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.

Initially identified in DNA damage repair, ATM-interactor (ATMIN) further functions as a transcriptional regulator of lung morphogenesis. Here we analyse three mouse mutants, Atmingpg6/gpg6, AtminH210Q/H210Q and Dynll1GT/GT, revealing how ATMIN and its transcriptional target dynein light chain LC8-type 1 (DYNLL1) are required for normal lung morphogenesis and ciliogenesis. Expression screening of ciliogenic genes confirmed Dynll1 to be controlled by ATMIN and further revealed moderately altered expression of known intraflagellar transport (IFT) protein-encoding loci in Atmin mutant embryos. Significantly, Dynll1GT/GT embryonic cilia exhibited shortening and bulging, highly similar to the characterised retrograde IFT phenotype of Dync2h1. Depletion of ATMIN or DYNLL1 in cultured cells recapitulated the in vivo ciliogenesis phenotypes and expression of DYNLL1 or the related DYNLL2 rescued the effects of loss of ATMIN, demonstrating that ATMIN primarily promotes ciliogenesis by regulating Dynll1 expression. Furthermore, DYNLL1 as well as DYNLL2 localised to cilia in puncta, consistent with IFT particles, and physically interacted with WDR34, a mammalian homologue of the Chlamydomonas cytoplasmic dynein 2 intermediate chain that also localised to the cilium. This study extends the established Atmin-Dynll1 relationship into a developmental and a ciliary context, uncovering a novel series of interactions between DYNLL1, WDR34 and ATMIN. This identifies potential novel components of cytoplasmic dynein 2 and furthermore provides fresh insights into the molecular pathogenesis of human skeletal ciliopathies.

Vertebrate organs show consistent left-right (L-R) asymmetry in placement and patterning. To identify genes involved in this process we performed an ENU-based genetic screen. Of 135 lines analyzed 11 showed clear single gene defects affecting L-R patterning, including 3 new alleles of known L-R genes and mutants in novel L-R loci. We identified six lines (termed "gasping") that, in addition to abnormal L-R patterning and associated cardiovascular defects, had complex phenotypes including pulmonary agenesis, exencephaly, polydactyly, ocular and craniofacial malformations. These complex abnormalities are present in certain human disease syndromes (e.g., HYLS, SRPS, VACTERL). Gasping embryos also show defects in ciliogenesis, suggesting a role for cilia in these human congenital malformation syndromes. Our results indicate that genes controlling ciliogenesis and left-right asymmetry have, in addition to their known roles in cardiac patterning, major and unexpected roles in pulmonary, craniofacial, ocular and limb development with implications for human congenital malformation syndromes. Developmental Dynamics 238:581-594, 2009.

BackgroundVertebrates show clear asymmetry in left-right (L-R) patterning of their organs and associated vasculature. During mammalian development a cilia driven leftwards flow of liquid leads to the left-sided expression of Nodal, which in turn activates asymmetric expression of the transcription factor Pitx2. While Pitx2 asymmetry drives many aspects of asymmetric morphogenesis, it is clear from published data that additional asymmetrically expressed loci must exist.ResultsA L-R expression screen identified the cytoskeletally-associated gene, actin binding lim protein 1 (Ablim1), as asymmetrically expressed in both the node and left lateral plate mesoderm (LPM). LPM expression closely mirrors that of Nodal. Significantly, Ablim1 LPM asymmetry was detected in the absence of detectable Nodal. In the node, Ablim1 was initially expressed symmetrically across the entire structure, resolving to give a peri-nodal ring at the headfold stage in a flow and Pkd2-dependent manner. The peri-nodal ring of Ablim1 expression became asymmetric by the mid-headfold stage, showing stronger right than left-sided expression. Node asymmetry became more apparent as development proceeded; expression retreated in an anticlockwise direction, disappearing first from the left anterior node. Indeed, at early somite stages Ablim1 shows a unique asymmetric expression pattern, in the left lateral plate and to the right side of the node.ConclusionLeft LPM Ablim1 is expressed in the absence of detectable LPM Nodal, clearly revealing existence of a Pitx2 and Nodal-independent left-sided signal in mammals. At the node, a previously unrecognised action of early nodal flow and Pkd2 activity, within the pit of the node, influences gene expression in a symmetric manner. Subsequent Ablim1 expression in the peri-nodal ring reveals a very early indication of L-R asymmetry. Ablim1 expression analysis at the node acts as an indicator of nodal flow. Together these results make Ablim1 a candidate for controlling aspects of L-R identity and patterning.

Meckel-Gruber syndrome (MKS) is a rare lethal autosomal recessive disease that is the most common cause of syndromic neural tube defects. There are five causative genes, mapped to six known disease loci, whose products encode proteins involved in formation and function of primary cilia. One of these genes, MKS3 (located on chromosome 8q22.1), is now well-characterized and a number of mutations in this gene have now been identified in MKS patients, and for the allelic condition Joubert syndrome. MKS3 encodes meckelin, a putative Frizzled-like transmembrane receptor that localizes to the ciliary membrane and apical cell surface in ciliated cells.MKS3 mutation screening of MKS patients has revealed a number of missense, in-frame deletion and synonymous sequence variants of unknown pathogenic potential. To assess the pathogenicity of these variants in functional assays, we determined the subcellular localization of mutated forms of HA epitopetagged meckelin, in comparison to a wild-type construct and endogenous meckelin. We introduced patient mutations using a standard site-directed mutagenesis strategy. Ciliated cells were transiently transfected with these constructs, immunostained with anti-HA and anti-calreticulin (an ER marker) and visualized by confocal microscopy. This revealed diverse meckelin localizations, which differ from the wild-type and endogenous, including aberrant ER aggregation. Our results demonstrate that mislocalization of meckelin is the probable pathogenic mechanism for a number of mutations in MKS.On-going work is assessing the consequences of these mutations on non-canonical Wnt signaling.Noto encodes a homeobox transcription factor that is expressed in the node and nascent notochord [Abdelkhalek et al., 2004]. Loss of Noto disrupts ciliogenesis in the embryonic node and node morphology [Beckers et al., 2007]. To further investigate the mechanisms that regulate ciliogenesis and node morphology, Noto is being analysed with respect to its regulation, as well as downstream effectors.To identify relevant regulatory sequences we used single copy insertions of promoter-reporter constructs into ES cells [Bronson et al., 1996] and analysed these in chimeric embryos. This analysis led to the localisation of the novel enhancer region NOCE (node and notochord-specific enhancer) that is necessary and suf-ficient to resemble the expression of endogenous Noto. NOCE is 500 bp long and contains the previously non-described regulatory sequence UFO (unknown factor binding site) that is necessary for its function. The ongoing analysis of NOCE and UFO should help to identify new upstream factors regulating node and notochordspecific gene expression.Furthermore Foxj1, another transcription factor that is essential for ciliogenesis in many tissues, was identified as an important downstream effector of Noto. By comparative analyses of Noto and Foxj1 mutants, as well as gene replacement, the specific functions of Noto and Foxj1 in the node are currently being delineated.The ENU-derived, embryonic lethal mouse ...