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The genetic basis of congenital heart malformations associated with disruption of left-right (L-R) asymmetry is broad and heterogenous, with variants in over 25 genes implicated thus far. Of these, deleterious variants in the Growth/Differentiation Factor 1 (GDF1) gene have been shown to cause heterotaxy with varied complex heart malformations of left-right patterning, in 23 individuals reported to date, either in monoallelic or biallelic state. We report three unrelated individuals exhibiting right isomerism with congenital heart defects, each originating from a consanguineous kindred of Arab-Muslim descent. Using whole exome sequencing, a shared novel homozygous truncating c.608G > A (p.W203*) variant in the GDF1 gene was revealed as the molecular basis of their disease. Subsequently, targeted sequencing of this variant showed full segregation with the disease in these families, with a total of over 15 reportedly affected individuals, enabling genetic counseling, prenatal diagnosis, and planning of future pregnancies. Our findings further confirm the association of biallelic GDF1 variants, heterotaxy and congenital heart defects of left-right patterning, and expand the previously described phenotypic spectrum and mutational profile. Moreover, we suggest targeted screening for the p.W203* variant in relevant clinical circumstances.

Cripto is a small glycosylphosphatidylinisitol (GPI)-anchored and secreted oncofetal protein that plays important roles in regulating normal physiological processes, including stem cell differentiation, embryonal development, and tissue growth and remodeling, as well as pathological processes such as tumor initiation and progression. Cripto functions as a co-receptor for TGF-β ligands such as Nodal, GDF1, and GDF3. Soluble and secreted forms of Cripto also exhibit growth factor-like activity and activate SRC/MAPK/PI3K/AKT pathways. Glucose-Regulated Protein 78 kDa (GRP78) binds Cripto at the cell surface and has been shown to be required for Cripto signaling via both TGF-β and SRC/MAPK/PI3K/AKT pathways. To provide a comprehensive overview of the scientific literature related to Cripto, we performed, for the first time, a bibliometric analysis of the biological roles of Cripto as reported in the scientific literature covering the last 10 years. We present different fields of knowledge in comprehensive areas of research on Cripto, ranging from basic to translational research, using a keyword-driven approach. Our ultimate aim is to aid the scientific community in conducting targeted research by identifying areas where research has been conducted so far and, perhaps more importantly, where critical knowledge is still missing.

The ability to differentiate into cells of different lineage, such as muscle, bone, cartilage and fat, is the chief value of adult mesenchymal stem cells (MSCs) which can be used with the final aim to regenerate damaged tissue. Due to potential use, as well as importance in tissue development, a number of questions have been raised regarding the molecular mechanisms of MSC differentiation. As one of the crucial mediators in organism development, transforming growth factor beta (TGF-beta) superfamily directs MSCs commitment in the selection of differentiation pathways. In this review we aim to give an overview of the current knowledge on the mechanisms of MSCs differentiation, on the involvement of TGF-beta superfamily in MSCs differentiation with additional insight into the mutual regulation of microRNAs and TGF-beta in MSCs differentiation. Particular focus has been given to the signaling and transcriptional networks governing the differentiation processes.

The TGF-beta signal Nodal is the major effector of left-right axis development. In mice, Nodal forms heterodimers with Gdf1 and is restricted to the left side by Cerl2/Dand5. Nodal expression then propagates up the lateral plate mesoderm (LPM) by autoinduction, while Lefty1 inhibition maintains it on the left. Studies in zebrafish have suggested some parallels, but also differences, between the modes of left-right patterning in mouse and zebrafish. To address these discrepancies, we generated single and double zebrafish mutants for spaw (the Nodal ortholog), dand5 (the Cerl2 ortholog) and lefty1, and performed biochemical and activity assays with Spaw and Vg1/Gdf3 (the Gdf1 ortholog). Contrary to previous findings, spaw mutants failed to initiate spaw expression in the LPM, and asymmetric heart looping was absent, similar to mouse Nodal mutants. In blastoderm assays, Vg1 and Spaw were interdependent for inducing target gene expression, and contrary to previous results, formed heterodimers. Loss of Dand5 or Lefty1 caused bilateral spaw expression, similar to mouse mutants, and premature expression and propagation of spaw in the LPM. Finally, Lefty1 inhibition of Nodal activity could be replaced by uniform exposure to a Nodal signaling inhibitor. Collectively, these results indicate that Dand5 activity biases Spaw-Vg1 heterodimer activity to the left, Spaw around Kupffer's vesicle induces the expression of spaw in the LPM, and global Nodal inhibition maintains the left bias of Spaw activity, demonstrating conservation between zebrafish and mouse mechanisms of left-right patterning.

