Loss of cilia causes embryonic lung hypoplasia,liver fibrosis,and cholestasis in the talpid3 ciliopathy mutant

Sonic hedgehog plays an essential role in maintaining hepatoblasts in a proliferative non-differentiating state during embryogenesis. Transduction of the Hedgehog signaling pathway is dependent on the presence of functional primary cilia and hepatoblasts, therefore, must require primary cilia for normal function. In congenital syndromes in which cilia are absent or non-functional (ciliopathies) hepatorenal fibrocystic disease is common and primarily characterized by ductal plate malformations which underlie the formation of liver cysts, as well as less commonly, by hepatic fibrosis, although a role for abnormal Hedgehog signal transduction has not been implicated in these phenotypes. We have examined liver, lung and rib development in the talpid³ chicken mutant, a ciliopathy model in which abnormal Hedgehog signaling is well characterized. We find that the talpid³ phenotype closely models that of human short-rib polydactyly syndromes which are caused by the loss of cilia, and exhibit hypoplastic lungs and liver failure. Through an analysis of liver and lung development in the talpid³ chicken, we propose that cilia in the liver are essential for the transduction of Hedgehog signaling during hepatic development. The talpid³ chicken represents a useful resource in furthering our understanding of the pathology of ciliopathies beyond the treatment of thoracic insufficiency as well as generating insights into the role Hedgehog signaling in hepatic development.     

The emerging face of primary cilia

Primary cilia are microtubule-based organelles that serve as hubs for the transduction of various developmental signaling pathways including Hedgehog, Wnt, FGF, and PDGF. Ciliary dysfunction contributes to a range of disorders, collectively known as the ciliopathies. Recently, interest has grown in these syndromes, particularly among craniofacial biologists, as many known and putative ciliopathies have severe craniofacial defects. Herein we discuss the current understanding of ciliary biology and craniofacial development in an attempt to gain insight into the molecular etiology for craniofacial ciliopathies, and uncover a characteristic ciliopathic craniofacial gestalt.     

Hedgehog signal activation coordinates proliferation and differentiation of fetal liver progenitor cells

Hedgehog (Hh) signaling plays crucial roles in development and homeostasis of various organs. In the adult liver, it regulates proliferation and/or viability of several types of cells, particularly under injured conditions, and is also implicated in stem/progenitor cell maintenance. However, the role of this signaling pathway during the normal developmental process of the liver remains elusive. Although Sonic hedgehog (Shh) is expressed in the ventral foregut endoderm from which the liver derives, the expression disappears at the onset of the liver bud formation, and its possible recurrence at the later stages has not been investigated. Here we analyzed the activation and functional relevance of Hh signaling during the mouse fetal liver development. At E11.5, Shh and an activation marker gene for Hh signaling, Gli1, were expressed in Dlk⁺ hepatoblasts, the fetal liver progenitor cells, and the expression was rapidly decreased thereafter as the development proceeded. In the culture of Dlk⁺ hepatoblasts isolated from the E11.5 liver, activation of Hh signaling stimulated their proliferation and this effect was cancelled by a chemical Hh signaling inhibitor, cyclopamine. In contrast, hepatocyte differentiation of Dlk⁺ hepatoblasts in vitro as manifested by the marker gene expression and acquisition of ammonia clearance activity was significantly inhibited by forced activation of Hh signaling. Taken together, these results demonstrate the temporally restricted manner of Hh signal activation and its role in promoting the hepatoblast proliferation, and further suggest that the pathway needs to be shut off for the subsequent hepatic differentiation of hepatoblasts to proceed normally.     

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies.     

Cauli: A Mouse Strain with an Ift140 Mutation That Results in a Skeletal Ciliopathy Modelling Jeune Syndrome

Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.     

Lack of centrioles and primary cilia in STIL−/− mouse embryos

Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL^−/− mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL^−/− cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novo generation of centrioles in STIL^−/− cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development.     

Time-resolved proteomics profiling of the ciliary Hedgehog response

The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein–coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type–specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.     

