Region-specific reversal of epidermal planar polarity in the rosette fancy mouse.

The planar cell polarity (PCP) pathway collectively orients cells with respect to a body axis. Hair follicles of the murine epidermis provide a striking readout of PCP activity in their uniform alignment across the skin. Here, we characterize, from the molecular to tissue-scale, PCP establishment in the rosette fancy mouse, a natural variant with posterior-specific whorls in its fur, to understand how epidermal polarity is coordinated across the tissue. We find that rosette hair follicles emerge with reversed orientations specifically in the posterior region, creating a mirror image of epidermal polarity. The rosette trait is associated with a missense mutation in the core PCP gene Fzd6, which alters a consensus site for N-linked glycosylation, inhibiting its membrane localization. Unexpectedly, the Fzd6 trafficking defect does not block asymmetric localization of the other PCP proteins. Rather, the normally uniform axis of PCP asymmetry rotates where the PCP-directed cell movements that orient follicles are reversed, suggesting the PCP axis rotates 180°. Collectively, our multiscale analysis of epidermal polarity reveals PCP patterning can be regionally decoupled to produce posterior whorls in the rosette fancy mouse.

A Van Gogh/Vangl tyrosine phosphorylation switch regulates its interaction with core Planar Cell Polarity factors Prickle and Dishevelled.

Epithelial tissues can be polarized along two axes: in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl in mammals). Here, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, we dissect how Vang/Vangl phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dishevelled (Dsh/Dvl1-3 in mammals) and Prickle (Pk/Pk1-3). We demonstrate that Pk and Dsh/Dvl bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl phosphorylation promotes its binding to Prickle, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dishevelled. Thus phosphorylation of this tyrosine appears to promote the formation of the mature Vang/Vangl-Pk complex during PCP establishment and conversely it inhibits the Vang interaction with the antagonistic effector Dishevelled. Intriguingly, the phosphorylation state of this tyrosine might thus serve as a switch between transient interactions with Dishevelled and stable formation of Vang-Pk complexes during PCP establishment.

New mouse models for high resolution and live imaging of planar cell polarity proteins in vivo.

The collective polarization of cellular structures and behaviors across a tissue plane is a near universal feature of epithelia known as planar cell polarity (PCP). This property is controlled by the core PCP pathway, which consists of highly conserved membrane-associated protein complexes that localize asymmetrically at cell junctions. Here, we introduce three new mouse models for investigating the localization and dynamics of transmembrane PCP proteins: Celsr1, Fz6 and Vangl2. Using the skin epidermis as a model, we characterize and verify the expression, localization and function of endogenously tagged Celsr1-3xGFP, Fz6-3xGFP and tdTomato-Vangl2 fusion proteins. Live imaging of Fz6-3xGFP in basal epidermal progenitors reveals that the polarity of the tissue is not fixed through time. Rather, asymmetry dynamically shifts during cell rearrangements and divisions, while global, average polarity of the tissue is preserved. We show using super-resolution STED imaging that Fz6-3xGFP and tdTomato-Vangl2 can be resolved, enabling us to observe their complex localization along junctions. We further explore PCP fusion protein localization in the trachea and neural tube, and discover new patterns of PCP expression and localization throughout the mouse embryo.

Overriding native cell coordination enhances external programming of collective cell migration.

As collective cell migration is essential in biological processes spanning development, healing, and cancer progression, methods to externally program cell migration are of great value. However, problems can arise if the external commands compete with strong, preexisting collective behaviors in the tissue or system. We investigate this problem by applying a potent external migratory cue-electrical stimulation and electrotaxis-to primary mouse skin monolayers where we can tune cell-cell adhesion strength to modulate endogenous collectivity. Monolayers with high cell-cell adhesion showed strong natural coordination and resisted electrotactic control, with this conflict actively damaging the leading edge of the tissue. However, reducing preexisting coordination in the tissue by specifically inhibiting E-cadherin-dependent cell-cell adhesion, either by disrupting the formation of cell-cell junctions with E-cadherin-specific antibodies or rapidly dismantling E-cadherin junctions with calcium chelators, significantly improved controllability. Finally, we applied this paradigm of weakening existing coordination to improve control and demonstrate accelerated wound closure in vitro. These results are in keeping with those from diverse, noncellular systems and confirm that endogenous collectivity should be considered as a key quantitative design variable when optimizing external control of collective migration.

