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Vascular associated endothelial cell (ECs) progenitors are still poorly studied and their role in the newly forming vasculature at embryonic or postnatal stage remains elusive. In the present work, we first defined a set of genes highly expressed during embryo development and strongly downregulated in the adult mouse. In this group, we then concentrated on the progenitor cell marker Peg3/PW1. By in vivo staining of the vasculature we found that only a subset of cells coexpressed endothelial markers and PW1. These cells were quite abundant in the embryo vasculature but declined in number at postnatal and adult stages. Using a reporter mouse for PW1 expression, we have been able to isolate PW1‐positive (PW1posECs) and negative endothelial cells (PW1negECs). PW1‐positive cells were highly proliferative in comparison to PW1negECs and were able to form colonies when seeded at clonal dilution. Furthermore, by RNAseq analysis, PW1posECs expressed endothelial cell markers together with mesenchymal and stem cell markers. When challenged by endothelial growth factors in vitro, PW1posECs were able to proliferate more than PW1negECs and to efficiently form new vessels in vivo. Taken together these data identify a subset of vessel associated endothelial cells with characteristics of progenitor cells. Considering their high proliferative potential these cells may be of particular importance to design therapies to improve the perfusion of ischemic tissues or to promote vascular repair. StemCells2017;35:1328–1340 (1): We identified a subpopulation of Endothelial Cells that specifically express the transcription factor PW1. These cells have a salt and pepper distribution along developing blood vessels. Scale bar = 20 μm. (2): Tacking advantage of the PW1nLacZ reporter mouse, we sorted out PW1 positive endothelial cells and characterized them. (2.1): RNAseq performed on PW1 positive ad PW1 negative endothelial cells revealed different gene expression profiles. (3.1): Isolated PW1 positive Endothelial cells seeded at clonal density were able to form endothelial cell colonies. Scale bar = 20 μm. (3.2): Isolated PW1 positive endothelial cells reimplanted in a Matrigel plug generated new blood vessels. Scale bar = 20 μm.

Pw1/Peg3is a parentally imprinted gene expressed in adult stem cells in every tissue thus far examined including the stem cells of the hair follicle. Using a Pw1/Peg3reporter mouse, we carried out a detailed dissection of the stem cells in the bulge, which is a major stem cell compartment of the hair follicle in mammalian skin. We observed that PW1/Peg3 expression initiates upon placode formation during fetal development, coincident with the establishment of the bulge stem cells. In the adult, we observed that PW1/Peg3 expression is found in both CD34+ and CD34− populations of bulge stem cells. We demonstrate that both populations can give rise to new hair follicles, reconstitute their niche, and self‐renew. These results demonstrate that PW1/Peg3 is a reliable marker of the full population of follicle stem cells and reveal a novel CD34− bulge stem‐cell population. StemCells2017;35:1015–1027 PW1 is expressed in the adult hair follicle stem cell niche (bulge). Cells purified from the bulge include both CD34+ and negative cells. When the bulge cells are enriched for PW1‐expressing cells, we confirm that the CD34+ cells have stem cell capacity and report here that the PW1+/CD34negative cells also have comparable stem cell capacity. These cells are capable of generating all components of the hair follicle including the dermal papilla.

In the mammalian cochlea, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are unidirectionally oriented. Development of this planar polarity is necessary for normal hearing as stereociliary bundles are only sensitive to vibrations in a single plane; however, the mechanisms governing their orientation are unknown. We report that Wnt signaling regulates the development of unidirectional stereociliary bundle orientation. In vitro application of Wnt7a protein or inhibitors of Wnt signaling, secreted Frizzled-related protein 1 or Wnt inhibitory factor 1, disrupts bundle orientation. Moreover, Wnt7a is expressed in a pattern consistent with a role in the polarization of the developing stereociliary bundles. We propose that Wnt signaling across the region of developing outer hair cells gives rise to planar polarity in the mammalian cochlea.

