Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation

Spindle orientation is often achieved by a complex of Pins/LGN, Mud/NuMa, Gαi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, laser ablation, and depletion of an adherens junction protein disrupted Pins planar polarity and spindle orientation. Furthermore, snail depletion, which abrogates ventral furrow forces, disrupted Pins polarization and spindle orientation, suggesting that morphogenetic movements and resulting forces transmitted through the tissue can polarize Pins and orient division. Thus, morphogenetic forces associated with mesoderm invagination result in planar polarized Pins to mediate division orientation at a distant region of the embryo during morphogenesis. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.

Abnormal larval neuromuscular junction morphology and physiology in Drosophila Prickle isoform mutants with defective axonal transport and adult seizure behavior

Previous studies have demonstrated that mutations of the Drosophila planar cell polarity gene prickle (pk) result in altered microtubule-mediated vesicular transport in larval motor axons, as well as adult neuronal circuit hyperexcitability and epileptic behavior. It is also known that mutant alleles of the prickle-prickle (pkpk) and prickle-spiny-legs (pksple) isoforms differ in phenotype but display isoform counterbalancing effects in heteroallelic pkpk/pksple flies to ameliorate adult motor circuit and behavioral hyperexcitability. We have further investigated the larval neuromuscular junction (NMJ) and uncovered robust phenotypes in both pkpk and pksple alleles (heretofore referred to as pk and sple alleles, respectively), including synaptic terminal overgrowth, as well as irregular motor axon terminal excitability, poor vesicle release synchronicity, and altered efficacy of synaptic transmission. We observed significant increase in whole-cell excitatory junctional potential (EJP) in pk homozygotes, which was restored to near WT level in pk/sple heterozygotes. We further examined motor terminal excitability sustained by presynaptic Ca2+ channels, under the condition of pharmacological blockade of Na+ and K+ channel function. Such manipulation revealed extreme Ca2+ channel-dependent nerve terminal excitability in both pk and sple mutants. However, when combined in pk/sple heterozygotes, such terminal hyper-excitability was restored to nearly normal. Focal recording from individual synaptic boutons revealed asynchronous vesicle release in both pk and sple homozygotes, which nevertheless persisted in pk/sple heterozygotes without indications of isoform counter-balancing effects. Similarly, the overgrowth at NMJs was not compensated in pk/sple heterozygotes, exhibiting an extremity comparable to that in pk and sple homozygotes. Our observations uncovered differential roles of the pk and sple isoforms and their distinct interactions in the various structural and functional aspects of the larval NMJ and adult neural circuits.

Loss of Planar Cell Polarity Effector Fuzzy Causes Renal Hypoplasia by Disrupting Several Signaling Pathways

In vertebrates, the planar cell polarity (PCP) pathway regulates tissue morphogenesis during organogenesis, including the kidney. Mutations in human PCP effector proteins have been associated with severe syndromic ciliopathies. Importantly, renal hypoplasia has been reported in some patients. However, the developmental disturbance that causes renal hypoplasia is unknown. Here, we describe the early onset of profound renal hypoplasia in mice homozygous for null mutation of the PCP effector gene, Fuzzy. We found that this phenotype is caused by defective branching morphogenesis of the ureteric bud (UB) in the absence of defects in nephron progenitor specification or in early steps of nephrogenesis. By using various experimental approaches, we show that the loss of Fuzzy affects multiple signaling pathways. Specifically, we found mild involvement of GDNF/c-Ret pathway that drives UB branching. We noted the deficient expression of molecules belonging to the Bmp, Fgf and Shh pathways. Analysis of the primary cilia in the UB structures revealed a significant decrease in ciliary length. We conclude that renal hypoplasia in the mouse Fuzzy mutants is caused by defective UB branching associated with dysregulation of ciliary and non-ciliary signaling pathways. Our work suggests a PCP effector-dependent pathogenetic mechanism that contributes to renal hypoplasia in mice and humans.

