Biology subjects

Moskowitz, I. P.

Publications and source records attributed to Moskowitz, I. P..

16 recordsLinked to original sources

bioRxiv2025

Receptor Allostery Promotes Context-Specific Sonic Hedgehog Signaling During Embryonic Development

Sonic Hedgehog (SHH) signaling functions in temporal- and context-dependent manners to pattern diverse tissues during embryogenesis. The signal transducer Smoothened (SMO) is activated by sterols, oxysterols, and arachidonic acid (AA) through binding pockets in its extracellular cysteine-rich domain (CRD) and 7-transmembrane (7TM) bundle. In vitro analyses suggest SMO signaling is allosterically enhanced by combinatorial ligand binding to these pockets but in vivo evidence of SMO allostery is lacking. Herein, we map an AA binding pocket at the top of the 7TM bundle and show that its disruption attenuates SHH and sterol-stimulated SMO induction. A knockin mouse model of compromised AA binding reveals that homozygous mutant mice are cyanotic, exhibit high perinatal lethality, and show congenital heart disease. Surviving mutants demonstrate pulmonary maldevelopment and fail to thrive. Neurodevelopment is unaltered in these mice, suggesting that context-dependent allosteric regulation of SMO signaling allows for precise tuning of pathway activity during cardiopulmonary development.

developmental biology↗

bioRxiv2024

Coordinated Tbx3 / Tbx5 transcriptional control of the adult ventricular conduction system

The cardiac conduction system (CCS) orchestrates the electrical impulses that enable coordinated contraction of the cardiac chambers. The T-box transcription factors TBX3 and TBX5 are required for cardiac conduction system development and associated with overlapping and distinct human cardiac conduction system diseases. We evaluated the coordinated role of Tbx3 and Tbx5 in the murine ventricular conduction system (VCS). We engineered a compound Tbx3:Tbx5 conditional knockout allele for both genes located in cis on mouse chromosome 5. Conditional deletion of both T-box transcriptional factors in the ventricular conduction system, using the VCS-specific MinK:Cre, caused loss of VCS function and molecular identity. Combined Tbx3 and Tbx5 deficiency in the adult VCS led to conduction defects, including prolonged PR and QRS intervals and elevated susceptibility to ventricular tachycardia. These electrophysiological defects occurred prior to detectable alterations in cardiac contractility or histologic morphology, indicative of a primary conduction system defect. Tbx3:Tbx5 double knockout VCS cardiomyocytes revealed a transcriptional shift towards non-CCS-specialized working myocardium, indicating a change to their cellular identity. Furthermore, optical mapping revealed a loss of VCS-specific conduction system propagation. Collectively, these findings indicate that Tbx3 and Tbx5 coordinate to control VCS molecular fate and function, with implications for understanding cardiac conduction disorders in humans.

developmental biology↗

bioRxiv2023

The cGAS-STING pathway is dispensable in a mouse model of LMNA-cardiomyopathy despite nuclear envelope rupture

Nuclear envelope (NE) ruptures are emerging observations in Lamin-related dilated cardiomyopathy, an adult-onset disease caused by loss-of-function mutations in Lamin A/C, a nuclear lamina component. Here, we tested a prevailing hypothesis that NE ruptures trigger pathological cGAS-STING cytosolic DNA-sensing pathway, using a mouse model of Lamin-cardiomyopathy. Reduction of Lamin A/C in cardiomyocytes of adult mice caused pervasive NE ruptures in cardiomyocytes, preceding inflammatory transcription, fibrosis, and fatal dilated cardiomyopathy. NE ruptures were followed by DNA damage accumulation without causing immediate cardiomyocyte death. However, cGAS-STING-dependent inflammatory signaling remained inactive. Deleting cGas or Sting did not rescue cardiomyopathy. The lack of cGAS-STING activation was likely due to the near absence of cGAS expression in adult cardiomyocytes at baseline. Instead, extracellular matrix (ECM) signaling was activated and predicted to initiate pro-inflammatory communication from Lamin-reduced cardiomyocytes to fibroblasts. Our work nominates ECM signaling, not cGAS-STING, as a potential inflammatory contributor in Lamin-cardiomyopathy.

