A poised chromatin platform for TGF-ß access to master regulators.

Specific chromatin marks keep master regulators of differentiation silent yet poised for activation by extracellular signals. We report that nodal TGF-ß signals use the poised histone mark H3K9me3 to trigger differentiation of mammalian embryonic stem cells. Nodal receptors induce the formation of companion Smad4-Smad2/3 and TRIM33-Smad2/3 complexes. The PHD-Bromo cassette of TRIM33 facilitates binding of TRIM33-Smad2/3 to H3K9me3 and H3K18ac on the promoters of mesendoderm regulators Gsc and Mixl1. The crystal structure of this cassette, bound to histone H3 peptides, illustrates that PHD recognizes K9me3, and Bromo binds an adjacent K18ac. The interaction between TRIM33-Smad2/3 and H3K9me3 displaces the chromatin-compacting factor HP1γ, making nodal response elements accessible to Smad4-Smad2/3 for Pol II recruitment. In turn, Smad4 increases K18 acetylation to augment TRIM33-Smad2/3 binding. Thus, nodal effectors use the H3K9me3 mark as a platform to switch master regulators of stem cell differentiation from the poised to the active state.

A unique form of collective epithelial migration is crucial for tissue fusion in the secondary palate and can overcome loss of epithelial apoptosis.

Tissue fusion frequently requires the removal of an epithelium that intervenes distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion support their importance in MES removal. However, genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES results in complete loss of cell death and cell extrusion, but successful removal of the MES. Novel static- and live-imaging approaches reveal that the MES is removed through streaming migration of epithelial trails and islands to reach the oral and nasal epithelial surfaces. Epithelial trail cells that express the basal epithelial marker ΔNp63 begin to express periderm markers, suggesting that migration is concomitant with differentiation. Live imaging reveals anisotropic actomyosin contractility within epithelial trails, and genetic ablation of actomyosin contractility results in dispersion of epithelial collectives and failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis, and reveal a crucial and unique form of collective epithelial migration during tissue fusion.

Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A.

Myostatin (MSTN) is a transforming growth factor-ß (TGF-ß) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-ß family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

Trim33 is required for appropriate development of pre-cardiogenic mesoderm.

Congenital cardiac malformations are among the most common birth defects in humans. Here we show that Trim33, a member of the Tif1 subfamily of tripartite domain containing transcriptional cofactors, is required for appropriate differentiation of the pre-cardiogenic mesoderm during a narrow time window in late gastrulation. While mesoderm-specific Trim33 mutants did not display noticeable phenotypes, epiblast-specific Trim33 mutant embryos developed ventricular septal defects, showed sparse trabeculation and abnormally thin compact myocardium, and died as a result of cardiac failure during late gestation. Differentiating embryoid bodies deficient in Trim33 showed an enrichment of gene sets associated with cardiac differentiation and contractility, while the total number of cardiac precursor cells was reduced. Concordantly, cardiac progenitor cell proliferation was reduced in Trim33-deficient embryos. ChIP-Seq performed using antibodies against Trim33 in differentiating embryoid bodies revealed more than 4000 peaks, which were significantly enriched close to genes implicated in stem cell maintenance and mesoderm development. Nearly half of the Trim33 peaks overlapped with binding sites of the Ctcf insulator protein. Our results suggest that Trim33 is required for appropriate differentiation of precardiogenic mesoderm during late gastrulation and that it will likely mediate some of its functions via multi-protein complexes, many of which include the chromatin architectural and insulator protein Ctcf.

Activin A receptor type 1-mediated BMP signaling regulates RANKL-induced osteoclastogenesis via canonical SMAD-signaling pathway.

