This review discusses the function of neural crest as they relate to cardiovascular defects. The cardiac neural crest cells are a subpopulation of cranial neural crest discovered nearly 30 years ago by ablation of premigratory neural crest. The cardiac neural crest cells are necessary for normal cardiovascular development. We begin with a description of the crest cells in normal development, including their function in remodeling the pharyngeal arch arteries, outflow tract septation, valvulogenesis, and development of the cardiac conduction system. The cells are also responsible for modulating signaling in the caudal pharynx, including the second heart field. Many of the molecular pathways that are known to influence specification, migration, patterning and final targeting of the cardiac neural crest cells are reviewed. The cardiac neural crest cells play a critical role in the pathogenesis of various human cardiocraniofacial syndromes such as DiGeorge, Velocardiofacial, CHARGE, Fetal Alcohol, Alagille, LEOPARD, and Noonan syndromes, as well as Retinoic Acid Embryopathy. The loss of neural crest cells or their dysfunction may not always directly cause abnormal cardiovascular development, but are involved secondarily because crest cells represent a major component in the complex tissue interactions in the head, pharynx and outflow tract. Thus many of the human syndromes linking defects in the heart, face and brain can be better understood when considered within the context of a single cardiocraniofacial developmental module with the neural crest being a key cell type that interconnects the regions.

Ca(2+) regulates cell differentiation and morphogenesis in a diversity of organisms and dysregulation of Ca(2+) signal transduction pathways leads to many cellular pathologies. In Dictyostelium Ca(2+) induces ecmB expression and stalk cell differentiation in vitro. Here we have analyzed the pattern of ecmB expression in intact and bisected slugs and the effect of agents that affect Ca(2+) levels or antagonize calmodulin (CaM) on this expression pattern. We have shown that Ca(2+) and CaM regulate ecmB expression and pstAB/pstB cell differentiation in vivo. Agents that increase intracellular Ca(2+) levels increased ecmB expression and/or pstAB and pstB cell differentiation, while agents that decrease intracellular Ca(2+) or antagonize CaM decreased it. In isolated slug tips agents that affect Ca(2+) levels and antagonize CaM had differential effect on ecmB expression and cell differentiation in the anterior versus posterior zones. Agents that increase intracellular Ca(2+) levels increased the number of ecmB expressing cells in the anterior region of slugs, while agents that decrease intracellular Ca(2+) levels or antagonize CaM activity increased the number of ecmB expressing cells in the posterior. We have also demonstrated that agents that affect Ca(2+) levels or antagonize CaM affect cells motility and regeneration of shape in isolated slug tips and backs and regeneration of tips in isolated slug backs. To our knowledge, this is the first study detailing the pattern of ecmB expression in regenerating slugs as well as the role of Ca(2+) and CaM in the regeneration process and ecmB expression.

Atrioventricular valve development commences with an EMT event whereby endocardial cells transform into mesenchyme. The molecular events that induce this phenotypic change are well understood and include many growth factors, signaling components, and transcription factors. Besides their clear importance in valve development, the role of these transformed mesenchyme and the function they serve in the developing prevalve leaflets is less understood. Indeed, we know that these cells migrate, but how and why do they migrate? We also know that they undergo a transition to a mature, committed cell, largely defined as an interstitial fibroblast due to their ability to secrete various matrix components including collagen type I. However, we have yet to uncover mechanisms by which the matrix is synthesized, how it is secreted, and how it is organized. As valve disease is largely characterized by altered cell number, cell activation, and matrix disorganization, answering questions of how the valves are built will likely provide us with information of real clinical relevance. Although expression profiling and descriptive or correlative analyses are insightful, to advance the field, we must now move past the simplicity of these assays and ask fundamental, mechanistic based questions aimed at understanding how valves are "built". Herein we review current understandings of atrioventricular valve development and present what is known and what isn't known. In most cases, basic, biological questions and hypotheses that were presented decades ago on valve development still are yet to be answered but likely hold keys to uncovering new discoveries with relevance to both embryonic development and the developmental basis of adult heart valve diseases. Thus, the goal of this review is to remind us of these questions and provide new perspectives on an old theme of valve development.

