DIVISION OF CELL DIFFERENTIATION

¹⁾CREST, JST Postdoctoral Fellow
²⁾Graduate School of Kyushu University
³⁾Graduate School of Tohoku University
⁴⁾Graduate School of Ehime University
⁵⁾Graduate University for Advanced Studies
⁶⁾Graduate School of Tokyo University
⁷⁾CREST, JST Technical Assitant
⁸⁾CREST, JST Secretary

Cell and tissue differentiation proceeds systematically based on orchestrated expressions of a battery of genes. The expressions commence successively along with the passage of time, and consequently a single fertilized egg develops into a variety of tissues and organs, which consist of specialized cells in terms of their structures and functions. Accordingly, it is reasonable to assume that investigation of the mechanisms underlying cell and tissue-specific gene expressions at a molecular level is essential for understanding molecular frameworks for genetic cascades to support cell and tissue differentiation. Based on the concept above, our division of Cell Differentiation has focussed on sex differentiation of the gonads and differentiation of the steroidogneic tissues form the aspect of functions of tissue-specific transcription factors and growth factors.

A number of transcription factors are involved in the process of gonadal differentiation. Some of these factors, such as SRY, WT-1, DAX-1, and SOX-9 have been identified as the products of genes responsible for human diseases that display structural and functional defects in tissues including the gonads. The crucial functions of the other transcription factors such as Ad4BP/SF-1, Emx-2, M33, and Lhx-9 were identified by phenotypes of their gene disrupted mice. In addition, the expression profiles with respect to their distribution and sexual dimorphism strongly suggested the functional significance at the early stage of gonadal differentiation. However, it remains to be clarified how the transcription factors above regulate their target genes transcription and how the genes encoding the transcription factors are regulated. When considering a gene regulatory cascade that supports differentiation and sex differentiation of the gonad, studies of the above two directions are quite important. Based on this concept, we investigated mainly the functions of Ad4BP/SF-1 and Dax-1, and the mechanism of gene regulation encoding these factors.

I. Gene regulatory cascade in the steroidogenic tissue differentiation

When a differentiation process of a tissue is considered, it is possible to assume that certain genes encoding transcription factors are involved in a gene regulatory cascade as the critical components. As a component in the cascades required for adrenocortical and gonadal differentiation, Ad4BP/SF-1 seems to be localized at the upstream of a set of tissue-specific genes including steroidogenic Cyp genes, while Ad4BP/SF-1 is localized at the downstream of other transcription factors regulating Ad4BP/SF-1 gene transcription. When considering that upregulation of the components occurs from upstream to downstream of the cascade along with the tissue differentiation and moreover Ad4BP/SF-1 is an essential transcription factor in the adrenocortical and gonadal cascade, identification of the components functioning with Ad4BP/SF-1 and regulating Ad4BP/SF-1 gene transcription is quite important for fully understanding the molecular mechanisms underlying differentiation of the adrenal cortex and gonad. Thus, we have investigated gene regulation of Ad4BP/SF-1 and Dax-1.

Tissue-Specific Enhancers on the Ad4BP/SF-1 Gene

Based on the aspect above, the regulatory region of the Ad4BP/SF-1 gene has been investigated in vivo. A mouse YAC clone (longer than 480 kb) containing whole Ad4BP/SF-1 gene was examined. The long fragment was subcloned into cosmid vector carrying Lac Z gene as a reporter gene, and subjected to transgenic assay to examine if they have tissues-specific enhancer element. Our survey for the genomic DNA revealed that particular regions in the Ad4BP/SF-1 gene are responsible for the gene expression specific for the adrenal cortex, ventromedial hypothalamic nucleus, and pituitary. Interestingly, all of them are localized at the intronic regions and the nucleotide sequences are conserved among animal species. The fine structures of these tissue-specific enhancers are investigating.

