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Ciliated epithelia are widespread in animals and play crucial roles in many developmental and physiological processes. Epithelia composed of multi-ciliated cells allow for directional fluid flow in the trachea, oviduct and brain cavities. Monociliated epithelia play crucial roles in vertebrate embryos, from the establishment of left–right asymmetry to the control of axis curvature via cerebrospinal flow motility in zebrafish. Cilia also have a central role in the motility and feeding of free-swimming larvae in a variety of marine organisms. These diverse functions rely on the coordinated orientation (rotational polarity) and asymmetric localization (translational polarity) of cilia and of their centriole-derived basal bodies across the epithelium, both being forms of planar cell polarity (PCP). Here, we review our current knowledge on the mechanisms of the translational polarity of basal bodies in vertebrate monociliated epithelia from the molecule to the whole organism. We highlight the importance of live imaging for understanding the dynamics of centriole polarization. We review the roles of core PCP pathways and of apicobasal polarity proteins, such as Par3, whose central function in this process has been recently uncovered. Finally, we emphasize the importance of the coordination between polarity proteins, the cytoskeleton and the basal body itself in this highly dynamic process. [ABSTRACT FROM AUTHOR]

Epithelial polarity is fundamental in maintaining barrier integrity and tissue protection. In cystic fibrosis (CF), apicobasal polarity of the airway epithelium is altered, resulting in increased apical fibronectin deposition and enhanced susceptibility to bacterial infections. Here, we evaluated the effect of highly effective modulator treatment (HEMT) on fibronectin apical deposition and investigated the intracellular mechanisms triggering the defect in polarity of the CF airway epithelium. To this end, primary cultures of CF (F508del variant) human airway epithelial cells (HAECs) and a HAEC line, Calu-3, knocked down for CFTR (CF transmembrane conductance regulator) were compared with control counterparts. We show that CFTR mutation in primary HAECs and CFTR knockdown cells promote the overexpression and oversecretion of TGF-β1 and DKK1 when cultured at an air–liquid interface. These dynamic changes result in hyperactivation of the TGF-β pathway and inhibition of the Wnt pathway through degradation of β-catenin leading to imbalanced proliferation and polarization. The abnormal interplay between TGF-β and Wnt signaling pathways is reinforced by aberrant Akt signaling. Pharmacological manipulation of TGF-β, Wnt, and Akt pathways restored polarization of the F508del CF epithelium, a correction that was not achieved by HEMT. Our data shed new insights into the signaling pathways that fine-tune apicobasal polarization in primary airway epithelial cells and may provide an explanation to the mitigated efficacy of HEMT on lung infection in people with CF. [ABSTRACT FROM AUTHOR]

Delivery to the correct membrane domain in polarized epithelial cells is a critical regulatory mechanism for transmembrane proteins. The trafficking of these proteins is directed by short amino acid sequences known as sorting motifs. In six basolaterally-localized proteins lacking the canonical tyrosine- and dileucine-based basolateral sorting motifs, a monoleucine-based sorting motif has been identified. This review will discuss these proteins with an identified monoleucine-based sorting motif, their conserved structural features, as well as the future directions of study for this non-canonical basolateral sorting motif. [ABSTRACT FROM AUTHOR]

Mitochondrial morphology dynamics regulate signaling pathways during epithelial cell formation and differentiation. The mitochondrial fission protein Drp1 affects the appropriate activation of EGFR and Notch signaling-driven differentiation of posterior follicle cells in Drosophila oogenesis. The mechanisms by which Drp1 regulates epithelial polarity during differentiation are not known. In this study,we show that Drp1-depleted follicle cells are constricted in early stages and present in multiple layers at later stages with decreased levels of apical polarity protein aPKC. These defects are suppressed by additional depletion of mitochondrial fusion protein Opa1. Opa1 depletion leads to mitochondrial fragmentation and increased reactive oxygen species (ROS) in follicle cells. We find that increasing ROS by depleting the ROS scavengers, mitochondrial SOD2 and catalase also leads to mitochondrial fragmentation. Further, the loss of Opa1, SOD2 and catalase partially restores the defects in epithelial polarity and aPKC, along with EGFR and Notch signaling in Drp1-depleted follicle cells. Our results show a crucial interaction between mitochondrial morphology, ROS generation and epithelial cell polarity formation during the differentiation of follicle epithelial cells in Drosophila oogenesis. [ABSTRACT FROM AUTHOR]

