Hidefumi Yoshioka

Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans.

Male embryonic mice with mutations in the X-linked aristaless-related homeobox gene (Arx) developed with small brains due to suppressed proliferation and regional deficiencies in the forebrain. These mice also showed aberrant migration and differentiation of interneurons containing γ-aminobutyric acid (GABAergic interneurons) in the ganglionic eminence and neocortex as well as abnormal testicular differentiation. These characteristics recapitulate some of the clinical features of X-linked lissencephaly with abnormal genitalia (XLAG) in humans. We found multiple loss-of-function mutations in ARX in individuals affected with XLAG and in some female relatives, and conclude that mutation of ARX causes XLAG. The present report is, to our knowledge, the first to use phenotypic analysis of a knockout mouse to identify a gene associated with an X-linked human brain malformation.

LXXLL-Related Motifs in Dax-1 Have Target Specificity for the Orphan Nuclear Receptors Ad4BP/SF-1 and LRH-1

ABSTRACT The orphan receptor Ad4BP/SF-1 (NR5A1) is a constitutive activator, and its activity is repressed by another orphan receptor, Dax-1 (NR0B1). In the present study, we investigated the molecular mechanisms underlying this repression by Dax-1. Yeast two-hybrid and transient-transfection assays confirmed the necessity of three LXXLL-related motifs in Dax-1 for interaction with and repression of Ad4BP/SF-1. In vitro pull-down experiments confirmed that Dax-1 interacts with Ad4BP/SF-1 and also with LRH-1 (NR5A2). The target specificity of the LXXLL-related motifs was indicated by the observations that Ad4BP/SF-1, ERα (NR3A1), LRH-1, ERR2 (NR3B2), and fly FTZ-F1 (NR5A3) interacted through their ligand binding domains with all the LXXLL-related motifs in Dax-1 whereas HNF4 (NR2A1) and RORα (NR1F1) did not. Transcriptional activities of the receptors whose DNA binding domains (DBDs) were replaced by the GAL4 DBD were repressed by Dax-1 to various levels, which correlated with the strength of interaction. Amino acid substitutions revealed that Ad4BP/SF-1 and LRH-1 preferentially interact with L(+1)XXLL-related motifs containing serine, tyrosine, serine, and threonine at positions −2, +2, +3, and +6, respectively. Taken together, our results indicate that the specificities of LXXLL-related motifs in Dax-1 based on their amino acid sequences play an important role in regulation of orphan receptors.

Mechanism of asymmetric ovarian development in chick embryos

In most animals, the gonads develop symmetrically, but most birds develop only a left ovary. A possible role for estrogen in this asymmetric ovarian development has been proposed in the chick, but the mechanism underlying this process is largely unknown. Here, we identify the molecular mechanism responsible for this ovarian asymmetry. Asymmetric PITX2 expression in the left presumptive gonad leads to the asymmetric expression of the retinoic-acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptors Ad4BP/SF-1 and estrogen receptor α in the right ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These data suggest that early asymmetric expression of PITX2 leads to asymmetric ovarian development through up- or downregulation of RALDH2, Ad4BP/SF-1, estrogen receptor α and cyclin D1.

LXXLL motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1.

The orphan receptor Ad4BP=SF-1 (NR5A1) is a constitutive activator and its activity is repressed by another orphan receptor, Dax-1 (NR0B1). In the present study, we investigated the molecular mechanisms underlying this repression by Dax-1. Yeast two-hybrid and transient transfection assays confirmed the necessity of three LXXLL motifs in Dax-1 for interaction with and repression against Ad4BP=SF-1. In vitro pull-down experiments confirmed that Dax-1 interacts with Ad4BP=SF-1 and also LRH-1 (NR5A2). The target specificity of the LXXLL motifs was manifested by the observations that Ad4BP=SF-1, ERa (NR3A1), LRH-1, ERR2 (NR3B2), and fly FTZ-F1 (NR5A3) interacted through their ligand binding domains with all the LXXLL motifs in Dax-1, whereas HNF4 (NR2A1) and RORa (NR1F1) did not. Transcriptional activities of the receptors whose DNA binding domains (DBDs) were replaced by GAL4 DBD were repressed by Dax-1 to varying levels, which correlated with the strength of interaction. Amino acid substitutions revealed that Ad4BP=SF-1 and LRH-1 preferentially interact with L(þ1)XXLL motifs containing serine, tyrosine, serine, and threonine at positions 2, þ2, þ3, and þ6, respectively. Taken together, our results indicate that the specificity of LXXLL motifs in Dax-1 based on their amino acid sequences plays an important role in regulation of orphan receptors.