Tissue morphodynamics: Translating planar polarity cues into polarized cell behaviors.

The ability of cells to collectively orient and align their behaviors is essential in multicellular organisms for unidirectional cilia beating, collective cell movements, oriented cell divisions, and asymmetric cell fate specification. The planar cell polarity pathway coordinates a vast and diverse array of collective cell behaviors by intersecting with downstream pathways that regulate cytoskeletal dynamics and intercellular signaling. How the planar polarity pathway translates directional cues to produce polarized cell behaviors is the focus of this review.

Planar cell polarity-dependent and independent functions in the emergence of tissue-scale hair follicle patterns.

Hair follicles of the mammalian epidermis display local order and global alignment, a complex pattern instructed by the core planar cell polarity (PCP) pathway. Here we address the contributions of core PCP genes, Van Gogh-like and Frizzled, to the establishment, local refinement, and global order of embryonic and postnatal hair follicles. We find that, similar to Fz6 mutants, the disordered hair patterns of Vangl2 mutants are refined over time and eventually corrected. In both mutants, we find that tissue-level reorientation occurs through locally coordinated follicle rotation at stereotyped locations. Strikingly, Vangl2 and Fz6 mutant follicles collectively rotate with opposing directionalities, suggesting that redundant core PCP signals contribute to their directed realignment. Consistently, global follicle alignment is not restored upon conditional ablation of both Vangl1 and Vangl2 genes. Instead, spatially distinct patterns of whorls and crosses emerge and persist even after a complete cycle of hair follicle regeneration. Thus, local refinement of hair follicles into higher order patterns can occur independently of the core PCP system, however, their global alignment with the body axes requires PCP function throughout morphogenesis, growth and regeneration.

Celsr1 coordinates the planar polarity of vestibular hair cells during inner ear development.

Vestibular hair cells of the inner ear are specialized receptors that detect mechanical stimuli from gravity and motion via the deflection of a polarized bundle of stereocilia located on their apical cell surfaces. The orientation of stereociliary bundles is coordinated between neighboring cells by core PCP proteins including the large adhesive G-protein coupled receptor Celsr1. We show that mice lacking Celsr1 have vestibular behavioral phenotypes including circling. In addition, we show that Celsr1 is asymmetrically distributed at cell boundaries between hair cells and neighboring supporting cells in the developing vestibular and auditory sensory epithelia. In the absence of Celsr1 the stereociliary bundles of vestibular hair cells are misoriented relative to their neighbors, a phenotype that is greatest in the cristae of the semicircular canals. Since horizontal semi-circular canal defects lead to circling in other mutant mouse lines, we propose that this PCP phenotype is the cellular basis of the circling behavior in Celsr1 mutants.

Dachsous1-Fat4 Signaling Controls Endothelial Cell Polarization During Lymphatic Valve Morphogenesis-Brief Report.

OBJECTIVE: The purpose of this study was to investigate the role of Fat4 and Dachsous1 signaling in the lymphatic vasculature. APPROACH AND RESULTS: Phenotypic analysis of the lymphatic vasculature was performed in mice lacking functional Fat4 or Dachsous1. The overall architecture of lymphatic vasculature is unaltered, yet both genes are specifically required for lymphatic valve morphogenesis. Valve endothelial cells (Prox1high [prospero homeobox protein 1] cells) are disoriented and failed to form proper valve leaflets. Using Lifeact-GFP (green fluorescent protein) mice, we revealed that valve endothelial cells display prominent actin polymerization. Finally, we showed the polarized recruitment of Dachsous1 to membrane protrusions and cellular junctions of valve endothelial cells in vivo and in vitro. CONCLUSIONS: Our data demonstrate that Fat4 and Dachsous1 are critical regulators of valve morphogenesis. This study highlights that valve defects may contribute to lymphedema in Hennekam syndrome caused by Fat4 mutations.

Structural Characterization and Statistical-Mechanical Model of Epidermal Patterns.