Continuum Theory for Planar Cell Polarity

Planar Cell Polarity (PCP), characterized by asymmetric localization of proteins at the cell membrane within the epithelial plane, plays essential roles in embryonic development and physiological functions. The significance of PCP can be appreciated by the outcomes of PCP failure in the form of defects in neural tube formation, tracheal malfunctions, organ shape misregulation, hair follicle misalignment etc. Extensive experimental works on PCP in fruit fly Drosophila melanogaster have classified the proteins involved in PCP into two modules: 'core' module, acting locally by inter-cellular protein interactions, and, 'global' module, responsible for the alignment of cell polarities with that of the tissue axis. Despite the involvement of different molecular players, the asymmetric localization of the proteins of the two modules on cell membrane primarily involve inter-cellular dimer formations. We have developed a continuum model of the localization of PCP proteins on the cell membrane and its regulation via intra- and inter-cellular protein-protein interactions. We have identified the conditions for the asymmetric protein localization, or PCP establishment, for uniform and graded protein expression levels in the tissue. We have found that in the absence of any tissue level expression gradient the polarized state of the tissue is not stable against finite length perturbations which is also a property of the active polar matter. However, in the presence of tissue level expression gradients of proteins the polarized state remains stable. We have also looked at the influence of the loss of PCP proteins from a select regions of the tissue on the polarization of the cells outside of that region. This continuum theory of the planar cell polarity can be coupled with the active matter hydrodynamics to study the cell flows and their regulation by genetic machinery.

Emergence of planar cell polarity from the interplay of asymmetric interaction and tissue-level gradients

Planar cell polarity (PCP) - asymmetric localization of proteins at cell-cell interface - is essential for embryonic development and physiological functions. Abnormalities in PCP can lead to neural tube closure defects, misalignment in hair follicles etc. Thus, decoding the mechanism responsible for PCP establishment and maintenance remains an open fundamental question. While various molecules-broadly classified into 'global' and 'local' modules have been well investigated; their necessity and sufficiency in explaining PCP and connecting their perturbations and defects in experimentally observed patterns has not been examined. Here, we develop a minimal model that captures the proposed features of these two modules- a tissue level gradient (global) and asymmetric localization of protein complexes (local). Our model results suggest that while polarity can emerge in absence of a gradient; the gradient can provide the direction of polarity as well as offer robustness for maintenance of PCP in presence of stochastic perturbations. We also recapitulated swirling patterns (seen experimentally) and the features of non-domineering autonomy; using only three free parameters in the model - protein binding rate; concentration of proteins forming heterodimer across cell boundaries and steepness of gradient. Our results explain how self-stabilizing asymmetric localisations in presence of tissue-level gradient can lead to robust PCP patterns in diverse biological systems and reveals the minimal design principles for a polarized system.

Tissue fluidity mediated by adherens junction dynamics promotes planar cell polarity-driven ommatidial rotation

The phenomenon of tissue fluidity—cells’ ability to rearrange relative to each other in confluent tissues—has been linked to several morphogenetic processes and diseases, yet few molecular regulators of tissue fluidity are known. Ommatidial rotation (OR), directed by planar cell polarity signaling, occurs during Drosophila eye morphogenesis and shares many features with polarized cellular migration in vertebrates. We utilize in vivo live imaging analysis tools to quantify dynamic cellular morphologies during OR, revealing that OR is driven autonomously by ommatidial cell clusters rotating in successive pulses within a permissive substrate. Through analysis of a rotation-specific nemo mutant, we demonstrate that precise regulation of junctional E-cadherin levels is critical for modulating the mechanical properties of the tissue to allow rotation to progress. Our study defines Nemo as a molecular tool to induce a transition from solid-like tissues to more viscoelastic tissues broadening our molecular understanding of tissue fluidity.

Actin Crosslinking Family Protein 7 Deficiency Does Not Impair Hearing in Young Mice

To enable hearing, the sensory hair cell contains specialized subcellular structures at its apical region, including the actin-rich cuticular plate and circumferential band. ACF7 (actin crosslinking family protein 7), encoded by the gene Macf1 (microtubule and actin crosslinking factor 1), is a large cytoskeletal crosslinking protein that interacts with microtubules and filamentous actin to shape cells. ACF7 localizes to the cuticular plate and the circumferential band in the hair cells of vertebrates. The compelling expression pattern of ACF7 in hair cells, combined with conserved roles of this protein in the cytoskeleton of various cell types in invertebrates and vertebrates, led to the hypothesis that ACF7 performs a key function in the subcellular architecture of hair cells. To test the hypothesis, we conditionally target Macf1 in the inner ears of mice. Surprisingly, our data show that in young, but mature, conditional knockout mice cochlear hair cell survival, planar cell polarity, organization of the hair cells within the organ of Corti, and capacity to hear are not significantly impacted. Overall, these results fail to support the hypothesis that ACF7 is an essential hair cell protein in young mice, and the purpose of ACF7 expression in the hair cell remains to be understood.

