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. Using fluorescence in situ hybridization and single nucleotide polymorphism studies, we identified an interval of approximately 200 kb that was deleted in all tested affected family members but retained in the unaffected members and in 100 control individuals. Comparative genomic analysis of this interval revealed eight highly conserved non-genic elements between 48 and 215 kb downstream of the SHOX gene. As mice do not have a Shox gene, we analysed the enhancer potential in chicken embryos using a green fluorescent protein reporter construct driven by the beta-globin promoter, by in ovo electroporation of the limb bud. We observed cis-regulatory activity in three of the eight non-genic elements in the developing limbs arguing for an extensive control region of this gene. These findings are consistent with the idea that the deleted region in the affected families contains several distinct elements that regulate Shox expression in the developing limb. Furthermore, the deletion of these elements in humans generates a phenotype apparently undistinguishable to those patients identified with mutations in the SHOX coding region and, for the first time, demonstrates the potential of an in vivo assay in chicken to monitor putative enhancer activity in relation to human disease.

During mouse pre-implantation development, extra-embryonic primitive endoderm (PrE) and pluripotent epiblast precursors are specified in the inner cell mass (ICM) of the early blastocyst in a ‘salt and pepper’ manner, and are subsequently sorted into two distinct layers. Positional cues provided by the blastocyst cavity are thought to be instrumental for cell sorting; however, the sequence of events and the mechanisms that control this segregation remain unknown. Here, we show that atypical protein kinase C (aPKC), a protein associated with apicobasal polarity, is specifically enriched in PrE precursors in the ICM prior to cell sorting and prior to overt signs of cell polarisation. aPKC adopts a polarised localisation in PrE cells only after they reach the blastocyst cavity and form a mature epithelium, in a process that is dependent on FGF signalling. To assess the role of aPKC in PrE formation, we interfered with its activity using either chemical inhibition or RNAi knockdown. We show that inhibition of aPKC from the mid blastocyst stage not only prevents sorting of PrE precursors into a polarised monolayer but concomitantly affects the maturation of PrE precursors. Our results suggest that the processes of PrE and epiblast segregation, and cell fate progression are interdependent, and place aPKC as a central player in the segregation of epiblast and PrE progenitors in the mouse blastocyst.

During neurogenesis in Xenopus, apicobasally polarised superficial and non-polar deep cells take up different fates: deep cells become primary neurons while superficial cells stay as progenitors. It is not known whether the proteins that affect cell polarity also affect cell fate and how membrane polarity information may be transmitted to the nucleus. Here, we examine the role of the polarity components, apically enriched aPKC and basolateral Lgl2, in primary neurogenesis. We report that a membrane-tethered form of aPKC (aPKC-CAAX) suppresses primary neurogenesis and promotes cell proliferation. Unexpectedly, both endogenous aPKC and aPKC-CAAX show some nuclear localisation. A constitutively active aPKC fused to a nuclear localisation signal has the same phenotypic effect as aPKC-CAAX in that it suppresses neurogenesis and enhances proliferation. Conversely, inhibiting endogenous aPKC with a dominant-negative form that is restricted to the nucleus enhances primary neurogenesis. These observations suggest that aPKC has a function in the nucleus that is important for cell fate specification during primary neurogenesis. In a complementary experiment, overexpressing basolateral Lgl2 causes depolarisation and internalisation of superficial cells, which form ectopic neurons when supplemented with a proneural factor. These findings suggest that both aPKC and Lgl2 affect cell fate, but that aPKC is a nuclear determinant itself that might shuttle from the membrane to the nucleus to control cell proliferation and fate; loss of epithelial cell polarity by Lgl2 overexpression changes the position of the cells and is permissive for a change in cell fate.

SUMMARYAs studies aim increasingly to understand key, evolutionarily conserved properties of biological systems, the ability to move transgenesis experiments efficiently between organisms becomes essential. DNA constructions used in transgenesis usually contain four elements, including sequences that facilitate transgene genome integration, a selectable marker and promoter elements driving a coding gene. Linking these four elements in a DNA construction, however, can be a rate-limiting step in the design and creation of transgenic organisms. In order to expedite the construction process and to facilitate cross-species collaborations, we have incorporated the four common elements of transgenesis into a modular, recombination-based cloning system called pTransgenesis. Within this framework, we created a library of useful coding sequences, such as various fluorescent protein, Gal4, Cre-recombinase and dominant-negative receptor constructs, which are designed to be coupled to modular, species-compatible selectable markers, promoters and transgenesis facilitation sequences. Using pTransgenesis in Xenopus, we demonstrate Gal4-UAS binary expression, Cre-loxP-mediated fate-mapping and the establishment of novel, tissue-specific transgenic lines. Importantly, we show that the pTransgenesis resource is also compatible with transgenesis in Drosophila, zebrafish and mammalian cell models. Thus, the pTransgenesis resource fosters a cross-model standardization of commonly used transgenesis elements, streamlines DNA construct creation and facilitates collaboration between researchers working on different model organisms.

We report the characterization of the nuclear localization signal (NLS) of the short stature homeobox gene SHOX. Mutations within the SHOX gene cause Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LD) as well as idiopathic short stature (ISS). Furthermore, haploinsufficiency of SHOX has also been implicated in Turner syndrome. SHOX has been shown to be a cell-type-specific transcriptional activator that localizes to the nucleus. The SHOX protein contains a central homeodomain that together with its transactivation domain regulates the transcription of its target sequences within the nucleus. The sequences for its nuclear localization have not been identified yet. Experimental characterization of SHOX-NLS by deletion mapping identified a non-classic type basic signal, AKCRK, in the recognition helix of the homeodomain. Fusion of this stretch of five amino acids to a cytoplasmic reporter protein resulted in its nuclear translocation. Functional analysis of a missense mutation R173C (C517T) affecting the identified SHOX-NLS in two families with LWS and LD showed that the mutated SHOX protein is unable to enter the nucleus. Conversely, we can demonstrate that insertion of the identified signal adjacent to the mutant site can restore its nuclear translocation. These results establish impairment of nuclear localization as a mechanistic basis for SHOX-related diseases.