Primary cilia are sensory cellular organelles crucial for organ development and homeostasis. Ciliogenesis in polarized epithelial cells requires Rab19-mediated clearing of apical cortical actin to allow the cilium to grow from the apically docked basal body into the extracellular space. Loss of the lysosomal membrane-tethering homotypic fusion and protein sorting (HOPS) complex disrupts this actin clearing and ciliogenesis, but it remains unclear how the ciliary function of HOPS relates to its canonical function in regulating late endosome-lysosome fusion. Here, we show that disruption of HOPSdependent lysosomal fusion indirectly impairs actin clearing and ciliogenesis by disrupting the targeting of Rab19 to the basal body, and that this effect is specific to polarized epithelial cells. We also find that Rab19 functions in endolysosomal cargo trafficking in addition to having its previously identified role in ciliogenesis. In summary, we show that inhibition of lysosomal fusion leads to the abnormal accumulation of Rab19 on late endosomes, thus depleting Rab19 from the basal body and thereby disrupting Rab19-mediated actin clearing and ciliogenesis in polarized epithelial cells. [ABSTRACT FROM AUTHOR]

Animal organs maintain tissue integrity and ensure removal of aberrant cells through several types of surveillance mechanisms. One prominent example is the elimination of polarity-deficient mutant cells within developing Drosophila imaginal discs. This has been proposed to require heterotypic cell competition dependent on the receptor tyrosine phosphatase PTP10D within the mutant cells. We report here experiments to test this requirement in various contexts and find that PTP10D is not obligately required for the removal of scribble (scrib) mutant and similar polarity-deficient cells. Our experiments used identical stocks with which another group can detect the PTP10D requirement, and our results do not vary under several husbandry conditions including high and low protein food diets. Although we are unable to identify the source of the discrepant results, we suggest that the role of PTP10D in polarity-deficient cell elimination may not be absolute.

Epithelial polarity is critical for proper functions of epithelial tissues, tumorigenesis, and metastasis. The evolutionarily conserved transmembrane protein Crumbs (Crb) is a key regulator of epithelial polarity. Both Crb protein and its transcripts are apically localized in epithelial cells. However, it remains not fully understood how they are targeted to the apical domain. Here, using Drosophila ovarian follicular epithelia as a model, we show that epithelial polarity is lost and Crb protein is absent in the apical domain in follicular cells (FCs) in the absence of Diamond (Dind). Interestingly, Dind is found to associate with different components of the dynactin‐dynein complex through co‐IP‐MS analysis. Dind stabilizes dynactin and depletion of dynactin results in almost identical defects as those observed in dind‐defective FCs. Finally, both Dind and dynactin are also required for the apical localization of crb transcripts in FCs. Thus our data illustrate that Dind functions through dynactin/dynein‐mediated transport of both Crb protein and its transcripts to the apical domain to control epithelial apico‐basal (A/B) polarity. [ABSTRACT FROM AUTHOR]

Delivery to the correct membrane domain in polarized epithelial cells is a critical regulatory mechanism for transmembrane proteins. The trafficking of these proteins is directed by short amino acid sequences known as sorting motifs. In six basolaterally-localized proteins lacking the canonical tyrosine- and dileucine-based basolateral sorting motifs, a monoleucine-based sorting motif has been identified. This review will discuss these proteins with an identified monoleucine-based sorting motif, their conserved structural features, as well as the future directions of study for this non-canonical basolateral sorting motif.

Branching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin β1 and inhibits its downstream signaling, and Integrin β1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin β1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct.

Ciliated epithelia are widespread in animals and play crucial roles in many developmental and physiological processes. Epithelia composed of multi-ciliated cells allow for directional fluid flow in the trachea, oviduct and brain cavities. Monociliated epithelia play crucial roles in vertebrate embryos, from the establishment of left–right asymmetry to the control of axis curvature via cerebrospinal flow motility in zebrafish. Cilia also have a central role in the motility and feeding of free-swimming larvae in a variety of marine organisms. These diverse functions rely on the coordinated orientation (rotational polarity) and asymmetric localization (translational polarity) of cilia and of their centriole-derived basal bodies across the epithelium, both being forms of planar cell polarity (PCP). Here, we review our current knowledge on the mechanisms of the translational polarity of basal bodies in vertebrate monociliated epithelia from the molecule to the whole organism. We highlight the importance of live imaging for understanding the dynamics of centriole polarization. We review the roles of core PCP pathways and of apicobasal polarity proteins, such as Par3, whose central function in this process has been recently uncovered. Finally, we emphasize the importance of the coordination between polarity proteins, the cytoskeleton and the basal body itself in this highly dynamic process.