Congenital heart disease is the most common birth defect in humans. Identifying factors that are critical to embryonic heart development could further our understanding of the disease and lead to new strategies of its prevention and treatment. Nitric oxide synthase-3 (NOS3) or endothelial nitric oxide synthase (eNOS) is known for many important biological functions including vasodilation, vascular homeostasis and angiogenesis. Over the past decade, studies from our lab and others have shown that NOS3 is required during heart development. More specifically, deficiency in NOS3 results in congenital septal defects, cardiac hypertrophy and postnatal heart failure. In addition, NOS3 is pivotal to the morphogenesis of major coronary arteries and myocardial capillary development. Interestingly, these effects of NOS3 are mediated through induction of transcription and growth factors that are crucial in the formation of coronary arteries. Finally, deficiency in NOS3 results in high incidences of bicuspid aortic valves, a disease in humans that often leads to complications with age including aortic valve stenosis or regurgitation, endocarditis, aortic aneurysm formation, and aortic dissection. In summary, these data suggest NOS3 plays a critical role in embryonic heart development and morphogenesis of coronary arteries and aortic valves.

During cardiac looping the heart tube elongates by addition of progenitor cells from adjacent pharyngeal mesoderm to the arterial and venous poles. This cell population, termed the second heart field, was first identified ten years ago and many studies in the intervening decade have refined our understanding of how heart tube elongation takes place and identified signaling pathways that regulate proliferation and differentiation during progressive contribution of second heart field cells to the embryonic heart. It has also become apparent that defective second heart field development results in common congenital heart anomalies affecting both the conotruncal region and venous pole of the heart, including atrial and atrioventricular septal defects. In this review we focus on a series of recent papers that have identified new regulators of second heart field development, in particular the retinoic acid signaling pathway and HOX, SIX and EYA transcription factors. We also discuss new findings concerning the regulation of fibroblast growth factor signaling during second heart field deployment and studies that have implicated FGF10 and FGF3 in outflow tract development in addition to FGF8. Second heart field derived parts of the heart share common progenitor cells in pharyngeal mesoderm with craniofacial skeletal muscles and recent findings from xenopus, zebrafish and the protochordate Ciona intestinalis provide insights into the evolution of the second heart field during vertebrate radiation.

The heart is a complex organ that is composed of numerous cell types, which must integrate their programs for proper specification, differentiation and cardiac morphogenesis. During cardiogenesis members of the Twist-family of basic helix-loop-helix (bHLH) transcription factors play distinct roles within cardiac lineages such as the endocardium and extra-cardiac lineages such as the cardiac neural crest (cNCC) and epicardium. While the study of these cell populations is often eclipsed by that of cardiomyocytes, the contributions of non-cardiomyocytes to development and disease are increasingly being appreciated as both dynamic and essential. This review summarizes what is known regarding Twist-family bHLH function in extra-cardiac cell populations and the endocardium, with a focus on regulatory mechanisms, downstream targets, and expression profiles. Improving our understanding of the molecular pathways that Twist-family bHLH factors mediate in these lineages will be necessary to ascertain how their dysfunction leads to congenital disease and adult pathologies such as myocardial infarctions and cardiac fibroblast induced fibrosis. Indeed, this knowledge will prove to be critical to clinicians seeking to improve current treatments.

Activation of Wnt/β-catenin signaling is crucial for the differentiation of pluripotent stem cells, namely the epiblast, embryonic stem, and embryonal carcinoma cells, into mesendoderm. However, downstream events of Wnt/β-catenin signaling that control the formation of mesendoderm are still unclear. In the present study, we used mouse P19 embryonal carcinoma cells as a model, and identified a homeodomain protein Nkx1-2 as a key regulator of mesendoderm formation. In the mouse embryo, Nkx1-2 was expressed in the primitive streak, in which the nascent mesendoderm emerges. In P19 cells, the expression of Nkx1-2 was activated by Wnt/β-catenin signaling independently of Brachyury, an evolutionary conserved early mesendoderm gene. In contrast, the expression of Nkx1-2 was both necessary and sufficient for the activation of Brachyury. Nkx1-2 acted as a transcriptional repressor to mediate the action of Wnt/β-catenin signaling to activate the Brachyury expression. We found Tcf3 as a potential target of gene repression by Nkx1-2, and the down-regulation of Tcf3 was partly required for effective activation of Brachyury by Wnt/β-catenin signaling. These results suggest that Nkx1-2 is a critical component of the gene regulatory network that operates downstream of Wnt/β-catenin signaling to regulate the formation of mesendoderm.

