Evidence for Two Subpopulations of Cerebrospinal Fluid-Contacting Neurons with Opposite GABAergic Signaling in Adult Mouse Spinal Cord.

Spinal cerebrospinal fluid-contacting neurons (CSF-cNs) form an evolutionary conserved bipolar cell population localized around the central canal of all vertebrates. CSF-cNs were shown to express molecular markers of neuronal immaturity into adulthood; however, the impact of their incomplete maturation on the chloride (Cl-) homeostasis as well as GABAergic signaling remains unknown. Using adult mice from both sexes, in situ hybridization revealed that a proportion of spinal CSF-cNs (18.3%) express the Na+-K+-Cl- cotransporter 1 (NKCC1) allowing intracellular Cl- accumulation. However, we did not find expression of the K+-Cl- cotransporter 2 (KCC2) responsible for Cl- efflux in any CSF-cNs. The lack of KCC2 expression results in low Cl- extrusion capacity in CSF-cNs under high Cl- load in whole-cell patch clamp. Using cell-attached patch clamp allowing recordings with intact intracellular Cl- concentration, we found that the activation of ionotropic GABAA receptors (GABAA-Rs) induced both depolarizing and hyperpolarizing responses in CSF-cNs. Moreover, depolarizing GABA responses can drive action potentials as well as intracellular calcium elevations by activating voltage-gated calcium channels. Blocking NKCC1 with bumetanide inhibited the GABA-induced calcium transients in CSF-cNs. Finally, we show that metabotropic GABAB receptors have no hyperpolarizing action on spinal CSF-cNs as their activation with baclofen did not mediate outward K+ currents, presumably due to the lack of expression of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Together, these findings outline subpopulations of spinal CSF-cNs expressing inhibitory or excitatory GABAA-R signaling. Excitatory GABA may promote the maturation and integration of young CSF-cNs into the existing spinal circuit.

Maternal Pre-Existing Diabetes: A Non-Inherited Risk Factor for Congenital Cardiopathies.

Congenital heart defects (CHDs) are the most common form of birth defects in humans. They occur in 9 out of 1000 live births and are defined as structural abnormalities of the heart. Understanding CHDs is difficult due to the heterogeneity of the disease and its multifactorial etiology. Advances in genomic sequencing have made it possible to identify the genetic factors involved in CHDs. However, genetic origins have only been found in a minority of CHD cases, suggesting the contribution of non-inherited (environmental) risk factors to the etiology of CHDs. Maternal pregestational diabetes is associated with a three- to five-fold increased risk of congenital cardiopathies, but the underlying molecular mechanisms are incompletely understood. According to current hypotheses, hyperglycemia is the main teratogenic agent in diabetic pregnancies. It is thought to induce cell damage, directly through genetic and epigenetic dysregulations and/or indirectly through production of reactive oxygen species (ROS). The purpose of this review is to summarize key findings on the molecular mechanisms altered in cardiac development during exposure to hyperglycemic conditions in utero. It also presents the various in vivo and in vitro techniques used to experimentally model pregestational diabetes. Finally, new approaches are suggested to broaden our understanding of the subject and develop new prevention strategies.

Transcriptome analysis of mouse and human sinoatrial node cells reveals a conserved genetic program.

The rate of contraction of the heart relies on proper development and function of the sinoatrial node, which consists of a small heterogeneous cell population, including Tbx3+ pacemaker cells. Here, we have isolated and characterized the Tbx3+ cells from Tbx3+/Venus knock-in mice. We studied electrophysiological parameters during development and found that Venus-labeled cells are genuine Tbx3+ pacemaker cells. We analyzed the transcriptomes of late fetal FACS-purified Tbx3+ sinoatrial nodal cells and Nppb-Katushka+ atrial and ventricular chamber cardiomyocytes, and identified a sinoatrial node-enriched gene program, including key nodal transcription factors, BMP signaling and Smoc2, the disruption of which in mice did not affect heart rhythm. We also obtained the transcriptomes of the sinoatrial node region, including pacemaker and other cell types, and right atrium of human fetuses, and found a gene program including TBX3, SHOX2, ISL1 and HOX family members, and BMP and NOTCH signaling components conserved between human and mouse. We conclude that a conserved gene program characterizes the sinoatrial node region and that the Tbx3+/Venus allele provides a reliable tool for visualizing the sinoatrial node, and studying its development and function.