Bone morphogenetic proteins (BMPs) are important mediators of osteoclast differentiation. Although accumulating evidence has implicated BMPs in osteoblastogenesis, the mechanisms by which BMPs regulate osteoclastogenesis remain unclear. Activin A receptor type 1 (ACVR1) is a BMP type 1 receptor essential for skeletal development. Here, we observed that BMP-7, which preferentially binds to ACVR1, promotes osteoclast differentiation, suggesting ACVR1 is involved in osteoclastogenesis. To investigate this further, we isolated osteoclasts from either Acvr1-floxed mice or mice with constitutively-activated Acvr1 (caAcvr1) carrying tamoxifen-inducible Cre driven by a ubiquitin promotor and induced Cre activity in culture. Osteoclasts from the Acvr1-floxed mice had reduced osteoclast numbers and demineralization activity, whereas those from the caAcvr1-mutant mice formed large osteoclasts and demineralized pits, suggesting that BMP signaling through ACVR1 regulates osteoclast fusion and activity. It is reported that BMP-2 binds to BMPR1A, another BMP type 1 receptor, whereas BMP-7 binds to ACVR1 to activate SMAD1/5/9 signaling. Here, Bmpr1a-disrupted osteoclasts displayed reduced phospho-SMAD1/5/9 (pSMAD1/5/9) levels when induced by BMP-2, whereas no impacts on pSMAD1/5/9 were observed when induced by BMP-7. In contract, Acvr1-disrupted osteoclasts displayed reduced pSMAD1/5/9 levels when induced either by BMP-2 or BMP-7, suggesting that ACVR1 is the major receptor for transducing BMP-7 signals in osteoclasts. Indeed, LDN-193189 and LDN-212854, which specifically block SMAD1/5/9 phosphorylation, inhibited osteoclastogenesis of caAcvr1-mutant cells. Moreover, increased BMP signaling promoted nuclear translocation of nuclear factor-activated T-cells 1 (NFATc1), which was inhibited by LDN treatments. Taken together, ACVR1-mediated BMP-SMAD signaling activates NFATc1, a regulatory protein crucial for receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis.

BmpR1A is a major type 1 BMP receptor for BMP-Smad signaling during skull development.

Craniosynostosis is caused by premature fusion of one or more sutures in an infant skull, resulting in abnormal facial features. The molecular and cellular mechanisms by which genetic mutations cause craniosynostosis are incompletely characterized, and many of the causative genes for diverse types of syndromic craniosynostosis have not yet been identified. We previously demonstrated that augmentation of BMP signaling mediated by a constitutively active BMP type IA receptor (ca-BmpR1A) in neural crest cells (ca1A hereafter) causes craniosynostosis and superimposition of heterozygous null mutation of Bmpr1a rescues premature suture fusion (ca1A;1aH hereafter). In this study, we superimposed heterozygous null mutations of the other two BMP type I receptors, Bmpr1b and Acvr1 (ca1A;1bH and ca1A;AcH respectively hereafter) to further dissect involvement of BMP-Smad signaling. Unlike caA1;1aH, ca1A;1bH and ca1A;AcH did not restore the craniosynostosis phenotypes. In our in vivo study, Smad-dependent BMP signaling was decreased to normal levels in mut;1aH mice. However, BMP receptor-regulated Smads (R-Smads; pSmad1/5/9 hereafter) levels were comparable between ca1A, ca1A;1bH and ca1A;AcH mice, and elevated compared to control mice. Bmpr1a, Bmpr1b and Acvr1 null cells were used to examine potential mechanisms underlying the differences in ability of heterozygosity for Bmpr1a vs. Bmpr1b or Acvr1 to rescue the mut phenotype. pSmad1/5/9 level was undetectable in Bmpr1a homozygous null cells while pSmad1/5/9 levels did not decrease in Bmpr1b or Acvr1 homozygous null cells. Taken together, our study indicates that different levels of expression and subsequent activation of Smad signaling differentially contribute each BMP type I receptor to BMP-Smad signaling and craniofacial development. These results also suggest differential involvement of each type 1 receptor in pathogenesis of syndromic craniosynostoses.

Scleraxis-Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon.

The pathologic development of heterotopic ossification (HO) is well described in patients with extensive trauma or with hyperactivating mutations of the bone morphogenetic protein (BMP) receptor ACVR1. However, identification of progenitor cells contributing to this process remains elusive. Here we show that connective tissue cells contribute to a substantial amount of HO anlagen caused by trauma using postnatal, tamoxifen-inducible, scleraxis-lineage restricted reporter mice (Scx-creERT2/tdTomatofl/fl ). When the scleraxis-lineage is restricted specifically to adults prior to injury marked cells contribute to each stage of the developing HO anlagen and coexpress markers of endochondral ossification (Osterix, SOX9). Furthermore, these adult preinjury restricted cells coexpressed mesenchymal stem cell markers including PDGFRα, Sca1, and S100A4 in HO. When constitutively active ACVR1 (caACVR1) was expressed in scx-cre cells in the absence of injury (Scx-cre/caACVR1fl/fl ), tendons and joints formed HO. Postnatal lineage-restricted, tamoxifen-inducible caACVR1 expression (Scx-creERT2/caACVR1fl/fl ) was sufficient to form HO after directed cardiotoxin-induced muscle injury. These findings suggest that cells expressing scleraxis within muscle or tendon contribute to HO in the setting of both trauma or hyperactive BMP receptor (e.g., caACVR1) activity. Stem Cells 2017;35:705-710.

