Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11.

The bones that comprise the axial skeleton have distinct morphological features characteristic of their positions along the anterior/posterior axis. We previously described a novel TGF-beta family member, myostatin (encoded by the gene Mstn, formerly Gdf8), that has an essential role in regulating skeletal muscle mass. We also identified a gene related to Mstn by low-stringency screening. While the work described here was being completed, the cloning of this gene, designated Gdf11 (also called Bmp11), was also reported by other groups. Here we show that Gdf11, a new transforming growth factor beta(TGFbeta) superfamily member, has an important role in establishing this skeletal pattern. During early mouse embryogenesis, Gdf11 is expressed in the primitive streak and tail bud regions, which are sites where new mesodermal cells are generated. Homozygous mutant mice carrying a targeted deletion of Gdf11 exhibit anteriorly directed homeotic transformations throughout the axial skeleton and posterior displacement of the hindlimbs. The effect of the mutation is dose dependent, as Gdf11+/- mice have a milder phenotype than Gdf11-/- mice. Mutant embryos show alterations in patterns of Hox gene expression, suggesting that Gdf11 acts upstream of the Hox genes. Our findings suggest that Gdf11 is a secreted signal that acts globally to specify positional identity along the anterior/posterior axis.     

MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression

MicroRNAs (miRNAs) are a class of short ( approximately 22-nt) noncoding RNA molecules that downregulate expression of their mRNA targets. Since their discovery as regulators of developmental timing in Caenorhabditis elegans, hundreds of miRNAs have been identified in both animals and plants. Here, we report a technique for visualizing detailed miRNA expression patterns in mouse embryos. We elucidate the tissue-specific expression of several miRNAs during embryogenesis, including two encoded by genes embedded in homeobox (Hox) clusters, miR-10a and miR-196a. These two miRNAs are expressed in patterns that are markedly reminiscent of those of Hox genes. Furthermore, miR-196a negatively regulates Hoxb8, indicating that its restricted expression pattern probably reflects a role in the patterning function of the Hox complex.     

SUMO modification is required for in vivo Hox gene regulation by the Caenorhabditis elegans Polycomb group protein SOP-2

Post-translational modification of proteins by the ubiquitin-like molecule SUMO (sumoylation) regulates their subcellular localization and affects their functional properties in vitro, but the physiological function of sumoylation in multicellular organisms is largely unknown. Here, we show that the C. elegans Polycomb group (PcG) protein SOP-2 interacts with the SUMO-conjugating enzyme UBC-9 through its evolutionarily conserved SAM domain. Sumoylation of SOP-2 is required for its localization to nuclear bodies in vivo and for its physiological repression of Hox genes. Global disruption of sumoylation phenocopies a sop-2 mutation by causing ectopic Hox gene expression and homeotic transformations. Chimeric constructs in which the SOP-2 SAM domain is replaced with that derived from fruit fly or mammalian PcG proteins, but not those in which the SOP-2 SAM domain is replaced with the SAM domains of non-PcG proteins, confer appropriate in vivo nuclear localization and Hox gene repression. These observations indicate that sumoylation of PcG proteins, modulated by their evolutionarily conserved SAM domain, is essential to their physiological repression of Hox genes.     

The mouse X chromosome is enriched for sex-biased genes not subject to selection by meiotic sex chromosome inactivation

Sex chromosomes are subject to sex-specific selective evolutionary forces. One model predicts that genes with sex-biased expression should be enriched on the X chromosome. In agreement with Rice's hypothesis, spermatogonial genes are over-represented on the X chromosome of mice and sex- and reproduction-related genes are over-represented on the human X chromosome. Male-biased genes are under-represented on the X chromosome in worms and flies, however. Here we show that mouse spermatogenesis genes are relatively under-represented on the X chromosome and female-biased genes are enriched on it. We used Spo11(-/-) mice blocked in spermatogenesis early in meiosis to evaluate the temporal pattern of gene expression in sperm development. Genes expressed before the Spo11 block are enriched on the X chromosome, whereas those expressed later in spermatogenesis are depleted. Inactivation of the X chromosome in male meiosis may be a universal driving force for X-chromosome demasculinization.     