T-box genes and retinoic acid signaling regulate the segregation of arterial and venous pole progenitor cells in the murine second heart field.

The arterial and venous poles of the mammalian heart are hotspots of congenital heart defects (CHD) such as those observed in 22q11.2 deletion (or DiGeorge) and Holt-Oram syndromes. These regions of the heart are derived from late differentiating cardiac progenitor cells of the Second Heart Field (SHF) located in pharyngeal mesoderm contiguous with the elongating heart tube. The T-box transcription factor Tbx1, encoded by the major 22q11.2 deletion syndrome gene, regulates SHF addition to both cardiac poles from a common progenitor population. Despite the significance of this cellular addition the mechanisms regulating the deployment of common progenitor cells to alternate cardiac poles remain poorly understood. Here we demonstrate that Tbx5, mutated in Holt-Oram syndrome and essential for venous pole development, is activated in Tbx1 expressing cells in the posterior region of the SHF at early stages of heart tube elongation. A subset of the SHF transcriptional program, including Tbx1 expression, is subsequently downregulated in Tbx5 expressing cells, generating a transcriptional boundary between Tbx1-positive arterial pole and Tbx5-positive venous pole progenitor cell populations. We show that normal downregulation of the definitive arterial pole progenitor cell program in the posterior SHF is dependent on both Tbx1 and Tbx5. Furthermore, retinoic acid (RA) signaling is required for Tbx5 activation in Tbx1-positive cells and blocking RA signaling at the time of Tbx5 activation results in atrioventricular septal defects at fetal stages. Our results reveal sequential steps of cardiac progenitor cell patterning and provide mechanistic insights into the origin of common forms of CHD.

Asb2α-Filamin A Axis Is Essential for Actin Cytoskeleton Remodeling During Heart Development.

RATIONALE: Heart development involves differentiation of cardiac progenitors and assembly of the contractile sarcomere apparatus of cardiomyocytes. However, little is known about the mechanisms that regulate actin cytoskeleton remodeling during cardiac cell differentiation. OBJECTIVE: The Asb2α (Ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2) CRL5 (cullin 5 RING E3 ubiquitin ligase) triggers polyubiquitylation and subsequent degradation by the proteasome of FLNs (filamins). Here, we investigate the role of Asb2α in heart development and its mechanisms of action. METHODS AND RESULTS: Using Asb2 knockout embryos, we show that Asb2 is an essential gene, critical to heart morphogenesis and function, although its loss does not interfere with the overall patterning of the embryonic heart tube. We show that the Asb2α E3 ubiquitin ligase controls Flna stability in immature cardiomyocytes. Importantly, Asb2α-mediated degradation of the actin-binding protein Flna marks a previously unrecognized intermediate step in cardiac cell differentiation characterized by cell shape changes and actin cytoskeleton remodeling. We further establish that in the absence of Asb2α, myofibrils are disorganized and that heartbeats are inefficient, leading to embryonic lethality in mice. CONCLUSIONS: These findings identify Asb2α as an unsuspected key regulator of cardiac cell differentiation and shed light on the molecular and cellular mechanisms determining the onset of myocardial cell architecture and its link with early cardiac function. Although Flna is known to play roles in cytoskeleton organization and to be required for heart function, this study now reveals that its degradation mediated by Asb2α ensures essential functions in differentiating cardiac progenitors.

Ectopic expression of Hoxb1 induces cardiac and craniofacial malformations.

Members of the large family of Hox transcription factors are encoded by genes whose tightly regulated expression in development and in space within different embryonic tissues confer positional identity from the neck to the tips of the limbs. Many structures of the face, head, and heart develop from cell populations expressing few or no Hox genes. Hoxb1 is the member of its chromosomal cluster expressed in the most rostral domain during vertebrate development, but never by the multipotent neural crest cell population anterior to the cerebellum. We have developed a novel floxed transgenic mouse line, CAG-Hoxb1,-EGFP (CAG-Hoxb1), which upon recombination by Cre recombinase conditionally induces robust Hoxb1 and eGFP overexpression. When induced within the neural crest lineage, pups die at birth. A variable phenotype develops from E11.5 on, associating frontonasal hypoplasia/aplasia, micrognathia/agnathia, major ocular and forebrain anomalies, and cardiovascular malformations. Neural crest derivatives in the body appear unaffected. Transcription of effectors of developmental signaling pathways (Bmp, Shh, Vegfa) and transcription factors (Pax3, Sox9) is altered in mutants. These outcomes emphasize that repression of Hoxb1, along with other paralog group 1 and 2 Hox genes, is strictly necessary in anterior cephalic NC for craniofacial, visual, auditory, and cardiovascular development.

