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.

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.

Ring Catalog: A resource for designing self-assembling RNA nanostructures.

Designing self-assembling RNA ring structures based on known 3D structural elements connected via linker helices is a challenging task due to the immense number of motif combinations, many of which do not lead to ring-closure. We describe an in silico solution to this design problem by combinatorial assembly of RNA 3-way junctions, bulges, and kissing loops, and tabulating the cases that lead to ring formation. The solutions found are made available in the form of a web-accessible Ring Catalog. As an example of a potential use of this resource, we chose a predicted RNA square structure consisting of five RNA strands and demonstrate experimentally that the self-assembly of those five strands leads to the formation of a square-like complex. This is a demonstration of a novel "design by catalog" approach to RNA nano-structure generation. The URL https://rnajunction.ncifcrf.gov/ringdb can be used to access the resource.

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.

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.

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.

Myosin 2-Induced Mitotic Rounding Enables Columnar Epithelial Cells to Interpret Cortical Spindle Positioning Cues.

During epithelial cell proliferation, planar alignment of the mitotic spindle allows the daughter cells to stay within the epithelium. Previous work has identified cortical cues that regulate spindle orientation and the division axis [1, 2]. One such cue is cortical Pins (LGN in vertebrates) [3-6], which recruits the conserved Mud/NuMA protein and the dynein/dynactin complex to the cortex. The dynein/dynactin motor complex pulls astral microtubules to orient the spindle. Cortical Pins can therefore dictate the division axis. In addition to cortical cues, cell shape can also serve as a division orientation cue [7-9]. Here, we investigated the interplay between cortical cues and cell shape in a proliferating tissue. We analyzed division orientation in the first mitotic divisions of the early Drosophila embryo, where groups of epithelial cells synchronously divide. Using chemical inhibitors, knockdowns, and mutants with known deficits in motor activity, we showed that the myosin 2 motor is required to orient cell division in the plane of a columnar epithelium. Disrupting myosin activity caused the division axis to orient perpendicular to the epithelial plane. This effect was independent of Pins cortical localization, which became uncoupled from spindle orientation. Instead, myosin motor activity was required for the formation of the actomyosin cortex and for cell rounding upon mitotic entry. We propose that mitotic cell rounding in columnar epithelia allows cells to properly interpret cortical cues that orient the spindle. In the absence of mitotic rounding, geometric cues imposed by tight cell packing prevail and cells divide along their long apical-basal axis.

Computational Generation of RNA Nanorings.

A variety of designed RNA ring structures (ranging from triangles to hexagonal rings) have been reported in the scientific literature. Designing self-assembling RNA ring structures from structural motifs is, however, a nontrivial problem as there are many combinations of motifs and linking helices. Moreover, most combinations of motifs and linker helices will not lead to ring closure. A solution to this problem was recently published using a "design-by-catalog" approach where motif combinations that lead to rings are precomputed and tabulated. Here we present a web-browser based workflow for creating RNA rings using Galaxy, a web-based platform that can be used for workflow management. An example of how these RNA rings are generated and processed to create a 3D model of the ring is discussed.

Effect of Cellular Therapy in Progression of Becker's Muscular Dystrophy: A Case Study.

Becker muscular dystrophy (BMD) is an inherited disorder due to deletions of the dystrophin gene that leads to muscle weakness. Effects of bone marrow mononuclear cell (BMMNC) transplantation in Muscular Dystrophy have shown to be safe and beneficial. We treated a 20-year-old male suffering from BMD with autologous BMMNC transplantation followed by multidisciplinary rehabilitation. He presented with muscle weakness and had difficulty in performing his activities. The BMMNCs were transplanted via intrathecal and intramuscular routes. The effects were measured on clinical and functional changes. Over 9 months, gradual improvement was noticed in muscle strength, respiratory functions and North Star Ambulatory Assessment Scale. Functional Independence Measure, Berg Balance Score, Brooke and Vignos Scale remained stable indicating halting of the progression. The case report suggests that cellular therapy combined with rehabilitation may have possibility of repairing and regenerating muscle fibers and decreasing the rate of progression of BMD.