In the mammalian cochlea, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are unidirectionally oriented. Development of this planar polarity is necessary for normal hearing as stereociliary bundles are only sensitive to vibrations in a single plane;however, the mechanisms governing their orientation are unknown. We report that Wnt signaling regulates the development of unidirectional stereociliary bundle orientation. In vitro application of Wnt7a protein or inhibitors of Wnt signaling, secreted Frizzled-related protein 1 or Wnt inhibitory factor 1,disrupts bundle orientation. Moreover, Wnt7a is expressed in a pattern consistent with a role in the polarization of the developing stereociliary bundles. We propose that Wnt signaling across the region of developing outer hair cells gives rise to planar polarity in the mammalian cochlea.

Homologue of Slimb (HOS) is the substrate-recognizing component of the SCFHOS-Roc1 E3 ubiquitin protein ligase. This ligase mediates ubiquitination of the inhibitor of NF-κB transcription factor (IκB). We have found that HOS is highly expressed in a number of human cancer cell lines. The rates of the HOSgene transcription as well as HOS mRNA and protein levels were up-regulated in cells treated with mitogens or transfected with the inducers of mitogen-activated protein kinase pathway. Conversely, mitogen withdrawal strikingly reduced HOS levels during differentiation of mouse myoblasts. Activators of mitogen-activated protein kinase accelerated IκBα degradation and increased NF-κB transcriptional activity. Inhibition of HOS function via expression of dominant negative HOS (HOSΔF) initiated mouse myoblast differentiation and prevented Ras-mediated acceleration of IκBα degradation as well as NF-κB trans-activation and transformation of NIH3T3 cells. These data link the induction of HOS in proliferating cells with mitogen-signaling-dependent inhibition of cell differentiation and promotion of cell transformation.

BOC is a receptor-like protein that, with the related factor CDO, belongs to a newly recognized subfamily within the immunoglobulin superfamily of cell-surface molecules. BOC and CDO form complexes that positively regulate myogenesis in vitro. To gain a better understanding of the role of boc during vertebrate embryogenesis and whether it could cooperate with cdo in vivo, we carried out an extensive in situ hybridization analysis of boc expression in mouse embryos from E7.0 to E17.5. Our results show that boc is widely expressed during murine embryogenesis, in a spatially and temporally restricted pattern. Overall, the highest levels of boc expression are detected between E10.5 and E15.5, with the strongest signals observed in the developing musculoskeletal and central nervous systems. At late stages of development, boc expression becomes more restricted and is limited primarily to regions harboring proliferating cells, undifferentiated cells, or both. This expression pattern is strikingly similar to that described for cdo (Mulieri et al., 2000). The overlapping expression patterns of cdo and boc in vivo, combined with the promotion of myogenesis by CDO/BOC complexes in cultured cells, strongly suggests that these proteins play a role together in the determination, differentiation, or both, of numerous cell types during vertebrate embryogenesis. © 2002 Wiley-Liss, Inc.

Members of the Siah (seven in absentia homolog) family of RING domain proteins are components of E3 ubiquitin ligase complexes that catalyze ubiquitination of proteins. We have determined the crystal structure of the substrate-binding domain (SBD) of murine Siah1a to 2.6 Å resolution. The structure reveals that Siah is a dimeric protein and that the SBD adopts an eight-stranded β-sandwich fold that is highly similar to the TRAF-C region of TRAF (TNF-receptor associated factor) proteins. The TRAF-C region interacts with TNF-α receptors and TNF-receptor associated death-domain (TRADD) proteins; however, our findings indicate that these interactions are unlikely to be mimicked by Siah. The Siah structure also reveals two novel zinc fingers in a region with sequence similarity to TRAF. We find that the Siah1a SBD potentiates TNF-α-mediated NF-κB activation. Therefore, Siah proteins share important similarities with the TRAF family of proteins, including their overall domain architecture, three-dimensional structure and functional activity.

