The BAF chromatin complex component SMARCC1 does not mediate GLI transcriptional repression of Hedgehog target genes in limb buds.

Transcriptional responses to the Hedgehog (HH) signaling pathway are primarily modulated by GLI repression in the mouse limb. Previous studies suggested a role for the BAF chromatin remodeling complex in mediating GLI repression. Consistent with this possibility, the core BAF complex protein SMARCC1 is present at most active limb enhancers including the majority of GLI enhancers. However, in contrast to GLI repression which reduces chromatin accessibility, SMARCC1 maintains chromatin accessibility at most enhancers, including those bound by GLI. Moreover, SMARCC1 binding at GLI-regulated enhancers occurs independently of GLI3. Consistent with previous studies, some individual GLI target genes are mis-regulated in Smarcc1 conditional knockouts, though most GLI target genes are unaffected. Moreover, SMARCC1 is not necessary for mediating constitutive GLI repression in HH mutant limb buds. We conclude that SMARCC1 does not mediate GLI3 repression, which we propose utilizes alternative chromatin remodeling complexes.

The BAF chromatin complex component SMARCC1 does not mediate GLI transcriptional repression of Hedgehog target genes in limb buds.

Transcriptional responses to the Hedgehog (HH) signaling pathway are primarily modulated by GLI repression in the mouse limb. Previous studies suggested a role for the BAF chromatin remodeling complex in mediating GLI repression. Consistent with this possibility, the core BAF complex protein SMARCC1 is present at most active limb enhancers including the majority of GLI enhancers. However, in contrast to GLI repression which reduces chromatin accessibility, SMARCC1 maintains chromatin accessibility at most enhancers, including those bound by GLI. Moreover, SMARCC1 binding at GLI-regulated enhancers occurs independently of GLI3. Consistent with previous studies, some individual GLI target genes are mis-regulated in Smarcc1 conditional knockouts, though most GLI target genes are unaffected. Moreover, SMARCC1 is not necessary for mediating constitutive GLI repression in HH mutant limb buds. We conclude that SMARCC1 does not mediate GLI3 repression, which we propose utilizes alternative chromatin remodeling complexes.

Hedgehog regulation of epithelial cell state and morphogenesis in the larynx.

The larynx enables speech while regulating swallowing and respiration. Larynx function hinges on the laryngeal epithelium which originates as part of the anterior foregut and undergoes extensive remodeling to separate from the esophagus and form vocal folds that interface with the adjacent trachea. Here we find that sonic hedgehog (SHH) is essential for epithelial integrity in the mouse larynx as well as the anterior foregut. During larynx-esophageal separation, low Shh expression marks specific domains of actively remodeling epithelium that undergo an epithelial-to-mesenchymal transition (EMT) characterized by the induction of N-Cadherin and movement of cells out of the epithelial layer. Consistent with a role for SHH signaling in regulating this process, Shh mutants undergo an abnormal EMT throughout the anterior foregut and larynx, marked by a cadherin switch, movement out of the epithelial layer and cell death. Unexpectedly, Shh mutant epithelial cells are replaced by a new population of FOXA2-negative cells that likely derive from adjacent pouch tissues and form a rudimentary epithelium. These findings have important implications for interpreting the etiology of HH-dependent birth defects within the foregut. We propose that SHH signaling has a default role in maintaining epithelial identity throughout the anterior foregut and that regionalized reductions in SHH trigger epithelial remodeling.

Timing is everything: Transcriptional repression is not the default mode for regulating Hedgehog signaling.

Hedgehog (HH) signaling is a conserved pathway that drives developmental growth and is essential for the formation of most organs. The expression of HH target genes is regulated by a dual switch mechanism where GLI proteins function as bifunctional transcriptional activators (in the presence of HH signaling) and transcriptional repressors (in the absence of HH signaling). This results in a tight control of GLI target gene expression during rapidly changing levels of pathway activity. It has long been presumed that GLI proteins also repress target genes prior to the initial expression of HH in a given tissue. This idea forms the basis for the limb bud pre-patterning model for regulating digit number. Recent findings indicate that GLI repressor proteins are indeed present prior to HH signaling but contrary to this model, GLI proteins are inert as they do not regulate transcriptional responses or enhancer chromatin modifications at this time. These findings suggest that GLI transcriptional repressor activity is not a default state as assumed, but is itself regulated in an unknown fashion. We discuss these findings and their implications for understanding pre-patterning, digit regulation, and HH-driven disease.