NSrp70 (nuclear speckle-related protein 70), a recently discovered protein and it belongs to the serine/arginine (SR) rich related protein family. NSrp70 is recognized as an important splicing factor comprising RNA recognition motif (RRM) and arginine/serine (RS)-like regions at the N- and C-terminus respectively, along with two coiled coil domains at each terminus. However, other functions of NSrp70 remain unelucidated. In this study, we investigated the role of NSrp70 in Xenopus embryogenesis and found that its maternal expression plays a critical role in embryonic development. Knockdown of NSrp70 resulted in dramatic reduction in the length of developing tadpoles and mild to severe malformation in Xenopus embryos. In addition, knockdown of NSrp70 resulted in an extremely short axis by blocking gastrulation and convergent extension. Further, animal cap assays along with activin A treatment revealed that NSrp70 is an essential factor for dorsal mesoderm induction as knockdown of NSrp70 caused a dramatic down-regulation of dorsal mesoderm specific genes and its loss significantly shortened the elongation region of animal caps. In conclusion, NSrp70 is crucial for early embryonic development, influencing gastrulation and mesoderm induction.

Nodal is a signaling molecule that belongs to the transforming growth factor-β superfamily that plays key roles during the early stages of development of animals. In vertebrates Nodal forms an heterodimer with a GDF1/3 protein to activate the Nodal pathway. Vertebrates have a paralog of nodal in their genomes labeled Nodal-related, but the evolutionary history of these genes is a matter of debate, mainly because of the presence of a variable numbers of genes in the vertebrate genomes sequenced so far. Thus, the goal of this study was to investigate the evolutionary history of the Nodal and Nodal-related genes with an emphasis in tracking changes in the number of genes among vertebrates. Our results show the presence of two gene lineages (Nodal and Nodal-related) that can be traced back to the ancestor of jawed vertebrates. These lineages have undergone processes of differential retention and lineage-specific expansions. Our results imply that Nodal and Nodal-related duplicated at the latest in the ancestor of gnathostomes, and they still retain a significant level of functional redundancy. By comparing the evolution of the Nodal/Nodal-related with GDF1/3 gene family, it is possible to infer that there are several types of heterodimers that can trigger the Nodal pathway among vertebrates.

Growth differentiation factors 1 (GDF1) and 3 (GDF3) are members of the transforming growth factor superfamily (TGF-β) that is involved in fundamental early-developmental processes that are conserved across vertebrates. The evolutionary history of these genes is still under debate due to ambiguous definitions of homologous relationships among vertebrates. Thus, the goal of this study was to unravel the evolution of the GDF1 and GDF3 genes of vertebrates, emphasizing the understanding of homologous relationships and their evolutionary origin. Our results revealed that the GDF1 and GDF3 genes found in anurans and mammals are the products of independent duplication events of an ancestral gene in the ancestor of each of these lineages. The main implication of this result is that the GDF1 and GDF3 genes of anurans and mammals are not 1:1 orthologs. In other words, genes that participate in fundamental processes during early development have been reinvented two independent times during the evolutionary history of tetrapods.

The Tbx6 transcription factor plays multiple roles during gastrulation, somite formation and body axis determination. One of the notable features of the Tbx6 homozygous mutant phenotype is randomization of left/right axis determination. Cilia of the node are morphologically abnormal, leading to the hypothesis that disrupted nodal flow is the cause of the laterality defect. However, Tbx6 is expressed around but not in the node, leading to uncertainty as to the mechanism of this effect. In this study, we have examined the molecular characteristics of the node and the genetic cascade determining left/right axis determination. We found evidence that a leftward nodal flow is generated in Tbx6 homozygous mutants despite the cilia defect, establishing the initial asymmetric gene expression in Dand5 around the node, but that the transduction of the signal from the node to the left lateral plate mesoderm is disrupted due to lack of expression of the Nodal coligand Gdf1 around the node. Gdf1 was shown to be a downstream target of Tbx6 and a Gdf1 transgene partially rescues the laterality defect., Summary: Tbx6 affects morphology of the cilia of the node, but a leftward nodal flow is still generated. Downstream of nodal flow, Tbx6 regulates the Nodal coligand Gdf1 leading to disruption of left/right axis determination.

