Abstract 1.? 4-[2((1R,2R)-2-Hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide (BLZ945) is a small molecule inhibitor of CSF-1R kinase activity within osteoclasts designed to prevent skeletal related events in metastatic disease. Key metabolites were enzymatically and structurally characterized to understand the metabolic fate of BLZ945 and pharmacological implications. The relative intrinsic clearances for metabolites were derived from in vitro studies using human hepatocytes, microsomes and phenotyped with recombinant P450 enzymes. 2.? Formation of a pharmacologically active metabolite (M9) was observed in human hepatocytes. The M9 metabolite is a structural isomer (diastereomer) of BLZ945 and is about 4-fold less potent. This isomer was enzymatically formed via P450 oxidation of the BLZ945 hydroxyl group, followed by aldo-keto reduction to the alcohol (M9). 3.? Two reaction phenotyping approaches based on fractional clearances were applied to BLZ945 using hepatocytes and liver microsomes. The fraction metabolized (fm) or contribution ratio was determined for each metabolic reaction type (oxidation, glucuronidation or isomerization) as well as for each metabolite. The results quantitatively illustrate contribution ratios of the involved enzymes and pathways, e.g. the isomerization to metabolite M9 accounted for 24% intrinsic clearance in human hepatocytes. In summary, contribution ratios for the Phase I and Phase II pathways can be determined in hepatocytes.
Holoprosencephaly (HPE) is the most common developmental defect of the forebrain characterized by inadequate or absent midline division of the forebrain into cerebral hemispheres, with concomitant midline facial defects in the majority of cases. Understanding the pathogenesis of HPE requires knowledge of the relationship between the developing brain and the facial structures during embryogenesis. A number of signaling pathways control and coordinate the development of the brain and face, including Sonic hedgehog, Bone morphogenetic protein, Fibroblast growth factor, and Nodal signaling. Mutations in these pathways have been identified in animal models of HPE and human patients. Because of incomplete penetrance and variable expressivity of HPE, patients carrying defined mutations may not manifest the disease at all, or have a spectrum of defects. It is currently unknown what drives manifestation of HPE in genetically at-risk individuals, but it has been speculated that other gene mutations and environmental factors may combine as cumulative insults. HPE can be diagnosed in utero by a high-resolution prenatal ultrasound or a fetal magnetic resonance imaging, sometimes in combination with molecular testing from chorionic villi or amniotic fluid sampling. Currently, there are no effective preventive methods for HPE. Better understanding of the mechanisms of gene-environment interactions in HPE would provide avenues for such interventions. For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.
The preservation of the bone-forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell-based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2-primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2-expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2-primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell-based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects.
Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7) in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of Twisted gastrulation (Twsg1), a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that Twsg1 null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs) and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. Twsg1 null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury.
Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-?1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-?1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-?1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-?1-ALK5 signaling. Conversely, TGF-?1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-?1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-?1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-?1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-?1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.
To understand life-long neurogenesis in the dentate gyrus (DG), characterizing dentate neural stem cells and the signals controlling their development are crucial. In the present study, we show that bone morphogenic protein (Bmp) signaling is a critical regulator of embryonic dentate development, required for initiating neurogenesis in embryonic DG progenitors and required for the establishment of dentate neural stem cells postnatally. We tested the hypothesis that Bmp signaling regulates dentate development in part by controlling the expression of Lef1, a Wnt responsive transcription factor expressed in dentate stem cells and absolutely required for dentate granule cell production. Bmp activation through the Acvr1 receptor induced Lef1 expression and neurogenesis in the embryonic DG. Ectopic expression of Bmp7 in the embryonic midline increased DG neurogenesis and inhibition of local Bmp signaling decreased embryonic DG neurogenesis. Mice with selective loss of Bmp expression due to defective meningeal development or with selective conditional deletion of meningeal Bmp7 also have dentate developmental defects. Conditional deletion of Activin receptor type I (Acvr1) or Smad4 (a downstream target nuclear effector of Bmp signaling) in DG neural stem cells resulted in defects in the postnatal subgranular zone and reduced neurogenesis. These results suggest that Acvr1-mediated meningeal Bmp signaling regulates Lef1 expression in the dentate, regulating embryonic DG neurogenesis, DG neural stem cell niche formation, and maintenance.
