Becoming a phenotypic male is ultimately determined by androgen-induced masculinization. Disorders of fetal masculinization, resulting in hypospadias or cryptorchidism, are common, but their cause remains unclear. Together with the adult-onset disorders low sperm count and testicular cancer, they can constitute a testicular dysgenesis syndrome (TDS). Although masculinization is well studied, no unifying concept explains normal male reproductive development and its abnormalities, including TDS. We exposed rat fetuses to either anti-androgens or androgens and showed that masculinization of all reproductive tract tissues was programmed by androgen action during a common fetal programming window. This preceded morphological differentiation, when androgen action was, surprisingly, unnecessary. Only within the programming window did blocking androgen action induce hypospadias and cryptorchidism and altered penile length in male rats, all of which correlated with anogenital distance (AGD). Androgen-driven masculinization of females was also confined to the same programming window. This work has identified in rats a common programming window in which androgen action is essential for normal reproductive tract masculinization and has highlighted that measuring AGD in neonatal humans could provide a noninvasive method to predict neonatal and adult reproductive disorders. Based on the timings in rats, we believe the programming window in humans is likely to be 8–14 weeks of gestation.
Michelle Welsh, Philippa T.K. Saunders, Mark Fisken, Hayley M. Scott, Gary R. Hutchison, Lee B. Smith, Richard M. Sharpe
In humans, hereditary inactivation of either p22phox or gp91phox leads to chronic granulomatous disease (CGD), a severe immune disorder characterized by the inability of phagocytes to produce bacteria-destroying ROS. Heterodimers of p22phox and gp91phox proteins constitute the superoxide-producing cytochrome core of the phagocyte NADPH oxidase. In this study, we identified the nmf333 mouse strain as what we believe to be the first animal model of p22phox deficiency. Characterization of nmf333 mice revealed that deletion of p22phox inactivated not only the phagocyte NADPH oxidase, but also a second cytochrome in the inner ear epithelium. As a consequence, mice of the nmf333 strain exhibit a compound phenotype consisting of both a CGD-like immune defect and a balance disorder caused by the aberrant development of gravity-sensing organs. Thus, in addition to identifying a model of p22phox-dependent immune deficiency, our study indicates that a clinically identifiable patient population with an otherwise cryptic loss of gravity-sensor function may exist. Thus, p22phox represents a shared and essential component of at least 2 superoxide-producing cytochromes with entirely different biological functions. The site of p22phox expression in the inner ear leads us to propose what we believe to be a novel mechanism for the control of vestibular organogenesis.
Yoko Nakano, Chantal M. Longo-Guess, David E. Bergstrom, William M. Nauseef, Sherri M. Jones, Botond Bánfi
Myocardin (Myocd) is a potent transcriptional coactivator that has been implicated in cardiovascular development and adaptation of the cardiovascular system to hemodynamic stress. To determine the function of myocardin in the developing cardiovascular system, MyocdF/F/Wnt1-Cre+ and MyocdF/F/Pax3-Cre+ mice were generated in which the myocardin gene was selectively ablated in neural crest–derived SMCs populating the cardiac outflow tract and great arteries. Both MyocdF/F/Wnt1-Cre+ and MyocdF/F/Pax3-Cre+ mutant mice survived to birth, but died prior to postnatal day 3 from patent ductus arteriosus (PDA). Neural crest–derived SMCs populating the ductus arteriosus (DA) and great arteries exhibited a cell autonomous block in expression of myocardin-regulated genes encoding SMC-restricted contractile proteins. Moreover, Myocd-deficient vascular SMCs populating the DA exhibited ultrastructural features generally associated with the SMC synthetic, rather than contractile, phenotype. Consistent with these findings, ablation of the Myocd gene in primary aortic SMCs harvested from Myocd conditional mutant mice caused a dramatic decrease in SMC contractile protein expression. Taken together, these data demonstrate that myocardin regulates expression of genes required for the contractile phenotype in neural crest–derived SMCs and provide new insights into the molecular and genetic programs that may underlie PDA.
Jianhe Huang, Lan Cheng, Jian Li, Mary Chen, Deying Zhou, Min Min Lu, Aaron Proweller, Jonathan A. Epstein, Michael S. Parmacek
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder associated with ciliary defects and situs inversus totalis, the complete mirror image reversal of internal organ situs (positioning). A variable incidence of heterotaxy, or irregular organ situs, also has been reported in PCD patients, but it is not known whether this is elicited by the PCD-causing genetic lesion. We studied a mouse model of PCD with a recessive mutation in Dnahc5, a dynein gene commonly mutated in PCD. Analysis of homozygous mutant embryos from 18 litters yielded 25% with normal organ situs, 35% with situs inversus totalis, and 40% with heterotaxy. Embryos with heterotaxy had complex structural heart defects that included discordant atrioventricular and ventricular outflow situs and atrial/pulmonary isomerisms. Variable combinations of a distinct set of cardiovascular anomalies were observed, including superior-inferior ventricles, great artery alignment defects, and interrupted inferior vena cava with azygos continuation. The surprisingly high incidence of heterotaxy led us to evaluate the diagnosis of PCD. PCD was confirmed by EM, which revealed missing outer dynein arms in the respiratory cilia. Ciliary dyskinesia was observed by videomicroscopy. These findings show that Dnahc5 is required for the specification of left-right asymmetry and suggest that the PCD-causing Dnahc5 mutation may also be associated with heterotaxy.
