Individuals with the birth defect synpolydactyly (SPD) have 1 or more digit duplicated and 2 or more digits fused together. One form of SPD is caused by polyalanine expansions in homeobox d13 (Hoxd13). Here we have used the naturally occurring mouse mutant that has the same mutation, the SPD homolog (Spdh) allele, and a similar phenotype, to investigate the molecular pathogenesis of SPD. A transgenic approach and crossing experiments showed that the Spdh allele is a combination of loss and gain of function. Here we identify retinaldehyde dehydrogenase 2 (Raldh2), the rate-limiting enzyme for retinoic acid (RA) synthesis in the limb, as a direct Hoxd13 target and show decreased RA production in limbs from Spdh/Spdh mice. Intrauterine treatment with RA restored pentadactyly in Spdh/Spdh mice. We further show that RA and WT Hoxd13 suppress chondrogenesis in mesenchymal progenitor cells, whereas Hoxd13 encoded by Spdh promotes cartilage formation in primary cells isolated from Spdh/Spdh limbs, and that this was associated with increased expression of Sox6/9. Increased Sox9 expression and ectopic cartilage formation in the interdigital mesenchyme of limbs from Spdh/Spdh mice suggest uncontrolled differentiation of these cells into the chondrocytic lineage. Thus, we propose that mutated Hoxd13 causes polydactyly in SPD by inducing extraneous interdigital chondrogenesis, both directly and indirectly, via a reduction in RA levels.
Pia Kuss, Pablo Villavicencio-Lorini, Florian Witte, Joachim Klose, Andrea N. Albrecht, Petra Seemann, Jochen Hecht, Stefan Mundlos
Based on extensive preclinical data, glycogen synthase kinase–3 (GSK-3) has been proposed to be a viable drug target for a wide variety of disease states, ranging from diabetes to bipolar disorder. Since these new drugs, which will be more powerful GSK-3 inhibitors than lithium, may potentially be given to women of childbearing potential, and since it has controversially been suggested that lithium therapy might be linked to congenital cardiac defects, we asked whether GSK-3 family members are required for normal heart development in mice. We report that terminal cardiomyocyte differentiation was substantially blunted in Gsk3b–/– embryoid bodies. While GSK-3α–deficient mice were born without a cardiac phenotype, no live-born Gsk3b–/– pups were recovered. The Gsk3b–/– embryos had a double outlet RV, ventricular septal defects, and hypertrophic myopathy, with near obliteration of the ventricular cavities. The hypertrophic myopathy was caused by cardiomyocyte hyperproliferation without hypertrophy and was associated with increased expression and nuclear localization of three regulators of proliferation — GATA4, cyclin D1, and c-Myc. These studies, which we believe are the first in mammals to examine the role of GSK-3α and GSK-3β in the heart using loss-of-function approaches, implicate GSK-3β as a central regulator of embryonic cardiomyocyte proliferation and differentiation, as well as of outflow tract development. Although controversy over the teratogenic effects of lithium remains, our studies suggest that caution should be exercised in the use of newer, more potent drugs targeting GSK-3 in women of childbearing age.
Risto Kerkela, Lisa Kockeritz, Katrina MacAulay, Jibin Zhou, Bradley W. Doble, Cara Beahm, Sarah Greytak, Kathleen Woulfe, Chinmay M. Trivedi, James R. Woodgett, Jonathan A. Epstein, Thomas Force, Gordon S. Huggins
Joubert syndrome is an autosomal recessive disorder characterized by congenital malformation of the cerebellum and brainstem, with abnormal decussation in the brain. Mutations in the Abelson helper integration site 1 gene, which encodes the protein AHI1, have been shown to cause Joubert syndrome. In this study, we found that mouse Ahi1 formed a stable complex with huntingtin-associated protein 1 (Hap1), which is critical for neonatal development and involved in intracellular trafficking. Hap1-knockout mice showed significantly reduced Ahi1 levels, defective cerebellar development, and abnormal axonal decussation. Suppression of Ahi1 also decreased the level of Hap1; and truncated Ahi1, which corresponds to the mutations in Joubert syndrome, inhibited neurite outgrowth in neuronal culture. Reducing Hap1 expression suppressed the level and internalization of TrkB, a neurotrophic factor receptor that mediates neurogenesis and neuronal differentiation, which led to decreased TrkB signaling. These findings provide insight into the pathogenesis of Joubert syndrome and demonstrate the critical role of the Ahi1-Hap1 complex in early brain development.
Guoqing Sheng, Xingshun Xu, Yung-Feng Lin, Chuan-En Wang, Juan Rong, Dongmei Cheng, Junmin Peng, Xiaoyan Jiang, Shi-Hua Li, Xiao-Jiang Li
Transcription factors regulate tissue patterning and cell fate determination during development; however, expression of early regulators frequently abates upon differentiation, suggesting that they may also play a role in maintaining an undifferentiated phenotype. The transcription factor paired box 3 (Pax3) is expressed by multipotent neural crest precursors and is implicated in neural crest disorders in humans such as Waardenburg syndrome. Pax3 is required for development of multiple neural crest lineages and for activation of lineage-specific programs, yet expression is generally extinguished once neural crest cells migrate from the dorsal neural tube and differentiate. Using a murine Cre-inducible system, we asked whether persistent Pax3 expression in neural crest derivatives would affect development or patterning. We found that persistent expression of Pax3 in cranial neural crest cells resulted in cleft palate, ocular defects, malformation of the sphenoid bone, and perinatal lethality. Furthermore, we demonstrated that Pax3 directly regulates expression of Sostdc1, a soluble inhibitor of bone morphogenetic protein (BMP) signaling. Persistent Pax3 expression renders the cranial crest resistant to BMP-induced osteogenesis. Thus, one mechanism by which Pax3 maintains the undifferentiated state of neural crest mesenchyme may be to block responsiveness to differentiation signals from the environment. These studies provide in vivo evidence for the importance of Pax3 downregulation during differentiation of multipotent neural crest precursors and cranial development.
Meilin Wu, Jun Li, Kurt A. Engleka, Bo Zhou, Min Min Lu, Joshua B. Plotkin, Jonathan A. Epstein
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
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