Neurons forming the human brain are generated during embryonic development by neural stem and progenitor cells via a process called neurogenesis. A crucial feature contributing to neural stem cell morphological and functional heterogeneity is cell polarity, defined as asymmetric distribution of cellular components. Cell polarity is built and maintained thanks to the interplay between polarity proteins and polarity-generating organelles, such as the endoplasmic reticulum (ER) and the Golgi apparatus (GA). ER and GA affect the distribution of membrane components and work as a hub where glycans are added to nascent proteins and lipids. In the last decades our knowledge on the role of polarity in neural stem and progenitor cells have increased tremendously. However, the role of traffic and associated glycosylation in neural stem and progenitor cells is still relatively underexplored. In this review, we discuss the link between cell polarity, architecture, identity and intracellular traffic, and highlight how studies on neurons have shaped our knowledge and conceptual framework on traffic and polarity. We will then conclude by discussing how a group of rare diseases, called congenital disorders of glycosylation (CDG) offers the unique opportunity to study the contribution of traffic and glycosylation in the context of neurodevelopment. [ABSTRACT FROM AUTHOR]

Neurons forming the human brain are generated during embryonic development by neural stem and progenitor cells via a process called neurogenesis. A crucial feature contributing to neural stem cell morphological and functional heterogeneity is cell polarity, defined as asymmetric distribution of cellular components. Cell polarity is built and maintained thanks to the interplay between polarity proteins and polarity-generating organelles, such as the endoplasmic reticulum (ER) and the Golgi apparatus (GA). ER and GA affect the distribution of membrane components and work as a hub where glycans are added to nascent proteins and lipids. In the last decades our knowledge on the role of polarity in neural stem and progenitor cells have increased tremendously. However, the role of traffic and associated glycosylation in neural stem and progenitor cells is still relatively underexplored. In this review, we discuss the link between cell polarity, architecture, identity and intracellular traffic, and highlight how studies on neurons have shaped our knowledge and conceptual framework on traffic and polarity. We will then conclude by discussing how a group of rare diseases, called congenital disorders of glycosylation (CDG) offers the unique opportunity to study the contribution of traffic and glycosylation in the context of neurodevelopment.

Collective migration is essential for development, wound repair, and cancer metastasis. For most collective systems, "leader cells" determine both the direction and the power of the migration. It has remained unclear, however, how the highly polarized vertebrate epithelium migrates directionally during branching morphogenesis. We show here that, unlike in other systems, front-rear polarity of the mammary epithelium is set up by preferential cell proliferation in the front in response to the FGF10 gradient. This leads to frontal stratification, loss of apicobasal polarity, and leader cell formation. Leader cells are a dynamic population and move faster and more directionally toward the FGF10 signal than do follower cells, partly because of their intraepithelial protrusions toward the signal. Together, our data show that directional migration of the mammary epithelium is a unique multistep process and that, despite sharing remarkable cellular and molecular similarities, vertebrate and invertebrate epithelial branching are fundamentally distinct processes.

BACKGROUND:Polarity is necessary for epithelial cells to perform distinct functions at their apical and basal surfaces. Oral epithelial cell-derived ameloblasts at secretory stage (SABs) synthesize large amounts of enamel matrix proteins (EMPs), largely amelogenins. EMPs are unidirectionally secreted into the enamel space through their apical cytoplasmic protrusions, or Tomes' processes (TPs), to guide the enamel formation. Little is known about the transcriptional regulation underlying the establishment of cell polarity and unidirectional secretion of SABs. RESULTS:The higher-order chromatin architecture of eukaryotic genome plays important roles in cell- and stage-specific transcriptional programming. A genome organizer, special AT-rich sequence-binding protein 1 (SATB1), was discovered to be significantly upregulated in ameloblasts compared to oral epithelial cells using a whole-transcript microarray analysis. The Satb1-/- mice possessed deformed ameloblasts and a thin layer of hypomineralized and non-prismatic enamel. Remarkably, Satb1-/- ameloblasts at the secretory stage lost many morphological characteristics found at the apical surface of wild-type (wt) SABs, including the loss of Tomes' processes, defective inter-ameloblastic adhesion, and filamentous actin architecture. As expected, the secretory function of Satb1-/- SABs was compromised as amelogenins were largely retained in cells. We found the expression of epidermal growth factor receptor pathway substrate 8 (Eps8), a known regulator for actin filament assembly and small intestinal epithelial cytoplasmic protrusion formation, to be SATB1 dependent. In contrast to wt SABs, EPS8 could not be detected at the apical surface of Satb1-/- SABs. Eps8 expression was greatly reduced in small intestinal epithelial cells in Satb1-/- mice as well, displaying defective intestinal microvilli. CONCLUSIONS:Our data show that SATB1 is essential for establishing secretory ameloblast cell polarity and for EMP secretion. In line with the deformed apical architecture, amelogenin transport to the apical secretory front and secretion into enamel space were impeded in Satb1-/- SABs resulting in a massive cytoplasmic accumulation of amelogenins and a thin layer of hypomineralized enamel. Our studies strongly suggest that SATB1-dependent Eps8 expression plays a critical role in cytoplasmic protrusion formation in both SABs and in small intestines. This study demonstrates the role of SATB1 in the regulation of amelogenesis and the potential application of SATB1 in ameloblast/enamel regeneration.