Growth/differentiation factor 1 (GDF1) is a secreted glycoprotein of the transforming growth factor-β (TGF-β) superfamily that mediates cell differentiation events during embryonic development. GDF1 is expressed in several tissues, including the heart. However, the functional role of GDF1 in myocardial infarction (MI)-induced cardiac remodelling and dysfunction is not known. Here, we performed gain-of-function and loss-of-function studies using cardiac-specific GDF1 transgenic (TG) and knockout (KO) mice to determine the role of GDF1 in the pathogenesis of functional and architectural cardiac remodelling after MI, which was induced by surgical left anterior descending coronary artery ligation. Our results demonstrate that overexpression of GDF1 in the heart causes a significant decrease in MI-derived mortality post-MI and leads to attenuated infarct size expansion, left ventricular (LV) dilatation, and cardiac dysfunction at 1 week and 4 weeks after MI injury. Compared with control animals, cardiomyocyte apoptosis, inflammation, hypertrophy, and interstitial fibrosis were all remarkably reduced in the GDF1-TG mice following MI. In contrast, GDF1 deficiency greatly exacerbated the pathological cardiac remodelling response after infarction. Further analysis of the in vitro and in vivo signalling events indicated that the beneficial role of GDF1 in MI-induced cardiac dysfunction and LV remodelling was associated with the inhibition of non-canonical (MEK-ERK1/2) and canonical (Smad) signalling cascades. Overall, our data reveal that GDF1 in the heart is a novel mediator that protects against the development of post-infarction cardiac remodelling via negative regulation of the MEK-ERK1/2 and Smad signalling pathways. Thus, GDF1 may serve as a valuable therapeutic target for the treatment of MI.

Establishment of asymmetry along the left-right (LR) body axis in vertebrates requires interplay between Nodal and Bmp signaling pathways. In the basal chordate amphioxus, the left-sided activity of the Nodal signaling has been attributed to the asymmetric morphogenesis of paraxial structures and pharyngeal organs, however the role of Bmp signaling in LR asymmetry establishment has not been addressed to date. Here, we show that Bmp signaling is necessary for the development of LR asymmetric morphogenesis of amphioxus larvae through regulation of Nodal signaling. Loss of Bmp signaling results in loss of the left-sided expression of Nodal, Gdf1/3, Lefty and Pitx and in gain of ectopic expression of Cerberus on the left side. As a consequence, the larvae display loss of the offset arrangement of axial structures, loss of the left-sided pharyngeal organs including the mouth, and ectopic development of the right-sided organs on the left side. Bmp inhibition thus phenocopies inhibition of Nodal signaling and results in the right isomerism. We conclude that Bmp and Nodal pathways act in concert to specify the left side and that Bmp signaling plays a fundamental role during LR development in amphioxus.

Pathological cardiac hypertrophy is a major risk factor for developing heart failure, the leading cause of death in the world. Growth/differentiation factor 1 (GDF1), a transforming growth factor-β family member, is a regulator of cell growth and differentiation in both embryonic and adult tissues. Evidence from human and animal studies suggests that GDF1 may play an important role in cardiac physiology and pathology. However, a critical role for GDF1 in cardiac remodelling has not been investigated. Here, we performed gain-of-function and loss-of-function studies using cardiac-specific GDF1 knockout mice and transgenic mice to determine the role of GDF1 in pathological cardiac hypertrophy, which was induced by aortic banding (AB). The extent of cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. Our results demonstrated that cardiac specific GDF1 overexpression in the heart markedly attenuated cardiac hypertrophy, fibrosis, and cardiac dysfunction, whereas loss of GDF1 in cardiomyocytes exaggerated the pathological cardiac hypertrophy and dysfunction in response to pressure overload. Mechanistically, we revealed that the cardioprotective effect of GDF1 on cardiac remodeling was associated with the inhibition of the MEK–ERK1/2 and Smad signaling cascades. Collectively, our data suggest that GDF1 plays a protective role in cardiac remodeling via the negative regulation of the MEK–ERK1/2 and Smad signaling pathways.

