OVOL1/2: drivers of epithelial differentiation in development, disease and reprogramming

OVOL proteins (OVOL1 and OVOL2), vertebrate homologs of Drosophila OVO, are critical regulators of epithelial lineage determination and differentiation during embryonic development in tissues such as kidney, skin, mammary epithelia, testis. OVOL inhibits EMT and can promote MET; moreover, they can regulate the stemness of cancer cells, thus playing an important role during cancer cell metastasis. Due to their central role in differentiation and maintenance of epithelial lineage, OVOL overexpression has been shown to be capable of reprogramming fibroblasts to epithelial cells. Here, we review the roles of OVOL mediated epithelial differentiation across multiple contexts – embryonic development, cancer progression, and cellular reprogramming.


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OVOL1 and OVOL2 are crucial regulators of epithelial lineages during embryonic 14 development and are involved in the maintenance of an epithelial state and terminal 15 differentiation during tissue homeostasis ( Fig. 1) [Mackay et al., 2006;Teng et al., 2007;Nair 16 et al., 2006;Kohn et al., 2014;Sun et al., 2019]. OVOL1/2 can inhibit epithelial-mesenchymal 17 transition (EMT) by directly repressing EMT inducing transcription factors (EMT-TFs) such as 18 ZEB1, ZEB2, TWIST and promote the reverse of EMT -mesenchymal-epithelial transition 19 (MET) by inducing the expression of cell-cell adhesion molecule E-cadherin [Kitazawa et al.,20 2019; Watanabe et al., 2019;Aue et al., 2015]. Thus, OVOL1/2 can be thought of as one of 21 MET-inducing transcription factors (MET-TFs), similar to GRHL2 [Frisch et al., 2017;Mooney 22 et al., 2017]. However, specific targets and functions of OVOL1 and OVOL2 are relatively 23 poorly understood. A deeper appreciation of how OVOL1 and OVOL2 regulate epithelial 24 differentiation and inhibit EMT will be required in the context of cancer metastasis, the leading 25 cause of cancer related deaths worldwide. OVO is a nuclear protein expressed specifically in the female germline, and is critical for 32 oogenesis and sex differentiation in Drosophila melanogaster [Mével-Ninio et al., 1995;33 Chidambaram et al., 1997;Oliver et al., 1987;Garfinkel et al., 1992]. OVO shares most of its 34 coding sequence with shavenbaby (SVB) which is involved in epidermal morphogenesis 35 [Payre et al., 1999;Mével-Ninio et al., 1995]. OVO/SVB forms a complex gene locus with 36 separate control regions performing two genetic functions: sexual differentiation and 37 epidermal differentiation [Garfinkel et al., 1992]. The OVO/SVB triggers F-actin redistribution 38 that initiates cytoskeleton remodelling, thus functioning as an important regulator of epidermal 39 differentiation [Delon et al., 2003]. The carboxyl terminal containing zinc finger domain (tetrad 40 of C2-H2) of OVO is known to be evolutionary conserved from metazoans to vertebrates. 41 Addition of various non-conserved sequences to primarily the N-terminus region of the gene 42 gave rise to different OVO-like (OVOL) genes during the course of evolution [Kumar et al., 43 2012].

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Mouse OVOL2 consists of 5 exons, two of which are alternatively used to form spliced variants 68 of OVOL2: OVOL2A (lacking 2 nd exon) and OVOL2B (lacking 1 st exon) [Li et al., 2002a]. The 69 human OVOL2 gene with six exons also have OVOL2A and OVOL2B transcript variants 70 similar to mouse [Li et al., 2002a]. OVOL2 null mutant mice showed expansion of 71 neuroectoderm causing failure of the closure of cranial neural tube [Mackay et al., 2006].

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Furthermore, defects in embryonic and extraembryonic vascularization along with improper 73 heart development was also observed [Unezaki et al., 2007]. In addition, OVOL2 has been 74 shown to function downstream of BMP signaling during neural/non-neural cell fate decision in 75 chick embryos [Zhang et al., 2013]. Moreover, OVOL2 is a downstream target of OVOL1: 76 OVOL1 represses transcription of OVOL2 by directly binding to its promoter [Tsuji et al., 77 2018]. In addition, OVOL1/2 recognize nearly identical DNA sequences for binding to their 78 target genes (Table 2), suggesting possible regulation of one another and/or themselves 79 [Wells et al., 2009;Lee et al., 2014]. 80 81 82 OVOL in embryonic development 83 84 OVOL1 has been shown to regulate proliferation of epidermal cells during embryonic 85 development in vivo using mouse models and in vitro using isolated keratinocytes from mouse 86 [Nair et al., 2006]. OVOL1 was crucial for the terminal differentiation and restricting the 87 proliferative potential of embryonic epidermal progenitor cells. In addition, cultured 88 keratinocytes lacking OVOL1 failed to restrict their growth under growth inhibitory signals.