Regulation of left-right patterning in mice by growth/differentiation factor-1

The transforming growth factor-beta (TGF-beta) superfamily encompasses a large group of structurally related polypeptides that are capable of regulating cell growth and differentiation in a wide range of embryonic and adult tissues. Growth/differentiation factor-1 (Gdf-1, encoded by Gdf1) is a TGF-beta family member of unknown function that was originally isolated from an early mouse embryo cDNA library and is expressed specifically in the nervous systemin late-stage embryos and adult mice. Here we show that at early stages of mouse development, Gdfl is expressed initially throughout the embryo proper and then most prominently in the primitive node, ventral neural tube, and intermediate and lateral plate mesoderm. To examine its biological function, we generated a mouse line carrying a targeted mutation in Gdf1. Gdf1-/- mice exhibited a spectrum of defects related to left-right axis formation, including visceral situs inversus, right pulmonary isomerism and a range of cardiac anomalies. In most Gdf1-/- embryos, the expression of Ebaf (formerly lefty-1) in the left side of the floor plate and Leftb (formerly lefty-2), nodal and Pitx2 in the left lateral plate mesoderm was absent, suggesting that Gdf1 acts upstream of these genes either directly or indirectly to activate their expression. Our findings suggest that Gdf1 acts early in the pathway of gene activation that leads to the establishment of left-right asymmetry.     

Herpesvirus vector gene transfer and expression of beta-glucuronidase in the central nervous system of MPS VII mice.

Genetic disorders affecting the central nervous system (CNS) can potentially be treated by gene transfer using vectors which infect and express genes in post-mitotic neurons. Herpesviruses establish latent infections in neurons during which only one viral gene (LAT) is expressed, thus the LAT promoter may express foreign genes in latently infected CNS cells. Expression of a beta-glucuronidase gene driven by the LAT promoter was tested in mice lacking this enzyme, which are a model for a human genetic disease affecting the CNS (mucopolysaccharidosis VII, Sly disease). Cells expressing the missing enzymatic activity were present in the trigeminal ganglia and brainstems of latently infected animals, up to four months post-inoculation, demonstrating the potential of this approach for the long-term expression of foreign genes in the CNS.     

A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure

Complex III (CIII; ubiquinol cytochrome c reductase of the mitochondrial respiratory chain) catalyzes electron transfer from succinate and nicotinamide adenine dinucleotide-linked dehydrogenases to cytochrome c. CIII is made up of 11 subunits, of which all but one (cytochrome b) are encoded by nuclear DNA. CIII deficiencies are rare and manifest heterogeneous clinical presentations. Although pathogenic mutations in the gene encoding mitochondrial cytochrome b have been described, mutations in the nuclear-DNA-encoded subunits have not been reported. Involvement of various genes has been indicated in assembly of yeast CIII (refs. 8-11). So far only one such gene, BCS1L, has been identified in human. BCS1L represents, therefore, an obvious candidate gene in CIII deficiency. Here, we report BCS1L mutations in six patients, from four unrelated families and presenting neonatal proximal tubulopathy, hepatic involvement and encephalopathy. Complementation study in yeast confirmed the deleterious effect of these mutations. Mutation of BCS1L would seem to be a frequent cause of CIII deficiency, as one-third of our patients have BCS1L mutations.     

Retinopathy and attenuated circadian entrainment in Crx-deficient mice

Crx, an Otx-like homeobox gene, is expressed specifically in the photoreceptors of the retina and the pinealocytes of the pineal gland. Crx has been proposed to have a role in the regulation of photoreceptor-specific genes in the eye and of pineal-specific genes in the pineal gland. Mutations in human CRX are associated with the retinal diseases, cone-rod dystrophy-2 (adCRD2; refs 3, 4, 5), retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), which all lead to loss of vision. We generated mice carrying a targeted disruption of Crx. Crx-/- mice do not elaborate photoreceptor outer segments and lacked rod and cone activity as assayed by electroretinogram (ERG). Expression of several photoreceptor- and pineal-specific genes was reduced in Crx mutants. Circadian entrainment was also affected in Crx-/- mice.     

Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production

The gene Ucp2 is a member of a family of genes found in animals and plants, encoding a protein homologous to the brown fat uncoupling protein Ucp1 (refs 1-3). As Ucp2 is widely expressed in mammalian tissues, uncouples respiration and resides within a region of genetic linkage to obesity, a role in energy dissipation has been proposed. We demonstrate here, however, that mice lacking Ucp2 following targeted gene disruption are not obese and have a normal response to cold exposure or high-fat diet. Expression of Ucp2 is robust in spleen, lung and isolated macrophages, suggesting a role for Ucp2 in immunity or inflammatory responsiveness. We investigated the response to infection with Toxoplasma gondii in Ucp2-/- mice, and found that they are completely resistant to infection, in contrast with the lethality observed in wild-type littermates. Parasitic cysts and inflammation sites in brain were significantly reduced in Ucp2-/- mice (63% decrease, P<0.04). Macrophages from Ucp2-/- mice generated more reactive oxygen species than wild-type mice (80% increase, P<0.001) in response to T. gondii, and had a fivefold greater toxoplasmacidal activity in vitro compared with wild-type mice (P<0.001 ), which was absent in the presence of a quencher of reactive oxygen species (ROS). Our results indicate a role for Ucp2 in the limitation of ROS and macrophage-mediated immunity.     

Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9.

The initial stages of pancreatic development occur early during mammalian embryogenesis, but the genes governing this process remain largely unknown. The homeodomain protein Pdx1 is expressed in the developing pancreatic anlagen from the approximately 10-somite stage, and mutations in the gene Pdx1 prevent the development of the pancreas. The initial stages of pancreatic development, however, still occur in Pdx1-deficient mice. Hlxb9 (encoding Hb9; ref. 6) is a homeobox gene that in humans has been linked to dominant inherited sacral agenesis and we show here that Hb9 is expressed at early stages of mouse pancreatic development and later in differentiated beta-cells. Hlxb9 has an essential function in the initial stages of pancreatic development. In absence of Hlxb9 expression, the dorsal region of the gut epithelium fails to initiate a pancreatic differentiation program. In contrast, the ventral pancreatic endoderm develops but exhibits a later and more subtle perturbation in beta-cell differentiation and in islet cell organization. Thus, dorsally Hlxb9 is required for specifying the gut epithelium to a pancreatic fate and ventrally for ensuring proper endocrine cell differentiation.     

Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome

Congenital central hypoventilation syndrome (CCHS or Ondine's curse; OMIM 209880) is a life-threatening disorder involving an impaired ventilatory response to hypercarbia and hypoxemia. This core phenotype is associated with lower-penetrance anomalies of the autonomic nervous system (ANS) including Hirschsprung disease and tumors of neural-crest derivatives such as ganglioneuromas and neuroblastomas. In mice, the development of ANS reflex circuits is dependent on the paired-like homeobox gene Phox2b. Thus, we regarded its human ortholog, PHOX2B, as a candidate gene in CCHS. We found heterozygous de novo mutations in PHOX2B in 18 of 29 individuals with CCHS. Most mutations consisted of 5-9 alanine expansions within a 20-residue polyalanine tract probably resulting from non-homologous recombination. We show that PHOX2B is expressed in both the central and the peripheral ANS during human embryonic development. Our data support an essential role of PHOX2B in the normal patterning of the autonomous ventilation system and, more generally, of the ANS in humans.     

Role of retrotransposon-derived imprinted gene, Rtl1, in the feto-maternal interface of mouse placenta

Eutherian placenta, an organ that emerged in the course of mammalian evolution, provides essential architecture, the so-called feto-maternal interface, for fetal development by exchanging nutrition, gas and waste between fetal and maternal blood. Functional defects of the placenta cause several developmental disorders, such as intrauterine growth retardation in humans and mice. A series of new inventions and/or adaptations must have been necessary to form and maintain eutherian chorioallantoic placenta, which consists of capillary endothelial cells and a surrounding trophoblast cell layer(s). Although many placental genes have been identified, it remains unknown how the feto-maternal interface is formed and maintained during development, and how this novel design evolved. Here we demonstrate that retrotransposon-derived Rtl1 (retrotransposon-like 1), also known as Peg11 (paternally expressed 11), is essential for maintenance of the fetal capillaries, and that both its loss and its overproduction cause late-fetal and/or neonatal lethality in mice.     

Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. The paternally expressed gene insulin-like growth-factor 2 (Igf2) is separated by approximately 100 kb from the maternally expressed noncoding gene H19 on mouse distal chromosome 7. Differentially methylated regions in Igf2 and H19 contain chromatin boundaries, silencers and activators and regulate the reciprocal expression of the two genes in a methylation-sensitive manner by allowing them exclusive access to a shared set of enhancers. Various chromatin models have been proposed that separate Igf2 and H19 into active and silent domains. Here we used a GAL4 knock-in approach as well as the chromosome conformation capture technique to show that the differentially methylated regions in the imprinted genes Igf2 and H19 interact in mice. These interactions are epigenetically regulated and partition maternal and paternal chromatin into distinct loops. This generates a simple epigenetic switch for Igf2 through which it moves between an active and a silent chromatin domain.     

Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality

By comparing mammalian genomes, we and others have identified actively transcribed Ty3/gypsy retrotransposon-derived genes with highly conserved DNA sequences and insertion sites. To elucidate the functions of evolutionarily conserved retrotransposon-derived genes in mammalian development, we produced mice that lack one of these genes, Peg10 (paternally expressed 10), which is a paternally expressed imprinted gene on mouse proximal chromosome 6. The Peg10 knockout mice showed early embryonic lethality owing to defects in the placenta. This indicates that Peg10 is critical for mouse parthenogenetic development and provides the first direct evidence of an essential role of an evolutionarily conserved retrotransposon-derived gene in mammalian development.