DIVISION OF CELL DIFFERENTIATION

¹⁾ CREST, JST Postdoctoral Fellow
²⁾ CREST, JST Technical Assitant
³⁾ CREST, JST Secretary
⁴⁾ Graduate School of Kyushu University
⁵⁾ Graduate School of Tohoku University
⁶⁾ Graduate School of Ehime University
⁷⁾ Graduate University for Advanced Studies
⁸⁾ Graduate School of Tokyo University
⁹⁾ Graduate School of Nagoya University
¹⁰⁾ Pfizer Inc.

Cell and tissue differentiation proceeds systematically based on orchestrated expressions of sets of genes. The expressions commence successively along with the passage of time. As the consequence, a single fertilised egg develops into a variety of tissues and organs, which consist of specialised cells in terms of their structures and functions. Accordingly, it is reasonable to assume that investigation of the mechanisms underlying the cell and tissue-specific gene expression at a molecular level is essential for understanding molecular frameworks for genetic cascades proceeding along with 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.

Several 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 responsible genes for various human diseases that display structural and functional defects in tissues including the gonads. The essential functions of 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 suggest 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 and how the genes encoding the transcription factors are regulated. When considering a gene regulatory cascade that supports differentiation of the gonadal tissues, studies of the above two directions are quite important. Based on this background, 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 the component in the cascades required for the steroidogenic adrenocortical and gonadal differentiation, Ad4BP/SF-1 is locates at the upstream of tissue-specific genes, including the steroidogenic CYP genes, and should locate at the downstream of other transcription factors regulating Ad4BP/SF-1 gene. When considering that the cascade flows from upstream to downstream 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 essential for fully understanding the molecular mechanisms underlying the tissue differentiation. Thus, some of the members in this division have investigated gene regulation of Ad4BP/SF-1 and Dax-1, both of which are quite important for the tissue differentiation.

Based on the aspect above, the regulatory region of the Ad4BP/SF-1 gene has been investigated in vivo by making transgenic mice. A genomic DNA fragment longer than 480 kb containing four genes including Ad4BP/SF-1 has been examined 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 and ventromedial hypothalamic nucleus.

Dax-1 is another transcription factor of our interest, which is also implicated in the steroidogenic tissue differentiation. Our previous study revealed that the factor acts as a suppressor against Ad4BP/SF-1. However, regulation of the suppressive effect has remained to be clarified at the molecular level. We recently uncovered the function of particular sequences, LXXLL motifs, located at the amino terminal half of Dax-1. When Dax-1 functions as the suppressor, the amino acid sequences in the repeats are essential for a protein-protein interaction with target nuclear receptors, and thereby the transcription mediated by the nuclear receptors are largely inhibited. The LXXLL motif was originally identified in coactivators as a motif essential for interaction with nuclear receptors. Thus, it is highly likely that the LXXLL motifs in Dax-1 compete with those of the coactivators for interaction with nuclear receptors such as Ad4BP/SF-1 (Fig 1). Although it remains unclear how the inhibitory activity is regulated in a variety of physiological conditions, interaction through the LXXLL motifs should be a crucial step for the functional regulation of the transcription facotors.

In addition to these transcription factors, it has been accepted that Sox-9, Wt-1, Emx-2, and GATA-4 are implicated in gonad differentiation through regulating transcription of gene essential for gonadal structures and functions. Although the essential functions of these transcription factors have been elucidated through symptoms of the genetic disorders and/or the phenotypes of the gene disrupted mice, characterization of their transcriptional regulation has not yet been performed enough. Therefore, we started to investigate it through characterizing proteins interacting with the transcription factors. In order to isolate the interacting factors, yeast two-hybrid screening has been performed using a cDNA library constructed with mRNA prepared from mouse fetal gonads. Extensive screening resulted in isolation of molecules including coactivators, other type of transcription factors, proteins carrying protein modification activities, and proteins carrying domains capable of signal transduction. In situ hybridization analyses with these particular molecules revealed that some of them are expressed in the developing gonads, and some of them showed sex dependent expression. For investigating their biological significance, gene-disruption study has been performed.