Myelodysplastic syndrome (MDS) is a clonal hematopoietic disorder characterized by ineffective hematopoiesis, cytopenias, and an increased risk of transformation to acute myeloid leukemia. New studies are urgently needed to understand the underlying processes that govern MDS and related bone marrow failure disorders and to develop better therapeutic modalities. Sphingolipids are an extensive classification of lipids which play prominent roles in cellular signaling in addition to being essential components of membranes. The most well studied sphingolipid is ceramide, which serves as a hypothetical center of sphingolipid metabolism. Ceramide is an important cellular signal that can lead to apoptosis. Overall ceramide biology is significant because novel metabolic routes that persist in MDS may be exploited for therapeutic development. Interestingly, the gene encoding ceramide synthase 1 is encoded from a bicistronic transcript that also encodes for GDF1. Little is known about the roles of GDF1 outside of cardiac development. However, GDF1 is in the TGF-beta superfamily of which many members have been attributed roles in stem cell biology. Therefore, a better understanding of the role of GDF1 and its regulation of ceramide metabolism and hematopoiesis may lead to better treatment approaches for MDS and other bone marrow failure syndromes. The current study tested an overarching hypothesis that GDF1 regulates SMAD and STAT signaling to promote ceramide generation and restore effective myelopoiesis in MDS. Initially, the expression of GDF1 was evaluated in hematopoietic cells isolated from transgenic murine models of MDS (Nup98-HoxD13; Srsf2P95H-mutant) as well as myeloproliferation (Flt3ITD). Interestingly, GDF1 expression was greatest in the bone marrow of MDS models. This was significant because we recently reported that nanoliposomal ceramide (Lip-C6), which delivers a bioactive ceramide analog, exerts unique therapeutic efficacy towards acute myeloid leukemia arising out of MDS (Barth et. al. Blood Advances 2019). Lip-C6 is a ceramide-based therapy that currently is in a clinical trial for solid tumor malignancies (ClinicalTrials.gov identifier: NCT02834611). Next, treatment of transgenic MDS mice with either recombinant GDF1 or Lip-C6 was shown to stimulate the expansion of erythroid progenitors. This was concomitant with a decrease in immature myeloid cells, which was revealed to be due to granulo-monocytopoietic differentiation. Mechanistic studies subsequently revealed that GDF1 increased SMAD2/3 phosphorylation while simultaneously down regulating STAT3 (Y705) phosphorylation, both in a TGF-beta receptor 1-dependent fashion. This dual effect was unique to GDF1, whereas TGF-beta or GDF3 only recapitulated individual the effects on SMAD2/3 and STAT3 signaling, respectively. Finally, STAT3 binding sites were identified in the promoter region of the ceramide detoxifying enzyme glucosylceramide synthase. This is important because glucosylceramide synthase expression was shown to be downregulated by recombinant GDF1 treatment. Therefore, GDF1-mediated downregulation of STAT3-dependent glucosylceramide synthase expression provides a mechanistic link where GDF1 can augment intracellular ceramide levels. Overall, this study enhances our understanding of a fundamental hematopoietic process where GDF1 regulates ceramide metabolism and myelopoiesis. This is impactful because the effects of GDF1, which are unique from other related factors including TGF-beta and GDF3, are due to a novel mechanism of action that both upregulates SMAD2/3 signaling while downregulating STAT3 signaling. More so, this study demonstrated that GDF1, and/or ceramide, can exert an anti-MDS therapeutic role by restoring normal aspects of myelopoiesis. This work was funded by NIH/NCI K22 CA190674 (B.M.B.) and University of New Hampshire COBRE Pilot Project Grant NIH/NIGMS P20 GM113131 (B.M.B.). The authors acknowledge US Provisional Patent 62/602,437, issued to B.M.B. and the University of New Hampshire. Disclosures Barth: University of New Hampshire: Patents & Royalties: US Provisional Patent 62/602,437; NIH (NCI and NIGMS): Research Funding. Loughran:Bioniz: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees.

The vertebrae mesoderm is a source of cells that forms a variety of tissues, including the heart, vasculature, and blood. Consequently, the derivation of various mesoderm-specific cell types from human embryonic stem cells (hESCs) has attracted the interest of many investigators owing to their therapeutic potential in clinical applications. However, the need for efficient and reliable methods of differentiation into mesoderm lineage cell types remains a significant challenge. Here, we demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3) is an essential first step toward efficient generation of the mesoderm. Under chemically defined conditions without additional growth factors/cytokines, short-term GSK inhibitor (GSKi) treatment effectively drives differentiation of hESCs into the primitive streak (PS), which can potentially commit toward the mesoderm when further supplemented with bone morphogenetic protein 4. Further analysis confirmed that the PS-like cells derived from GSKi treatment are bipotential, being able to specify toward the endoderm as well. Our findings suggest that the bipotential, PS/mesendoderm-like cell population exists only at the initial stages of GSK-3 inhibition, whereas long-term inhibition results in an endodermal fate. Lastly, we demonstrated that our differentiation approach could efficiently generate lateral plate (CD34(+)KDR(+)) and paraxial (CD34(-)PDGFRα(+)) mesoderm subsets that can be further differentiated along the endothelial and smooth muscle lineages, respectively. In conclusion, our study presents a unique approach for generating early mesoderm progenitors in a chemically directed fashion through the use of small-molecule GSK-3 inhibitor, which may be useful for future applications in regenerative medicine.