Wnt4 Signal for Gonad Sex Differentiation

Dax-1 is an orphan nuclear receptor acting as a suppressor of Ad4BP/SF-1, and as an anti-Sry factor in the process of gonadal sex differentiation. The roles of these nuclear receptors in the differentiation of the gonads and the adrenal cortex have been established through studies of the mutant phenotype in both mice and humans. However, the mechanisms underlying transcriptional regulation of these genes remain largely unknown. We examined the relationship between Dax-1 gene transcription and the Wnt4 pathway. Reporter gene analysis revealed that Dax-1 gene transcription was activated by b-catenin, a key signal-transducing protein in the Wnt pathway, acting in synergy with Ad4BP/SF-1. Interaction between b-catenin and Ad4BP/SF-1 was observed using yeast two-hybrid and in vitro pull-down assays. The region of Ad4BP/SF-1 essential for this interaction consists of an acidic amino acid cluster, which resides in the first helix of the ligand binding domain. Mutation of the amino acid cluster impaired transcriptional activation of Dax-1 as well as interaction of Ad4BP/SF-1 with b-catenin. These results were supported by in vivo observations using Wnt4 gene disrupted mice, where Dax-1 gene expression was decreased significantly in sexually differentiating female gonads. We thus conclude that Wnt4 signaling mediates the increased expression of Dax-1 as the ovary becomes sexually differentiated.

II. Characterization of factors interacting with Ad4BP/SF-1

ARX is the responsible gene for XLAG
The structural and functional defects observed in the mutant and the chromosomal localization of ARX on Xp22.13 suggested ARX as a plausible candidate gene for XLAG (X-linked lissencephaly with abnormal genitalia). To investigate this possibility, we determined nucleotide sequence of the ARX in eight XLAG probands, and detected eight mutations. Overall, we found one nonsense mutation, four frame-shift mutations, two misssense mutations, and one larger deletion. Two of the truncated ARX proteins contain the homeodomain but lack the C-peptide (aristaless) domain conserved in prd-like homeoproteins. Although the function of the domain remains unknown, physiological significance is suggested by our results. Moreover, we identified two missense mutations that lead to amino acid substitutions, R322H and L343Q. Since both amino acids are located in the homeodomain and are highly conserved among the family, it is reasonable to assume that the mutations result in functional failure such as DNA binding.

Publication List:

Asoy, R., Mellgren, G., Morohashi, K. and Lund, J. (2002) Activation of cAMP-dependent Protein Kinase increases the protein level of Steroidogenic Factor-1. Endocrinol. 143, 295-303.

Kitamura, K., Yanazawa, M., Sugiyama, N., Miura, H., Iizuka-Kogo, A., Kusaka, M., Suzuki, R., Kato-Fukui, Y., Kamiirisa, K., Omichi, K., Kasahara, M., Yoshioka, H., Ogata, T., Fukuda, T., Kondo, I., Kato, M., Dobyns, W. B.. Yokoyama M., and Morohashi K. (2002) Mutations of Arx/ARX cause abnormal migration and differentiation of GABAergic interneurons and abnormal development of testes in mice, and X-linked lissencephaly with abnormal genitalia in humans. Nature Genet. 32, 359-369,

Meeks, J. J., Crawford, S. E., Russell, T. A. Morohashi, K., Capel, B., Weiss, J. and Jameson, J. L. (2003) Dax1 is required for patterning of testis cords during gonadal differentiation. Development. (in press)

Mukai, T., Kusaka, M., Kawabe, K., Goto, K., Nawata, H., Fujieda, K. and Morohashi K. (2002) Sexually dimorphic expression of Dax-1 in the adrenal cortex. Genes Cells 7, 717-729

Suzuki, T., Kasahara, M., Yoshioka, H., Morohashi, K. and Umesono K. (2003) LXXLL motifs in Dax-1 have target specificity for the orphan receptors Ad4BP/SF-1 and LRH-1. Mol. Cell. Biol. 23, 238-249

Wang, D., Kobayashi, T., Senthilkumaran, B., Sakai, F., Sudhakumari, C. C., Suzuki, T., Yoshikuni, M., Matsuda, M., Morohashi, K. and Nagahama Y. (2002) Molecular cloning of DAX-1 and SHP cDNAs and their expression patterns in the Nile tilapia, Oreochromis niloticus. Biochem. Biophys. Res. Commun. 297, 632-640,

Yokoi, H., Kobayashi, T., Tanaka, M., Nagahama, Y., Wakamatsu, Y., Takeda, H., Araki, K., Morohashi, K. and Ozato K. (2002) sox9 in a teleost fish, medaka (Oryzias latipes): evidence for diversified function of Sox9 in gonad differentiation. Mol. Reprod. Dev. 63, 5-16