Emmanuèle Délot

Up-Regulation of WNT-4 Signaling and Dosage-Sensitive Sex Reversal in Humans

Wnt-4, a member of the Wnt family of locally acting secreted growth factors, is the first signaling molecule shown to influence the sex-determination cascade. In mice, a targeted deletion of Wnt-4 causes the masculinization of XX pups. Therefore, WNT-4, the human homologue of murine Wnt-4, is a strong candidate gene for sex-reversal phenotypes in humans. In this article, we show that, in testicular Sertoli and Leydig cells, Wnt-4 up-regulates Dax1, a gene known to antagonize the testis-determining factor, Sry. Furthermore, we elucidate a possible mechanism for human XY sex reversal associated with a 1p31-p35 duplication including WNT-4. Overexpression of WNT-4 leads to up-regulation of DAX1, which results in an XY female phenotype. Thus, WNT-4, a novel sex-determining gene, and DAX1 play a concerted role in both the control of female development and the prevention of testes formation. These observations suggest that mammalian sex determination is sensitive to dosage, at multiple steps in its pathway.

BMP signaling is required for septation of the outflow tract of the mammalian heart

Bone morphogenetic proteins (BMPs) constitute a family of ∼20 growth factors involved in a tremendous variety of embryonic inductive processes. BMPs elicit dose-dependent effects on patterning during gastrulation and gradients of BMP activity are thought to be established through regulation of the relative concentrations of BMP receptors, ligands and antagonists. We tested whether later developmental events also are sensitive to reduced levels of BMP signaling. We engineered a knockout mouse that expresses a BMP type II receptor that lacks half of the ligand-binding domain. This altered receptor is expressed at levels comparable with the wild-type allele, but has reduced signaling capability. Unlike Bmpr2-null mice, mice homozygous for this hypomorphic receptor undergo normal gastrulation, providing genetic evidence of the dose-dependent effects of BMPs during mammalian development. Mutants, however, die at midgestation with cardiovascular and skeletal defects, demonstrating that the development of these tissues requires wild-type levels of BMP signaling. The most striking defects occur in the outflow tract of the heart, with absence of septation of the conotruncus below the valve level and interrupted aortic arch, a phenotype known in humans as persistent truncus arteriosus (type A4). In addition, semilunar valves do not form in mutants, while the atrioventricular valves appear unaffected. Abnormal septation of the heart and valve anomalies are the most frequent forms of congenital cardiac defects in humans; however, most mouse models display broad defects throughout cardiac tissues. The more restricted spectrum of cardiac anomalies in Bmpr2ΔE2 mutants makes this strain a key murine model to understand the embryonic defects of persistent truncus arteriosus and impaired semilunar valve formation in humans.

Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome

IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.

Zebrafish Radar: A new member of the TGF-β superfamily defines dorsal regions of the neural plate and the embryonic retina

Proper development of metazoan embryos requires cell to cell communications. In many instances, these communications involve diffusible molecules, particularly members of the Transforming Growth Factor beta superfamily. In an effort to identify new members of this superfamily involved in the control of early zebrafish embryogenesis, we have isolated a gene, Radar, which appears to be conserved throughout vertebrate evolution and defines a new subfamily within the superfamily. Its pattern of expression suggests that Radar plays a role in the dorso-ventral polarity of the neural plate, blood islands formation, blood cells differentiation, the establishment of retinal dorso-ventral polarity and/or proper axonal retinotectal projections. Radar expression in ntl homozygous mutants indicates that notochord and hypochord development are intimately linked.

Exome Sequencing for the Diagnosis of 46,XY Disorders of Sex Development

CONTEXT Disorders of sex development (DSD) are clinical conditions where there is a discrepancy between the chromosomal sex and the phenotypic (gonadal or genital) sex of an individual. Such conditions can be stressful for patients and their families and have historically been difficult to diagnose, especially at the genetic level. In particular, for cases of 46,XY gonadal dysgenesis, once variants in SRY and NR5A1 have been ruled out, there are few other single gene tests available. OBJECTIVE We used exome sequencing followed by analysis with a list of all known human DSD-associated genes to investigate the underlying genetic etiology of 46,XY DSD patients who had not previously received a genetic diagnosis. DESIGN Samples were either submitted to the research laboratory or submitted as clinical samples to the UCLA Clinical Genomic Center. Sequencing data were filtered using a list of genes known to be involved in DSD. RESULTS We were able to identify a likely genetic diagnosis in more than a third of cases, including 22.5% with a pathogenic finding, an additional 12.5% with likely pathogenic findings, and 15% with variants of unknown clinical significance. CONCLUSIONS Early identification of the genetic cause of a DSD will in many cases streamline and direct the clinical management of the patient, with more focused endocrine and imaging studies and better-informed surgical decisions. Exome sequencing proved an efficient method toward such a goal in 46,XY DSD patients.