Palatogenesis is a complex process implying growth, elevation and fusion of the two lateral palatal shelves during embryogenesis. This process is tightly controlled by genetic and mechanistic cues that also coordinate the growth of other orofacial structures. Failure at any of these steps can result in cleft palate, which is a frequent craniofacial malformation in humans. To understand the etiology of cleft palate linked to the BMP signaling pathway, we studied palatogenesis in Bmp7-deficient mouse embryos. Bmp7 expression was found in several orofacial structures including the edges of the palatal shelves prior and during their fusion. Bmp7 deletion resulted in a general alteration of oral cavity morphology, unpaired palatal shelf elevation, delayed shelf approximation, and subsequent lack of fusion. Cell proliferation and expression of specific genes involved in palatogenesis were not altered in Bmp7-deficient embryos. Conditional ablation of Bmp7 with Keratin14-Cre or Wnt1-Cre revealed that neither epithelial nor neural crest-specific loss of Bmp7 alone could recapitulate the cleft palate phenotype. Palatal shelves from mutant embryos were able to fuse when cultured in vitro as isolated shelves in proximity, but not when cultured as whole upper jaw explants. Thus, deformations in the oral cavity of Bmp7-deficient embryos such as the shorter and wider mandible were not solely responsible for cleft palate formation. These findings indicate a requirement for Bmp7 for the coordination of both developmental and mechanistic aspects of palatogenesis.
We investigated the relative effect of amenorrhea and insulin-like growth factor-I (sIGF-I) levels on cancellous and cortical bone density and size. We investigated 66 adult women with anorexia nervosa. Lumbar spine and proximal femur bone mineral density was measured by DXA. We calculated bone mineral apparent density. Structural geometry of the spine and the hip was determined from DXA images. Weight and BMI, but not height, as well as bone mineral content and density, but not area and geometry parameters, were lower in patients with anorexia nervosa as compared with the control group. Amenorrhea, disease duration, and sIGF-I were significantly associated with lumbar spine and proximal femur BMD. In a multiple regression model, we found that sIGF-I was the only significant independent predictor of proximal femur BMD, while duration of amenorrhea was the only factor associated with lumbar spine BMD. Finally, femoral neck bone mineral apparent density, but not hip geometry variables, was correlated with sIGF-I. In anorexia nervosa, spine BMD was related to hypogonadism, whereas sIGF-I predicted proximal femur BMD. The site-specific effect of sIGF-I could be related to reduced volumetric BMD rather than to modified hip geometry.
Bone morphogenetic protein (BMP) signalling regulates lymphopoiesis in bone marrow and thymus via the interaction of haemato-lymphoid progenitors with the stroma microenvironment. Despite increasing functional evidence for the role of BMP signalling in lymphopoiesis, little is known of the spatial distribution of BMP/BMP antagonists in the thymus and of how BMP signals exert specific functions in developing lymphocytes. We analysed expression of BMP/BMP antagonists in the thymus and bone marrow and determined the topology of BMP/BMP antagonist expression using lacZ reporter mice. Bmp4, Bmp7, Gremlin and Twisted gastrulation (Twsg1) are all expressed in the thymus and expression was clearly different for each gene investigated. Expression was seen both in cortical and medullary regions suggesting that BMP signals regulate all stages of T-cell development. Two genes in particular, Bmp7 and Twsg1, were dynamically expressed in developing T and B lymphocytes. Their conditional ablation in all haematopoietic cells surprisingly did not affect the steady state of B-cell and T-cell development. This indicates that both lymphoid cell-derived BMP7 and TWSG1 are dispensable for normal lymphopoiesis and that bone-marrow stroma-derived TWSG1 is responsible for the lymphoid defects observed in Twsg1 null mice. In summary our data demonstrate a complex network of lymphoid and stroma derived BMP signals involved in the orchestration of lymphopoiesis in both bone marrow and thymus.
