Hox genes define distinct progenitor sub-domains within the second heart field

AbstractMuch of the heart, including the atria, right ventricle and outflow tract (OFT) is derived from a progenitor cell population termed the second heart field (SHF) that contributes progressively to the embryonic heart during cardiac looping. Several studies have revealed anterior-posterior patterning of the SHF, since the anterior region (anterior heart field) contributes to right ventricular and OFT myocardium whereas the posterior region gives rise to the atria. We have previously shown that Retinoic Acid (RA) signal participates to this patterning. We now show that Hoxb1, Hoxa1, and Hoxa3, as downstream RA targets, are expressed in distinct sub-domains within the SHF. Our genetic lineage tracing analysis revealed that Hoxb1, Hoxa1 and Hoxa3-expressing cardiac progenitor cells contribute to both atria and the inferior wall of the OFT, which subsequently gives rise to myocardium at the base of pulmonary trunk. By contrast to Hoxb1Cre, the contribution of Hoxa1-enhIII-Cre and Hoxa3Cre-labeled cells is restricted to the distal regions of the OFT suggesting that proximo-distal patterning of the OFT is related to SHF sub-domains characterized by combinatorial Hox genes expression. Manipulation of RA signaling pathways showed that RA is required for the correct deployment of Hox-expressing SHF cells. This report provides new insights into the regulatory gene network in SHF cells contributing to the atria and sub-pulmonary myocardium

Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells

The growth and repair of skeletal muscle after birth depends on satellite cells that are characterized by the expression of Pax7. We show that Pax3, the paralogue of Pax7, is also present in both quiescent and activated satellite cells in many skeletal muscles. Dominant-negative forms of both Pax3 and -7 repress MyoD, but do not interfere with the expression of the other myogenic determination factor, Myf5, which, together with Pax3/7, regulates the myogenic differentiation of these cells. In Pax7 mutants, satellite cells are progressively lost in both Pax3-expressing and -nonexpressing muscles. We show that this is caused by satellite cell death, with effects on the cell cycle. Manipulation of the dominant-negative forms of these factors in satellite cell cultures demonstrates that Pax3 cannot replace the antiapoptotic function of Pax7. These findings underline the importance of cell survival in controlling the stem cell populations of adult tissues and demonstrate a role for upstream factors in this context

A Retinoic Acid Responsive Hoxa3 Transgene Expressed in Embryonic Pharyngeal Endoderm, Cardiac Neural Crest and a Subdomain of the Second Heart Field

A transgenic mouse line harbouring a β-galacdosidase reporter gene controlled by the proximal 2 kb promoter of Hoxa3 was previously generated to investigate the regulatory cues governing Hoxa3 expression in the mouse. Examination of transgenic embryos from embryonic day (E) 8.0 to E15.5 revealed regionally restricted reporter activity in the developing heart. Indeed, transgene expression specifically delineated cells from three distinct lineages: a subpopulation of the second heart field contributing to outflow tract myocardium, the cardiac neural crest cells and the pharyngeal endoderm. Manipulation of the Retinoic Acid (RA) signaling pathway showed that RA is required for correct expression of the transgene. Therefore, this transgenic line may serve as a cardiosensor line of particular interest for further analysis of outflow tract development

Retinoic acid deficiency alters second heart field formation

Retinoic acid (RA), the active derivative of vitamin A, has been implicated in various steps of cardiovascular development. The retinaldehyde dehydrogenase 2 (RALDH2) enzyme catalyzes the second oxidative step in RA biosynthesis and its loss of function creates a severe embryonic RA deficiency. Raldh2(−/−) knockout embryos fail to undergo heart looping and have impaired atrial and sinus venosus development. To understand the mechanism(s) producing these changes, we examined the contribution of the second heart field (SHF) to pharyngeal mesoderm, atria, and outflow tract in Raldh2(−/−) embryos. RA deficiency alters SHF gene expression in two ways. First, Raldh2(−/−) embryos exhibited a posterior expansion of anterior markers of the SHF, including Tbx1, Fgf8, and the Mlc1v-nlacZ-24/Fgf10 reporter transgene as well as of Islet1. This occurred at early somite stages, when cardiac defects became irreversible in an avian vitamin A-deficiency model, indicating that endogenous RA is required to restrict the SHF posteriorly. Explant studies showed that this expanded progenitor population cannot differentiate properly. Second, RA up-regulated cardiac Bmp expression levels at the looping stage. The contribution of the SHF to both inflow and outflow poles was perturbed under RA deficiency, creating a disorganization of the heart tube. We also investigated genetic cross-talk between Nkx2.5 and RA signaling by generating double mutant mice. Strikingly, Nkx2.5 deficiency was able to rescue molecular defects in the posterior region of the Raldh2(−/−) mutant heart, in a gene dosage-dependent manner

A severe clinical phenotype of Noonan syndrome with neonatal hypertrophic cardiomyopathy in the second case worldwide with RAF1 S259Y neomutation

