Michihiko Ito

A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis.

In the XX/XY sex-determining system, the Y-linked SRY genes of most mammals and the DMY/Dmrt1bY genes of the teleost fish medaka have been characterized as sex-determining genes that trigger formation of the testis. However, the molecular mechanism of the ZZ/ZW-type system in vertebrates, including the clawed frog Xenopus laevis, is unknown. Here, we isolated an X. laevis female genome-specific DM-domain gene, DM-W, and obtained molecular evidence of a W-chromosome in this species. The DNA-binding domain of DM-W showed a strikingly high identity (89%) with that of DMRT1, but it had no significant sequence similarity with the transactivation domain of DMRT1. In nonmammalian vertebrates, DMRT1 expression is connected to testis formation. We found DMRT1 or DM-W to be expressed exclusively in the primordial gonads of both ZZ and ZW or ZW tadpoles, respectively. Although DMRT1 showed continued expression after sex determination, DM-W was expressed transiently during sex determination. Interestingly, DM-W mRNA was more abundant than DMRT1 mRNA in the primordial gonads of ZW tadpoles early in sex determination. To assess the role of DM-W, we produced transgenic tadpoles carrying a DM-W expression vector driven by ≈3 kb of the 5′-flanking sequence of DM-W or by the cytomegalovirus promoter. Importantly, some developing gonads of ZZ transgenic tadpoles showed ovarian cavities and primary oocytes with both drivers, suggesting that DM-W is crucial for primary ovary formation. Taken together, these results suggest that DM-W is a likely sex (ovary)-determining gene in X. laevis.

JSAP1, a novel Jun N-terminal protein kinase (JNK)-binding protein that functions as a scaffold factor in the JNK signaling pathway

ABSTRACT The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38α), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.

Genome evolution in the allotetraploid frog Xenopus laevis

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of ‘fossil’ transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17–18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Phosphorylation-dependent Scaffolding Role of JSAP1/JIP3 in the ASK1-JNK Signaling Pathway A NEW MODE OF REGULATION OF THE MAP KINASE CASCADE

JSAP1 (also termed JIP3) is a scaffold protein that interacts with specific components of the JNK signaling pathway. Apoptosis signal-regulating kinase (ASK) 1 is a MAP kinase kinase kinase that activates the JNK and p38 mitogen-activated protein (MAP) kinase cascades in response to environmental stresses such as reactive oxygen species. Here we show that JSAP1 bound ASK1 and enhanced ASK1- and H2O2-induced JNK activity. ASK1 phosphorylated JSAP1 in vitro and in vivo, and the phosphorylation facilitated interactions of JSAP1 with SEK1/MKK4, MKK7 and JNK3. Furthermore, ASK1-dependent phosphorylation was required for JSAP1 to recruit and thereby activate JNK in response to H2O2. We thus conclude that JSAP1 functions not only as a simple scaffold, but it dynamically participates in signal transduction by forming a phosphorylation-dependent signaling complex in the ASK1-JNK signaling module.

The Xenopus Sox3 gene expressed in oocytes of early stages

We have isolated an SRY-type HMG box (Sox) cDNA, XSox3, from a Xenopus immature ovary cDNA library. The XSox3 cDNA contains an open reading frame (ORF) of 309 amino-acid residues, showing 62.7 and 77.0% homology with human and chicken Sox3 proteins, respectively. We also showed that the XSox3 gene is composed of a single exon by sequence analysis of the genomic clone and the determination of the transcription start site of the XSox3. The XSox3 mRNA was detected only in ovary and was at a higher level in immature ovary than in mature ovary. During oocyte maturation, the XSox3 mRNA was most abundant in stage I oocytes, and the XSox3 protein was detected in stage I and II oocytes. Recombinant XSox3 protein produced in Escherichia coli bound specifically to sequences containing the binding motif for the HMG box of SRY or SOX proteins, AACAAT or AACAAAG, demonstrating its sequence-specific DNA binding property. Taken together, these results indicate that the XSox3 protein may participate in early oogenesis of Xenopus as a transcription factor.

A gene that is related to SRY and is expressed in the testes encodes a leucine zipper-containing protein.