Holoprosencephaly (HPE) is a heterogeneous craniofacial and neural developmental anomaly characterized in its most severe form by the failure of the forebrain to divide. In humans, HPE is associated with disruption of Sonic hedgehog and Nodal signaling pathways, but the role of other signaling pathways has not yet been determined. In this study, we analyzed mice which, due to the lack of the Bmp antagonist Noggin, exhibit elevated Bmp signaling. Noggin(-/-) mice exhibited a solitary median maxillary incisor that developed from a single dental placode, early midfacial narrowing as well as abnormalities in the developing hyoid bone, pituitary gland and vomeronasal organ. In Noggin(-/-) mice, the expression domains of Shh, as well as the Shh target genes Ptch1 and Gli1, were reduced in the frontonasal region at key stages of early facial development. Using E10.5 facial cultures, we show that excessive BMP4 results in reduced Fgf8 and Ptch1 expression. These data suggest that increased Bmp signaling in Noggin(-/-) mice results in downregulation of the hedgehog pathway at a critical stage when the midline craniofacial structures are developing, which leads to a phenotype consistent with a microform of HPE.
Bone morphogenetic protein (BMP) signaling is increasingly implicated in immune cell differentiation and function; however, direct in vivo evidence for such a role is still missing. In this article, we report that Twisted gastrulation (TWSG1), an extracellular regulator of BMP signaling, is expressed in activated B cells and regulates T-independent B cell responses in the mouse. Twsg1-deficient B cells mount stronger T-independent type 2 responses reflected as increased IgM levels and numbers of Ag-specific IgM-secreting cells. BCR stimulation of Twsg1-deficient B cells results in hyperproliferation, hyperresponsiveness, and decreased apoptosis, whereas TLR stimulation results in hyperproliferation and increased IgG3 production. These changes are reflected on the molecular level by increased transcription of Bcl-6, Pax5, and the BMP-responsive gene Id-2. The TWSG1 effects on B cells appear to be cell intrinsic, suggesting that Twsg1 expression in B cells serves to interpret BMP signals on a per-cell basis. In summary, our observations on the role of TWSG1 in B cell function is opening new paths toward the exploration of the role of BMP signaling in immunological processes.
In previous molecular phylogenetic analyses of the freshwater mussel family Unionidae (Bivalvia: Unionoida), the Afrotropical genus Coelatura had been recovered in various positions, generally indicating a paraphyletic Unionidae. However that result was typically poorly supported and in conflict with morphology-based analyses. We set out to test the phylogenetic position of Coelatura by sampling tropical lineages omitted from previous studies. Forty-one partial 28S nuclear rDNA and partial COI mtDNA sequences (1130 total aligned nucleotides) were analyzed separately and in combination under both maximum parsimony and likelihood, as well as Bayesian inference. There was significant phylogenetic incongruence between the character sets (partition homogeneity test, p<0.01), but a novel heuristic for comparing bootstrap values among character sets analyzed separately and in combination illustrated that the observed conflict was due to homoplasy rather than separate gene histories. Phylogenetic analyses robustly supported a monophyletic Unionidae, with Coelatura recovered as part of a well-supported Africa-India clade (=Parreysiinae). The implications of this result are discussed in the context of Afrotropical freshwater mussel evolution and the classification of the family Unionidae.
We examined the hypothesis that high FGF-23 levels early after transplantation contribute to the onset of hypophosphatemia, independently of parathyroid hormone (PTH) and other factors regulating phosphate metabolism.
