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Hematopoietic progenitors are enriched in the endocardial cushion and contribute, in a Nkx2-5-dependent manner, to tissue macrophages required for the remodeling of cardiac valves and septa. However, little is known about the molecular mechanism of endocardial-hematopoietic transition. In the current study, we identified the regulatory network of endocardial hematopoiesis. Signal network analysis from scRNA-seq datasets revealed that genes in Notch and retinoic acid (RA) signaling are significantly downregulated in Nkx2-5-null endocardial cells. In vivo and ex vivo analyses validate that the Nkx2-5-Notch axis is essential for the generation of both hemogenic and cushion endocardial cells, and the suppression of RA signaling via Dhrs3 expression plays important roles in further differentiation into macrophages. Genetic ablation study revealed that these macrophages are essential in cardiac valve remodeling. In summary, the study demonstrates that the Nkx2-5/Notch/RA signaling plays a pivotal role in macrophage differentiation from hematopoietic progenitors.

BackgroundThe somatopleure serves as the primordium of the amnion, an extraembryonic membrane surrounding the embryo. Recently, we have reported that amniogenic somatopleural cells (ASCs) not only form the amnion but also migrate into the embryo and differentiate into cardiomyocytes and vascular endothelial cells. However, detailed differentiation processes and final distributions of these intra-embryonic ASCs (hereafter referred to as iASCs) remain largely unknown.ResultsBy quail-chick chimera analysis, we here show that iASCs differentiate into various cell types including cardiomyocytes, smooth muscle cells, cardiac interstitial cells, and vascular endothelial cells. In the pharyngeal region, they distribute selectively into the thyroid gland and differentiate into vascular endothelial cells to form intra-thyroid vasculature. Explant culture experiments indicated sequential requirement of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) signaling for endothelial differentiation of iASCs. Single-cell transcriptome analysis further revealed heterogeneity and the presence of hemangioblast-like cell population within ASCs, with a switch from FGF to VEGF receptor gene expression.ConclusionThe present study demonstrates novel roles of ASCss especially in heart and thyroid development. It will provide a novel clue for understanding the cardiovascular development of amniotes from embryological and evolutionary perspectives.

Sex differences in the development and progression of cardiovascular disease manifest across multiple life stages. These differences are associated with variations in cardiovascular morphology and function between the sexes. Although estrogens and sex hormones are associated with sex differences in cardiovascular diseases in reproductive adults, the molecular mechanisms of cardiovascular sex differences during development are largely unknown. Thus, we investigated sex differences in cardiovascular development. We used a newly developed coronary arteriogram system to visualize the morphology of the coronary arteries in murine anterior surface ventricles at embryonic day 17.5 by injecting nanoparticle ink at a constant pressure. No sex difference was found in the length of ventricle. Based on the boundary value of the distribution of that length, the hearts were divided into "long" and "short" groups and the diameters of the left coronary arteries were analyzed. The mean diameter of the coronary arteries was significantly smaller in females than in males only in the group with the longer length of ventricle. This ventricular size-specific sex difference was observed in the presence of vasodilators such as NOC7 (1-Hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene). When NOC7 was perfused into the left coronary arteries of embryonic day 17.5 mice, females with longer ventricles showed larger left coronary arteries than males. These sex differences in vasodilation capacity suggest that factors related to drug reactivity such as signaling pathways are present at a late embryonic stage. These results indicate that sex differences in the functional morphology of the left coronary arteries exist at a late embryonic stage in mice. NEW & NOTEWORTHY: This study introduces a novel coronary angiography method for analyzing murine embryonic hearts, revealing sex differences in coronary artery morphology and contractile function in the late stage of the fetal period. By categorizing heart components based on size, we unveil nuanced insights into sexual dimorphism during this critical fetal period. This work contributes insights into the early origins of sexual dimorphism in coronary vessels, laying the foundation for further understanding of cardiovascular development. [ABSTRACT FROM AUTHOR]

Mutations of G protein-coupled receptors (GPCRs) cause various human diseases, but the mechanistic details are limited. Here, we establish p.E303K in the gene encoding the endothelin receptor type A ([ET.sub.A]R/EDNRA) as a recurrent mutation causing mandibulofacial dysostosis with alopecia (MFDA), with craniofacial changes similar to those caused by p.Y129F. Mouse models carrying either of these missense mutations exhibited a partial maxillary-to-mandibular transformation, which was rescued by deleting the ligand endothelin 3 (ET3/EDN3). Pharmacological experiments confirmed the causative [ET.sub.A]R mutations as gain of function, dependent on ET3. To elucidate how an amino acid substitution far from the ligand binding site can increase ligand affinity, we used molecular dynamics (MD) simulations. E303 is located at the intracellular end of transmembrane domain 6, and its replacement by a lysine increased flexibility of this portion of the helix, thus favoring G protein binding and leading to G protein-mediated enhancement of agonist affinity. The Y129F mutation located under the ligand binding pocket reduced the sodium-water network, thereby affecting the extracellular portion of helices in favor of ET3 binding. These findings provide insight into the pathogenesis of MFDA and into allosteric mechanisms regulating GPCR function, which may provide the basis for drug design targeting GPCRs., Introduction G protein-coupled receptor-mediated (GPCR-mediated) signal transduction plays essential roles in various pathophysiological and physiological events. Recent progress in structural biology has accelerated the mechanistic understanding of GPCR activation. In [...]

The somatopleure is the amniotic primordium in amniote development, but its boundary to the embryonic body at early embryonic stages and the fate of cells constituting this structure are not well characterized. It also remains unclear how cells behave during the demarcation between intra- and extra-embryonic tissues. Here we identify cellular alignments, which indicate two streams towards the sites of dorsal amniotic closure and ventral thoracic wall formation. A subpopulation of mesodermal cells moving ventrally from the somatopleural region adjacent to the base of the head fold enter the body of the embryo and distribute to the thoracic wall, pharyngeal arches and heart. These cells are induced to differentiate into vascular endothelial cells and cardiomyocytes possibly by FGF and BMP signaling, respectively. These results indicate that the somatopleure acting as the amniotic primordium also serves as a source of embryonic cells, which may contribute to cardiovascular development.

Background: The somatopleure serves as the primordium of the amnion, an extraembryonic membrane surrounding the embryo. Recently, we have reported that amniogenic somatopleural cells (ASCs) not only form the amnion but also migrate into the embryo and differentiate into cardiomyocytes and vascular endothelial cells. However, detailed differentiation processes and final distributions of these intra‐embryonic ASCs (hereafter referred to as iASCs) remain largely unknown. Results: By quail‐chick chimera analysis, we here show that iASCs differentiate into various cell types including cardiomyocytes, smooth muscle cells, cardiac interstitial cells, and vascular endothelial cells. In the pharyngeal region, they distribute selectively into the thyroid gland and differentiate into vascular endothelial cells to form intra‐thyroid vasculature. Explant culture experiments indicated sequential requirement of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) signaling for endothelial differentiation of iASCs. Single‐cell transcriptome analysis further revealed heterogeneity and the presence of hemangioblast‐like cell population within ASCs, with a switch from FGF to VEGF receptor gene expression. Conclusion: The present study demonstrates novel roles of ASCss especially in heart and thyroid development. It will provide a novel clue for understanding the cardiovascular development of amniotes from embryological and evolutionary perspectives. Key Findings: Here, we show that amniogenic somatopleure cells (ASCs) differentiate into various cell types constituting the cardiovascular system, with some populations having a molecular background similar to that of hemangioblasts. Among them, those contributing to the thyroid vascular network were suggested to differentiate into vascular endothelial cells with FGF‐specification and VEGF‐induced maturation. This study will provide a novel clue for understanding the cardiovascular development of amniotes from embryological and evolutionary perspectives. [ABSTRACT FROM AUTHOR]

Macrophages contribute to the development of atherosclerosis through pinocytotic deposition of native LDL-derived cholesterol in macrophages in the vascular wall. Inhibiting macrophage-mediated lipid deposition may have protective effects in atheroprone vasculature, and identifying mechanisms that potentiate this process may inform potential therapeutic interventions for atherosclerosis. Here, we report that dysregulation of exon junction complex-driven (EJC-driven) mRNA splicing confers hyperpinocytosis to macrophages during atherogenesis. Mechanistically, we determined that inflammatory cytokines induce an unconventional nonproteolytic calpain, calpain-6 (CAPN6), which associates with the essential EJC-loading factor CWC22 in the cytoplasm. This association disturbs the nuclear localization of CWC22, thereby suppressing the splicing of target genes, including those related to Rac1 signaling. CAPN6 deficiency in LDL receptor-deficient mice restored CWC22/EJC/Rac1 signaling, reduced pinocytotic deposition of native LDL in macrophages, and attenuated macrophage recruitment into the lesions, generating an atheroprotective phenotype in the aorta. In macrophages, the induction of CAPN6 in the atheroma interior limited macrophage movements, resulting in a decline in cell clearance from the lesions. Consistent with this finding, we observed that myeloid CAPN6 contributed to atherogenesis in a murine model of bone marrow transplantation. Furthermore, macrophages from advanced human atheromas exhibited increased CAPN6 induction and impaired CWC22 nuclear localization. Together, these results indicate that CAPN6 promotes atherogenicity in inflamed macrophages by disturbing CWC22/EJC systems., Introduction Atherosclerosis is a vascular disease characterized by intimal and subintimal cholesterol deposition in which cholesterol forms an initial fatty streak that is followed by an extended atheroma (1). Rupture [...]