The immune system plays a key role in eliminating cancer cells in the body. However, even in fully immune-competent bodies cancers can evade anti-tumor immune action. There is increasing evidence that epithelial cancers can actively suppress anti-tumor immune responses by creating an immune-suppressive micro-environment. It has been reported that epithelial cancers can express immune genes/proteins not normally expressed by their parental tissues, including a variety of cytokines/receptors, immune transcription factors and Ig motifs in cell surface molecules. Recently we observed increased expression of immune genes, including immune-suppressive genes, by prostate epithelial cancers. In view of the above, we propose that immune-suppressive activity of epithelial cancers may stem from their acquisition of immune properties via a transdifferentiation process, we term "Epithelial Immune Cell-like Transition"(EIT), similar to neuroendocrine-like transdifferentiation of prostate adenocarcinoma cells. We propose that the acquired immune properties enable the cancer cells to "communicate" with immune cells, leading to suppression of anti-cancer immune activity in their micro-environment and facilitation of the expansion and malignant progression of the disease. Acquired immune properties of epithelial cancers, which might be quite common, could provide novel targets for reducing cancer-generated immune-suppressive activity and enhancing anti-tumor immune activity. This proposed paradigm shift could lead to novel therapeutic approaches with improved efficacy and broad application.

Rhabdomyosarcoma is a soft tissue sarcoma mainly seen in children. Despite considerable progress within the last few years, therapeutic approaches for this type of tumor are still limited. The respective tumor cells originate from myogenic precursor cells and are characterized by a blockade in their differentiation program. Interestingly, there is a direct inverse correlation between the differentiation status of a specific rhabdomyosarcoma cell and its metastatic potential. Thus, here, we tested whether the ubiquitous transcription factor NF-κB, which regulates myogenic differentiation and is also a promising therapeutic target in the treatment of other types of tumors, might be an interesting candidate for the development of novel rhabdomyosarcoma treatment strategies. For this purpose, we analyzed NF-κB activity (classical pathway) in myoblasts with different differentiation potential, specifically in three different rhabdomyosarcoma cell lines. In addition, we inhibited NF-κB activity in these cells and analyzed the effects on myogenic differentiation. We show that after the induction of differentiation, NF-κB activity declines rapidly in normal myoblasts, but only slightly in rhabdomyosarcoma cells. However, after treatment of the cells with two different small-molecule NF-κB-inhibiting compounds, the IKK inhibitor curcumin and the proteasome inhibitor lactacystin, we found that neither curcumin nor lactacystin promoted myogenic differentiation in either normal myoblasts or rhabdomyosarcoma cells. Taken together, our data suggest that treatment with curcumin or lactacystin might not be a suitable approach in the treatment of rhabdomyosarcoma.

The present study evaluated the human mesenchymal stem cells (hMSCs) isolated from skin (hSMSC), bone marrow (hBMSC) and dental follicle (hDFMSC) tissues on their in vitro and in vivo osteogenic potential using demineralized bone matrix (DBM) and fibrin glue scaffold. Cells originated from three distinct tissues showed positive expressions of CD44, CD73, CD90, CD105 and vimentin, and differentiation ability into osteocytes, adipocytes and chondrocytes. hMSCs from all tissues co-cultured with a mixed DBM and fibrin glue scaffold in non-osteogenic induction media were positively stained by von Kossa and expressed osteoblast-related genes, such as osteocalcin (OC), osteonectin (ON), runt-related transcription factor 2 (Runx2) and osterix. For in vivo osteogenic evaluation, PKH26 labeled hMSCs were implanted into the subcutaneous spaces of athymic mice with a mixed scaffold. At 4 weeks of implantation, PKH26 labeled cells were detected in all hMSC-implanted groups. Bone formation with OC expression and radio-opacity intensity were observed around DBM scaffold in all hMSC-implanted groups. Interestingly, hDFMSCs-implanted group showed the highest OC expression and calcium content. These findings demonstrated that hDFMSCs could be a potential alternative autologous cell source for bone tissue engineering.

