Hedgehog (Hh) signaling plays a major role in multiple aspects of embryonic development. A key issue in Hh signaling is to elucidate the molecular mechanism by which a Hh protein morphogen gradient is formed despite its membrane association. In this study, we used a combination of genetic, cellular, and biochemical approaches to address the role of lipid modifications in long-range vertebrate Hh signaling. Our molecular analysis of knockout mice deficient in Skn, the murine homolog of the Drosophila ski gene, which catalyzes Hh palmitoylation, and gene-targeted mice producing a nonpalmitoylated form of Shh indicates that Hh palmitoylation is essential for its activity as well as the generation of a protein gradient in the developing embryos. Furthermore, our biochemical data show that Hh lipid modifications are required for producing a soluble multimeric protein complex, which constitutes the major active component for Hh signaling. These results suggest that soluble Hh multimeric complex travels in the morphogenetic field to activate Hh signaling in distant Hh-responsive cells.[Keywords: Cholesterol; palmitoylation; Hedgehog; lipid modification; signaling; multimerization] Supplemental material is available at http://www.genesdev.org.

A central question in Hedgehog (Hh) signaling is how evolutionarily conserved components of the pathway might use the primary cilium in mammals but not fly. We focus on Suppressor of fused (Sufu), a major Hh regulator in mammals, and reveal that Sufu controls protein levels of full-length Gli transcription factors, thus affecting the production of Gli activators and repressors essential for graded Hh responses. Surprisingly, despite ciliary localization of most Hh pathway components, regulation of Gli protein levels by Sufu is cilium-independent. We propose that Sufu-dependent processes in Hh signaling are evolutionarily conserved. Consistent with this, Sufu regulates Gli protein levels by antagonizing the activity of Spop, a conserved Gli-degrading factor. Furthermore, addition of zebrafish or fly Sufu restores Gli protein function in Sufu-deficient mammalian cells. In contrast, fly Smo is unable to translocate to the primary cilium and activate the mammalian Hh pathway. We also uncover a novel positive role of Sufu in regulating Hh signaling, resulting from its control of both Gli activator and repressor function. Taken together, these studies delineate important aspects of cilium-dependent and ciliumindependent Hh signal transduction and provide significant mechanistic insight into Hh signaling in diverse species.[Keywords: Hedgehog; signal transduction; evolution; primary cilium; Sufu; Gli] Supplemental material is available at http://www.genesdev.org. In particular, the primary cilium, an ancient and evolutionarily conserved organelle, is essential for mammalian Hh signal transduction but dispensable for Hh signaling in Drosophila. The extent of Hh pathway divergence in different organisms is a major unresolved issue. Delineating cilium-dependent and cilium-independent processes of Hh signal transduction is crucial to understanding how the mammalian Hh pathway has evolved. Insight into this question will not only advance our mechanistic understanding of Hh signaling but also serve as a paradigm for investigating the evolution of signal transduction pathways.Most vertebrate cells possess a nonmotile primary cilium (Huangfu and Anderson 2005). Primary cilia contain a long microtubular axoneme that extends from the basal body and is surrounded by an external membrane that is continuous with the plasma membrane (Rosenbaum and Witman 2002). Assembly and maintenance of the primary cilium are mediated by a process called intraflagellar transport (IFT), which involves bidirectional movement of IFT particles powered by anterograde kinesin (Kif3a, Kif3b, and Kif3c) and retrograde dynein motors (Rosenbaum and Witman 2002). Mouse ethylnitrosourea (ENU) mutants in genes encoding IFT proteins, or the respective motors, have defective Hh signaling (Huangfu et al. 2003), providing strong evidence that the primary cilium plays a key role in mammalian Hh signaling. Moreover, most core mammalian 3 These authors contributed equally to this work. 4 Corresponding author. E-MAIL pao-tien.chuang@ucsf.edu; FAX (415) 476-8173. Arti...

Pulmonary neuroendocrine cells (PNECs) are proposed to be the first specialized cell type to appear in the lung, but their ontogeny remains obscure. Although studies of PNECs have suggested their involvement in a number of lung functions, neither their in vivo significance nor the molecular mechanisms underlying them have been elucidated. Importantly, PNECs have long been speculated to constitute the cells of origin of human small-cell lung cancer (SCLC) and recent mouse models support this hypothesis. However, a genetic system that permits tracing the early events of PNEC transformation has not been available. To address these key issues, we developed a genetic tool in mice by introducing a fusion protein of Cre recombinase and estrogen receptor (CreER) into the calcitonin gene-related peptide (CGRP) locus that encodes a major peptide in PNECs. The CGRP CreER mouse line has enabled us to manipulate gene activity in PNECs. Lineage tracing using this tool revealed the plasticity of PNECs. PNECs can be colabeled with alveolar cells during lung development, and following lung injury, PNECs can contribute to Clara cells and ciliated cells. Contrary to the current model, we observed that elimination of PNECs has no apparent consequence on Clara cell recovery. We also created mouse models of SCLC in which CGRP CreER was used to ablate multiple tumor suppressors in PNECs that were simultaneously labeled for following their fate. Our findings suggest that SCLC can originate from differentiated PNECs. Together, these studies provide unique insight into PNEC lineage and function and establish the foundation of investigating how PNECs contribute to lung homeostasis, injury/repair, and tumorigenesis.progenitor | naphthalene | cell of origin | tumor suppressor gene