The respiratory epithelium, particularly the airway epithelium, is the primary infection site for respiratory pathogens. The apical surface of epithelial cells is constantly exposed to external stimuli including invading pathogens. Efforts have been made to establish organoid cultures to recapitulate the human respiratory tract. However, a robust and simple model with an easily accessible apical surface would benefit respiratory research. Here, we report the generation and characterization of apical-out airway organoids from the long-term expandable lung organoids that we previously established. The apical-out airway organoids morphologically and functionally recapitulated the human airway epithelium at a comparable level to the apical-in airway organoids. Moreover, apical-out airway organoids sustained productive and multicycle replication of SARS-CoV-2, and accurately recapitulated the higher infectivity and replicative fitness of the Omicron variants BA.5 and B.1.1.529 and an ancestral virus. In conclusion, we established a physiologically relevant and convenient apical-out airway organoid model for studying respiratory biology and diseases. [ABSTRACT FROM AUTHOR]

Microbiome is an integral part of the gut and is essential for its proper function. Imbalances of the microbiota can be devastating and have been linked with several gastrointestinal conditions. Current gastrointestinal models do not fully reflect the in vivo situation. Thus, it is important to establish more advanced in vitro models to study host-microbiome/pathogen interactions. Here, we developed for the first time an apical-out human small intestinal organoid model in hypoxia, where the apical surface is directly accessible and exposed to a hypoxic environment. These organoids mimic the intestinal cell composition, structure and functions and provide easy access to the apical surface. Co-cultures with the anaerobic strains Lactobacillus casei and Bifidobacterium longum showed successful colonization and probiotic benefits on the organoids. These novel hypoxia-tolerant apical-out small intestinal organoids will pave the way for unraveling unknown mechanisms related to host-microbiome interactions and serve as a tool to develop microbiome-related probiotics and therapeutics. [ABSTRACT FROM AUTHOR]

The loss of apicobasal polarity during the epithelial-to-mesenchymal transition (EMT) is a hallmark of cancer and metastasis. The key feature of this polarity in epithelial cells is the subdivision of the plasma membrane into apical and basolateral domains, with each orchestrating specific intra- and extracellular functions. Epithelial transport and signaling capacities are thought to be determined largely by the quality, quantity, and nanoscale organization of proteins residing in these membrane domains, the apicobasal surfaceomes. Despite its implications for cancer, drug uptake, and infection, our current knowledge of how the polarized surfaceome is organized and maintained is limited. Here, we used chemoproteomic surfaceome scanning to establish proteotype maps of apicobasal surfaceomes and reveal quantitative distributions of, i.e., surface proteases, phosphatases, and tetraspanins as potential key regulators of polarized cell functionality. We show further that the tumor suppressor PTEN regulates polarized surfaceome architecture and uncover a potential role in collective cell migration. Our differential surfaceome analysis provides a molecular framework to elucidate polarized protein networks regulating epithelial functions and PTEN-associated cancer progression. [ABSTRACT FROM AUTHOR]

Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, β-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes. • Inactivation of calpain2 expression by CRISPR/Cas9 reduces cell migration and delays cell scattering induced by HGF/SF. • Cells lacking calpain2 establish tight junctions faster than control cells. • Cells lacking calpain2 are resistant to morphological transformation induced by ERK2 overexpression. [ABSTRACT FROM AUTHOR]

Epithelial polarization and polarized cargo transport are highly coordinated and interdependent processes. In our search for novel regulators of epithelial polarization and protein secretion, we used a genome-wide CRISPR/Cas9 screen and combined it with an assay based on fluorescence-activated cell sorting (FACS) to measure the secretion of the apical brush-border hydrolase dipeptidyl peptidase 4 (DPP4). In this way, we performed the first CRISPR screen to date in human polarized epithelial cells. Using high-resolution microscopy, we detected polarization defects and mislocalization of DPP4 to late endosomes/lysosomes after knockout of TM9SF4, anoctamin 8, and ARHGAP33, confirming the identification of novel factors for epithelial polarization and apical cargo secretion. Thus, we provide a powerful tool suitable for studying polarization and cargo secretion in epithelial cells. In addition, we provide a dataset that serves as a resource for the study of novel mechanisms for epithelial polarization and polarized transport and facilitates the investigation of novel congenital diseases associated with these processes.