molecular biology↗

bioRxiv2023

Tbx5 maintains atrial identity by regulating an atrial enhancer network

Understanding how the atrial and ventricular chambers of the heart maintain their distinct identity is a prerequisite for treating chamber-specific diseases. Here, we selectively inactivated the transcription factor Tbx5 in the atrial working myocardium of the neonatal mouse heart to show that it is required to maintain atrial identity. Atrial Tbx5 inactivation downregulated highly chamber specific genes such as Myl7 and Nppa, and conversely, increased the expression of ventricular identity genes including Myl2. Using combined single nucleus transcriptome and open chromatin profiling, we assessed genomic accessibility changes underlying the altered atrial identity expression program, identifying 1846 genomic loci with greater accessibility in control atrial cardiomyocytes compared to KO aCMs. 69% of the control-enriched ATAC regions were bound by TBX5, demonstrating a role for TBX5 in maintaining atrial genomic accessibility. These regions were associated with genes that had higher expression in control aCMs compared to KO aCMs, suggesting they act as TBX5-dependent enhancers. We tested this hypothesis by analyzing enhancer chromatin looping using HiChIP and found 510 chromatin loops that were sensitive to TBX5 dosage. Of the loops enriched in control aCMs, 73.7% contained anchors in control-enriched ATAC regions. Together, these data demonstrate a genomic role for TBX5 in maintaining the atrial gene expression program by binding to atrial enhancers and preserving tissue-specific chromatin architecture of atrial enhancers.

cell biology↗

bioRxiv2022

Frem1 activity regulated by Sonic Hedgehog signaling in the cranial neural crest mesenchyme guides midfacial morphogenesis

The Frem/Fras family of extracellular matrix proteins has been linked to human face shape variation and malformation, but little is known about their regulation and biological roles in facial development. During midfacial morphogenesis in mice, we observed Frem1 expression in the embryonic growth centers that form the median upper lip, nose, and palate. Expansive spatial gradients of Frem1 expression in the cranial neural crest cell (cNCC) mesenchyme of these tissues suggested transcriptional regulation by a secreted morphogen. Accordingly, Frem1 expression paralleled that of the conserved Sonic Hedgehog (Shh) target gene Gli1 in the cNCC mesenchyme. Suggesting direct transcriptional regulation by Shh signaling, we found that Frem1 expression is induced by SHH ligand stimulation or downstream pathway activation in cNCCs and observed GLI transcription factor binding at the Frem1 transcriptional start site during midfacial morphogenesis. Shh pathway antagonism reduced Frem1 expression during pathogenesis of midfacial hypoplasia, and FREM1 was sufficient to induce cNCC proliferation in a concentration-dependent manner. These findings provide novel insight into the mechanism by which the Shh pathway drives midfacial morphogenesis and reveal a functional role for Frem1 in cNCC biology that establishes the developmental basis for FREM1-associated face shape variation and malformation.

developmental biology↗

bioRxiv2021

The cardiac lncRNA Chantico directly regulates Cxcl1 chemokine transcription

Withdrawal StatementThe corresponding author has withdrawn this preprint owing to inability to reproduce some of the data, instances of inappropriate data exclusion, and loss of much of the primary experimental records/data. Specifically, Figures 2B,E,H; 3; 4; 5A,B,D; 6; S2A,C; S4A; S5; and S6A,B and attendant text contain analyses for which the primary record and/or raw data no longer exist; the analyses, where still available, suffer from inappropriate data exclusion and thus should not be construed to be an accurate reflection of the experiments. Attempts by others in the lab to repeat several of the experiments in these indicated panels have failed reproduce the presented effects, despite showing much greater precision. Therefore the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

molecular biology↗

bioRxiv2021

ETV2 primes hematoendothelial gene enhancers prior to hematoendothelial fate commitment

