and osteoblast-like UMR-108 cells.