A limb bud morphogen bites the dust.

Hedgehog signaling has traditionally been considered to be a morphogen for digits. In this issue of Developmental Cell, Zhu et al. show that a brief exposure to Sonic Hedgehog is sufficient for digit specification, and this finding suggests that it is not acting as a direct morphogen but rather as an initiator of this process.

GLI transcriptional repression is inert prior to Hedgehog pathway activation.

The Hedgehog (HH) pathway regulates a spectrum of developmental processes through the transcriptional mediation of GLI proteins. GLI repressors control tissue patterning by preventing sub-threshold activation of HH target genes, presumably even before HH induction, while lack of GLI repression activates most targets. Despite GLI repression being central to HH regulation, it is unknown when it first becomes established in HH-responsive tissues. Here, we investigate whether GLI3 prevents precocious gene expression during limb development. Contrary to current dogma, we find that GLI3 is inert prior to HH signaling. While GLI3 binds to most targets, loss of Gli3 does not increase target gene expression, enhancer acetylation or accessibility, as it does post-HH signaling. Furthermore, GLI repression is established independently of HH signaling, but after its onset. Collectively, these surprising results challenge current GLI pre-patterning models and demonstrate that GLI repression is not a default state for the HH pathway.

GLI transcriptional repression regulates tissue-specific enhancer activity in response to Hedgehog signaling.

Transcriptional repression needs to be rapidly reversible during embryonic development. This extends to the Hedgehog pathway, which primarily serves to counter GLI repression by processing GLI proteins into transcriptional activators. In investigating the mechanisms underlying GLI repression, we find that a subset of GLI binding regions, termed HH-responsive enhancers, specifically loses acetylation in the absence of HH signaling. These regions are highly enriched around HH target genes and primarily drive HH-specific transcriptional activity in the mouse limb bud. They also retain H3K27ac enrichment in limb buds devoid of GLI activator and repressor, indicating that their activity is primarily regulated by GLI repression. Furthermore, the Polycomb repression complex is not active at most of these regions, suggesting it is not a major mechanism of GLI repression. We propose a model for tissue-specific enhancer activity in which an HDAC-associated GLI repression complex regulates target genes by altering the acetylation status at enhancers.

Regulation of terminal hypertrophic chondrocyte differentiation in Prmt5 mutant mice modeling infantile idiopathic scoliosis.

Idiopathic scoliosis (IS) is the most common type of musculoskeletal defect affecting children worldwide, and is classified by age of onset, location and degree of spine curvature. Although rare, IS with onset during infancy is the more severe and rapidly progressive form of the disease, associated with increased mortality due to significant respiratory compromise. The pathophysiology of IS, in particular for infantile IS, remains elusive. Here, we demonstrate the role of PRMT5 in the infantile IS phenotype in mouse. Conditional genetic ablation of PRMT5 in osteochondral progenitors results in impaired terminal hypertrophic chondrocyte differentiation and asymmetric defects of endochondral bone formation in the perinatal spine. Analysis of these several markers of endochondral ossification revealed increased type X collagen (COLX) and Ihh expression, coupled with a dramatic reduction in Mmp13 and RUNX2 expression, in the vertebral growth plate and in regions of the intervertebral disc in the Prmt5 conditional mutant mice. We also demonstrate that PRMT5 has a continuous role in the intervertebral disc and vertebral growth plate in adult mice. Altogether, our results establish PRMT5 as a critical promoter of terminal hypertrophic chondrocyte differentiation and endochondral bone formation during spine development and homeostasis.This article has an associated First Person interview with the first author of the paper.

PRMT5 is necessary to form distinct cartilage identities in the knee and long bone.