Chorion, amnion and villi are reservoirs of mesenchymal stromal cells (StC) and the hypothesis that StC from fetal tissues retain higher plasticity compared to adult StC has been suggested. Aimed at investigating this aspect, a series of in vitro experiments were performed with StC isolated from first trimester human chorionic villi (CVStC). CVStC were cultured in:(i) standard mesenchymal medium (MM) and (ii) AmniomaxII(®)(AM), specifically designed to grow amnion-derived cells in prenatal diagnostic procedures. Cells were then exposed to distinct differentiation treatments and distinguished according to morphology, immunophenotype and molecular markers. Human StC obtained from adult bone marrow (BMStC) were used as control. CVStC cultured either in MM or AM presented stromal morphology and immunophenotype, were negative for pluripotency factors (Nanog, Oct-4 and Sox-2), lacked detectable telomerase activity and retained high genomic stability. In AM, however, CVStC exhibited a faster proliferation rate compared to BMStC or CVStC kept in MM. During differentiation, CVStC were less efficient than BMStC in acquiring adipocytes and osteocytes features; the cardiomyogenic conversion occurred at low efficiency in both cell types. Remarkably, in the presence of pro-angiogenic factors, CVStC reprogrammed toward an endothelial-like phenotype at significantly higher efficiency than BMStC. This effect was particularly evident in CVStC expanded in AM. Mechanistically, the reduced CVStC expression of anti-angiogenic microRNA could support this process. The present study demonstrates that, despite of fetal origin, CVStC exhibit restricted plasticity, distinct from that of BMStC and predominantly directed toward the endothelial lineage.

The reciprocal influence and bidirectional cross-talk between bone and energy metabolism is a recent finding, since the discovery that the product of osteoblasts osteocalcin increases pancreatic β-cell proliferation, insulin secretion and sensitivity. Conversely, the anabolic effect of insulin is crucial for osteoblast function, as suggested by severe osteopenia and increased incidence of fracture in insulin-deficient diabetic patients. The Insulin Receptor (IR) tyrosine kinase, which is commonly expressed in the insulin-sensitive liver, muscle, and adipose tissues, is also found in animal and human bone. Here we show that in human bone two insulin receptor isoforms (IR-A and IR-B) are differently expressed. Mature human osteoblasts predominantly express IR-B, whereas IR-A is mainly expressed in osteoblast precursors, and IR-B/IR-A mRNA ratio significantly increases along the osteogenic differentiation of mesenchymal stromal precursors. Moreover, transfected osteoprogenitors overexpressing IR-A show an increased proliferation rate. In contrast, when transfected with and overexpressing IR-B, their proliferation rate is reduced, corresponding to a more differentiated phenotype. In conclusion, the fine regulation of the expression of different isoforms of IR during osteogenic differentiation confirms the important role played by IR in bone homeostasis, providing the basis for new perspectives on the various involvements of IR isoforms in bone pathophysiology.

Embryonic stem (ES) cells are pluripotent cells that can differentiate into all three main germ layers: endoderm, mesoderm, and ectoderm. Although a number of methods have been developed to differentiate ES cells into neuronal phenotypes such as sensory and motor neurons, the efficient generation of GABAergic interneurons from ES cells still presents an ongoing challenge. Because the main output of inhibitory GABAergic interneurons is the gamma-aminobutyric-acid (GABA), a neurotransmitter whose controlled homeostasis is required for normal brain function, the efficient generation in culture of functional interneurons may have future implications on the treatment of neurological disorders such as epilepsy, autism, and schizophrenia. The goal of this work was to examine the generation of GABAergic neurons from mouse ES cells by comparing an embryoid body-based methodology versus a hydrogel-based encapsulation protocol that involves the use of all-trans-retinoid acid (RA). We observed that (1) there was a 2-fold increase in neuronal differentiation in encapsulated versus non-encapsulated cells and (2) there was an increase in the specificity for interneuronal differentiation in encapsulated cells, as assessed by mRNA expression and electrophysiology approaches. Furthermore, our results indicate that most of the neurons obtained from encapsulated mouse ES cells are GABA-positive (∼87%). Thus, these results suggest that combining encapsulation of ES cells and RA treatment provide a more efficient and scalable differentiation strategy for the generation in culture of functional GABAergic interneurons. This technology may have implications for future cell replacement therapies and the treatment of CNS disorders.