The lineage-determining transcription factor ETV2 is necessary and sufficient for hematoendothelial fate commitment. We investigated how ETV2-driven gene regulatory networks promote hematoendothelial fate commitment. We resolved the sequential determination steps of hematoendothelial versus cardiac differentiation from mouse embryonic stem cells. Etv2 was strongly induced and bound to the enhancers of hematoendothelial genes in a common cardiomyocyte-hematoendothelial mesoderm progenitor. However, only Etv2 itself and Tal1, not other ETV2-bound genes, were induced. Despite ETV2 genomic binding and Etv2 and Tal1 expression, cardiomyogenic fate potential was maintained. A second wave of ETV2-bound target genes was up-regulated during the transition from the common cardiomyocyte-hematoendothelial progenitor to the committed hematoendothelial population. A third wave of ETV-bound genes were subsequently expressed in the committed hematoendothelial population for sub-lineage differentiation. The shift from ETV2 binding to productive transcription, not ETV2 binding to target gene enhancers, drove hematoendothelial fate commitment. This work identifies mechanistic phases of ETV2-dependent gene expression that distinguish hematoendothelial specification, commitment, and differentiation. HIGHLIGHTSO_LIThe hematoendothelial master TF ETV2 is expressed in a multipotent mesoderm progenitor. C_LIO_LIETV2 binds to target genes in the mesoderm progenitor without target gene activation. C_LIO_LIETV2 binding in the mesoderm progenitor does not diminish alternative cardiac fate potential. C_LIO_LIETV2-bound target genes are activated upon hematoendothelial fate commitment. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/449981v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@371c3dorg.highwire.dtl.DTLVardef@1ee8e7org.highwire.dtl.DTLVardef@14ca731org.highwire.dtl.DTLVardef@1748ff7_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2021

TBX5 drives Aldh1a2 expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development

The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. We show that Tbx5 regulates an evolutionarily conserved retinoic acid (RA)-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary development. We demonstrate that Tbx5 directly maintains expression of the RA-synthesizing enzyme Aldh1a2 in the foregut lateral plate mesoderm via an intronic enhancer that is evolutionarily conserved among terrestrial vertebrates. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module and restricting FGF activity to the anterior. Tbx5/Aldh1a2-dependent RA signaling also directly activates Shh transcription in the adjacent foregut endoderm through the conserved MACS1 enhancer. Epithelial Hedgehog then signals back to the mesoderm, where together with Tbx5 it activates expression of Wnt2/2b that ultimately induce pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing cardiopulmonary evolution and birth defects. KEY FINDINGSO_LITbx5 regulates second heart field patterning and pulmonary development via retinoic acid (RA) and Hedgehog (Hh) signaling. C_LIO_LITbx5 directly maintains transcription of the RA-synthesizing enzyme Aldh1a2 in the posterior second heart field mesoderm via an evolutionarily conserved intronic enhancer. C_LIO_LIDownstream of Tbx5, RA directly promotes Shh transcription through the evolutionarily conserved MACS1 endoderm enhancer. C_LIO_LIDownstream of Tbx5, RA suppresses FGF signaling to pattern the second heart field while promoting a Hedgehog-Wnt2/2b signaling cascade that induces pulmonary fate. C_LI SUMMARY STATEMENTTbx5-dependent Retinoic Acid signaling regulates an evolutionarily conserved gene regulatory network that coordinates cardiac and pulmonary development.

developmental biology↗

bioRxiv2019

A Hedgehog-FGF signaling axis patterns anterior mesoderm during gastrulation

The application of single cell technologies to early development holds promise for resolving complex developmental phenotypes. Here we define a novel role for Hedgehog (Hh) signaling for the formation of anterior mesoderm lineages during gastrulation. Single-cell transcriptome analysis of Hh-deficient mesoderm revealed selective deficits in anterior mesoderm populations that later translate to physical defects to anterior embryonic structures including the first pharyngeal arch, heart, and anterior somites. We found that Hh-dependent anterior mesoderm defects were cell non-autonomous to Hh-signal reception. Transcriptional profiling of Hh-deficient mesoderm during gastrulation revealed disruptions to both transcriptional patterning of the mesoderm and a key FGF signaling pathway for mesoderm migration. FGF4 protein application was able to restore cellular migration during gastrulation that was decreased by Hh pathway antagonism. These findings implicate that primitive streak-mediated regulation of anterior mesoderm patterning is controlled by a multicomponent signaling hierarchy activated by Hh signaling and executed by FGF signal transduction.\n\nSIGNIFICANCE STATEMENTHow signaling events during gastrulation pattern the mesoderm is a fascinating developmental process. Although Hedgehog signaling has been implicated in early mesoderm development, its mechanistic role has not been described. We applied single cell sequencing to describe mesodermal defects in Hedgehog pathway mutants--revealing selective defects in anterior mesoderm populations. Transcriptional profiling of gastrulating Hedgehog mutants indicated that several pathways essential for primitive streak function, including FGF, required Hh signaling. Blocking Hedgehog signaling abrogated cell migration during gastrulation, which could be mitigated by addition of FGF4 ligand. This work uncovers a novel Hedgehog to FGF signaling event and describes a unique mechanism by which signals from the node impact to anterior mesoderm formation through the modulation of primitive streak function.

