The Hedgehog signalling pathway is essential for the development of diverse tissues during embryogenesis. Signalling is activated by binding of Hedgehog protein to the multipass membrane protein Patched (Ptc). We have now identified a novel component in the vertebrate signalling pathway, which we name Hip (for Hedgehog-interacting protein) because of its ability to bind Hedgehog proteins. Hip encodes a membrane glycoprotein that binds to all three mammalian Hedgehog proteins with an affinity comparable to that of Ptc-1. Hip-expressing cells are located next to cells that express each Hedgehog gene. Hip expression is induced by ectopic Hedgehog signalling and is lost in Hedgehog mutants. Thus, Hip, like Ptc-1, is a general transcriptional target of Hedgehog signalling. Overexpression of Hip in cartilage, where Indian hedgehog (Ihh) controls growth, leads to a shortened skeleton that resembles that seen when Ihh function is lost (B. St-Jacques, M. Hammerschmidt & A.P.M., in preparation). Our findings support a model in which Hip attenuates Hedgehog signalling as a result of binding to Hedgehog proteins: a negative regulatory feedback loop established in this way could thus modulate the responses to any Hedgehog signal.

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

Hip1 encodes a membrane-bound protein that directly binds all mammalian Hh proteins (Chuang and McMahon 1999). Like Ptch1, Hip1 is transcriptionally activated in response to Hh signaling, overlapping the expression domains of Ptch1 (Goodrich et al. 1996;Chuang and McMahon 1999). Further, gain-of-function experiments indicate that Hip1 binding of Hh ligands attenuates Hh signaling (Chuang and McMahon 1999). Here we demonstrate that loss-of-function mutants in Hip1 result in an up-regulation of Hh signaling in the mouse embryo, disrupting cell interactions essential for the normal morphogenesis of the lung and skeleton (see Supplemental Material). Results and Discussion Targeted disruption of Hip1 results in neonatal lethality with respiratory failureTo generate a null allele of the Hip1 gene in mice, a standard positive/negative targeting vector was constructed. The details are described in the Supplemental Material and Supplementary Figure 1A. Loss of Hip1 activity leads to recessive postnatal lethality. The ratio of Hip1+/+:Hip1+/−:Hip1−/− (55:118:51) newborn pups approximates a 1:2:1 Mendelian distribution (Supplementary Fig. 1B), but all homozygous Hip1 mutant pups die a few hours after birth due to respiratory failure. Hip1 mutants are superficially identical to their wild-type littermates, indicating that Hip1 activity does not appear to be essential for normal patterning of limbs, hair, or whisker, all of which are regulated by Hh signaling (Supplementary Fig. 1C; McMahon et al. 2003). Histological analysis revealed that dorsal-ventral patterning of the neural tube, development of the somites, and the organization of most internal organs appeared grossly normal in Hip1 mutants (data not shown). In contrast, Hip1 mutants have only one right and one left lung lobe rather than the five lobes (four on the right side and one

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Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP -/-limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP -/-embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP -/-embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP -/-embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/β-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy.

We show that a functional component of the C. elegans mitotic machinery regulates X chromosome gene expression. This protein, MIX-1, is a member of the dosage compensation complex that associates specifically with hermaphrodite X chromosomes to reduce their gene expression during interphase. MIX-1 also associates with all mitotic chromosomes to ensure their proper segregation. Both dosage compensation and mitosis are severely disrupted by mix-1 mutations. MIX-1 belongs to the SMC protein family required for mitotic chromosome condensation and segregation in yeast and frogs. Thus, an essential, conserved component of mitotic chromosomes has been recruited to the dosage compensation process. Rather than dosage compensation and mitosis being achieved by two separate sets of related genes, these two processes share an identical component, indicating a common mechanism for establishing higher order chromosome structure and proper X chromosome gene expression.