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In mammals, the developing reproductive tract primordium of male and female fetuses consists of the Wolffian duct and the Müllerian duct (MD), two epithelial tube pairs surrounded by mesenchyme. During male development, mesenchyme-epithelia interactions mediate MD regression to prevent its development into a uterus, oviduct and upper vagina. It is well established that transforming growth factor-beta family member anti-Müllerian hormone (AMH) secreted from the fetal testis and its type 1 and 2 receptors expressed in MD mesenchyme regulate MD regression. However, little is known about the molecular network regulating downstream actions of AMH signaling. To identify potential AMH-induced genes and regulatory networks controlling MD regression in a global non-biased manner, we examined transcriptome differences in MD mesenchyme between males (AMH signaling on) and females (AMH signaling off) by RNA-Seq analysis of purified fetal MD mesenchymal cells. This analysis found 82 genes up-regulated in males during MD regression and identifiedOsterix (Osx)/Sp7, a key transcriptional regulator of osteoblast differentiation and bone formation, as a novel downstream effector of AMH signaling during MD regression. Osx/OSX was expressed in a male-specific pattern in MD mesenchyme during MD regression. OSX expression was lost in mutant males without AMH signaling. In addition, transgenic mice ectopically expressing human AMH in females induced a male pattern ofOsxexpression. Together these results indicate that AMH signaling is necessary and sufficient forOsxexpression in the MD mesenchyme. In addition, MD regression was delayed inOsxnull males, identifyingOsxas a new factor that regulates MD regression.SignificanceIn mammals, each embryo forms both male and female reproductive tract organ progenitor tissues. Anti-Müllerian hormone (AMH) secreted by fetal testes acts on mesenchyme cells adjacent to the Müllerian duct (MD) epithelium, the progenitor tissue of the female reproductive tract, to induce MD regression. While AMH and early AMH signaling components are elucidated, downstream gene networks directing this process are largely unknown. A global non-biased approach using whole transcriptome sequencing of fetal MD mesenchymal cells identified 82 factors as potential target genes of AMH includingOsterix (Osx). Our findings providein vivoevidenceOsxis an AMH-induced gene that regulates MD regression. Identification ofOsxmay provide key insights into gene regulatory networks underlying MD regression and male sex differentiation.

Purkinje cells are one of the major types of neurons that form the neural circuitry in the cerebellum essential for fine control of movement and posture. During development, Purkinje cells also are critically involved in the regulation of proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. The process that controls differentiation of Purkinje cells from their early precursors is poorly understood. Here we report that two closely related LIM-homeobox genes, Lhx1 and Lhx5 , were expressed in the developing Purkinje cells soon after they exited the cell cycle and migrated out of the cerebellar ventricular zone. Double-mutant mice lacking function of both Lhx1 and Lhx5 showed a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1 , which encodes a cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. Our studies thus provide evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum.

Aminopeptidase N (APN, CD13; EC 3.4.11.2) is a transmembrane metalloprotease with several functions, depending on the cell type and tissue environment. In tumor vasculature, APN is overexpressed in the endothelium and promotes angiogenesis. However, there have been no reports ofin vivoinactivation of the APN gene to validate these findings. Here we evaluated, by targeted disruption of the APN gene, whether APN participates in blood vessel formation and function under normal conditions. Surprisingly, APN-null mice developed with no gross or histological abnormalities. Standard neurological, cardiovascular, metabolic, locomotor, and hematological studies revealed no alterations. Nonetheless, in oxygen-induced retinopathy experiments, APN-deficient mice had a marked and dose-dependent deficiency of the expected retinal neovascularization. Moreover, gelfoams embedded with growth factors failed to induce functional blood vessel formation in APN-null mice. These findings establish that APN-null mice develop normally without physiological alterations and can undergo physiological angiogenesis but show a severely impaired angiogenic response under pathological conditions. Finally, in addition to vascular biology research, APN-null mice may be useful reagents in other medical fields such as malignant, cardiovascular, immunological, or infectious diseases.

Potential repair by cell grafting or mobilizing endogenous cells holds particular attraction in heart disease, where the meager capacity for cardiomyocyte proliferation likely contributes to the irreversibility of heart failure. Whether cardiac progenitors exist in adult myocardium itself is unanswered, as is the question whether undifferentiated cardiac precursor cells merely fuse with preexisting myocytes. Here we report the existence of adult heart-derived cardiac progenitor cells expressing stem cell antigen-1. Initially, the cells express neither cardiac structural genes nor Nkx2.5 but differentiate in vitro in response to 5′-azacytidine, in part depending on Bmpr1a , a receptor for bone morphogenetic proteins. Given intravenously after ischemia/reperfusion, cardiac stem cell antigen 1 cells home to injured myocardium. By using a Cre/Lox donor/recipient pair (α MHC-Cre / R26R ), differentiation was shown to occur roughly equally, with and without fusion to host cells.

