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¹ Division of Sex Differentiation, National Institute for Basic Biology, Okazaki 444-8787, Japan
² School of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan
³ Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan
⁴ Laboratory of Molecular and Cellular Biology, Department of Life Science, Faculty of Bioresources, Mie University, Tsu 514-8507, Japan
⁵ Technology and Development Team for Mammalian Cellular Dynamics, BioResource Center, RIKEN Tsukuba Institute, Tsukuba 305-0074, Japan
⁶ Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan

	Abstract

Top Abstract Introduction Results Discussion Experimental procedures References

 
Recent loss-of-function and gain-of-function studies have revealed that transcription factor Sox9 is required for testis formation by governing Sertoli cell differentiation, and thereafter regulating transcription of Sertoli marker genes. In the present study, we identified a novel isoform of Vinexin, which is expressed in somatic cells but not germ cells of sexually indifferent stages of fetal gonads. After the sex is determined, the expression continues in testicular Sertoli cells. Immunohistochemical analyses with a specific antibody to Vinexin indicated that Vinexin is localized in the cytoplasm. Functional studies with C3H10T1/2 cells showed that Vinexin acted as a scaffold protein to activate MEK and ERK through interaction with c-Raf and ERK. Ultimately, Sox9 transcription was induced by Vinexin . This up-regulation of Sox9 expression disappeared when the cells were treated with a specific MEK inhibitor, U0126. To determine the role of Vinexin during gonad formation, the gene was disrupted by targeted mutagenesis. The phenotype displayed by the mice indicated that ERK activation was decreased in the Vinexin ^–/– XY gonads, and Sox9 expression was down-regulated. Thus, Vinexin seems to be implicated in regulation of Sox9 gene expression by modulating MAPK cascade in mouse fetal gonads.

	Introduction

Top Abstract Introduction Results Discussion Experimental procedures References

 
In mammals, sexes are determined by the presence or absence of Sry, the sex determining gene on the Y chromosome (Koopman et al. 1991; Sinclair 1998). Early gonads are morphologically identical between XX and XY individuals until embryonic day 11.5 (E11.5). However, through its transient expression from E10.5 and E12.5, Sry initiates the developmental processes necessary to shape the XY indifferent gonad into a testis (Swain & Lovell-Badge 1999). Thereafter, multiple transcriptional regulators such as Sox9 (Foster et al. 1994; Wagner et al. 1994), M33 (Katoh-Fukui et al. 1998), Wt-1 (Hammes et al. 2001), Gata-4 (Tevosian et al. 2002), and Dax-1 (Meeks et al. 2003a, 2003b) function to develop sexually differentiated gonads. In particular, based on the temporal and spatial correlation between Sox9 and Sry, it has been proposed that Sry directly regulates Sox9 gene expression (Albrecht & Eicher 2001; Canning & Lovell-Badge 2002; Sekido et al. 2004). Structurally, Sox9 is characterized by the presence of an HMG DNA-binding domain (Bowles et al. 2000), and it has been functionally shown to activate the expression of a testicular marker gene, Mullerian-inhibiting substance (MIS), in Sertoli cells (Arango et al. 1999). Based on these observations, it was predictable that over-expression of Sox9 induced XX sex reversal (Huang et al. 1999; Vidal et al. 2001), and homozygous deletion of Sox9 in the XY gonads interfered with testicular development (Chaboissier et al. 2004).

Recently, increasing numbers of growth factors, such as fibroblast growth factor 9 (Fgf9) (Colvin et al. 2001; Schmahl et al. 2004), Wnt4 (Vainio et al. 1999; Jeays-Ward et al. 2003), Desert hedgehog (Dhh) (Pierucci-Alves et al. 2001; Yao et al. 2002), growth factor receptors such as platelet-derived growth factor receptor alpha (Pdgfr) (Brennan et al. 2003), and insulin/insulin-like growth factor receptors (Ir, Igf1r, Irr) (Nef et al. 2003) have been shown to be necessary to induce testicular development. A certain group of growth factors, including Fgf, mediates the development of various tissues through receptor tyrosine kinase signaling (Schlessinger 2000). In this signaling process, initiation of Ras activation is known to lead to sequential phosphorylation of protein kinases, Raf, MEK, and ERK (canonical MAPK cascade) (Schaeffer & Weber 1999). Eventually, the activated ERK enters into the nucleus to transduce signals to the transcriptional machinery (Hazzalin & Mahadevan 2002). In vertebrates, a number of gain-of-function and loss-of-function studies have implicated MAPK signaling in developmental processes such as gastrulation, convergent extension, somite boundary formation, limb development, and neural development (Martin 1998; Curran & Grainger 2000; Elowe et al. 2001; Sawada et al. 2001; Shinya et al. 2001). However, the function of growth factor-mediated MAPK signaling is largely unknown in the process of gonad development.

Recently, Vinexin was identified as a Vinculin binding protein localized at focal adhesions and cell-cell junctions in cultured cells (Kioka et al. 1999). The Vinexin gene produces two isoforms, Vinexin and ß, both of which are characterized by three tandemly aligned SH3 domains at the C-terminal region (Kioka et al. 1999). The first and second SH3 domains bind to Vinculin (Kioka et al. 1999), while the third one binds to Sos, a guanine nucleotide exchange factor for Ras and Rac (Akamatsu et al. 1999). Through these protein interactions, Vinexin ß regulates the epidermal growth factor (EGF)-induced activation of c-jun N-terminal kinase (JNK) and ERK2 in certain cellular conditions (Akamatsu et al. 1999; Suwa et al. 2002). In addition to the functions as the signal mediator, it was recently reported that Vinexin stimulates hormone-induced transcriptional activity of steroid receptors via physical interaction (Tujague et al. 2004).