The costimulatory molecules in the B7-CD28 families are important in the regulation of T cell activation and tolerance. The butyrophilin family of proteins shares sequence and structure homology with B7 family molecules; however, the function of the butyrophilin family in the immune system has not been defined. In this study, we performed an analysis on multiple butyrophilin molecules and found that butyrophilin-like (BTNL)1 molecule functions to dampen T cell activation. BTNL1 mRNA was broadly expressed, but its protein was only found in APCs and not T cells. The putative receptor for BTNL1 was found on activated T cells and APCs. Also, recombinant BTNL1 molecule inhibited T cell proliferation by arresting cell cycle progression. The administration of neutralizing Abs against BTNL1 provoked enhanced T cell activation and exacerbated disease in autoimmune and asthma mouse models. Therefore, BTNL1 is a critical inhibitory molecule for T cell activation and immune diseases.
The Notch signalling pathway is an evolutionarily conserved intercellular signalling mechanism that is essential for cell fate specification and proper embryonic development. We have analysed the expression, regulation and function of the jagged 2 (Jag2) gene, which encodes a ligand for the Notch family of receptors, in developing mouse teeth. Jag2 is expressed in epithelial cells that give rise to the enamel-producing ameloblasts from the earliest stages of tooth development. Tissue recombination experiments showed that its expression in epithelium is regulated by mesenchyme-derived signals. In dental explants cultured in vitro, the local application of fibroblast growth factors upregulated Jag2 expression, whereas bone morphogenetic proteins provoked the opposite effect. Mice homozygous for a deletion in the Notch-interaction domain of Jag2 presented a variety of severe dental abnormalities. In molars, the crown morphology was misshapen, with additional cusps being formed. This was due to alterations in the enamel knot, an epithelial signalling structure involved in molar crown morphogenesis, in which Bmp4 expression and apoptosis were altered. In incisors, cytodifferentiation and enamel matrix deposition were inhibited. The expression of Tbx1 in ameloblast progenitors, which is a hallmark for ameloblast differentiation and enamel formation, was dramatically reduced in Jag2(-/-) teeth. Together, these results demonstrate that Notch signalling mediated by Jag2 is indispensable for normal tooth development.
Glioblastoma cells with stem-like properties control brain tumour growth and recurrence. Here, we show that endogenous neural precursor cells perform an anti-tumour response by specifically targeting stem-like brain tumour cells. In vitro, neural precursor cells predominantly express bone morphogenetic protein-7; bone morphogenetic protein-7 is constitutively released from neurospheres and induces canonical bone morphogenetic protein signalling in stem-like glioblastoma cells. Exposure of human and murine stem-like brain tumour cells to neurosphere-derived bone morphogenetic protein-7 induces tumour stem cell differentiation, attenuates stem-like marker expression and reduces self-renewal and the ability for tumour initiation. Neurosphere-derived or recombinant bone morphogenetic protein-7 reduces glioblastoma expansion from stem-like cells by down-regulating the transcription factor Olig2. In vivo, large numbers of bone morphogenetic protein-7-expressing neural precursors encircle brain tumours in young mice, induce canonical bone morphogenetic protein signalling in stem-like glioblastoma cells and can thereby attenuate tumour formation. This anti-tumour response is strongly reduced in older mice. Our results indicate that endogenous neural precursor cells protect the young brain from glioblastoma by releasing bone morphogenetic protein-7, which acts as a paracrine tumour suppressor that represses proliferation, self-renewal and tumour-initiation of stem-like glioblastoma cells.
The increasing demand for drinking water and its stricter quality requirements have resulted in an exponentially expanding industry of membrane filtration processes. Currently, reverse osmosis (RO) is the most common method of desalination, able to produce water that is virtually free of pollutants and pathogenic micro-organisms. Biofouling of these devices however is a significant limitation. Here we present a 3D simulation of RO membrane biofouling based on a lattice Boltzmann (LB) platform that we subsequently favorably compare with experimental data. This data consists of temporally (and spatially) resolved velocity measurements acquired for a RO membrane using magnetic resonance techniques. The effect of biofilm cohesive strength on system pressure drop is then explored; weaker biomass is observed to have a reduced impact on pressure drop (per unit biomass accumulated).
The family of bone morphogenetic proteins (BMPs) comprises approximately 30 secreted cytokines that signal through transmembrane serine/threonine kinase receptors. The BMP signaling pathways are fine-tuned on multiple levels: Extracellular antagonists modify ligand activity; several co-receptors enhance or inhibit downstream signaling events through multiple mechanisms; and intracellular molecules further regulate the signaling outcome and mediate crosstalk with other pathways. BMPs affect structures and processes throughout the entire body, ranging from embryonic patterning and development through stem cells and their niches, to tissue homeostasis and regeneration. This comprehensive involvement in various tissues had not been expected by Marshall Urist, who initially discovered the ability of an unknown factor in bone to induce bone growth in muscle and subsequently suggested the name "bone morphogenetic protein." Today, recombinant BMPs are used in clinical practice for the treatment of bone and kidney disorders, and new genetically modified BMPs are emerging as promising tools in regenerative medicine and tissue engineering. Clearly, the functions of BMPs within the body are more versatile than initially suspected. To discuss modern trends in BMP signaling, leaders in the field met for the First International BMP Workshop in Berlin in September 2009. Here, we summarize new insights on the roles of BMPs in various tissues and highlight recent findings in cell, structural, and developmental biology as well as the therapeutic potential of BMPs. Finally, we conclude that BMPs today deserve to be called body morphogenetic proteins.
Teeth arise from sequential and reciprocal interactions between the oral epithelium and the underlying cranial neural crest-derived mesenchyme. Their formation involves a precisely orchestrated series of molecular and morphogenetic events, and gives us the opportunity to discover and understand the nature of the signals that direct cell fates and patterning. For that reason, it is important to elucidate how signaling factors work together in a defined number of cells to generate the diverse and precise patterned structures of the mature functional teeth. Over the last decade, substantial research efforts have been directed toward elucidating the molecular mechanisms that control cell fate decisions during tooth development. These efforts have contributed toward the increased knowledge on dental stem cells, and observation of the molecular similarities that exist between tooth development and regeneration.
The cloaca/urogenital sinus and its adjacent region differentiate into the urogenital/reproductive organs. Caudal regression syndrome (CRS; including mermaid syndrome), a type of severe cloacal malformation displays hindlimb fusion and urogenital organ defects, thus suggesting that such defects are caused by several morphogenetic alterations during early development. The attenuation of bone morphogenetic protein (Bmp) signaling at the posterior primitive streak of embryos leads to the caudal dysmorphogenesis including the cloaca and fusion of both hindlimbs. Genetic tissue lineage studies indicate the presence of coordinated organogenesis. Hedgehog (HH)-responding cells derived from peri-cloacal mesenchyme (PCM) contribute to the urogenital/reproductive organs. These findings indicate the existence of developmental programs for the coordinated organogenesis of urogenital/reproductive tissues based on growth factor function and crosstalk.
T cell activation and tolerance are delicately regulated by costimulatory molecules. Although B and T lymphocyte attenuator (BTLA) has been shown as a negative regulator for T cell activation, its role in peripheral T cell tolerance induction in vivo has not been addressed. In this study, we generated a novel strain of BTLA-deficient mice and used three different models to characterize the function of BTLA in controlling T cell tolerance. In an oral tolerance model, BTLA-deficient mice were found resistant to the induction of T cell tolerance to an oral Ag. Moreover, compared with wild-type OT-II cells, BTLA(-/-) OT-II cells were less susceptible to tolerance induction by a high-dose OVA peptide administered i.v. Finally, BTLA(-/-) OT-I cells caused autoimmune diabetes in RIP-mOVA recipient mice. Our results thus demonstrate an important role for BTLA in the induction of peripheral tolerance of both CD4(+) and CD8(+) T cells in vivo.
