Cultured Pig Thyroid Follicular Cells: Electrical Evaluation of Epithelial Integrity.

Thyroid epithelial cells organize as enclosed follicles containing thyroid hormone precursor, iodinated thyroglobulin, with lumina bordered by the cellular apices. Transepithelial transport determines composition of compartmental milieu essential for both prohormone formation and its downstream conversion to thyroxine. Hence, not only do follicular lumina function as storage vessels but also as physiological reaction chambers into which reactive components, together with the proper salts and water, are secreted. Polarized, two-dimensional cultures of pig thyroid epithelia, prepared using established protocols, provide a convenient system for assessing transport processes subserving hormone production. This chapter details established methods for growing and evaluating integrity of primary pig thyroid cultures for downstream analysis of transport and other key physiological functions.

A comprehensive model of Drosophila epithelium reveals the role of embryo geometry and cell topology in mechanical responses.

In order to understand morphogenesis, it is necessary to know the material properties or forces shaping the living tissue. In spite of this need, very few in vivo measurements are currently available. Here, using the early Drosophila embryo as a model, we describe a novel cantilever-based technique which allows for the simultaneous quantification of applied force and tissue displacement in a living embryo. By analyzing data from a series of experiments in which embryonic epithelium is subjected to developmentally relevant perturbations, we conclude that the response to applied force is adiabatic and is dominated by elastic forces and geometric constraints, or system size effects. Crucially, computational modeling of the experimental data indicated that the apical surface of the epithelium must be softer than the basal surface, a result which we confirmed experimentally. Further, we used the combination of experimental data and comprehensive computational model to estimate the elastic modulus of the apical surface and set a lower bound on the elastic modulus of the basal surface. More generally, our investigations revealed important general features that we believe should be more widely addressed when quantitatively modeling tissue mechanics in any system. Specifically, different compartments of the same cell can have very different mechanical properties; when they do, they can contribute differently to different mechanical stimuli and cannot be merely averaged together. Additionally, tissue geometry can play a substantial role in mechanical response, and cannot be neglected.

Epithelial apoptotic pattern emerges from global and local regulation by cell apical area.

Geometry is a fundamental attribute of biological systems, and it underlies cell and tissue dynamics. Cell geometry controls cell-cycle progression and mitosis and thus modulates tissue development and homeostasis. In sharp contrast and despite the extensive characterization of the genetic mechanisms of caspase activation, we know little about whether and how cell geometry controls apoptosis commitment in developing tissues. Here, we combined multiscale time-lapse microscopy of developing Drosophila epithelium, quantitative characterization of cell behaviors, and genetic and mechanical perturbations to determine how apoptosis is controlled during epithelial tissue development. We found that early in cell lives and well before extrusion, apoptosis commitment is linked to two distinct geometric features: a small apical area compared with other cells within the tissue and a small relative apical area with respect to the immediate neighboring cells. We showed that these global and local geometric characteristics are sufficient to recapitulate the tissue-scale apoptotic pattern. Furthermore, we established that the coupling between these two geometric features and apoptotic cells is dependent on the Hippo/YAP and Notch pathways. Overall, by exploring the links between cell geometry and apoptosis commitment, our work provides important insights into the spatial regulation of cell death in tissues and improves our understanding of the mechanisms that control cell number and tissue size.

Non-Bilaterians as Model Systems for Tissue Mechanics.

In animals, epithelial tissues are barriers against the external environment, providing protection against biological, chemical, and physical damage. Depending on the organism's physiology and behavior, these tissues encounter different types of mechanical forces and need to provide a suitable adaptive response to ensure success. Therefore, understanding tissue mechanics in different contexts is an important research area. Here, we review recent tissue mechanics discoveries in three early divergent non-bilaterian systems-Trichoplax adhaerens, Hydra vulgaris, and Aurelia aurita. We highlight each animal's simple body plan and biology and unique, rapid tissue remodeling phenomena that play a crucial role in its physiology. We also discuss the emergent large-scale mechanics in these systems that arise from small-scale phenomena. Finally, we emphasize the potential of these non-bilaterian animals to be model systems in a bottom-up approach for further investigation in tissue mechanics.

Genomic targets of the IRE1-XBP1s pathway in mediating metabolic adaptation in epithelial plasticity.