A large permissive regulatory domain exclusively controls Tbx3 expression in the cardiac conduction system.

RATIONALE: The evolutionary conserved Tbx3/Tbx5 gene cluster encodes T-box transcription factors that play crucial roles in the development and homeostasis of the cardiac conduction system in human and mouse. Both genes are expressed in overlapping patterns and function in strictly tissue-specific and dose-dependent manners, yet, their regulation is poorly understood. OBJECTIVE: To analyze the mechanism underlying the complex regulation of the Tbx3/Tbx5 cluster. METHODS AND RESULTS: By probing the 3-dimensional architecture of the Tbx3/Tbx5 cluster using high-resolution circular chromosome conformation capture sequencing in vivo, we found that its regulatory landscape is in a preformed conformation similar in embryonic heart, limbs, and brain. Tbx3 and its flanking gene desert form a 1 Mbp loop between CCCTC-binding factor (CTCF)-binding sites that is separated from the neighboring Tbx5 loop. However, Ctcf inactivation did not result in transcriptional regulatory interaction between Tbx3 and Tbx5. Multiple sites within the Tbx3 locus contact the promoter, including sites corresponding to regions known to contain variations in the human genome influencing conduction. We identified an atrioventricular-specific enhancer and a pan-cardiac enhancer that contact the promoter and each other and synergize to activate transcription in the atrioventricular conduction system. CONCLUSIONS: We provide a high-resolution model of the 3-dimensional structure and function of the Tbx3/Tbx5 locus and show that the locus is organized in a preformed, permissive structure. The Tbx3 locus forms a CTCF-independent autonomous regulatory domain with multiple combinatorial regulatory elements that control the precise pattern of Tbx3 in the cardiac conduction system.

GATA-dependent transcriptional and epigenetic control of cardiac lineage specification and differentiation.

Heart progenitor cells differentiate into various cell types including pacemaker and working cardiomyocytes. Cell-type specific gene expression is achieved by combinatorial interactions between tissue-specific transcription factors (TFs), co-factors, and chromatin remodelers and DNA binding elements in regulatory regions. Dysfunction of these transcriptional networks may result in congenital heart defects. Functional analysis of the regulatory DNA sequences has contributed substantially to the identification of the transcriptional network components and combinatorial interactions regulating the tissue-specific gene programs. GATA TFs have been identified as central players in these networks. In particular, GATA binding elements have emerged as a platform to recruit broadly active histone modification enzymes and cell-type-specific co-factors to drive cell-type-specific gene programs. Here, we discuss the role of GATA factors in cell fate decisions and differentiation in the developing heart.

A mutation in the Kozak sequence of GATA4 hampers translation in a family with atrial septal defects.

Atrial septal defect (ASD) is the most common congenital heart defect clinically characterized by an opening in the atrial septum. Mutations in GATA4, TBX5, and NKX2-5 underlie this phenotype. Here, we report on the identification of a novel -6 G>C mutation in the highly conserved Kozak sequence in the 5'UTR of GATA4 in a small family presenting with two different forms of ASD. This is the first time a mutation in the Kozak sequence has been linked to heart disease. Functional assays demonstrate reduced GATA4 translation, though the GATA4 transcript levels remain normal. This leads to a reduction of GATA4 protein level, consequently diminishing the ability of GATA4 to transactivate target genes, as demonstrated by using the GATA4-driven Nppa (ANF) promoter. In conclusion, we identified a mutation in the GATA4 Kozak sequence that likely contributes to the pathogenesis of ASD. In general, it points to the importance of accurate protein level regulation during heart development and emphasizes the need to analyze the entire transcribed region when screening for mutations.

Understanding the cell fate and behavior of progenitors at the origin of the mouse cardiac mitral valve.

Congenital heart malformations include mitral valve defects, which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal undergoes an endothelial-to-mesenchymal transition to give rise to mitral valvular cells. However, the identity and fate decisions of these progenitors as well as the behavior and distribution of their derivatives in valve leaflets remain unknown. We used single-cell RNA sequencing (scRNA-seq) of genetically labeled endocardial cells and microdissected mouse embryonic and postnatal mitral valves to characterize the developmental road. We defined the metabolic processes underlying the specification of the progenitors and their contributions to subtypes of valvular cells. Using retrospective multicolor clonal analysis, we describe specific modes of growth and behavior of endocardial cell-derived clones, which build up, in a proper manner, functional valve leaflets. Our data identify how both genetic and metabolic mechanisms specifically drive the fate of a subset of endocardial cells toward their distinct clonal contribution to the formation of the valve.