Whereas Vinexin ß is expressed ubiquitously (Kioka et al. 1999; Kawauchi et al. 2001), the form shows tissue preference for expression in adult mice (Kioka et al. 1999; Tujague et al. 2004). Likewise, in mouse fetuses Vinexin is expressed in the outflow tract and atrioventricular canal of the heart, ventral part of the pons, and gonads of both sexes (Kawauchi et al. 2001). Although the tissue preferential expression seems to be correlated with tissue functions and developmental processes, functional studies of Vinexin in vivo remain to be performed.

In this study, we isolated a novel isoform, Vinexin , from mouse fetal gonads and investigated its functions by examining the phenotype of gene-disrupted mice, in addition to in vitro analyses. These studies revealed that Vinexin is implicated in transcriptional regulation of Sox9 gene in the gonad by modulating the MAPK cascade.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
Novel Vinexin isoform isolated from mouse fetal gonads

Two isoforms of Vinexin, Vinexin and ß, had previously been identified (Kioka et al. 1999), and Vinexin was indicated to be expressed in gonads of both sexes (Kawauchi et al. 2001). To investigate its functions during gonadal development, we screened a cDNA library prepared from E11.5–13.5 mouse fetal gonads (Mizusaki et al. 2003) and unexpectedly a novel isoform, Vinexin , was isolated. Structurally, Vinexin is composed of 680 amino acids corresponding to the 54th to 733th amino acids of Vinexin (76 kDa) (Fig. 1A,B). Analyses of the gene structure indicated that this difference is due to alternative usage of the first exon (data not shown). In silico screening revealed the presence of Vinexin in other animal species: human SCAM-1 (SH3-Containing Adaptor Molecule-1) (GenBank^TM accession number AF037261) and rat SCAM-1 (Tujague et al. 2004).

View larger version (22K): [in this window] [in a new window]   Figure 1  Schematic diagram of three isoforms of Vinexin. (A) ORFs of Vinexin (82 kDa/733 amino acids), ß (37 kDa/329 amino acids), and (76 kDa/680 amino acids) are shown by the boxes. The gray boxes within the ORFs indicate three SH3 domains. Untranslated regions of the three isoforms of Vinexin are also shown by lines. The 5' non-coding regions are unique to three isoforms of Vinexin. The positions of the cDNA fragments used as the probes for whole-mount in situ hybridization are shown by bold lines (see Figs 2 and 7). (B) The N-terminal (1st to 60th) amino acid sequences of Vinexin isoforms are shown. Arrowheads with and indicate translation start sites of Vinexin and , respectively.

Vinexin is expressed in sexually differentiating gonads

To investigate the expression profile of Vinexin , whole-mount in situ hybridization was performed with mouse fetal gonads. Vinexin expression was not evident in developing gonads and mesonephroi of both sexes at E10.5 (data not shown); however, expression became evident in the gonads by E11.5 (Fig. 2A,B). At E12.5, Vinexin continued to be expressed at a high level in the developing testes, with the transcripts being localized to the cord of the testis (Fig. 2C) and to whole of the ovaries (Fig. 2D). The expression was weakened in the gonads of both sexes by E14.5 (Fig. 2E,F). Although the in situ probe used in these studies potentially detects both Vinexin and (/ probe shown in Fig. 1), the signal detected corresponds to the expression of Vinexin as described below.

View larger version (44K): [in this window] [in a new window]   Figure 2  Vinexin expression during mouse gonad development. (A–F) Whole-mount in situ hybridization analyses of the Vinexin gene using the / probe (see Figure 1). At E11.5, Vinexin is present in the indifferent gonadal region of the male (A) and female (B), while at E12.5, it is expressed in the testicular cord of the male (C), and in the whole gonad of the female (D). At E14.5, Vinexin expression is weakened in both sexes (E, F). (G) Somatic cell-specific expression of the Vinexin gene. Total RNAs were purified from the somatic cells and germ cells of E12.5 fetal gonads, and subjected to RT-PCR analyses with sets of primers for Vinexin , Ad4BP/Sf-1, Oct3/4, and GAPDH as described in Experimental procedures. Vinexin was detected in somatic cells but not germ cells. Ad4BP/Sf-1 and Oct-3/4 are the representative markers of the somatic cells and germ cells, respectively.

Vinexin

Vinexin is localized in cytoplasm in the developing gonads

The expression of Vinexin was examined further with a polyclonal antibody raised to a recombinant Vinexin . First, we investigated whether Vinexin could be separated from Vinexin on SDS-PAGE. Each isoform of Vinexin was expressed in CV-1 cells and the cell lysate was subjected to Western blotting. As shown in Fig. 3A, Vinexin and were observed as discrete signals in spite of their similar molecular weights. Thus, we examined which isoforms of Vinexin were expressed in mouse adult tissues. Vinexin ß isoform was expressed ubiquitously (Fig. 3A) as previously described (Kioka et al. 1999), while the isoform was expressed in the lung and testis. The isoform was expressed in heart, lung, testis and ovary but not in kidney (Fig. 3A). In the E12.5 fetal gonads, Vinexin and ß were detected, whereas Vinexin was not detected.The expression of Vinexin in the developing gonads was examined immunohistochemically. Since the antibody was able to detect all Vinexin isoforms as described above, the immunohistochemical analysis revealed a mixed distribution of the two isoforms, Vinexin ß and . Vinexins started to be expressed at around E11.0 in both sexes (data not shown), and were detected in somatic cells in the XX and XY gonads at E11.5 (Fig. 3B,C). At E12.5, Vinexin expression was observed in Sertoli cells of the XY gonads (Fig. 3D) and in the somatic cells of the XX gonads (Fig. 3E). In addition, the antibody gave weak signals in the interstitial and coelomic epithelial cells of the testis. By E14.5, Vinexin expression was weakened in both sexes (data not shown). Vinexins were localized in the cytoplasm but not in the nuclei at all stages examined.