The cardiac conduction system is a complex network of cells that together orchestrate the rhythmic and coordinated depolarization of the heart. The molecular mechanisms regulating the specification and patterning of cells that form this conductive network are largely unknown. Studies in avian models have suggested that components of the cardiac conduction system arise from progressive recruitment of cardiomyogenic progenitors, potentially influenced by inductive effects from the neighboring coronary vasculature. However, relatively little is known about the process of conduction system development in mammalian species, especially in the mouse, where even the histological identification of the conductive network remains problematic. We have identified a line of transgenic mice where lacZ reporter gene expression delineates the developing and mature murine cardiac conduction system, extending proximally from the sinoatrial node to the distal Purkinje fibers. Optical mapping of cardiac electrical activity using a voltage-sensitive dye confirms that cells identified by the lacZ reporter gene are indeed components of the specialized conduction system. Analysis of lacZ expression during sequential stages of cardiogenesis provides a detailed view of the maturation of the conductive network and demonstrates that patterning occurs surprisingly early in embryogenesis. Moreover, optical mapping studies of embryonic hearts demonstrate that a murine His-Purkinje system is functioning well before septation has completed. Thus, these studies describe a novel marker of the murine cardiac conduction system that identifies this specialized network of cells throughout cardiac development. Analysis of lacZ expression and optical mapping data highlight important differences between murine and avian conduction system development. Finally, this line of transgenic mice provides a novel tool for exploring the molecular circuitry controlling mammalian conduction system development and should be invaluable in studies of developmental mutants with potential structural or functional conduction system defects.

The molecular basis underlying the establishment of the myogenic lineage, subsequent differentiation, and the establishment of specific fiber types (i.e., fast versus slow) is becoming well understood. In contrast, the regulation of the general properties of a specific anatomical muscle group (e.g., leg versus jaw muscles) and the regulation of muscle-fiber properties within a particular group are less well characterized. We have investigated the potential role of the homeobox-containing gene, Engrailed-2 (En-2), in the mouse, which is specifically expressed in myoblasts in the first arch and maintained in the muscles of mastication in the adult. We have generated mice that ectopically express En-2 in all muscles during early development and primarily in fast muscles in the adult. Ectopic En-2 in nonjaw muscles leads to a decrease in fiber size, whereas overexpression in the jaw muscles leads to a shift in fiber metabolic properties as well as a decrease in fiber size. In contrast, loss of En-2 in the jaw leads to a shift in fiber metabolic properties in the jaw of female mice only. Jaw muscles are sexually dimorphic, and we propose that the function of En-2 and mechanisms guiding sexual dimorphism of the jaw muscles are integrated. We conclude that the specific expression of En-2 in the jaw therefore plays a role in specifying muscle-fiber characteristics that contribute to the physiologic properties of specific muscle groups.

CDO is a cell‐surface protein of the immunoglobulin/fibronectin type III repeat family that positively regulates myogenic differentiation in vitro. To gain a better understanding of the role of cdoduring vertebrate development, we carried out an extensive in situ hybridization study to characterize its expression pattern from postimplantation to late stages of mouse embryogenesis and in rat brain from E13 to adult. Our results show a broad pattern of cdoexpression that is spatially and temporally restricted during embryogenesis. In the central nervous system (CNS), cdoexpression is detected as early as E7.5 and maintained in the dorsal ventricular zones of the brain and spinal cord, becoming increasingly restricted in the adult. High levels of cdoare detected in developing sensory organs, such as the eye and ear. Outside the CNS, cdois expressed mainly in neural crest and mesodermal derivatives, including skeletal muscle precursors. Overall, the highest levels of cdoexpression are seen from E9.0 to E15.5. The temporal onset and restricted expression of cdosuggest that cdoplays a role in the determination and/or differentiation of a number of cell types during embryogenesis. © 2000 Wiley‐Liss, Inc.

Introduction:Myocardial infarction (MI) induces a repair response that ultimately generates a stable fibrotic scar. Although the scar prevents cardiac rupture, an excessive profibrotic response impairs optimal recovery.Hypothesis:To explore the regulation of fibroblasts proliferation through a paracrine action of cardiac stromal cells post-MIMethods:We carried out a bioinformatic secretome analysis of cardiac stromal PW1+cells isolated from normal and post-MI mouse hearts to identify novel secreted proteins. Functional assays were used to screen secreted proteins that promote fibroblast proliferation. The expressions of secreted proteins candidates were subsequently analyzed in mouse and human hearts and plasmas. The relation between levels of circulating protein candidates and adverse post-MI cardiac remodeling was examined in a cohort of 80 patients with a first ST-elevation MI and serial cardiac magnetic resonance imaging (MRI) evaluations.Results:Cardiac stromal PW1+cells undergo a change in paracrine behavior post-MI and secrete factors that promote fibroblast proliferation. Among these factors, growth differentiation factor 3 (GDF3), a member of the transforming growth factor-β family, was markedly upregulated in the ischemic hearts and induced fibroblast proliferation at high level. In humans, GDF3 was detected in the plasma at day 4 post-MI and GDF3 circulating levels were significantly associated with an increased risk of adverse remodeling 6-month post-MI (adjusted Odds Ratio (OR) = 1.76 [1.03 - 3.00], p = 0.037).Conclusions:Our findings define a mechanism for the pro-fibrotic action of cardiac stromal cells through secreted cardiokines, such as GDF3, a candidate marker of adverse fibrotic remodeling following MI.