Hedgehog (Hh) signaling is essential for multiple aspects of embryogenesis1 , 2. In Drosophila, Hh transduction is mediated by a cytoplasmic signaling complex3 -5 that includes the putative serinethreonine kinase Fused (Fu) and the kinesin Costal 2 (Cos2), yet Fu does not play a conserved role in Hh signaling in mammals6 , 7. Mouse Fu mutants are viable and appear to respond normally to Hh signaling. Here we show that mouse Fu is essential for construction of the central pair (CP) apparatus of motile, 9+2 cilia and offers a novel model of human primary ciliary dyskinesia. We found that mouse Fu physically interacts with Kif27, a mammalian Cos2 ortholog8, and linked Fu to known structural components of the CP apparatus, providing evidence for the first regulatory component involved in CP construction. We also demonstrated that zebrafish Fu is required both for Hh signaling and cilia biogenesis in Kupffer's vesicle. Mouse Fu rescued both Hh-dependent and independent defects in zebrafish. Our results delineate a novel pathway for CP apparatus assembly, identify common regulators of Hh signaling and motile ciliogenesis, and add insight into evolution of the Hh cascade.To further investigate the role of Fu in mammalian Hh signaling, we asked whether Hhdependent Smo localization to the primary cilium is affected in the absence of Fu. Primary cilia, which have a "9+0" arrangement of 9 outer doublet microtubules (MTs), are required for Hh responses and contain several Hh pathway components2 , 9. We found that Fu -/-mouse embryonic fibroblasts (MEFs) formed primary cilia normally, trafficked Smo to the primary cilium in response to Hh ligand, and exhibited a typical Gli transcriptional response (Supplementary Fig. 1 , 2; data not shown). This suggests that the single mammalian Fu ortholog is dispensable for Hh signaling. To explore the function of Fu in mice, we examined its expression in postnatal tissues by in situ hybridization. Fu transcript was expressed strongly in the respiratory epithelium, the ependymal lining of the ventricles in the brain, and in oviduct and testis ( Fig. 1a-c; data not shown). These expression patterns are reminiscent of genes involved in biogenesis of motile cilia, which function in these tissues to propel mucus, fluid and cells. In contrast to the primary cilium, the classical "9+2" motile cilium consists of 9 outer doublet MTs and two singlet central pair (CP) MTs10. The CP apparatus plays a key role in regulating ciliary motility but its formation is poorly understood since the centriole-derived basal body, from which the cilia axoneme extends, does not provide a template for CP outgrowth. Disruption of human motile cilia function leads to primary ciliary dyskinesia (PCD), which is associated with recurrent respiratory infection, hydrocephalus and infertility11 -13. To determine whether motile cilia function is * These authors contributed equally to this work.

Both the Wnt/␤-catenin and Ihh signaling pathways play essential roles in crucial aspects of endochondral ossification: osteoblast differentiation, chondrocyte proliferation and hypertrophy. To understand the genetic interaction between these two signaling pathways, we have inactivated the ␤-catenin gene and upregulated Ihh signaling simultaneously in the same cells during endochondral skeletal development using ␤-catenin and patched 1 floxed alleles. We uncovered previously unexpected roles of Ihh signaling in synovial joint formation and the essential function of Wnt/␤-catenin signaling in regulating chondrocyte survival. More importantly, we found that Wnt and Ihh signaling interact with each other in distinct ways to control osteoblast differentiation, chondrocyte proliferation, hypertrophy, survival and synovial joint formation in the developing endochondral bone. ␤-catenin is required downstream of Ihh signaling and osterix expression for osteoblast differentiation. But in chondrocyte survival, ␤-catenin is required upstream of Ihh signaling to inhibit chondrocyte apoptosis. In addition, Ihh signaling can inhibit chondrocyte hypertrophy and synovial joint formation independently of ␤-catenin. However, there is a strong synergistic interaction between Wnt/␤-catenin and Ihh signaling in regulating synovial joint formation.

Activation of Hedgehog (Hh) signaling requires the transmembrane protein Smoothened (Smo), a member of the G-protein coupled receptor superfamily. In mammals, Smo translocates to the primary cilium upon binding of Hh ligands to their receptor, Patched (Ptch1), but it is unclear if ciliary trafficking of Smo is sufficient for pathway activation. Here, we demonstrate that cyclopamine and jervine, two structurally related inhibitors of Smo, force ciliary translocation of Smo. Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation. Further, activation of protein kinase A, either directly or through activation of Gαs, causes Smo to translocate to a proximal region of the primary cilium. We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

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BackgroundDNA methylation is an epigenetic mechanism central to development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited.ResultsWe use whole genome bisulfite sequencing, and find that differentiation of mouse colonic intestinal stem cells to intestinal epithelium is not associated with major changes in DNA methylation. However, we detect extensive dynamic epigenetic changes in intestinal stem cells and their progeny during the suckling period, suggesting postnatal epigenetic development in this stem cell population. We find that postnatal DNA methylation increases at 3′ CpG islands (CGIs) correlate with transcriptional activation of glycosylation genes responsible for intestinal maturation. To directly test whether 3′ CGI methylation regulates transcription, we conditionally disrupted two major DNA methyltransferases, Dnmt1 or Dnmt3a, in fetal and adult intestine. Deficiency of Dnmt1 causes severe intestinal abnormalities in neonates and disrupts crypt homeostasis in adults, whereas Dnmt3a loss was compatible with intestinal development. These studies reveal that 3′ CGI methylation is functionally involved in the regulation of transcriptional activation in vivo, and that Dnmt1 is a critical regulator of postnatal epigenetic changes in intestinal stem cells. Finally, we show that postnatal 3′ CGI methylation and associated gene activation in intestinal epithelial cells are significantly altered by germ-free conditions.ConclusionsOur results demonstrate that the suckling period is critical for epigenetic development of intestinal stem cells, with potential important implications for lifelong gut health, and that the gut microbiome guides and/or facilitates these postnatal epigenetic processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0763-5) contains supplementary material, which is available to authorized users.