developmental biology↗

bioRxiv2019

Phosphorylated Lamin A targets active enhancers and is associated with abnormal transcriptional activation in progeria

LMNA encodes nuclear lamin A/C that tethers lamina-associated heterochromatin domains (LADs) to the nuclear periphery. Point mutations in LMNA cause degenerative disorders including the premature aging disorder Hutchinson-Gilford progeria, but the mechanisms are unknown. We report that Ser22-phosphorylated Lamin A/C (pS22-Lamin A/C) was localized to the interior of the nucleus in human fibroblasts throughout the cell cycle. pS22-Lamin A/C interacted with a specific subset of putative active enhancers, not LADs, primarily at locations co-bound by the transcriptional activator c-Jun. In progeria-patient fibroblasts, a subset of pS22-Lamin A/C-binding sites were lost whereas new pS22-Lamin A/C-binding sites emerged in normally quiescent loci. These new pS22-Lamin A/C-binding sites displayed increased histone acetylation and c-Jun binding, implying increased enhancer activity. The genes near these new binding sites, implicated in clinical components of progeria including carotid artery diseases, hypertension, and cardiomegaly, were upregulated in progeria. These results suggest that Lamin A/C regulates gene expression by direct enhancer binding in the nuclear interior. Disruption of the gene regulatory rather than LAD function of Lamin A/C presents a novel mechanism for disorders caused by LMNA mutations including progeria. HIGHLIGHTSO_LIpS22-Lamin A/C is present in the nuclear interior throughout interphase. C_LIO_LIpS22-Lamin A/C associates with active enhancers, not lamina-associated domains. C_LIO_LIpS22-Lamin A/C-genomic binding sites are co-bound by the transcriptional activator c-Jun. C_LIO_LINew pS22-Lamin A/C binding in progeria accompanies upregulation of disease-related genes. C_LI

genetics↗

bioRxiv2019

Enhancer transcription identifies cis-regulatory elements for photoreceptor cell types

Identification of the cis-regulatory elements (CREs) that regulate gene expression in specific cell types is critical for defining the gene regulatory networks (GRNs) that control normal physiology and disease states. We previously utilized non-coding RNA (ncRNA) profiling to define CREs that comprise a GRN in the adult mouse heart1. Here, we applied ncRNA profiling to the mouse retina in the presence and absence of Nrl, a rod photoreceptor-specific transcription factor required for rod versus cone photoreceptor cell fate. Differential expression of Nrl-dependent ncRNAs positively correlated with differential expression of Nrl-dependent local genes. Two distinct Nrl-dependent regulatory networks were discerned in parallel: Nrl-activated ncRNAs were enriched for accessible chromatin in rods but not cones whereas Nrl-repressed ncRNAs were enriched for accessible chromatin in cones but not rods. Furthermore, differential Nrl-dependent ncRNA expression levels quantitatively correlated with photoreceptor cell type-specific ATAC-seq read density. Direct assessment of Nrl-dependent ncRNA-defined loci identified functional cone photoreceptor CREs. This work supports differential ncRNA profiling as a platform for identifying context-specific regulatory elements and provides insight into the networks that define photoreceptor cell types.

molecular biology↗

bioRxiv2018

Gata4 drives Hh-signaling for second heart field migration and outflow tract development

