Hedgehog signaling controls progenitor differentiation timing during heart development

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.