© 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev180398. doi:10.1242/dev.180398 RESEARCH ARTICLE A cellular atlas of Pitx2-dependent cardiac development Matthew C. Hill1, Zachary A. Kadow1, Lele Li2, Tien T. Tran2, Joshua D. Wythe1,2,4 and James F. Martin1,2,3,4,* ABSTRACT confers left-sided morphogenesis onto all organs in the body The Pitx2 gene encodes a homeobox transcription factor that is (Logan et al., 1998; Piedra et al., 1998; Yoshioka et al., 1998). β required for mammalian development. Disruption of PITX2 expression Nodal is a Tgf family signaling molecule that participates in the in humans causes congenital heart diseases and is associated early break in symmetry in mammalian embryos and Nodal- with atrial fibrillation; however, the cellular and molecular processes mediated regulation of Pitx2 takes place via an asymmetric cis- dictated by Pitx2 during cardiac ontogeny remain unclear. To regulatory element located within the Pitx2 gene body. As a characterize the role of Pitx2 during murine heart development we downstream effector of LRA signaling, Pitx2 plays an essential sequenced over 75,000 single cardiac cell transcriptomes between two function at the late stages of LRA through mechanisms that remain key developmental timepoints in control and Pitx2 null embryos. We poorly understood, particularly in the developing heart. found that cardiac cell composition was dramatically altered in mutants During heart development, Pitx2 has two main functions: at both E10.5 and E13.5. Interestingly, the differentiation dynamics of morphogenesis of the outflow tract (OFT) and left-right both anterior and posterior second heart field-derived progenitor cells specification of the atria. Pitx2 is required for complete OFT were disrupted in Pitx2 mutants. We also uncovered evidence for septation (Liu et al., 2001). Conditional mutagenesis revealed that defects in left-right asymmetry within atrial cardiomyocyte populations. Pitx2 functions in the second heart field (SHF) to regulate Furthermore, we were able to detail defects in cardiac outflow tract and proliferation of OFT myocardium, and that Pitx2 was dispensable in valve development associated with Pitx2. Our findings offer insight into the cardiac neural crest (Ai et al., 2006). In the left atrium, Pitx2 Pitx2 function and provide a compilation of gene expression signatures confers left atrial morphology (Liu et al., 2001). Pitx2 null mutant for further detailing the complexities of heart development that will left atria have right-sided morphologic characteristics including serve as the foundation for future studies of cardiac morphogenesis, venous valves and trabeculated myocardium (Liu et al., 2001). congenital heart disease and arrhythmogenesis. Moreover, Pitx2-deficient embryos also possess bilateral or ectopic sinoatrial nodes (SANs), which may explain the predisposition to KEY WORDS: Pitx2, Left-right asymmetry, Single cell RNA-seq, AF that is observed in adult animals with decreased Pitx2 Cardiac development expression (Ammirabile et al., 2012; Mommersteeg et al., 2007; Wang et al., 2010). In addition to OFT morphogenesis, Pitx2 has INTRODUCTION also been implicated in atrioventricular valve development. Further, Pitx2 encodes a paired related homeodomain transcription factor morphogenesis of both the AV cushions and the dorsal mesenchymal that is essential for both human and mouse development. protrusion are defective in Pitx2 null embryos, suggesting an essential Investigations aimed at dissecting the biological role of Pitx2 are function for Pitx2 during ventricular septation. important, especially given that Pitx2 has been implicated in several Here, we used single cell transcriptomics to inspect Pitx2 human diseases, including Rieger syndrome, ocular dysgenesis with function in cardiac development and left-right cellular glaucoma, acute appendicitis and atrial fibrillation (AF), the most specification. Deployment of a high-throughput single cell RNA- common sustained human arrhythmia (Ellinor et al., 2010; seq (scRNA-seq) platform on cardiac tissue dissected from both Gudbjartsson et al., 2007; Lin et al., 1999; Lu et al., 1999; control and Pitx2 null embryos at embryonic day (E)10.5 and E13.5 Semina et al., 1996; Syeda et al., 2017). In postnatal cardiomyocytes was carried out to characterize all deviations in cell composition, (CMs), Pitx2 regulates genes that are important for the cellular cellular state and differentiation trajectories. Our data revealed that response to reactive oxygen species (ROS), and is itself a target of the cell fates of SHF progenitors in Pitx2-deficient hearts are Nrf2 (also known as Nfe2l2), a master transcriptional regulator of severely affected at the transcriptional level. We also uncovered