Epicardial HDAC3 Promotes Myocardial Growth Through a Novel MicroRNA Pathway.

BACKGROUND: Establishment of the myocardial wall requires proper growth cues from nonmyocardial tissues. During heart development, the epicardium and epicardium-derived cells instruct myocardial growth by secreting essential factors including FGF (fibroblast growth factor) 9 and IGF (insulin-like growth factor) 2. However, it is poorly understood how the epicardial secreted factors are regulated, in particular by chromatin modifications for myocardial formation. The current study is to investigate whether and how HDAC (histone deacetylase) 3 in the developing epicardium regulates myocardial growth. METHODS: Various cellular and mouse models in conjunction with biochemical and molecular tools were employed to study the role of HDAC3 in the developing epicardium. RESULTS: We deleted Hdac3 in the developing murine epicardium, and mutant hearts showed ventricular myocardial wall hypoplasia with reduction of epicardium-derived cells. The cultured embryonic cardiomyocytes with supernatants from Hdac3 knockout (KO) mouse epicardial cells also showed decreased proliferation. Genome-wide transcriptomic analysis revealed that Fgf9 and Igf2 were significantly downregulated in Hdac3 KO mouse epicardial cells. We further found that Fgf9 and Igf2 expression is dependent on HDAC3 deacetylase activity. The supplementation of FGF9 or IGF2 can rescue the myocardial proliferation defects treated by Hdac3 KO supernatant. Mechanistically, we identified that microRNA (miR)-322 and miR-503 were upregulated in Hdac3 KO mouse epicardial cells and Hdac3 epicardial KO hearts. Overexpression of miR-322 or miR-503 repressed FGF9 and IGF2 expression, while knockdown of miR-322 or miR-503 restored FGF9 and IGF2 expression in Hdac3 KO mouse epicardial cells. CONCLUSIONS: Our findings reveal a critical signaling pathway in which epicardial HDAC3 promotes compact myocardial growth by stimulating FGF9 and IGF2 through repressing miR-322 or miR-503, providing novel insights in elucidating the etiology of congenital heart defects and conceptual strategies to promote myocardial regeneration.

Arrhythmia Mechanism and Dynamics in a Humanized Mouse Model of Inherited Cardiomyopathy Caused by Phospholamban R14del Mutation.

BACKGROUND: Arginine (Arg) 14 deletion (R14del) in the calcium regulatory protein phospholamban (hPLNR14del) has been identified as a disease-causing mutation in patients with an inherited cardiomyopathy. Mechanisms underlying the early arrhythmogenic phenotype that predisposes carriers of this mutation to sudden death with no apparent structural remodeling remain unclear. METHODS: To address this, we performed high spatiotemporal resolution optical mapping of intact hearts from adult knock-in mice harboring the human PLNWT (wildtype [WT], n=12) or the heterozygous human PLNR14del mutation (R14del, n=12) before and after ex vivo challenge with isoproterenol and rapid pacing. RESULTS: Adverse electrophysiological remodeling was evident in the absence of significant structural or hemodynamic changes. R14del hearts exhibited increased arrhythmia susceptibility compared with wildtype. Underlying this susceptibility was preferential right ventricular action potential prolongation that was unresponsive to ß-adrenergic stimulation. A steep repolarization gradient at the left ventricular/right ventricular interface provided the substrate for interventricular activation delays and ultimately local conduction block during rapid pacing. This was followed by the initiation of macroreentrant circuits supporting the onset of ventricular tachycardia. Once sustained, these circuits evolved into high-frequency rotors, which in their majority were pinned to the right ventricle. These rotors exhibited unique spatiotemporal dynamics that promoted their increased stability in R14del compared with wildtype hearts. CONCLUSIONS: Our findings highlight the crucial role of primary electric remodeling caused by the hPLNR14del mutation. These inherently arrhythmogenic features form the substrate for adrenergic-mediated VT at early stages of PLNR14del induced cardiomyopathy.

Nkx2-5 defines a subpopulation of pacemaker cells and is essential for the physiological function of the sinoatrial node in mice.