View larger version (118K): [in this window] [in a new window]   Figure 3  Tissue-specific expression of the three isoforms of Vinexin and localization in the fetal gonads. (A) Western blot analyses with an antibody to Vinexin. Total cell lysates were prepared from CV-1 cells transfected with empty (vec) or Vinexin expression vectors (, ß, ). Total tissue lysates were prepared from heart, lung, kidney, testis, and ovary of adult mice and fetal gonads at E12.5 of () and (). Five µg of cell lysates or 20 µg of tissue lysates were analyzed by Western blotting with anti-Vinexin antibody. Arrows with , ß, or indicate Vinexin , ß, or , respectively. The molecular weight marker is on the right. (B–E) Vinexin was detected in the fetal gonads by immunohistochemical analyses. Sagittal sections of the E11.5 male (B) and female (C), and E12.5 male (D) and female (E) fetal gonads were prepared. They were stained with anti-Vinexin antibody. The higher magnification (insets) reveals the cytoplasmic distribution of Vinexin protein. Scale bar: B–E, 50 µm; inset, 10 µm.

Vinexin activates the MEK-ERK cascade in vitro

Recently, it was reported that Vinexin ß regulates EGF-induced phosphorylation of c-jun N-terminal kinase (JNK) and anchorage-dependent phosphorylation of ERK2 (Akamatsu et al. 1999; Suwa et al. 2002). Therefore, we examined whether Vinexin regulates the MAPK cascade in a mouse embryonic fibroblast cell line, C3H10T1/2. The cells were transiently transfected with empty or Vinexin expression plasmids, and cultured for 3 h in serum-free medium. Thereafter, they were cultured for the indicated times in medium containing 10% serum (Fig. 4A). Phosphorylation of ERK, MEK, and c-Raf (Ser338) was examined at each time point. Phosphorylation of ERK and MEK was found to be up-regulated significantly from 6 h after the transfection of Vinexin while that of c-Raf was not up-regulated. In contrast, Vinexin did not affect phosphorylation of JNK and p38 (data not shown). Moreover, by using a specific inhibitor of MEK activation, U0126, we examined whether activation of ERK phosphorylation by Vinexin was dependent on MEK activity. The increase of ERK phosphorylation was prevented by pretreatment with the reagent, suggesting that the site of action of Vinexin is localized upstream of MEK in the MAPK cascade (Fig. 4B).

View larger version (78K): [in this window] [in a new window]   Figure 4  Implication of Vinexin into regulation of the MEK-ERK cascade. (A) Regulation of MEK and ERK phosphorylation by Vinexin . C3H10T1/2 cells were transiently transfected with empty vector (–) or pCMX-Vinexin (+). The cells were cultured for the indicated periods of times after transfection. Ten µg of total cell lysates were analyzed by Western blotting using antibodies that recognize the phosphorylated (p-ERK, p-MEK, and p-c-Raf) and total (ERK, MEK, and c-Raf) MAPK. (B) Vinexin induced MAPK activation inhibited by MEK inhibitor, U0126. C3H10T1/2 cells were transiently transfected with empty vector (–) or pCMX-Vinexin (+). The cells were incubated without (DMSO) or with 10 µM U0126 for 30 min before addition of 10% serum. Twenty-four hours after transfection, 10 µg of total cell lysates were analyzed by Western blotting using antibodies that recognize p-ERK and ERK.

Interaction between Vinexin and components of the MAPK cascade

As previously described (Mitsushima et al. 2004), Vinexin ß is phosphorylated by ERK through direct interaction. Since Vinexin was also implicated in regulation by the MAPK cascade, we examined the interaction between Vinexin and components of the MAPK cascade. C3H10T1/2 cells were transfected with flag-tagged Vinexin , and the cell lysates were immunoprecipitated using anti-flag antibody, followed by Western blotting using anti-ERK, MEK, and c-Raf antibodies. As shown in Fig. 5A, Vinexin interacted with endogenous ERK and c-Raf but not MEK. The interaction with c-Raf appeared to be stronger than that with ERK. To identify the binding region of Vinexin , deletion constructs carrying the N-terminus [1–368] or C-terminus [352–680] of Vinexin were used for immunoprecipitation assays. Interaction with c-Raf was observed with the N-terminal Vinexin but not with the C-terminal (Fig. 5B).

View larger version (37K): [in this window] [in a new window]   Figure 5  Interaction of Vinexin with ERK and c-Raf but not MEK. (A) Interaction between Vinexin and components of the MAPK cascade. C3H10T1/2 cells were transiently transfected with empty (–) or flag-tagged Vinexin (+). Twenty-four hours after transfection, the cell lysates were used for immunoprecipitation using anti-flag antibody, and the resulting immunoprecipitates were subjected to Western blotting using anti-MAPK (ERK, MEK, and c-Raf) antibodies. Interaction of Vinexin with ERK and c-Raf was detected, whereas interaction with MEK was not detected. (B) Interaction with c-Raf through the N-terminus of Vinexin . A schematic diagram of deletion constructs is presented. C3H10T1/2 cells were transiently transfected with empty (–) or flag-tagged full-length (+), N-terminus [1–368] (N), and C-terminus [352–680] (C) Vinexin . Twenty-four hours after transfection, the cell lysates were subjected to immunoprecipitation using anti-flag antibody, and detected by Western blotting using anti-c-Raf and anti-flag antibodies.