A genetically female brain is required for a regular reproductive cycle in chicken brain chimeras

Sexual differentiation leads to structural and behavioural differences between males and females. Here we investigate the intrinsic sex identity of the brain by constructing chicken chimeras in which the brain primordium is switched between male and female identities before gonadal development. We find that the female chimeras with male brains display delayed sexual maturation and irregular oviposition cycles, although their behaviour, plasma concentrations of sex steroids and luteinizing hormone levels are normal. The male chimeras with female brains show phenotypes similar to typical cocks. In the perinatal period, oestrogen concentrations in the genetically male brain are higher than those in the genetically female brain. Our study demonstrates that male brain cells retain male sex identity and do not differentiate into female cells to drive the normal oestrous cycle, even when situated in the female hormonal milieu. This is clear evidence for a sex-specific feature that develops independent of gonadal steroids.

Mesonephric Wnt Signaling Associate with a Formation of an Adreno‐Gonadal Primordium in Chick Embryos

The adrenal cortex as well as the reproductive tract, kidney, and gonad is derived from the intermediate mesoderm. In addition, the mesonephros essential for the early adrenal development also originates from the intermediate mesoderm. A contribution of mesonephros to adrenal formation through expression of growth factors has been largely unknown. Moreover, Ad4 binding protein/steroidogenic factor 1[Ad4BP/SF-1(NR5A1)], and Dmrt-1(doublesex- and mab-3-related transcription factor) are expressed in adrenal and gonadal development chick embryos. What growth factor(s) regulate the expression of Ad4BP/SF-1 and Dmrt-1, has not been determined. Here we show with chick embryos that Wnt4 is expressed in only certain parts of the mesonephric tubules, while the expression was also detected in the mesenchyme at stage 21. Moreover, Ad4BP/SF-1 and Dmrt-1 were expressed in the developing adreno-gonadal and gonadal primordial at the same stage adjacent to the expressions of Wnt4. To examine the possibility that Ad4BP/ SF-1 and Dmrt-1 were influenced by Wnt4, we performed with misexpression. Our studies showed that Wnt4 expanded marker gene expression for the adreno-gonadal primordium, whereas Wnt4 does not stimulated gonad formation. We conclude that Wnt signaling is involved in the formation of the adreno-gonadal primordium.

Contribution of the coelomic epithelial cells specific to the left testis in the chicken embryo

Background: The left male gonad in the chicken embryo has a thickened cortical layer, but it eventually becomes flattened after the onset of testicular development. Because the destination of the cortical cells migrating from the left gonad remains unclear, we examined this issue herein. Results: The testis‐inducing gene doublesex‐ and mab‐3‐related transcription factor 1 (DMRT1) was detected in a proportion of the columnar and cubic epithelial cells in the cortex of the left testis as well as Sertoli cells in both testes. Interestingly, some of the DMRT1‐expressing cortical cells were contiguous with Sertoli cells in the testis cord. Some cortical cells exhibited a vimentin‐positive cytoplasm that was elongated all the way to the medulla. In addition, a desmosome‐like structure was observed between the elongated cytoplasm in these cells and the adjacent Sertoli cell. After the organ culture, a few cells labeled with a fluorescent dye that stained only the cortical cells at the beginning of the culture were located in the testis cord of the left testis. Conclusions: Some cortical cells expressing DMRT1 were suggested to contribute to the Sertoli cells in the testis cord only after the onset of testicular development and only in the left testis. Developmental Dynamics 246:148–156, 2017.