Mutation analysis and embryonic expression of the HLXB9 Currarino syndrome gene.

The HLXB9 homeobox gene was recently identified as a locus for autosomal dominant Currarino syndrome, also known as hereditary sacral agenesis (HSA). This gene specifies a 403-amino acid protein containing a homeodomain preceded by a very highly conserved 82-amino acid domain of unknown function; the remainder of the protein is not well conserved. Here we report an extensive mutation survey that has identified mutations in the HLXB9 gene in 20 of 21 patients tested with familial Currarino syndrome. Mutations were also detected in two of seven sporadic Currarino syndrome patients; the remainder could be explained by undetected mosaicism for an HLXB9 mutation or by genetic heterogeneity in the sporadic patients. Of the mutations identified in the 22 index patients, 19 were intragenic and included 11 mutations that could lead to the introduction of a premature termination codon. The other eight mutations were missense mutations that were significantly clustered in the homeodomain, resulting, in each patient, in nonconservative substitution of a highly conserved amino acid. All of the intragenic mutations were associated with comparable phenotypes. The only genotype-phenotype correlation appeared to be the occurrence of developmental delay in the case of three patients with microdeletions. HLXB9 expression was analyzed during early human development in a period spanning Carnegie stages 12-21. Signal was detected in the basal plate of the spinal cord and hindbrain and in the pharynx, esophagus, stomach, and pancreas. Significant spatial and temporal expression differences were evident when compared with expression of the mouse Hlxb9 gene, which may partly explain the significant human-mouse differences in mutant phenotype.

SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development.

SRY, SOX9, and DAX1 are key genes in human sex determination, by virtue of their associated male-to-female sex reversal phenotypes when mutated (SRY, SOX9) or over-expressed (DAX1). During human sex determination, SRY is expressed in 46,XY gonads coincident with sex cord formation, but also persists as nuclear protein within Sertoli cells at 18 weeks gestation. High-level SOX9 expression in the sex cords of the testis parallels that seen during mouse development, however in humans, SOX9 transcripts also are detected in the developing ovary. Low-level DAX1 expression predates peak SRY expression by at least 10 days, and persists in Sertoli cells throughout the entire sex determination period. In Dosage Sensitive Sex reversal, the anti-testis properties of DAX1 over-expression could act prior to the peak effects of SRY and continue during the period of SOX9 expression. These findings highlight expression differences for the SRY, SOX9, and DAX1 genes during sex determination in humans and mice. These results provide a direct framework for future investigation into the mechanisms underlying normal and abnormal human sex determination.

Expression of steroidogenic factor 1 and Wilms' tumour 1 during early human gonadal development and sex determination.

The transcription factors SF-1 and WT1 play pivotal roles in mammalian gonadal development and sexual differentiation. In human embryos, both SF-1 and WT1 are expressed when the indifferent gonadal ridge first forms at 32 days post-ovulation. As the sex cords develop - providing morphological evidence of testis differentiation - SF-1 localises predominantly to developing Sertoli cells in the sex cords, whereas WT1 retains a broader pattern of expression. Later, SF-1 localises predominantly to steroidogenic Leydig cells, and WT1 localises to the sex cords. In the ovary, SF-1 and WT1 transcripts persist in the gonadal ridge from the earliest developmental stages throughout the critical period of sex determination. These studies, which delineate for the first time the sequential expression profiles of SF-1 and WT1 during human gonadal development, provide a framework for understanding human sex reversal phenotypes associated with their mutations.

A homeobox gene, HLXB9, is the major locus for dominantly inherited sacral agenesis.

Partial absence of the sacrum is a rare congenital defect which also occurs as an autosomal dominant trait; association with anterior meningocoele, presacral teratoma and anorectal abnormalities constitutes the Currarino triad (MIM 176450). Malformation at the caudal end of the developing notochord at approximately Carnegie stage 7 (16 post-ovulatory days), which results in aberrant secondary neurulation, can explain the observed pattern of anomalies. We previously reported linkage to 7q36 markers in two dominantly inherited sacral agenesis families. We now present data refining the initial subchromosomal localization in several additional hereditary sacral agenesis (HSA) families. We excluded several candidate genes before identifying patient-specific mutations in a homeobox gene, HLXB9, which was previously reported to map to 1q41-q42.1 and to be expressed in lymphoid and pancreatic tissues.