Left-right patterning is among the least well understood of the three axes defining the body plan, and yet it is of no less importance, with left-right patterning defects causing structural birth defects with high morbidity and mortality, such as in complex congenital heart disease, biliary atresia, or intestinal malrotation. The cell signaling pathways that govern left-right asymmetry are highly conserved and involved multiple components of the transforming growth factor beta (TGFβ) superfamily of cell signaling molecules. Central to left-right patterning is the differential activation of Nodal on the left and bone morphogenetic protein (BMP) signaling on the right. In addition, a plethora of other cell signaling pathways including sonic hedgehog (Shh), fibroblast growth factor (FGF), and Notch also contribute to the regulation of left-right patterning. In vertebrate embryos such as the mouse, frog, or zebrafish, the specification of left-right identity requires the left-right organizer (LRO) containing cells with motile and primary cilia. Cilia-generated flow plays an important role in the left-sided propagation of Nodal signaling. Ultimately, it is the left-sided expression of the transcription factor paired-like homeodomain 2 (Pitx2) that drives visceral organ asymmetry. Interestingly, while this overall scheme for left-right patterning is well conserved evolutionarily, are striking differences that suggests caution in broadly generalizing conclusions on the molecular pathways regulating left-right patterning.

During normal cardiovascular development, the outflow tract becomes septated and rotates so that the separate aorta and pulmonary trunk are correctly aligned with the left and right ventricles, respectively. However, when this process goes wrong, the aorta and pulmonary trunk are incorrectly positioned resulting in oxygenated blood being directly returned to the lungs, with deoxygenated blood being delivered to the systemic circulation. This is termed transposition of the great arteries (TGA). The precise etiology of TGA is not known, but the use of animal models has elucidated that genes involved in left–right determination of the embryonic body play key roles. Other factors such as retinoic acid levels are also crucial. This chapter reviews the animal models that can be manipulated genetically or with exogenous agents to present with TGA.

Dextro-transposition of the great arteries (d-TGA) is one of the rare congenital heart diseases (CHD) which benefits from early neonatal diagnosis because d-TGA requires rapid postnatal catheter procedure. In that respect, detecting parental genetic predisposing factors would contribute to focusing prenatal echographical attention to the early detection of d-TGA cases. A high male to female ratio and a high recurrence risk of d-TGA in the context of heterotaxy suggest the impact of genetic factors although familial cases of d-TGA are exceptional. Since the late 1990s, a growing list of genes and chromosomal regions was associated with d-TGA among which the ZIC3 gene. Although this gene is located on the X chromosome, ZIC3 (Zic family member 3) does not explain the male preponderance in d-TGA. d-TGA causal genes are involved in many different cellular pathways and can be provisionally sorted in two groups: those which disrupt the function of the embryonic node cilia and those which are downstream of this major embryological process of lateralization. Many more genes or gene factors remain to be discovered in d-TGA and related CHD because only a small percentage of d-TGA is yet genetically resolved.

The transforming growth factor β (TGF-β) superfamily of growth and differentiation factors plays diverse roles in embryonic development and adult tissue homeostasis, and leads to a range of human diseases when misregulated. In this article we will discuss the main components of the TGF-β superfamily signaling pathways and their regulation. We will start by discussing the production and release of ligands, their binding to and activation of cell surface receptors and the subsequent activity of the downstream effectors of the pathway, the SMADs. We will end with a discussion of the role of TGF-β superfamily signaling in human disease.

Heart failure is a leading cause of death worldwide. Studies of stem cell biology are essential for developing efficient treatments. Recently, we established and characterized c-kit-positive cardiac stem cells from the adult rat heart. Using a MethoCult culture system with a methyl-cellulose-based medium, stem-like left-atrium-derived pluripotent cells could be regulated to differentiate into skeletal/cardiac myocytes or adipocytes with almost 100% purity. Microarray and pathway analyses of these cells showed that transforming growth factor-β1 (TGF-β1) and noggin were significantly involved in the differentiation switch. Furthermore, TGF-β1 may act as a regulator for this switch because it simultaneously inhibits adipogenesis and activates myogenesis in a dose-dependent manner. However, the effect of TGF-β varies with developmental stage, dosage, and timing of treatment. In the present review, the findings of recent studies, in particular the use of c-kit-positive cardiac stem cells, are discussed. The effects of the TGF-β superfamily on differentiation, especially on adipogenesis and/or myogenesis, have important implications for future regenerative medicine.