Introduction:The epicardium is a source of the coronary vasculature and stroma during heart development. Although quiescent in the physiological adult heart, increasing evidences indicate the potential epicardium activation and differentiation into its progeny after cardiac injury; however, this remains debated.Hypothesis:We hypothesize that in vivo hypoxia exposure can mimic the embryonic cardiac profile and prime the adult epicardial progenitor cell to a more immature profile.Methods:Perinatal and adult BL6 and Pw1nLaczhearts were used to track the expression of PW1 - progenitor cell marker - together with cell surface proteins. In vivo hypoxia (10% O2) exposure was used to stimulate the adult heart. Histology, protein expression profiles and single cell analysis were assessed. Freshly FACS isolated Gp38+PW1+epicardial cells were cultured to determine their progenitor cell potential (clonogenicity, self-renewal and cell fate potential).Results:Here, we report that PW1 expression decreases during postnatal life, but is maintained in the cardiac hypoxic niche epicardium and subepicardium. Adult mice exhibit increased PW1 expression in epicardial/subepicardial cells and endothelial cells in response to hypoxia. The epicardium and subepicardium are activated to undergo cell proliferation and re-express a subset of embryonic genes in response to hypoxia. We established a new in vitro model to study purified PW1+/Gp38+epicardial cells, and we found properties of clonogenicity, self-renewal and multipotency (fibroblast, smooth muscle and endothelial cells) at single cell level in the developing epicardial cells. Cell fate potential towards an endothelial profile is dependent upon low levels of oxygen, and the in vitro ability to grow in culture is restore after in vivo priming of the adult heart.Conclusions:In conclusion our data support that the resident PW1+/Gp38+epicardial cells are a reminiscent progenitor population in adult mammalian heart that can be reactivated by exposure to hypoxia, highlighting the potential therapeutic role of hypoxia in regenerative medicine.

The cellular and molecular processes leading to the establishment of the skeletal muscle lineage in the vertebrate are not well understood. The MyoD-related family of myogenic regulatory factors (MRFs) are expressed during somitogenesis although cells with myogenic capacity are present prior to gastrulation. We propose that regulatory genes exist that guide the skeletal muscle lineage during early development. In an effort to identify these regulatory genes, we performed a differential screening to isolate transcripts that are present in myogenic cells and in the embryo prior to MRF expression but absent in nonmyogenic fibroblasts. We report here the identification of Pw1. The Pw1 transcript is ∼8.5 kb long and encodes a large protein containing 12 widespread C2H2 zinc fingers and 3 motifs containing periodic prolines and acidic residues. Consistent with the possibility that Pw1 is a transcription factor, we observe nuclear localization of the protein. Pw1 is strongly expressed upon gastrulation and subsequently becomes restricted to skeletal muscle and subregions of the central nervous system. Pw1 is initially expressed in all mesodermal cells early in development; however, its maintained expression in adult differentiated muscle suggests a specific role in the skeletal muscle lineage. Pw1 expression is cell cycle specific with levels highest during late M-phase. The gene is intronless which may facilitate transcription during cell division. At present, the precise function of Pw1 is not understood; however, we note that Pw1 maps to the proximal region of chromosome 7 near the axial segmentation mutantpudgywhich shows severe perturbation of axial skeletal and muscle structures.