Epithelial mesenchymal plasticity (EMP) is a complex cellular reprogramming event that plays a major role in tissue homeostasis. Recently we observed the unfolded protein response (UPR) triggers EMP through the inositol-requiring protein 1 (IRE1α)-X-box-binding protein 1 spliced (XBP1s) axis, enhancing glucose shunting to protein N glycosylation. To better understand the genomic targets of XBP1s, we identified its genomic targets using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) of a FLAG-epitope tagged XBP1s in RSV infection. CUT&RUN identified 7086 binding sites in chromatin that were enriched in AP-1 motifs and GC-sequences. Of these binding sites, XBP1s peaks mapped to 4827 genes controlling Rho-GTPase signaling, N-linked glycosylation and ER-Golgi transport. Strikingly, XBP1s peaks were within 1 kb of transcription start sites of 2119 promoters. In addition to binding core mesenchymal transcription factors SNAI1 and ZEB1, we observed that hexosamine biosynthetic pathway (HBP) enzymes were induced and contained proximal XBP1s peaks. We demonstrate that IRE1α -XBP1s signaling is necessary and sufficient to activate core enzymes by recruiting elongation-competent phospho-Ser2 CTD modified RNA Pol II. We conclude that the IRE1α-XBP1s pathway coordinately regulates mesenchymal transcription factors and hexosamine biosynthesis in EMP by a mechanism involving recruitment of activated pSer2-Pol II to GC-rich promoters.

A mesothelium divides the subarachnoid space into functional compartments.

The central nervous system is lined by meninges, classically known as dura, arachnoid, and pia mater. We show the existence of a fourth meningeal layer that compartmentalizes the subarachnoid space in the mouse and human brain, designated the subarachnoid lymphatic-like membrane (SLYM). SLYM is morpho- and immunophenotypically similar to the mesothelial membrane lining of peripheral organs and body cavities, and it encases blood vessels and harbors immune cells. Functionally, the close apposition of SLYM with the endothelial lining of the meningeal venous sinus permits direct exchange of small solutes between cerebrospinal fluid and venous blood, thus representing the mouse equivalent of the arachnoid granulations. The functional characterization of SLYM provides fundamental insights into brain immune barriers and fluid transport.

Novel analytical tools reveal that local synchronization of cilia coincides with tissue-scale metachronal waves in zebrafish multiciliated epithelia.

Motile cilia are hair-like cell extensions that beat periodically to generate fluid flow along various epithelial tissues within the body. In dense multiciliated carpets, cilia were shown to exhibit a remarkable coordination of their beat in the form of traveling metachronal waves, a phenomenon which supposedly enhances fluid transport. Yet, how cilia coordinate their regular beat in multiciliated epithelia to move fluids remains insufficiently understood, particularly due to lack of rigorous quantification. We combine experiments, novel analysis tools, and theory to address this knowledge gap. To investigate collective dynamics of cilia, we studied zebrafish multiciliated epithelia in the nose and the brain. We focused mainly on the zebrafish nose, due to its conserved properties with other ciliated tissues and its superior accessibility for non-invasive imaging. We revealed that cilia are synchronized only locally and that the size of local synchronization domains increases with the viscosity of the surrounding medium. Even though synchronization is local only, we observed global patterns of traveling metachronal waves across the zebrafish multiciliated epithelium. Intriguingly, these global wave direction patterns are conserved across individual fish, but different for left and right noses, unveiling a chiral asymmetry of metachronal coordination. To understand the implications of synchronization for fluid pumping, we used a computational model of a regular array of cilia. We found that local metachronal synchronization prevents steric collisions, i.e., cilia colliding with each other, and improves fluid pumping in dense cilia carpets, but hardly affects the direction of fluid flow. In conclusion, we show that local synchronization together with tissue-scale cilia alignment coincide and generate metachronal wave patterns in multiciliated epithelia, which enhance their physiological function of fluid pumping.

Couple stresses and discrete potentials in the vertex model of cellular monolayers.

The vertex model is widely used to simulate the mechanical properties of confluent epithelia and other multicellular tissues. This inherently discrete framework allows a Cauchy stress to be attributed to each cell, and its symmetric component has been widely reported, at least for planar monolayers. Here, we consider the stress attributed to the neighbourhood of each tricellular junction, evaluating in particular its leading-order antisymmetric component and the associated couple stresses, which characterise the degree to which individual cells experience (and resist) in-plane bending deformations. We develop discrete potential theory for localised monolayers having disordered internal structure and use this to derive the analogues of Airy and Mindlin stress functions. These scalar potentials typically have broad-banded spectra, highlighting the contributions of small-scale defects and boundary layers to global stress patterns. An affine approximation attributes couple stresses to pressure differences between cells sharing a trijunction, but simulations indicate an additional role for non-affine deformations.

