{"paper_id":"03ca3a09-6d8e-4a0d-a005-b49c51ae89a5","body_text":"1 \nTitle: Placental cytotrophoblast microvillar stabilization is required for cell-cell fusion \nRunning title: Trophoblast fusion needs microvilli \nAuthors: Wendy K. Duan1, Sumaiyah Z. Shaha1, Khushali J. Patel2, Ivan K. Domingo2, \nMeghan R. Riddell1,2 \nAffiliations: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada1 5 \nDepartment of Obstetrics and Gynecology, University of Alberta, Edmonton, Alberta, Canada2 \nCorresponding Author Email: mriddell@ualberta.ca \nKeywords: trophoblast, cell fusion, ezrin, microvilli, placenta \nSummary statement:  \nFusion-competent cytotrophoblasts undergo dynamic changes in cell morphology including 10 \nthe acquisition of apically localized microvilli. Microvillar stabilization facilitates effective fusion \nand differentiation. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 2 \nAbstract: \nThe placenta is an essential organ of pregnancy required for maternal-fetal transport and 15 \ncommunication. The surface of the placenta facing the maternal blood is formed by a single \ngiant multinucleate cell: the syncytiotrophoblast. The syncytiotrophoblast is formed and \nmaintained via fusion of progenitor cytotrophoblasts. Cell-cell fusion is a tightly regulated \nprocess, and in non-trophoblastic cells is accompanied by stereotypical alterations in cell \nshape by cells that have attained fusion-competence. The most prominent feature is the 20 \nformation of actin-based membrane protrusions, but whether stereotypic morphological \nchanges occur in fusion-competent cytotrophoblasts has not been characterized.  Using a \nhuman placental explant model, we characterized cell shape factors associated with the \nattainment of cytotrophoblast fusion competence. We found that fusion-competent \ncytotrophoblasts are hypertrophic, elongated cells, that form microvilli at the apical 25 \nmembrane. The actin-membrane cross linker protein ezrin was found to have highly polarized \nexpression within cytotrophoblast microvilli. Inhibition of ezrin activation destabilized \ncytotrophoblast microvilli and prevented cytotrophoblast fusion. Thus, we propose that the \npolarized activation of ezrin within apical microvilli and actin-mediated changes in membrane \ndynamics are necessary for cytotrophoblast fusion.  30 \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 3 \nIntroduction: \nThe placenta is an embryonically derived organ of pregnancy that carries out critical functions \nsuch as maternal-fetal nutrient exchange, hormone production, and protection from \npathogens and maternal leukocytes.  Trophoblasts are the placental epithelial lineage that 35 \ncomprise the maternal blood-facing placental surface. In humans, the syncytiotrophoblast \n(ST), a giant multinucleate single cell, covers nearly the entire maternal blood-facing surface. \nTherefore, this embryonically derived single cell is both a fetal sentinel and nutrient exchanger \nwithin the maternal compartment since it is bathed in maternal blood on its apical surface. By \nthe end of gestation, the ST has been estimated to be ~12m2 and contain billions of nuclei 40 \n(Benirschke, 2000; Simpson et al., 1992). Importantly, the ST is post-mitotic and it is therefore \nmaintained and expands via the incorporation of its underlying progenitor villous \ncytotrophoblasts (vCT) via cell-cell fusion. Adequate formation and renewal of the ST via vCT \nfusion is critical for proper placental function. Hence, altered vCT fusion is a feature of the \ncommon pregnancy complications intrauterine growth restriction (IUGR) and preeclampsia 45 \n(Gauster et al., 2009; Langbein et al., 2008; Ruebner et al., 2010), which have a shared \netiology of placental malformation. But much remains to be understood about the regulation of \nvCT fusion.  \n \nCell-to-cell fusion (hereafter referred to as cell fusion) is a relatively uncommon but essential 50 \nevent in human biology. It occurs between gametes when the sperm and oocyte fuse, in \nmyoblasts to maintain and form myofibers, and in the aforementioned fusion between cells of \nthe villous trophoblast lineage.  Fusion is a dramatic cellular event that is highly regulated to \nensure it occurs between the right cells at the correct location within a tissue, and requires the \nexpression of fusogens, proteins that catalyze the local disruption of the lipid bilayers and 55 \ntheir rejoining. It has been proposed that fusion can be broken down into three stages: 1): \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 4 \nattainment of fusion competence; 2) commitment to fusing; 3) the fusion event (Aguilar et al., \n2013). In myoblasts, these stages have been extensively characterized in both mammalian \nand Drosophila cells (Abmayr and Pavlath, 2012; Kim and Chen, 2019; Petrany and Millay, \n2019), but our understanding of the regulation and execution of trophoblast fusion is less 60 \ndeveloped.  \n \nSingle cell-RNA-seq and single nuclei-seq analyses have revealed that multiple \ntranscriptionally distinct populations of vCT exist at one time in the first trimester placenta \n(Arutyunyan et al., 2023; Shannon et al., 2022; Wang et al., 2024). These analyses have 65 \nidentified vCT with higher expression of ST marker genes that are assumed to be a \npopulation that has attained fusion competence due to high expression of the placental \nfusogen syncytin-2 (ERVFRD-1) (Arutyunyan et al., 2023; Shannon et al., 2022; Wang et al., \n2024). Similarly, ultrastructural examination of first trimester and term placenta has revealed a \nsub-population of vCT that more strongly resemble the ST in their nuclear structure and 70 \norganellular composition (Jones and Fox, 1991). It is well established that commitment to \nfusion is accompanied by changes in cell shape. For example, myoblasts are known to \nchange their cell shape to laterally align with the myofiber with which they will fuse (Lehka and \nRedowicz, 2020), and the appearance of membrane protrusions on the cell surface of fusion \ncompetent cells is also a highly conserved feature amongst fusogenic cell types (Brukman et 75 \nal., 2019; Wilson and Snell, 1998). These shape changes appear to have the purpose of \nbringing fusing membranes into closer approximation (Brukman et al., 2019; Kim and Chen, \n2019; Wilson and Snell, 1998), and alterations in cell surface area immediately after fusion \nhas been proposed to regulate transcriptional commitment to a differentiated state via an \nAMPK-YAP1 signaling pathway (Feliciano et al., 2021). Bundled filamentous-actin (F-actin) is 80 \na core cytoskeletal component of membrane protrusions observed in fusion competent \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 5 \nmyoblasts where the formation of invasive membrane podosomes allows for efficient fusogen \nengagement and mechanical coordination between the membranes of apposing cells (Kim \nand Chen, 2019; Kim et al., 2015; Petrany and Millay, 2019; Sens et al., 2010; Shilagardi et \nal., 2013). Membrane projections have been observed by brightfield live cell imaging between 85 \nfusing BeWo trophoblastic choriocarcinoma cells (Wang et al., 2014) and the variable \npresence of membrane protrusions in primary vCT undergoing spontaneous fusion has been \nobserved by scanning electron microscopy (SEM) (Bax et al., 1989), but the cytoskeletal \ncomponents driving these protrusions and characterization of other shape factors that may \ndistinguish fusion competent vCT have not been identified.  90 \n \nThe actin-membrane linker protein ezrin has been shown to play an important, though \ncomplex, role in the formation and stabilization of membrane protrusions (Saotome et al., \n2004; Welf et al., 2020) and is particularly important for the maintenance of more stable \nmembrane structures like epithelial microvilli (Pelaseyed and Bretscher, 2018). Ezrin requires 95 \nactivation via phosphorylation on its Thr-567 residue in order to attain an open conformation \nand bind both the F-actin filament and overlying membrane (Zhu et al., 2007). Interestingly, \nezrin has been shown to function as an A-kinase anchoring protein (AKAP) in fusing 2D \nprimary vCT cultures and serves as a scaffolding protein to protein kinase A (PKA), an \nimportant regulator of vCT fusogen expression (Gerbaud et al., 2015; Pidoux et al., 2014) but 100 \nwhether it also functions as a regulator of vCT shape alterations to enable fusion remains to \nbe examined.  \n \nHere, we characterized cellular shape parameters of the vCT layer in intact human first \ntrimester placental tissue. To identify the shape characteristics that are associated with fusion 105 \ncommitment, we exploited a first trimester explant model where widespread spontaneous vCT \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 6 \nfusion occurs after the removal of the overlying ST layer (Baczyk et al., 2009; Baczyk et al., \n2013; Miller et al., 2005). We identified that the population of vCTs stimulated to fuse by the \nremoval of the ST are enriched in cells that accumulate surface area in the apical region and \nproduce F-actin and ezrin containing microvilli. Chemical inhibition of ezrin activation in ST-110 \nstripped explants strongly abrogated vCT fusion, altered the pattern of F-actin on the apical \nsurface, and reduced expression of ST marker genes. Therefore, we have uncovered that \nvCT fusion requires the polarized accumulation and activation of ezrin within apically localized \nmicrovilli.  \n 115 \nResults: \nST regeneration explant model recapitulates syncytialization in vitro  \nTo establish a model that would allow for the accumulation of fusion-competent vCT, we used \na first trimester human placental explant model. Tissue was briefly trypsinized to promote ST \ndenudation and then cultured to allow for ST shedding and regeneration. Unlike established 120 \ntwo-dimensional trophoblast stem cell culture (Okae et al., 2018), primary cultured vCT \n(Pidoux et al., 2014), and organoid models (Haider et al., 2018; Sheridan et al., 2020; Turco \net al., 2018) this model allows for the observation of three dimensional spontaneous \nsyncytialization from pre-existing vCT subpopulations on an intact basement membrane. Near \ncomplete removal of the pre-existing ST was achieved by 24hrs post-trypsinization (Fig. 1A), 125 \nexposing a mononucleate anti-E-cadherin and anti-integrin-α-6 positive vCT layer \n(Aghababaei et al., 2015; Aplin, 1993; Longtine et al., 2012). By 72hrs post-trypsinization, a \nmultinucleated structure displaying clear anti-β-human chorionic gonadotropin (β-hCG) signal \n(ST marker) atop or interspersed with anti-integrin-⍺-6-positive or anti-E-cadherin-positive \nmononucleated vCTs was consistently observed (Fig. 1A). The formation of multinucleated 130 \nstructures often led to the exhaustion of the vCT layer, resulting in regions of ST without \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 7 \nunderlying vCTs; however, cases where vCTs were present underneath a regenerated ST \nwere also observed. The degree of ST regeneration was quantified by a significant increase in \nthe relative ST:vCT coverage between 24hrs and 72hrs post-trypsinization (Fig. 1B).  Thus, \nthis model enriches vCT into a fusion competent state to allow for widespread synchronized 135 \nST regeneration.  \n \nST denudation leads to enrichment of a morphologically distinct vCT subpopulation \nSince the removal of the ST layer led to depletion of the vCT layer and regeneration of a \nmultinucleate ST, we surmised that there was a time-dependent enrichment in fusion 140 \ncompetent vCT, allowing for the identification of their characteristic morphology. Therefore, we \nacquired high magnification z-stack confocal images of uncultured placental tissue and \ncultured explants at 24hrs post-trypsinization from the same donor placentas after whole-\nmount staining with anti-E-cadherin, phalloidin, and a nuclear dye (Fig. 1C) to quantify \npopulation-wide shifts in morphological parameters observed with ST denudation. ST-145 \ndenuded explants were enriched with hypertrophic vCTs that resulted in an upward shift in the \nmedian cell length (Fig. 1D) and median cell volume (Fig. 1E) compared to ST-intact placental \ntissue. Accompanying this growth, ST-denuded vCT populations shifted towards an elongated \nand less spherical cell shape, resulting in a downward shift in shape factor (Fig. 1F). The \nmedian position of the vCT nucleus in ST-intact placental tissue was found to be halfway 150 \nbetween the apical and basal membrane; however, the median nuclear position in ST-\ndenuded vCTs shifted closer to the basal membrane (Fig. 1G). On the apical surface, ST-\ndenuded vCTs were found to have significantly increased apical volumes compared to ST-\nintact vCTs (Fig.1H). Thus, ST denudation pushes vCTs to undergo morphological changes \nincluding changes in cell size, shape, and polarity, thereby enriching a structurally distinct vCT 155 \nsubpopulation prior to fusion. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 8 \n \nMicrovilli decorate the vCT apical surface prior to fusion in vitro and in vivo \nOne of the most striking and highly enriched features of vCT in denuded explants was an \nincrease in apical volume that was mostly attributable to an increased occurrence of F-actin-160 \npositive hair-like projections at the apical surface (Fig. 1C, 2A). Bundled F-actin in fusion-\ncompetent cells has been established as central to membrane protrusion formation (Kim and \nChen, 2019; Kim et al., 2015; Petrany and Millay, 2019; Sens et al., 2010; Shilagardi et al., \n2013), therefore we hypothesized that membrane projections were accumulating on the apical \nsurface of vCT in our explant model. Scanning electron micrographs (SEM) of the vCT 165 \nsurface at 24hrs post-trypsinization revealed varying amounts of membranous protrusions \n~100nm in diameter on the apical surface of individual vCTs and bridging the intercellular \ngaps between individual cells (Fig. 2B). A wide variation in the amount of these structures on \nindividual cells was observed within these micrographs. Most vCTs were moderately to \nheavily decorated on their apical surface, although vCTs devoid of these structures were also 170 \nobserved. Berryman et al. previously reported immunogold labelling of ezrin, a core \ncomponent of epithelial microvilli, within apical membrane projections of sporadic vCT in \nintact placenta of an unreported gestational age using transmission electron microscopy \n(TEM) (Berryman et al., 1993). Therefore, we stained ST-denuded explants with an anti-ezrin \nantibody to confirm whether a similar signal pattern of apically accumulated ezrin existed in 175 \nour model. As expected, anti-ezrin signal strongly localized to the apical surface of both \nmononucleated vCTs and fused binucleated regenerated STs (Fig. 2C). Based on the relative \ndiameter of the apical membrane structures and the presence of F-actin and ezrin within them \nwe concluded that these membrane structures are microvilli as opposed to the invasive \npodosomes characteristic of fusion competent myocytes.  Altogether, these data suggest that 180 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 9 \nthe formation of extensive microvilli on the apical surface of the vCT surface represents a \nmorphological adaptation associated with a fusion competent state. \n \nWe then sought to confirm the observations of Berryman et al. to identify whether vCT with \napical F-actin and ezrin containing structures could be observed in intact first trimester 185 \nplacenta.  Microvilliation of the vCT apical (ST-facing) membrane of a subset of cells has also \nbeen observed in additional TEM studies of both term and first trimester placenta (Jones and \nFox, 1991; Tashev et al., 2022).  As expected, we observed vCT with apical membrane \nprojections at the vCT-ST interface with approximately the same diameter of those observed \non the apical surface of vCT in our denuded explant model in TEM micrographs (Fig. 3A), 190 \nthough extensive microvilli were not clearly visible in the majority of vCT, aligning with \npublished data (Jones and Fox, 1991; Tashev et al., 2022). Confocal z-stack images of whole-\nmount intact placental tissue revealed that individual vCT possess varying degrees of apical \nhair-like F-actin (phalloidin) signal (Fig. 3B), similar to the ST regeneration model. \nInterestingly, towards the tip of individual villi there was a visible increase in the occurrence of 195 \nhighly branched and fine apical phalloidin signal in vCT while along the length of the villus \nvCT more often displayed a largely smooth, unbranched apical phalloidin staining pattern. \nConsistent with the findings in our in vitro model, anti-ezrin signal variably localized to the \napical surface of individual vCT and, as previously reported, in a discontinuous pattern at the \napical surface of the ST (Fig. 3C) (Berryman et al., 1993; Patel et al., 2023). Therefore, we 200 \nconfirmed that apical microvilli are present in a subset of vCT in the first trimester placenta. In \ncombination with our data showing the accumulation of cells with this feature in denuded \nplacental explants it suggests that the assembly of apical microvilli is a previously \nunrecognized stage in fusion commitment for vCT.   \n 205 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 10 \n \nMicrovillar stabilization via activated ezrin is needed for vCT fusion and differentiation \nSince vCT microvilliation is a conserved feature in vivo and in vitro, we hypothesized that \nformation of apical microvilli may be obligate for trophoblast fusion. To target microvilli without \ndisrupting whole cell actin dynamics, we selected to treat vCT in our ST-regeneration explant 210 \nmodel with NSC668394, which binds directly to ezrin to prevent its phosphorylation at the Thr-\n567 residue that is necessary for ezrin activation, membrane binding, and microvilli \nstabilization (Viswanatha et al., 2012; Welf et al., 2020).  When ST-denuded explants were \npulsed with ezrin inhibitor for 2hrs, there was a smoothening of the actin signal at the apical \nmembrane and a loss of the hair-like F-actin projections (Fig. 4A), showing that disruption of 215 \nezrin activation leads to the destabilization of apical microvilli. In addition, ezrin inhibitor dose-\ndependently impaired vCT fusion when explants were treated for 48hrs (Fig. 4B, C). While \nexposure to 5µM and 10µM of inhibitor had variable effects on fusion, 20µM and 50µM doses \nresulted in a consistent 63% and 86% decrease in fusion respectively when compared to \nvehicle controls. Therefore, blocking ezrin activation strongly inhibits vCT fusion and leads to 220 \na loss of apical microvilli.   \n \nCell shape, or more specifically, a decrease in cell surface area immediately after fusion, has \nrecently been shown to reinforce transcriptional regulation of cell differentiation in fused cells \n(Feliciano et al., 2021). Multiple studies examining trophoblast fusion and differentiation using 225 \nthe BeWo choriocarcinoma cell line and primary vCT have identified that trophoblast fusion \nand functional differentiation into ST, or at least the production of β-hCG, are isolatable \nprocesses (Collett et al., 2012; Johnstone et al., 2005; Orendi et al., 2010). Therefore, we \nquestioned whether ezrin inhibitor treatment would also block β-hCG expression and the \nexpression of other vCT differentiation and ST marker genes. Ezrin inhibitor treatment led to 230 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 11 \nan almost a complete loss of anti-β-hCG signal compared to vehicle controls (Fig. 5A), and a \nconcomitant decrease in CGB mRNA (Fig. 5B). Additional markers of vCT differentiation and \nST were also assessed by RT-PCR. GCM1 is present nearly exclusively in the human \nplacenta (Baczyk et al., 2013; Nait-Oumesmar et al., 2000) and is a key placental \ntranscription factor for vCT differentiation into ST (Baczyk et al., 2009; Jeyarajah et al., 2022; 235 \nWoods et al., 2018), and its levels increase as syncytialization progresses (Jeyarajah et al., \n2022). With ezrin inhibition, GCM1 expression in explant lysates was significantly decreased \ncompared to paired controls (Fig. 5C). Syncytin-1 (ERVW-1) and syncytin-2 (ERVFRD-1) are \nvCT fusogens (Malassine et al., 2007; Mi et al., 2000; Muir et al., 2006) and therefore \nessential for mediating vCT fusion(Blond et al., 2000; Frendo et al., 2003; Mi et al., 2000; 240 \nVargas et al., 2009). A significant increase in relative ERVFRD-1 expression (Fig. 5D) and a \nnon-significant increase in ERVW-1 abundance (Fig. 5E) was observed with ezrin inhibition, \nindicating that blocking ezrin activation stalled vCT differentiation into ST. When taken \ntogether with the significant decrease in cell fusion observed in Fig. 4, these data suggest a \nloss of fusogen efficacy, not expression, occurs in the absence of ezrin activation. In 245 \nsummary, ezrin activity and stabilization of microvillar structure is necessary for fusion and \nloss of ezrin activity effectively arrests syncytialization.  \n \nDiscussion: \nProper formation and maintenance of the ST throughout gestation is critical for placental 250 \nfunction and significantly contributes to healthy pregnancy progression. Diminished vCT \nfusion capacity is a feature of devastating pregnancy complications like IUGR and \npreeclampsia (Gauster et al., 2009; Langbein et al., 2008; Ruebner et al., 2010), which are \nassociated with increased perinatal morbidity and mortality and lifelong increased risk of \ncardiovascular and neurological disease for affected infants (Burton et al., 2016). Therefore, 255 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 12 \nthorough understanding of the molecular regulation of vCT fusion could allow for the \ndevelopment of treatments for harmful placental pathologies and improve lifelong health. \nHere, we found that the stabilization of microvillar membrane protrusions within vCT is \nnecessary for successful fusion between neighbouring cells for ST regeneration. We also \npresent data that suggests that stabilized vCT microvilli may be necessary for fusion with 260 \nintact ST, thereby confirming that trophoblasts require protrusive F-actin-based membrane \nstructures to successfully catalyse cell-cell fusion as has been seen in all other fusing cell \ntypes.  \n \nOur data shows that the membrane binding protein ezrin plays a critical role in regulating vCT 265 \nmembrane structure and fusion. Importantly, ezrin has previously been shown to regulate vCT \nfusion through its role as a scaffolding protein for PKA and the gap junction protein connexin-\n43 (Pidoux et al., 2014). PKA phosphorylation of connexin-43 allows for the formation of gap \njunctions between adjacent cells to facilitate signal synchronization before fusion occurs. \nUsing primary 2D vCT culture and ezrin Thr-567 mutant overexpression, Pidoux et al. have 270 \nshown that the active form of ezrin is required for it to serve as a PKA scaffold. Therefore, we \ncannot rule out that the significant effects we observed with ezrin inhibitor treatment on fusion \nare not at least partially due to disruption in PKA/connexin-43 binding and our results are \nunlikely to be due to the loss of microvillar structures alone. Irrespective of the relative \ncontribution of microvillar structure versus PKA scaffolding on fusion, the combination of our 275 \nanalyses and this past work highlight that vCT accumulation of ezrin within microvillar \nstructures localizes critical pro-fusogenic signaling within a highly specialized cellular \ncompartment within the apical region of the cell. Intestinal microvilli are known to contain their \nown sub-proteome (McConnell et al., 2011) and the polarized localization of proteins within \nhighly specialized structures may be driven by the differential composition of the microvillar 280 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 13 \nlipid bilayer within highly curved and tightly packed structures (Cebecauer, 2021; Ikenouchi et \nal., 2013). The concept of microvilli as signaling centres is established in diverse non-fusing \ncell types like intestinal epithelia and T-cells (Kim et al., 2018; McConnell et al., 2009). \nTherefore, stabilized microvilli within the vCT seem likely to serve as a signaling platform to \nenrich the necessary components for fusion in proximity to the overlying ST and warrant 285 \nfurther investigation. \n \nIn addition, other fusing cell systems have shown the importance of increasing membrane-\nmembrane contact sites for fusion (Brukman et al., 2019; Kim and Chen, 2019; Wilson and \nSnell, 1998). Therefore, microvilli may serve to increase the apical surface area of vCT for this 290 \npurpose. E-cadherin signal was strongly accumulated in proximity to the observed apical ezrin \nsignal in our experiments, and others have observed the gap junction protein connexin-43 in a \nsimilar pattern (Cronier et al., 2002), thus microvilli may facilitate increased contact points by \nconcentrating junctional protein interactions between the vCT and overlying ST.  