During skeletal development, limb progenitors become specified as chondrocytes and subsequently differentiate into specialized cartilage compartments. We previously showed that the arginine dimethyl transferase, PRMT5, is essential for regulating the specification of progenitor cells into chondrocytes within early limb buds. Here, we report that PRMT5 regulates the survival of a separate progenitor domain that gives rise to the patella. Independent of its role in knee development, PRMT5 regulates several distinct types of chondrocyte differentiation within the long bones. Chondrocytes lacking PRMT5 have a striking blockage in hypertrophic chondrocyte differentiation and are marked by abnormal gene expression. PRMT5 remains important for articular cartilage and hypertrophic cell identity during adult stages, indicating an ongoing role in homeostasis of these tissues. We conclude that PRMT5 is required for distinct steps of early and late chondrogenic specialization and is thus a critical component of multiple aspects of long bone development and maintenance.

Correction: Foxf Genes Integrate Tbx5 and Hedgehog Pathways in the Second Heart Field for Cardiac Septation.

[This corrects the article DOI: 10.1371/journal.pgen.1004604.].

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud.

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4 Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis.

An interdigit signalling centre instructs coordinate phalanx-joint formation governed by 5'Hoxd-Gli3 antagonism.

The number of phalanges and joints are key features of digit 'identity' and are central to limb functionality and evolutionary adaptation. Prior chick work indicated that digit phalanges and their associated joints arise in a different manner than the more sparsely jointed long bones, and their identity is regulated by differential signalling from adjacent interdigits. Currently, there is no genetic evidence for this model, and the molecular mechanisms governing digit joint specification remain poorly understood. Using genetic approaches in mouse, here we show that functional 5'Hoxd-Gli3 antagonism acts indirectly, through Bmp signalling from the interdigital mesenchyme, to regulate specification of joint progenitors, which arise in conjunction with phalangeal precursors at the digit tip. Phalanx number, although co-regulated, can be uncoupled from joint specification. We propose that 5'Hoxd genes and Gli3 are part of an interdigital signalling centre that sets net Bmp signalling levels from different interdigits to coordinately regulate phalanx and joint formation.

The Pluripotency Factor NANOG Binds to GLI Proteins and Represses Hedgehog-mediated Transcription.

The Hedgehog (HH) signaling pathway is essential for the maintenance and response of several types of stem cells. To study the transcriptional response of stem cells to HH signaling, we searched for proteins binding to GLI proteins, the transcriptional effectors of the HH pathway in mouse embryonic stem (ES) cells. We found that both GLI3 and GLI1 bind to the pluripotency factor NANOG. The ectopic expression of NANOG inhibits GLI1-mediated transcriptional responses in a dose-dependent fashion. In differentiating ES cells, the presence of NANOG reduces the transcriptional response of cells to HH. Finally, we found thatGli1andNanogare co-expressed in ES cells at high levels. We propose that NANOG acts as a negative feedback component that provides stem cell-specific regulation of the HH pathway.

Turning publicly available gene expression data into discoveries using gene set context analysis.

Gene Set Context Analysis (GSCA) is an open source software package to help researchers use massive amounts of publicly available gene expression data (PED) to make discoveries. Users can interactively visualize and explore gene and gene set activities in 25,000+ consistently normalized human and mouse gene expression samples representing diverse biological contexts (e.g. different cells, tissues and disease types, etc.). By providing one or multiple genes or gene sets as input and specifying a gene set activity pattern of interest, users can query the expression compendium to systematically identify biological contexts associated with the specified gene set activity pattern. In this way, researchers with new gene sets from their own experiments may discover previously unknown contexts of gene set functions and hence increase the value of their experiments. GSCA has a graphical user interface (GUI). The GUI makes the analysis convenient and customizable. Analysis results can be conveniently exported as publication quality figures and tables. GSCA is available at https://github.com/zji90/GSCA. This software significantly lowers the bar for biomedical investigators to use PED in their daily research for generating and screening hypotheses, which was previously difficult because of the complexity, heterogeneity and size of the data.

Spatiotemporal regulation of GLI target genes in the mammalian limb bud.

GLI proteins convert Sonic hedgehog (Shh) signaling into a transcriptional output in a tissue-specific fashion. The Shh pathway has been extensively studied in the limb bud, where it helps regulate growth through a SHH-FGF feedback loop. However, the transcriptional response is still poorly understood. We addressed this by determining the gene expression patterns of approximately 200 candidate GLI-target genes and identified three discrete SHH-responsive expression domains. GLI-target genes expressed in the three domains are predominately regulated by derepression of GLI3 but have different temporal requirements for SHH. The GLI binding regions associated with these genes harbor both distinct and common DNA motifs. Given the potential for interaction between the SHH and FGF pathways, we also measured the response of GLI-target genes to inhibition of FGF signaling and found the majority were either unaffected or upregulated. These results provide the first characterization of the spatiotemporal response of a large group of GLI-target genes and lay the foundation for a systems-level understanding of the gene regulatory networks underlying SHH-mediated limb patterning.

