Ciliogenesis and Hedgehog signalling are suppressed downstream of KRAS during acinar-ductal metaplasia in mouse.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths worldwide, but has a 5-year survival rate of only 7% primarily due to late diagnosis and ineffective therapies. To treat or even prevent PDAC, it is vital that we understand the initiating events that lead to tumour onset. PDAC develops from preneoplastic lesions, most commonly pancreatic intraepithelial neoplasias (PanINs), driven by constitutive activation of KRAS.

The Meckel syndrome- associated protein MKS1 functionally interacts with components of the BBSome and IFT complexes to mediate ciliary trafficking and hedgehog signaling.

The importance of primary cilia in human health is underscored by the link between ciliary dysfunction and a group of primarily recessive genetic disorders with overlapping clinical features, now known as ciliopathies. Many of the proteins encoded by ciliopathy-associated genes are components of a handful of multi-protein complexes important for the transport of cargo to the basal body and/or into the cilium. A key question is whether different complexes cooperate in cilia formation, and whether they participate in cilium assembly in conjunction with intraflagellar transport (IFT) proteins.

Primary Cilia and Mammalian Hedgehog Signaling.

It has been a decade since it was discovered that primary cilia have an essential role in Hedgehog (Hh) signaling in mammals. This discovery came from screens in the mouse that identified a set of genes that are required for both normal Hh signaling and for the formation of primary cilia. Since then, dozens of mouse mutations have been identified that disrupt cilia in a variety of ways and have complex effects on Hedgehog signaling.

Lineage specificity of primary cilia in the mouse embryo.

Primary cilia are required for vertebrate cells to respond to specific intercellular signals. Here we define when and where primary cilia appear in the mouse embryo using a transgenic line that expresses ARL13B-mCherry in cilia and Centrin 2-GFP in centrosomes. Primary cilia first appear on cells of the epiblast at E6.

The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the cilium tip compartment.

Mammalian Hedgehog (Hh) signal transduction requires a primary cilium, a microtubule-based organelle, and the Gli-Sufu complexes that mediate Hh signalling, which are enriched at cilia tips. Kif7, a kinesin-4 family protein, is a conserved regulator of the Hh signalling pathway and a human ciliopathy protein. Here we show that Kif7 localizes to the cilium tip, the site of microtubule plus ends, where it limits cilium length and controls cilium structure.

Generation of mice with functional inactivation of talpid3, a gene first identified in chicken.

Specification of digit number and identity is central to digit pattern in vertebrate limbs. The classical talpid(3) chicken mutant has many unpatterned digits together with defects in other regions, depending on hedgehog (Hh) signalling, and exhibits embryonic lethality. The talpid(3) chicken has a mutation in KIAA0586, which encodes a centrosomal protein required for the formation of primary cilia, which are sites of vertebrate Hh signalling.

3D expression patterns of cell cycle genes in the developing chick wing and comparison with expression patterns of genes implicated in digit specification.

Sonic hedgehog (Shh) signalling controls integrated specification of digit pattern and growth in the chick wing but downstream gene networks remain to be unravelled. We analysed 3D expression patterns of genes encoding cell cycle regulators using Optical Projection Tomography. Hierarchical clustering of spatial matrices of gene expression revealed a dorsal layer of the wing bud, in which almost all genes were expressed, and that genes encoding positive cell cycle regulators had similar expression patterns while those of N-myc and CyclinD2 were distinct but closely related.

Foxj1 regulates floor plate cilia architecture and modifies the response of cells to sonic hedgehog signalling.

Sonic hedgehog signalling is essential for the embryonic development of many tissues including the central nervous system, where it controls the pattern of cellular differentiation. A genome-wide screen of neural progenitor cells to evaluate the Shh signalling-regulated transcriptome identified the forkhead transcription factor Foxj1. In both chick and mouse Foxj1 is expressed in the ventral midline of the neural tube in cells that make up the floor plate.

Identification of genes downstream of the Shh signalling in the developing chick wing and syn-expressed with Hoxd13 using microarray and 3D computational analysis.

Sonic hedgehog (Shh) signalling by the polarizing region at the posterior margin of the chick wing bud is pivotal in patterning the digits but apart from a few key downstream genes, such as Hoxd13, which is expressed in the posterior region of the wing that gives rise to the digits, the genes that mediate the response to Shh signalling are not known. To find genes that are co-expressed with Hoxd13 in the posterior of chick wing buds and regulated in the same way, we used microarrays to compare gene expression between anterior and posterior thirds of wing buds from normal chick embryos and from polydactylous talpid³ mutant chick embryos, which have defective Shh signalling due to lack of primary cilia. We identified 1070 differentially expressed gene transcripts, which were then clustered.

Human intronic enhancers control distinct sub-domains of Gli3 expression during mouse CNS and limb development.

Background: The zinc-finger transcription factor GLI3 is an important mediator of Sonic hedgehog signaling and crucial for patterning of many aspects of the vertebrate body plan. In vertebrates, the mechanism of SHH signal transduction and its action on target genes by means of activating or repressing forms of GLI3 have been studied most extensively during limb development and the specification of the central nervous system. From these studies it has emerged, that Gli3 expression must be subject to a tight spatiotemporal regulation.

Enhancer elements upstream of the SHOX gene are active in the developing limb.

Léri-Weill Dyschondrosteosis (LWD) is a dominant skeletal disorder characterized by short stature and distinct bone anomalies. SHOX gene mutations and deletions of regulatory elements downstream of SHOX resulting in haploinsufficiency have been found in patients with LWD. SHOX encodes a homeodomain transcription factor and is known to be expressed in the developing limb.

The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation.

The chicken talpid(3) mutant, with polydactyly and defects in other embryonic regions that depend on hedgehog (Hh) signalling (e.g. the neural tube), has a mutation in KIAA0568.

Long-range conserved non-coding SHOX sequences regulate expression in developing chicken limb and are associated with short stature phenotypes in human patients.

Defects in long-range regulatory elements have recently emerged as previously underestimated factors in the genesis of human congenital disorders. Léri-Weill dyschondrosteosis is a dominant skeletal malformation syndrome caused by mutations in the short stature homeobox gene SHOX. We have analysed four families with Léri-Weill dyschondrosteosis with deletions in the pseudoautosomal region but still with an intact SHOX coding region.

Expression of the short stature homeobox gene Shox is restricted by proximal and distal signals in chick limb buds and affects the length of skeletal elements.

SHOX is a homeobox-containing gene, highly conserved among species as diverse as fish, chicken and humans. SHOX gene mutations have been shown to cause idiopathic short stature and skeletal malformations frequently observed in human patients with Turner, Leri-Weill and Langer syndromes. We cloned the chicken orthologue of SHOX, studied its expression pattern and compared this with expression of the highly related Shox2.

The chicken talpid3 gene encodes a novel protein essential for Hedgehog signaling.

Talpid3 is a classical chicken mutant with abnormal limb patterning and malformations in other regions of the embryo known to depend on Hedgehog signaling. We combined the ease of manipulating chicken embryos with emerging knowledge of the chicken genome to reveal directly the basis of defective Hedgehog signal transduction in talpid3 embryos and to identify the talpid3 gene. We show in several regions of the embryo that the talpid3 phenotype is completely ligand independent and demonstrate for the first time that talpid3 is absolutely required for the function of both Gli repressor and activator in the intracellular Hedgehog pathway.

A robust system for RNA interference in the chicken using a modified microRNA operon.

RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells.