Planar cell polarity proteins determine basal cell height in the later stage embryonic mouse epidermis'.

Background: Complex organ formation requires the coordinated morphogenesis of adjacent tissue layers. Here, a role for the planar cell polarity (PCP) proteins Fz6 and Celsr1 in generating squamous basal cells in the later stage embryonic epidermis of the mouse is reported, which impacts upon the shape of overlying suprabasal cells. Methods: The depth of the epidermis and basal layer as well as cell proliferation index was scored from immunostained wax sections taken from different mouse embryos mutant in planar cell polarity signalling and their wild-type littermates. Orientation of epidermal cell division in Celsr1 Crash/Crash mutants was determined from thick frozen immunostained sections. Immunostained wax sections of wild-type skin explants cultured using the Lumox method enabled any changes in epidermal and basal layer depth to be measured following the release of surface tension upon dissection of skin away from the whole embryo.   Results: Increased numbers of columnar and cuboidal basal epidermal cells were observed in fz6 and Celsr1 mouse mutants including Celsr1 Crash/Crash which correlated with more rounded suprabasal cells and a thicker epidermis. Conclusions: Altogether these data support tissue intrinsic roles for PCP proteins in 'outside-in' (radial) skin architecture.

The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.

A role for core planar polarity proteins in cell contact-mediated orientation of planar cell division across the mammalian embryonic skin.

The question of how cell division orientation is determined is fundamentally important for understanding tissue and organ shape in both healthy or disease conditions. Here we provide evidence for cell contact-dependent orientation of planar cell division in the mammalian embryonic skin. We propose a model where the core planar polarity proteins Celsr1 and Frizzled-6 (Fz6) communicate the long axis orientation of interphase basal cells to neighbouring basal mitoses so that they align their horizontal division plane along the same axis. The underlying mechanism requires a direct, cell surface, planar polarised cue, which we posit depends upon variant post-translational forms of Celsr1 protein coupled to Fz6. Our hypothesis has parallels with contact-mediated division orientation in early C. elegans embryos suggesting functional conservation between the adhesion-GPCRs Celsr1 and Latrophilin-1. We propose that linking planar cell division plane with interphase neighbour long axis geometry reinforces axial bias in skin spreading around the mouse embryo body.

Epiboly generates the epidermal basal monolayer and spreads the nascent mammalian skin to enclose the embryonic body.

Epiboly is a morphogenetic process that is employed in the surface ectoderm of anamniotes during gastrulation to cover the entire embryo. We propose here that mammals also utilise this process to expand the epidermis and enclose the body cavity and spinal cord with a protective surface covering. Our data supports a model whereby epidermal spreading is driven by the primary establishment of the epidermal basal progenitor monolayer through radial cell intercalation of a multi-layered epithelium towards the basal lamina. By using a suspension organotypic culture strategy, we find that this process is fibronectin-dependent and autonomous to the skin. The radial cell rearrangements that drive epidermal spreading also require ROCK activity but are driven by cell protrusions and not myosin II contractility. Epidermal progenitor monolayer formation and epidermal spreading are delayed in Crash mice, which possess a dominant mutation in Celsr1, an orthologue of the core planar cell polarity (PCP) Drosophila protein Flamingo (also known as Stan). We observe a failure of ventral enclosure in Crash mutants suggesting that defective epidermal spreading might underlie some ventral wall birth defects.

Basal enrichment within neuroepithelia suggests novel function(s) for Celsr1 protein.

A characteristic of the 7TM-cadherins, Flamingo and Celsr1, is their asymmetric protein distribution and polarized activity at neighboring epithelial cell interfaces along defined axes of planar cell polarity. Here, we describe a novel distribution of Celsr1 protein to the basal surface of neuroepithelial cells within both the early neural tube and a less well-defined group of ventricular zone cells at the midline of the developing spinal cord. Importantly, this basal enrichment is lost in embryos homozygous for a mutant Celsr1 allele. We also demonstrate an intimate association between basal enrichment of Celsr1 protein and dorsal sensory tract morphogenesis, an intriguing spatio-temporal organization of Celsr1 protein along the apico-basal neuroepithelial axis suggestive of multiple Celsr1 protein isoforms and the existence of distinct cell surface Celsr1 protein species with direct signaling potential. Together, these data raise compelling new questions concerning the role of Celsr1 during neural development.

The PCP genes Celsr1 and Vangl2 are required for normal lung branching morphogenesis.

The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1(Crsh) and Vangl2(Lp) mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies.

7TM-Cadherins: developmental roles and future challenges.

The 7TM-Cadherins, Celsr/Flamingo/Starry night, represent a unique subgroup of adhesion-GPCRs containing atypical cadherin repeats, capable of homophilic interaction, linked to the archetypal adhesion-GPCR seven-transmembrane domain. Studies in Drosophila provided a first glimpse of their functional properties, most notably in the regulation of planar cell polarity (PCP) and in the formation of neural architecture. Many of the developmental functions identified in flies are conserved in vertebrates with PCP predicted to influence the development of multiple organ systems. Details of the molecular and cellular functions of 7TM-Cadherins are slowly emerging but many questions remain unanswered. Here the developmental roles of 7TM-Cadherins are discussed and future challenges in understanding their molecular and cellular roles are explored.

Combinatorial activity of Flamingo proteins directs convergence and extension within the early zebrafish embryo via the planar cell polarity pathway.

The seven-transmembrane protocadherin, Flamingo, functions in a number of processes during Drosophila development, including planar cell polarity (PCP). To assess the role(s) of Flamingo1/Celsr1 (Fmi1) during vertebrate embryogenesis we have exploited the zebrafish system, identifying two Fmi1 orthologues (zFmi1a and zFmi1b) and employing morpholinos to induce mis-splicing of zebrafish fmi1 mRNAs, to both imitate mutations identified in Drosophila flamingo and generate novel aberrant Flamingo proteins. We demonstrate that in the zebrafish gastrula, Fmi1 proteins function in concert with each other and with the vertebrate PCP proteins, Wnt11 and Strabismus, to mediate convergence and extension during gastrulation, without altering early dorso-ventral patterning. We show that zebrafish Fmi1a promotes extension of the entire antero-posterior axis of the zebrafish gastrula including prechordal plate and ventral diencephalic precursors. However, while we show that control over axial extension is autonomous, we find that Fmi1a is not required within lateral cells undergoing dorsal convergence.

Expression of the Celsr/flamingo homologue, c-fmi1, in the early avian embryo indicates a conserved role in neural tube closure and additional roles in asymmetry and somitogenesis.

Flamingo is one of a core group of proteins that regulate planar cell polarity of epithelial structures within the Drosophila embryo while their vertebrate counterparts have been implicated in the coordination of convergent extension movements during gastrulation and in neural tube closure, suggesting that planar polarity mechanisms also function in these processes. Failure of neural tube closure is one of the most common human birth defects, and a murine flamingo (fmi) homologue, Celsr1/fmi-1, was identified as the defective gene in two mouse mutants exhibiting failure of closure 1 of the neural tube. This failure resulted in craniorachischisis in which the neural tube is open from the midbrain posteriorly. The avian embryo provides a tractable system to study neural tube closure. We have identified a chick Celsr1/fmi-1 orthologue, c-fmi1 and provide the first study of expression of an avian flamingo gene. We show that expression is highly dynamic in the early embryo and that c-fmi1 transcripts become enriched within the avian neural epithelium at the initiation of neural tube closure, suggesting a conserved function for Flamingo proteins in this process. Our data also suggest a role for c-fmi1 in myotome development.