Physiological and Pathological Secretion of Cartilage Oligomeric Matrix Protein by Cells in Culture

Abnormalities in cartilage oligomeric matrix protein (COMP), a pentameric structural protein of the cartilage extracellular matrix, have been identified in pseudoachondroplasia and multiple epiphyseal dysplasia, two human autosomal dominant osteochondrodysplasias. However, the function of the protein remains unknown. With the goal of establishing a model to study the mechanisms by which COMP mutations cause disease, we have analyzed synthesis and secretion of COMP in cultured chondrocytes, tendon, and ligament cells. Pentameric protein detected inside of control cells suggested that pentamerization is an intracellular process. Patient cells expressed mutant and normal RNA and secreted COMP at levels similar to controls, suggesting that abnormal pentamers are likely to be found in the extracellular matrix. Inclusions within patient cartilage stained with anti-COMP antibodies, and cultured cells presented similar inclusions, indicating that presumably abnormal COMP pentamers are less efficiently secreted than normal molecules. We conclude that the COMP disorders are likely to result from a combination of a decreased amount of COMP in the matrix and a dominant negative effect due to the presence of abnormal pentamers in cartilage.

Targeted massively parallel sequencing provides comprehensive genetic diagnosis for patients with disorders of sex development

Arboleda VA, Lee H, Sánchez FJ, Délot EC, Sandberg DE, Grody WW, Nelson SF, Vilain E. Targeted massively parallel sequencing provides comprehensive genetic diagnosis for patients with disorders of sex development.

THE BMP-RELATED PROTEIN RADAR : A MAINTENANCE FACTOR FOR DORSAL NEUROECTODERM CELLS?

We have previously cloned several members of the TGF-beta superfamily of growth factors in zebrafish, one of which, Radar, belongs to the Dpp-Vg1-related (DVR) subgroup, with highest homology to GDF6. The pattern of expression of Radar suggested a possible involvement in several induction steps during embryogenesis including in the dorsal neural tube, red blood cells, the dorsal fin and the retina. We have analyzed the pattern of expression of Radar in comparison with that of a marker of dorsal neural tube structures, msxC and show that Radar and msxC are expressed in similar and/or adjacent tissues throughout embryogenesis. In order to demonstrate a functional relationship between these two proteins, we have generated a full-length cDNA for Radar and shown that Radar overexpression by DNA injection maintains expression of msxC in tissues where it is normally expressed then turned off, in particular in the dorsal neurectoderm. Study of the phenotype of a mutant carrying a deletion of Radar shows a loss of identity and death of the cells of the dorsal neural tube. Taken together these results suggest that Radar could be involved in maintaining the identity of cells of the dorsal-most neural tube and of at least a subset of neural crest cells.

Membrane β‐catenin and adherens junctions in early gonadal patterning

Background: Mechanisms involved in early patterning of the mammalian gonad as it develops from a bipotential state into a testis or an ovary are as yet not well understood. Sex‐specific vascularization is essential in this process, but more specific mechanisms required to, for example, establish interstitial vs. cord compartments in the testis or ovigerous cords in the ovary have not been reported. Adherens junctions (AJs) are known for their roles in morphogenesis; we, therefore, examined expression of AJ components including β‐catenin, p120 catenin, and cadherins for possible involvement in sex‐specific patterning of the gonad. Results: We show that, at the time of early gonadal sex differentiation, membrane‐associated β‐catenin and p120 catenin colocalize with cell‐specific cadherins in both sex‐nonspecific and sex‐specific patterns. These expression patterns are consistent with an influence of AJs in overall patterning of the testis vs. ovary through known AJ mechanisms of cell–cell adhesion, cell sorting, and boundary formation. Conclusions: Together these complex and dynamic patterns of AJ component expression precisely mirror patterning of tissues during gonadogenesis and raise the possibility that AJs are essential effectors of patterning within the developing testis and ovary. Developmental Dynamics 241:1782–1798, 2012.

Abnormal venous and arterial patterning in chordin mutants

Classic dye injection methods yielded amazingly detailed images of normal and pathological development of the cardiovascular system. However, because these methods rely on the beating heart of diffuse the dyes, the vessels visualized have been limited to the arterial tree, and our knowledge of vein development is lagging. In order to solve this problem, we injected pigmented methylsalicylate resins in mouse embryos after they were fixed and made transparent. This new technique allowed us to image the venous system and prompted the discovery of multiple venous anomalies in Chord−/− mutant mice. Genetic inactivation of Chordin, an inhibitor of the Bone Morphogenetic Protein signaling pathway, results in neural crest defects affecting heart and neck organs, as seen in DiGeorge syndrome patients. Injection into the descending aorta of Chrd−/− mutants demonstrated how a very severe early phenotype of the aortic arches develops into persistent truncus arteriosus. In addition, injection into the atrium revealed several patterning defects of the anterior cardinal veins and their tributaries, including absence of segments, looping and midline defects. The signals that govern the development of the individual cephalic veins are unknown, but our results show that the Bone Morphogenetic Protein pathway is necessary for the process. Developmental Dynamics 236:2586–2593, 2007.