in-vivo-crisprcas9-screening-to-simultaneously-evaluate-gene-function

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to ...

Tel Aviv University

tel aviv university

Actomyosin contractility is a highly regulated process that affects many fundamental biological processes in each and every cell in our body. In this Cell Science at a Glance article and the accompanying poster, we mined the literature and databases to map the contractome of non-muscle cells. Actomyosin contractility is involved in at least 49 distinct cellular functions that range from providing cell architecture to signal transduction and nuclear activity. Containing over 100 scaffolding and regulatory proteins, the contractome forms a highly complex network with more than 230 direct interactions between its components, 86 of them involving phosphorylation. Mapping these interactions, we identify the key regulatory pathways involved in the assembly of actomyosin structures and in activating myosin to produce contractile forces within non-muscle cells at the exact time and place necessary for cellular function. 

The contractome--a systems view of actomyosin contractility in non-muscle cells.

Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway.

Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis.

The ability of cells to generate, maintain, and repair tissues with complex architecture, in which distinct cells function as coherent units, relies on polarity cues. Polarity can be described as an asymmetry along a defined axis, manifested at the molecular, structural, and functional levels. Several types of cell and tissue polarities were described in the literature, including front-back, apical-basal, anterior-posterior, and left-right polarity. Extensive research provided insights into the specific regulators of each polarization process, as well as into generic elements that affect all types of polarities. The actin cytoskeleton and the associated adhesion structures are major regulators of most, if not all, known forms of polarity. Actin filaments exhibit intrinsic polarity and their ability to bind many proteins including the mechanosensitive adhesion and motor proteins, such as myosins, play key roles in cell polarization. The actin cytoskeleton can generate mechanical forces and together with the associated adhesions, probe the mechanical, structural, and chemical properties of the environment, and transmit signals that impact numerous biological processes, including cell polarity. In this article we highlight novel mechanisms whereby the mechanical forces and actin-adhesion complexes regulate cell and tissue polarity in a variety of natural and experimental systems.

Multiscale View of Cytoskeletal Mechanoregulation of Cell and Tissue Polarity.

The establishment of epithelial architecture is a complex process involving cross-talk between cells and the basement membrane. Basement membrane assembly requires integrin activity but the role of the associated actomyosin cytoskeleton is poorly understood. Here, we identify the actin-bundling protein T-plastin (Pls3) as a regulator of basement membrane assembly and epidermal morphogenesis. In utero depletion of  transcripts in mouse embryos caused basement membrane and polarity defects in the epidermis but had little effect on cell adhesion and differentiation. Loss-of-function experiments demonstrated that the apicobasal polarity defects were secondary to the disruption of the basement membrane. However, the basement membrane itself was profoundly sensitive to subtle perturbations in the actin cytoskeleton. We further show that Pls3 localized to the cell cortex, where it was essential for the localization and activation of myosin II. Inhibition of myosin II motor activity disrupted basement membrane organization. Our results provide insights into the regulation of cortical actomyosin and its importance for basement membrane assembly and skin morphogenesis.

T-plastin is essential for basement membrane assembly and epidermal morphogenesis.

Mutations in key transcription factors SOX2 and P63 were linked with developmental defects and postnatal abnormalities such as corneal opacification, neovascularization, and blindness. The latter phenotypes suggest that SOX2 and P63 may be involved in corneal epithelial regeneration. Although P63 has been shown to be a key regulator of limbal stem cells, the expression pattern and function of SOX2 in the adult cornea remained unclear. Here, we show that SOX2 regulates P63 to control corneal epithelial stem/progenitor cell function. SOX2 and P63 were co-expressed in the stem/progenitor cell compartments of the murine cornea in vivo and in undifferentiated human limbal epithelial stem/progenitor cells in vitro. In line, a new consensus site that allows SOX2-mediated regulation of P63 enhancer was identified while repression of SOX2 reduced P63 expression, suggesting that SOX2 is upstream to P63. Importantly, knockdown of SOX2 significantly attenuated cell proliferation, long-term colony-forming potential of stem/progenitor cells, and induced robust cell differentiation. However, this effect was reverted by forced expression of P63, suggesting that SOX2 acts, at least in part, through P63. Finally, miR-450b was identified as a direct repressor of SOX2 that was required for SOX2/P63 downregulation and cell differentiation. Altogether, we propose that SOX2/P63 pathway is an essential regulator of corneal stem/progenitor cells while mutations in SOX2 or P63 may disrupt epithelial regeneration, leading to loss of corneal transparency and blindness. Stem Cells 2019;37:417-429.

SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium.

Development of the skin epidermis requires tight spatiotemporal control over the activity of several signaling pathways; however, the mechanisms that orchestrate these events remain poorly understood. Here, we identify a key role for the Wave complex proteins ABI1 and Wave2 in regulating signals that control epidermal shape and growth. In utero RNAi-mediated silencing of  or  induced cellular hyperproliferation and defects in architecture of the interfollicular epidermis (IFE) and delayed hair follicle growth. Unexpectedly, SOX9, a hair follicle growth regulator, was aberrantly expressed throughout the IFE of the mutant embryos, and its forced overexpression mimicked the Wave complex loss-of-function phenotype. Moreover, Wnt signaling, which regulates SOX9 cell specification, was up-regulated in Wave complex loss-of-function IFE. Importantly, we show that the Wave complex regulates filamentous actin content and that a decrease in actin levels is sufficient to elevate Wnt/β-catenin signaling. Our results identify a novel role for Wave complex- and actin-regulated signaling via Wnt and SOX9 in skin development.

The Wave complex controls epidermal morphogenesis and proliferation by suppressing Wnt-Sox9 signaling.

Animal cells exhibit a wide range of shapes that reflect their diverse functions. Cell shape is determined by a balance between internal and external forces and therefore involves the cytoskeleton and its associated adhesion structures. Cell shape dynamics during development and homeostasis are tightly regulated and closely coordinated with cell fate determination. Defects in cell shape are a hallmark of many pathological conditions including cancer and skin diseases. This review highlights the links between cell shape and cell fate in the epidermis, which have been studied for over 40 years both in vitro and in vivo. Briefly discussing seminal experiments showing the strong coupling between keratinocyte cell shape and their fate we primarily focus on recent studies uncovering novel cellular and molecular mechanisms linking epidermal cell shape with cell growth, differentiation, asymmetric division, and delamination.

From cell shape to cell fate via the cytoskeleton - Insights from the epidermis.

Melanoma is the deadliest skin cancer due to its high rate of metastasis, frequently to the brain. Brain metastases are incurable; therefore, understanding melanoma brain metastasis is of great clinical importance. We used a mouse model of spontaneous melanoma brain metastasis to study the interactions of melanomas with the brain microenvironment. We find that CXCL10 is upregulated in metastasis-associated astrocytes in mice and humans and is functionally important for the chemoattraction of melanoma cells. Moreover, CXCR3, the receptor for CXCL10, is upregulated in brain-tropic melanoma cells. Targeting melanoma expression of CXCR3 by nanoparticle-mediated siRNA delivery or by shRNA transduction inhibits melanoma cell migration and attenuates brain metastasis in vivo. These findings suggest that the instigation of pro-inflammatory signaling in astrocytes is hijacked by brain-metastasizing tumor cells to promote their metastatic capacity and that the CXCL10-CXCR3 axis may be a potential therapeutic target for the prevention of melanoma brain metastasis.

Inflammatory Activation of Astrocytes Facilitates Melanoma Brain Tropism via the CXCL10-CXCR3 Signaling Axis.

The WASP-family verprolin-homologous (Wave) complex enhances actin-related protein 2/3 (Arp2/3)-mediated actin polymerization. Recently, we have identified a novel Wave complex-actin-Wnt/β-catenin-Sox9 pathway that regulates epidermal morphogenesis and proliferation. These findings highlight a mechanism by which actin polymerization impacts crucial signaling events in vivo.