Congenital limb reduction defects occurring in isolation of other developmental abnormalities continue to be an important medical problem in which little progress has been made. Herein we generated transgenic mice expressing Dkk1 in an appendicular mesodermal pattern. Prx1-Dkk1 mice recapitulate a full spectrum of human congenital limb reduction defects, without other developmental issues, and have normal life-spans. Importantly, a close examination of the inheritance pattern suggests that there is a significant degree of incomplete penetrance as progeny of phenotypically positive or phenotypically negative, but genotypically positive Prx1-Dkk1 mice, consistently give rise to both phenotypically positive mice and phenotypically normal-appearing mice. Thus, this heterogeneous phenotype is reproducible with each generation regardless of the phenotype of the parents. We further go on to identify that mesenchymal stem cells from Prx1-Dkk1 mice have limited proliferative ability, but normal differentiation potential, which may explain the mechanism for the limb reduction defects observed. We believe Prx1-Dkk1 mice may prove useful in the future to study the mechanisms underlying the development of congenital limb reduction defects.

FGFs and BMPs act in concert to regulate a wide range of processes in vertebrate development. In most cases, FGFs and BMPs have opposing effects, and specific developmental outcomes arise out of a balance between the two growth factors. We and others have previously demonstrated that signaling pathways activated by FGFs and BMPs interact via inhibitory crosstalk. Here we demonstrate a role for the BMP effector TGF-β Activated Kinase 1 (TAK1) in the maintenance of Smad1 activity in Xenopus embryos, via the inhibition of erk MAPK. Up- or downregulation of TAK1 levels produces an inverse alteration in the amount of activated erk MAPK. The inhibition of erk MAPK by TAK1 is mediated by p38 and a corresponding decrease in phosphorylation of MEK. TAK1 morphant embryos show a decrease in the nuclear accumulation of Smad1. Conversely, reduction of erk MAPK activity via overexpression of MAP Kinase Phosphatase1 (MKP1) leads to an increase in nuclear Smad1. Both TAK1 morphant ectoderm and ectoderm treated with FGF show a decrease in the expression of several Smad1-inducible genes. Neural-specific gene expression is inhibited in isolated ectoderm coexpressing noggin and TAK1, suggesting that TAK1 is sufficient to inhibit neural specification. Introduction of TAK1 morpholino oligonucleotide expands the expression of organizer genes, disrupts formation of the boundary between organizer and non-organizer mesoderm, and increases the spatial range of MAPK activation in response to localized FGF. Our results indicate that inhibitory interactions between FGF and BMP4 effector pathways increase the robustness of BMP signaling via a feed-forward mechanism.

Several fundamental questions regarding cell growth and development can be answered by recording and analyzing the history of cells and their progeny. Herein, long-term and large-field live cell imaging was used to study the process of megakaryopoiesis at the single cell level (n=9300) from human CD34(+) cord blood (CB) in the presence of thrombopoietin (TPO) or the cytokine cocktail BS1 with or without nicotinamide (NIC). Comparative analyses revealed that the cocktail BS1 increased the mitotic and proplatelet rate of diploid and polyploid cells, respectively. Conversely, only NIC treatment increased the endomitotic rate of megakaryocytes (MKs) leading to the formation of CB-MKs with ploidy level frequently observed with BM-MKs. However, NIC failed to enhance platelet production. Rather, a 7- and 31-fold reduction in proplatelet formation was observed in tetraploid and octaploid CB-MKs, respectively, and ex vivo platelet production output was reduced by half due to a reduction in MK output in NIC cultures. Unexpectedly, a significant fraction of di- and polyploid CB-MKs were seen to undergo complete proplatelet regression. Though rare (<0.6%), proplatelet reversal led to the formation of regular round cells that could at times resume normal development. The cell tracking data was then used to investigate the impact of "developmental fate" and ploidy on cell cycling time, and to identify potential developmental patterns. These analyses revealed that cell fate and ploidy level have major impacts on the cell cycling time of the cells, and that four recurrent cell lineage patterns could be identified for CD34(+) cells undergoing MK differentiation.

