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Failure of proper ventricular trabeculation is often associated with congenital heart disease. Support from endocardial cells, including the secretion of extracellular matrix and growth factors is critical for trabeculation. However, it is poorly understood how the secretion of extracellular matrix and growth factors is initiated and regulated by endocardial cells. We find that genetic knockout of histone deacetylase 3 in the endocardium in mice results in early embryo lethality and ventricular hypotrabeculation. Single cell RNA sequencing identifies significant downregulation of extracellular matrix components in histone deacetylase 3 knockout endocardial cells. Secretome from cultured histone deacetylase 3 knockout mouse cardiac endothelial cells lacks transforming growth factor ß3 and shows significantly reduced capacity in stimulating cultured cardiomyocyte proliferation, which is remarkably rescued by transforming growth factor ß3 supplementation. Mechanistically, we identify that histone deacetylase 3 knockout induces transforming growth factor ß3 expression through repressing microRNA-129-5p. Our findings provide insights into the pathogenesis of congenital heart disease and conceptual strategies to promote myocardial regeneration. Lack of trabeculation compromises heart structure and function. How myocardial trabeculation is regulated by nonmyocytes is poorly understood. Researchers found that histone deacetylase 3 in the developing endocardial cells guides myocardial trabeculation by inducing growth signals. [ABSTRACT FROM AUTHOR]

Cardiomyocyte (CM) nuclei are constantly exposed to mechanical stress, but how they maintain their nuclear shape remains unknown. In this study, we found that ventricular CM nuclei acquire characteristic prominent spatial organizations of heterochromatin (SOH), which are disrupted by high-level expression of H2B-mCherry in mice. SOH disruption was associated with nuclear softening, leading to extreme elongation and rupture under unidirectional mechanical stress. Loosened chromatin then leaks into the cytosol, causing severe inflammation and cardiac dysfunction. Although SOH disruption was accompanied by loosened higher-order genomic structures, the change in gene expression before nuclear deformation was mild, suggesting that SOH play major roles in nuclear structural integrity. Aged CM nuclei consistently exhibited scattered SOH and marked elongation. Furthermore, we provide mechanistic insight into the development and maintenance of SOH driven by chromatin compaction and condensate formation. These results highlight SOH as a safeguard of nuclear shape and genomic integrity against mechanical stress. [Display omitted] • Cardiomyocytes acquire characteristic spatial organizations of heterochromatin (SOH) • High levels of H2B-mCherry disrupt SOH, leading to nuclear elongation in cardiomyocytes • SOH disruption minimally impacts gene expression despite loosening global genome structure • SOH alter with aging, leading to nuclear deformation in ventricular cardiomyocytes Cardiomyocytes exhibit characteristic prominent spatial organizations of heterochromatin (SOH). Fujiwara et al. found that the replacement of endogenous H2B with H2B-mCherry disrupts SOH, leading to extreme elongation of ventricular cardiomyocyte nuclei, resulting in severe heart failure, demonstrating that SOH play a structural role in maintaining nuclear shape against mechanical stress. [ABSTRACT FROM AUTHOR]

Organs consist of the parenchyma and stroma, the latter of which coordinates the generation of organotypic structures. Despite recent advances in organoid technology, induction of organ-specific stroma and recapitulation of complex organ configurations from pluripotent stem cells (PSCs) have remained challenging. By elucidating the in vivo molecular features of the renal stromal lineage at a single-cell resolution level, we herein establish an in vitro induction protocol for stromal progenitors (SPs) from mouse PSCs. When the induced SPs are assembled with two differentially induced parenchymal progenitors (nephron progenitors and ureteric buds), the completely PSC-derived organoids reproduce the complex kidney structure, with multiple types of stromal cells distributed along differentiating nephrons and branching ureteric buds. Thus, integration of PSC-derived lineage-specific stroma into parenchymal organoids will pave the way toward recapitulation of the organotypic architecture and functions. Organs consist of parenchyma and stroma. Nishinakamura and colleagues induce renal stromal progenitors from mouse pluripotent stem cells (PSCs), and generate completely PSC-derived organoids that reproduce complex kidney structure. [ABSTRACT FROM AUTHOR]

