The transforming growth factor-beta (TGF-beta) superfamily encompasses a large group of growth and differentiation factors playing important roles in regulating embryonic development and in maintaining tissue homeostasis in adult animals. Using degenerate polymerase chain reaction, we have identified a new murine TGF-beta family member, growth/differentiation factor-8 (GDF-8), which is expressed specifically in developing and adult skeletal muscle. During early stages of embryogenesis, GDF-8 expression is restricted to the myotome compartment of developing somites. At later stages and in adult animals, GDF-8 is expressed in many different muscles throughout the body. To determine the biological function of GDF-8, we disrupted the GDF-8 gene by gene targeting in mice. GDF-8 null animals are significantly larger than wild-type animals and show a large and widespread increase in skeletal muscle mass. Individual muscles of mutant animals weigh 2-3 times more than those of wild-type animals, and the increase in mass appears to result from a combination of muscle cell hyperplasia and hypertrophy. These results suggest that GDF-8 functions specifically as a negative regulator of skeletal muscle growth.

Hypoxia is an essential developmental and physiological stimulus that plays a key role in the pathophysiology of cancer, heart attack, stroke, and other major causes of mortality. Hypoxia-inducible factor 1 (HIF-1) is the only known mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia. We now report that in Hif1a −/− embryonic stem cells that did not express the O 2 -regulated HIF-1␣ subunit, levels of mRNAs encoding glucose transporters and glycolytic enzymes were reduced, and cellular proliferation was impaired. Vascular endothelial growth factor mRNA expression was also markedly decreased in hypoxic Hif1a −/− embryonic stem cells and cystic embryoid bodies. Complete deficiency of HIF-1␣ resulted in developmental arrest and lethality by E11 of Hif1a −/− embryos that manifested neural tube defects, cardiovascular malformations, and marked cell death within the cephalic mesenchyme. In Hif1a +/+ embryos, HIF-1␣ expression increased between E8.5 and E9.5, coincident with the onset of developmental defects and cell death in Hif1a −/− embryos. These results demonstrate that HIF-1␣ is a master regulator of cellular and developmental O 2 homeostasis.

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.

The metabolism of poly(ADP-ribose) (PAR) is critical for genomic stability in multicellular eukaryotes. Here, we show that the failure to degrade PAR by means of disruption of the murine poly(ADPribose) glycohydrolase (PARG) gene unexpectedly causes early embryonic lethality and enhanced sensitivity to genotoxic stress. This lethality results from the failure to hydrolyze PAR, because PARG null embryonic day (E) 3.5 blastocysts accumulate PAR and concurrently undergo apoptosis. Moreover, embryonic trophoblast stem cell lines established from early PARG null embryos are viable only when cultured in medium containing the poly(ADPribose) polymerase inhibitor benzamide. Cells lacking PARG also show reduced growth, accumulation of PAR, and increased sensitivity to cytotoxicity induced by N-methyl-N -nitro-N-nitrosoguanidine and menadione after benzamide withdrawal. These results provide compelling evidence that the failure to degrade PAR has deleterious consequences. Further, they define a role for PARG in embryonic development and a protective role in the response to genotoxic stress.poly(ADP-ribose) glycohydrolase ͉ poly(ADP-ribose) polymerase ͉ apoptosis

No neurological involvement has been detected in X-ALD mice up to 6 months. We conclude that X-ALD mice exhibit biochemical defects equivalent to those found in human X-ALD and thus provide an experimental system for testing therapeutic intervention.

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.

Overexpression of the gene encoding the beta-amyloid precursor protein (APP) may have a key role in the pathogenesis of both Alzheimer's disease (AD) and Down Syndrome (DS). We have therefore introduced a 650 kilobase (kb) yeast artificial chromosome (YAC) that contains the entire, unrearranged 400 kb human APP gene into mouse embryonic stem (ES) cells by lipid-mediated transfection. ES lines were generated that contain a stably integrated, unrearranged human APP gene. Moreover, we demonstrate germ line transmission of the APP YAC in transgenic mice and expression of human APP mRNA and protein at levels comparable to endogenous APP. This transgenic strategy may prove invaluable for the development of mouse models for AD and DS.

Deficiency of ornithine-delta-aminotransferase (OAT) in humans causes hyperornithinaemia and gyrate atrophy (GA), a blinding chorioretinal degeneration. Surprisingly, OAT-deficient mice produced by gene targeting exhibit neonatal hypoornithinaemia and lethality, rescuable by short-term arginine supplementation. Post-weaning, these mice develop hyperornithinaemia similar to human GA patients. Subsequent studies in one human GA infant also showed transient hypoornithinaemia. Thus, the OAT reaction plays opposite roles in neonatal and adult mammals. Over several months, OAT-deficient mice develop a retinal degeneration with involvement of photoreceptors and pigment epithelium. OAT-deficient mice appear to be an excellent model of human GA.