International audienceNoonan syndrome and related disorders are a group of clinically and genetically heterogeneous conditions caused by mutations in genes of the RAS/MAPK pathway. Noonan syndrome causes multiple congenital anomalies, which are frequently accompanied by hypertrophic cardiomyopathy (HCM). We report here a Tunisian patient with a severe phenotype of Noonan syndrome including neonatal HCM, facial dysmorphism, severe failure to thrive, cutaneous abnormalities, pectus excavatum and severe stunted growth, who died in her eighth month of life. Using whole exome sequencing, we identified a de novo mutation in exon 7 of the RAF1 gene: c.776C > A (p.Ser259Tyr). This mutation affects a highly conserved serine residue, a main mediator of Raf-1 inhibition via phosphorylation. To our knowledge the c.776C > A mutation has been previously reported in only one case with prenatally diagnosed Noonan syndrome. Our study further supports the striking correlation of RAF1 mutations with HCM and highlights the clinical severity of Noonan syndrome associated with a RAF1 p.Ser259Tyr mutation

Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.We thank the Leducq Fondation for supporting Tui Neri, and funding this research under the framework of the MITRAL network and for generously awarding us for the equipment of our cell imaging facility in the frame of their program “Equipement de Recherche et Plateformes Technologiques” (ERPT to M.P.), the Genopole at Evry and the Fondation de la recherche Medicale (grant DEQ20100318280) for supporting the laboratory of Michel Puceat. Part of this work in South Carolina University was conducted in a facility constructed with support from the National Institutes of Health, Grant Number C06 RR018823 from the Extramural Research Facilities Program of the National Center for Research Resources. Other funding sources: National Heart Lung and Blood Institute: RO1-HL33756 (R.R.M.), COBRE P20RR016434–07 (R.R.M., R.A. N.), P20RR016434–09S1 (R.R.M. and R.A.N.); American Heart Association: 11SDG5270006 (R.A.N.); National Science Foundation: EPS-0902795 (R.R.M. and R.A. N.); American Heart Association: 10SDG2630130 (A.C.Z.), NIH: P01HD032573 (A.C. Z.), NIH: U54 HL108460 (A.C.Z), NCATS: UL1TR000100 (A.C.Z.); EH was supported by a fellowship of the Ministere de la recherche et de l’éducation in France.TM-M was supported by a fellowship from the Fondation Foulon Delalande and the Leducq Foundation. P.v.V. was sponsored by a UC San Diego Cardiovascular Scholarship Award and a Postdoctoral Fellowship from the California Institute for Regenerative Medicine (CIRM) Interdisciplinary Stem Cell Training Program II. S.M.E. was funded by a grant from the National Heart, Lung, and Blood Institute (HL-117649). A.T. is supported by the National Heart, Lung, and Blood Institute (R01-HL134664).S

ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation

The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations

Telomeric Trans-Silencing in Drosophila melanogaster: Tissue Specificity, Development and Functional Interactions between Non-Homologous Telomeres

BACKGROUND: The study of P element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE), a homology-dependent repression mechanism by which a P-transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences, "TAS") has the capacity to repress in trans, in the female germline, a homologous P-lacZ transgene located in euchromatin. TSE can show variegation in ovaries, displays a maternal effect as well as an epigenetic transmission through meiosis and involves heterochromatin and RNA silencing pathways. PRINCIPAL FINDINGS: Here, we analyze phenotypic and genetic properties of TSE. We report that TSE does not occur in the soma at the adult stage, but appears restricted to the female germline. It is detectable during development at the third instar larvae where it presents the same tissue specificity and maternal effect as in adults. Transgenes located in TAS at the telomeres of the main chromosomes can be silencers which in each case show the maternal effect. Silencers located at non-homologous telomeres functionally interact since they stimulate each other via the maternally-transmitted component. All germinally-expressed euchromatic transgenes tested, located on all major chromosomes, were found to be repressed by a telomeric silencer: thus we detected no TSE escaper. The presence of the euchromatic target transgene is not necessary to establish the maternal inheritance of TSE, responsible for its epigenetic behavior. A single telomeric silencer locus can simultaneously repress two P-lacZ targets located on different chromosomal arms. CONCLUSIONS AND SIGNIFICANCE: Therefore TSE appears to be a widespread phenomenon which can involve different telomeres and work across the genome. It can explain the P cytotype establishment by telomeric P elements in natural Drosophila populations

Mechanisms of retinoic acid signaling during cardiogenesis

International audienceSubstantial 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

Hox and Tale transcription factors in heart development and disease

Hox genes are highly conserved transcription factors with critical functions during development, in particular for patterning the antero-posterior axis of the embryo. Their action is very often associated with cofactors including the TALE family transcription factors. From Drosophila to vertebrates, Hox genes have been shown to have a major role in heart development. In this review, we focus on the increasing evidence implicating the anterior Hoxgenes and the Tale family members during heart development both in the cardiac mesoderm and in neural crest cells. Congenital heart defects are the leading cause of death in the first year of life and a better understanding of the role of Hox and Tale factors is highly relevant to human pathologies and will provide novel mechanistic insights into the underlying defects