Ciliary Hedgehog signaling patterns the digestive system to generate mechanical forces driving elongation

The mechanisms underlying tubular organ elongation remain poorly understood. Here, the authors show that primary cilia interpret Hedgehog signals to pattern the developing gut and that smooth muscle in the gut wall generates mechanical forces that direct longitudinal growth. How tubular organs elongate is poorly understood. We found that attenuated ciliary Hedgehog signaling in the gut wall impaired patterning of the circumferential smooth muscle and inhibited proliferation and elongation of developing intestine and esophagus. Similarly, ablation of gut-wall smooth muscle cells reduced lengthening. Disruption of ciliary Hedgehog signaling or removal of smooth muscle reduced residual stress within the gut wall and decreased activity of the mechanotransductive effector YAP. Removing YAP in the mesenchyme also reduced proliferation and elongation, but without affecting smooth muscle formation, suggesting that YAP interprets the smooth muscle-generated force to promote longitudinal growth. Additionally, we developed an intestinal culture system that recapitulates the requirements for cilia and mechanical forces in elongation. Pharmacologically activating YAP in this system restored elongation of cilia-deficient intestines. Thus, our results reveal that ciliary Hedgehog signaling patterns the circumferential smooth muscle to generate radial mechanical forces that activate YAP and elongate the gut.Peer reviewe

Deciphering transcriptional and epigenomic regulation of early cardiogenesis

Transcriptional networks governing early cardiac precursor cell (CPC) specification are incompletely understood due in part to the difficulty of distinguishing CPCs from their mesoderm germ layer of origin in early gastrulation. Cardiogenesis in the gastrulating embryo begins when mesoderm progenitor cells emerge from the primitive streak and migrate anterior-laterally to coalesce at the anterior midline. Errors during CPC specification and patterning can cause devastating Congenital Heart Defects (CHDs). Occurring in 1-2% of live births, CHDs often require surgical interventions and can result in secondary heart disease. The genetic etiology of CHDs indicates that genes encoding transcription factors (TFs) are overrepresented as causative and are predominantly haploinsufficient, indicating that fine dysregulation of gene expression is a driving mechanism for disease. Thus, understanding the transcriptional regulatory networks governing early cardiac specification is paramount for understanding CHDs and necessary to develop novel therapeutic strategies. Our comprehension of transcriptional regulation at the initiation of cardiogenesis is hindered in part by the paucity of molecular tools capable of distinguishing the emerging cardiac lineage from the surrounding mesoderm. Prior studies leveraged lineage tracing from the basic-helix-loop-helix (bHLH) TF Mesp1, however as this lineage contributes to other mesodermal derivatives beyond the heart the method is insufficient for isolation of early CPCs. To overcome this challenge and investigate the cardiac lineage distinctly from the surrounding mesoderm, we leveraged a pan-cardiac enhancer transgene reporter, Smarcd3-F6, that restrictively marks emerging, early CPC populations within the mesoderm. We utilized bioinformatic detection of fluorescent reporter transgenes tracking both the Mesp1 lineage and Smarcd3-F6 expression in whole embryo single cell transcriptomic data to interrogate the heterogeneity of CPC transcriptional profiles in an in vivo mouse gastrulation time course. The dataset we generated towards this goal represents a valuable resource for investigations of the early cardiac mesoderm and for broader questions of cell fate allocation from germ layers during gastrulation. We further leveraged the Smarcd3-F6 enhancer sequence as an experimental discovery platform for the identification of regulatory network logic during early cardiogenesis. We identified specific GATA and T-box motif sites necessary for a minimal Smarcd3-F6 sub-region’s enhancer activity. This in vivo enhancer study provides a framework for functional characterization of transcriptional regulatory networks during development. Lastly, we utilized single cell transcriptomic and chromatin accessibility sequencing to define the resilience and vulnerability of cardiac specification in embryos deficient for Mesp1, the early-expressed and often-posited ‘cardiac master regulator’. Our results distinguish Mesp1-independent and dependent processes in early cardiogenesis, showing that Mesp1 deficient CPCs progress through cardiogenesis until lateral plate mesoderm stages, at which point their disrupted regulatory landscape prohibits maturation further into patterned cardiac progenitor and cardiomyocyte fates. Collectively, these results illustrate the complex transcriptional and epigenomic interdependence of regulatory networks during lineage specification and further advance our fundamental understanding of the processes governing cardiac specification in vivo at single cell resolution. The investigative frameworks and the interpretations of findings described in this dissertation illuminate generalizable principles for the regulatory logic guiding the allocation and subsequent differentiation of precursor cells towards distinct, functional cell types during gastrulation

Hedgehog signaling drives medulloblastoma growth via CDK6

Medulloblastoma, an aggressive cancer of the cerebellum, is among the most common pediatric brain tumors. Approximately one-third of medulloblastomas are associated with misactivation of the Hedgehog (Hh) pathway. GLI family zinc finger 2 (GLI2) coordinates the Hh transcriptional program; however, the GLI2 targets that promote cancer cell proliferation are unknown. Here, we incorporated a Gli2-EGFP allele into 2 different genetic mouse models of Hh-associated medulloblastoma. Hh signaling induced GLI2 binding to the Cdk6 promoter and activated Cdk6 expression, thereby promoting uncontrolled cell proliferation. Genetic or pharmacological inhibition of CDK6 in mice repressed the growth of Hh-associated medulloblastoma and prolonged survival through inhibition of cell proliferation. In human medulloblastoma, misactivation of Hh signaling was associated with high levels of CDK6, pointing to CDK6 as a direct transcriptional target of the Hh pathway. These results suggest that CDK6 antagonists may be a promising therapeutic approach for Hh-associated medulloblastoma in humans

Cilia-associated oxysterols activate smoothened

Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11β2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.This work was supported by grants from the NIH (HL007731 and CA212279-01), the UCSF Physician Scientist Scholar Program, the American Society of Clinical Oncology, the Rally Foundation for Childhood Cancer Research, and the American Brain Tumor Association to D.R.R.; the NIH program Project Grant to Molecular Genetics (HL20948) to J.G.M.; Cancer Research UK (C20724 and A14414) and the European Research Council (647278) to C.S.; the NIH (AR065409 and HD092659) to S.Y.W. and L.X., respectively; the NIH (R01GM102498), the Ludwig Cancer Institute, and the Howard Hughes Medical Institute to P.A.B.; and the NIH (AR054396 and GM095941), the Burroughs Wellcome Fund, and the Packard Foundation to J.F.R.Peer reviewe