GLI1+ cells are a source of repair-supportive mesenchymal cells (RSMCs) during airway epithelial regeneration.

Repair-supportive mesenchymal cells (RSMCs) have been recently reported in the context of naphthalene (NA)-induced airway injury and regeneration. These cells transiently express smooth muscle actin (Acta2) and are enriched with platelet-derived growth factor receptor alpha (Pdgfra) and fibroblast growth factor 10 (Fgf10) expression. Genetic deletion of Ctnnb1 (gene coding for beta catenin) or Fgf10 in these cells using the Acta2-Cre-ERT2 driver line after injury (defined as NA-Tam condition; Tam refers to tamoxifen) led to impaired repair of the airway epithelium. In this study, we demonstrate that RSMCs are mostly captured using the Acta2-Cre-ERT2 driver when labeling occurs after (NA-Tam condition) rather than before injury (Tam-NA condition), and that their expansion occurs mostly between days 3 and 7 following NA treatment. Previous studies have shown that lineage-traced peribronchial GLI1+ cells are transiently amplified after NA injury. Here, we report that Gli1 expression is enriched in RSMCs. Using lineage tracing with Gli1Cre-ERT2 mice combined with genetic inactivation of Fgf10, we show that GLI1+ cells with Fgf10 deletion fail to amplify around the injured airways, thus resulting in impaired airway epithelial repair. Interestingly, Fgf10 expression is not upregulated in GLI1+ cells following NA treatment, suggesting that epithelial repair is mostly due to the increased number of Fgf10-expressing GLI1+ cells. Co-culture of SCGB1A1+ cells with GLI1+ cells isolated from non-injured or injured lungs showed that GLI1+ cells from these two conditions are similarly capable of supporting bronchiolar organoid (or bronchiolosphere) formation. Single-cell RNA sequencing on sorted lineage-labeled cells showed that the RSMC signature resembles that of alveolar fibroblasts. Altogether, our study provides strong evidence for the involvement of mesenchymal progenitors in airway epithelial regeneration and highlights the critical role played by Fgf10-expressing GLI1+ cells in this context.

Following Ussing's legacy: from amphibian models to mammalian kidney and brain.

Professor Hans H. Ussing (1911-2000) was one of the founding members of the field of epithelial cell biology. He is most famous for the electrophysiological technique that he developed to measure electrogenic ion flux across epithelial tissues. Ussing-style electrophysiology has been applied to multiple tissues and has informed fields as diverse as amphibian biology and medicine. In the latter, this technique has contributed to a basic understanding of maladies such as hypertension, polycystic kidney disease, cystic fibrosis, and diarrheal diseases to mention but a few. In addition to this valuable contribution to biological methods, Prof. Ussing also provided strong evidence for the concept of active transport several years before the elucidation of Na+K+ATPase. In addition, he provided cell biologists with the important concept of polarized epithelia with specific and different transporters found in the apical and basolateral membranes, thus providing these cells with the ability to conduct directional, active and passive transepithelial transport. My studies have used Ussing chamber electrophysiology to study the toad urinary bladder, an amphibian cell line, renal cell lines, and, most recently, choroid plexus cell lines. This technique has formed the basis of our in vitro mechanistic studies that are used in an iterative manner with animal models to better understand disease progress and treatment. I was honored to be invited to deliver the 2022 Hans Ussing Lecture sponsored by the Epithelial Transport Group of the American Physiological Society. This manuscript is a version of the material presented in that lecture.

Fine-tuning of epithelial taste bud organoid to promote functional recapitulation of taste reactivity.

Taste stem/progenitor cells from posterior mouse tongues have been used to generate taste bud organoids. However, the inaccessible location of taste receptor cells is observed in conventional organoids. In this study, we established a suspension-culture method to fine-tune taste bud organoids by apicobasal polarity alteration to form the accessible localization of taste receptor cells. Compared to conventional Matrigel-embedded organoids, suspension-cultured organoids showed comparable differentiation and renewal rates to those of taste buds in vivo and exhibited functional taste receptor cells and cycling progenitor cells. Accessible taste receptor cells enabled the direct application of calcium imaging to evaluate the taste response. Moreover, suspension-cultured organoids can be genetically altered. Suspension-cultured taste bud organoids harmoniously integrated with the recipient lingual epithelium, maintaining the taste receptor cells and gustatory innervation capacity. We propose that suspension-cultured organoids may provide an efficient model for taste research, including taste bud development, regeneration, and transplantation.