The sinoatrial node (SAN), the primary cardiac pacemaker, consists of a head domain and a junction/tail domain that exhibit different functional properties. However, the underlying molecular mechanism defining these two pacemaker domains remains elusive. Nkx2-5 is a key transcription factor essential for the formation of the working myocardium, but it was generally thought to be detrimental to SAN development. However, Nkx2-5 is expressed in the developing SAN junction, suggesting a role for Nkx2-5 in SAN junction development and function. In this study, we present unambiguous evidence that SAN junction cells exhibit unique action potential configurations intermediate to those manifested by the SAN head and the surrounding atrial cells, suggesting a specific role for the junction cells in impulse generation and in SAN-atrial exit conduction. Single-cell RNA-seq analyses support this concept. Although Nkx2-5 inactivation in the SAN junction did not cause a malformed SAN at birth, the mutant mice manifested sinus node dysfunction. Thus, Nkx2-5 defines a population of pacemaker cells in the transitional zone. Despite Nkx2-5 being dispensable for SAN morphogenesis during embryogenesis, its deletion hampers atrial activation by the pacemaker.

Novel Myh11 Dual Reporter Mouse Model Provides Definitive Labeling and Identification of Smooth Muscle Cells-Brief Report.

OBJECTIVE: Myh11 encodes a myosin heavy chain protein that is specifically expressed in smooth muscle cells (SMCs) and is important for maintaining vascular wall stability. The goal of this study is to generate a Myh11 dual reporter mouse line for definitive visualization of MYH11+ SMCs in vivo. Approach and Results: We generated a Myh11 knock-in mouse model by inserting LoxP-nlacZ-4XpolyA-LoxP-H2B-GFP-polyA-FRT-Neo-FRT reporter cassette into the Myh11 gene locus. The nuclear (n) lacZ-4XpolyA cassette is flanked by 2 LoxP sites followed by H2B-GFP (histone 2B fused green fluorescent protein). Upon Cre-mediated recombination, nlacZ-stop cassette is removed thereby permitting nucleus localized H2B-GFP expression. Expression of the nuclear localized lacZ or H2B-GFP is under control of the endogenous Myh11 promoter. Nuclear lacZ was expressed specifically in SMCs at embryonic and adult stages. Following germline Cre-mediated deletion of nuclear lacZ, H2B-GFP was specifically expressed in the nuclei of SMCs. Comparison of nuclear lacZ expression with Wnt1Cre and Mef2cCre mediated-H2B-GFP expression revealed heterogenous origins of SMCs from neural crest and second heart field in the great arteries and coronary vessels adjacent to aortic root. CONCLUSIONS: The Myh11 knock-in dual reporter mouse model offers an exceptional genetic tool to visualize and trace the origins of SMCs in mice.

Pkm2 Regulates Cardiomyocyte Cell Cycle and Promotes Cardiac Regeneration.

BACKGROUND: The adult mammalian heart has limited regenerative capacity, mostly attributable to postnatal cardiomyocyte cell cycle arrest. In the last 2 decades, numerous studies have explored cardiomyocyte cell cycle regulatory mechanisms to enhance myocardial regeneration after myocardial infarction. Pkm2 (Pyruvate kinase muscle isoenzyme 2) is an isoenzyme of the glycolytic enzyme pyruvate kinase. The role of Pkm2 in cardiomyocyte proliferation, heart development, and cardiac regeneration is unknown. METHODS: We investigated the effect of Pkm2 in cardiomyocytes through models of loss (cardiomyocyte-specific Pkm2 deletion during cardiac development) or gain using cardiomyocyte-specific Pkm2 modified mRNA to evaluate Pkm2 function and regenerative affects after acute or chronic myocardial infarction in mice. RESULTS: Here, we identify Pkm2 as an important regulator of the cardiomyocyte cell cycle. We show that Pkm2 is expressed in cardiomyocytes during development and immediately after birth but not during adulthood. Loss of function studies show that cardiomyocyte-specific Pkm2 deletion during cardiac development resulted in significantly reduced cardiomyocyte cell cycle, cardiomyocyte numbers, and myocardial size. In addition, using cardiomyocyte-specific Pkm2 modified RNA, our novel cardiomyocyte-targeted strategy, after acute or chronic myocardial infarction, resulted in increased cardiomyocyte cell division, enhanced cardiac function, and improved long-term survival. We mechanistically show that Pkm2 regulates the cardiomyocyte cell cycle and reduces oxidative stress damage through anabolic pathways and ß-catenin. CONCLUSIONS: We demonstrate that Pkm2 is an important intrinsic regulator of the cardiomyocyte cell cycle and oxidative stress, and highlight its therapeutic potential using cardiomyocyte-specific Pkm2 modified RNA as a gene delivery platform.

Generation of Pecam1 endothelial specific dual reporter mouse model.