The type-II TGFβ receptor mediates many of the biological responses to TGFβ. An examination of the expression of the type-II TGFβ receptor during mouse embryogenesis therefore provides specific information about the role of TGFβ during embryogenesis than has been available to date. We have isolated the genomic murine homologue of the human type-II TGFβ receptor corresponding to exon 2. The murine and human sequences show a high degree of homology. Using the murine probe, we found that type-II TGFβ receptor expression is regulated in both a spatial and a temporal fashion by using in situ hybridization and ribonuclease protection assays. Type-II TGFβ receptor expression is localized to the mesenchyme during critical interactions with adjacent epithelium such as developing hair follicles, whisker follicles and tooth anlage. In the central nervous system, type-II TGFβ receptor expression is highly restricted to the floor plate. Strong expression is also detected in migrating neural crest cells, meninges, and choroid plexus. Specific mesenchymal localization of type-II TGFβ receptor is also observed in lung, kidney, intestine, stomach, and bladder. The restricted expression of type-II TGFβ receptor in mesenchymal cells at sites of epithelial-mesenchymal interactions suggests that type-II TGFβ receptor plays a major role in mediating the establishment of embryonic organ systems. The highly restricted expression of type-II TGFβ receptor in the developing CNS suggests an important role for a serine/threonine kinase in patterning of the nervous system.

Tumor necrosis factor (TNF) mediates a variety of biological activities including cell proliferation, differentiation and programmed cell death. The specific response to TNF depends upon cell type and reflects the presence of specific regulatory proteins that participate in the TNF response pathway. TNF signal transduction is mediated by TRAF2 which binds the TNF Receptor2 (TNFR2) and activates NFκB. We previously identified a gene Pw1, which encodes a large zinc-finger containing protein1. We have determined that Pw1 is identical to Peg3, a paternally expressed gene of unknown function2(and will therefore be referred to as Peg3 throughout this text). We report here that Peg3 associates specifically with TRAF2 but not with other TRAF family members. Peg3 expression activates NFκB via IκB-NFκB dissociation and acts synergistically with TRAF2. Transfection of a truncated Peg3 containing the TRAF2 interaction site, abolishes NFκB activation by TRAF2 and/or TNF. We conclude that Peg3 is a regulator of the TNF response. These data reveal the involvement of an imprinted gene in this pathway.

The elucidation of the myogenic regulatory factors (MRFs) has resulted in a surge of research activity that promises to bridge molecular and embryological approaches to muscle research. In this review, we consider the arguments that would place MRFs in a key determination role for the skeletal muscle lineage in the vertebrate embryo. Amphibian, avian, and murine systems are compared and recent developments in the molecular embryology of myogenesis are reviewed. A model is proposed to account for the apparent discrepancies in conclusions drawn from studies using cultured cells that indicate a role for the MRFs in determination, and studies of MRF and muscle gene expression in vivo that indicate a role in differentiation but not in lineage determination. Copyright 1993, 1999 Academic Press

Homeobox-containing genes play an important role in patterning processes that occur during embryogenesis. Programmed cell death is a key process during pattern formation. The mechanisms by which programmed cell death is spatially regulated are not well characterized. Msx1 and Msx2 are two closely related homeobox-containing genes that are expressed at sites where cellular proliferation and programmed cell death occur, including the developing limb and the cephalic neural crest. Tissue interactions are necessary for the maintenance of Msx1 and Msx2 expression and programmed cell death. It has been demonstrated that BMP4 can regulate cell death at these same sites as well as induce Msx expression. These observations lead to the hypothesis that Msx2 is a key regulator of cell death in the BMP-mediated pathway. Embryonic stem cell lines will undergo processes typical of early embryogenesis upon aggregation and have recently been shown to provide a model system for programmed cell death. In contrast to ES cells, we see that P19 cells do not undergo pronounced cell death upon aggregation; however, constitutive ectopic Msx2 expression in P19 cells results in a marked increase in apoptosis induced upon aggregation but has no effect when cells are grown as a monolayer. If aggregates are allowed to interact with a substrate, the process of programmed cell death is completely inhibited. Addition of BMP4 to aggregated P19 cells also results in cell death; however, BMP4 does not increase levels of cell death in Msx2-expressing cells. Addition of BMP4 to P19 cells results in an induction of Msx2 transcription consistent with its proposed role in cell death in the embryo. Our data support a model by which BMP4 induces programmed cell death via an Msx2-mediated pathway and provide direct functional evidence that Msx2 expression is a regulator of this process.