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

OBJECTIVE: Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2, and Alk3 in mouse retinal vessels. APPROACH AND RESULTS: Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2. Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. CONCLUSIONS: Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels.

proBDNF and p75NTR Control Excitability and Persistent Firing of Cortical Pyramidal Neurons.

Persistent firing of entorhinal cortex (EC) pyramidal neurons is a key component of working and spatial memory. We report here that a pro-brain-derived neurotrophic factor (proBDNF)-dependent p75NTR signaling pathway plays a major role in excitability and persistent activity of pyramidal neurons in layer V of the EC. Using electrophysiological recordings, we show that proBDNF suppresses persistent firing in entorhinal slices from wild-type mice but not from p75NTR-null mice. Conversely, function-blocking proBDNF antibodies enhance excitability of pyramidal neurons and facilitate their persistent firing, and acute exposure to function-blocking p75NTR antibodies results in enhanced firing activity of pyramidal neurons. Genetic deletion of p75NTR specifically in neurons or during adulthood also induces enhanced excitability and persistent activity, indicating that the proBDNF-p75NTR signaling cascade functions within adult neurons to inhibit pyramidal activity. Phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive transient receptor potential canonical channels play a critical role in mediating persistent firing in the EC and we hypothesized that proBDNF-dependent p75NTR activation regulates PIP2 levels. Accordingly, proBDNF decreases cholinergic calcium responses in cortical neurons and affects carbachol-induced depletion of PIP2. Further, we show that the modulation of persistent firing by proBDNF relies on a p75NTR-Rac1-PI4K pathway. The hypothesis that proBDNF and p75NTR maintain network homeostasis in the adult CNS was tested in vivo and we report that p75NTR-null mice show improvements in working memory but also display an increased propensity for severe seizures. We propose that the proBDNF-p75NTR axis controls pyramidal neuron excitability and persistent activity to balance EC performance with the risk of runaway activity. SIGNIFICANCE STATEMENT: Persistent firing of entorhinal cortex (EC) pyramidal neurons is required for working memory. We report here that pro-brain-derived neurotrophic factor (proBDNF) activates p75NTR to induce a Rac1-dependent and phosphatidylinositol 4,5-bisphosphate-dependent signaling cascade that suppresses persistent activity. Conversely, using loss-of-function approaches, we find that endogenous proBDNF or p75NTR activation strongly decreases pyramidal neuron excitability and persistent firing, suggesting that a physiological role of this proBDNF-p75NTR cascade may be to regulate working memory in vivo. Consistent with this, mice rendered null for p75NTR during adulthood show improvements in working memory but also display an increased propensity for severe seizures. We propose that by attenuating EC network performance, the proBDNF-p75NTR signaling cascade reduces the probability of epileptogenesis.

Differential regenerative capacity of neonatal mouse hearts after cryoinjury.

Neonatal mouse hearts fully regenerate after ventricular resection similar to adult zebrafish. We established cryoinjury models to determine if different types and varying degrees of severity in cardiac injuries trigger different responses in neonatal mouse hearts. In contrast to ventricular resection, neonatal mouse hearts fail to regenerate and show severe impairment of cardiac function post transmural cryoinjury. However, neonatal hearts fully recover after non-transmural cryoinjury. Interestingly, cardiomyocyte proliferation does not significantly increase in neonatal mouse hearts after cryoinjuries. Epicardial activation and new coronary vessel formation occur after cryoinjury. The profibrotic marker PAI-1 is highly expressed after transmural but not non-transmural cryoinjuries, which may contribute to the differential scarring. Our results suggest that regenerative medicine strategies for heart injuries should vary depending on the nature of the injury.

Tak1, Smad4 and Trim33 redundantly mediate TGF-ß3 signaling during palate development.