II. Wnt4 signal for the gonad sex differentiation

As reported previously, Ad4BP/SF-1 is an indispensable component for Dax-1 gene transcription. In fact, multiple binding sites recognized by Ad4BP/SF-1 at the upstream region of Dax-1 gene are necessary for transcriptional activation. The in vitro observation using reporter gene assays was confirmed subsequently by an in vivo study using Ad4BP/SF-1 gene disrupted mice, which lacked Dax-1 expression in the developing genital ridge. Although these results strongly indicated that Ad4BP/SF-1 gene is genetically located upstream from the Dax-1 gene, their expression profiles in terms of distribution and sexual dimorphism do not necessarily agree with our findings. In this regard, it should be noticed that a recent gene disruption study indicated implication of Wnt4 in gonadal sex differentiation. Normally, steroidogenic 3b-HSD gene and MIS gene are expressed in the developing fetal gonads of males but not females. Interestingly, however, the expression was detected in the fetal ovary of the gene-disrupted mice, suggesting that the Wnt4 represses 3b-HSD and MIS gene transcription in the fetal ovaries of the wild type. If considering that some of Wnt signals activate downstream gene transcription through stabilization of b-catenine, it is unlikely that the signal represses the 3b-HSD and MIS gene transcription.

To provide a rational explanation, we hypothesized that Wnt4 expressed in the developing gonad upregulates a suppressor molecule and thereby downregulates 3b-HSD and MISgene transcription. Since transcription of both genes is regulated in a positive fashion by Ad4BP/SF-1, it was reasonable to assume that Dax-1 plays a role as the suppressor. To confirm this assumption, we examined if b-catenin activates the Dax-1 gene transcription. As the result, Dax-1gene transcription was activated in the presence of b-catenin in a dose-dependent manner. Interestingly the action of b-catenin is further upregulated in the presence of Ad4BP/SF-1, indicating that the two factors, b-catenin and Ad4BP/SF-1, synergistically activate the Dax-1 gene transcription. Showing a good correlation, interaction between Ad4BP/SF-1 and b-catenin was confirmed with protein-pull down and yeast tow-hybrid analyses.

The mechanisms of Dax-1 gene regulation governing its sexually dimorphic and spatial characteristics are summarized in Fig. 2. As described previously, it is difficult to explain the whole regulatory mechanism of the Dax-1 gene transcription by Ad4BP/SF-1 alone. For instance, Ad4BP/SF-1 is expressed in the male developing gonads more abundantly than in the female. Nevertheless, the amount of Dax-1 in the female developing gonad is higher than that in the male gonad. In the case of Wnt4 expression in the developing gonads and mesonephros, in situ examination revealed that the amount expressed in the female tissues is higher than in the male. With respect to the distribution of Dax-1, strong signals were detected in the gonadal regions facing the mesonephros although such expression domain was not observed in the case of Ad4BP/SF-1. In such inconsistent distribution, it is interesting to note that the expression of Wnt4 in the gonads was more abundant at the region proximal to than that distal to mesonephros. Therefore, to understand the mechanism underlying Dax-1 expression, we propose that Ad4BP/SF-1 plays basal and fundamental roles and that the Wnt4 signal modulates the transcription mediated by Ad4BP/SF-1. Although the regulation above is likely to function in the sexually differentiating gonads of both sexes, the mechanisms of other transcription factors such as Sox-9 and Emx-2 are not fully understood. In addition, it should be noted that other growth factors as well as other forms of Wnt molecules are expressed in the developing gonads and mesonephros. Further studies of the functional relationship between growth factors and transcription factors should identify the fine and sophisticated mechanisms underlying the sex differentiation of the gonads.

Publication List:

Asoy, R., Mellgren, G., Morohashi, K., Lund, J. Activation of cAMP-dependent Protein Kinase increases the protein level of Steroidogenic Factor-1. Endocrinol. in press.

Ikeda, Y., Takeda, Y., Shikayama, T., Mukai, T., Hisano, S., Morohashi, K. (2001) Comparative localization of Dax-1 and Ad4BP/SF-1 during development of the hypothalamic-pituitary-gonadal axis implies their closely related and distinct functions. Develop. Dynam. 220, 363-376.

Shibata, H., Ikeda, Y., Mukai, T., Morohashi, K., Kurihara, I., Ando, T., Suzuki, T., Kobayashi, S., Murai, M., Saito, I., Saruta, T. (2001) Expression profiles of COUP-TF, DAX-1 and SF-1 in the human adrenal gland and adrenocortical tumors: Possible implications in steroidogenesis. Mol. Genet. Metab. 74, 206-216.

Suzuki, T., Mizusaki, H., Kawabe, K., Kasahara, M., Yoshioka, H., Morohashi, K. Concerted Regulation of Gonadal Differentiation by Transcription Factors and Growth Factors. In 'The Genetics and Biology of Sex Determination' John Wiley & Sons Ltd. in press.