We report here that Msx1 (formerly Hox-7.1) is expressed at high levels in uterine epithelial cells of the non-pregnant adult. These cells undergo pronounced changes in morphology in response to embryo implantation and show a concomitant decrease in Msx1 levels. While Msx1 is restricted to the uterus in adulthood, we observe Msx1 expression throughout the entire perinatal Müllerian duct epithelium in the prospective uterus, cervix and vagina. Through analysis of tissue recombinants, the expression of Msx1 in the epithelium was shown to be dependent upon an interaction with the underlying mesenchyme of uterine origin. The capacity of uterine mesenchyme to support or induce Msx1 expression in Müllerian epithelium is correlated with mesenchymal expression of Wnt-5a. Whereas Msx1 expression in the epithelium results from interaction with uterine mesenchyme, Wnt-5a expression is an intrinsic property of the uterine mesenchyme and does not depend upon the epithelium. The observation that Msx1 is expressed in the adult uterine epithelium and that conversion of the presumptive vaginal epithelium to uterine epithelium can be elicited only during the first week of postnatal development when Msx1 expression is detected suggests that, in addition to regulating various aspects of uterine epithelial morphology and function (e.g. gestation), this homeobox-containing gene plays a role in maintaining the uterus in a morphogenic and developmentally responsive state prerequisite for its unique function.

Myogenic helix-loop-helix (HLH) proteins, such as myo-genin and MyoD, can activate muscle-specific transcription when introduced into a variety of nonmuscle cell types. Whereas cells of mesodermal origin are especially permissive to the actions of these myogenic regulators, many other cell types are refractory to myogenic conversion by them. Here we describe a novel home-odomain protein, MHox, that binds an A+T-rich element in the muscle creatine kinase (MCK) enhancer that is essential for muscle-specific transcription and trans-activation by myogenic HLH proteins. MHox is completely restricted to mesodermally derived cell types during embryogenesis and to established cell lines of mesodermal origin. In contrast to most other homeobox genes, MHox expression is excluded from the nervous system, with the highest levels observed in limb bud and visceral arches. In adult mice, MHox is expressed at high levels in skeletal muscle, heart and uterus. The DNA-binding properties and pattern of MHox expression are unique among homeobox genes and suggest a role for MHox as a transcriptional regulator that participates in the establishment of diverse mesodermal cell types.

The murine female reproductive tract differentiates along the anteroposterior axis during postnatal development. This process is marked by the emergence of distinct cell types in the oviduct, uterus, cervix and vagina and is dependent upon specific mesenchymal-epithelial interactions as demonstrated by earlier heterografting experiments. Members of the Wnt family of signaling molecules have been recently identified in this system and an early functional role in reproductive tract development has been demonstrated. Mice were generated using ES-mediated homologous recombination for the Wnt-7a gene (Parr, B. A. and McMahon, A. P. (1995) Nature 374, 350-353). Since Wnt-7a is expressed in the female reproductive tract, we examined the developmental consequences of lack of Wnt-7a in the female reproductive tract. We observe that the oviduct lacks a clear demarcation from the anterior uterus, and acquires several cellular and molecular characteristics of the uterine horn. The uterus acquires cellular and molecular characteristics that represent an intermediate state between normal uterus and vagina. Normal vaginas have stratified epithelium and normal uteri have simple columnar epithelium, however, mutant uteri have stratified epithelium. Additionally, Wnt-7a mutant uteri do not form glands. The changes observed in the oviduct and uterus are accompanied by a postnatal loss of hoxa-10 and hoxa-11 expression, revealing that Wnt-7a is not required for early hoxa gene expression, but is required for maintenance of expression. These clustered hox genes have been shown to play a role in anteroposterior patterning in the female reproductive tract. In addition to this global posterior shift in the female reproductive tract, we note that the uterine smooth muscle is disorganized, indicating development along the radial axis is affected. Changes in the boundaries and levels of other Wnt genes are detectable at birth, prior to changes in morphologies. These results suggest that a mechanism whereby Wnt-7a signaling from the epithelium maintains the molecular and morphological boundaries of distinct cellular populations along the anteroposterior and radial axes of the female reproductive tract.