Wave of the future: involvement of actin polymerization in the regulation of tissue growth and shape.

p16 (CDKN2A) is a central tumor suppressor, which induces cell-cycle arrest and senescence. Cells expressing p16 accumulate in aging tissues and appear in premalignant lesions, yet their physiologic effects are poorly understood. We found that prolonged expression of transgenic p16 in the mouse epidermis induces hyperplasia and dysplasia, involving high proliferation rates of keratinocytes not expressing the transgene. Continuous p16 expression increases the number of epidermal papillomas formed after carcinogen treatment. Wnt-pathway ligands and targets are activated upon prolonged p16 expression, and Wnt inhibition suppresses p16-induced hyperplasia. Senolytic treatment reduces p16-expressing cell numbers, and inhibits Wnt activation and hyperplasia. In human actinic keratosis, a precursor of squamous cell carcinoma, p16-expressing cells are found adjacent to dividing cells, consistent with paracrine interaction. These findings reveal that chronic p16 expression is sufficient to induce hyperplasia through Wnt-mediated paracrine stimulation, and suggest that this tumor suppressor can promote early premalignant epidermal lesion formation.

Chronic expression of p16 in the epidermis induces Wnt-mediated hyperplasia and promotes tumor initiation.

Striatin, a subunit of the serine/threonine phosphatase PP2A, is a core member of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complexes. The protein is expressed in the cell junctions between epithelial cells, which play a role in maintaining cell-cell adhesion. Since the cell junctions are crucial for the function of the mammalian inner ear, we examined the localization and function of striatin in the mouse cochlea. Our results show that in neonatal mice, striatin is specifically expressed in the cell-cell junctions of the inner hair cells, the receptor cells in the mammalian cochlea. Auditory brainstem response measurements of striatin-deficient mice indicated a progressive, high-frequency hearing loss, suggesting that striatin is essential for normal hearing. Moreover, scanning electron micrographs of the organ of Corti revealed a moderate degeneration of the outer hair cells in the middle and basal regions, concordant with the high-frequency hearing loss. Additionally, striatin-deficient mice show aberrant ribbon synapse maturation. Loss of the outer hair cells, combined with the aberrant ribbon synapse distribution, may lead to the observed auditory impairment. Together, these results suggest a novel function for striatin in the mammalian auditory system.

Article	Year
The contractome--a systems view of actomyosin contractility in non-muscle cells. Journal of cell science\| PubMed ID: 26021351	2015
Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis. PLoS genetics\| PubMed ID: 27736875	2016
Multiscale View of Cytoskeletal Mechanoregulation of Cell and Tissue Polarity. Handbook of experimental pharmacology\| PubMed ID: 27807694	2017
T-plastin is essential for basement membrane assembly and epidermal morphogenesis. Science signaling\| PubMed ID: 28559444	2017
SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium. Stem cells (Dayton, Ohio)\| PubMed ID: 30548157	2019
The Wave complex controls epidermal morphogenesis and proliferation by suppressing Wnt-Sox9 signaling. The Journal of cell biology\| PubMed ID: 30867227	2019
From cell shape to cell fate via the cytoskeleton - Insights from the epidermis. Experimental cell research\| PubMed ID: 30872138	2019
Inflammatory Activation of Astrocytes Facilitates Melanoma Brain Tropism via the CXCL10-CXCR3 Signaling Axis. Cell reports\| PubMed ID: 31412247	2019
Wave of the future: involvement of actin polymerization in the regulation of tissue growth and shape. Molecular & cellular oncology\| PubMed ID: 31528690	2019
Chronic expression of p16 in the epidermis induces Wnt-mediated hyperplasia and promotes tumor initiation. Nature communications\| PubMed ID: 32483135	2020
Striatin Is Required for Hearing and Affects Inner Hair Cells and Ribbon Synapses. Frontiers in cell and developmental biology\| PubMed ID: 32766247	2020

Chen Luxenburg

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