Vinexin regulates the Sox9 gene by the MAPK cascade in vitro

It was previously reported that Fgfs activate Sox9 gene expression in C3H10T1/2 and primary chondrocytes (Murakami et al. 2000). Since the up-regulation of Sox9 by Fgfs was inhibited by U0126, this strongly implicated the MAPK cascade in regulation of Sox9 gene expression in chondrocytes. Thus, we hypothesized that Vinexin is involved in regulation of Sox9 gene expression through interaction with components of the MAPK cascade. Based on this hypothesis, we investigated whether Sox9 gene expression is induced by Vinexin in C3H10T1/2. As shown in Fig. 6A, Vinexin increased Sox9 mRNA levels from 12 h after transfection. We then tested whether activation of the MEK-ERK pathway leads to the increase in Sox9 mRNA levels. When a constitutively active form of MEK1 was transfected (Fukuda et al. 1997), Sox9 expression was up-regulated (Fig. 6B). Conversely, when the cells were treated with U0126, the reagent markedly inhibited up-regulation of Sox9 gene expression by Vinexin . Phosphorylation of ERK was also inhibited (Fig. 6C). These observations strongly suggested that Vinexin regulates Sox9 gene expression by activating the MEK-ERK cascade.

View larger version (71K): [in this window] [in a new window]   Figure 6  Up-regulation of Sox9 gene by Vinexin through the MAPK cascade. (A) Sox9 gene expression enhanced by the presence of Vinexin . C3H10T1/2 cells were transiently transfected with empty vector (–) or pCMX-Vinexin (+). The cells were cultured for the indicated periods of time after transfection. Ten µg of total RNA were used for Northern blot analyses to measure Sox9 mRNA levels. Gapdh was used as an internal control. (B) Up-regulation of Sox9 gene by constitutively active MEK1 mutant (MEK). C3H10T1/2 cells were transiently transfected with empty vector (–) or MEK (+). Twenty-four hours after transfection, 10 µg of total RNA were used for Northern blot analyses as described above. (C) Vinexin induced Sox9 expression inhibited by the MEK inhibitor, U0126. C3H10T1/2 cells were transiently transfected with empty vector (–) or pCMX-Vinexin (+). The cells were incubated without (DMSO) or with 10 µM U0126 for 30 min before addition of 10% serum. Twenty-four hours after transfection, 10 µg of total RNA were used for Northern blot analyses as described above. Ten µg total cell lysates were analyzed by Western blotting using the antibodies for p-ERK and ERK.

Generation of Vinexin -disrupted mice

To investigate the functions of Vinexin in vivo, we generated Vinexin gene-disrupted mice. Clones that cover approximately 30 kb of the Vinexin gene were isolated from a murine 129/SvEv genomic library. Structural analyses of these clones revealed that the Vinexin gene is encoded by 23 exons (data not shown). In order to exclude the possibility that the three forms of Vinexin are transcribed from distinct but quite similar genes, we investigated whether the Vinexin locus was single or not. Fluorescent in situ hybridization clearly gave a single Vinexin signal on Chromosome 14C (data not shown), which is consistent with the location indicated in public DNA databases. Moreover, the first exons specific for Vinexin , ß, and were indeed localized at the locus. Based on these observations, we generated a Vinexin -specific targeting vector by deleting the form-specific first exon together with the upstream region (Fig. 7A). The targeting vector was electroporated into ES cells and homologous recombinants were obtained (Fig. 7B). After they were transmitted into the germ-line, homozygous mutant mice were produced. These mice were maintained in a mixed 129/C57BL6 background. Representative genotypings by Southern blot and PCR analyses of the offspring are shown in Fig. 7C,D.

View larger version (57K): [in this window] [in a new window]   Figure 7  Targeted disruption of Vinexin (A) A diagram of the Vinexin genomic locus (wild-type), targeting vector, and targeted allele. Black boxes and open boxes indicate common and or specific exons, respectively. Probes for Southern blot (5' probe and 3' probe) and PCR primers (wt, rv, and neo) are shown. Insertion of the neo gene introduced HindIII (H) and EcoRI (E) sites that were used in conjunction with the indicated external 5'- and 3' probe to distinguish the wild-type (5.6 k, 7.3 k) and mutant (4.5 k, 5.1 k) by Southern blot analyses. (B, C) Southern blot analyses of DNAs from ES clones (B) and tails from offspring (C). The 5' probe detected the 5.6 kb and 4.5 kb HindIII fragments in the wild-type and mutant allele, respectively. The 3' probe detected the 7.3 kb and 5.1 kb EcoRI fragments in the wild-type and mutant allele, respectively. (D) PCR genotyping of offspring. PCR with primers described above amplified 328-bp and 252-bp fragments from the wild-type and mutant allele, respectively. (E) Western blot analyses of extracts from wild-type and mutant fetal gonads at E12.5. Total tissue lysates were prepared from the wild-type (+/+) and mutant (–/–) fetal gonads of and Ten µg of total tissue lysates were analyzed by Western blotting using anti-Vinexin antibody. (F–I) Whole-mount in situ hybridization analyses of Vinexin gene expression. Vinexin +/– (F, H) and Vinexin –/– (G, I) XY fetal gonads at E12.5 were subjected to whole-mount in situ hybridization probed with / Vinexin probe (F, G) and /ß/ Vinexin probe (H, I). Arrowheads indicate that the expression of Vinexin was barely detected at the inside of the Vinexin –/– XY testicular cord.