Orthotopic Transplantation of Mouse Mammary Epithelial Cells.

The orthotopic transplantation assay has provided important insights into mammary development, stem cell function, and tumorigenesis. Technically, it consists in grafting mammary tissue fragments, organoids, mammospheres, or isolated cells into the fat pads of prepubertal mice from which the endogenous epithelium has been surgically removed, thereby allowing growth and differentiation of mammary epithelial cells in their physiological environment. Here, we describe how is conducted transplantation of epithelial fragments and cells isolated from mouse mammary glands, report the various approaches currently used to evaluate the regeneration and self-renewal properties of mammary stem cells, and highlight the strengths and limitations of this in vivo grafting assay.

Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages.

Interactions of developing T cells with Aire+ medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEChi) are critical for the induction of central tolerance in the thymus. In turn, thymocytes regulate the cellularity of Aire+ mTEChi. However, it remains unknown whether thymocytes control the precursors of Aire+ mTEChi that are contained in mTEClo cells or other mTEClo subsets that have recently been delineated by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen presentation between mTECs and CD4+ thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4+ thymocytes induce key transcriptional regulators in mTEClo and control the composition of mTEClo subsets, including Aire+ mTEChi precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEClo is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4+ thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4+ thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.

Cyclic activity of signal transduction pathways in fimbrial epithelium of the human fallopian tube.

INTRODUCTION: The local environment of the fallopian tube represents the optimal conditions for reproductive processes. To maintain tissue homeostasis, signal transduction pathways are thought to play a pivotal role. Enhancing our understanding of functional signal transduction pathway activity is important to be able to clarify the role of aberrant signal transduction pathway activity leading to female subfertility and other tubal diseases. Therefore, in this study we investigate the influence of the hormonal cycle on the activity of key signal transduction pathways in the fimbrial epithelium of morphologically normal fallopian tubes. MATERIAL AND METHODS: We included healthy pre- (n = 17) and postmenopausal (n = 8) patients who had surgical interventions for benign gynecologic conditions. Histologic sections of the fallopian tubes were reviewed by two pathologists and, for the premenopausal patients, hormone serum levels and sections of the endometrium were examined to determine the hormonal phase (early follicular [n = 4], late follicular [n = 3], early luteal [n = 5], late luteal [n = 5]). After laser capture microdissection, total mRNA was extracted from the fimbrial epithelium and real-time quantitative reverse transcription-PCR was performed to determine functional signal transduction pathway activity of the androgen receptor (AR), estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor-beta (TGF-ß) and canonical wingless-type MMTV integration site (Wnt) pathways. RESULTS: The early luteal phase demonstrated high AR and ER pathway activity in comparison with the late luteal phase (p = 0.016 and p = 0.032, respectively) and low PI3K activity compared with the late follicular phase (p = 0.036), whereas the late luteal phase showed low activity of HH and Wnt compared with the early follicular phase (both p = 0.016). Signal transduction pathway activity in fimbrial epithelium from postmenopausal patients was most similar to the early follicular and/or late luteal phase with regard to the AR, ER and PI3K pathways. Wnt pathway activity in postmenopausal patients was comparable to the late follicular and early luteal phase. We observed no differences in HH and TGF-ß pathway activity between pre- and postmenopausal samples. The cyclic changes in signal transduction pathway activity suggest a stage-specific function which may affect the morphology and physiology of the human fallopian tube. CONCLUSIONS: We demonstrated cyclic changes in activity of the AR, ER, PI3K, HH and Wnt pathways throughout the hormonal cycle.

Developing a reproducible protocol for culturing functional confluent monolayers of differentiated equine oviduct epithelial cells†.

We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air-liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux, and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed <1% ciliated EOECs). Occasionally (5-10%), re-differentiated monolayers with 11-27% EOECs with secondary cilia in a diffuse pattern were obtained. Additionally, nuclear progesterone receptor expression was found to be inhibited by simulated luteal phase hormone concentrations, and sperm binding to cilia was higher for re-differentiated EOEC monolayers exposed to estrogen-progesterone concentrations mimicking the follicular rather than luteal phase. Overall, a functional equine oviduct model was established with close morphological resemblance to in vivo oviduct epithelium.

Autonomous epithelial folding induced by an intracellular mechano-polarity feedback loop.

