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.

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 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.

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.

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.

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.

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 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.

Celsr1 and Celsr2 exhibit distinct adhesive interactions and contributions to planar cell polarity.

Cadherin EGF LAG seven-pass G-type receptor (Celsr) proteins 1-3 comprise a subgroup of adhesion GPCRs whose functions range from planar cell polarity (PCP) signaling to axon pathfinding and ciliogenesis. Like its Drosophila ortholog, Flamingo, mammalian Celsr1 is a core component of the PCP pathway, which, among other roles, is responsible for the coordinated alignment of hair follicles across the skin surface. Although the role of Celsr1 in epidermal planar polarity is well established, the contribution of the other major epidermally expressed Celsr protein, Celsr2, has not been investigated. Here, using two new CRISPR/Cas9-targeted Celsr1 and Celsr2 knockout mouse lines, we define the relative contributions of Celsr1 and Celsr2 to PCP establishment in the skin. We find that Celsr1 is the major Celsr family member involved in epidermal PCP. Removal of Celsr1 function alone abolishes PCP protein asymmetry and hair follicle polarization, whereas epidermal PCP is unaffected by loss of Celsr2. Further, elimination of both Celsr proteins only minimally enhances the Celsr1 -/- phenotype. Using FRAP and junctional enrichment assays to measure differences in Celsr1 and Celsr2 adhesive interactions, we find that compared to Celsr1, which stably enriches at junctional interfaces, Celsr2 is much less efficiently recruited to and immobilized at junctions. As the two proteins seem equivalent in their ability to interact with core PCP proteins Vangl2 and Fz6, we suggest that perhaps differences in homophilic adhesion contribute to the differential involvement of Celsr1 and Celsr2 in epidermal PCP.

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.

Celsr1 adhesive interactions mediate the asymmetric organization of planar polarity complexes.

To orchestrate collective polarization across tissues, planar cell polarity (PCP) proteins localize asymmetrically to cell junctions, a conserved feature of PCP that requires the atypical cadherin Celsr1. We report that mouse Celsr1 engages in both trans- and cis-interactions, and organizes into dense and highly stable punctate assemblies. We provide evidence suggesting that PCP-mutant variant of Celsr1, Celsr1Crsh, selectively impairs lateral cis-interactions. Although Celsr1Crsh mediates cell adhesion in trans, it displays increased mobility, diminishes junctional enrichment, and fails to engage in homophilic adhesion with the wild-type protein, phenotypes that can be rescued by ectopic cis-dimerization. Using biochemical and super-resolution microscopy approaches, we show that although Celsr1Crsh physically interacts with PCP proteins Frizzled6 and Vangl2, it fails to organize these proteins into asymmetric junctional complexes. Our results suggest mammalian Celsr1 functions not only as a trans-adhesive homodimeric bridge, but also as an organizer of intercellular Frizzled6 and Vangl2 asymmetry through lateral, cis-interactions.

A Live-Cell Screen for Altered Erk Dynamics Reveals Principles of Proliferative Control.

Complex, time-varying responses have been observed widely in cell signaling, but how specific dynamics are generated or regulated is largely unknown. One major obstacle has been that high-throughput screens are typically incompatible with the live-cell assays used to monitor dynamics. Here, we address this challenge by screening a library of 429 kinase inhibitors and monitoring extracellular-regulated kinase (Erk) activity over 5 h in more than 80,000 single primary mouse keratinocytes. Our screen reveals both known and uncharacterized modulators of Erk dynamics, including inhibitors of non-epidermal growth factor receptor (EGFR) receptor tyrosine kinases (RTKs) that increase Erk pulse frequency and overall activity. Using drug treatment and direct optogenetic control, we demonstrate that drug-induced changes to Erk dynamics alter the conditions under which cells proliferate. Our work opens the door to high-throughput screens using live-cell biosensors and reveals that cell proliferation integrates information from Erk dynamics as well as additional permissive cues.

Epithelial geometry regulates spindle orientation and progenitor fate during formation of the mammalian epidermis.

The control of cell fate through oriented cell division is imperative for proper organ development. Basal epidermal progenitor cells divide parallel or perpendicular to the basement membrane to self-renew or produce differentiated stratified layers, but the mechanisms regulating the choice between division orientations are unknown. Using time-lapse imaging to follow divisions and fates of basal progenitors, we find that mouse embryos defective for the planar cell polarity (PCP) gene, Vangl2, exhibit increased perpendicular divisions and hyperthickened epidermis. Surprisingly, this is not due to defective Vangl2 function in the epidermis, but to changes in cell geometry and packing that arise from the open neural tube characteristic of PCP mutants. Through regional variations in epidermal deformation and physical manipulations, we show that local tissue architecture, rather than cortical PCP cues, regulates the decision between symmetric and stratifying divisions, allowing flexibility for basal cells to adapt to the needs of the developing tissue.

Counter-rotational cell flows drive morphological and cell fate asymmetries in mammalian hair follicles.

Organ morphogenesis is a complex process coordinated by cell specification, epithelial-mesenchymal interactions and tissue polarity. A striking example is the pattern of regularly spaced, globally aligned mammalian hair follicles, which emerges through epidermal-dermal signaling and planar polarized morphogenesis. Here, using live-imaging, we discover that developing hair follicles polarize through dramatic cell rearrangements organized in a counter-rotational pattern of cell flows. Upon hair placode induction, Shh signaling specifies a radial pattern of progenitor fates that, together with planar cell polarity, induce counter-rotational rearrangements through myosin and ROCK-dependent polarized neighbour exchanges. Importantly, these cell rearrangements also establish cell fate asymmetry by repositioning radial progenitors along the anterior-posterior axis. These movements concurrently displace associated mesenchymal cells, which then signal asymmetrically to maintain polarized cell fates. Our results demonstrate how spatial patterning and tissue polarity generate an unexpected collective cell behaviour that in turn, establishes both morphological and cell fate asymmetry.

Trans-endocytosis of Planar Cell Polarity Complexes during Cell Division.

To coordinate epithelial architecture with proliferation, cell polarity proteins undergo extensive remodeling during cell division [1-3]. A dramatic example of polarity remodeling occurs in proliferative basal cells of mammalian epidermis whereupon cell division, transmembrane planar cell polarity (PCP) proteins are removed from the cell surface via bulk endocytosis [4]. PCP proteins form intercellular complexes, linked by Celsr1-mediated homophilic adhesion, that coordinate polarity non-autonomously between cells [5, 6]. Thus, the mitotic reorganization of PCP proteins must alter not only proteins intrinsic to the dividing cell but also their interacting partners on neighboring cells. Here, we show that intercellular Celsr1 complexes that connect dividing cells with their neighbors remain intact during mitotic internalization, resulting in an uptake of Celsr1 protein from interphase neighbors. Trans-internalized Celsr1 carries with it additional core PCP proteins, including the posteriorly enriched Fz6 and anteriorly enriched Vangl2. Cadherin-mediated homophilic adhesion is necessary for trans-endocytosis, and adhesive junctional PCP complexes appear to be destined for degradation upon internalization. Surprisingly, whereas Fz6 and Vangl2 both internalize in trans, Vangl2 proteins intrinsic to the dividing cell remain associated with the plasma membrane. Persistent Vangl2 stabilizes Celsr1 and impedes its internalization, suggesting that dissociation of Vangl2 from Celsr1 is a prerequisite for Celsr1 endocytosis. These results demonstrate an unexpected transfer of PCP complexes between neighbors and suggest that the Vangl2 population that persists at the membrane during cell division could serve as an internal cue for establishing PCP in new daughter cells.

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.