Vinexin

Vinexin

Vinexin -disrupted mice were obtained at the predicted Mendelian ratios and were fertile in both sexes (data not shown). Moreover, the XX and XY fetal gonads of the disrupted mice were morphologically normal (data not shown). Since Vinexin ß was still expressed in the Vinexin ^–/– gonads, it seemed possible that Vinexin ß might overcome the defects that might otherwise occur in Vinexin ^–/– gonads. Thus, we investigated which cell types express Vinexin ß in fetal gonads by whole-mount in situ hybridization using the /ß/ probe (shown in Fig. 1). When the Vinexin ^+/– XY gonad at E12.5 was examined, the staining pattern was distinct from that with the / probe (compare Fig. 7F,H). Indeed, since the expression was distributed in the whole gonad, the expression observed at the testicular cord with the / probe became obscure with the /ß/ probe. Moreover, expression in the mesonephros was detected although it was weaker than that in the gonad (Fig. 7H). This differential expression profile seemed to be due to the expression of Vinexin ß in cells other than Sertoli cells. This is clearly indicated by in situ hybridization with the Vinexin ^–/– XY gonad. Since the Vinexin ^–/– XY gonad lacks expression of both Vinexin and as revealed by Western blot analysis, any signals detected in the gonad with the /ß/ probe should correspond to the expression of Vinexin ß. As shown in Fig. 7I, the signal was detected outside of the testicular cord. We further examined localization of Vinexin ß in the Vinexin ^–/– XY gonads at E12.5 immunohistochemically. No signals could be detected in the Vinexin ^–/– XY Sertoli cells (data not shown). Taken together, since Vinexin ß is not expressed in Sertoli cells, it is unlikely that the residual Vinexin ß overcomes the defects of the Vinexin ^–/– fetal gonads.

ERK activation was decreased in the Vinexin ^–/– XY gonads

In order to examine whether MAPK activation occurs in mouse fetal gonads, Western blot analyses with mouse fetal gonads were performed using antibodies to phosphorylated forms of MAPK (ERK, JNK, and p38). Interestingly, ERK was phosphorylated, whereas JNK and p38 were not phosphorylated in the fetal gonads at E12.5 (Fig. 8A). We then examined ERK phosphorylation in the Vinexin ^–/– fetal gonads. As shown in Fig. 8B, ERK phosphorylation was decreased in the Vinexin ^–/– XY gonads when compared to the Vinexin ^+/– XY gonads at E12.5. In contrast, there was no significant difference in ERK phosphorylation between the Vinexin ^+/– and Vinexin ^–/– XX gonads (Fig. 8B). These sexually distinct effects on ERK phosphorylation strongly suggested that the phosphorylation mediated by Vinexin occurs in the male but not in the female gonad, even though Vinexin is expressed in the gonads of both sexes (Fig. 8B). To examine the effects of decreased phosphorylation of ERK on the phosphorylation states of MAPK, JNK and p38, Western blot analyses were performed. No change in phosphorylation state was induced in the Vinexin ^–/– gonads. Given that the lack of Vinexin correlated with decreased ERK phosphorylation, it was expected that the phosphorylation state would not be affected in tissues where Vinexin was not expressed. Since Vinexin was not expressed in fetal limb buds, the state of ERK phosphorylation was investigated in this tissue. As expected, ERK phosphorylation was not affected in the Vinexin ^–/– fetal limb buds (Fig. 8C), strongly suggesting that the decreased ERK phosphorylation in the Vinexin ^–/– XY gonad is tightly coupled with the loss of Vinexin .

View larger version (32K): [in this window] [in a new window]   Figure 8  Decreased ERK activation in Vinexin –/– XY gonads. (A) Western blot analyses of extracts from mouse fetal gonads. Total cell lysates were prepared from 293 cells treated with UV (+) or without UV (–), while total tissue lysates were prepared from the wild-type male () and female () gonads at E12.5. Five µg of total cell lysates or 20 µg of total tissue lysates were analyzed by Western blotting using antibodies that recognize the phosphorylated MAPKs (p-ERK, p-JNK, and p-p38) and ERK. (B) Western blot analyses of phosphorylated MAPKs in Vinexin +/– and Vinexin –/– fetal gonads at E12.5. 5 µg () or 2.5 µg () total tissue lysates were analyzed. (C) Western blot analyses of phosphorylated ERK in Vinexin +/– and Vinexin –/– fetal limb buds at E12.5. Ten µg of total tissue lysates were analyzed by antibodies for p-ERK, ERK, and Vinexin.