Members of the transforming growth factor-β (TGF-β) superfamily have significant roles in the regulation of a wide variety of physiological processes. In our present work, phylogenetic tree analysis showed that human GDF3 (Growth and differentiation factor 3) and human GDF1 formed a subgroup of closely related molecules. Through quantitative real-time PCR analysis in different human tissues, GDF1 and GDF3 expression level had a big difference in brain. GDF3 could activate downstream signaling through associating with ALK7 (Activin receptor-like kinase 7) in a Cripto-dependent fashion. A CHO cell line stably transfected with the encoding sequence of GDF3, named CHO-GDF3, was established. Western blotting analysis demonstrated that GDF3 protein could be secreted into the medium from CHO cells and immunofluorescence experiment showed that GDF3 was mainly distributed in cytoplasm of the stable cell line, the primary hippocampal neurons, and brain tissues. Furthermore, the conditioned medium from CHO-GDF3 could reduce PC12 cell growth and induce cell differentiation. All these findings bring new insights into the functional study of GDF3.

Right atrial isomerism (RAI) is a heterotaxy syndrome with disturbances in the left-right axis development, resulting in complex heart malformations and abnormal lateralization of other thoracic and abdominal organs. Although autosomal-recessive inheritance of heterotaxy syndrome is seen in multiple families, underlying gene defects have remained unknown. Here we identify the molecular genetic basis of a kindred with five siblings with RAI. Linkage analysis and positional candidate gene approach showed that the affected children were compound heterozygotes for truncating mutations in the growth/differentiation factor 1 (GDF1) gene. Individuals heterozygous for the mutations were clinically healthy. This finding, supported by the similar phenotype in Gdf1 knockout mouse, provides firm evidence that RAI can occur as a recessively inherited condition, with GDF1 as the culprit gene. The results will shed light on the biological basis of human laterality defects and facilitate molecular diagnosis of RAI.

SMAD4 serves as a common mediator for signaling of TGF-β superfamily. Previous studies illustrated that SMAD4-null mice die at embryonic day 6.5 (E6.5) due to failure of mesoderm induction and extraembryonic defects; however, functions of SMAD4 in each germ layer remain elusive. To investigate this, we disrupted SMAD4 in the visceral endoderm and epiblast, respectively, using a Cre-loxP mediated approach. We showed that mutant embryos lack of SMAD4 in the visceral endoderm (Smad4(Co/Co);TTR-Cre) died at E7.5-E9.5 without head-fold and anterior embryonic structures. We demonstrated that TGF-β regulates expression of several genes, such as Hex1, Cer1, and Lim1, in the anterior visceral endoderm (AVE), and the failure of anterior embryonic development in Smad4(Co/Co);TTR-Cre embryos is accompanied by diminished expression of these genes. Consistent with this finding, SMAD4-deficient embryoid bodies showed impaired responsiveness to TGF-β-induced gene expression and morphological changes. On the other hand, embryos carrying Cre-loxP mediated disruption of SMAD4 in the epiblasts exhibited relatively normal mesoderm and head-fold induction although they all displayed profound patterning defects in the later stages of gastrulation. Cumulatively, our data indicate that SMAD4 signaling in the epiblasts is dispensable for mesoderm induction although it remains critical for head patterning, which is significantly different from SMAD4 signaling in the AVE, where it specifies anterior embryonic patterning and head induction.

The cyclopic and laterality phenotypes in model organisms linked to disturbances in the generation or propagation of Nodal-like signals are potential examples of similar impairments resulting in birth defects in humans. However, the types of gene mutation(s) and their pathogenetic combinations in humans are poorly understood. Here we describe a mutational analysis of the human NODAL gene in a large panel of patients with phenotypes compatible with diminished NODAL ligand function. Significant reductions in the biological activity of NODAL alleles are detected among patients with congenital heart defects (CHD), laterality anomalies (e.g. left-right mis-specification phenotypes), and only rarely holoprosencephaly (HPE). While many of these NODAL variants are typical for family-specific mutations, we also report the presence of alleles with significantly reduced activity among common population variants. We propose that some of these common variants act as modifiers and contribute to the ultimate phenotypic outcome in these patients; furthermore, we draw parallels with strain-specific modifiers in model organisms to bolster this interpretation.

