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 gamma-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.

Two 21-hydroxylase [P450(C21)] genes have been isolated from a human genomic library using a bovine P-450(C21) cDNA. The insert DNAs containing the P450(C21) genes were also hybridized with the sequences of the 5' or 3' end regions of human C4 cDNA, indicating a close linkage of the P450(C21) gene to the C4 gene. Sequence analysis has revealed that the two P450(C21) genes are both 3.4 kilobases long and split into 10 exons. Comparing the two sequences, we found that the two genes are highly homologous including their introns and flanking sequences, but that three mutations render one of the two P450(C21) genes nonfunctional-i base insertion, an 8-base deletion, and a transition mutation-all of which may cause premature termination of the translation.

Signaling molecules such as Activin, Sonic hedgehog, Nodal, Lefty, and Vg1 have been found to be involved in determination of left-right (L-R) asymmetry in the chick, mouse, or frog. However, a common signaling pathway has not yet been identified in vertebrates. We report that Pitx2, a bicoid-type homeobox gene expressed asymmetrically in the left lateral plate mesoderm, may be involved in determination of L-R asymmetry in both mouse and chick. Since Pitx2 appears to be downstream of lefty-1 in the mouse pathway, we examined whether mouse Lefty proteins could affect the expression of Pitx2 in the chick. Our results indicate that a common pathway from lefty-1 to Pitx2 likely exists for determination of L-R asymmetry in vertebrates.

Dax-1 [dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (NR0B1)] is an orphan nuclear receptor acting as a suppressor of Ad4 binding protein/steroidogenic factor 1 [Ad4BP/SF-1 (NR5A1)] 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. Here, 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 beta-catenin, a key signal-transducing protein in the Wnt pathway, acting in synergy with Ad4BP/SF-1. Interaction between beta-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 beta-catenin. These results were supported by in vivo observations using Wnt4 gene-disrupted mice, in which 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.

No abstract

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 ␣ 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 ␣ and cyclin D1.

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, ER␣ (NR3A1), LRH-1, ERR2 (NR3B2), and fly FTZ-F1 (NR5A3) interacted through their ligand binding domains with all the LXXLLrelated motifs in Dax-1 whereas HNF4 (NR2A1) and ROR␣ (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.More than 70 molecular species of the nuclear receptor superfamily have been identified to date in animals ranging from hydras to humans (47). These transcription factors are generally characterized by the presence of two conserved structural features, a DNA binding domain (DBD) composed of two zinc fingers and a ligand binding domain (LBD) located at the C-terminal region. Various lipophilic ligands interact with the cognate LBDs in apo-type receptors, converting them into an active holo-type conformation that can dynamically regulate transcription (14, 38).The general structure of the LBD is composed predominantly of 12 helices. Interaction with ligand induces allosteric changes in conformation, especially in the configuration of helix 12 at the C terminus of the LBD, leading to transcriptional activation (3, 9) or repression (4, 57). Helix 12 is often referred to as the AF-2 core, which in some receptors is a conserved domain essential for ligand-dependent transcriptional activation (14, 37). Transcriptional coactivators mediate activating signals by binding to nuclear receptors in a ligand-dependent manner (reference 67 and references therein). For this receptor-coactivator interaction, conserved sequences containing a short signature motif of LXXLL (where L is leucine and X can be any amino acid) have been implicated (18,31,60). The conserved leucines in these so-called LXXLL motifs, or NR-boxes, appear to be indispensable for hydrophobic interaction with nuclear receptors (10,46,57). In addition, amino acid residues flanking the core LXXLL are known to influence the binding specificity (8,17,29,36,39,65). In the absence of li...

No abstract