An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis

Here, we describe a protocol for single-cell isolation from the primary culture of normal human epidermal keratinocytes derived from neonatal foreskin. The cell culture conditions have been optimized for inducing expression of keratinocyte differentiation markers. Cells are cultured in the absence or presence of a bioactive lipid lysophosphatidic acid (LPA). Single cells are isolated by Fluidigm C1 system. This is followed by cDNA library preparation using Takara SMART-Seq v4 Ultra and Illumina Nextera XT kit for RNA sequencing

T cell–intrinsic prostaglandin E₂-EP2/EP4 signaling is critical in pathogenic T_H17 cell–driven inflammation

PGE2経路による病因細胞Th17の増殖機構を解明 --乾癬の慢性的な皮膚炎症を改善する新しい治療薬開発に向けて--. 京都大学プレスリリース. 2018-06-20.Background: IL-23 is the key cytokine for generation of pathogenic IL-17–producing helper T (TH17) cells, which contribute critically to autoimmune diseases. However, how IL-23 generates pathogenic TH17 cells remains to be elucidated. Objectives: We sought to examine the involvement, molecular mechanisms, and clinical implications of prostaglandin (PG) E2–EP2/EP4 signaling in induction of IL-23–driven pathogenic TH17 cells. Methods: The role of PGE2 in induction of pathogenic TH17 cells was investigated in mouse TH17 cells in culture in vitro and in an IL-23–induced psoriasis mouse model in vivo. Clinical relevance of the findings in mice was examined by using gene expression profiling of IL-23 and PGE2-EP2/EP4 signaling in psoriatic skin from patients. Results: IL-23 induces Ptgs2, encoding COX2 in TH17 cells, and produces PGE2, which acts back on the PGE receptors EP2 and EP4 in these cells and enhances IL-23–induced expression of an IL-23 receptor subunit gene, Il23r, by activating signal transducer and activator of transcription (STAT) 3, cAMP-responsive element binding protein 1, and nuclear factor κ light chain enhancer of activated B cells (NF-κB) through cyclic AMP–protein kinase A signaling. This PGE2 signaling also induces expression of various inflammation-related genes, which possibly function in TH17 cell–mediated pathology. Combined deletion of EP2 and EP4 selectively in T cells suppressed accumulation of IL-17A+ and IL-17A+IFN-γ+ pathogenic Th17 cells and abolished skin inflammation in an IL-23–induced psoriasis mouse model. Analysis of human psoriatic skin biopsy specimens shows positive correlation between PGE2 signaling and the IL-23/TH17 pathway. Conclusions: T cell–intrinsic EP2/EP4 signaling is critical in IL-23–driven generation of pathogenic TH17 cells and consequent pathogenesis in the skin

Single-cell RNA sequencing identifies a migratory keratinocyte subpopulation expressing THBS1 in epidermal wound healing

Keratinocyte differentiation is an intricate process that is regulated by multiple mediators. Using cultured human keratinocytes, we found that lysophosphatidic acid (LPA) induced the differentiation of a previously unsuspected keratinocyte subpopulation expressing the extracellular matrix protein, thrombospondin-1 (THBS1). This action of LPA was mediated by the RHO/ROCK-SRF signaling downstream of LPA₁ and LPA₅ receptors and required ERK activity. Suppression of THBS1 in vitro suggested a migratory role of THBS1⁺ keratinocytes. Moreover, we analyzed publicly deposited single-cell RNA sequencing dataset and identified Thbs1-expressing keratinocytes in the mouse wound skin. Immunohistochemistry analysis revealed that Thbs1⁺ keratinocytes were apparently differentiated from basal keratinocytes upon wounding, subsequently polarized and migrated suprabasally toward the wound front, and eventually underwent terminal differentiation in the neo-epidermis. Importantly, inhibition of Erk activity suppressed Thbs1⁺ keratinocyte differentiation in wound healing. Based on these findings, we suggest that THBS1⁺ keratinocyte is a migratory keratinocyte subpopulation that facilitates epidermal wound healing

Deficiency of mDia, an Actin Nucleator, Disrupts Integrity of Neuroepithelium and Causes Periventricular Dysplasia

During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that mDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both mDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells

A role for mDia, a Rho-regulated actin nucleator, in tangential migration of interneuron precursors.

神経細胞の配置メカニズムを解明－抑制性神経前駆細胞に特有の移動の機構が明らかに. 京都大学プレスリリース. 2012-1-16.In brain development, distinct types of migration, radial migration and tangential migration, are shown by excitatory and inhibitory neurons, respectively. Whether these two types of migration operate by similar cellular mechanisms remains unclear. We examined neuronal migration in mice deficient in mDia1 (also known as Diap1) and mDia3 (also known as Diap2), which encode the Rho-regulated actin nucleators mammalian diaphanous homolog 1 (mDia1) and mDia3. mDia deficiency impaired tangential migration of cortical and olfactory inhibitory interneurons, whereas radial migration and consequent layer formation of cortical excitatory neurons were unaffected. mDia-deficient neuroblasts exhibited reduced separation of the centrosome from the nucleus and retarded nuclear translocation. Concomitantly, anterograde F-actin movement and F-actin condensation at the rear, which occur during centrosomal and nuclear movement of wild-type cells, respectively, were impaired in mDia-deficient neuroblasts. Blockade of Rho-associated protein kinase (ROCK), which regulates myosin II, also impaired nuclear translocation. These results suggest that Rho signaling via mDia and ROCK critically regulates nuclear translocation through F-actin dynamics in tangential migration, whereas this mechanism is dispensable in radial migration

Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility

ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles

Rho-associated kinase (ROCK) I mediates signaling from Rho to the actin cytoskeleton. To investigate the in vivo functions of ROCK-I, we generated ROCK-I–deficient mice. Loss of ROCK-I resulted in failure of eyelid closure and closure of the ventral body wall, which gave rise to the eyes open at birth and omphalocele phenotypes in neonates. Most ROCK-I−/− mice died soon after birth as a result of cannibalization of the omphalocele by the mother. Actin cables that encircle the eye in the epithelial cells of the eyelid were disorganized and accumulation of filamentous actin at the umbilical ring was impaired, with loss of phosphorylation of the myosin regulatory light chain (MLC) at both sites, in ROCK-I−/− embryos. Stress fiber formation and MLC phosphorylation induced by EGF were also attenuated in primary keratinocytes from ROCK-I−/− mice. These results suggest that ROCK-I regulates closure of the eyelids and ventral body wall through organization of actomyosin bundles

Neural Crest Cell Survival Is Dependent on Rho Kinase and Is Required for Development of the Mid Face in Mouse Embryos

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia

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

© 2016. Published by The Company of Biologists Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.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.Peer reviewedFinal Published versio