Mouse hepatoblasts at distinct developmental stages are characterized by expression of EpCAM and DLK1: Drastic change of EpCAM expression during liver development

Hepatoblasts are hepatic progenitor cells that expand and give rise to either hepatocyte or cholangiocytes during liver development. We previously reported that delta-like 1 homolog (DLK1) is expressed in the mouse liver primordium at embryonic day (E) 10.5 and that DLK1⁺ cells in E14.5 liver contain high proliferative and bipotential hepatoblasts. While the expression of epithelial cell adhesion molecule (EpCAM) in hepatic stem/progenitor cells has been reported, its expression profile at an early stage of liver development remains unknown. In this study, we show that EpCAM is expressed in mouse liver bud at E9.5 and that EpCAM⁺DLK1⁺ hepatoblasts form hepatic cords at the early stage of hepatogenesis. DLK1⁺ cells of E11.5 liver were fractionated into EpCAM⁺ and EpCAM⁻ cells; one forth of EpCAM⁺DLK1⁺ cells formed a colony in vitro whereas EpCAM⁻DLK1⁺ cells rarely did it. Moreover, EpCAM⁺DLK1⁺ cells contained cells capable of forming a large colony, indicating that EpCAM⁺DLK1⁺ cells in E11.5 liver contain early hepatoblasts with high proliferation potential. Interestingly, EpCAM expression in hepatoblasts was dramatically reduced along with liver development and the colony-forming capacities of both EpCAM⁺DLK1⁺ and EpCAM⁻DLK1⁺ cells were comparable in E14.5 liver. It strongly suggested that most of mouse hepatoblasts are losing EpCAM expression at this stage. Moreover, we provide evidence that EpCAM⁺DLK1⁺ cells in E11.5 liver contain extrahepatic bile duct cells as well as hepatoblasts, while EpCAM⁻DLK1⁺ cells contain mesothelial cell precursors. Thus, the expression of EpCAM and DLK1 suggests the developmental pathways of mouse liver progenitors.     

The emerging functions of intraflagellar transport 52 in ciliary transport and ciliopathies

Ciliary transport in eukaryotic cells is an intricate and conserved process involving the coordinated assembly and functioning of a multiprotein intraflagellar transport (IFT) complex. Among the various IFT proteins, intraflagellar transport 52 (IFT52) plays a crucial role in ciliary transport and is implicated in various ciliopathies. IFT52 is a core component of the IFT-B complex that facilitates movement of cargoes along the ciliary axoneme. Stable binding of the IFT-B1 and IFT-B2 subcomplexes by IFT52 in the IFT-B complex regulates recycling of ciliary components and maintenance of ciliary functions such as signal transduction and molecular movement. Mutations in the IFT52 gene can disrupt ciliary trafficking, resulting in dysfunctional cilia and affecting cellular processes in ciliopathies. Such ciliopathies caused by IFT52 mutations exhibit a wide range of clinical features, including skeletal developmental abnormalities, retinal degeneration, respiratory failure and neurological abnormalities in affected individuals. Therefore, IFT52 serves as a promising biomarker for the diagnosis of various ciliopathies, including short-rib thoracic dysplasia 16 with or without polydactyly. Here, we provide an overview of the IFT52-mediated molecular mechanisms underlying ciliary transport and describe the IFT52 mutations that cause different disorders associated with cilia dysfunction.     

A novel Cre‐inducible knock‐in ARL13B‐tRFP fusion cilium reporter

Cilia play a major role in the regulation of numerous signaling pathways and are essential for embryonic development. Mutations in genes affecting ciliary function can cause a variety of diseases in humans summarized as ciliopathies. To facilitate the detection and visualization of cilia in a temporal and spatial manner in mouse tissues, we generated a Cre‐inducible cilium‐specific reporter mouse line expressing an ARL13B‐tRFP fusion protein driven by a CMV enhancer/chicken β actin promotor (pCAG) from the Hprt locus. We detected bright and specific ciliary signals by immunostainings of various mono‐ and multiciliated tissues and by time‐lapse live‐cell analysis of cultured embryos and organ explant cultures. Additionally, we monitored cilium assembly and disassembly in embryonic fibroblast cells using live‐cell imaging. Thus, the ARL13B‐tRFP reporter mouse strain is a valuable tool for the investigation of ciliary structure and function in a tissue‐specific manner to understand processes, such as ciliary protein trafficking or cilium‐dependent signaling in vitro and in vivo.     