Sox9 expression was down-regulated in Vinexin ^–/– XY gonads

Since ERK phosphorylation was shown to lead to up-regulation of Sox9 expression (Murakami et al. 2000), Sox9 expression was expected to be affected by Vinexin gene disruption. Thus, Sox9 expression in the gonads was examined by whole-mount in situ hybridization. Although there was no difference in Sox9 expression between the Vinexin ^+/+ and Vinexin ^+/– XY gonads (6/6; data not shown), Sox9 expression was affected in the Vinexin ^–/– XY gonads at E12.5 (Fig. 9A–D). However, the decreased level of Sox9 varied among the Vinexin ^–/– XY gonads. Indeed, among 11 individual gonads, Sox9 expression was either similar to the wild-type (n = 2: Fig. 9B), decreased modestly (n = 7: Fig. 9C), or decreased severely (n = 2: Fig. 9D). However, Sox9 expression did not completely disappear from any of the Vinexin ^–/– XY gonads. Interestingly, the decreased Sox9 expression was observed with higher frequency at E11.5 (4/6; data not shown), and with lower frequency at E13.5 (1/6) and E14.5 (1/6) (data not shown). Sox9 expression eventually recovered during the late fetal period. The expression of other gonadal marker genes such as Ad4BP/Sf-1, 3ß-HSD, Wt-1, Gata-4 and Mis was investigated at E12.5. However, the expression of these genes was not affected (Fig. 9E,F for Ad4BP/Sf-1; Fig. 9G,H for 3ß-HSD; data for Wt-1, Gata-4, and Mis are not shown). As a control for altered Sox9 expression in the gonad, Sox9 expression was investigated in the limb buds where Vinexin is not expressed. As expected, no difference in Sox9 gene expression was observed between the Vinexin ^+/– and Vinexin ^–/– hind limb buds (Fig. 9I,J).

View larger version (50K): [in this window] [in a new window]   Figure 9  Down-regulation of Sox9 gene expression in Vinexin –/– XY gonads. (A–H) Whole-mount in situ hybridization analyses of Sox9, Ad4BP/Sf-1 and 3ß-HSD gene in Vinexin +/– and Vinexin –/– XY fetal gonads at E12.5. Sox9 expression in the Vinexin +/– (A) and Vinexin –/– (B–D) XY fetal gonads are shown. Expression of Ad4BP/Sf-1 (E, F) and 3ß-HSD (G, H) was examined with Vinexin +/– (E, G) and Vinexin –/– (F, H) XY fetal gonads. (I, J) Sox9 expression in hind limb buds. The expression of Sox9 were examined with the Vinexin +/– (I) and Vinexin –/– (J) hind limb buds. (K–M) Northern blot analyses to measure Sox9 and Ad4BP/Sf-1 mRNA levels in Vinexin +/– and Vinexin –/– XY fetal gonads at E12.5. Total RNA was prepared from the male () or female () gonads and mesonephroi at E12.5. Twenty µg of total RNA were used for Northern blot analyses. Gapdh was used as an internal control. The expression levels are given as the ratios of the Sox9 (L) and Ad4BP/Sf-1 (M) gene to the Gapdh gene. These data are mean ± SEM of the result of three experiments. The asterisk indicates that the difference between Vinexin +/– and Vinexin –/– was statistically significant (P < 0.05) as determined by Student's t-test.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

 
Northern blot analyses previously showed that Vinexin is expressed preferentially in several tissues, while Vinexin ß is expressed ubiquitously (Kioka et al. 1999; Tujague et al. 2004). In the present study, we identified a novel isoform of Vinexin, Vinexin . Although the two isoforms, Vinexin and , are hardly distinguishable by Northern blot analysis because of their similar mRNA lengths, our Western blot analyses distinguished the two molecules. Thus, we successfully confirmed that Vinexin but not Vinexin is expressed in the fetal gonads of both sexes. Likewise, expression of Vinexin in the fetal gonads was confirmed by whole-mount in situ hybridization. Based on these observations, we hypothesized that Vinexin plays crucial roles in the process of gonadal development.

Vinexin acts as a scaffold protein through the MAPK cascade

The MAPK cascade plays a critical role in transduction of many growth and differentiation signals. Upon stimulation, all of the components of the MAPK cascade are neatly organized by scaffolding proteins that ensure the efficiency and fidelity of signal transduction by joining the pathway components (Kolch 2000; Morrison & Davis 2003). In this study, we found that Vinexin interacts with ERK and c-Raf, but not with MEK. This observation is consistent with a recent report that Vinexin ß interacts with active ERK but not with MEK (Mitsushima et al. 2004). In addition, the present study clearly showed that Vinexin activated the phosphorylation of both MEK and ERK. Together with its interaction properties, these observations strongly suggested that Vinexin is involved in regulation of the MAPK cascade as a scaffold protein (Fig. 10).

View larger version (30K): [in this window] [in a new window]   Figure 10  Model for Vinexin functions.Vinexin binds to Sos through the third SH3 domain. Upon stimulation by growth factors, phosphorylation of Sos is induced, and thereafter Vinexin is released from the complex (Akamatsu et al. 1999). Vinexin in cytoplasm was indicated to act as a scaffold protein to activate MEK and ERK through interaction with c-Raf and ERK. As a modulator of MAPK signaling, Vinexin is involved in Sox9 gene regulation in a developing male fetal gonad.

Vinexin regulates the Sox9 gene through the MAPK cascade in fetal male gonads

It was reported previously that up-regulation of the Sox9 gene is mediated by the MAPK cascade during chondrogenesis (Murakami et al. 2000, 2004). In this study, we showed that Vinexin increased the Sox9 mRNA level in correlation with ERK phosphorylation in cultured cells. These studies with cultured cells were strongly supported by the current study with the Vinexin gene-disrupted mice. Similar to the culture cell studies, Sox9 gene expression was decreased simultaneously with ERK phosphorylation in the Vinexin ^–/– XY gonads. Interestingly, however, Vinexin -dependent ERK phosphorylation is male specific, regardless of the Vinexin expression in both sexes. Accordingly, the Vinexin -dependent sexually dimorphic phosphorylation of ERK might be elucidated by the presence of sex-dependent signaling (Fig. 10). In this context, it is noteworthy that nuclear localization of Fgf receptor 2 (Fgfr2) is induced by Fgf9 signal in the male gonad but not female (Schmahl et al. 2004). Ultimately, the male specific nuclear localization of the receptor mediates up-regulation of Sox9. Although the nuclear translocation of Fgfr2 may not correlate with that of the MAPK cascade, the Fgf9 signal is transduced in a male-specific manner. Thus, it is understandable that the effect of Vinexin gene disruption on MAPK signaling is manifested in a male-specific manner.