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb mesenchyme that has been demonstrated to support limb outgrowth and proper limb development. The homeobox gene, Max-1, is associated with the distal limb mesenchyme (progress zone) and its expression depends upon the presence of the AER in chick limbs. We demonstrate here that the expression of Max-1 is dependent upon the limb ectoderm in the mouse, but that the inductive capacity of murine limb ectoderm is not restricted to the AER. Msx-1 can also he maintained in limb mesenchyme by the substitution of FGF 4 for the ectoderm; however, we see that local cell-cell interactions are required for high levels of expression. Disruption of cell-call interactions in the limb mesenchyme results in a dramatic decrease in Msx-1 levels and a precocious expression of MyoD1, suggesting that the limb environment represses differentiation and promotes cell proliferation during early development. BMP 4 and FGF 2 can also maintain Msx-1 expression in limb mesenchyme as well as retinoic acid which is usually associated with polarizing activity in the early limb. Max-2 expression does not appear to be dependent upon cell-cell interactions as measured in these experiments. Taken together, our data suggest that the expression of Max-1, but not Max-2, not only requires factors from the limb ectoderm, but also relies upon cues from local cell interactions and that the spatial distribution of inductive capacities in limb ectoderm differs between the avian and murine systems.

Among the first tissues to differentiate in the mammalian embryo are cardiac and subsequently skeletal striated muscle. We have developed specific cRNA probes corresponding to the 5′ noncoding regions of α-cardiac and α-skeletal actin mRNAs in order to investigate myogenesis in the mouse embryo. Transcripts coding for cardiac actin which is the major isoform of the adult heart can first be detected between 7·5 and 7·8 days p.c. in the developing heart and are observed in all somites as they are formed. In addition, α-skeletal actin transcripts are accumulated at much lower levels in cardiac tissue and newly formed somites; both heart and skeletal muscle show co expression of this actin gene pair at all stages of development examined. The fact that cardiac actin transcripts can be observed in the myotomal portion of the somite prior to muscle fibre differentiation indicates that cardiac actin transcripts (and to a lesser extent skeletal actin transcripts) are markers not only of striated muscle tissue, but also of earlier stages of the myogenic programme in vivo.

We report that during mouse fetal development transcripts of Msx1 and Msx2 become progressively restricted to cells that will form more distal digit structures; the Msx2 expression domain is always more distal than Msx1. At birth both Msx1 and Msx2 are expressed in cells of the nail bed and hair follicle. We have found that the regenerative ability of mouse digit tips is restricted to levels in which the amputation plane is within the region of Msx1, but not Msx2, expression in early fetal digits and to levels where both Msx1 and Msx2 are expressed in late fetal and neonatal digits. Fetal digit tip regeneration is rapid and completed by birth, whereas neonatal digit tip regeneration requires 4 weeks and is sometimes imperfect. In both fetal and neonatal digits, we find that both Msx1 and Msx2 are expressed during regeneration, but not during wound healing associated with proximal amputations where no regenerative response is observed. These data support the hypothesis that the expression of Msx genes are important for digit cells to initiate and participate in a regenerative response.

The int-3 oncogene was identified as a frequent target in Mouse Mammary Tumor Virus (MMTV)-induced mammary carcinomas and encodes the intracellular domain of a novel mouse Notch gene. To investigate the role of the int-3 proto-oncogene in mouse development and carcinogenesis, we isolated cDNA clones corresponding to the entire coding potential of the int-3 proto-oncogene. We propose to name this gene Notch4 and reserve the int-3 nomenclature for references to the oncogenic form. The deduced amino acid sequence of Notch4 contains conserved motifs found in Notch proteins; however Notch4 has fewer epidermal growth factor (EGF)-like repeats and a shorter intracellular domain than other mouse Notch homologues. Comparison of the coding potential of the int-3 gene to that of Notch4 suggests that loss of the extracellular domain of Notch4 leads to constitutive activation of this murine Notch protein. In situ hybridization revealed that Notch4 transcripts are primarily restricted to endothelial cells in embryonic and adult life. Truncated Notch4 transcripts were detected in post-meiotic male germ cells. The distinct Notch4 protein features and its restricted expression pattern suggests a specific role for Notch4 during development of vertebrate endothelium.

An approach to investigate the role of cellular senescence in organismal aging has been to abrogate signaling pathways known to induce cellular senescence and to assess the effects in mouse models of premature aging. Recently, we reported the effect of loss of function of p21, a gene implicated in p53-induced cellular senescence, in the background of the Ku80-/- premature aging mouse (Zhao et al., EMBO reports, 2009). Here, we provide an overview of the effects of p21 deletion in different models of premature aging.

