Hedgehog signaling activates a heterochronic gene regulatory network to control differentiation timing across lineages

Abstract

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.

LRP2 controls sonic hedgehog-dependent differentiation of cardiac progenitor cells during outflow tract formation

“…Data presented herein argue that LRP2 acts as agonist to promote responsiveness of SHF progenitor cells to SHH signals during OFT formation. This hypothesis is supported by a recent study documenting a crucial role for SHH in activating progenitor gene expression and in inhibiting premature differentiation, thereby maintaining the progenitor cell population in the SHF (58). We also show that LRP2 ensures the correct migration pattern of SHH-responsive SHF progenitors during OFT elongation.…”

WITHDRAWN: The cardiac lncRNA Chantico directly regulatesCxcl1chemokine transcription

“…Since the influence of Tbx5 in Chantico-Cxcl1 gene regulation is absent in naïve mESCs but critical in mature cardiomyocytes, we evaluated the regulation of Chantico and Cxcl1 through discrete stages of cardiac differentiation. To accomplish this, we directed the differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes (mESC-CMs) (Kattman et al, 2011;Rowton et al, 2020) and measured chromatin accessibility by ATAC-seq (Buenrostro et al, 2013) every 24 hours during differentiation and RNA abundance by RT-qPCR (Figure 5C-D).…”

“…A2Lox.cre mES cells were maintained and differentiated into cardiomyocytes as previously described (Kattman et al, 2011;Rowton et al, 2020). A2Lox.cre mES cells derive from E14Tg2a.4, which are male.…”

“…A2Lox.cre mES cells were maintained and differentiated into cardiomyocytes as previously described (Kattman et al, 2011;Rowton et al, 2020). RNA was harvested from two replicate samples every 24 hours of the differentiation from the mESC (2i) stage to D8, in addition to D10 and D15 (cardiomyocyte stage).…”

Gene-teratogen interactions influence the penetrance of birth defects by altering Hedgehog signaling strength

“…All of the Mosmo -/- embryos had conotruncal heart defects, most commonly Transposition of the Great Arteries (TGA) ( Figure 3A and Table S2 ). Hh signaling plays a critical role in multiple aspects of outflow tract development including: (1) maintenance of cardiac progenitor proliferation and identity within the secondary heart field (which contributes to the outflow tract) (Dyer and Kirby, 2009; Rowton et al, 2020), (2) survival of migratory cardiac neural crest cells (Washington Smoak et al, 2005), and (3) proper septation of the outflow tract (Goddeeris et al, 2007). To determine if the conotruncal heart defects observed in the Mosmo -/- embryos are due to elevated Hh signaling, we first needed to identify the critical time window during gestation when outflow tract development is sensitive to vismodegib.…”