In embryonic stem cells, Oct-4 concentration is critical in determining the development of endoderm, mesoderm, and trophectoderm. Although Oct-4 expression is essential for mesoderm development, it is unclear whether it has a role in the development of specific mesodermal tissues. In this study, we have examined the importance of Oct-4 in the generation of hematopoietic cells using an inducible Oct-4 ESC line. We demonstrate that Oct-4 has a role in supporting hematopoiesis after specifying brachyury-positive mesoderm. When we suppressed Oct-4 expression before or after mesoderm specification, no hematopoietic cells are detected. However, hematopoiesis can be rescued in the absence of Oct-4 after mesoderm specification if the essential hematopoietic transcription factor stem cell leukemia is expressed. Our results suggest that, for hematopoiesis to occur, Oct-4 is required for the initial specification of mesoderm and subsequently is required for the development of hematopoietic cells from uncommitted mesoderm.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo, including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunohistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3-were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

Vg1, a member of the TGF-beta superfamily of ligands, has been implicated in the induction of mesoderm, formation of primitive streak, and left-right patterning in Xenopus and chick embryos. In mice, GDF1 and GDF3 - two TGF-beta superfamily ligands that share high sequence identity with Vg1 - have been shown to independently mimic distinct aspects of Vg1's functions. However, the extent to which the developmental processes controlled by GDF1 and GDF3 and the underlying signaling mechanisms are evolutionarily conserved remains unclear. Here we show that phylogenetic and genomic analyses indicate that Gdf1 is the true Vg1 ortholog in mammals. In addition, and similar to GDF1, we find that GDF3 signaling can be mediated by the type I receptor ALK4, type II receptors ActRIIA and ActRIIB, and the co-receptor Cripto to activate Smad-dependent reporter genes. When expressed in heterologous cells, the native forms of either GDF1 or GDF3 were incapable of inducing downstream signaling. This could be circumvented by using chimeric constructs carrying heterologous prodomains, or by co-expression with the Furin pro-protein convertase, indicating poor processing of the native GDF1 and GDF3 precursors. Unexpectedly, co-expression with Nodal - another TGF-beta superfamily ligand involved in mesoderm formation - could also expose the activities of native GDF1 and GDF3, suggesting a potentially novel mode of cooperation between these ligands. Functional complementarity between GDF1 and GDF3 during embryonic development was investigated by analyzing genetic interactions between their corresponding genes. This analysis showed that Gdf1(-/-);Gdf3(-/-) compound mutants are more severely affected than either Gdf1(-/-) or Gdf3(-/-) single mutants, with defects in the formation of anterior visceral endoderm and mesoderm that recapitulate Vg1 loss of function, suggesting that GDF1 and GDF3 together represent the functional mammalian homologs of Vg1.

Congenital heart defects (CHDs) are among the most common birth defects in humans (incidence 8-10 per 1,000 live births). Although their etiology is often poorly understood, most are considered to arise from multifactorial influences, including environmental and genetic components, as well as from less common syndromic forms. We hypothesized that disturbances in left-right patterning could contribute to the pathogenesis of selected cardiac defects by interfering with the extrinsic cues leading to the proper looping and vessel remodeling of the normally asymmetrically developed heart and vessels. Here, we show that heterozygous loss-of-function mutations in the human GDF1 gene contribute to cardiac defects ranging from tetralogy of Fallot to transposition of the great arteries and that decreased TGF- beta signaling provides a framework for understanding their pathogenesis. These findings implicate perturbations of the TGF- beta signaling pathway in the causation of a major subclass of human CHDs.

The evolution of mesoderm was important for the development of complex body plans as well as key organ systems. Genetic and molecular studies in the fruitfly, Drosophila melanogaster, have provided the majority of information concerning mesoderm development in arthropods. In Drosophila, twist is necessary for the specification and correct morphogenesis of mesoderm and myocyte enhancing factor 2 (mef2) is involved downstream of twist to activate muscle differentiation. In Drosophila, mesoderm is defined by positional cues in the blastoderm embryo, while in another arthropod group, the amphipod crustaceans, cell lineage plays a greater role in defining the mesoderm. It is not known how different mechanistic strategies such as positional information vs. cell-lineage-dependent development affect the timing and use of gene networks. Here we describe the development of the mesoderm in a malacostracan crustacean, Parhyale hawaiensis, and characterize the expression of Parhyale twist and mef2 orthologues. In Parhyale, the mesoderm of the post-mandibular segments arises mainly through the asymmetric division of mesoteloblasts as the germband elongates. Ph-twist expression is seen in a subset of segmental mesoderm during germband development, but not during early cleavages when the specific mesodermal cell lineages first arise. ph-mef2 expression starts after the segmental mesoderm begins to proliferate and persists in developing musculature. While the association of these genes with mesoderm differentiation appears to be conserved across the animal kingdom, the timing of expression and relationship with different mechanisms of mesoderm development may give us greater insight into the ancestral use of these genes during mesoderm differentiation.