Serena Y. Tan, Julie Rosenthal, Xiao-Qing Zhao, Richard J. Francis, Bishwanath Chatterjee, Steven L. Sabol, Kaari L. Linask, Luciann Bracero, Patricia S. Connelly, Mathew P. Daniels, Qing Yu, Heymut Omran, Linda Leatherbury, Cecilia W. Lo
The discovery of fetal mRNA transcripts in the maternal circulation holds great promise for noninvasive prenatal diagnosis. To identify potential fetal biomarkers, we studied whole blood and plasma gene transcripts that were common to 9 term pregnant women and their newborns but absent or reduced in the mothers postpartum. RNA was isolated from peripheral or umbilical blood and hybridized to gene expression arrays. Gene expression, paired Student’s t test, and pathway analyses were performed. In whole blood, 157 gene transcripts met statistical significance. These fetal biomarkers included 27 developmental genes, 5 sensory perception genes, and 22 genes involved in neonatal physiology. Transcripts were predominantly expressed or restricted to the fetus, the embryo, or the neonate. Real-time RT-PCR amplification confirmed the presence of specific gene transcripts; SNP analysis demonstrated the presence of 3 fetal transcripts in maternal antepartum blood. Comparison of whole blood and plasma samples from the same pregnant woman suggested that placental genes are more easily detected in plasma. We conclude that fetal and placental mRNA circulates in the blood of pregnant women. Transcriptional analysis of maternal whole blood identifies a unique set of biologically diverse fetal genes and has a multitude of clinical applications.
Jill L. Maron, Kirby L. Johnson, Donna Slonim, Chao-Qiang Lai, Marco Ramoni, Gil Alterovitz, Zina Jarrah, Zinger Yang, Diana W. Bianchi
In humans, loss-of-function mutations in the gene encoding Wnt1 inducible signaling pathway protein 3 (WISP3) cause the autosomal-recessive skeletal disorder progressive pseudorheumatoid dysplasia (PPD). However, in mice there is no apparent phenotype caused by Wisp3 deficiency or overexpression. Consequently, the in vivo activities of Wisp3 have remained elusive. We cloned the zebrafish ortholog of Wisp3 and investigated its biologic activity in vivo using gain-of-function and loss-of-function approaches. Overexpression of zebrafish Wisp3 protein inhibited bone morphogenetic protein (BMP) and Wnt signaling in developing zebrafish. Conditioned medium–containing zebrafish and human Wisp3 also inhibited BMP and Wnt signaling in mammalian cells by binding to BMP ligand and to the Wnt coreceptors low-density lipoprotein receptor–related protein 6 (LRP6) and Frizzled, respectively. Wisp3 proteins containing disease-causing amino acid substitutions found in patients with PPD had reduced activity in these assays. Morpholino-mediated inhibition of zebrafish Wisp3 protein expression in developing zebrafish affected pharyngeal cartilage size and shape. These data provide a biologic assay for Wisp3, reveal a role for Wisp3 during zebrafish cartilage development, and suggest that dysregulation of BMP and/or Wnt signaling contributes to cartilage failure in humans with PPD.
Yukio Nakamura, Gilbert Weidinger, Jennifer O. Liang, Allisan Aquilina-Beck, Keiko Tamai, Randall T. Moon, Matthew L. Warman
Forkhead box O (Foxo) transcription factors govern metabolism and cellular differentiation. Unlike Foxo-dependent metabolic pathways and target genes, the mechanisms by which these proteins regulate differentiation have not been explored. Activation of Notch signaling mimics the effects of Foxo gain of function on cellular differentiation. Using muscle differentiation as a model system, we show that Foxo physically and functionally interacts with Notch by promoting corepressor clearance from the Notch effector Csl, leading to activation of Notch target genes. Inhibition of myoblast differentiation by constitutively active Foxo1 is partly rescued by inhibition of Notch signaling while Foxo1 loss of function precludes Notch inhibition of myogenesis and increases myogenic determination gene (MyoD) expression. Accordingly, conditional Foxo1 ablation in skeletal muscle results in increased formation of MyoD-containing (fast-twitch) muscle fibers and altered fiber type distribution at the expense of myogenin-containing (slow-twitch) fibers. Notch/Foxo1 cooperation may integrate environmental cues through Notch with metabolic cues through Foxo1 to regulate progenitor cell maintenance and differentiation.