Bone Morphogenetic Proteins (BMPs) play multiple and important roles in embryonic development as well as in homeostasis and tissue repair in the adult. Bmp7 has been implicated in developmental disorders and in a variety of diseases, but functional studies to elucidate its role so far have been hampered, since mice deficient in BMP7 die around or just after birth. To facilitate such studies, we generated mice in which the Bmp7 gene has been rendered conditional-null by flanking its first coding exon with loxP sites. To this end, we adapted the two-loxP site strategy to Bacterial Homologous Recombination to create a Bacterial Artificial Chromosome-based vector for direct targeting in mouse embryonic stem cells. Functional analysis showed that in vivo, the conditional-null Bmp7(flx/flx) mice are phenotypically wild type, whereas post Cre-mediated recombination, the resulting Bmp7(delta/delta) mice are phenotypically null. Thus, this study validates the usefulness of the Bmp7(flx/flx) mouse which in turn should empower in vivo studies aimed at elucidating the roles of Bmp7 in postnatal development, homeostasis and disease.
The mandibular arch (BA1) is critical for craniofacial development. The distal region of BA1, which gives rise to most of the mandible, is dependent upon an optimal level of bone morphogenetic protein (BMP) signaling. BMP activity is modulated in the extracellular space by BMP-binding proteins such as Twisted gastrulation (TWSG1). Twsg1(-/-) mice have a spectrum of craniofacial phenotypes, including mandibular defects that range from micrognathia to agnathia. At E9.5, the distal region of the mutant BA1 was prematurely and variably fused with loss of distal markers eHand and Msx1. Expression of proximal markers Fgf8 and Barx1 was expanded across the fused BA1. The expression of Bmp4 and Msx2 was preserved in the distal region, but shifted ventrally. While wild type embryos showed a gradient of BMP signaling with higher activity in the distal region of BA1, this gradient was disrupted and shifted ventrally in the mutants. Thus, loss of TWSG1 results in disruption of the BMP4 gradient at the level of signaling activity as well as mRNA expression. Altered distribution of BMP signaling leads to a shift in gene expression and increase in apoptosis. The extent of apoptosis may account for the variable degree of mandibular defects in Twsg1 mutants.
Sequential and reciprocal epithelial-mesenchymal interactions govern the development of most tissues and organs of the craniofacial region. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of secreted signaling molecules that have long been implied to have a significant contribution in this process. However, evidence for such a role during craniofacial development is largely missing. Using a lacZ reporter mouse we mapped the spatiotemporal expression of BMP7 in the developing craniofacial region. The observed pattern suggested a potential involvement of BMP7 in epithelial-mesenchymal interactions and thus a direct role for this molecule in the development of ectodermal appendages (teeth, hair follicle, lachrymal and sweat glands, taste buds) and, furthermore, palatal formation. To correlate the expression to function we analyzed germline deleted conditional BMP7-deficient embryos for malformations. We found developmental defects in many craniofacial structures such as teeth, eyes, whiskers, hair follicles, salivary glands, and palate. These findings place BMP7 as a central mediator of epithelial-mesenchymal interactions that are necessary for the correct development of structures belonging to the orofacial complex.
Nitrous oxide (N(2)O) is a major radiative forcing and stratospheric ozone-depleting gas emitted from terrestrial and aquatic ecosystems. It can be transformed to nitrogen gas (N(2)) by bacteria and archaea harboring the N(2)O reductase (N(2)OR), which is the only known N(2)O sink in the biosphere. Despite its crucial role in mitigating N(2)O emissions, knowledge of the N(2)OR in the environment remains limited. Here, we report a comprehensive phylogenetic analysis of the nosZ gene coding the N(2)OR in genomes retrieved from public databases. The resulting phylogeny revealed two distinct clades of nosZ, with one unaccounted for in studies investigating N(2)O-reducing communities. Examination of N(2)OR structural elements not considered in the phylogeny revealed that the two clades differ in their signal peptides, indicating differences in the translocation pathway of the N(2)OR across the membrane. Sequencing of environmental clones of the previously undetected nosZ lineage in various environments showed that it is widespread and diverse. Using quantitative PCR, we demonstrate that this clade was most often at least as abundant as the other, thereby more than doubling the known extent of the overall N(2)O-reducing community in the environment. Furthermore, we observed that the relative abundance of nosZ from either clade varied among habitat types and environmental conditions. Our results indicate a physiological dichotomy in the diversity of N(2)O-reducing microorganisms, which might be of importance for understanding the relationship between the diversity of N(2)O-reducing microorganisms and N(2)O reduction in different ecosystems.