Cilia in hereditary cerebral anomalies

Ciliopathies are complex genetic multi‐system disorders causally related to abnormal assembly or function of motile or non‐motile cilia. While most human cells possess a non‐motile sensory/primary cilium (PC) during development and/or in adult tissues, motile cilia are restricted to specialised cells. As a result, PC‐associated ciliopathies are characterised by high phenotypic variability with extensive clinical and genetic overlaps. In the present review, we have focused on cerebral developmental anomalies, which are commonly found in PC‐associated ciliopathies and which have mostly been linked to Hedgehog signalling defects. In addition, we have reviewed emerging evidence that PC dysfunctions could be directly or indirectly involved in the mechanisms underlying malformations of cerebral cortical development including primary microcephaly.     

TCTN3 Mutations Cause Mohr-Majewski Syndrome

Orofaciodigital syndromes (OFDSs) consist of a group of heterogeneous disorders characterized by abnormalities in the oral cavity, face, and digits and associated phenotypic abnormalities that lead to the delineation of 13 OFDS subtypes. Here, by a combined approach of homozygozity mapping and exome ciliary sequencing, we identified truncating TCTN3 mutations as the cause of an extreme form of OFD associated with bone dysplasia, tibial defect, cystic kidneys, and brain anomalies (OFD IV, Mohr-Majewski syndrome). Analysis of 184 individuals with various ciliopathies (OFD, Meckel, Joubert, and short rib polydactyly syndromes) led us to identify four additional truncating TCTN3 mutations in unrelated fetal cases with overlapping Meckel and OFD IV syndromes and one homozygous missense mutation in a family with Joubert syndrome. By exploring roles of TCTN3 in human ciliary related functions, we found that TCTN3 is necessary for transduction of the sonic hedgehog (SHH) signaling pathway, as revealed by abnormal processing of GLI3 in patient cells. These results are consistent with the suggested role of its murine ortholog, which forms a complex at the ciliary transition zone with TCTN1 and TCTN2, both of which are also implicated in the transduction of SHH signaling. Overall, our data show the involvement of the transition zone protein TCTN3 in the regulation of the key SHH signaling pathway and that its disruption causes a severe form of ciliopathy, combining features of Meckel and OFD IV syndromes.     

Zebrafish inositol polyphosphate kinases: New effectors of cilia and developmental signaling

We described here our recent findings that Ipk1 catalyzed production of IP₆ regulates LR-axis specification (Sarmah et al., 2005) and that IP₆ is an essential effector of ciliary beating and length maintenance in zebrafish (Sarmah et al., 2007). We have also uncovered a novel role for the IP-kinase IP₆k2 in craniofacial development, neural crest cell migration, and hedgehog signal transduction (B.S. and S.R.W., unpublished). Together, these findings place IP production as a key mediator for cellular signaling mechanisms that regulate vital cellular and developmental processes. How these and other IPs are integrated with cell–cell signaling networks during complex processes, such as, tissue morphogenesis and maintenance of cell fate and function? We propose that with its enormous resource and unique set of structural, functional, and sensory attributes, cilium provides a platform for executing IP-based signaling functions. Given the evolutionary conservation of the IP repertoire and pathways, the developmental and molecular events uncovered in our studies in the zebrafish system could be applicable in other vertebrates including humans. This unbiased approach of systematic identification of IP functions in cilia and development will aid in understanding of multiple disease pathologies including ciliopathies and dysmorphic syndromes.     

ICK is essential for cell type‐specific ciliogenesis and the regulation of ciliary transport

Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK‐deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK‐deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT‐A, IFT‐B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT‐B, but not IFT‐A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia.     

5‐HT6R null mutatrion induces synaptic and cognitive defects

Serotonin 6 receptor (5‐HT6R) is a promising target for a variety of human diseases, such as Alzheimer''s disease (AD) and schizophrenia. However, the detailed mechanism underlying 5‐HT6R activity in the central nervous system (CNS) is not fully understood. In the present study, 5‐HT6R null mutant (5‐HT6R^−/−) mice were found to exhibit cognitive deficiencies and abnormal anxiety levels. 5‐HT6R is considered to be specifically localized on the primary cilia. We found that the loss of 5‐HT6R affected the Sonic Hedgehog signaling pathway in the primary cilia. 5‐HT6R^−/− mice showed remarkable alterations in neuronal morphology, including dendrite complexity and axon initial segment morphology. Neurons lacking 5‐HT6R exhibited increased neuronal excitability. Our findings highlight the complexity of 5‐HT6R functions in the primary ciliary and neuronal physiology, supporting the theory that this receptor modulates neuronal morphology and transmission, and contributes to cognitive deficits in a variety of human diseases, such as AD, schizophrenia, and ciliopathies.