In proliferating epithelia of mammalian skin, cells of irregular polygon-like shapes pack into complex, nearly flat two-dimensional structures that are pliable to deformations. In this work, we employ various sensitive correlation functions to quantitatively characterize structural features of evolving packings of epithelial cells across length scales in mouse skin. We find that the pair statistics in direct space (correlation function) and Fourier space (structure factor) of the cell centroids in the early stages of embryonic development show structural directional dependence (statistical anisotropy), which is a reflection of the fact that cells are stretched, which promotes uniaxial growth along the epithelial plane. In the late stages, the patterns tend toward statistically isotropic states, as cells attain global polarization and epidermal growth shifts to produce the skin's outer stratified layers. We construct a minimalist four-component statistical-mechanical model involving effective isotropic pair interactions consisting of hard-core repulsion and extra short-range soft-core repulsion beyond the hard core, whose length scale is roughly the same as the hard core. The model parameters are optimized to match the sample pair statistics in both direct and Fourier spaces. By doing this, the parameters are biologically constrained. In contrast with many vertex-based models, our statistical-mechanical model does not explicitly incorporate information about the cell shapes and interfacial energy between cells; nonetheless, our model predicts essentially the same polygonal shape distribution and size disparity of cells found in experiments, as measured by Voronoi statistics. Moreover, our simulated equilibrium liquid-like configurations are able to match other nontrivial unconstrained statistics, which is a testament to the power and novelty of the model. The array of structural descriptors that we deploy enable us to distinguish between normal, mechanically deformed, and pathological skin tissues. Our statistical-mechanical model enables one to generate tissue microstructure at will for further analysis. We also discuss ways in which our model might be extended to better understand morphogenesis (in particular the emergence of planar cell polarity), wound healing, and disease-progression processes in skin, and how it could be applied to the design of synthetic tissues.

Functional screening in Drosophila identifies Alzheimer's disease susceptibility genes and implicates Tau-mediated mechanisms.

Using a Drosophila model of Alzheimer's disease (AD), we systematically evaluated 67 candidate genes based on AD-associated genomic loci (P < 10(-4)) from published human genome-wide association studies (GWAS). Genetic manipulation of 87 homologous fly genes was tested for modulation of neurotoxicity caused by human Tau, which forms neurofibrillary tangle pathology in AD. RNA interference (RNAi) targeting 9 genes enhanced Tau neurotoxicity, and in most cases reciprocal activation of gene expression suppressed Tau toxicity. Our screen implicates cindr, the fly ortholog of the human CD2AP AD susceptibility gene, as a modulator of Tau-mediated disease mechanisms. Importantly, we also identify the fly orthologs of FERMT2 and CELF1 as Tau modifiers, and these loci have been independently validated as AD susceptibility loci in the latest GWAS meta-analysis. Both CD2AP and FERMT2 have been previously implicated with roles in cell adhesion, and our screen additionally identifies a fly homolog of the human integrin adhesion receptors, ITGAM and ITGA9, as a modifier of Tau neurotoxicity. Our results highlight cell adhesion pathways as important in Tau toxicity and AD susceptibility and demonstrate the power of model organism genetic screens for the functional follow-up of human GWAS.

Integrin-dependent anchoring of a stem-cell niche.

Interactions between stem cells and their surrounding microenvironment, or niche, are critical for the establishment and maintenance of stem-cell properties. The adult Drosophila testis contains a morphologically discrete stem-cell niche, the 'hub'. The small cluster of non-dividing, somatic hub cells at the anterior tip of the fly testis is contacted by the germline stem cells (GSCs), which retain their stem-cell character through the direct association with the hub. Here we show that integrin-mediated adhesion is important for maintaining the correct position of embryonic hub cells during gonad morphogenesis. The misplaced hub in integrin-deficient embryos directs the orientation of cell divisions in the presumptive GSCs, a hallmark of the active germline stem-cell niche. A decrease in integrin-mediated adhesion in adult testes, which resulted in a loss of the hub and the stem-cell population, revealed the importance of hub-cell anchoring. Finally, we show that an extracellular matrix (ECM) is present around the gonad during late embryogenesis and that this ECM is defective in integrin-deficient gonads. On the basis of our data, we propose that integrins are required for the attachment of the hub cells to the ECM, which is essential for maintaining the stem-cell niche.

Evaluating Planar Cell Polarity in the Developing Mouse Epidermis.