PAX6 is an evolutionarily conserved transcription factor that plays a critical role in vertebrate and invertebrate eye formation. Heterozygous null mutations in the PAX6 gene result in aniridia in humans and a distinct small eye syndrome in rodents. Vertebrates primarily express two alternatively spliced isoforms of Pax6 that differ by the presence or absence of exon 5a (e5A) that encodes an additional 14 aa residues within the paired domain. The e5a-containing isoform, PAX6(5a), is specific to and conserved in vertebrates. To determine the role of PAX6(5a), we have generated mice that lack e5a of the Pax6 gene. Unlike Pax6 null mice that exhibit anopthalmia with central nervous system defects and lethality, 5a isoform-null mice have iris hypoplasia and defects in the cornea, lens, and retina. Although invertebrates have structures that respond to light intensity and act to restrict light exposure of the eyes, a significant and distinct feature of the vertebrate eye is its ability to regulate the amount of incoming light through contractile pupils. This feature of the eye not only allows vertebrates to see in various light conditions but also enhances image resolution. The requirement of the 5a isoform in iris formation suggests that the evolution of this isoform contributed to advanced features of the vertebrate eye.

Receptors for bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGFbeta) superfamily, are persistently expressed during cardiac development, yet mice lacking type II or type IA BMP receptors die at gastrulation and cannot be used to assess potential later roles in creation of the heart. Here, we used a Cre/lox system for cardiac myocyte-specific deletion of the type IA BMP receptor, ALK3. ALK3 was specifically required at mid-gestation for normal development of the trabeculae, compact myocardium, interventricular septum, and endocardial cushion. Cardiac muscle lacking ALK3 was specifically deficient in expressing TGFbeta2, an established paracrine mediator of cushion morphogenesis. Hence, ALK3 is essential, beyond just the egg cylinder stage, for myocyte-dependent functions and signals in cardiac organogenesis. ispartof: Proceedings of the National Academy of Sciences of the United States of America vol:99 issue:5 pages:2878-83 ispartof: location:United States status: published

In humans, SOX9 heterozygous mutations cause the severe skeletal dysmorphology syndrome campomelic dysplasia. Except for clinical descriptions, little is known about the pathogenesis of this disease. We have generated heterozygous Sox9 mutant mice that phenocopy most of the skeletal abnormalities of this syndrome. The Sox9 +/− mice died perinatally with cleft palate, as well as hypoplasia and bending of many skeletal structures derived from cartilage precursors. In embryonic day (E)14.5 heterozygous embryos, bending of radius, ulna, and tibia cartilages was already prominent. In E12.5 heterozygotes, all skeletal elements visualized by using Alcian blue were smaller. In addition, the overall levels of Col2a1 RNA at E10.5 and E12.5 were lower than in wild-type embryos. We propose that the skeletal abnormalities observed at later embryonic stages were caused by delayed or defective precartilaginous condensations. Furthermore, in E18.5 embryos and in newborn heterozygotes, premature mineralization occurred in many bones, including vertebrae and some craniofacial bones. Because Sox9 is not expressed in the mineralized portion of the growth plate, this premature mineralization is very likely the consequence of allele insufficiency existing in cells of the growth plate that express Sox9 . Because the hypertrophic zone of the heterozygous Sox9 mutants was larger than that of wild-type mice, we propose that Sox9 also has a role in regulating the transition to hypertrophic chondrocytes in the growth plate. Despite the severe hypoplasia of cartilages, the overall organization and cellular composition of the growth plate were otherwise normal. Our results suggest the hypothesis that two critical steps of the chondrocyte differentiation pathway are sensitive to Sox9 dosage. First, an early step presumably at the stage of mesenchymal condensation of cartilage primordia, and second, a later step preceding the transition of chondrocytes into hypertrophic chondrocytes.

Transition nuclear proteins (TPs), the major proteins found in chromatin of condensing spermatids, are believed to be important for histone displacement and chromatin condensation during mammalian spermatogenesis. We generated mice lacking the major TP, TP1, by targeted deletion of the Tnp1 gene in mouse embryonic stem cells. Surprisingly, testis weights and sperm production were normal in the mutant mice, and only subtle abnormalities were observed in sperm morphology. Electron microscopy revealed large rod-like structures in the chromatin of mutant step 13 spermatids, in contrast to the fine chromatin fibrils observed in wild type. Steps 12–13 spermatid nuclei from the testis of Tnp1- null mice contained, in place of TP1, elevated levels of TP2 and some protamine 2 (P2) precursor. Most of the precursor was processed to mature P2, but high levels of incompletely processed forms remained in epididymal spermatozoa. Sperm motility was reduced severely, and ≈60% of Tnp1 -null males were infertile. We concluded that TP1 is not essential for histone displacement or chromatin condensation. The absence of TP1 may partially be compensated for by TP2 and P2 precursor, but this dysregulation of nucleoprotein replacement results in an abnormal pattern of chromatin condensation and in reduced fertility.

