Control of cardiomyocyte differentiation timing by intercellular signaling pathways.

Congenital Heart Disease (CHD), malformations of the heart present at birth, is the most common class of life-threatening birth defect (Hoffman (1995) [1], Gelb (2004) [2], Gelb (2014) [3]). A major research challenge is to elucidate the genetic determinants of CHD and mechanistically link CHD ontogeny to a molecular understanding of heart development. Although the embryonic origins of CHD are unclear in most cases, dysregulation of cardiovascular lineage specification, patterning, proliferation, migration or differentiation have been described (Olson (2004) [4], Olson (2006) [5], Srivastava (2006) [6], Dunwoodie (2007) [7], Bruneau (2008) [8]). Cardiac differentiation is the process whereby cells become progressively more dedicated in a trajectory through the cardiac lineage towards mature cardiomyocytes. Defects in cardiac differentiation have been linked to CHD, although how the complex control of cardiac differentiation prevents CHD is just beginning to be understood. The stages of cardiac differentiation are highly stereotyped and have been well-characterized (Kattman et al. (2011) [9], Wamstad et al. (2012) [10], Luna-Zurita et al. (2016) [11], Loh et al. (2016) [12], DeLaughter et al. (2016) [13]); however, the developmental and molecular mechanisms that promote or delay the transition of a cell through these stages have not been as deeply investigated. Tight temporal control of progenitor differentiation is critically important for normal organ size, spatial organization, and cellular physiology and homeostasis of all organ systems (Raff et al. (1985) [14], Amthor et al. (1998) [15], Kopan et al. (2014) [16]). This review will focus on the action of signaling pathways in the control of cardiomyocyte differentiation timing. Numerous signaling pathways, including the Wnt, Fibroblast Growth Factor, Hedgehog, Bone Morphogenetic Protein, Insulin-like Growth Factor, Thyroid Hormone and Hippo pathways, have all been implicated in promoting or inhibiting transitions along the cardiac differentiation trajectory. Gaining a deeper understanding of the mechanisms controlling cardiac differentiation timing promises to yield insights into the etiology of CHD and to inform approaches to restore function to damaged hearts.

Hedgehog-FGF signaling axis patterns anterior mesoderm during gastrulation.

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-specific Fgf4/Fgf8 double-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.

Gata4 potentiates second heart field proliferation and Hedgehog signaling for cardiac septation.

GATA4, an essential cardiogenic transcription factor, provides a model for dominant transcription factor mutations in human disease. Dominant GATA4 mutations cause congenital heart disease (CHD), specifically atrial and atrioventricular septal defects (ASDs and AVSDs). We found that second heart field (SHF)-specific Gata4 heterozygote embryos recapitulated the AVSDs observed in germline Gata4 heterozygote embryos. A proliferation defect of SHF atrial septum progenitors and hypoplasia of the dorsal mesenchymal protrusion, rather than anlage of the atrioventricular septum, were observed in this model. Knockdown of the cell-cycle repressor phosphatase and tensin homolog (Pten) restored cell-cycle progression and rescued the AVSDs. Gata4 mutants also demonstrated Hedgehog (Hh) signaling defects. Gata4 acts directly upstream of Hh components: Gata4 activated a cis-regulatory element at Gli1 in vitro and occupied the element in vivo. Remarkably, SHF-specific constitutive Hh signaling activation rescued AVSDs in Gata4 SHF-specific heterozygous knockout embryos. Pten expression was unchanged in Smoothened mutants, and Hh pathway genes were unchanged in Pten mutants, suggesting pathway independence. Thus, both the cell-cycle and Hh-signaling defects caused by dominant Gata4 mutations were required for CHD pathogenesis, suggesting a combinatorial model of disease causation by transcription factor haploinsufficiency.

Evolutionarily new genes in humans with disease phenotypes reveal functional enrichment patterns shaped by adaptive innovation and sexual selection.

New genes (or young genes) are structural novelties pivotal in mammalian evolution. Their phenotypic impacts on humans, however, remain elusive due to the technical and ethical complexities in functional studies. Through combining gene age dating with Mendelian disease phenotyping, our research reveals a steady integration of new genes with biomedical phenotypes into the human genome over macroevolutionary timescales (~0.07% per million years). Despite this stable pace, we observe distinct patterns in phenotypic enrichment, pleiotropy, and selective pressures shaped by different gene ages. Notably, young genes show significant enrichment in the male reproductive system, indicating strong sexual selection. Young genes also exhibit functions in tissues and systems potentially linked to human phenotypic innovations, such as increased brain size, musculoskeletal phenotypes, and color vision. Our findings further reveal increasing levels of pleiotropy over evolutionary time, which accompanies stronger selective constraints. We propose a "pleiotropy-barrier" model that delineates different potentials for phenotypic innovation between young and older genes subject to natural selection. Our study demonstrates that evolutionary new genes are critical in influencing human reproductive evolution and adaptive phenotypic innovations driven by sexual and natural selection, with low pleiotropy as a selective advantage.

Hedgehog signaling activates a mammalian heterochronic gene regulatory network controlling differentiation timing across lineages.

Many developmental signaling pathways have been implicated in lineage-specific differentiation; however, mechanisms that explicitly control differentiation timing remain poorly defined in mammals. We report that murine Hedgehog signaling is a heterochronic pathway that determines the timing of progenitor differentiation. Hedgehog activity was necessary to prevent premature differentiation of second heart field (SHF) cardiac progenitors in mouse embryos, and the Hedgehog transcription factor GLI1 was sufficient to delay differentiation of cardiac progenitors in vitro. GLI1 directly activated a de novo progenitor-specific network in vitro, akin to that of SHF progenitors in vivo, which prevented the onset of the cardiac differentiation program. A Hedgehog signaling-dependent active-to-repressive GLI transition functioned as a differentiation timer, restricting the progenitor network to the SHF. GLI1 expression was associated with progenitor status across germ layers, and it delayed the differentiation of neural progenitors in vitro, suggesting a broad role for Hedgehog signaling as a heterochronic pathway.