Transforming growth factor-beta3 (TGF-ß3) plays a critical role in palatal epithelial cells by inducing palatal epithelial fusion, failure of which results in cleft palate, one of the most common birth defects in humans. Recent studies have shown that Smad-dependent and Smad-independent pathways work redundantly to transduce TGF-ß3 signaling in palatal epithelial cells. However, detailed mechanisms by which this signaling is mediated still remain to be elucidated. Here we show that TGF-ß activated kinase-1 (Tak1) and Smad4 interact genetically in palatal epithelial fusion. While simultaneous abrogation of both Tak1 and Smad4 in palatal epithelial cells resulted in characteristic defects in the anterior and posterior secondary palate, these phenotypes were less severe than those seen in the corresponding Tgfb3 mutants. Moreover, our results demonstrate that Trim33, a novel chromatin reader and regulator of TGF-ß signaling, cooperates with Smad4 during palatogenesis. Unlike the epithelium-specific Smad4 mutants, epithelium-specific Tak1:Smad4- and Trim33:Smad4-double mutants display reduced expression of Mmp13 in palatal medial edge epithelial cells, suggesting that both of these redundant mechanisms are required for appropriate TGF-ß signal transduction. Moreover, we show that inactivation of Tak1 in Trim33:Smad4 double conditional knockouts leads to the palatal phenotypes which are identical to those seen in epithelium-specific Tgfb3 mutants. To conclude, our data reveal added complexity in TGF-ß signaling during palatogenesis and demonstrate that functionally redundant pathways involving Smad4, Tak1 and Trim33 regulate palatal epithelial fusion.

BMP signaling mediated by constitutively active Activin type 1 receptor (ACVR1) results in ectopic bone formation localized to distal extremity joints.

BMP signaling mediated by ACVR1 plays a critical role for development of multiple structures including the cardiovascular and skeletal systems. While deficient ACVR1 signaling impairs normal embryonic development, hyperactive ACVR1 function (R206H in humans and Q207D mutation in mice, ca-ACVR1) results in formation of heterotopic ossification (HO). We developed a mouse line, which conditionally expresses ca-ACVR1 with Nfatc1-Cre(+) transgene. Mutant mice developed ectopic cartilage and bone at the distal joints of the extremities including the interphalangeal joints and hind limb ankles as early as P4 in the absence of trauma or exogenous bone morphogenetic protein (BMP) administration. Micro-CT showed that even at later time points (up to P40), cartilage and bone development persisted at the affected joints most prominently in the ankle. Interestingly, this phenotype was not present in areas of bone outside of the joints - tibia are normal in mutants and littermate controls away from the ankle. These findings demonstrate that this model may allow for further studies of heterotopic ossification, which does not require the use of stem cells, direct trauma or activation with exogenous Cre gene administration.

Activin-like kinase 2 functions in peri-implantation uterine signaling in mice and humans.

Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP) ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2), during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice). In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein ß (Cebpb) expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC) and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP) analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3' UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR). Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization.

Generation of Evc2/Limbin global and conditional KO mice and its roles during mineralized tissue formation.

Ellis-van Creveld (EvC) syndrome (OMIM 225500) is an autosomal recessive disease characterized with chondrodysplastic dwarfism in association with abnormalities in oral cavity. Ciliary proteins EVC and EVC2 have been identified as causative genes and they play an important role on Hedgehog signal transduction. We have also identified a causative gene LIMBIN for bovine chondrodysplastic dwarfism (bcd) that is later identified as the bovine ortholog of EVC2. Here, we report generation of conventional and conditional mutant Evc2/Limbin alleles that mimics mutations found in EvC patients and bcd cattle. Resulted homozygous mice showed no ciliary localization of EVC2 and EVC and displayed reduced Hedgehog signaling activity in association with skeletal and oral defects similar to the EvC patients. Cartilage-specific disruption of Evc2/Limbin resulted in similar but milder skeletal defects, whereas osteoblast-specific disruption did not cause overt changes in skeletal system. Neural crest-specific disruption of Evc2/Limbin resulted in defective incisor growth similar to that seen in conventional knockouts; however, differentiation of amelobolasts was relatively normal in the conditional knockouts. These results showcased functions of EVC2/LIMBIN during formation of mineralized tissues. Availability of the conditional allele for this gene should facilitate further detailed analyses of the role of EVC2/LIMBIN in pathogenesis of EvC syndrome. genesis 53:612-626, 2015. © 2015 Wiley Periodicals, Inc.

AcvR1-mediated BMP signaling in second heart field is required for arterial pole development: implications for myocardial differentiation and regional identity.