The Xenopus cerberus gene encodes a secreted factor that is expressed in the anterior endomesoderm of gastrula stage embryos and can induce the formation of ectopic heads when its mRNA is injected into Xenopus embryos [Bouwmeester, T., Kim, S., Lu, B. & De Robertis, E. M. (1996) Nature (London) 382, 595–601]. Here we describe the existence of a cerberus -related gene, Cerr1 , in the mouse. Cerr1 encodes a putative secreted protein that is 48% identical to cerberus over a 110-amino acid region. Analysis of a mouse interspecific backcross panel demonstrated that Cerr1 mapped to the central portion of mouse chromosome 4. In early gastrula stage mouse embryos, Cerr1 is expressed in the anterior visceral endoderm and in the anterior definitive endoderm. In somite stage embryos, Cerr1 expression is restricted to the most recently formed somites and in the anterior presomitic mesoderm. Germ layer explant recombination assays demonstrated that Cerr1 -expressing somitic-presomitic mesoderm, but not older Cerr1 -nonexpressing somitic mesoderm, was able to mimic the anterior neuralizing ability of anterior mesendoderm and maintain Otx2 expression in competent ectoderm. In most Lim1 −/− headless embryos, Cerr1 expression in the anterior endoderm was weak or absent. These results suggest that Cerr1 may play a role in anterior neural induction and somite formation during mouse development.

USF1 and USF2 are ubiquitously expressed transcription factors implicated as antagonists of the c-Myc protooncoprotein in the control of cellular proliferation. To determine the biological role of the USF proteins, mutant mice were generated by homologous recombination in embryonic stem cells. USF1-null mice were viable and fertile, with only slight behavioral abnormalities. However, these mice contained elevated levels of USF2, which may compensate for the absence of USF1. In contrast, USF2-null mice contained reduced levels of USF1 and displayed an obvious growth defect: they were 20–40% smaller at birth than their wild-type or heterozygous littermates and maintained a smaller size with proportionate features throughout postnatal development. Some of the USF-deficient mice, especially among the females, were prone to spontaneous epileptic seizures, suggesting that USF is important in normal brain function. Among the double mutants, an embryonic lethal phenotype was observed for mice that were homozygous for the Usf2 mutation and either heterozygous or homozygous for the Usf1 mutation, demonstrating that the USF proteins are essential in embryonic development.

Cancer is a clonal disease, initiating from a single cell that has accumulated sufficient genetic damage to cause uncontrolled growth. The malignant daughter cells within a clone interact with each other and their normal neighboring cells, the so-called microenvironment, influencing tumor progression (1). The time required to clonally expand a single malignant cell to a clinically detectable tumor can take many years. During this time, other genetic changes occur, leading to the evolution of the tumor phenotype. One fundamental mechanism that triggers cancer formation is the mutation of tumor suppressor genes followed by loss of heterozygosity (2). Tumor suppressor gene mutations in the mouse have been used extensively to model human cancer (3). However, current mouse models, including germ-line mutations and tissue-specific knockouts, have not been able to reproduce the clonal nature of human cancer (Fig. 1 Left and Center). In this issue of PNAS, Muzumdar et al. (4) and Wang et al. (5) exploit genetic technologies modeled after systems in Drosophila to induce a second hit in a tumor suppressor gene in very few somatic cells in mice (Fig. 1 Right). The exciting findings show that the resulting few homozygous mutant cells surrounded by otherwise normal cells behave differently than in mice in which every cell of the body or a particular organ is mutant. The ability to generate clones of mutant cells and follow their behaviors (e.g., cell cycle status or tumor formation) in the mouse offers new opportunities to determine the cellular mechanisms of tumor formation and more accurately model human cancer.

Previous experiments have demonstrated that the human beta-globin gene is correctly regulated in transgenic mice. The beta-globin gene is not expressed in yolk sac-derived erythroid cells in early embryonic development but is expressed concomitantly with the adult mouse beta-globin genes in 14- to 16-day fetal liver and adult reticulocytes. In an attempt to localize sequences that direct erythroid-specific expression, fragments of the human beta-globin gene were inserted in the opposite orientation 200 base pairs (bp) upstream of an intact human A gamma marker gene, which is not expressed on its own in mouse fetal liver. In the experiments reported here, two beta-globin 3' sequences activated the marker gene specifically in fetal liver. One sequence is located in a 250-bp Pst I fragment 550-800 bp downstream from the poly(A) site; the other is located near an EcoRI site in the third exon. These two sequences are active individually, and their combined effect is greater than their effects alone. beta-Globin 5' sequences from -815 to -50 were also analyzed for activity in this assay. The 5' sequences did not activate the marker gene when tested alone but did stimulate expression that was already directed to adult erythroid tissue by the two 3' sequences. These results suggest that three separate sequences are involved in human beta-globin gene regulation. The two 3' sequences act as adult erythroid enhancers and the 5' sequence stimulates expression that is already determined to be erythroid specific.