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OVOL1-deficient suprabasal cells showed up-regulated c-MYC, which was shown to be 90 transcriptionally repressed by OVOL1 by directly binding to its promoter, suggesting that 91 OVOL1 is required to repress c-MYC for restricting cell proliferation [Nair et al., 2006 it [Zhang et al., 2013]. Ectopic expression of OVOL2 in the neural plate represses the 101 expression of SOX2, a definitive marker of neural plate [Zhang et al., 2013]. Thus, OVOL2 102 seems to control proper germ layer development in multiple model organisms. 103

2a) Epidermal/skin differentiation 104 105
OVOL1 was first characterized functionally in mouse (gene: movo1) where it was shown to 106 express in differentiating cells of epidermis and hair follicles, similar to its fly counterpart. It 107 was found to be involved in hair and sperm formation as well, movo1 -/mice produce aberrant 108 hair and exhibit a limited ability to reproduce [Dai et al., 1998]. OVOL1 mutant mice show 109 perinatal lethality accompanied with epithelial cysts in kidney of embryonic onset and delayed 110 acquisition of skin barrier [Teng et al., 2007]. Loss of OVOL1 was compensated by OVOL2 111 which was upregulated in OVOL1-deficient epidermis. This effect can be mediated by direct 112 repression of OVOL2 by OVOL1 through two OVOL1 recognition sequence in its promoter 113 (CCGTTA) [Teng et al., 2007]. OVOL2-deficient mice display compromised wound healing 114 characterized by aberrant epidermal cell migration and proliferation and defects in hair follicle 115 matrix cell proliferation and differentiation [Hong et al., 2015].

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The reciprocal regulation of epithelial and mesenchymal genes, as seen in EMT/MET, such 118 as a mutually inhibitory feedback loop between OVOL1/2 and ZEB1 [Roca et al., 2013], is also 119 implicated during the differentiation of embryonic ectoderm to neuro-ectoderm and surface 120 ectoderm. Similar reciprocal inhibitory loops is a hallmark of multiple cell-fate decisions during 121 embryonic development [Zhou, and Huang, 2011]. While neuroectoderm derivatives 122 expressed various mesenchymal genes, the corneal epithelial cells (CECs) arising from 123 surface ectoderm have higher expression of epithelial genes such as CDH1, CLDN1, KRT3 124 and downregulated mesenchymal genes such as ZEB1 and ZEB2. OVOL2 is an important 125 regulator of the human CEC transcriptional program [Kitazawa et al., 2016]. The 126 OVOL1/OVOL2-ZEB1 axis seems to be relevant in epidermal progenitor cells too: 127 OVOL1/OVOL2 conditional double knockout mice had blocked terminal differentiation and 128 increased proliferation of embryonic epidermal progenitor cells with defects in α-catenin driven 129 actin cytoskeleton reorganisation and adhesive maturation [Lee et al., 2014]. In addition, 130 OVOL1/ovo2-deficient epidermal cells displayed increased expression of c-MYC and p63: 131 markers of stemness which were shown to be transcriptionally repressed by OVOL2 and 132 exhibited changes similar to EMT which was reversed upon inhibition of EMT regulator ZEB1 133 [Lee et al., 2014]. Contrarily, another study has suggested the transient expansion of HaCaT 134 keratinocytes upon OVOL2 depletion with a loss of long term proliferation potential and 135 suppression of terminal differentiation [Wells et al., 2009], suggesting a potential context-136 dependent mediation of the functions of OVOL2. 137 138 Deletion of ZEB1 has also been shown to restore the directional migration of OVOL2-deficient 139 bulge hair follicle stem cells (Bu-HSCs) which display characteristics of reduced proliferation 140 and enhanced EMT [Hong et al., 2015]. Overexpression of OVOL2 in epidermal basal cells 141 disrupts cytoskeleton structure and display defective basal keratin network and defects in their 142 association with hemidesmosomes, adhesion structures which anchors keratin filaments to 143 cell/ basement membrane, resulting into skin blistering [Lee et al., 2017]. Human placental epithelium consists of cytotrophoblast which either proliferates to maintain 171 sufficient reservoir of mononuclear progenitor cells or can differentiate that leads to the fusion 172 of differentiated cells with the overlying syncytium forming syncytiotrophoblast [Renaud et al., 173 2015]. Syncytiotrophoblast forms the principal epithelial barrier between maternal and fetal 174 blood and is important for nutrient, gas, waste and water exchange between the two blood 175 circulations and synthesizes various hormones for fetal development and maintenance of 176 pregnancy [Renaud et al., 2015]. OVOL1 has been shown to be robustly expressed in human 177 placenta upon induction of trophoblast differentiation and is crucial for the differentiation of 178 cytotrophoblast to syncytiotrophoblast that remains largely epithelial [Vićovac, and Aplin, 179 1996]. OVOL1 repressed genes involved in maintaining the progenitor cell state of 180 cytotrophoblast such as MYC, ID1, TP63, and ASCL2 which have OVOL1 recognition 181 sequence (CCGTTA) within their proximal promoter regions. Deletion of OVOL1 abrogated 182 cytotrophoblast fusion and inhibited genes involved in trophoblast cell fusion and 183 hormonogenesis [Renaud et al., 2015]. Similarly, OVOL2 has been shown to be critical for the 184 development of functional placenta in mice and OVOL2 depletion shows embryonic lethality. 185 Mice placenta and trophoblast stem cells show higher expression of OVOL2 which is 186 implicated in trophoblast differentiation. OVOL2 deletion inhibits differentiation of trophoblast 187 stem cells such that they express higher levels of stem cell related genes such as EOMES, 188 ESRRB, ID2 and genes involved in differentiation such as GCM1, TPBPA, PRL3B1, SYNA 189 are downregulated. In addition, ectopic expression of OVOL2 results into precocious 190 differentiation of trophoblast stem cells [Jeyarajah et al., 2020] [Roca et al., 2013]. Importantly, ESRP1 and E-cadherin can 210 also form mutually inhibitory loops with ZEB1, thus repressing EMT [Jolly et al., 2018b;211 Schmalhofer et al., 2009]. In addition, expression of OVOL1 and OVOL2 in mesenchymal 212 prostate cancer cells decreased their metastatic potential in orthotopic mouse models of 213 prostate cancer [Roca et al., 2013]. Similarly, OVOL2 has been shown to antagonize TGF-β 214 signaling and inhibited EMT during mammary tumor metastasis by repressing SMAD4 215 expression and interfering in SMAD4 and SMAD2/3 complex formation . Thus, 216 OVOL1/2-ZEB1 axis can form an important axis of regulation of EMT in cancer progression, 217 as observed for Cancer Cell Line Encyclopedia (CCLE) cohort [Barretina et al., 2012] (Fig. 2). 218 progression of AK to cSCC is rare. AK display suppressed EMT owing to higher levels of 237 OVOL1/OVOL2 and lower levels of ZEB1, vimentin contrary to higher levels of ZEB1 and 238 vimentin in cSCC and lower levels of OVOL1/OVOL2. 239 Furthermore, in A431 SCC cells, OVOL1 and OVOL2 knockdown increased the mRNA and 240 protein levels of ZEB1. Also, OVOL2 knockdown increased the invasive capability of cells, 241 suggesting OVOL2/ZEB1 crosstalk in modulating AK/cSCC progression [Murata et al., 2020].