Intestinal organoids recapitulate many features of the in vivo gastrointestinal tract and have revolutionized in vitro studies of intestinal function and disease. However, the restricted accessibility of the apical surface of the organoids facing the central lumen (apical-in) limits studies related to nutrient uptake and drug absorption and metabolism. Here, we demonstrate that pluripotent stem cell (PSC)-derived intestinal organoids with reversed epithelial polarity (apical-out) can successfully recapitulate tissue-specific functions. In particular, these apical-out organoids show strong epithelial barrier formation with all the major junctional complexes, nutrient transport and active lipid metabolism. Furthermore, the organoids express drug-metabolizing enzymes and relevant apical and basolateral transporters. The scalable and robust generation of functional, apical-out intestinal organoids lays the foundation for a completely new range of organoid-based high-throughput/high-content in vitro applications in the fields of nutrition, metabolism and drug discovery.

The respiratory epithelium, particularly the airway epithelium, is the primary infection site for respiratory pathogens. The apical surface of epithelial cells is constantly exposed to external stimuli including invading pathogens. Efforts have been made to establish organoid cultures to recapitulate the human respiratory tract. However, a robust and simple model with an easily accessible apical surface would benefit respiratory research. Here, we report the generation and characterization of apical-out airway organoids from the long-term expandable lung organoids that we previously established. The apical-out airway organoids morphologically and functionally recapitulated the human airway epithelium at a comparable level to the apical-in airway organoids. Moreover, apical-out airway organoids sustained productive and multicycle replication of SARS-CoV-2, and accurately recapitulated the higher infectivity and replicative fitness of the Omicron variants BA.5 and B.1.1.529 and an ancestral virus. In conclusion, we established a physiologically relevant and convenient apical-out airway organoid model for studying respiratory biology and diseases.

In multicellular organisms, epithelial cells are key elements of tissue organization. In developing tissues, cellular proliferation and differentiation are under the tight regulation of morphogenetic programs, that ensure the correct organ formation and functioning. In these processes, mitotic rates and division orientation are crucial in regulating the velocity and the timing of the forming tissue. Division orientation, specified by mitotic spindle placement with respect to epithelial apico-basal polarity, controls not only the partitioning of cellular components but also the positioning of the daughter cells within the tissue, and hence the contacts that daughter cells retain with the surrounding microenvironment. Daughter cells positioning is important to determine signal sensing and fate, and therefore the final function of the developing organ. In this review, we will discuss recent discoveries regarding the mechanistics of planar divisions in mammalian epithelial cells, summarizing technologies and model systems used to study oriented cell divisions in vitro such as three-dimensional cysts of immortalized cells and intestinal organoids. We also highlight how misorientation is corrected in vivo and in vitro, and how it might contribute to the onset of pathological conditions.

Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, our understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, we have monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). We show that the scrib mutation impacts the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch-dedicated transcription factor. The Notch-dependent neoplasms require, however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm(namely AP-1, Stat92E, Ftz-F1 and basic leucine zipper factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally, our work highlights some Notch/scrib specificities, in particular the role of the PAR domain-containing basic leucine zipper transcription factor and Notch direct target Pdp1 for neoplastic growth. [ABSTRACT FROM AUTHOR]

Cell polarity is a fundamental property of most animal cells and is critical during development and for most cell and tissue functions. Epithelial cells are organized into apical and basolateral compartments, and this intrinsic cellular asymmetry is essential for all functions that are carried out by epithelial tissue. The establishment of a polarized epithelial phenotype is orchestrated by major rearrangements of the cell cytoskeleton, polarized membrane trafficking, the formation and maturation of epithelial cell junctions, cell signaling pathways, and the generation of cortical phospholipid asymmetry. These processes need to be coordinated precisely in time and space and integrated with physical and chemical signals from the environment, failure of which leads to severe developmental disorders and various human diseases. At the heart of this regulatory network are the evolutionarily conserved polarity modules Par, Crumbs, and Scribble, whose components engage in complex cooperative and antagonistic interactions to compartmentalize and functionalize the epithelial cell cortex and to control the spatiotemporal activity of downstream polarity effectors. In this review, we will discuss recent insights into the organization and regulation of the mammalian Par and Crumbs modules and outline a hypothetical framework of how these proteins orchestrate epithelial polarity development, HIPPO signaling, and actomyosin activity at the apical–lateral border. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the huntingtin gene (HTT). Although HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG lengthdependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced activin signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFβ signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of activin response. This is due to defects in the apicobasal polarization in the context of the polarized epitheliumof the 2D gastruloid, leading to ectopic subcellular localization of TGFβ receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first 2 weeks of human development, as modeled by our 2D gastruloids. [ABSTRACT FROM AUTHOR]