The poles of the heart and branchiomeric muscles of the face and neck are formed from the cardiopharyngeal mesoderm within the pharyngeal apparatus. They are disrupted in patients with 22q11.2 deletion syndrome, due to haploinsufficiency of TBX1, encoding a T-box transcription factor. Here, using single cell RNA-sequencing, we now identify a multilineage primed population within the cardiopharyngeal mesoderm, marked by Tbx1, which has bipotent properties to form cardiac and branchiomeric muscle cells. The multilineage primed cells are localized within the nascent mesoderm of the caudal lateral pharyngeal apparatus and provide a continuous source of cardiopharyngeal mesoderm progenitors. Tbx1 regulates the maturation of multilineage primed progenitor cells to cardiopharyngeal mesoderm derivatives while restricting ectopic non-mesodermal gene expression. We further show that TBX1 confers this balance of gene expression by direct and indirect regulation of enriched genes in multilineage primed progenitors and downstream pathways, partly through altering chromatin accessibility, the perturbation of which can lead to congenital defects in individuals with 22q11.2 deletion syndrome. Perturbations of the cardiopharyngeal mesoderm can lead to congenital defects in individuals with 22q11.2 deletion syndrome. Here the authors use single cell RNA-sequencing to identify a multilineage primed population within the mesoderm, marked by Tbx1, which has bipotent properties to form cardiac and branchiomeric muscle cells. [ABSTRACT FROM AUTHOR]

The RNA-binding protein QKI belongs to the hnRNP K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders. Qki is found highly expressed in developing and adult hearts. By employing the human embryonic stem cell (hESC) to cardiomyocyte differentiation system and generating QKI-deficient hESCs (hESCs-QKIdel) using CRISPR/Cas9 gene editing technology, we analyze the physiological role of QKI in cardiomyocyte differentiation, maturation, and contractile function. hESCs-QKIdel largely maintain normal pluripotency and normal differentiation potential for the generation of early cardiogenic progenitors, but they fail to transition into functional cardiomyocytes. In this work, by using a series of transcriptomic, cell and biochemical analyses, and the Qki-deficient mouse model, we demonstrate that QKI is indispensable to cardiac sarcomerogenesis and cardiac function through its regulation of alternative splicing in genes involved in Z-disc formation and contractile physiology, suggesting that QKI is associated with the pathogenesis of certain forms of cardiomyopathies. RNA binding protein Quaking (QKI) is known for its broad function in pre-mRNA splicing and modification and its association with several neurodevelopmental disorders. Here the authors reveal that QKI-mediated regulation of RNA splicing is indispensable to cardiac development and contractile physiology. [ABSTRACT FROM AUTHOR]

The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of TBX1, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of Tbx1 in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical Wnt/β-catenin has major roles in cardiac OFT development that may act upstream of Tbx1. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in β-catenin loss of function embryos compared to Tbx1 loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of Tbx1 and one allele of β-catenin were inactivated in the Mef2c-AHF-Cre domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, β-catenin was increased in expression when Tbx1 was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and Tbx1 in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS. [ABSTRACT FROM AUTHOR]

The correct version of the legend now states 'Tamoxifen was injected at E11.5 and the mice were analyzed at E15.5' The HTML has been updated to include a corrected version of the Supplementary Information. Correction to: I Nature Communications i https://doi.org/10.1038/s41467-022-28226-7, published online 01 February 2022 The original version of the Supplementary Information associated with this Article contained an error in the legend of Supplementary Fig. [Extracted from the article]