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Outflow Tract Formation-Embryonic Origins of Conotruncal Congenital Heart Disease.

Anomalies in the cardiac outflow tract (OFT) are among the most frequent congenital heart defects (CHDs). During embryogenesis, the cardiac OFT is a dynamic structure at the arterial pole of the heart. Heart tube elongation occurs by addition of cells from pharyngeal, splanchnic mesoderm to both ends. These progenitor cells, termed the second heart field (SHF), were first identified twenty years ago as essential to the growth of the forming heart tube and major contributors to the OFT. Perturbation of SHF development results in common forms of CHDs, including anomalies of the great arteries. OFT development also depends on paracrine interactions between multiple cell types, including myocardial, endocardial and neural crest lineages. In this publication, dedicated to Professor Andriana Gittenberger-De Groot and her contributions to the field of cardiac development and CHDs, we review some of her pioneering studies of OFT development with particular interest in the diverse origins of the many cell types that contribute to the OFT. We also discuss the clinical implications of selected key findings for our understanding of the etiology of CHDs and particularly OFT malformations.

Cardiac regeneration: still a 21st century challenge in search for cardiac progenitors from stem cells and embryos.

Regeneration of the heart after a stroke would be the best biologic response to restore its function. However, although this phenomenon occurs in primitive organisms, the regenerative potential is lost in mammals. Thus, the search for an appropriate cardiac progenitor with the potential to differentiate into a functional cardiomyocyte in vitro and in vivo has been the subject of intensive investigation. We summarize the cardiogenic transcriptional pathway that constitutes the molecular scaffold to drive pluripotent stem cells toward a cardiac progenitor fate. Then we overview the literature on derivation of cardiac progenitors from both embryos and stem cells.

[The dual role of OCT4]. / L'octamanie continue : le double jeu de OCT4.

OCT4 encoded by pou5f1 is one of the most ancient and early transcription factors identified in the embryo. It has been longwise recognized as a gatekeeper for pluripotency of embryonic stem (ES) cell. Uncovered twenty years ago, its fame was built up from its key role in maintaining embryonic stem cell pluripotency in 1998. Since, OCT4 was reported to also instruct stem cell fate through a gene dosage effect. It reached recently a novel glorious hit with its master role in reprogramming somatic cells.

Gradual differentiation and confinement of the cardiac conduction system as indicated by marker gene expression.

The components of the cardiac conduction system, responsible for coordinated activation of the heart chambers, are well defined and their cells differ in gene expression profile and phenotype from those of the surrounding working myocardium. Yet, when and on what basis the myocardium of each of the conduction system components become distinguishable from other myocardium during heart development has not been well established. To identify and assess cell type-specific expression profiles and differentiation markers, we performed transcriptome analysis on fluorescence activated cell sorted purified conduction system (Venus+) and chamber myocardial cells (Katushka+) of Tbx3+/Venus;TgNppb(Katushka) double transgenic mouse fetuses. We found that transcripts associated with nervous system development and ion channel activity were enriched in Tbx3+ conduction system cells, whereas transcripts associated with mitochondrial function, muscle contraction and fatty acid metabolism were enriched in the Nppb+ working myocardium. We analyzed spatio-temporal expression patterns of several candidate markers (Cacna2d2, Cacna1g, Ephb3, Tnni1), reviewed those of established conduction system markers (Tbx3, Hcn4, Gja5, Cntn2), and placed the patterns in the context of conduction system development. The overview indicates that different properties of conduction system components develop gradually and at different developmental stages, and that chamber myocardium gradually differentiates and diverges from conduction system myocardium until after birth.

Hox-dependent coordination of mouse cardiac progenitor cell patterning and differentiation.

