The mechanisms used by embryos to pattern tissues across their axes has fascinated developmental biologists since the founding of embryology. Here, using single-cell technology, we interrogate complex patterning defects and define a Hedgehog (Hh)–fibroblast growth factor (FGF) signaling axis required for anterior mesoderm lineage development during gastrulation. Single-cell transcriptome analysis of Hh-deficient mesoderm revealed selective deficits in anterior mesoderm populations, culminating in defects to anterior embryonic structures, including the pharyngeal arches, heart, and anterior somites. Transcriptional profiling of Hh-deficient mesoderm during gastrulation revealed disruptions to both transcriptional patterning of the mesoderm and FGF signaling for mesoderm migration. Mesoderm-specificFgf4/Fgf8double-mutants recapitulated anterior mesoderm defects and Hh-dependent GLI transcription factors modulated enhancers at FGF gene loci. Cellular migration defects during gastrulation induced by Hh pathway antagonism were mitigated by the addition of FGF4 protein. These findings implicate a multicomponent signaling hierarchy activated by Hh ligands from the embryonic node and executed by FGF signals in nascent mesoderm to control anterior mesoderm patterning.

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Hedgehog (Hh) signaling acts as a developmental morphogen that contributes to the diversification of cell fates and tissue patterning in multiple embryonic contexts, as well as regulating the proliferation of adult tissue stem cells1-9. Here, we report a novel function of Hh signaling GLI transcription factors (TFs) in directly governing the timing of cellular differentiation, independent of a role in specification or proliferation. Disruption of active Hh signaling in the embryo resulted in reduced expression of a progenitor-specific transcription factor network and the inappropriate activation of cardiac differentiation-specific gene expression programs. Expression of the activating Hh transcription factor, GLI1, a marker and effector of active Hh signaling, is correlated with stem and progenitor states during the differentiation of all three germ layers in mouse and human. Transient induction of GLI1 in mouse embryonic stem cell (mESC)-derived cardiac and neural progenitors delayed the onset of the cardiomyocyte and neuron differentiation programs, respectively, while activating progenitor-specific gene expression. GLI1 expression in cardiac progenitors promoted a shift in chromatin accessibility towards a progenitor-like profile at distal regulatory elements near Hh-dependent genes. Manipulating the balance of active to repressive GLI TF predominance unveiled a molecular switch that determined the activity patterns of progenitor-specific distal cis-regulatory elements in vitro and in vivo. Overriding this switch through forced expression of a repressive GLI TF in cardiac progenitors in vivo caused precocious cardiomyocyte differentiation and Congenital Heart Disease (CHD). Our data suggest that a GLI TF switch at distal regulatory elements maintains progenitor cell status and inhibits premature differentiation through the activation and maintenance of a progenitor-specific regulatory network, thus controlling progenitor differentiation timing. We propose a novel molecular paradigm for progenitor maintenance in diverse cellular contexts by signal-dependent TFs with implications for organ development, regenerative potential, and Hh-driven cancers.

The first heart field (FHF) and the second heart field (SHF) comprise the major progenitor pools for the developing vertebrate heart. We investigated the delayed differentiation of the SHF relative to the FHF, a major distinguishing feature of these fields. Single-cell transcriptional profiling of the SHF revealed a differentiation trajectory of SHF progenitors to cardiomyocytes. Hedgehog (Hh) signaling was highly enriched in the progenitor state in signaling pathway analysis, suggesting a possible role in cardiomyocyte differentiation control. Transcriptional profiling of the SHF following removal of active Hh signaling in vivo revealed inappropriate cardiomyocyte gene expression. We observed precocious cardiomyocyte differentiation in the SHF in vivo in Hh mutants, which led to Congenital Heart Disease (CHD). Modeling active Hh signaling in a novel mouse embryonic stem cell (mESC) line through transient expression of the activating Hh transcription factor (TF), GLI1, in cardiac progenitors delayed the onset of cardiomyocyte differentiation. GLI1 directly activated a progenitor-specific gene regulatory network, dominated by repressive TFs, that prevented the acquisition of the cardiomyocyte gene regulatory network. Maintained expression of one GLI1 target TF, FOXF1, repressed the cardiomyocyte differentiation program. FOXF1 binding sites were identified at putative regulatory elements near repressed cardiac genes involved in contraction, electrical impulse propagation and transcriptional regulation. Finally, FOXF1 repressed the activity of these elements in vitro , indicating that FOXF1 can directly repress the activation of genes essential for cardiomyocyte differentiation. Together, these results indicate that a Hh-dependent gene regulatory network including transcriptional repressors directly delays the onset of cardiomyocyte gene expression to delay SHF differentiation. Abrogation of Hh signaling and the resultant premature differentiation of cardiac progenitors provides a molecular mechanism for the ontogeny of some CHD.

During development, the enteric nervous system (ENS) arises from neural crest cells that emerge from the neural tube, migrate to and along the gut, and colonize the entire intestinal tract. While much of the ENS arises from vagal neural crest cells that originate from the caudal hindbrain, there is a second contribution from the sacral neural crest that migrates from the caudal end of the spinal cord to populate the post-umbilical gut. By coupling single cell transcriptomics with axial-level specific lineage tracing in avian embryos, we compared the contributions between embryonic vagal and sacral neural crest cells to the ENS. The results show that the two neural crest populations form partially overlapping but also complementary subsets of neurons and glia in distinct ganglionic units. In particular, the sacral neural crest cells appear to be the major source of adrenergic/dopaminergic and serotonergic neurons, melanocytes and Schwann cells in the post-umbilical gut. In addition to neurons and glia, the results also reveal sacral neural crest contributions to connective tissue and mesenchymal cells of the gut. These findings highlight the specific properties of the sacral neural crest population in the hindgut and have potential implications for understanding development of the complex nervous system in the hindgut environment that may influence congenital neuropathies.