the the cellular antioxidant response (Tao et al., 2016). However, less is transcriptional consequences associated with the loss of proper known about the cell type-specific direct targets for Pitx2 during left-right specification in atrial CM populations. Finally, we detailed cardiac development. the transcriptional and cellular compositional shifts associated Early during embryogenesis, Pitx2 is directly regulated by the with abnormal cardiac cushion remodeling, valvulogenesis and Nodal-mediated left-right asymmetry (LRA) pathway, which vasculature development observed in Pitx2-deficient hearts. RESULTS 1 Program in Developmental Biology, Baylor College of Medicine, Houston, Pitx2 TX 77030, USA. 2Department of Molecular Physiology and Biophysics, Baylor Profiling of control and -deficient E10.5 cardiac tissue College of Medicine, Houston, TX 77030, USA. 3Texas Heart Institute, Houston, To characterize the precise cellular and molecular events dictated by TX 77030, USA. 4Cardiovascular Research Institute, Baylor College of Medicine, Pitx2 during cardiac ontogeny, we first focused on E10.5, when Houston, TX 77030, USA. Pitx2 is highly expressed and atrial septation, valvulogenesis, *Author for correspondence ( [email protected]) atrioventricular junction formation and OFT remodeling begin to occur. We performed droplet-based scRNA-seq on E10.5 murine L.L., 0000-0002-3607-3821; J.D.W., 0000-0002-3225-2937; J.F.M., 0000-0002- hd−/− 7842-9857 cardiac tissue derived from control and Pitx2 null (Pitx2 ) animals (Fig. 1A). After computational processing, we carried out Received 3 December 2018; Accepted 13 May 2019 graph-based clustering followed by dimensionality reduction using DEVELOPMENT 1 RESEARCH ARTICLE Development (2019) 146, dev180398. doi:10.1242/dev.180398 Fig. 1. Single cell profiling of Pitx2hd−/− cardiac tissue at E10.5. (A) Schematic of the study. (B) UMAP representation of single cell transcriptomes derived from E10.5 control and Pitx2 null cardiac tissue. (C) Heatmap showing the average expression for the top differentially expressed genes between E10.5 cardiac cell clusters (n=1062). (D) Force-directed graph of PAGA-initialized embedding (left). PAGA graph showing the relationships between all the E10.5 clusters (right). (E) UMAP feature plots showing the expression of individual genes. High expression is dark red, medium expression is yellow and no expression is gray. Encircled clusters are colored according to Fig. 1B. CM-AVC, atrioventricular cardiomyocytes; CM-LSV, cardiomyocytes of left ventricular septum; CM-LV, cardiomyocytes of left ventricle; CM-OFT, cardiomyocytes of outflow tract; CM-RA, right atrial cardiomyocytes; CP, cardiac progenitors; EndoC, endocardial cells; EndoV, endocardial valve cells; Epi, epithelial cells; EpiC, epicardial cells; Gata1+, Gata1-expressing immune cells; LA, left atrium; LV, left ventricle; Mes, mesenchymal cells; MΦ, macrophage; OFT, outflow tract; PV, pulmonary vein; RA, right atrium; RV, right ventricle. DEVELOPMENT 2 RESEARCH ARTICLE Development (2019) 146, dev180398. doi:10.1242/dev.180398 Uniform Manifold Approximation and Projection (UMAP) (Fig. S2C). Thus, we subset these cell populations along with closely (McInnes and Healy, 2018). Overall, we captured 22,797 cells interconnected clusters and performed iterative clustering before which belonged to 18 clusters that we assigned cell identities to UMAP dimensionality reduction to gain further insight into the based on their top differentially expressed genes (Fig. 1B,C, Fig. mutant phenotype (Fig. 2A). Differential expression analysis of these S1A and Table S1). Overall, we were able to identify a total of 1062 clusters provided us with a gene list from which we were able to more differentially expressed genes and several interesting markers for all accurately assign cell identities to CPs (Fig. 2B, and Table S2) cardiac cell types and cell states present at E10.5. We detected all (reviewed by Meilhac and Buckingham, 2018). And again, the major cardiac cell types, including CMs, endocardial cells PAGA-initialized cell embeddings were in close agreement with our (EndoCs), mesenchymal cells (Mes), epicardial cells (EpiC), UMAP results (Fig. 2C). Indeed, we found Tbx18-positive posterior macrophages (MΦ), and cardiac progenitor cells (CPs). second heart field (pSHF) progenitors, and two clusters of Interestingly, the cardiac progenitors were heterogenous and were Isl1-expressing cells that resembled anterior second heart field more similar to epicardial cells. Moreover, we detected several CM (aSHF) progenitors (Fig. 2D and Fig. S2D). Strikingly, the aSHF2 subtypes, which we categorized according to previously detailed population, which appeared to be actively differentiating into markers for E10.5