A mouse model that mediates temporal, specific, and efficient myocardial deletion with Cre-LoxP technology will be a valuable tool to determine the function of genes during heart formation. Mhy6 encodes a cardiac muscle specific protein: alpha-myosin heavy chain. Here, we generated a new Myh6-MerCreMer (Myh6MerCreMer/+) inducible Cre knock-in mouse by inserting a MerCreMer cassette into the Myh6 start codon. By crossing knock-in mice with Rosa26 reporter lines, we found the Myh6MerCreMer/+ mice mediate complete Cre-LoxP recombination in cardiomyocytes after tamoxifen induction. X-gal staining and immunohistochemistry analysis revealed that Myh6-driven Cre recombinase was specifically activated in cardiomyocytes at embryonic and adult stages. Furthermore, echocardiography showed that Myh6MerCreMer/+ mice maintained normal cardiac structure and function before and after tamoxifen administration. These results suggest that the new Myh6MerCreMer/+ mouse can serve as a robust tool to dissect the roles of genes in heart development and function. Additionally, myocardial progeny during heart development and after cardiac injury can be traced using this mouse line. [ABSTRACT FROM AUTHOR]

The sinoatrial node (SAN) is essential for rhythmic beating of the heart; however, our understanding of what controls proper functioning of the SAN remains primitive. To explore molecular control of SAN function, we specifically deleted Baf250a, a key regulatory component of the ATP-dependent chromatin remodeling complex SWI/SNF, in the SAN. Deletion of Baf250a in the SAN led to sinus bradycardia. Time series analysis of dysregulated genes after deletion of Baf250a reveals a transcriptional hierarchy maintaining pacemaker cell identity, i.e., Baf250a activates the expression of Tbx3, and Baf250a, Tbx3 and histone deacetylase 3 coordinately repress the expression of Nkx2.5. Disruption of this repressive pathway switches on expression of Nkx2.5, which stimulates expression of Gata4 and Tbx5. These three cardiac transcription factors further turn on a contractile cardiomyocyte program in the SAN, which eventually leads to sick sinus disease (SSD). Our study suggests that disruption of key genetic pathways regulating cardiac lineage segregation may cause SSD and cardiac arrhythmias in general. [ABSTRACT FROM AUTHOR]

Proper formation of ureteral smooth muscle cells (SMCs) during embryogenesis is essential for ureter peristalsis that propels urine from the kidney to the bladder in mammals. Currently the molecular factors that regulate differentiation of ureteral mesenchymal cells into SMCs are incompletely understood. A recent study has reported that Smad4 deficiency reduces the number of ureteral SMCs. However, its precise role in the ureteral smooth muscle development remains largely unknown. Here, we used Tbx18:Cre knock-in mouse line to delete Smad4 to examine its requirement in the development of ureteral mesenchyme and SMC differentiation. We found that mice with specific deletion of Smad4 in Tbx18-expressing ureteral mesenchyme exhibited hydroureter and hydronephrosis at embryonic day (E) 16.5, and the mutant mesenchymal cells failed to differentiate into SMCs with increased apoptosis and decreased proliferation. Molecular markers for SMCs including alpha smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) were absent in the mutant ureters. Moreover, disruption of Smad4 significantly reduced the expression of genes, including Sox9, Tbx18 and Myocardin associated with SMC differentiation. These findings suggest that Smad4 is essential for initiating the SMC differentiation program during ureter development. [ABSTRACT FROM AUTHOR]

OBJECTIVE--The generation of mature cell types during pancreatic development depends on the expression of many regulatory and signaling proteins. In this study, we tested the hypothesis that the transcriptional regulator Islet-1 (Isl-1), whose expression is first detected in the mesenchyme and epithelium of the developing pancreas and is later restricted to mature islet cells, is involved in the terminal differentiation of islet cells and maintenance of islet mass. RESEARCH DESIGN AND METHODS--To investigate the role of Isl-1 in the pancreatic epithelium during the secondary transition, Isl-1 was conditionally and specifically deleted from embryonic day 13.5 onward using Cre/LoxP technology. RESULTS--Isl-1-deficient endocrine precursors failed to mature into functional islet cells. The postnatal expansion of endocrine cell mass was impaired, and consequently Isl-1 deficient mice were diabetic. In addition, MafA, a potent regulator of the Insulin gene and β-cell function, was identified as a direct transcriptional target of Isl-1. CONCLUSIONS--These results demonstrate the requirement for Isl-1 in the maturation, proliferation, and survival of the second wave of hormone-producing islet cells. Diabetes 58: 2059-2069, 2009 [ABSTRACT FROM AUTHOR]