Phenotypic variation and moderate defects displayed by the Vinexin -disrupted mice

In order to reveal the function of Vinexin during gonadal development, we generated a Vinexin -specific gene-disrupted mouse. Unexpectedly, the homozygous mutant mice of both sexes were fertile with morphologically normal gonads. The mice showed decreased Sox9 expression, and the decreased levels varied among individuals. Indeed, even in the most affected individuals, we failed to detect either complete disappearance of Sox9 expression or developmental defects in the XY gonad. Since other MAPKs, JNK and p38, were not activated ectopically in the Vinexin ^–/– fetal XY gonad, it is unlikely that the Vinexin -dependent function of ERK was displaced by the other MAPKs. Together, the moderate phenotype displayed by the Vinexin ^–/– gonad suggested that MAPK activation is not critical for testis formation. Consistent with our observations, it was recently reported that a MAP kinase inhibitor, PD98059, did not inhibit testis cord formation in gonadal organ culture (Uzumcu et al. 2002). However, further studies should be needed to draw a definite conclusion concerning contribution of MAPK cascade to the gonadal sex differentiation.

Alternatively, the relatively moderate phenotype displayed by the Vinexin -disrupted mice might be due to the following two possibilities. Firstly, Vinexin and Vinexin ß might function redundantly. Indeed, all isoforms share three tandem repeats of SH3 domains at their C-termini, while the and isoforms share a sorbin domain in their N-terminal halves. Moreover Vinexin ß was still evident in the gonad of Vinexin -disrupted mice. However, given that Vinexin contains its specific N-terminal half and the expression in the fetal testis does not appear to overlap with that of Vinexin ß, it is unlikely that the function of Vinexin ß is completely redundant with that of Vinexin . Secondly, the functions of two closely related proteins, Arg-binding protein 2 (ArgBP2) and c-Cbl-associated protein (CAP/ponsin/SH3P12), should be noted (Wang et al. 1997; Ribon et al. 1998; Mandai et al. 1999; Kioka et al. 2002). Structurally, besides the presence of three SH3 domains in common at their C-termini, these proteins share a sorbin domain with Vinexin in their N-terminal halves. Moreover, expression of ArgBP2 and CAP/ponsin/SH3P12 was detected in the fetal gonads (E12.5) of both sexes by RT-PCR and whole-mount in situ hybridization, although these signals were weak (data not shown). Thus, it might be possible that their features functionally and spatially overlapped with Vinexin failed to display drastic gonadal defects in the Vinexin -disrupted mice.

Recently, a conditional knockout study indicated that ablation of Sox9 affected XY testis cord formation and Sertoli cell differentiation, and that this phenotype was reinforced by concomitant deletion of Sox8 (Chaboissier et al. 2004). In the Vinexin ^–/– XY fetal gonad, Sox9 expression was obviously decreased. However, the decreased Sox9 expression soon recovered, and thereafter the expression was maintained at the same level as the wild-type. This Sox9 down-regulation at the restricted period was likely responsible for the moderate gonadal phenotype.

In conclusion, we identified a novel isoform of Vinexin, Vinexin , and characterized it as a scaffold protein for the MAPK cascade. Through regulating the activation of MEK–ERK via interaction with c-Raf, Vinexin is implicated in Sox9 gene expression. The gene disruption study revealed that Vinexin regulates the process of testis differentiation through modulating the MAPK cascade.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Materials and antibodies

The Vinexin nucleotide sequence data is available from GENBANK/EMBL/DDBJ under accession No. AB190911. A MEK inhibitor, U0126 was purchased from Cell Signaling Technology (Beverly, MA, USA). The following antibodies were used for Western blot analyses: Flag M2 (Sigma Chemical Co., St. Louis, MO, USA), c-Raf (BD Transduction Laboratories, San Jose, CA, USA), phospho-c-Raf (Cell Signaling), MEK1/2 (BD Transduction), phospho-MEK1/2 (Cell Signaling), ERK1/2 (E-4, Santa Cruz Biotechnology, Santa Cruz, CA), phospho-ERK1/2 (Cell Signaling), phospho-JNK (Cell Signaling), and phospho-p38 (Cell Signaling).

Whole-mount in situ hybridization

SacII/PstI (181–791 bp) and PstI/XhoI (1556–2126 bp) fragments of Vinexin cDNA were cloned into pBluescriptSKII (+) (Stratagene, La Jolla, CA, USA). Since the former and latter contain regions common to Vinexin and , and common to Vinexin , ß and , they were designated / probe and /ß/ probe, respectively (shown in Fig. 1). Digoxigenin-labeled anti-sense and sense riboprobes from Vinexin, Ad4BP/Sf-1, 3ß-HSD, Gata-4, Wt-1, Mis, and Sox9 (kindly provided by Dr Peter Koopman) were generated using T7 or T3 RNA polymerase (Promega Corp., Madison, WI, USA). Whole-mount in situ hybridization was performed as previously described (Kawabe et al. 1999).