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OVOL2 mediated suppression of ZEB1 and promotion of MET is also reported in 243 osteosarcoma [Liu et al., 2018]. A recent study has shown OVOL1 mediated suppression of 244 proliferation, invasion and migration in oral squamous cell carcinoma (SCC-152) cells by 245 inhibiting ZEB1 expression by directly binding to its promoter [Xu et al., 2019], further 246 endorsing that OVOL2/ZEB1 feedback loop controls epithelial-mesenchymal plasticity across 247 carcinomas. 248 Mathematical modeling of the feedback loop between ZEB1 and OVOL2 has revealed that in 249 addition to epithelial (high OVOL2, low ZEB1) and mesenchymal (low OVOL2, high ZEB1), 250 cells can acquire one or more hybrid epithelial/mesenchymal (medium OVOL2, medium 251 ZEB1) states [Jolly et al., 2015;Hong et al., 2015] which can be the most plastic and 252 aggressive state [Jolly et al., 2018a]. In H1975 lung cancer cells that can display a hybrid E/M 253 stably over multiple passages, knockdown of OVOL2 increased the levels of mesenchymal 254 markers, impaired collective cell migration and drove a complete EMT [Jolly et al., 2016], 255 reminiscent of its role reported in mammary morphogenesis. In collective migration of terminal 256 end buds during mammary development, and that of lung cancer cell lines, OVOL2 can be 257 thought of as a "phenotypic stability factor" [Jolly et al., 2016] that can prevent "cells that have 258 gained partial plasticity from crossing the line to undergo complete EMT" [Watanabe et al.,259 2014], thus acting as a gatekeeper of epithelial phenotype. 260 Similarly, in A549 cells, OVOL2 can suppress EMT by inhibiting the transcriptional activity of 261 TWIST [Wang et al., 2017]. Low OVOL1 and high ZEB1 and FN1 expression was also seen 262 in tumor buds of oral SCCs which form the invasive front and known to display EMT features 263 [Jensen et al., 2015]. Low OVOL2 expression has been shown to be associated with worse 264 overall survival in hepatocellular and colon carcinoma patients and correlated with EMT 265 progression in patient HCC tissue samples [Fu et al., 2016;Ye et al., 2016]. 266