Ontogeny of pro-opiomelanocortin (POMC) gene expression and translated products adrenocorticotrophin (ACTH) and alpha-melanocyte stimulating hormone (alpha-MSH) in the ovine fetal pituitary gland.

This study sought to determine the ontogeny of pro-opiomelanocortin (POMC) mRNA by in situ hybridization, and the expression of alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotrophin (ACTH) by immunohistochemistry, in the ovine fetal pituitary gland. Detection of POMC mRNA, and of ACTH and alpha-MSH immunoreactivity was first noted at Day 40 of gestation in the pars intermedia (PI) and the pars distalis (PD). After Day 70, alpha-MSH immunoreactivity was confined to the PI whereas POMC mRNA and ACTH immunostaining remained in both the PI and the PD. Increases (P < 0.05) of POMC mRNA expression were observed in the PI between Days 50-70 and levels then remained constant to Day 141 (term, 145 days). In the basal region of the PD, POMC mRNA expression was high at Day 40, declined (P < 0.01) by Day 50 and then increased progressively to Day 141 of gestation. The proportion of ACTH-immunopositive cells in the PD also fell from 14% at Day 40 to 11.4% at Day 70 and then increased to 15.3% by Day 141. In contrast, neither the level of POMC mRNA expression nor the percentage of corticotrophs changed in the region of the PD immediately adjacent to the PI. These data provide evidence for differential processing of POMC in a tissue-specific manner during early fetal pituitary development.

Dopaminergic regulation of adrenocorticotrophic hormone, alpha-melanocyte-stimulating hormone and cortisol secretion in the ovine fetus.

During ovine fetal development there is a progressive maturation of the hypothalamo-pituitary-adrenal axis which culminates in the onset of birth. The role and regulation of the intermediate lobe of the fetal pituitary gland in relation to this maturational process remains controversial. To test the hypothesis that the pars intermedia of the ovine fetal pituitary is under tonic inhibitory dopaminergic control we treated fetal sheep at day 131 of gestation with a 3-day intravenous infusion of one of the following: the dopamine antagonist sulpiride (0.3 mg/ 0.5 ml/h; n = 12), the dopamine agonist bromocriptine (0.03 mg/0.5 ml/h; n = 7) or vehicle (0.1 M tartaric acid in saline; n = 8) alone. Fetal plasma concentrations of alpha-MSH were significantly (P < 0.01) increased by treatment with sulpiride and decreased (P < 0.05) by bromocriptine. The increase in alpha-MSH after sulpiride was characterised by an increase in the amplitude of alpha-MSH pulses whereas bromocriptine virtually abolished all pulses of alpha-MSH. Immunoreactive ACTH (IR-ACTH) concentrations were significantly (P < 0.05) elevated after sulpiride but were unaffected by bromocriptine. There were no changes in the pulsatile characteristics of IR-ACTH secretion. Cortisol concentrations were unchanged by either treatment. We conclude that fetal alpha-MSH and IR-ACTH are secreted in a pulsatile fashion and are tonically inhibited by dopaminergic pathways. The lack of an effect of endogenously raised alpha-MSH on plasma cortisol concentrations provides evidence that this POMC-derived peptide is not responsible for the regulation of cortisol biosynthesis and/or secretion.

Neuroendocrine regulation of pituitary-adrenal function during fetal life.

During late gestation there is a rise in the concentration of corticosteroids in the fetal circulation that is essential for the coordinated maturation of many fetal organ systems and is a key component in the endocrine pathway leading to the onset of birth. Fetal plasma concentrations of adrenocorticotrophin (ACTH) increase during late gestation and this rise precedes the increase in plasma corticosteroids. Paradoxically, ACTH and cortisol concentrations increase concomitantly even though cortisol would normally be expected to exert negative feedback effects to inhibit pituitary ACTH secretion. Elucidating the neuroendocrine signals that cause the increase in fetal ACTH, despite the elevated concentrations of cortisol at this time, will therefore provide vital clues as to the trigger for fetal organ maturation and birth. This article describes the normal ontogeny of the hypothalamo-pituitary-adrenal axis, discusses the neuroendocrine signals that trigger the increase in fetal ACTH secretion and provides potential explanations for the concomitant rise in ACTH and cortisol.