Bundled with various kinds of adhesion molecules and anchored to the basement membrane, the epithelium has historically been considered as an immotile tissue and, to migrate, it first needs to undergo epithelial-mesenchymal transition (EMT). Since its initial description more than half a century ago, the EMT process has fascinated generations of developmental biologists and, more recently, cancer biologists as it is believed to be essential for not only embryonic development, organ formation, but cancer metastasis. However, recent progress shows that epithelium is much more motile than previously realized. Here, we examine the emerging themes in epithelial collective migration and how this has impacted our understanding of EMT.

During kidney development, nephron epithelia arise de novo from fate-committed mesenchymal progenitors through a mesenchymal-to-epithelial transition (MET). Downstream of fate specification, transcriptional mechanisms that drive establishment of epithelial morphology are poorly understood. We used human iPSC-derived renal organoids, which recapitulate nephrogenesis, to investigate mechanisms controlling renal MET. Multi-ome profiling via snRNA-seq and ATAC-seq of organoids identified dynamic changes in gene expression and chromatin accessibility driven by activators and repressors throughout MET. CRISPR interference identified that paired box 8 (PAX8) is essential for initiation of MET in human renal organoids, contrary to in vivo mouse studies, likely by activating a cell-adhesion program. While Wnt/β-catenin signaling specifies nephron fate, we find that it must be attenuated to allow hepatocyte nuclear factor 1-beta (HNF1B) and TEA-domain (TEAD) transcription factors to drive completion of MET. These results identify the interplay between fate commitment and morphogenesis in the developing human kidney, with implications for understanding both developmental kidney diseases and aberrant epithelial plasticity following adult renal tubular injury. [Display omitted] • Human iPSC-derived kidney organoids faithfully recapitulate renal MET • Dynamic activity of transcriptional activators and repressors controls progression of MET • PAX8 initiates renal MET downstream of Wnt/β-catenin signaling • Wnt signaling must be downregulated for proper completion of MET via HNF1B and TEAD How the genome encodes human nephron structure and function is incompletely understood. Using multi-omic analysis of human renal organoids and fetal kidneys, Ng-Blichfeldt et al. map transcriptional dynamics controlling the emergence of nephron epithelia from mesenchymal progenitors during human kidney development, revealing critical roles for PAX8 and transient Wnt signaling. [ABSTRACT FROM AUTHOR]

Summary: Visualizing mRNA in real time in vivo at high resolution is critical for a full understanding of the spatiotemporal dynamics of gene regulation and function. Here, using a PP7/PCP-based mRNA-tagging approach, we construct a collection of tissue-specific and differentially expressed toolkit strains for visualizing mRNAs encoding apical, basolateral, and junctional proteins in Caenorhabditis elegans epithelia. We precisely delineate the spatiotemporal organization and dynamics of these transcripts across multiple subcellular compartments and tissues. Remarkably, all the transcripts exhibit an asymmetric, membrane-associated localization during epithelial polarization and maturation, which suggests that mRNA localization is a prerequisite for epithelial polarization and function. Single-particle tracking reveals striking features of the transport dynamics of the mRNAs in a gene-specific, compartment-linked, and time-resolved manner. The toolkit can be used to identify the cis-regulatory elements and trans-acting factors for mRNA localization. This study provides a valuable resource to investigate complex RNA dynamics in epithelial polarity and morphogenesis.

Molecular events that drive the development of precancerous lesions in the bronchial epithelium, which are precursors of lung squamous cell carcinoma (LUSC), are poorly understood. We demonstrate that disruption of epithelial cellular polarity, via the conditional deletion of the apical determinant Crumbs3 (Crb3), initiates and sustains precancerous airway pathology. The loss of Crb3 in adult luminal airway epithelium promotes the uncontrolled activation of the transcriptional regulators YAP and TAZ, which stimulate intrinsic signals that promote epithelial cell plasticity and paracrine signals that induce basal-like cell growth. We show that aberrant polarity and YAP/TAZ-regulated gene expression associates with human bronchial precancer pathology and disease progression. Analyses of YAP/TAZ-regulated genes further identified the ERBB receptor ligand Neuregulin-1 (NRG1) as a key transcriptional target and therapeutic targeting of ERBB receptors as a means of preventing and treating precancerous cell growth. Our observations offer important molecular insight into the etiology of LUSC and provides directions for potential interception strategies of lung cancer. [ABSTRACT FROM AUTHOR]