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OVOL, plasticity and stemness 268 269 Hybrid E/M cells can facilitate collective cell migration as observed in clusters of circulating 270 tumor cells (CTCs). Relative to individually migrating CTCs, CTC clusters tend to have 271 increased survival of cancer cells in blood circulation [Saxena et al., 2020]. Moreover, in CTC 272 clusters, the binding sites for stemness associated factors such as OCT4, NANOG and SOX2 273 are more hypomethylated [Gkountela et al., 2019]. Together, these factors may contribute to 274 the disproportionately high metastatic propensity of CTC clusters [Aceto et al., 2014].

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A mechanism based mathematical model coupling OVOL with EMT (miR-200/ZEB) and 277 stemness (LIN28/let-7) circuit predicted that OVOL can not only stabilize a hybrid E/M 278 phenotype, but also increase the stemness associated with hybrid E/M and/or epithelial 279 phenotypes, while decreasing the stemness associated with mesenchymal state. Thus, OVOL 280 can fine tune the positioning of "stemness window" on "EMT axis" [Jolly et al., 2015]. Similarly, 281 an increase in stemness by OVOL2 has been shown in nasopharyngeal cancer, where OVOL2 282 ectopic expression in mesenchymal cells could only induce partial epithelial character. In 283 addition, inhibition of ZEB1 in OVOL2-deficient cells decreased stemness without affecting 284 their invasiveness, suggesting a crucial role of OVOL2 in stabilizing hybrid E/M phenotype and 285 conferring stemness to it [Qi et al., 2018]. The transient receptor potential vanilloid 1 (TRPV1), 286 a non-selective cation channel, has been shown to modulate the plasticity of hepatocyte.

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TRPV1 downregulation has been shown to be associated with activation of ZEB1 and 288 inhibition of OVOL2 which promoted hepatocarcinogenesis in a SOX10-dependent manner 289 [Xie et al., 2019]. Coordinated regulation of cell state by ZEB1-OVOL2-GRHL2 axis has been 290 shown to be crucial for corneal endothelial cells and alteration in this axis results into posterior 291 polymprphous corneal dystrophy (PPCD) characterized by aberrant activation of Wnt 292 pathway, highlighting the role of ZEB1-OVOL2-GRHL2 axis in mediating cellular plasticity in 293 corneal tissue homeostasis [Chung et al., 2019].

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OVOL and cellular reprogramming 296 297 OVOLs are known to be critical regulators of epidermal fate determination and differentiation 298 indicating their capability to confer epidermal phenotype to other cell types by reprogramming 299 their gene expression [Kagawa et al., 2019]. OVOL1 in combination with some or all of the 300 selected critical regulators of epidermal fate (PRDM1, p63, KLF4, ZNF750, and GRHL3) has 301 been shown to be sufficient for the rapid conversion of human dermal fibroblasts to an induced 302 keratinocyte phenotype (iKP) expressing keratinocyte specific genes: KRT14 and GJB2 [Chen 303 et al., 2014]. Furthermore, OVOL2 has been shown to cooperatively promote MET in 304 fibroblasts to keratinocytes induced by epithelial lineage promoting transcription factors such 305 as HNF1A, TP63, and KLF4 by inducing genes involved in epithelial phenotype and repressing 306 fibroblast-associated genes [Watanabe et al., 2019]. Similarly, reprogramming of human 307 fibroblast cells to corneal epithelial cells (CECs) was done by overexpressing core CEC 308 network consisting of PAX6, OVOL2 and KLF4. In addition, OVOL2 was sufficient to direct 309 reprogram neural ectoderm to a more epithelial cell state: surface ectoderm [Kitazawa et al.,310 2019]. 311

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Conclusion 313 314 OVOL (OVOL1/OVOL2) are critical regulators of epithelial differentiation (Fig. 3). They 315 function downstream of growth signaling such as Wnt/β-catenin and BMP-SMAD. OVOL play 316 an important role in cancer where they inhibit EMT and promote MET and has been implicated 317 as prognostic marker for patient survival. OVOL1 and OVOL2 regulate many common targets, 318 perform many redundant functions, and can compensate for each other, however, few targets 319 and functions are specific to OVOL1 and OVOL2. Looking at the diverse roles of OVOL during 320 embryonic development, cancer and reprogramming, it is important to identify and 321 characterize upstream and downstream targets of OVOL and understand how various OVOL 322 mediated pathways converge to perform similar functions.