Perturbation of addition of second heart field (SHF) cardiac progenitor cells to the poles of the heart tube results in congenital heart defects (CHD). The transcriptional programs and upstream regulatory events operating in different subpopulations of the SHF remain unclear. Here, we profile the transcriptome and chromatin accessibility of anterior and posterior SHF sub-populations at genome-wide levels and demonstrate that Hoxb1 negatively regulates differentiation in the posterior SHF. Spatial mis-expression of Hoxb1 in the anterior SHF results in hypoplastic right ventricle. Activation of Hoxb1 in embryonic stem cells arrests cardiac differentiation, whereas Hoxb1-deficient mouse embryos display premature cardiac differentiation. Moreover, ectopic differentiation in the posterior SHF of embryos lacking both Hoxb1 and its paralog Hoxa1 results in atrioventricular septal defects. Our results show that Hoxb1 plays a key role in patterning cardiac progenitor cells that contribute to both cardiac poles and provide new insights into the pathogenesis of CHD.

Mechanisms of retinoic acid signaling during cardiogenesis.

Substantial experimental and epidemiological data have highlighted the interplay between nutritional and genetic factors in the development of congenital heart defects. Retinoic acid (RA), a derivative of vitamin A, plays a key role during vertebrate development including the formation of the heart. Retinoids bind to RA and retinoid X receptors (RARs and RXRs) which then regulate tissue-specific genes. Here, we will focus on the roles of RA signaling and receptors in gene regulation during cardiogenesis, and the consequence of deregulated retinoid signaling on heart formation and congenital heart defects.

Hoxa1 and Hoxb1 are required for pharyngeal arch artery development.

Hox transcription factors play critical roles during early vertebrate development. Previous studies have revealed an overlapping function of Hoxa1 and Hoxb1 during specification of the rhombomeres from which neural crest cells emerge. A recent study on Hoxa1 mutant mice documented its function during cardiovascular development, however, the role of Hoxb1 is still unclear. Here we show using single and compound Hoxa1;Hoxb1 mutant embryos that reduction of Hoxa1 gene dosage in Hoxb1-null genetic background is sufficient to result in abnormal pharyngeal aortic arch (PAA) development and subsequently in great artery defects. Endothelial cells in the 4th PAAs of compound mutant differentiate normally whereas vascular smooth muscle cells of the vessels are absent in the defective PAAs. The importance of Hoxa1 and Hoxb1, and their interaction during specification of cardiac NCCs is demonstrated. Together, our data reveal a critical role for anterior Hox genes during PAA development, providing new mechanistic insights into the etiology of congenital heart defects.

A cohesin-OCT4 complex mediates Sox enhancers to prime an early embryonic lineage.

Short- and long-scales intra- and inter-chromosomal interactions are linked to gene transcription, but the molecular events underlying these structures and how they affect cell fate decision during embryonic development are poorly understood. One of the first embryonic cell fate decisions (that is, mesendoderm determination) is driven by the POU factor OCT4, acting in concert with the high-mobility group genes Sox-2 and Sox-17. Here we report a chromatin-remodelling mechanism and enhancer function that mediate cell fate switching. OCT4 alters the higher-order chromatin structure at both Sox-2 and Sox-17 loci. OCT4 titrates out cohesin and switches the Sox-17 enhancer from a locked (within an inter-chromosomal Sox-2 enhancer/CCCTC-binding factor CTCF/cohesin loop) to an active (within an intra-chromosomal Sox-17 promoter/enhancer/cohesin loop) state. SALL4 concomitantly mobilizes the polycomb complexes at the Soxs loci. Thus, OCT4/SALL4-driven cohesin- and polycombs-mediated changes in higher-order chromatin structure mediate instruction of early cell fate in embryonic cells.

GATA-dependent regulatory switches establish atrioventricular canal specificity during heart development.

The embryonic vertebrate heart tube develops an atrioventricular canal that divides the atrial and ventricular chambers, forms atrioventricular conduction tissue and organizes valve development. Here we assess the transcriptional mechanism underlying this localized differentiation process. We show that atrioventricular canal-specific enhancers are GATA-binding site-dependent and act as switches that repress gene activity in the chambers. We find that atrioventricular canal-specific gene loci are enriched in H3K27ac, a marker of active enhancers, in atrioventricular canal tissue and depleted in H3K27ac in chamber tissue. In the atrioventricular canal, Gata4 activates the enhancers in synergy with Bmp2/Smad signalling, leading to H3K27 acetylation. In contrast, in chambers, Gata4 cooperates with pan-cardiac Hdac1 and Hdac2 and chamber-specific Hey1 and Hey2, leading to H3K27 deacetylation and repression. We conclude that atrioventricular canal-specific enhancers are platforms integrating cardiac transcription factors, broadly active histone modification enzymes and localized co-factors to drive atrioventricular canal-specific gene activity.