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. In patients, PanINs are associated with regions of acinar-to-ductal metaplasia (ADM) where, in response to inflammation, acini dedifferentiate to a pancreatic progenitor-like fate. In healthy tissue this process is reversible leading to regeneration of the pancreas; however, in the presence of oncogenic KRAS, regeneration is blocked and ADM can give rise to PanIN lesions. Here, we used a 3D mouse acinar culture that recapitulates ADM in vitro to explore how KRAS prevents regeneration. Regeneration is regulated by Hedgehog (Hh) signalling, which is transduced via the primary cilium. In wild-type acini, cilia assemble upon ADM and Hh target gene expression is upregulated; however, ciliogenesis and Hh signalling are suppressed during ADM in cells expressing oncogenic KRAS. We show that ciliogenesis fails due to ectopic activation of the cilium disassembly pathway, which is mediated by AurkA, a direct transcriptional target of KRAS. Inhibition of AurkA is able to rescue primary cilia and restore Hh signalling. We suggest that this could be used as a mechanism to prevent the formation of early lesions and thereby prevent progression to PDAC.

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. To examine how ciliopathy protein complexes might function together, we have analyzed double mutants of an allele of the Meckel syndrome (MKS) complex protein MKS1 and the BBSome protein BBS4. We find that Mks1; Bbs4 double mutant mouse embryos exhibit exacerbated defects in Hedgehog (Hh) dependent patterning compared to either single mutant, and die by E14.5. Cells from double mutant embryos exhibit a defect in the trafficking of ARL13B, a ciliary membrane protein, resulting in disrupted ciliary structure and signaling. We also examined the relationship between the MKS complex and IFT proteins by analyzing double mutant between Mks1 and a hypomorphic allele of the IFTB component Ift172. Despite each single mutant surviving until around birth, Mks1; Ift172avc1 double mutants die at mid-gestation, and exhibit a dramatic failure of cilia formation. We also find that Mks1 interacts genetically with an allele of Dync2h1, the IFT retrograde motor. Thus, we have demonstrated that the MKS transition zone complex cooperates with the BBSome to mediate trafficking of specific trans-membrane receptors to the cilium. Moreover, the genetic interaction of Mks1 with components of IFT machinery suggests that the transition zone complex facilitates IFT to promote cilium assembly and structure.

The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formationThe Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation' Development, vol 136, no. 4, pp. 655-64. General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. INTRODUCTION The chicken talpid 3 mutant has a complex phenotype, including polydactylous limbs with many unpatterned digits, vascular defects, hypoteleorism, abnormal dorsoventral patterning of the neural tube, loss of endochondral bone formation and embryonic lethality (Davey et al. Development of all the regions affected in the talpid 3 mutant embryo requires Hedgehog (Hh) signalling and analysis of the developing mutant limb bud and neural tube has shown that it is the response to Hh signalling that is defective (Lewis et al., 1999). Expression of some downstream Shh target genes in talpid 3 mutant limb buds, head, neural tube and somites is lost, whereas other genes are expressed more widely, suggesting that there is failure both to activate gene expression in response to Shh and to repress gene expression in its absence (Buxton et al., 2004; Davey et al., 2006; Lewis et al., 1999). These opposing effects can be understood in terms of the bifunctionality of the Gli proteins, the transcriptional effectors of vertebrate Hedgehog (Hh) signalling, which function as activators (mainly Gli1, Gli2) or are processed to short forms (mainly Gli3) that function as repressors (Aza-Blanc et al.

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. Here, we summarize the genetic and developmental studies used to deduce how Hedgehog signal transduction is linked to cilia and the complex effects that perturbation of cilia structure can have on Hh signaling. We conclude by describing the current status of our understanding of the cell-type-specific regulation of ciliogenesis and how that determines the ability of cells to respond to Hedgehog ligands.

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 E6.0 and are subsequently present on all derivatives of the epiblast. In contrast, extraembryonic cells of the visceral endoderm and trophectoderm lineages have centrosomes but no cilia. Stem cell lines derived from embryonic lineages recapitulate the in vivo pattern: epiblast stem cells are ciliated, whereas trophoblast stem cells and extraembryonic endoderm (XEN) stem cells lack cilia. Basal bodies in XEN cells are mature and can form cilia when the AURKA-HDAC6 cilium disassembly pathway is inhibited. The lineage-dependent distribution of cilia is stable throughout much of gestation, defining which cells in the placenta and yolk sac are able to respond to Hedgehog ligands.