Dominant mutations of Gata4, an essential cardiogenic transcription factor (TF), cause outflow tract (OFT) defects in both human and mouse. We investigated the molecular mechanism underlying this requirement. Gata4 happloinsufficiency in mice caused OFT defects including double outlet right ventricle (DORV) and conal ventricular septum defects (VSDs). We found that Gata4 is required within Hedgehog (Hh)-receiving second heart field (SHF) progenitors for normal OFT alignment. Increased Pten-mediated cell-cycle transition, rescued atrial septal defects but not OFT defects in Gata4 heterozygotes. SHF Hh-receiving cells failed to migrate properly into the proximal OFT cushion in Gata4 heterozygote embryos. We find that Hh signaling and Gata4 genetically interact for OFT development. Gata4 and Smo double heterozygotes displayed more severe OFT abnormalities including persistent truncus arteriosus (PTA) whereas restoration of Hedgehog signaling rescued OFT defects in Gata4-mutant mice. In addition, enhanced expression of the Gata6 was observed in the SHF of the Gata4 heterozygotes. These results suggested a SHF regulatory network comprising of Gata4, Gata6 and Hh-signaling for OFT development. This study indicates that Gata4 potentiation of Hh signaling is a general feature of Gata4-mediated cardiac morphogenesis and provides a model for the molecular basis of CHD caused by dominant transcription factor mutations.\n\nAuthor SummaryGata4 is an important protein that controls the development of the heart. Human who possess a single copy of Gata4 mutation display congenital heart defects (CHD), including the double outlet right ventricle (DORV). DORV is an alignment problem in which both the Aorta and Pulmonary Artery originate from the right ventricle, instead of originating from the left and the right ventricles, respectively. To study how Gata4 mutation causes DORV, we used a Gata4 mutant mouse model, which displays DORV. We showed that Gata4 is required in the cardiac precursor cells for the normal alignment of the great arteries. Although Gata4 mutation inhibits the rapid increase in number of the cardiac precursor cells, rescuing this defects does not recover the normal alignment of the great arteries. In addition, there is a movement problem of the cardiac precursor cells when migrating toward the great arteries during development. We further showed that a specific molecular signaling, Hh-signaling, is responsible to the Gata4 action in the cardiac precursor cells. Importantly, over-activating the Hh-signaling rescues the DORV in the Gata4 mutant embryos. This study provides an explanation for the ontogeny of CHD.

developmental biology↗

bioRxiv2018

A genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics

Biomechanical cues dynamically control major cellular processes but whether genetic variants actively participate in mechano-sensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies have identified chromosome 1p32.2 as one of the most strongly associated loci with CAD/IS; however, the causal mechanism related to this locus remains unknown. Employing statistical analyses, ATAC-seq, and H3K27ac/H3K4me2 ChIP-Seq in human aortic endothelium (HAEC), our results demonstrate that rs17114036, a common noncoding polymorphism at the 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR/Cas9-based genome editing shows that rs17114036-containing region promotes endothelial quiescence under unidirectional flow by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays further demonstrate that CAD/IS protective allele at rs17114036 in PLPP3 intron 5 confers an increased endothelial enhancer activity. ChIPPCR and luciferase assays show that CAD/IS protective allele at rs17114036 creates a binding site for transcription factor Kruppel-like factor 2, which increases the enhancer activity under unidirectional flow. These results demonstrate for the first time that a human single-nucleotide polymorphism contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related cisregulatory elements provide a previously unappreciated layer of regulatory control in cellular mechano-sensing mechanisms.\n\nSignificance StatementBiomechanical stimuli control major cellular functions and play critical roles in the pathogenesis of diverse human diseases. Although recent studies have implicated genetic variation in regulating key biological processes, whether human genetic variants contribute to the cellular mechano-sensing mechanisms remains unclear. This study provides the first line of evidence supporting an underappreciated role of genetic predisposition in cellular mechanotransduction mechanisms. Employing epigenomic profiling, genome-editing, and latest human genetics approaches, our data demonstrate that rs17114036, a common noncoding polymorphism implicated in coronary artery disease and ischemic stroke by genome-wide association studies, dynamically regulates endothelial responses to blood flow (hemodynamics) related to atherosclerosis via regulation of an intronic enhancer. The results provide new molecular insights linking disease-associated genetic variants to cellular mechanobiology.

genomics↗

bioRxiv2018

A promoter interaction map for cardiovascular disease genetics

Over 500 genetic loci have been associated with risk of cardiovascular diseases (CVDs), however most loci are located in gene-distal non-coding regions and their target genes are not known. Here, we generated high-resolution promoter capture Hi-C (PCHi-C) maps in human induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes (CMs) to provide a resource for identifying and prioritizing the functional targets of CVD associations. We validate these maps by demonstrating that promoters preferentially contact distal sequences enriched for tissue-specific transcription factor motifs and are enriched for chromatin marks that correlate with dynamic changes in gene expression. Using the CM PCHi-C map, we linked 1,999 CVD-associated SNPs to 347 target genes. Remarkably, more than 90% of SNP-target gene interactions did not involve the nearest gene, while 40% of SNPs interacted with at least two genes, demonstrating the importance of considering long-range chromatin interactions when interpreting functional targets of disease loci.

genetics↗

bioRxiv2018

Depolymerized lamins link nuclear envelope breakdown to mitotic transcriptional quiescence

The nuclear envelope, a defining feature of eukaryotic cells, restricts DNA-dependent processes including gene transcription to the nucleus. The nuclear lamina is an integral component of the animal nuclear envelope, composed of polymers of nuclear lamin proteins1,2. Upon mitosis, the nuclear lamina disassembles, the nuclear envelope breaks down, and transcription becomes quiescent3,4. We report here a direct molecular link between nuclear lamina disassembly and mitotic transcriptional quiescence. We found that, at the G2 cell-cycle phase immediately preceding mitosis, nuclear lamin A/C (LMNA) became phosphorylated at Ser22 and depolymerized from the nuclear lamina. Depolymerized LMNA accumulated in the nuclear interior and physically associated with active cis-regulatory elements genome-wide. Depolymerized LMNA-associated sites were overrepresented near genes repressed by LMNA, suggesting that depolymerized LMNA participates in transcriptional repression at G2. Consistently, depolymerized LMNA-target genes underwent a steep expression decline from S to G2/M. Furthermore, LMNA deletion caused inappropriate RNA Polymerase II (Pol II) accumulation downstream of Pol II pause sites at promoters and enhancers genome-wide, leading to inappropriate and excessive transcriptional elongation. A subset of depolymerized LMNA-target genes were upregulated in fibroblasts of patients with Hutchinson-Gilford progeria, a premature aging disorder caused by LMNA mutations5, raising the possibility that defects in depolymerized LMNA-mediated mitotic transcriptional quiescence contribute to progeria pathogenesis. These observations support a model in which depolymerized LMNA targets active regulatory elements to promote RNA Pol II pausing preceding mitosis, coupling nuclear envelope breakdown to mitotic transcriptional quiescence.

molecular biology↗

bioRxiv2018

Hedgehog signaling controls progenitor differentiation timing during heart development

Heterochrony, defined as differences in the timing of developmental processes, impacts organ development, homeostasis, and regeneration. The molecular basis of heterochrony in mammalian tissues is poorly understood. We report that Hedgehog signaling activates a heterochronic pathway that controls differentiation timing in multiple lineages. A differentiation trajectory from second heart field cardiac progenitors to first heart field cardiomyocytes was identified by single-cell transcriptional profiling in mouse embryos. A survey of developmental signaling pathways revealed specific enrichment for Hedgehog signaling targets in cardiac progenitors. Removal of Hh signaling caused loss of progenitor and precocious cardiomyocyte differentiation gene expression in the second heart field in vivo. Introduction of active Hh signaling to mESC-derived progenitors, modelled by transient expression of the Hh-dependent transcription factor GLI1, delayed differentiation in cardiac and neural lineages in vitro. A shared GLI1-dependent network in both cardiac and neural progenitors was enriched with FOX family transcription factors. FOXF1, a GLI1 target, was sufficient to delay onset of the cardiomyocyte differentiation program in progenitors, by epigenetic repression of cardiomyocyte-specific enhancers. Removal of active Hh signaling or Foxf1 expression from second heart field progenitors caused precocious cardiac differentiation in vivo, establishing a mechanism for resultant Congenital Heart Disease. Together, these studies suggest that Hedgehog signaling directly activates a gene regulatory network that functions as a heterochronic switch to control differentiation timing across developmental lineages.

developmental biology↗