Vangl2-environment interaction causes severe neural tube defects, without abnormal neuroepithelial convergent extension

Planar cell polarity (PCP) signalling is vital for initiation of mouse neurulation, with diminished convergent extension (CE) cell movements leading to craniorachischisis, a severe neural tube defect (NTD). Some humans with NTDs also have PCP gene mutations but these are heterozygous, not homozygous as in mice. Other genetic or environmental factors may interact with partial loss of PCP function in human NTDs. We found that reduced sulfation of glycosaminoglycans interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause craniorachischisis in cultured mouse embryos, with rescue by exogenous sulphate. We hypothesised this glycosaminoglycan-PCP interaction may regulate CE but, surprisingly, DiO labeling of the embryonic node demonstrates no abnormality of midline axial extension in sulfation-depleted Lp/+ embryos. Positive-control Lp/Lp embryos show severe CE defects. Abnormalities were detected in the size and shape of somites that flank the closing neural tube in sulfation-depleted Lp/+ embryos. We conclude that failure of closure initiation can arise by a mechanism other than faulty neuroepithelial CE, with possible involvement of matrix-mediated somite expansion, adjacent to the closing neural tube.

Frizzled3 inhibits Vangl2-Prickle3 association to establish planar cell polarity in the vertebrate neural plate

The orientation of epithelial cells in the plane of the tissue, known as planar cell polarity (PCP), is regulated by interactions of asymmetrically localized PCP protein complexes. In the Xenopus neural plate, Van Gogh-like2 (Vangl2) and Prickle3 (Pk3) proteins form a complex at the anterior cell boundaries, but how this complex is regulated in vivo remains largely unknown. Here we use proximity biotinylation and crosslinking approaches to show that Vangl2-Pk3 association is inhibited by Frizzled3 (Fz3), a core PCP protein that is specifically expressed in the neuroectoderm and is essential for the establishment of PCP in this tissue. This inhibition required Fz3-dependent Vangl2 phosphorylaton. Consistent with our observations, the complex of Pk3 with nonphosphorylatable Vangl2 did not polarize in the neural plate. These findings provide evidence for in vivo regulation of Vangl2-Pk3 complex formation and localization by a Frizzled receptor.

The Drosophila RNA binding protein Nab2 patterns dendritic arbors and axons via the planar cell polarity pathway

RNA binding proteins support neurodevelopment by modulating numerous steps in post-transcriptional regulation, including splicing, export, translation, and turnover of mRNAs that can traffic into axons and dendrites. One such RBP is ZC3H14, which is lost in an inherited intellectual disability. The Drosophila melanogaster ZC3H14 ortholog, Nab2, localizes to neuronal nuclei and cytoplasmic ribonucleoprotein granules, and is required for olfactory memory and proper axon projection into brain mushroom bodies. Nab2 can act as a translational repressor in conjunction with the Fragile-X mental retardation protein homolog Fmr1 and shares target RNAs with the Fmr1-interacting RBP Ataxin-2. However, neuronal signaling pathways regulated by Nab2 and their potential roles outside of mushroom body axons remain undefined. Here, we demonstrate that Nab2 restricts branching and projection of larval sensory dendrites via the planar cell polarity pathway, and that this link may provide a conserved mechanism through which Nab2/ZC3H14 modulates projection of both axons and dendrites. Planar cell polarity proteins are enriched in a Nab2-regulated brain proteomic dataset. Complementary genetic data indicate that Nab2 guides dendrite and axon growth through the planar-cell-polarity pathway. Analysis of the core planar cell polarity protein Vang, which is depleted in the Nab2 mutant whole-brain proteome, uncovers selective and dramatic loss of Vang within axon/dendrite-enriched brain neuropil relative to brain regions containing cell bodies. Collectively, these data demonstrate that Nab2 regulates dendritic arbors and axon projection by a planar-cell-polarity-linked mechanism and identify Nab2 as required for accumulation of the core planar cell polarity factor Vang in distal neuronal projections.