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. Purified recombinant Kif7 binds the plus ends of growing microtubules in vitro, where it reduces the rate of microtubule growth and increases the frequency of microtubule catastrophe. Kif7 is not required for normal intraflagellar transport or for trafficking of Hh pathway proteins into cilia. Instead, a central function of Kif7 in the mammalian Hh pathway is to control cilium architecture and to create a single cilium tip compartment, where Gli-Sufu activity can be correctly regulated.

Specification of digit number and identity is central to digit pattern in vertebrate limbs. The classical talpid3 chicken mutant has many unpatterned digits together with defects in other regions, depending on hedgehog (Hh) signalling, and exhibits embryonic lethality. The talpid3 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. The highly conserved exons 11 and 12 of KIAA0586 are essential to rescue cilia in talpid3 chicken mutants. We constitutively deleted these two exons to make a talpid3–/– mouse. Mutant mouse embryos lack primary cilia and, like talpid3 chicken embryos, have face and neural tube defects but also defects in left/right asymmetry. Conditional deletion in mouse limb mesenchyme results in polydactyly and in brachydactyly and a failure of subperisoteal bone formation, defects that are attributable to abnormal sonic hedgehog and Indian hedgehog signalling, respectively. Like talpid3 chicken limbs, the mutant mouse limbs are syndactylous with uneven digit spacing as reflected in altered Raldh2 expression, which is normally associated with interdigital mesenchyme. Both mouse and chicken mutant limb buds are broad and short. talpid3–/– mouse cells migrate more slowly than wild-type mouse cells, a change in cell behaviour that possibly contributes to altered limb bud morphogenesis. This genetic mouse model will facilitate further conditional approaches, epistatic experiments and open up investigation into the function of the novel talpid3 gene using the many resources available for mice.

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. We compared these patterns computationally with those of genes implicated in digit specification and Ptch1, 50 genes in total. Nineteen genes have similar posterior expression to Ptch1, including Hoxd13, Sall1, Hoxd11, and Bmp2, all likely Gli targets in mouse limb, and cell cycle genes, N-myc, CyclinD2. We suggest that these genes contribute to a network integrating digit specification and growth in response to Shh.

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. Consistent with the role of Foxj1 in the formation of long motile cilia, floor plate cells produce cilia that are longer than the primary cilia found elsewhere in the neural tube, and forced expression of Foxj1 in neuroepithelial cells is sufficient to increase cilia length. In addition, the expression of Foxj1 in the neural tube and in an Shh-responsive cell line attenuates intracellular signalling by decreasing the activity of Gli proteins, the transcriptional mediators of Shh signalling. We show that this function of Foxj1 depends on cilia. Nevertheless, floor plate identity and ciliogenesis are unaffected in mouse embryos lacking Foxj1 and we provide evidence that additional transcription factors expressed in the floor plate share overlapping functions with Foxj1. Together, these findings identify a novel mechanism that modifies the cellular response to Shh signalling and reveal morphological and functional features of the amniote floor plate that distinguish these cells from the rest of the neuroepithelium.

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. Two clusters contained genes predominantly expressed in posterior thirds of normal wing buds; in one cluster, genes including Hoxd13, were expressed at high levels in anterior and posterior thirds in talpid3 wing buds, in the other cluster, genes including Ptc1, were expressed at low levels in anterior and posterior thirds in talpid3 wing buds. Expression patterns of genes in these two clusters were validated in normal and talpid3 mutant wing buds by in situ hybridisation and demonstrated to be responsive to application of Shh. Expression of several genes in the Hoxd13 cluster was also shown to be responsive to manipulation of protein kinase A (PKA) activity, thus demonstrating regulation by Gli repression. Genes in the Hoxd13 cluster were then sub-clustered by computational comparison of 3D expression patterns in normal wing buds to produce syn-expression groups. Hoxd13 and Sall1 are syn-expressed in the posterior region of early chick wing buds together with 6 novel genes which are likely to be functionally related and represent secondary targets of Shh signalling. Other groups of syn-expressed genes were also identified, including a group of genes involved in vascularisation.

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. However, the genetic mechanisms and the cis-acting elements controlling the expression of Gli3 remained largely unknown. Results: Here, we demonstrate in chicken and mouse transgenic embryos that human GLI3-intronic conserved noncoding sequence elements (CNEs) autonomously control individual aspects of Gli3 expression. Their combined action shows many aspects of a Gli3-specific pattern of transcriptional activity. In the mouse limb bud, different CNEs enhance Gli3-specific expression in evolutionary ancient stylopod and zeugopod versus modern skeletal structures of the autopod. Limb bud specificity is also found in chicken but had not been detected in zebrafish embryos. Three of these elements govern central nervous system specific gene expression during mouse embryogenesis, each targeting a subset of endogenous Gli3 transcription sites. Even though fish, birds, and mammals share an ancient repertoire of gene regulatory elements within Gli3, the functions of individual enhancers from this catalog have diverged significantly. During evolution, ancient broad-range regulatory elements within Gli3 attained higher specificity, critical for patterning of more specialized structures, by abolishing the potential for redundant expression control. Conclusion: These results not only demonstrate the high level of complexity in the genetic mechanisms controlling Gli3 expression, but also reveal the evolutionary significance of cis-acting regulatory networks of early developmental regulators in vertebrates. Background Zinc-finger proteins of the GLI family, GLI1, GLI2, and GLI3 act as transcriptional mediators integrating various upstream patterning signals in a context dependent combinatorial and cooperative fashion to direct a multitude of developmental programs. GLI2 and GLI3 can serve both as transcriptional activators or repressors, whereas GLI1, whose expression is transcriptionally regulated by GLI2 and GLI3, appears to play a secondary role, e.g. in potentiating response to the secreted protein sonic hedgehog (SHH) [1]. Mutations in the human GLI3 gene cause a variety of dominant developmental syndromes subsumed as "GLI3 morphopathies" [2,3], including Greig cephalopolysyndactyly syndrome (GCPS) [3-5], Pallister Hall syndrome (PHS) [6], postaxial polydactyly type A (PAPA) [7], and preaxial polydactyly type IV (PPD-IV) [2]. Mutations affecting murine Gli3, such as extra toes (Xt), anterior digit deformity (add), and polydactyly Nagoya (Pdn), serve as models for GLI3 morphopathies. Mouse embryos with homozygous Gli3 deficiency show pleiotropic and lethal congenital malformations with distinct * Correspondence: abbasiam@qau.edu.pk 1 Department of Human Genetics, Philipps-Universität Marburg, 35037

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. However, the genetic mechanisms and the cis-acting elements controlling the expression of Gli3 remained largely unknown.

involves a major collaboration between groups in Edinburgh, Dublin, Bath and London. This database will be based on EMAGE and cross-referenced to the mouse through orthologous gene pairs (http://www.emouseatlas.org/testemage/home.php). Throughout this project, the data and framework will be used to identify groups of genes that are co-expressed in important signalling regions. Conservation of these genes will be examined in the chick and mouse. This database will be made publicly available (http://www.echickatlas.org/) and will be a valuable resource to the developmental community.

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. Similar phenotypes are seen in mice and in human syndromes with mutations in genes that encode centrosomal or intraflagella transport proteins. Such mutations lead to defects in primary cilia, sites where Hh signalling occurs. Here, we show that cells of talpid(3) mutant embryos lack primary cilia and that primary cilia can be rescued with constructs encoding Talpid3. talpid(3) mutant embryos also develop polycystic kidneys, consistent with widespread failure of ciliogenesis. Ultrastructural studies of talpid(3) mutant neural tube show that basal bodies mature but fail to dock with the apical cell membrane, are misorientated and almost completely lack ciliary axonemes. We also detected marked changes in actin organisation in talpid(3) mutant cells, which may explain misorientation of basal bodies. KIAA0586 was identified in the human centrosomal proteome and, using an antibody against chicken Talpid3, we detected Talpid3 in the centrosome of wild-type chicken cells but not in mutant cells. Cloning and bioinformatic analysis of the Talpid3 homolog from the sea anemone Nematostella vectensis identified a highly conserved region in the Talpid3 protein, including a predicted coiled-coil domain. We show that this region is required to rescue primary cilia formation and neural tube patterning in talpid(3) mutant embryos, and is sufficient for centrosomal localisation. Thus, Talpid3 is one of a growing number of centrosomal proteins that affect both ciliogenesis and Hh signalling.

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. We have now analyzed the regulatory significance of the region upstream of the SHOX gene. By comparative genomic analyses, we identified several conserved non-coding elements, which subsequently were tested in an in ovo enhancer assay in both chicken limb bud and cornea, where SHOX is also expressed. In this assay, we found three enhancers to be active in the developing chicken limb, but none were functional in the developing cornea. A screening of 60 LWD patients with an intact SHOX coding and downstream region did not yield any deletion of the upstream enhancer region. Thus, we speculate that SHOX upstream deletions occur at a lower frequency because of the structural organization of this genomic region and/or that SHOX upstream deletions may cause a phenotype that differs from the one observed in LWD.