Endothelial cells are specialized epithelium lining the interior surface of vessels and play fundamental roles in angiogenesis, vascular permeability, and immune response. To identify endothelial cells in vivo, we constructed a Pecam1nlacZ-H2B-GFP/+ knock-in mouse model in which the endothelial cells are labeled by nuclear LacZ (nlacZ) expression. When Pecam1nlacZ-H2B-GFP/+ mice are bred with germline Cre deleter mice, Pecam1H2B-GFP/+ line is created with native nuclear GFP (H2B-GFP) expression in the endothelium of various organs. This dual reporter mouse provides us with a powerful genetic tool for definitive identification of endothelial cells and monitoring this important cell population throughout development, homeostasis, and disease conditions in mammals.

CDC42 is required for epicardial and pro-epicardial development by mediating FGF receptor trafficking to the plasma membrane.

The epicardium contributes to multiple cardiac lineages and is essential for cardiac development and regeneration. However, the mechanism of epicardium formation is unclear. This study aimed to establish the cellular and molecular mechanisms underlying the dissociation of pro-epicardial cells (PECs) from the pro-epicardium (PE) and their subsequent translocation to the heart to form the epicardium. We used lineage tracing, conditional deletion, mosaic analysis and ligand stimulation in mice to determine that both villous protrusions and floating cysts contribute to PEC translocation to myocardium in a CDC42-dependent manner. We resolved a controversy by demonstrating that physical contact of the PE with the myocardium constitutes a third mechanism for PEC translocation to myocardium, and observed a fourth mechanism in which PECs migrate along the surface of the inflow tract to reach the ventricles. Epicardial-specific Cdc42 deletion disrupted epicardium formation, and Cdc42 null PECs proliferated less, lost polarity and failed to form villous protrusions and floating cysts. FGF signaling promotes epicardium formation in vivo, and biochemical studies demonstrated that CDC42 is involved in the trafficking of FGF receptors to the cell membrane to regulate epicardium formation.

Reduced dosage of ß-catenin provides significant rescue of cardiac outflow tract anomalies in a Tbx1 conditional null mouse model of 22q11.2 deletion syndrome.

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/ß-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 ß-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 ß-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, ß-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.

Myocardial-specific R-spondin3 drives proliferation of the coronary stems primarily through the Leucine Rich Repeat G Protein coupled receptor LGR4.

Coronary artery anomalies are common congenital disorders with serious consequences in adult life. Coronary circulation begins when the coronary stems form connections between the aorta and the developing vascular plexus. We recently identified the WNT signaling modulator R-spondin 3 (Rspo3), as a crucial regulator of coronary stem proliferation. Using expression analysis and tissue-specific deletion we now demonstrate that Rspo3 is primarily produced by cardiomyocytes. Moreover, we have employed CRISPR/Cas9 technology to generate novel Lgr4-null alleles that showed a significant decrease in coronary stem proliferation and thus phenocopied the coronary artery defects seen in Rspo3 mutants. Interestingly, Lgr4 mutants displayed slightly hypomorphic right ventricles, an observation also made after myocardial specific deletion of Rspo3. These results shed new light on the role of Rspo3 in heart development and demonstrate that LGR4 is the principal R-spondin 3 receptor in the heart.

Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development.

Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation remains incompletely understood. In this study, using a Tbx18:Cre (Tbx18Cre/+) gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 (Smad4f/f ) in the limbs of mice. We found that the Smad4-deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including Col2a1 and Acan, in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy-related markers Col10a1 and Panx3 were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 (Runx2), a critical mediator of chondrocyte hypertrophy, was also down-regulated in Smad4-deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the Runx2 promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating Runx2 expression during skeletal development. The regulatory mechanism involving Smad4-mediated Runx2 activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia.

Cardiac Sca-1+ Cells Are Not Intrinsic Stem Cells for Myocardial Development, Renewal, and Repair.

BACKGROUND: For more than a decade, Sca-1+ cells within the mouse heart have been widely recognized as a stem cell population with multipotency that can give rise to cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and after cardiac grafting. However, the developmental origin and authentic nature of these cells remain elusive. METHODS: Here, we used a series of high-fidelity genetic mouse models to characterize the identity and regenerative potential of cardiac resident Sca-1+ cells. RESULTS: With these novel genetic tools, we found that Sca-1 does not label cardiac precursor cells during early embryonic heart formation. Postnatal cardiac resident Sca-1+ cells are in fact a pure endothelial cell population. They retain endothelial properties and exhibit minimal cardiomyogenic potential during development, normal aging and upon ischemic injury. CONCLUSIONS: Our study provides definitive insights into the nature of cardiac resident Sca-1+ cells. The observations challenge the current dogma that cardiac resident Sca-1+ cells are intrinsic stem cells for myocardial development, renewal, and repair, and suggest that the mechanisms of transplanted Sca-1+ cells in heart repair need to be reassessed.

A unique stylopod patterning mechanism by Shox2-controlled osteogenesis.

Vertebrate appendage patterning is programmed by Hox-TALE factor-bound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.

The Elusive Progenitor Cell in Cardiac Regeneration: Slip Slidin' Away.

The adult human heart is unable to regenerate after various forms of injury, suggesting that this organ lacks a biologically meaningful endogenous stem cell pool. However, injecting the infarcted area of the adult mammalian heart with exogenously prepared progenitor cells of various types has been reported to create new myocardium by the direct conversion of these progenitor cells into cardiomyocytes. These reports remain controversial because follow-up studies from independent laboratories failed to observe such an effect. Also, the exact nature of various putative myocyte-producing progenitor cells remains elusive and undefined across laboratories. By comparison, the field has gradually worked toward a consensus viewpoint that proposes that the adult mammalian myocardium can undergo a low level of new cardiomyocyte renewal of ≈1% per year, which is primarily because of proliferation of existing cardiomyocytes but not from the differentiation of putative progenitor cells. This review will weigh the emerging evidence, suggesting that the adult mammalian heart lacks a definable myocyte-generating progenitor cell of biological significance.

Maternal and zygotic Zfp57 modulate NOTCH signaling in cardiac development.

Zfp57 is a maternal-zygotic effect gene that maintains genomic imprinting. Here we report that Zfp57 mutants exhibited a variety of cardiac defects including atrial septal defect (ASD), ventricular septal defect (VSD), thin myocardium, and reduced trabeculation. Zfp57 maternal-zygotic mutant embryos displayed more severe phenotypes with higher penetrance than the zygotic ones. Cardiac progenitor cells exhibited proliferation and differentiation defects in Zfp57 mutants. ZFP57 is a master regulator of genomic imprinting, so the DNA methylation imprint was lost in embryonic heart without ZFP57. Interestingly, the presence of imprinted DLK1, a target of ZFP57, correlated with NOTCH1 activation in cardiac cells. These results suggest that ZFP57 may modulate NOTCH signaling during cardiac development. Indeed, loss of ZFP57 caused loss of NOTCH1 activation in embryonic heart with more severe loss observed in the maternal-zygotic mutant. Maternal and zygotic functions of Zfp57 appear to play redundant roles in NOTCH1 activation and cardiomyocyte differentiation. This serves as an example of a maternal effect that can influence mammalian organ development. It also links genomic imprinting to NOTCH signaling and particular developmental functions.

A series of robust genetic indicators for definitive identification of cardiomyocytes.

Definitively identifying the cell type of newly generated cells in the heart and defining their origins are central questions in cardiac regenerative medicine. Currently, it is challenging to ascertain the myocardial identity and to track myocardial progeny during heart development and disease due to lack of proper genetic tools. This may lead to many misinterpretations of the findings in cardiac regenerative biology. In this study, we developed a set of novel mouse models by inserting double reporter genes nlacZ/H2B-GFP, mGFP/H2B-mCherry into the start codon of Tnnt2 and Myh6. nlacZ (nuclear lacZ) and mGFP (membrane GFP) are flanked by two LoxP sites in these animals. We found that the reporter genes faithfully recapitulated Tnnt2 and Myh6 cardiac expression from embryonic stage and adulthood. The reporter mice provide unprecedented robustness and fidelity for visualizing and tracing cardiomyocytes with nuclear or cell membrane localization signals. These animal models offer superior genetic tools to meet a critical need in studies of heart development, cardiac stem cell biology and cardiac regenerative medicine.

Tbx20 acts upstream of Wnt signaling to regulate endocardial cushion formation and valve remodeling during mouse cardiogenesis.

Cardiac valves are essential to direct forward blood flow through the cardiac chambers efficiently. Congenital valvular defects are prevalent among newborns and can cause an immediate threat to survival as well as long-term morbidity. Valve leaflet formation is a rigorously programmed process consisting of endocardial epithelial-mesenchymal transformation (EMT), mesenchymal cell proliferation, valve elongation and remodeling. Currently, little is known about the coordination of the diverse signals that regulate endocardial cushion development and valve elongation. Here, we report that the T-box transcription factor Tbx20 is expressed in the developing endocardial cushions and valves throughout heart development. Ablation of Tbx20 in endocardial cells causes severe valve elongation defects and impaired cardiac function in mice. Our study reveals that endocardial Tbx20 is crucial for valve endocardial cell proliferation and extracellular matrix development, but is not required for initiation of EMT. Elimination of Tbx20 also causes aberrant Wnt/ß-catenin signaling in the endocardial cushions. In addition, Tbx20 regulates Lef1, a key transcriptional mediator for Wnt/ß-catenin signaling, in this developmental process. Our study suggests a model in which Tbx20 regulates the Wnt pathway to direct endocardial cushion maturation and valve elongation, and provides new insights into the etiology of valve defects in humans.

Loss of Asxl1 leads to myelodysplastic syndrome-like disease in mice.

ASXL1 is mutated/deleted with high frequencies in multiple forms of myeloid malignancies, and its alterations are associated with poor prognosis. De novo ASXL1 mutations cause Bohring-Opitz syndrome characterized by multiple congenital malformations. We show that Asxl1 deletion in mice led to developmental abnormalities including dwarfism, anophthalmia, and 80% embryonic lethality. Surviving Asxl1(-/-) mice lived for up to 42 days and developed features of myelodysplastic syndrome (MDS), including dysplastic neutrophils and multiple lineage cytopenia. Asxl1(-/-) mice had a reduced hematopoietic stem cell (HSC) pool, and Asxl1(-/-) HSCs exhibited decreased hematopoietic repopulating capacity, with skewed cell differentiation favoring granulocytic lineage. Asxl1(+/-) mice also developed mild MDS-like disease, which could progress to MDS/myeloproliferative neoplasm, demonstrating a haploinsufficient effect of Asxl1 in the pathogenesis of myeloid malignancies. Asxl1 loss led to an increased apoptosis and mitosis in Lineage(-)c-Kit(+) (Lin(-)c-Kit(+)) cells, consistent with human MDS. Furthermore, Asxl1(-/-) Lin(-)c-Kit(+) cells exhibited decreased global levels of H3K27me3 and H3K4me3 and altered expression of genes regulating apoptosis (Bcl2, Bcl2l12, Bcl2l13). Collectively, we report a novel ASXL1 murine model that recapitulates human myeloid malignancies, implying that Asxl1 functions as a tumor suppressor to maintain hematopoietic cell homeostasis. Future work is necessary to clarify the contribution of microenvironment to the hematopoietic phenotypes observed in the constitutional Asxl1(-/-) mice.

Generation of a tamoxifen inducible Tnnt2MerCreMer knock-in mouse model for cardiac studies.

Tnnt2, encoding thin-filament sarcomeric protein cardiac troponin T, plays critical roles in heart development and function in mammals. To develop an inducible genetic deletion strategy in myocardial cells, we generated a new Tnnt2:MerCreMer (Tnnt2(MerCreMer/+)) knock-in mouse. Rosa26 reporter lines were used to examine the specificity and efficiency of the inducible Cre recombinase. We found that Cre was specifically and robustly expressed in the cardiomyocytes at embryonic and adult stages following tamoxifen induction. The knock-in allele on Tnnt2 locus does not impact cardiac function. These results suggest that this new Tnnt2(MerCreMer/+) mouse could be applied towards the temporal genetic deletion of genes of interests in cardiomyocytes with Cre-LoxP technology. The Tnnt2(MerCreMer/+) mouse model also provides a useful tool to trace myocardial lineage during development and repair after cardiac injury.

Tbx18 is essential for normal development of vasculature network and glomerular mesangium in the mammalian kidney.

Tbx18 has been shown to be essential for ureteral development. However, it remains unclear whether it plays a direct role in kidney development. Here we addressed this by focusing on examining the pattern and contribution of Tbx18+ cells in the kidney and its role in kidney vascular development. Expression studies and genetic lineage tracing revealed that Tbx18 is expressed in renal capsule, vascular smooth muscle cells and pericytes and glomerular mesangial cells in the kidney and that Tbx18-expressing progenitors contribute to these cell types. Examination of Tbx18(-/-) kidneys revealed large reduction in vasculature density and dilation of glomerular capillary loops. While SMA+ cells were reduced in the mutant, PDGFRß+ cells were seen in early capillary loop renal corpuscles in the mutant, but fewer than in the controls, and further development of the mesangium failed. Analysis of kidney explants cultured from E12.5 excluded the possibility that the defects observed in the mutant were caused by ureter obstruction. Reduced proliferation in glomerular tuft and increased apoptosis in perivascular mesenchyme were observed in Tbx18(-/-) kidneys. Thus, our analyses have identified a novel role of Tbx18 in kidney vasculature development.