In human glandular endometrial epithelial cells, desmosomal and adherens junction proteins have been shown to extend from a subapically restricted lateral position to the entire lateral membrane during the implantation window of the menstrual cycle. Similarly, a menstrual cycle stage-dependent redistribution of the extracellular matrix adhesion protein α6-integrin has been reported. These changes are believed to be important for endometrial receptiveness and successful embryo implantation. To prove the hypothesis that steroid hormones and human choriogonadotropin can induce the redistribution of these adhesion molecules, we used the human endometrial cell line Ishikawa in a 3D culture system. Gland-like spheroids were grown in reconstituted basement membrane (Matrigel™). The lumen-bearing spheroids were treated for 2 or 4 days with ovarian steroids or human choriogonadotropin and then assessed by immunofluorescence microscopy. In addition, human endometrial biopsies were obtained from patients, who were in therapy for assisted reproductive technology, and were examined in parallel. Lateral redistribution of the desmosomal plaque protein desmoplakin 1 was observed in the spheroids treated either with progesterone, medroxyprogesterone acetate or human choriogonadotropin. Furthermore, the extracellular matrix adhesion protein α6-integrin showed an increased lateral membrane localization upon gestagen stimulation in the 3D culture system. The results of this study demonstrate that the 3D endometrial Ishikawa cell culture might be suited as an experimental model system to prove the effect of hormonal changes like those occurring during the window of implantation. [ABSTRACT FROM AUTHOR]

Introduction: In vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell‐ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood. Methods: Here, using in vitro assay with engineered MDCK cells expressing H2B‐mCherry (nucleus) and gp135/Podocalyxin‐GFP (apical marker), we show in multi‐dimensions that such coordination for epithelial morphogenesis can be determined by cell‐soluble ECM interaction in the fluidic phase. Results: The coordination depends on the native topology of ECM components such as sheet‐like basement membrane (BM) and type I collagen (COL) fibres: scaffold formed by BM (COL) facilitates a close‐ended (open‐ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time‐lapse two‐photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135‐GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi‐lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment. Conclusion: Our results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell‐soluble ECM interaction and self‐assembly. [ABSTRACT FROM AUTHOR]

For decades, recycling of membrane proteins has been represented in figures by arrows between the "endosome" and the plasma membrane, but recently there has been an explosion in the understanding of the mechanisms and protein complexes required to facilitate protein recycling. Here, some key discoveries will be introduced, including assigning function to a number of recently recognized protein complexes and linking their function to protein recycling. Furthermore, the importance of lipid interactions and links to diseases and epithelial polarity will be summarized. [ABSTRACT FROM AUTHOR]

The choroid plexus (CP) is an extensively vascularized neuroepithelial tissue that projects into the brain ventricles. The restriction of transepithelial transport across the CP establishes the blood-cerebrospinal fluid (CSF) barrier that is fundamental to the homeostatic regulation of the central nervous system microenvironment. However, the molecular mechanisms that control this process remain elusive. Here we show that the genetic ablation of Sox9 in the hindbrain CP results in a hyperpermeable blood-CSF barrier that ultimately upsets the CSF electrolyte balance and alters CSF protein composition. Mechanistically, SOX9 is required for the transcriptional up-regulation of Col9a3 in the CP epithelium. The reduction of Col9a3 expression dramatically recapitulates the blood-CSF barrier defects of Sox9 mutants. Loss of collagen IX severely disrupts the structural integrity of the epithelial basement membrane in the CP, leading to progressive loss of extracellular matrix components. Consequently, this perturbs the polarized microtubule dynamics required for correct orientation of apicobasal polarity and thereby impedes tight junction assembly in the CP epithelium. Our findings reveal a pivotal cascade of SOX9- dependent molecular events that is critical for construction of the blood-CSF barrier. [ABSTRACT FROM AUTHOR]

Summary: Epithelial cells are polarized along their apical-basal axis by the action of the small GTPase Cdc42, which is known to activate the aPKC kinase at the apical domain. However, loss of aPKC kinase activity was reported to have only mild effects on epithelial cell polarity. Here, we show that Cdc42 also activates a second kinase, Pak1, to specify apical domain identity in Drosophila and mammalian epithelia. aPKC and Pak1 phosphorylate an overlapping set of polarity substrates in kinase assays. Inactivating both aPKC kinase activity and the Pak1 kinase leads to a complete loss of epithelial polarity and morphology, with cells losing markers of apical polarization such as Crumbs, Par3/Bazooka, or ZO-1. This function of Pak1 downstream of Cdc42 is distinct from its role in regulating integrins or E-cadherin. Our results define a conserved dual-kinase mechanism for the control of apical membrane identity in epithelia.