SRY-related cDNA encoding a protein with a high-mobility-group (HMG) box and a leucine zipper motif, which was designated SOX-LZ, was isolated from a rainbow trout testis cDNA library. Comparison of this cDNA with the mouse homologous cDNA isolated from a testis cDNA library exhibits an overall amino acid sequence identity of 77%, which is in striking contrast to the abrupt loss of amino acid sequence homology outside the HMG box found among mammalian SRY genes. In both rainbow trout and mice, Northern (RNA) blot analyses have revealed the presence of a testis-specific 3-kb-long SOX-LZ mRNA, and this transcript appeared coincidentally with the protamine mRNA, suggesting its expression in the germ line. A recombinant HMG box region protein encoded by SOX-LZ could bind strongly with an oligonucleotide containing an AACAAT sequence, which is also recognized by mouse Sry and Sox-5. Upon cotransfection into CHO cells, SOX-LZ transactivated transcription through its binding motif when the region including the leucine zipper motif was deleted [SOX-LZ (D105-356)]; however, the intact SOX-LZ failed to transactivate. The intact SOX-LZ could form homodimers through the leucine zipper, which resulted in inhibition of DNA binding by the HMG box, while SOX-LZ (D105-356), which was incapable of dimerization, showed specific binding with the AACAAT sequence. Thus, the repressed transactivation of the intact SOX-LZ in CHO cells was primarily attributable to the low level of DNA binding of SOX-LZ homodimers.

Rainbow trout SOX9: cDNA cloning, gene structure and expression.

We have isolated and characterized rainbow trout SOX9 cDNA and genomic clones. The cDNA encodes a protein of 488 amino acids (aa) with an HMG box and has 70% aa sequence identity with human SOX9. The rainbow trout and human SOX9 genes show a similar gene structure, and the two introns in the coding region are located at conserved positions. In rainbow trout, the SOX9 mRNA was prominent in testis and brain.

A ZZ/ZW-type sex determination in Xenopus laevis

Genetic sex‐determining systems in vertebrates include two basic types of heterogamety, which are represented by the XX/XY and ZZ/ZW types. Both types occur among amphibian species. Little is known, however, about the molecular mechanisms underlying amphibian sex determination. Recently, a W‐linked gene, DM‐W, was isolated as a paralog of DMRT1 in the African clawed frog Xenopus laevis, which has a female heterogametic ZZ/ZW‐type sex‐determining system. The DNA‐binding domain of DM‐W shows high sequence identity with that of DMRT1, but DM‐W does not contain a domain with homology to DMRT1’s transactivation domain. Importantly, phenotypic analysis of transgenic individuals bearing a DM‐W‐expression or ‐knockdown vector strongly suggested that DM‐W acts as a female sex‐determining gene in this species. In this minireview, we briefly describe the sex‐determining systems in amphibians, discuss recent findings from the discovery of the DM‐W gene in terms of its molecular evolution and its function in sex determination and ovary formation, and introduce a new model for the ZZ/ZW‐type sex determination elicited by DM‐W and DMRT1 in X. laevis. Finally, we discuss sex‐determining systems and germ‐cell development during vertebrate evolution, especially in view of a conserved role of DMRT1 in gonadal masculinization.

Diversity in the origins of sex chromosomes in anurans inferred from comparative mapping of sexual differentiation genes for three species of the Raninae and Xenopodinae

Amphibians employ genetic sex determination systems with male and female heterogamety. The ancestral state of sex determination in amphibians has been suggested to be female heterogamety; however, the origins of the sex chromosomes and the sex-determining genes are still unknown. In Xenopus laevis, chromosome 3 with a candidate for the sex- (ovary-) determining gene (DM-W) was recently identified as the W sex chromosome. This study conducted comparative genomic hybridization for X. laevis and Xenopus tropicalis and FISH mapping of eight sexual differentiation genes for X. laevis, X. tropicalis, and Rana rugosa. Three sex-linked genes of R. rugosa—AR,SF-1/Ad4BP, and Sox3—are all localized to chromosome 10 of X. tropicalis, whereas AR and SF-1/Ad4BP are mapped to chromosome 14 and Sox3 to chromosome 11 in X. laevis. These results suggest that the W sex chromosome was independently acquired in the lineage of X. laevis, and the origins of the ZW sex chromosomes are different between X. laevis and R. rugosa. Cyp17, Cyp19, Dmrt1, Sox9, and WT1 were localized to autosomes in X. laevis and R. rugosa, suggesting that these five genes probably are not candidates for the sex-determining genes in the two anuran species.

cDNA cloning of a new member of the FTZ-F1 subfamily from a rainbow trout

We describe here cDNA cloning of an orphan nuclear receptor family member, tFZR1, which has a FTZ-F1 box. The amino acid sequences of the zinc finger domain and the FTZ-F1 box has 92.8% and 100% identity, respectively, with those of zebrafish FTZ-F1. On the other hand, the overall homology between tFZR1 and zebrafish FTZ-F1 is low (33.0%). The results indicate that tFZR1 is a new member of fushitarazu factor 1 (FTZ-F1) subfamily.