Epidermal tissues are among the most striking examples of planar polarity. Insect bristles, fish scales, and mammalian fur are all uniformly oriented along an animal's body axis. The collective alignment of epidermal structures provides a valuable system to interrogate the signaling mechanisms that coordinate cellular behaviors at both local and tissue-levels. Here, we provide methods to analyze the planar organization of hair follicles within the mouse epidermis. Hair follicles are specified and bud into the underlying dermis during embryonic development. Shortly after, follicle cells dynamically rearrange to orient each follicle toward the anterior of the animal. When directional signaling is disrupted, hair follicles become misoriented. In this chapter, we describe how to create a spatial map of hair follicle orientations to reveal tissue-scale patterns in both embryonic and postnatal skin. Additionally, we provide a live imaging protocol that can be used to monitor cell movements in embryonic skin explants to reveal the cellular behaviors that polarize the hair follicle itself.

Vangl-dependent mesenchymal thinning shapes the distal lung during murine sacculation.

The planar cell polarity (PCP) complex is speculated to function in murine lung development, where branching morphogenesis generates an epithelial tree whose distal tips expand dramatically during sacculation. Here, we show that PCP is dispensable in the airway epithelium for sacculation. Rather, we find a Celsr1-independent role for the PCP component Vangl in the pulmonary mesenchyme: loss of Vangl1/2 inhibits mesenchymal thinning and expansion of the saccular epithelium. Further, loss of mesenchymal Wnt5a mimics sacculation defects observed in Vangl2-mutant lungs, implicating mesenchymal Wnt5a/Vangl signaling as a key regulator of late lung morphogenesis. A computational model predicts that sacculation requires a fluid mesenchymal compartment. Lineage-tracing and cell-shape analyses are consistent with the mesenchyme acting as a fluid tissue, suggesting that loss of Vangl1/2 impacts the ability of mesenchymal cells to exchange neighbors. Our data thus identify an explicit function for Vangl and the pulmonary mesenchyme in actively shaping the saccular epithelium.

An mTurq2-Col4a1 mouse model allows for live visualization of mammalian basement membrane development.

Basement membranes (BMs) are specialized sheets of extracellular matrix that underlie epithelial and endothelial tissues. BMs regulate the traffic of cells and molecules between compartments, and participate in signaling, cell migration, and organogenesis. The dynamics of mammalian BMs, however, are poorly understood, largely due to a lack of models in which core BM components are endogenously labeled. Here, we describe the mTurquoise2-Col4a1 mouse in which we fluorescently tag collagen IV, the main component of BMs. Using an innovative planar-sagittal live imaging technique to visualize the BM of developing skin, we directly observe BM deformation during hair follicle budding and basal progenitor cell divisions. The BM's inherent pliability enables dividing cells to remain attached to and deform the BM, rather than lose adhesion as generally thought. Using FRAP, we show BM collagen IV is extremely stable, even during periods of rapid epidermal growth. These findings demonstrate the utility of the mTurq2-Col4a1 mouse to shed new light on mammalian BM developmental dynamics.

Cell polarity: How to build an asymmetric bridge.

A new study provides key insights into planar cell polarity (PCP) establishment through the discovery of molecular asymmetries in the homotypic adhesive interactions of the PCP cadherin, Flamingo, resulting in the formation of asymmetric, intercellular bridges.

Mesenchymal Vangl facilitates airway elongation and widening independently of the planar cell polarity complex.

A hallmark of mammalian lungs is the fractal nature of the bronchial tree. In the adult, each successive generation of airways is a fraction of the size of the parental branch. This fractal structure is physiologically beneficial, as it minimizes the energy needed for breathing. Achieving this pattern likely requires precise control of airway length and diameter, as the branches of the embryonic airways initially lack the fractal scaling observed in those of the adult lung. In epithelial monolayers and tubes, directional growth can be regulated by the planar cell polarity (PCP) complex. Here, we comprehensively characterized the roles of PCP-complex components in airway initiation, elongation, and widening during branching morphogenesis of the murine lung. Using tissue-specific knockout mice, we surprisingly found that branching morphogenesis proceeds independently of PCP-component expression in the developing airway epithelium. Instead, we found a novel, Celsr1-independent role for the PCP component Vangl in the pulmonary mesenchyme. Specifically, mesenchymal loss of Vangl1/2 leads to defects in branch initiation, elongation, and widening. At the cellular level, we observe changes in the shape of smooth muscle cells that indicate a potential defect in collective mesenchymal rearrangements, which we hypothesize are necessary for lung morphogenesis. Our data thus reveal an explicit function for Vangl that is independent of the core PCP complex, suggesting a functional diversification of PCP components in vertebrate development. These data also reveal an essential role for the embryonic mesenchyme in generating the fractal structure of airways of the mature lung.