Reverse transcription-polymerase chain reaction cDNAs were synthesized with RNAs prepared from the somatic and germ cells of E12.5 fetal gonads as previously described (Ohbo et al. 2003; Mitsunaga et al. 2004). RT-PCR was performed using the following primers; Vinexin-fw (5'-GCA GCT GGA CTG GAC CTT GGA-3') and Vinexin-rv (5'-CAG GAC CTC AAT GGG CTG TGC-3'), Ad4BP-fw (5'-GTA CGG CAA GGA AGA CAG CAT-3') and Ad4BP-rv (5'-CCA CCA GGC ACA ATA GCA ACT-3'), Oct3/4-fw (5'-GCG GAG GGA TGG CAT ACT GTG-3') and Oct3/4-rv (5'-AAG AGA ACG CCC AGG GTG AGC-3'), GAPDH-fw (5'-GGC ATG GCC TTC CGT GTT CCT-3') and GAPDH-rv (5'-TCC TTG CTG GGG TGG GTG GTC-3'). PCR reactions were performed using the following parameters: 94 °C for 30 s, 60 °C 30 s and 72 °C 30 s; 35 cycles.

Immunohistochemistry

A prokaryotic expression vector for Vinexin was constructed by insertion of the full-length Vinexin cDNA into pET-11a (Stratagene). Preparation of recombinant Vinexin and immunization of a rabbit were previously described (Morohashi et al. 1993). Anti-Vinexin antibody was purified using an antigen-conjugated NHS-activated Sepharose4 Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's protocol. Immunohistochemistry was performed as previously described (Nomura et al. 1998). Briefly, mouse fetal gonads were fixed with 4% paraformaldehyde (PFA) in PBS at 4 °C. Five-µm-thick sections were prepared and mounted for immunostaining using the purified anti-Vinexin antibody (1/1000).

Cell culture and transfection

Mammalian expression vectors for Vinexin , ß, and were constructed by insertion of the full-length cDNAs into pRSV, pCMX, and p3xFlag-CMX10 (Sigma) vectors. Murine embryonic fibroblast line C3H10T1/2 (obtained from Health Science Research Resources Bank, Osaka, Japan) was grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin-glutamine at 37 °C in 5% CO₂. The cells were seeded at 9 x 10⁵ per 100-mm dish one day before transfection. Transfection was performed using lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

Northern and Western blotting

Northern blot analyses were performed as previously described (Kanai & Koopman 1999; Murakami et al. 2000), as were Western blot analyses (Morohashi et al. 1994). Briefly, cells were lyzed with a cell-lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% SDS, 50 mM NaF, 40 mMß-glycerophosphate, 2 mM Na₃VO₄, 10 mM pyrophosphate Na, and 1 x Complete ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA). Mouse adult tissues and fetal gonads were lyzed with a tissue-lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 50 mM MgCl₂, 0.1% Tween 20, 50 mM NaF, 40 mMß-glycerophosphate, 2 mM Na₃VO₄, 10 mM pyrophosphate Na, and 1 x Complete EDTA-free protease inhibitor cocktail. The lysates were loaded, transferred, and subjected to Western blotting with specific antibodies.

Immunoprecipitation

After C3H10T1/2 cells were transfected with expression plasmids for Vinexin and its truncated derivatives in p3xFlag-CMX10, they were cultured for 24 h. The cells were lyzed with IP buffer (1% Triton X, 50 mM NaF, 40 mMß-glycerophosphate, and 1 mM Na₃VO₄ in PBS) containing 1 x Complete EDTA-free protease inhibitor cocktail, and thus subjected to immunoprecipitation with anti-Flag antibody at 4 °C. The immunoprecipitates were washed with ice-cold 1% Triton X/PBS. The co-precipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blotting with specific antibodies.

Generation of Vinexin -disrupted mice

Bacteriophage clones containing the Vinexin gene were obtained by screening a 129/SvEv genomic library with the Vinexin cDNA probe. The 5' 2.3 kb fragment and 3' 3.7 kb fragment of the Vinexin genomic DNA (Fig. 7A, bold line) were ligated into a PGKneobpA-loxP positive selection marker cassette (Arango et al. 1999) to delete the Vinexin -specific exon. An MC1DTpA negative selection marker cassette (Yagi et al. 1993) was added on to the 5' homologous arm to enrich homologous recombinants. The targeting vector was linearized by NotI digestion at the outside of the homologous region. Electroporation into AB1 ES cells (McMahon & Bradley 1990) was performed as previously described (Shimono et al. 2003). G418-resistant clones obtained were initially screened via HindIII digestion and hybridization with a unique external 5' probe. The targeted clones were subsequently examined by EcoRI digestion and hybridization with a unique 3' external probe to verify homologous recombination. Seven of 392 were identified as clones correctly targeted, of which two were found to be capable of contributing to the germ-line in mouse chimeras. These mice were maintained in a mixed 129/C57BL6 background. PCR genotyping was performed using the following primers: wt (5'-CTC CTG CCG CAT TCA TCT CAG-3'), rv (5'-CAC GGA GGG AGG GAC TCT ACG-3'), and neo (5'-ATG GCT TCT GAG GCG GAA AGA-3').

	Acknowledgements

 
We thank Drs A. Bradly for AB1 ES and SNL 76/7 STO cell lines, R.R. Behringer for the pPGKneobpA-loxP plasmid, T. Yagi for the pMCDTpA plasmid, Y. Gotoh for constitutively active MEK1 plasmid, and P. Koopman and Y. Kanai for the Sox9 probe. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

	Footnotes

 
Communicated by: Yo-ichi Nabeshima

^* Correspondence: E-mail: moro{at}nibb.ac.jp

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