Epithelial tissues form folded structures during embryonic development and organogenesis. Whereas substantial efforts have been devoted to identifying mechanical and biochemical mechanisms that induce folding, whether and how their interplay synergistically shapes epithelial folds remains poorly understood. Here we propose a mechano-biochemical model for dorsal fold formation in the early Drosophila embryo, an epithelial folding event induced by shifts of cell polarity. Based on experimentally observed apical domain homeostasis, we couple cell mechanics to polarity and find that mechanical changes following the initial polarity shifts alter cell geometry, which in turn influences the reaction-diffusion of polarity proteins, thus forming a feedback loop between cell mechanics and polarity. This model can induce spontaneous fold formation in silico, recapitulate polarity and shape changes observed in vivo, and confer robustness to tissue shape change against small fluctuations in mechanics and polarity. These findings reveal emergent properties of a developing epithelium under control of intracellular mechano-polarity coupling.

Cystic Fibrosis: Systems Biology Analysis from Homozygous p.Phe508del Variant Patients' Samples Reveals Perturbations in Tissue-Specific Pathways.

Cystic fibrosis (CF) is an autosomal recessive disorder, caused by diverse genetic variants for the CF transmembrane conductance regulator (CFTR) protein. Among these, p.Phe508del is the most prevalent variant. The effects of this variant on the physiology of each tissue remains unknown. This study is aimed at predicting cell signaling pathways present in different tissues of fibrocystic patients, homozygous for p.Phe508del. The study involved analysis of two microarray datasets, E-GEOD-15568 and E-MTAB-360 corresponding to the rectal and bronchial epithelium, respectively, obtained from the ArrayExpress repository. Particularly, differentially expressed genes (DEGs) were predicted, protein-protein interaction (PPI) networks were designed, and centrality and functional interaction networks were analyzed. The study reported that p.Phe508del-mutated CFTR-allele in homozygous state influenced the whole gene expression in each tissue differently. Interestingly, gene ontology (GO) term enrichment analysis revealed that only "neutrophil activation" was shared between both tissues; however, nonshared DEGs were grouped into the same GO term. For further verification, functional interaction networks were generated, wherein no shared nodes were reported between these tissues. These results suggested that the p.Phe508del-mutated CFTR-allele in homozygous state promoted tissue-specific pathways in fibrocystic patients. The generated data might further assist in prediction diagnosis to define biomarkers or devising therapeutic strategies.

Response of Esophageal Epithelium to Acute and Chronic Stress in Rabbits.

We studied electrophysiological changes in rabbit esophageal epithelium following acute (AS) and chronic stress (CS). Esophageal tissue was placed in Ussing chamber and the potential difference U between the luminal and abluminal sides, the short-circuit current Isc, as well as the tissue resistance R were measured. The initial values of these parameters for each sample were determined after the samples were stabilized in Ringer solution. Then, the tissues were exposed for 1 h to normal Ringer solution or Ringer solution with pH 4.0 and pH 1.7 with or without pepsin (0.25 mg/ml). Fluorescein was added to the luminal side of the sample to measure its permeability. In the AS group, U at Ringer solution (pH 1.7)+pepsin was significantly decreased in comparison with the baseline and control values (by 46 and 22%, respectively, p<0.05). R decreased by 74% in comparison with baseline, which little differed from the decrease in control samples exposed to Ringer solution (pH 1.7)+pepsin (by 62%). CS did not change U relative to baseline values, while changes in R were similar to those in the AS group. In the AS group, the permeability of the esophageal tissue perfused with Ringer solution (pH 1.7)+pepsin was significantly higher than in both the control and CS groups. AS, but not CS, made the esophageal epithelium more sensitive to the effects of noxious agents, disrupted barrier properties, and increased permeability. The effects of stress on gastroesophageal reflux disease symptoms can be related to severe exposure to acid and/or pepsin; however, the mechanisms other than epithelial defense should be evaluated.

3D in vitro culture system to study collective migration in mammary organoid epithelium.

We recently established an in vitro culture system in which mammary gland organoid undergoes directional migration in response to an FGF10 concentration gradient. Here, we describe a step-by-step protocol for preparing organoids, the setup of the 3D culture system, and the image acquisition approach. The technical difficulties in conducting the 3D migration assay are choosing epithelial organoids of appropriate sizes and manually paring organoids and beads pre-soaked in FGF10 within a desirable distance (∼100 µm). For complete details on the use and execution of this protocol, please refer to Lu et al. (2020).