A complex system of multiple signaling molecules often produce differential gene expression patterns in animal embryos. In the ascidian embryo, four signaling ligands, Ephrin-A.d (Efna.d), Fgf9/16/20, Admp, and Gdf1/3-r, coordinately induce Otx expression in the neural lineage at the 32-cell stage. However, it has not been determined whether differential inputs of all of these signaling pathways are really necessary. It is possible that differential activation of one of these signaling pathways is sufficient and the remaining signaling pathways are activated in all cells at similar levels. To address this question, we developed a parameter-free method for determining a Boolean function for Otx expression in the present study. We treated activities of signaling pathways as Boolean values, and we also took all possible patterns of signaling gradients into consideration. We successfully determined a Boolean function that explains Otx expression in the animal hemisphere of wild-type and morphant embryos at the 32-cell stage. This Boolean function was not inconsistent with three sensing patterns, which represented whether or not individual cells received sufficient amounts of the signaling molecules. These sensing patterns all indicated that differential expression of Otx in the neural lineage is primarily determined by Efna.d, but not by differential inputs of Fgf9/16/20, Admp, and Gdf1/3-r signaling. To confirm this hypothesis experimentally, we simultaneously knocked-down Admp, Gdf1/3-r, and Fgf9/16/20, and treated this triple morphant with recombinant bFGF and BMP4 proteins, which mimic Fgf9/16/20 and Admp/Gdf1/3-r activity, respectively. Although no differential inputs of Admp, Gdf1/3-r and Fgf9/16/20 signaling were expected under this experimental condition, Otx was expressed specifically in the neural lineage. Thus, direct cell–cell interactions through Efna.d play a critical role in patterning the ectoderm of the early ascidian embryo., Author Summary It is often difficult to understand a complex system of multiple signaling molecules in animal embryos only with experimental procedures. Although theoretical analysis might solve this problem, it is often difficult to precisely determine parameters for signaling gradients and kinetics of signaling molecules. In the present study, we developed a parameter-free method for determining a Boolean function for understanding a complex signaling system using gene expression patterns of signaling molecules and geometrical configurations of individual cells within the embryo. In the ascidian embryo, four signaling ligands, Ephrin-A.d (Efna.d), Fgf9/16/20, Admp, and Gdf1/3-r, coordinately induce Otx expression in the neural lineage at the 32-cell stage. In addition to determining a Boolean function, our method determined sensing patterns, which represented whether or not individual cells received sufficient amounts of the signaling molecules. The sensing patterns predicted that differential expression of Otx in the neural lineage is primarily determined by Efna.d, but not by differential inputs of Fgf9/16/20, Admp, and Gdf1/3-r. We confirmed this prediction by an experiment. As a result, we found that only Efna.d signaling pathway is differentially activated between ectodermal cells and the remaining signaling pathways are activated in all ectodermal cells at similar levels.

The interplay between different levels of gene regulation in modulating developmental transcriptional programs, such as histone modifications and chromatin remodeling, is not well understood. Here, we show that the chromatin remodeling factor Brg1 is required for enhancer activation in mesoderm induction. In an embryonic stem cell-based directed differentiation assay, the absence of Brg1 results in a failure of cardiomyocyte differentiation and broad deregulation of lineage-specific gene expression during mesoderm induction. We find that Brg1 co-localizes with H3K27ac at distal enhancers and is required for robust H3K27 acetylation at distal enhancers that are activated during mesoderm induction. Brg1 is also required to maintain Polycomb-mediated repression of non-mesodermal developmental regulators, suggesting cooperativity between Brg1 and Polycomb complexes. Thus, Brg1 is essential for modulating active and repressive chromatin states during mesoderm lineage commitment, in particular the activation of developmentally important enhancers. These findings demonstrate interplay between chromatin remodeling complexes and histone modifications that, together, ensure robust and broad gene regulation during crucial lineage commitment decisions., SUMMARY: The chromatin remodeling factor Brg1 is essential for mesoderm induction and, by modulating active and repressive chromatin states, is involved in promoting the activation of dynamic enhancers.