The establishment of uterine receptivity is a prerequisite for embryo implantation and begins with the transformation of the luminal epithelium. MicroRNAs (miRNAs) have been widely reported to be involved in the regulation of embryo implantation, but their roles in establishing uterine receptivity remain unclear. In this study, through small RNA sequencing analysis, we showed that a low level of miR-192-5p is essential for initiating implantation in mice, and transient upregulation of miR-192-5p led to implantation failure. In situ hybridization results revealed that miR-192-5p was primarily expressed in the endometrial epithelium, and dysregulation of miR-192-5p interfered with the performance of the luminal epithelium, resulting in inadequate receptivity. By manipulating miR-192-5p expression in mouse uterus and an endometrial epithelial cell line, we showed that miR-192-5p maintains cell polarity through stabilizing adherens junction protein E-cadherin, thereby preventing epithelial-mesenchymal transition. Furthermore, miR-192-5p preserved the pattern of microvilli as well as Muc1 expression on the apical membrane of epithelial cells, thereby avoiding embryo adhesion. Moreover, miR-192-5p was found to be regulated by ovarian steroids. Collectively, this study demonstrated that the physiological role of miR-192-5p in mouse uterus is to maintain the nonreceptive state of epithelial cells and prevent their transformation to the receptive state. Thus, a sustained high level of miR-192-5p is detrimental to embryo implantation. These findings help elucidate the mechanisms involved in miRNA-based regulation of uterine physiology in early pregnancy, and may even contribute to the diagnosis and treatment of infertility. • A minimum uterine expression of miR-192-5p is essential for the onset of embryo implantation. • miR-192-5p impeded epithelial transformation in establishing uterine receptivity. • miR-192-5p sustained epithelial polarity via the maintenance of E-cadherin. • miR-192-5p preserved anti-adhesive properties on the apical membrane of epithelial cells. • The downregulation of uterine miR-192-5p levels in mouse early pregnancy was caused by ovarian steroids. [ABSTRACT FROM AUTHOR]

Tight junctions (TJs) are intercellular junctions critical for building the epithelial barrier and maintaining epithelial polarity. The claudin family of membrane proteins play central roles in TJ structure and function. However, recent findings have uncovered claudin-independent aspects of TJ structure and function, and additional players including junctional adhesion molecules (JAMs), membrane lipids, phase separation of the zonula occludens (ZO) family of scaffolding proteins, and mechanical force have been shown to play important roles in TJ structure and function. In this review, we discuss how these new findings have the potential to transform our understanding of TJ structure and function, and how the intricate network of TJ proteins and membrane lipids dynamically interact to drive TJ assembly. Tight junction strand formation and membrane apposition formation are differentially regulated. Claudins form charge-selective small pores, while junctional adhesion molecules regulate the formation of size-selective large pores. Tight junction proteins regulate epithelial polarity, although how tight junctions form a membrane fence remains unclear. Tight junction associated membrane proteins regulate tight junction assembly in conjunction with zonula occludens protein phase separation, membrane lipids, mechanical force, and polarity signaling proteins. [ABSTRACT FROM AUTHOR]

Drosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress.

Polarized epithelial cells adhere to each other at apical junctions that connect to the apical F-actin belt. Regulated remodeling of apical junctions supports morphogenesis, while dysregulated remodeling promotes diseases such as cancer. We have documented that branched actin regulator, WAVE, and apical junction protein, Cadherin, assemble together in developing C. elegans embryonic junctions. If WAVE is missing in embryonic epithelia, too much Cadherin assembles at apical membranes, and yet apical F-actin is reduced, suggesting the excess Cadherin is not fully functional. We proposed that WAVE supports apical junctions by regulating the dynamic accumulation of Cadherin at membranes. To test this model, here we examine if WAVE is required for Cadherin membrane enrichment and apical–basal polarity in a maturing epithelium, the post-embryonic C. elegans intestine. We find that larval and adult intestines have distinct apicobasal populations of Cadherin, each with distinct dependence on WAVE branched actin. In vivo imaging shows that loss of WAVE components alters post-embryonic E-cadherin membrane enrichment, especially at apicolateral regions, and alters the lateral membrane. Analysis of a biosensor for PI(4,5)P2 suggests loss of WAVE or Cadherin alters the polarity of the epithelial membrane. EM (electron microscopy) illustrates lateral membrane changes including separations. These findings have implications for understanding how mutations in WAVE and Cadherin may alter cell polarity.

The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity was observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now. [ABSTRACT FROM AUTHOR]

The morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. Here, we show that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis.