Search

The Pitx2 gene encodes a homeobox transcription factor that is required for mammalian development. Disruption of PITX2 expression in humans causes congenital heart diseases and is associated with atrial fibrillation; however, the cellular and molecular processes dictated by Pitx2 during cardiac ontogeny remain unclear. To characterize the role of Pitx2 during murine heart development we sequenced over 75,000 single cardiac cell transcriptomes between two key developmental timepoints in control and Pitx2 null embryos. We found that cardiac cell composition was dramatically altered in mutants at both E10.5 and E13.5. Interestingly, the differentiation dynamics of both anterior and posterior second heart field-derived progenitor cells were disrupted in Pitx2 mutants. We also uncovered evidence for defects in left-right asymmetry within atrial cardiomyocyte populations. Furthermore, we were able to detail defects in cardiac outflow tract and valve development associated with Pitx2. Our findings offer insight into Pitx2 function and provide a compilation of gene expression signatures for further detailing the complexities of heart development that will serve as the foundation for future studies of cardiac morphogenesis, congenital heart disease and arrhythmogenesis.

Loss of the paired-like homeodomain transcription factor 2 (Pitx2) in cardiomyocytes predisposes mice to atrial fibrillation and compromises neonatal regenerative capacity. In addition, Pitx2 gain-of-function protects mature cardiomyocytes from ischemic injury and promotes heart repair. Here, we characterized the long-term myocardial phenotype following myocardial infarction (MI) in Pitx2 conditional-knockout (Pitx2 CKO) mice. We found adipose-like tissue in Pitx2 CKO hearts 60 days after MI induced surgically at postnatal day 2 but not at day 8. Molecular and cellular analyses showed the onset of adipogenic signaling in mutant hearts after MI. Lineage tracing experiments showed a non-cardiomyocyte origin of the de novo adipose-like tissue. Interestingly, we found that Pitx2 promotes mitochondrial function through its gene regulatory network, and that the knockdown of a key mitochondrial Pitx2 target gene, Cox7c, also leads to the accumulation of myocardial fat tissue. Single-nuclei RNA-seq revealed that Pitx2-deficient hearts were oxidatively stressed. Our findings reveal a role for Pitx2 in maintaining proper cardiac cellular composition during heart regeneration via the maintenance of proper mitochondrial structure and function.

Congenital heart disease (CHD) is a devastating anomaly that affects ∼1% of live births. Defects of the outflow tract (OFT) make up a large percentage of human CHD. We investigated Bmp signaling in mouse OFT development by conditionally deleting both Bmp4 and Bmp7 in the second heart field (SHF). SHF Bmp4/7 deficiency resulted in defective epithelial to mesenchymal transition (EMT) and reduced cardiac neural crest ingress, with resultant persistent truncus arteriosus. Using a candidate gene approach, we found that Vegfa was upregulated in the Bmp4/7 mutant hearts. To determine if Vegfa is a downstream Bmp effector during EMT, we examined whether Vegfa is transcriptionally regulated by the Bmp receptor-regulated Smad. Our findings indicate that Smad directly binds to Vegfa chromatin and represses Vegfa transcriptional activity. We also found that Vegfa is a direct target for the miR-17-92 cluster, which is also regulated by Bmp signaling in the SHF. Deletion of miR-17-92 reveals similar phenotypes to Bmp4/7 SHF deletion. To directly address the function of Vegfa repression in Bmp-mediated EMT, we performed ex vivo explant cultures from Bmp4/7 and miR-17-92 mutant hearts. EMT was defective in explants from the Bmp4/7 double conditional knockout (dCKO; Mef2c-Cre;Bmp4/7f/f) and miR-17-92 null. By antagonizing Vegfa activity in explants, EMT was rescued in Bmp4/7 dCKO and miR-17-92 null culture. Moreover, overexpression of miR-17-92 partially suppressed the EMT defect in Bmp4/7 mutant embryos. Our study reveals that Vegfa levels in the OFT are tightly controlled by Smad- and microRNA-dependent pathways to modulate OFT development.

We performed an in depth analysis of Bmp4, a critical regulator of development, disease, and evolution, in cranial neural crest (CNC). Conditional Bmp4 overexpression, using a tetracycline-regulated Bmp4 gain-of-function allele, resulted in facial skeletal changes that were most dramatic after an E10.5 Bmp4 induction. Expression profiling uncovered a signature of Bmp4-induced genes (BIG) composed predominantly of transcriptional regulators that control self-renewal, osteoblast differentiation and negative Bmp autoregulation. The complimentary experiment, CNC inactivation of Bmp2, Bmp4 and Bmp7, resulted in complete or partial loss of multiple CNC-derived skeletal elements, revealing a crucial requirement for Bmp signaling in membranous bone and cartilage development. Importantly, the BIG signature was reduced in Bmp loss-of-function mutants, indicating Bmp-regulated target genes are modulated by Bmp dose. Chromatin immunoprecipitation (ChIP) revealed a subset of the BIG signature, including Satb2, Smad6, Hand1, Gadd45γ and Gata3, that was bound by Smad1/5 in the developing mandible, revealing direct Smad-mediated regulation. These data support the hypothesis that Bmp signaling regulates craniofacial skeletal development by balancing self-renewal and differentiation pathways in CNC progenitors.

Chondrocyte differentiation is strictly regulated by various transcription factors, including Runx2 and Runx3; however, the physiological role of Runx1 in chondrocyte differentiation remains unknown. To examine the role of Runx1, we generated mesenchymal-cell-specific and chondrocyte-specific Runx1-deficient mice [Prx1 Runx1(f/f) mice and alpha1(II) Runx1(f/f) mice, respectively] to circumvent the embryonic lethality of Runx1-deficient mice. We then mated these mice with Runx2 mutant mice to obtain mesenchymal-cell-specific or chondrocyte-specific Runx1; Runx2 double-mutant mice [Prx1 DKO mice and alpha1(II) DKO mice, respectively]. Prx1 Runx1(f/f) mice displayed a delay in sternal development and Prx1 DKO mice completely lacked a sternum. By contrast, alpha1(II) Runx1(f/f) mice and alpha1(II) DKO mice did not show any abnormal sternal morphogenesis or chondrocyte differentiation. Notably, Runx1, Runx2 and the Prx1-Cre transgene were co-expressed specifically in the sternum, which explains the observation that the abnormalities were limited to the sternum. Histologically, mesenchymal cells condensed normally in the prospective sternum of Prx1 DKO mice; however, commitment to the chondrocyte lineage, which follows mesenchymal condensation, was significantly impaired. In situ hybridization analyses demonstrated that the expression of alpha1(II) collagen (Col2a1 - Mouse Genome Informatics), Sox5 and Sox6 in the prospective sternum of Prx1 DKO mice was severely attenuated, whereas Sox9 expression was unchanged. Molecular analyses revealed that Runx1 and Runx2 induce the expression of Sox5 and Sox6, which leads to the induction of alpha1(II) collagen expression via the direct regulation of promoter activity. Collectively, these results show that Runx1 and Runx2 cooperatively regulate sternal morphogenesis and the commitment of mesenchymal cells to become chondrocytes through the induction of Sox5 and Sox6.

The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. Although emerging evidence shows that fibroblast growth factor(FGF) is essential for OFT development, the downstream pathways mediating FGF signaling in cardiac progenitors remain poorly understood. Here, we report that FRS2α (FRS2), an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates crucial aspects of FGF-dependent OFT development in mouse. Ablation of Frs2α in mesodermal OFT progenitor cells that originate in the second heart field (SHF) affects their expansion into the OFT myocardium, resulting in OFT misalignment and hypoplasia. Moreover, Frs2α mutants have defective endothelial-to-mesenchymal transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. These results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors.

The role of the Hippo signaling pathway in cranial neural crest (CNC) development is poorly understood. We used the Wnt1Cre and Wnt1Cre2SOR drivers to conditionally ablate both Yap and Taz in the CNC of mice. When using either Cre driver, Yap and Taz deficiency in the CNC resulted in enlarged, hemorrhaging branchial arch blood vessels and hydrocephalus. However, Wnt1Cre2SOR embryos had an open cranial neural tube phenotype that was not evident in Wnt1Cre embryos. In O9-1 CNC cells, the loss of Yap and Taz impaired smooth muscle cell differentiation. RNA-sequencing data indicated that Yap and Taz regulate genes encoding Fox transcription factors, specifically Foxc1. Proliferation was reduced in the branchial arch mesenchyme of Yap and Taz CNC conditional knockout (CKO) embryos. Moreover, Yap and Taz CKO embryos had cerebellar aplasia similar to Dandy Walker spectrum malformations observed in human patients and mouse embryos with mutations in Foxc1. In embryos and O9-1 cells deficient for Yap and Taz, Foxc1 expression was significantly reduced. Analysis of Foxc1 regulatory regions revealed a conserved recognition element for the Yap and Taz DNA binding co-factor Tead. ChIP-pcr experiments further supported the conclusion that Foxc1 is directly regulated by the Yap/Tead complex. Our findings uncover important roles for Yap and Taz in CNC diversification and development.

In humans, atrial fibrillation is often triggered by ectopic pacemaking activity in the myocardium sleeves of the pulmonary vein (PV) and systemic venous return. However, the genetic programs that abnormally reinforce pacemaker properties at these sites and how this relates to normal sinoatrial node (SAN) development remain uncharacterized. It was noted previously that Nkx2-5, which is expressed in the PV myocardium and reinforces a chamber-liker myocardial identity in the PV, is lacking in the SAN. Here we present evidence that Shox2 antagonizes the transcription output of Nkx2-5 in the PV myocardium and in a functional Nkx2-5+ domain within the SAN to determine the cell fate. Shox2 deletion in the Nkx2-5+ domain of the SAN caused sick sinus syndrome, associated with the loss of pacemaker program. Explanted Shox2+ cells from the embryonic PV myocardium exhibited pacemaker characteristics including node-like electrophysiological properties and the capability to pace surrounding Shox2− cells. Shox2 deletion led to Hcn4 obliteration in the developing PV myocardium. Nkx2-5 hypomorphism rescued the requirement for Shox2 for the expression of genes essential for SAN development in Shox2 mutants. Similarly, the pacemaker-like phenotype induced in the PV myocardium in Nkx2-5 hypomorphs reverted back to a working myocardial phenotype when Shox2 was simultaneously deleted. A similar mechanism is also adopted in differentiated embryoid-bodies. Moreover, we found that Shox2 interacts with Nkx2-5 directly, and discovered a substantial genome wide co-occupancy of Shox2, Nkx2-5, and Tbx5, further supporting a pivotal role for Shox2 in the core myogenic program orchestrating venous pole and pacemaker development.

We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKO(ob)) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKO(ob) mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKO(ob) osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA(+) MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.