Understanding the origins and roles of cardiac progenitor cells is important for elucidating the pathogenesis of congenital and acquired heart diseases. Moreover, manipulation of cardiac myocyte progenitors has potential for cell-based repair strategies for various myocardial disorders. Here we report the identification in mouse of a previously unknown cardiac myocyte lineage that derives from the proepicardial organ. These progenitor cells, which express the T-box transcription factor Tbx18, migrate onto the outer cardiac surface to form the epicardium, and then make a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Tbx18-expressing cardiac progenitors also give rise to cardiac fibroblasts and coronary smooth muscle cells. The pluripotency of Tbx18 proepicardial cells provides a theoretical framework for applying these progenitors to effect cardiac repair and regeneration. [ABSTRACT FROM AUTHOR]

The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease. [ABSTRACT FROM AUTHOR]

The regeneration capacity of neonatal mouse heart is controversial. In addition, whether epicardial cells provide a progenitor pool for de novo heart regeneration is incompletely defined. Following apical resection of the neonatal mouse heart, we observed limited regeneration potential. Fate-mapping of Tbx18 MerCreMer mice revealed that newly formed coronary vessels and a limited number of cardiomyocytes were derived from the T-box transcription factor 18 (Tbx18) lineage. However, further lineage tracing with SM-MHC CreERT2 and Nfactc1 Cre mice revealed that the new smooth muscle and endothelial cells are in fact derivatives of pre-existing coronary vessels. Our data show that neonatal mouse heart can regenerate but that its potential is limited. Moreover, although epicardial cells are multipotent during embryogenesis, their contribution to heart repair through "stem" or "progenitor" cell conversion is minimal after birth. These observations suggest that early embryonic heart development and postnatal heart regeneration are distinct biological processes. Multipotency of epicardial cells is significantly decreased after birth. • Limited regeneration occurs in the neonatal mouse heart upon ventricular apex amputation • Progenitor cell differentiation from epicardium is very minimal during heart repair • New coronary vessels are generated through angiogenesis after injury The regeneration potential of the newborn mouse heart is controversial, and whether epicardial cells provide progenitors for coronary vascular regeneration is unclear. Cai et al. demonstrate a limited regeneration capacity of the neonatal heart upon injury. Epicardial cells do not convert into functional cardiac cells, including coronary vessels, during repair. [ABSTRACT FROM AUTHOR]

Replying to: V. M. Christoffels et al. 458, 10.1038/nature07916 (2009)We recognized the importance of confirming data from Cre lineage studies with an alternative approach, and thus used a fluorescent dye to lineage-label epicardial cells. This approach confirmed that epicardial cells contributed to myocytes within the heart. In our experiments, we never observed Tbx18 mRNA within ventricles at embryonic day E10.5 to E11.5, although our RNA signal in epicardium at these stages is at least as strong as that observed by Christoffels et al.. We note a discrepancy between amount of RNA staining (broadly throughout the middle of the ventricle) and protein staining (much more restricted) that Christoffels et al. observe at early stages. Reasons for discrepancies between our RNA in situ data and that of Christoffels et al. are unclear. Christoffels et al. detect Tbx18 intronic sequences by RT–PCR of E11.5 IVS, but do not provide data to demonstrate mature Tbx18 mRNA. Notably, we did observe active expression of Tbx18 in non-myocytes after E11.5 (ref. 1). [ABSTRACT FROM AUTHOR]