BMP signaling plays an essential role in second heart field-derived heart and arterial trunk development, including myocardial differentiation, right ventricular growth, and interventricular, outflow tract and aortico-pulmonary septation. It is mediated by a number of different BMP ligands, and receptors, many of which are present simultaneously. The mechanisms by which they regulate morphogenetic events and degree of redundancy amongst them have still to be elucidated. We therefore assessed the role of BMP Type I receptor AcvR1 in anterior second heart field-derived cell development, and compared it with that of BmpR1a. By removing Acvr1 using the driver Mef2c[AHF]-Cre, we show that AcvR1 plays an essential role in arterial pole morphogenesis, identifying defects in outflow tract wall and cushion morphology that preceded a spectrum of septation defects from double outlet right ventricle to common arterial trunk in mutants. Its absence caused dysregulation in gene expression important for myocardial differentiation (Isl1, Fgf8) and regional identity (Tbx2, Tbx3, Tbx20, Tgfb2). Although these defects resemble to some degree those in the equivalent Bmpr1a mutant, a novel gene knock-in model in which Bmpr1a was expressed in the Acvr1 locus only partially restored septation in Acvr1 mutants. These data show that both BmpR1a and AcvR1 are needed for normal heart development, in which they play some non-redundant roles, and refine our understanding of the genetic and morphogenetic processes underlying Bmp-mediated heart development important in human congenital heart disease.

Smad4 and Trim33/Tif1γ redundantly regulate neural stem cells in the developing cortex.

During central nervous system (CNS) development, proliferation and differentiation of neural stem cells (NSCs) have to be regulated in a spatio-temporal fashion. Here, we report different branches of the transforming growth factor ß (TGFß) signaling pathway to be required for the brain area-specific control of NSCs. In the midbrain, canonical TGFß signaling via Smad4 regulates the balance between proliferation and differentiation of NSCs. Accordingly, Smad4 deletion resulted in horizontal expansion of NSCs due to increased proliferation, decreased differentiation, and decreased cell cycle exit. In the developing cortex, however, ablation of Smad4 alone did not have any effect on proliferation and differentiation of NSCs. In contrast, concomitant mutation of both Smad4 and Trim33 led to an increase in proliferative cells in the ventricular zone due to decreased cell cycle exit, revealing a functional redundancy of Smad4 and Trim33. Furthermore, in Smad4-Trim33 double mutant embryos, cortical NSCs generated an excess of deep layer neurons concurrent with a delayed and reduced production of upper layer neurons and, in addition, failed to undergo the neurogenic to gliogenic switch at the right developmental stage. Thus, our data disclose that in different regions of the developing CNS different aspects of the TGFß signaling pathway are required to ensure proper development.

TiF1-gamma plays an essential role in murine hematopoiesis and regulates transcriptional elongation of erythroid genes.

Transcriptional regulators play critical roles in the regulation of cell fate during hematopoiesis. Previous studies in zebrafish have identified an essential role for the transcriptional intermediary factor TIF1γ in erythropoiesis by regulating the transcription elongation of erythroid genes. To study if TIF1γ plays a similar role in murine erythropoiesis and to assess its function in other blood lineages, we generated mouse models with hematopoietic deletion of TIF1γ. Our results showed a block in erythroid maturation in the bone marrow following tif1γ deletion that was compensated with enhanced spleen erythropoiesis. Further analyses revealed a defect in transcription elongation of erythroid genes in the bone marrow. In addition, loss of TIF1γ resulted in defects in other blood compartments, including a profound loss of B cells, a dramatic expansion of granulocytes and decreased HSC function. TIF1γ exerts its functions in a cell-autonomous manner as revealed by competitive transplantation experiments. Our study therefore demonstrates that TIF1γ plays essential roles in multiple murine blood lineages and that its function in transcription elongation is evolutionally conserved.

TGF-ß-activated kinase 1 (Tak1) mediates agonist-induced Smad activation and linker region phosphorylation in embryonic craniofacial neural crest-derived cells.