Previous work, verified here, showed that colchicine affects Dictyostelium pattern formation, disrupts morphogenesis, inhibits spore differentiation and induces terminal stalk cell differentiation. Here we show that colchicine specifically induces ecmB expression and enhances accumulation of ecmB-expressing cells at the posterior end of multicellular structures. Colchicine did not induce a nuclear translocation of DimB, a DIF-1 responsive transcription factor in vitro. It also induced terminal stalk cell differentiation in a mutant strain that does not produce DIF-1 (dmtA(-)) and after the treatment of cells with DIF-1 synthesis inhibitor cerulenin (100μM). This suggests that colchicine induces the differentiation of ecmB-expressing cells independent of DIF-1 production and likely through a signaling pathway that is distinct from the one that is utilized by DIF-1. Depending on concentration, colchicine enhanced random cell motility, but not chemotaxis, by 3-5 fold (10-50mM colchicine, respectively) through a Ca(2+)-mediated signaling pathway involving phospholipase C, calmodulin and heterotrimeric G proteins. Colchicine's effects were not due to microtubule depolymerization as other microtubule-depolymerizing agents did not have these effects. Finally normal morphogenesis and stalk and spore cell differentiation of cells treated with 10mM colchicine were rescued through chelation of Ca(2+) by BAPTA-AM and EDTA and calmodulin antagonism by W-7 but not PLC inhibition by U-73122. Morphogenesis or spore cell differentiation of cells treated with 50mM colchicine could not be rescued by the above treatments but terminal stalk cell differentiation was inhibited by BAPTA-AM, EDTA and W-7, but not U-73122. Thus colchicine disrupts morphogenesis and induces stalk cell differentiation through a Ca(2+)-mediated signaling pathway involving specific changes in gene expression and cell motility.

Oscillatory mechanical stimulation at relatively high frequencies (0.1Hz) has been shown to inhibit adipogenic and promote osteogenic differentiation of mesenchymal stem cells. However, for physiological interpretations and ease of implementation it is of interest to know whether different rates of mechanical stimulation can produce similar results. We hypothesized that relatively low frequency mechanical stimulation (0.01Hz) can inhibit adipogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells, even in a potent adipogenic differentiation medium. C3H10T1/2 cells were cultured in adipogenic medium under control (non-mechanically stimulated) conditions and under oscillatory surface stretch with 10% amplitude and 0.01Hz frequency for 6h per day for up to 5 days. Cell population was assessed by counting and adipogenic differentiation was assessed by real-time quantitative PCR (qPCR) analysis of peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid binding protein 4 (FABP4) after 3 and 5 days. Involvement of the ERK signaling pathway was assessed by Western blot. Low frequency mechanical stimulation significantly decreased expression of PPARγ after 3 days and FABP4 after 3 and 5 days versus non-stimulated culture. ERK signaling was decreased in mechanically-stimulated culture, indicating a role in the inhibition of adipogenic differentiation. Application of this study: Low frequency mechanical stimulation may provide a technically simple means for control of mesenchymal stem cell differentiation in cell-based therapies, particularly for inhibition of differentiation toward undesired adipogenic lineages.

While the pathologies associated with in utero smoke exposure are well established, their underlying molecular mechanisms are incompletely understood. We differentiated human embryonic stem cells in the presence of physiological concentrations of tobacco smoke and nicotine. Using post hoc microarray analysis, quantitative PCR, and immunoblot analysis, we demonstrated that tobacco smoke has lineage- and stage-specific effects on human embryonic stem cell differentiation, through both nicotine-dependent and -independent pathways. We show that three major stem cell pluripotency/differentiation pathways, Notch, canonical Wnt, and transforming growth factor-β, are affected by smoke exposure, and that Nodal signaling through SMAD2 is specifically impacted by effects on Lefty1, Nodal, and FoxH1. These events are associated with upregulation of microRNA-302a, a post-transcriptional silencer of Lefty1. The described studies provide insight into the mechanisms by which tobacco smoke influences fetal development at the cellular level, and identify specific transcriptional, post-transcriptional, and signaling pathways by which this likely occurs.