Tadahiro Kitamura, Yukari Ido Kitamura, Yasuhiro Funahashi, Carrie J. Shawber, Diego H. Castrillon, Ramya Kollipara, Ronald A. DePinho, Jan Kitajewski, Domenico Accili
Holoprosencephaly (HPE) is a clinically heterogeneous developmental anomaly affecting the CNS and face, in which the embryonic forebrain fails to divide into distinct halves. Numerous genetic loci and environmental factors are implicated in HPE, but mutation in the sonic hedgehog (Shh) gene is an established cause in both humans and mice. As growth arrest–specific 1 (Gas1) encodes a membrane glycoprotein previously identified as a Shh antagonist in the somite, we analyzed the craniofacial phenotype of mice harboring a targeted Gas1 deletion. Gas1–/– mice exhibited microform HPE, including midfacial hypoplasia, premaxillary incisor fusion, and cleft palate, in addition to severe ear defects; however, gross integrity of the forebrain remained intact. These defects were associated with partial loss of Shh signaling in cells at a distance from the source of transcription, suggesting that Gas1 can potentiate hedgehog signaling in the early face. Loss of a single Shh allele in a Gas1–/– background significantly exacerbated the midline craniofacial phenotype, providing genetic evidence that Shh and Gas1 interact. As human GAS1 maps to chromosome 9q21.3–q22, a region previously associated with nonsyndromic cleft palate and congenital deafness, our results establish GAS1 as a potential locus for several human craniofacial malformations.
Maisa Seppala, Michael J. Depew, David C. Martinelli, Chen-Ming Fan, Paul T. Sharpe, Martyn T. Cobourne
Ewing sarcoma gene EWS encodes a putative RNA-binding protein with proposed roles in transcription and splicing, but its physiological role in vivo remains undefined. Here, we have generated Ews-deficient mice and demonstrated that EWS is required for the completion of B cell development and meiosis. Analysis of Ews–/– lymphocytes revealed a cell-autonomous defect in precursor B lymphocyte (pre–B lymphocyte) development. During meiosis, Ews-null spermatocytes were deficient in XY bivalent formation and showed reduced meiotic recombination, resulting in massive apoptosis and complete arrest in gamete maturation. Inactivation of Ews in mouse embryonic fibroblasts resulted in premature cellular senescence, and the mutant animals showed hypersensitivity to ionizing radiation. Finally, we showed that EWS interacts with lamin A/C and that loss of EWS results in a reduced lamin A/C expression. Our findings reveal essential functions for EWS in pre–B cell development and meiosis, with proposed roles in DNA pairing and recombination/repair mechanisms. Furthermore, we demonstrate a novel role of EWS in cellular senescence, possibly through its interaction and modulation of lamin A/C.
Hongjie Li, Wendy Watford, Cuiling Li, Alissa Parmelee, Mark A. Bryant, Chuxia Deng, John O’Shea, Sean Bong Lee
Insig-1 and Insig-2 are regulatory proteins that restrict the cholesterol biosynthetic pathway by preventing proteolytic activation of SREBPs and by enhancing degradation of HMG-CoA reductase. Here, we created Insig–double-knockout (Insig-DKO) mice that are homozygous for null mutations in Insig-1 and Insig-2. After 18.5 days of development, 96% of Insig-DKO embryos had defects in midline facial development, ranging from cleft palate (52%) to complete cleft face (44%). Middle and inner ear structures were abnormal, but teeth and skeletons were normal. The animals were lethargic and runted; they died within 1 day of birth. The livers and heads of Insig-DKO embryos overproduced sterols, causing a marked buildup of sterol intermediates. Treatment of pregnant mice with the HMG-CoA reductase inhibitor lovastatin reduced sterol synthesis in Insig-DKO embryos and reduced the pre-cholesterol intermediates. This treatment ameliorated the clefting syndrome so that 54% of Insig-DKO mice had normal faces, and only 7% had cleft faces. We conclude that buildup of pre-cholesterol sterol intermediates interferes with midline fusion of facial structures in mice. These findings have implications for the pathogenesis of the cleft palate component of Smith-Lemli-Opitz syndrome and other human malformation syndromes in which mutations in enzymes catalyzing steps in cholesterol biosynthesis produce a buildup of sterol intermediates.
Luke J. Engelking, Bret M. Evers, James A. Richardson, Joseph L. Goldstein, Michael S. Brown, Guosheng Liang
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