Simply due \nto the proximity of vCT microvilli to the ST membrane, it is tempting to suggest that fusion 295 \npores may also arise in microvillar tips. Syncytin-2 has been shown to have a non-polarized \nmembrane localization in select vCT in vivo (Esnault et al., 2008), but as a class I fusogen \n(Vance and Lee, 2020), it requires engagement with its receptor major facilitator superfamily \ndomain containing 2 (MFSD2) (Esnault et al., 2008) allowing for its extended conformation \nand subsequent insertion into the adjacent cell membrane to facilitate formation of a fusion 300 \npore. Therefore, microvilliation may allow for more efficient syncytin-2/MFSD2 engagement \ndue to the diminished space between vCT and ST membranes and increased contact sites. \nMFSD2 is expressed on both the ST and CT (Esnault et al., 2008), therefore increasing the \ncontact points between vCT syncytin-2 and ST MFSD2 would increase the likelihood of \nfusion.  305 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 14 \n \nOur ST denuded explant model takes advantage of the tendency of vCT to spontaneously \nfuse when overlying syncytium is lost. Though not a perfect physiologic system, regions of \nlocalized ST denudation and subsequent fibrin deposition are a feature of both healthy and \npathologic placentas (Scifres and Nelson, 2009), therefore this model more accurately 310 \nrepresents vCT fusion for ST regeneration than ST maintenance. Importantly, though our cell \nshape factor analyses revealed that ST denudation shifts vCT population medians for the \nanalysed factors by increasing the proportion of cells that lie on the higher or lower extremes \nof a population, the majority of vCT in the denuded tissue still fall within the limits of those \nobserved in intact tissue. Thus, the cellular features associated with fusion competence are 315 \nlikely accentuated by a lack of negative feedback from the ST.  \n \nInterestingly, the tendency for vCT to increase in length in the apical-basal axis, the \npositioning of the nucleus in a more basal position with ST removal, and the polarized \naccumulation of ezrin at the apical surface support a model whereby the initiation of an apical-320 \nbasal polarization network amplifies a pro-fusogenic vCT state and dictates the position of the \nnewly formed ST. Ezrin localization in 2D cultured primary vCT was not found to be polarized \nand generally localized throughout the membrane (Pidoux et al., 2014), suggesting that vCT \ninteractions with the extracellular matrix of the basement membrane may allow for \npolarization, and thereby enhance fusion. Critically, apparent microvillar projections have 325 \nbeen observed by others during 2D vCT fusion (Bax et al., 1989), indicating that polarization \nof ezrin within the cell is not obligate for vCT fusion, but supporting that the formation of \nmicrovilli likely is. Work with trophoblast organoids indirectly supports our hypothesis that vCT \npolarity and extracellular matrix interactions direct the position of ST formation. In Matrigel \nembedded organoids where the extracellular matrix is surrounding a ball of progenitor CTs, 330 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 15 \nCTs spontaneously form ST in a cystic core instead of on the outer surface (Haider et al., \n2018; Turco et al., 2018). Our previous work has shown that the canonical apical-basal \npolarity regulating factor atypical protein kinase-c (aPKC) does not have a highly polarized \nstaining pattern within first trimester vCT (Shaha et al., 2022), suggesting an alternative \npathway may be activated to reinforce vCT polarity during differentiation. Understanding the 335 \nfactors that initiate vCT polarity and microvillus formation will be an interesting future \ndirection.  \n \nWe chose to perform our analyses in first trimester tissue explants due to the continuous \nnature of the vCT layer at this point in gestation, thereby allowing us to analyze the greatest 340 \nnumber of cells. With advancing gestation vCT change from a continuous layer of cuboidal \ncells to a discontinuous layer of flattened cells (Jones and Fox, 1991). Microvilliated vCT in \nterm tissue have been observed in intact term placental sections with TEM (Jones and Fox, \n1991), supporting that this may be a conserved feature of vCT fusion competence across \ngestation. Other shape parameters we examined may not translate to later gestational stages. 345 \nFuture work is necessary to explore whether fusion competent vCT beyond the first trimester \nhave shared morphological features with first trimester cells. Differences in the way vCT \nexecute fusion across gestation are possible and understanding their conserved and \ndivergent regulation would inform development of treatments in the future. \n 350 \nIn summary, we show that stabilization of vCT microvilli is critical for fusion and formation of \nST. The microvillus is a conserved site of fusion throughout evolution seen in cell-fusion \nevents in species as diverse as sea urchin and Drosophila, yet understanding of why these \nstructures are necessary and how their emergence and stabilization is regulated to control \none of the most dramatic events in cell biology is lacking. We identified that ezrin and F-actin 355 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 16 \nare contained in vCT membrane protrusions when fusion is stimulated, therefore future work \nto address the temporal and spatial regulation of the cortical actin cytoskeleton and its \ninterplay with ezrin and membrane proteins to regulate microvilli emergence and localization \nwill be particularly important.    \n  360 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 17 \n \nMaterials and Methods: \nTissue collection  \n9-12 weeks gestational age human placental samples were collected by methods approved \nby the University of Alberta Human Research Ethics Board (Pro00089293). First trimester 365 \nplacental tissue was obtained from elective pregnancy terminations following informed \nconsent from the patients.  \n \nST regeneration model and treatments \nPlacental samples were cut into 2-3mm3 sized explants in ice-cold phosphate-buffered saline 370 \n(PBS) and then either fixed immediately as uncultured pre-trypsin controls or trypsinized and \ncultured. For trypsinization, tissue was digested with 0.25% trypsin-EDTA (Gibco; Burlington, \nON, Canada; cat:25200-072) for seven minutes at 37˚C. The reaction was quenched using \n10% heat-inactivated fetal bovine serum (FBS, Wisent; Saint-Jean-Baptiste, QC, Canada; \ncat:098150), and a single explant was placed in each well of a 48 well plate in medium 375 \nconsisting of Iscove's Modified Dulbecco's Medium (IMDM) (Gibco, cat:12440-053), 5% heat-\ninactivated FBS (Wisent, cat:098150), 50µg/mL gentamicin (Gibco, cat:15710-064), and 1X \nITS-X supplement (Gibco, cat:51500-056) at 37˚C 5% CO2. Medium was changed 24hrs post-\ntrypsinization to remove stripped ST debris. For ezrin inhibitor treatments, 5-50µM, of \nNSC668394 (EMD Millipore; Oakville, ON, Canada; cat:341216, lot:3934373) or vehicle 380 \ncontrol (DMSO) was added to medium 24hrs post-trypsinization with 3-12 explants per \ntreatment group for 2-48hrs.  \n \n \n 385 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 18 \nTissue staining  \nPlacental tissue (uncultured and cultured explants) was fixed using 2% paraformaldehyde \n(PFA) overnight at 4˚C or 4% PFA on ice for two hours. When necessary, heat-induced \nantigen retrieval was conducted using 0.01M sodium citrate buffer at pH 6.0. Fixed tissue was \nblocked and permeabilized in blocking buffer (5% normal donkey serum, 0.5% Triton-X100). 390 \nTissue was then incubated overnight at 4˚C in blocking buffer with anti-E-cadherin (R&D \nSystems; Minneapolis, MN, USA; cat:MAB1838), anti-integrin-⍺-6 (Stem Cell Technologies; \nVancouver, BC, Canada; cat:60037), anti-β-human-chorionic-gonadotrophin (β-hCG) (Abcam; \nCambridge, UK; cat:ab9582), and anti-Ezrin (Invitrogen; Burlington, ON, Canada; cat: PA5-\n18541). For further antibody information, please see Appendix Table 1. Tissue was then 395 \nwashed with PBS and 0.05% Tween (PBST) and PBS, and incubated with the appropriate \nfluorescent secondary antibodies AlexaFluor 488 (Invitrogen), AlexaFluor 594 (Invitrogen), \nand/or stained with 0.17µM phalloidin-iFluor 594 (AAT Bioquest; Pleasanton, CA, USA; \ncat:23122). Tissue was then incubated with 10µg/mL Hoechst 33342 (Thermo Fisher \nScientific; Burlington, ON, Canada; cat:H3570) followed by PBS washes and whole-mounted 400 \nwith imaging spacers and Fluoromount-G mounting medium (Southern Biotech; Burlington, \nON, Canada; cat: 0100-01).  \n \nConfocal microscopy image capture and analyses \nTriplicate z-stack images were acquired with a Zeiss LSM 700 confocal microscope using a 405 \nZeiss Plan Apochromat-20X/0.8 M27 or Zeiss Plan Apochromat-63X/1.4 M27 oil lens. 15-\n30µm z-stack images with a 0.6µm step-size were taken at 63x magnification. 60-120µm z-\nstack images with a 4µm step-size and single XY-plane snapshots were taken at 20x \nmagnification.  ST structures were identified as apically located multinucleated structures. \nvCTs were identified as E-cadherin-positive or integrin-⍺-6-positive mononucleated cells.  410 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 19 \n \nvCT cell characteristics were measured using 63x z-stack confocal microscopy images on \nplacental samples stained with anti-E-cadherin, phalloidin, and Hoechst. vCT cell length, vCT \nvolume, vCT shape factor, relative nuclear positioning within vCT, and vCT apical volume was \nquantified using Volocity Imaging software (Quorum Technologies Inc., version 7.0.0). 415 \nRelative positioning of the nucleus was calculated along the apical-basal axis by measuring \nthe length between the center of the nucleus to the basement membrane and then dividing it \nby the total length of the cell (equation 1).  \n \nST coverage and fusion capacity was measured using 20x single-plane confocal microscopy 420 \nimages on placental samples stained with anti-E-cadherin, phalloidin, and Hoechst. ST \ncoverage was quantified using Volocity Imaging software by measuring the ratio of the cross-\nsectional area of the ST compared to the vCT (ST:vCT) (equation 2). Time dependent relative \nchange in ST coverage was calculated as the relative ratio of ST:vCT coverage at 72hrs \ncompared to 24hrs (equation 3). Relative fusion in ezrin-inhibitor-treated explants was 425 \nnormalized to vehicle treated explants.  \n \nEquation 1 \n𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑛𝑢𝑐𝑙𝑒𝑎𝑟 𝑝𝑜𝑠𝑖𝑡𝑖𝑜𝑛 = 𝐿𝑒𝑛𝑔𝑡ℎ 𝑓𝑟𝑜𝑚 𝑐𝑒𝑛𝑡𝑒𝑟 𝑜𝑓 𝑛𝑢𝑐𝑙𝑒𝑢𝑠 𝑡𝑜 𝑏𝑎𝑠𝑒𝑚𝑒𝑛𝑡 𝑚𝑒𝑚𝑏𝑟𝑎𝑛𝑒\n𝑇𝑜𝑡𝑎𝑙 𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑡ℎ𝑒 𝑐𝑒𝑙𝑙  \n 430 \nEquation 2 \n𝑆𝑇 𝑐𝑜𝑣𝑒𝑟𝑎𝑔𝑒 =  𝑐𝑟𝑜𝑠𝑠 𝑠𝑒𝑐𝑡𝑖𝑜𝑛𝑎𝑙 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑆𝑇\n𝑐𝑟𝑜𝑠𝑠 𝑠𝑒𝑐𝑡𝑖𝑜𝑛𝑎𝑙 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑣𝐶𝑇 \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 20 \nEquation 3 \n𝐹𝑢𝑠𝑖𝑜𝑛 =  ∆𝑆𝑇 𝑐𝑜𝑣𝑒𝑟𝑎𝑔𝑒 = 𝑆𝑇 𝑐𝑜𝑣𝑒𝑟𝑎𝑔𝑒 𝑎𝑡 72ℎ𝑟𝑠\n𝑆𝑇 𝑐𝑜𝑣𝑒𝑟𝑎𝑔𝑒 𝑎𝑡 24ℎ𝑟𝑠 435 \n \n \nScanning electron microscopy (SEM) \nAt 24 hrs post-trypsinization, explants were collected, washed in PBS, and fixed in EM fixative \n(2.5% glutaraldehyde, 2% PFA in PBS). Tissue was then washed with PBS and treated with 440 \n1% osmium tetroxide in PBS for one hour. After washing, samples were dehydrated by \nincubation in a graded ethanol series and then in a graded series of increasing \nhexamethyldisilazane (HMDS) and decreasing ethanol concentrations. Samples were \nincubated in 100% HMDS and then airdried overnight. Tissue was then mounted onto \nstandard aluminium stubs and sputter coated with Au/Pd using a Hummer 6.2 Sputter Coater 445 \n(Anatech). Images were acquired using a Zeiss EVO 10 scanning electron microscope \noperating at 15kV and SmartSEM software (Zeiss, version 6.06). \n \nTransmission electron microscopy (TEM) \nPBS washed and trimmed placental tissue was fixed in EM fixative (2.5% glutaraldehyde, 2% 450 \nPFA in PBS) upon collection. Tissue was then washed with PBS and treated with 1% osmium \ntetroxide in PBS for one hour. After washing, tissue was then dehydrated by incubation in a \ngraded ethanol series followed by incubation in 1:1 ethanol:Spurr resin (Electron Microscopy \nSciences; Hatfield, PA, USA) overnight. The following day the tissue was embedded in 100% \nresin and cured overnight at 70˚C. Using a Reichert-Jung Ultracut_E Ultramicrotome, the 455 \nresin blocks were cut into 70-90nm sections. Sections were stained with uranyl acetate and \nlead citrate on 75 mesh grids. Images were acquired using a Philips/FEI (Morgagni) \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 21 \ntransmission electron microscope operating at 80kV with Gatan camera and Digital \nMicrograph software (version, 1.81.78). \n 460 \nReverse-transcriptase polymerase chain reaction (RT-PCR)  \nAfter 48 hrs of incubation (+/- 50µM ezrin inhibitor), explant cultured tissue was collected, \nrinsed in PBS, and then homogenized in TRIzol (Thermo Fisher Scientific) using a tissue lyser \nand RNA was extracted using TRIzol-chloroform extraction. RNA was isolated and purified \nusing a PureLink RNA Mini Kit (Ambion; Carlsbad, CA, USA ; cat:12183025). Conversion into 465 \ncDNA was performed using reverse transcription (iScript cDNA Synthesis Kit; Bio-Rad \nLaboratories, Mississauga, ON, Canada; cat:1708890) with 250ng RNA. Quantitative RT-PCR \nwas performed using 3uL diluted cDNA (1:5) per reaction and Applied Biosystems SYBR \nGreen PCR Master Mix (Thermo Fisher Scientific; cat:4309155) on a QuantStudio 3 Real-\nTime PCR System (Thermo Fisher Scientific). Primer sequences are indicated in Appendix 470 \nTable 2. Relative change in mRNA expression was calculated using the 2-^∆∆CT method to \npaired samples using the mean CT from CYC1 and TOP1 as housekeeping genes (Kaitu'u-\nLino et al., 2014; Livak and Schmittgen, 2001; Shaha et al., 2022).  \n \nStatistical analyses 475 \nStatistical analyses were conducted using GraphPad Prism (version 9.3.1) with a p<0.05 \nthreshold for significance. Exact statistical analyses are within the figure legends. Statistical \noutliers were determined using a ROUT outlier analyses in GraphPad Prism. All graphs and \nrepresentative images are representative of three to six biological replicates (placental tissue \nfrom different patients) and at least three technical replicates.  480 \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 22 \nAcknowledgements: We would like to thank all the tissue donors and the staff of Women’s \nHealth Options Clinic without whom this work would not have been possible. We would also 485 \nlike to thank Dr. Kacie Norton and Arlene Oatway of the University of Alberta Biological \nSciences Imaging Facility for their expert technical advice for the electron microscopy sample \npreparation and imaging.  \n \nCompeting Interests: The authors declare no competing interests for this work. 490 \n \nFunding: This work was supported by the Natural Sciences and Engineering Research \nCouncil of Canada [RGPIN-2021-02807; to M.R.]; W.D was supported by studentships from \nthe Natural Sciences and Engineering Research Council of Canada and the Women and \nChildren’s Health Research Institute and their donors at the Alberta Women’s Health 495 \nFoundation. S.Z.S was supported by an Alberta Innovates Graduate Student Scholarship. \n \nData Availability: All relevant data can be found within the article and supplementary \ninformation. \n 500 \nDiversity and Inclusion Statement:  \nThe authors W.D (she/her), S.Z.S (she/her), I.D (he/him), and K.P (she/her) have lived \nexperience as racial and ethnic minorities in Canada intersecting with our respective diverse \ngender identities and sexual orientations. Ensuring equity, diversity, and inclusion in the field \nof developmental biology is more than moral obligation but also necessary to enrich existing 505 \nand future knowledge and to promote excellence in research and healthcare. As a lab we are \ncommitted to integrating these diverse perspectives into our current understanding and \nproviding equitable opportunities.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 23 \nReferences: \n Abmayr, S. M. and Pavlath, G. K. (2012). Myoblast fusion: lessons from flies and 510 \nmice. Development 139, 641-56. \n Aghababaei, M., Hogg, K., Perdu, S., Robinson, W. P. and Beristain, A. G. (2015). \nADAM12-directed ectodomain shedding of E-cadherin potentiates trophoblast fusion. Cell \nDeath Differ 22, 1970-84. \n Aguilar, P. S., Baylies, M. K., Fleissner, A., Helming, L., Inoue, N., Podbilewicz, B., 515 \nWang, H. and Wong, M. (2013). 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Regulation of Placental 695 \nDevelopment and Its Impact on Fetal Growth-New Insights From Mouse Models. Front \nEndocrinol (Lausanne) 9, 570. \n Zhu, L., Zhou, R., Mettler, S., Wu, T., Abbas, A., Delaney, J. and Forte, J. G. (2007). \nHigh turnover of ezrin T567 phosphorylation: conformation, activity, and cellular function. Am \nJ Physiol Cell Physiol 293, C874-84. 700 \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 29 \nFigures: \n \nFigure 1: ST regeneration model enriches a fusion-competent vCT subpopulation and 705 \nsynchronizes ST development. A) Representative single XY-plane confocal microscopy \nimages of placental tissue in pre-trypsinized non-cultured controls, at 24hrs post-\ntrypsinization, and 72hrs post-trypsinization; top row was stained with phalloidin (F-actin; \nmagenta), anti-E-cadherin (green), and Hoechst 33342 (nuclei; blue); bottom row was stained \nwith anti-β-hCG (magenta), anti-integrin-⍺-6 (green), Hoechst 33342 (nuclei; blue); white 710 \narrows indicate ST regions; arrowheads indicate vCT regions; B) Summary data of ST \ncoverage (ST:vCT) at 24hrs and 72hrs normalized to pre-trypsin controls; mean ± S.E.M., \nunpaired parametric student’s t-test, n=3; ST coverage (ST:vCT) was calculated using \nequation 2 (Methods); C) Representative single XY-plane confocal microscopy images of pre-\ntrypsinized control tissue and tissue 24hrs post-trypsinization stained with phalloidin 715 \n(magenta), anti-E-cadherin (green), and Hoechst 33342 (nuclei; blue); D) Summary data of \nvCT cell length (µm); npretrypsin=210; n24hrs=178; E) Summary data of vCT cell volume (µm3); \nnpretrypsin=237; n24hrs=290; F) Summary data of vCT shape factor; npretrypsin=237; n24hrs=290; G) \nSummary data for the relative position of the nucleus along the apical-basal axis; \nnpretrypsin=210; n24hrs=178; calculated using equation 1 (Methods); H) Summary data of vCT 720 \napical volume (µm3); npretrypsin=195; n24hrs=220; D) - H) unpaired Mann-Whitney test; bold line \n= median; dashed lines = quartile 1 and quartile 3. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 30 \nFigure 2: ST denudation increases accumulation of microvilli on the vCT apical \nmembrane. A) Representative single ZY-plane (i), XY-plane (ii, iii), and 3D reconstituted (iv) 725 \nconfocal microscopy images of tissue 24hrs post-trypsinization; merged image of phalloidin \n(greyscale) and Hoechst 33342 (nuclei; blue) signals; iii = higher magnification image of \nindicated region in ii; B) Representative SEM images of explant tissue 24hrs post-\ntrypsinization; bottom panels = false-coloured images with colours marking individual vCT; C) \nRepresentative single XY-plane confocal microscopy images (i, ii) of tissue 24hrs post-730 \ntrypsinization; i = merged image of anti-ezrin (magenta), anti-E-cadherin (green), and Hoechst \n33342 (nuclei; blue) signals; ii = isolated anti-ezrin (greyscale) and Hoechst 33342 (nuclei; \nblue) signals; iii= ZY plane isolated anti-ezrin (greyscale) and Hoechst 33342 (nuclei; blue) \nsignals. \n 735 \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 31 \nFigure 3: Apical microvilli of are present in vCT in intact placental tissue.  \nA) Representative TEM images of uncultured placental tissue; ii = higher magnification image \nof indicated region in i. B) Representative single XY-plane confocal microscopy images of \nuncultured placental tissue; i = merged images of phalloidin (magenta), anti-E-cadherin 740 \n(green), and Hoechst 33342 (nuclei; blue) signals; ii = isolated anti-E-cadherin (greyscale) \nand Hoechst 33342 (nuclei; blue) signals; iii = isolated phalloidin (greyscale) and Hoechst \n33342 (nuclei; blue) signals; iv = higher magnification images of indicated regions of isolated \nphalloidin (greyscale) and Hoechst 33342 (nuclei; blue) signals; C) Representative single XY-\nplane confocal microscopy images of uncultured placental tissue; i = merged images of anti-745 \nezrin (magenta), anti-E-cadherin (green), and Hoechst 33342 (nuclei; blue); ii = isolated anti-\nezrin (greyscale) and Hoechst 33342 signals; iii = higher magnification image of indicated \nregion in ii.  \n \n  750 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 32 \nFigure 4: Ezrin inhibitor treatment impairs vCT fusion and alters protrusive actin \nfilament accumulation on the apical surface. A) Representative single XY-plane confocal \nmicroscopy images of explants pulsed for 2hrs ± 50µM ezrin inhibitor at 24hrs post-\ntrypsinization; i = merged image of phalloidin (magenta), anti-E-cadherin (green), and \nHoechst 33342 (nuclei; blue) signals; ii = isolated phalloidin (greyscale) and Hoechst 33342 755 \n(nuclei; blue) signals; iii = higher magnification image of regions indicated in ii; B) \nRepresentative single XY-plane and extended focus confocal microscopy images of explants \ncultured for 72hrs total ± 50µM ezrin inhibitor added at 24hrs post-trypsinization stained with \nphalloidin (magenta), anti-E-cadherin (green), and Hoechst 33342 (nuclei; blue); white arrows \nindicate regenerated ST regions; arrowheads indicate unfused vCT regions; C) Summary 760 \ndata of dose-dependent ezrin inhibitor treatment on fusion (change in ST:vCT) normalized to \nvehicle controls; mean ± S.E.M., unpaired Kruskal-Wallis test with Dunnett’s multiple \ncomparisons, n=6; fusion (change in ST:vCT) is calculated using equation 3 (Methods).  \n \n  765 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n 33 \nFigure 5: Ezrin inhibitor treatment impairs vCT functional differentiation.  \nA) Representative single XY-plane confocal microscopy images of explants cultured for 48hrs \n± 50µM ezrin inhibitor added 24hrs post-trypsinization; i = merged images of anti-β-hCG \n(magenta), anti-integrin-⍺-6 (green), and Hoechst 33342 (nuclei; blue) signals; ii = isolated \nanti-β-hCG (greyscale) and Hoechst 33342 (nuclei; blue) signals; white arrows indicate 770 \nregenerated ST regions; arrowheads indicate unfused vCT regions; B) Relative CGB \nexpression C) Relative GCM1 expression D) Relative ERVFRD-1 expression, and E) Relative \nERVW-1 expression in explants cultured for 48hrs total ± 50µM ezrin inhibitor added 24hrs \npost-trypsinization; Data represented as 2^-ΔΔCT to mean of TOP1 and CYC1 normalized to \ncontrols; mean ± S.E.M., one-sample t-test, n=4-5. 775 \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\nFigure 1\nPre-trypsin 24hrs 72hrs\nPhall/E-cad/ Nucβ-hCG/ITGA-6/Nuc\nPre-trypsin 24hrs\n10µm 10µm\n50µm 50µm 50µm\n50µm 50µm 50µm\nA)\nB) C)\nD) E)\nH)\nF)\nG)\nPhall/E-cad/Nuc\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted February 24, 2024. ; https://doi.org/10.1101/2024.02.22.581647doi: bioRxiv preprint \n\n2µm\n2µm\n2µm\n2µm\n2µm\n2µm\nFigure 2\nPhall/Nuc\nB)\nC)Ezrin/E-cad/Nuc\nA)\nEzrin/Nuc\n10µm10µm\n10µm\n5µm\nii iii iv\ni ii\ni\niii\n1 unit = 10.2µm\n10µm\n10µm\n\nFigure 3\nB)\nA)\n0.5 µmPhall/E-cad/NucE-cad/NucPhall/Nuc\n10µm\n10µm\nPhall/Nuc\n10µm\n10µm\n10µm\n10µm\n5µm\n5µm\nvCTST\nC)Ezrin/E-cad/NucEzrin/Nuc\n10µm10µm\n 5µm\nEzrin/Nuc\ni ii iii iv\ni ii iii\ni ii\nvCT\nST\nVillous TipVillous Length\ni ii iii iv\n\nFigure 4\nB)Ezrin inhibitorControl\nC)\n50µm\n50µm\n50µm\n50µm\nA)Phall/E-cad/Nuc\nControlEzrin inhibitor\nPhall/Nuc\nPhall/Nuc\nXY plane\nXY plane\nExtended focus\nExtended focus\nE-cad/Nuc\n10µm5µm\n5µm10µm10µm\n10µm\ni ii iii\ni ii iii\nPhall/E-cad/Nuc\n\nFigure 5A)\nB)\nEzrin inhibitorControl\nβ-hCG/ITGA-6/Nuc\n50µm50µm\n 50µm50µm\nβ-hCG/Nucleus\ni ii\nC)D)E)\ni iiEzrin inhibitorControl","source_license":"CC-BY-4.0","license_restricted":false}