Foxf genes integrate tbx5 and hedgehog pathways in the second heart field for cardiac septation.

The Second Heart Field (SHF) has been implicated in several forms of congenital heart disease (CHD), including atrioventricular septal defects (AVSDs). Identifying the SHF gene regulatory networks required for atrioventricular septation is therefore an essential goal for understanding the molecular basis of AVSDs. We defined a SHF Hedgehog-dependent gene regulatory network using whole genome transcriptional profiling and GLI-chromatin interaction studies. The Forkhead box transcription factors Foxf1a and Foxf2 were identified as SHF Hedgehog targets. Compound haploinsufficiency for Foxf1a and Foxf2 caused atrioventricular septal defects, demonstrating the biological relevance of this regulatory network. We identified a Foxf1a cis-regulatory element that bound the Hedgehog transcriptional regulators GLI1 and GLI3 and the T-box transcription factor TBX5 in vivo. GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro. This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps. Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

Dynamics of BMP signaling in limb bud mesenchyme and polydactyly.

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.

Transcriptional regulation of graded Hedgehog signaling.

The Hedgehog (Hh) pathway plays conserved roles in regulating a diverse spectrum of developmental processes. In some developmental contexts, a gradient of Hh protein specifies multiple cell types in a dose-dependent fashion, thereby acting as a morphogen. Hh signaling ultimately acts on the transcriptional level through GLI proteins. In the presence of Hh signaling full length GLI proteins act as transcriptional activators of target genes. Conversely, in the absence of Hh, GLI proteins act as transcriptional repressors. This review will highlight mechanisms contributing to how graded Hh signaling might translate to differential GLI activity and be interpreted into distinct transcriptional responses.

A Gli silencer is required for robust repression of gremlin in the vertebrate limb bud.

The transcriptional response to the Hedgehog (Hh) pathway is mediated by Gli proteins, which function as context-dependent transcriptional activators or repressors. However, the mechanism by which Gli proteins regulate their target genes is poorly understood. Here, we have performed the first genetic characterization of a Gli-dependent cis-regulatory module (CRM), focusing on its regulation of Grem1 in the mouse limb bud. The CRM, termed GRE1 (Gli responsive element 1), can act as both an enhancer and a silencer. The enhancer activity requires sustained Hh signaling. As a Gli-dependent silencer, GRE1 prevents ectopic transcription of Grem1 driven through additional CRMs. In doing so, GRE1 works with additional GREs to robustly regulate Grem1. We suggest that multiple Gli CRMs may be a general mechanism for mediating a robust transcriptional response to the Hh pathway.

Manipulating gene expression and signaling activity in cultured mouse limb bud cells.

BACKGROUND: The vertebrate limb bud is a well-established system for studying the mechanisms driving growth and patterning of an embryonic tissue. However, approaches for manipulating gene expression are currently limited to time-consuming methods. Culturing primary limb bud cells could potentially be used as a quicker assay. However, limb cells in culture quickly differentiate into cartilage under normal conditions, and approaches delivering DNA and siRNA into primary limb cells in culture are limited. These technical limitations have restricted the utility of limb buds for investigating problems that require higher-throughput approaches. RESULTS: In this report, we describe adaptations to a method for culturing primary limb bud cells in a pre-chondrogenic state, and generate a population of mouse primary limb cells that are responsive to Hedgehog (Hh) signaling. Hh-stimulated cells upregulate Hh target genes as well as an exogenous Hh-responsive reporter. We then describe a method for highly efficient delivery of plasmids and siRNAs into cultured primary limb bud cells in a 96-well format. CONCLUSIONS: Cultures of primary limb bud cells are amenable to gene manipulation under conditions that maintain the limb cells in an Hh-responsive, undifferentiated state. This approach provides a medium-throughput system to manipulate gene expression, and test DNA regulatory elements.