Bone morphogenetic proteins (BMPs) are involved in embryonic mammary gland (MG) development and can be dysregulated in breast cancer. However, the role BMPs play in the postnatal MG remains virtually unknown. BMPs are potent morphogens that are involved in cell fate determination, proliferation, apoptosis and adult tissue homeostasis. Twisted gastrulation (TWSG1) is a secreted BMP binding protein that modulates BMP ligand availability in the extracellular space. Here we investigate the consequences of TWSG1 deletion on development of the postnatal MG. At puberty, Twsg1 is expressed in the myoepithelium and in a subset of body cells of the terminal end buds. In the mature duct, Twsg1 expression is primarily restricted to the myoepithelial layer. Global deletion of Twsg1 leads to a delay in ductal elongation, reduced secondary branching, enlarged terminal end buds, and occluded lumens. This is associated with an increase in luminal epithelial cell number and a decrease in apoptosis. In the MG, pSMAD1/5/8 level and the expression of BMP target genes are reduced, consistent with a decrease in BMP signaling. GATA-3, which is required for luminal identity, is reduced in Twsg1(-/-) MGs, which may explain why K14 positive cells, which are normally restricted to the myoepithelial layer, are found within the luminal compartment and shed into the lumen. In summary, regulation of BMP signaling by TWSG1 is required for normal ductal elongation, branching of the ductal tree, lumen formation, and myoepithelial compartmentalization in the postnatal MG.
Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs). As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5), we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx.
Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-?3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-?/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-?3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-?3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf-?/Bmp activity.
Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4(flox/flox)) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4(flox/flox) conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.
Generation and interpretation of biotransformation data on drugs, i.e. identification of physiologically relevant metabolites, defining metabolic pathways and elucidation of metabolite structures, have become increasingly important to the drug development process. Profiling using (14)C or (3)H radiolabel is defined as the chromatographic separation and quantification of drug-related material in a given biological sample derived from an in vitro, preclinical in vivo or clinical study. Metabolite profiling is a very time intensive activity, particularly for preclinical in vivo or clinical studies which have defined limitations on radiation burden and exposure levels. A clear gap exists for certain studies which do not require specialized high volume automation technologies, yet these studies would still clearly benefit from automation. Use of radiolabeled compounds in preclinical and clinical ADME studies, specifically for metabolite profiling and identification are a very good example. The current lack of automation for measuring low level radioactivity in metabolite profiling requires substantial capacity, personal attention and resources from laboratory scientists. To help address these challenges and improve efficiency, we have innovated, developed and implemented a novel and flexible automation platform that integrates a robotic plate handling platform, HPLC or UPLC system, mass spectrometer and an automated fraction collector.
The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial-mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues.
Bone morphogenetic proteins (BMPs) are considered important regulators of neural development. However, results mainly from a wide set of in vitro gain-of-function experiments are conflicting since these show that BMPs can act either as inhibitors or promoters of neurogenesis. Here, we report a specific and non-redundant role for BMP7 in cortical neurogenesis in vivo using knockout mice. Bmp7 is produced in regions adjacent to the developing cortex; the hem, meninges, and choroid plexus, and can be detected in the cerebrospinal fluid. Bmp7 deletion results in reduced cortical thickening, impaired neurogenesis, and loss of radial glia attachment to the meninges. Subsequent in vitro analyses of E14.5 cortical cells revealed that lack of Bmp7 affects neural progenitor cells, evidenced by their reduced proliferation, survival and self-renewal capacity. Addition of BMP7 was able to rescue these proliferation and survival defects. In addition, at the developmental stage E14.5 Bmp7 was also required to maintain Ngn2 expression in the subventricular zone. These data demonstrate a novel role for Bmp7 in the embryonic mouse cortex: Bmp7 nurtures radial glia cells and regulates fundamental properties of neural progenitor cells that subsequently affect Ngn2-dependent neurogenesis.
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