Bacterial DNA on the skin surface overrepresents the viable skin microbiome.

The skin microbiome provides vital contributions to human health. However, the spatial organization and viability of its bacterial components remain unclear. Here, we apply culturing, imaging, and molecular approaches to human and mouse skin samples, and find that the skin surface is colonized by fewer viable bacteria than predicted by bacterial DNA levels. Instead, viable skin-associated bacteria are predominantly located in hair follicles and other cutaneous invaginations. Furthermore, we show that the skin microbiome has a uniquely low fraction of viable bacteria compared to other human microbiome sites, indicating that most bacterial DNA on the skin surface is not associated with viable cells Additionally, a small number of bacterial families dominate each skin site and traditional sequencing methods overestimate both the richness and diversity of the skin microbiome. Finally, we performed an in vivo skin microbiome perturbation-recovery study using human volunteers. Bacterial 16S rRNA gene sequencing revealed that, while the skin microbiome is remarkably stable even in the wake of aggressive perturbation, repopulation of the skin surface is driven by the underlying viable population. Our findings help explain the dynamics of skin microbiome perturbation as bacterial DNA on the skin surface can be transiently perturbed but is replenished by a stable underlying viable population. These results address multiple outstanding questions in skin microbiome biology with significant implications for future efforts to study and manipulate it.

A Window into Mammalian Basement Membrane Development: Insights from the mTurq2-Col4a1 Mouse Model.

Basement membranes (BMs) are specialized sheets of extracellular matrix that underlie epithelial and endothelial tissues. BMs regulate traffic of cells and molecules between compartments, and participate in signaling, cell migration and organogenesis. The dynamics of mammalian BMs, however, are poorly understood, largely due to a lack of models in which core BM components are endogenously labelled. Here, we describe the mTurquoise2-Col4a1 mouse, in which we fluorescently tag collagen IV, the main component of BMs. Using an innovative Planar-Sagittal live imaging technique to visualize the BM of developing skin, we directly observe BM deformation during hair follicle budding and basal progenitor cell divisions. The BM's inherent pliability enables dividing cells to remain attached to and deform the BM, rather than lose adhesion as generally thought. Using FRAP, we show BM collagen IV is extremely stable, even during periods of rapid epidermal growth. These findings demonstrate the utility of the mTurq2-Col4a1 mouse to shed new light on mammalian BM developmental dynamics.

Region-specific reversal of epidermal planar polarity in the fancy rosette mouse.

The planar cell polarity (PCP) pathway collectively orients thousands of cells with respect to a body axis to direct cellular behaviors that are essential for embryonic morphogenesis. Hair follicles of the murine epidermis provide a striking readout of PCP activity in their uniform alignment along the entire skin surface. Here, we characterize, from the molecular to tissue-scale, PCP establishment in the rosette fancy mouse, a natural variant with posterior-specific whorls in its fur, to understand how epidermal polarity is coordinated across the tissue. We find that embryonic hair follicles of rosette mutants emerge with reversed orientations specifically in the posterior region, creating a mirror image of epidermal polarity. The rosette trait is associated with a missense mutation in the core PCP gene Fzd6 , which alters a consensus site for N-linked glycosylation and inhibits its membrane localization. Unexpectedly, this defect in Fzd6 trafficking, observed across the entire dorsal epidermis, does not interfere with the ability of other core PCP proteins to localize asymmetrically. Rather, the normally uniform axis of PCP asymmetry is disrupted and rotated in the posterior region such that polarity is reflected on either side of a transition zone. The result is a reversal of polarized cell movements that orient nascent follicles, specifically in the posterior of the embryo. Collectively, our multiscale analysis of epidermal polarity reveals PCP patterning can be regionally decoupled to produce the unique posterior whorls of the fancy rosette mouse. Summary: Region-specific rotation of the Planar Cell Polarity axis reverses posterior hair follicles in the fancy rosette mouse.

A squash and a squeeze.

Advanced imaging techniques reveal details of the interactions between the two layers of the embryonic midgut that influence its ultimate shape.