The synthetic estrogen diethylstilbestrol (DES) was administered to pregnant women between the 1940s and the mid‐1970s and is believed to be responsible for numerous uterine/cervical/vaginal malformations and cancers that appeared after birth and in young adult life. This medical tragedy has served as one of the prototypical examples of a phenomenon known as “endocrine disruption,” in which either environmental agents or other compounds disrupt normal hormonal signaling in the body. Whereas DES signals through estrogen receptors, the subsequent molecular targets were largely unknown. We had identified Wnt7a as a target in this pathway and have used genetic analyses of mutant mice to demonstrate that disruption of Wnt7a is the key event leading to the DES phenotypes and cancers. We find that Wnt7a expression is only transiently deregulated in response to DES exposure, leading to the conclusion that critical events during early reproductive tract development results in a permanent change or “reprogramming” in subsequent development. Birth Defects Research (Part A), 2005. © 2005 Wiley‐Liss, Inc.

Skeletal muscle denervation leads to myofibre atrophy with fibrosis and fatty infiltration of muscle-resident fibroadipogenic progenitors (FAPs). A study shows that on denervation, FAPs activate pathogenic STAT3–IL-6 signalling. Inhibition of this pathway prevents atrophy and points to potential therapeutic targets.

Introduction:Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling and neomuscularization. We identified pulmonary vascular progenitor cells, characterized by PW1, CD34 and PDGFR? expression, which proliferate and differentiate into smooth muscle cells (SMCs), and participate in pulmonary vessels neomuscularisation early during chronic hypoxia (CH), a mouse pulmonary hypertension model (PH).Hypothesis:To identify the pathways that regulate their recruitment during CH, we addressed the role of PDGFR?.Methods:PW1nLacZ+/-reporter mice were used to follow the lineage of PW1+ cells and PDGFR? receptor was inhibited by Imatinib or PDGFR? blocking antibody. Constitutive activation of PDGFR? was induced by Tamoxifen administration in RosaCRE-ERT2xPDGFR?+/(S)Kmice.Results:In vivo, inhibition of PDGFR? by Imatinib or blocking antibody administration totally prevented the early CH-induced increase in PW1+ progenitor cell proliferation and differentiation into vascular SMC (without inducing apoptosis) and reduced pulmonary vessel neomuscularisation (p<0.05 vs control). Conversely, constitutive activation of PDGFR? after tamoxifen injection induced pulmonary vessel neomuscularization similar to CH (p<0.05 PDGFR?+/(S)Kvs PDGFR?+/+) followed by PH development with an increased right ventricular pressure (27?2.7 vs 17.9?1.3 mmHg in controls) and a right ventricular hypertrophy. In vitro, PW1+ progenitor cell differentiation into SMC was inhibited by blocking antibody administration without induction of apoptosis, while PDGFR? ligands, PDGF-AA and AB, induced PW1+ progenitor cell proliferation.Conclusions:These results demonstrate that blocking of the PDGFR? pathway prevents pulmonary vessels neomuscularization by inhibiting PW1+ progenitor cells proliferation and differentiation. They suggest that this pathway is a major new regulator of the lung vascular remodeling process and PH development.

A recent study shows that skeletal muscle responds to tumor-secreted factors by undergoing a change in fatty acid metabolism, and that blocking this metabolic response in mice inhibits muscle wasting.

The article discusses the responsibilities of fiduciaries in dealing with problems associated with climate change. Climate change is a risk that cannot be managed and money managers are in powerful position to safeguard the financial security for whom they manage money. It is inferred that fiduciaries are surrounded by an undeniable and unavoidable flood of information and significant material developments. Reports are provided to fiduciaries as first-step-by-step how-to-guide to address climate change risks prepared by the investment consultant and institutional investors.

Everyone in power is entitled to a honeymoon period. Unsurprisingly, Tony Blair's government has had a rather longer honeymoon stint with the public than most other governments. Liz Allen, Policy and Research Officer with Education Network — who addressed BEMAS South-East on 7 March 1998 — would like to think that this has been so for positive reasons. Others would argue that it is entirely because Labour has been out in the cold for 18 long years.