Megumi Kasahara

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.

Distinctive inhibitory activity of docosahexaenoic acid against sphingosine-induced apoptosis.

The effect of supplementation of docosahexaenoic acid (DHA) on the apoptosis of HL60 cells was examined using N-acetyl sphingosine (C2-ceramide) and sphingosine as apoptosis-inducing agents. Although C2-ceramide-induced apoptosis was not affected by DHA supplementation, sphingosine-induced apoptosis was reduced almost to the background level by preincubation with 10 microM DHA for 24 h. Among the fatty acids, only DHA appeared to be endowed with the ability to reduce sphingosine-induced apoptosis, whereas, other unsaturated fatty acids, such as arachidonic acid (AA) and eicosapentaenoic acid (EPA), did not show this activity. Incubation of HL60 with DHA within 6 h did not affect the apoptosis, suggesting that DHA probably expressed the inhibitory activity after modulation of the membrane fatty acid composition. DHA also attenuated the apoptosis induced by dimethylsphingosine and H-7, but not by calphostin C, indicating that enrichment of DHA in membranous phospholipid does not necessarily prevent all of the apoptosis associated with the inhibition of protein kinase C. The mechanism of the inhibition against sphingosine-induced apoptosis by DHA remains to be further explored. However, the inhibition of cytosolic phospholipase A2 (cPLA2) may be involved in the mechanism, because distinctive inhibitory activity of DHA against cPLA2 has been demonstrated [M. Shikano, Y. Masuzawa, K. Yazawa, K. Takayama, I. Kudo, K. Inoue, Biochim. Biophys. Acta, 1212, 1994, 211-216], and arachidonyl trifluoromethylketone, a specific inhibitor of cPLA2, attenuated the apoptosis induced by sphingosine.

Evaluation of a trans configuration for the apoptosis-inducing activity of ceramide

The requirement of a trans double bond for the biological action of ceramide was assessed by comparing the apoptosis-inducing activity of various ceramide analogs. The cis isomer and an acetylene type derivative of sphingosine were chemically synthesized, and the 2-amino moiety was acylated with hexanoic acid. These cell-permeable ceramide derivatives were compared with N-hexanoyl sphingosine (C6-Cer) or N-hexanoyl dihydrosphingosine (C6-DH-Cer) in their activity to induce apoptosis of HL60. Either the cis isomer of C6-Cer (C6-cis-Cer) or a triple bond derivative (C6-TRP-Cer) induced apoptosis when assessed by fluorescence microscopy of the morphological changes and electrophoretic analysis of DNA C6-TRP-Cer yielded the highest percentage of apoptotic cells corresponding to three times that was induced by C6-Cer. C6-cis-Cer also showed stronger activity than C6-Cer. The minimum amounts of C6-TRP-Cer and C6-cis derivative required to induce apoptosis were 0.1 and 0.5 microM, respectively, while 1 microM C6-Cer was required to exhibit the activity. C6-DH-Cer showed very low but significant activity above 10 microM. N-acetyl-sphingosine (C2-Cer) induced more apoptotic cells than C6-Cer, and C2-TRP-Cer was much more potent than C2-Cer. These observations suggest that the trans configuration of ceramide is not necessarily essential for the activity to induce apoptosis. In addition, distinctive activity of C6- or C2-TRP-Cer suggests that this ceramide analog might be useful for developing a new type of antitumor drug.

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.

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.