BACKGROUND: The role of Smad-independent TGF-ß signaling in craniofacial development is poorly elucidated. RESULTS: In craniofacial mesenchymal cells, Tak1 regulates both R-Smad C-terminal and linker region phosphorylation in TGF-ß signaling. CONCLUSION: Tak1 plays an irreplaceable role in craniofacial ecto-mesenchyme during embryogenesis. SIGNIFICANCE: Understanding the mechanisms of TGF-ß signaling contributes to knowledge of pathogenetic mechanisms underlying common craniofacial birth defects. Although the importance of TGF-ß superfamily signaling in craniofacial growth and patterning is well established, the precise details of its signaling mechanisms are still poorly understood. This is in part because of the concentration of studies on the role of the Smad-dependent (so-called "canonical") signaling pathways relative to the Smad-independent ones in many biological processes. Here, we have addressed the role of TGF-ß-activated kinase 1 (Tak1, Map3k7), one of the key mediators of Smad-independent (noncanonical) TGF-ß superfamily signaling in craniofacial development, by deleting Tak1 specifically in the neural crest lineage. Tak1-deficient mutants display a round skull, hypoplastic maxilla and mandible, and cleft palate resulting from a failure of palatal shelves to appropriately elevate and fuse. Our studies show that in neural crest-derived craniofacial ecto-mesenchymal cells, Tak1 is not only required for TGF-ß- and bone morphogenetic protein-induced p38 Mapk activation but also plays a role in agonist-induced C-terminal and linker region phosphorylation of the receptor-mediated R-Smads. Specifically, we demonstrate that the agonist-induced linker region phosphorylation of Smad2 at Thr-220, which has been shown to be critical for full transcriptional activity of Smad2, is dependent on Tak1 activity and that in palatal mesenchymal cells TGFßRI and Tak1 kinases mediate both overlapping and distinct TGF-ß2-induced transcriptional responses. To summarize, our results suggest that in neural crest-derived ecto-mesenchymal cells, Tak1 provides a critical point of intersection in a complex dialogue between the canonical and noncanonical arms of TGF-ß superfamily signaling required for normal craniofacial development.

Cell cycle arrest in node cells governs ciliogenesis at the node to break left-right symmetry.

Cilia at the node generate a leftward fluid flow that breaks left-right symmetry. However, the molecular mechanisms that regulate ciliogenesis at the node are largely unknown. Here, we show that the epiblast-specific deletion of the gene encoding the BMP type 1 receptor (Acvr1) compromised development of nodal cilia, which results in defects in leftward fluid flow and, thus, abnormalities in left-right patterning. Acvr1 deficiency in mouse embryonic fibroblasts (MEFs) resulted in severe defects in their quiescence-induced primary cilia. Although the induction of quiescence in wild-type MEFs leads to an increase in the level of the cyclin-dependent kinase inhibitor p27(Kip1) and to rapid p27(Kip1) phosphorylation on Ser(10), MEFs deficient in Acvr1 show a reduction in both p27(Kip1) protein levels and in p27(Kip1) Ser(10) phosphorylation. The observed defects in cilium development were rescued by the introduction of p27(Kip1) into Acvr1-deficient MEFs, implying that BMP signaling positively controls p27(Kip1) stability in the G0 phase via p27(Kip1) Ser(10) phosphorylation, which is a prerequisite for induction of primary cilia. Importantly, in control embryos, p27(Kip1) protein is clearly present and strongly phosphorylated on Ser(10) in cells on the quiescent ventral surface of the node. By contrast, the corresponding cells in the node of Acvr1 mutant embryos were proliferative and showed a dramatic attenuation in both p27(Kip1) protein levels and phosphorylation on Ser(10). Our data suggest that cell quiescence controlled by BMP signaling via ACVR1 is required for transient formation of nodal cilia, and provide insight into the fundamental question of how the node represents the mechanistic `node' that regulates the development of left-right symmetry in vertebrates.

Notch signaling prevents mucous metaplasia in mouse conducting airways during postnatal development.

Goblet cell metaplasia and mucus overproduction contribute to the pathogenesis of chronic lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). Notch signaling regulates cell fate decisions and is crucial in controlling goblet cell differentiation in the gut epithelium. Little is known, however, about how endogenous Notch signaling influences the goblet cell differentiation program that takes place in the postnatal lung. Using a combination of genetic and in vitro approaches here we provide evidence of a novel role for Notch in restricting goblet cell differentiation in the airway epithelium during the postnatal period. Conditional inactivation of the essential Notch pathway component Pofut1 (protein O-fucosyltransferase1) in Tgfb3-Cre-expressing mice resulted in an aberrant postnatal airway phenotype characterized by marked goblet cell metaplasia, decreased Clara cell number and increase in ciliated cells. The presence of the same phenotype in mice in which the Notch transcriptional effector Rbpjk was deleted indicated the involvement of the canonical Notch pathway. Lineage study in vivo suggested that goblet cells originated from a subpopulation of Clara cells largely present in proximal airways in which Notch was disrupted. The phenotype was confirmed by a panel of goblet cell markers, showed no changes in cell proliferation or altered expression of proinflammatory cytokines and was associated with significant downregulation of the bHLH transcriptional repressor Hes5. Luciferase reporter analysis suggested that Notch directly repressed MUC5AC transcription in lung epithelial cells. The data suggested that during postnatal life Notch is required to prevent Clara cells from differentiating into goblet cells.