The mechanical influence of densification on epithelial architecture.

Epithelial tissues are the most abundant tissue type in animals, lining body cavities and generating compartment barriers. The function of a monolayered epithelial tissue-whether protective, secretory, absorptive, or filtrative-relies on the side-by-side arrangement of its component cells. The mechanical parameters that determine the shape of epithelial cells in the apical-basal plane are not well-understood. Epithelial tissue architecture in culture is intimately connected to cell density, and cultured layers transition between architectures as they proliferate. This prompted us to ask to what extent epithelial architecture emerges from two mechanical considerations: A) the constraints of densification and B) cell-cell adhesion, a hallmark feature of epithelial cells. To address these questions, we developed a novel polyline cell-based computational model and used it to make theoretical predictions about epithelial architecture upon changes to density and cell-cell adhesion. We tested these predictions using cultured cell experiments. Our results show that the appearance of extended lateral cell-cell borders in culture arises as a consequence of crowding-independent of cell-cell adhesion. However, cadherin-mediated cell-cell adhesion is associated with a novel architectural transition. Our results suggest that this transition represents the initial appearance of a distinctive epithelial architecture. Together our work reveals the distinct mechanical roles of densification and adhesion to epithelial layer formation and provides a novel theoretical framework to understand the less well-studied apical-basal plane of epithelial tissues.

Automated Layer Analysis (ALAn): An Image Analysis Tool for the Unbiased Characterization of Mammalian Epithelial Architecture in Culture.

Cultured mammalian cells are a common model system for the study of epithelial biology and mechanics. Epithelia are often considered as pseudo-two dimensional and thus imaged and analyzed with respect to the apical tissue surface. We found that the three-dimensional architecture of epithelial monolayers can vary widely even within small culture wells, and that layers that appear organized in the plane of the tissue can show gross disorganization in the apical-basal plane. Epithelial cell shapes should be analyzed in 3D to understand the architecture and maturity of the cultured tissue to accurately compare between experiments. Here, we present a detailed protocol for the use of our image analysis pipeline, Automated Layer Analysis (ALAn), developed to quantitatively characterize the architecture of cultured epithelial layers. ALAn is based on a set of rules that are applied to the spatial distributions of DNA and actin signals in the apical-basal (depth) dimension of cultured layers obtained from imaging cultured cell layers using a confocal microscope. ALAn facilitates reproducibility across experiments, investigations, and labs, providing users with quantitative, unbiased characterization of epithelial architecture and maturity. Key features • This protocol was developed to spatially analyze epithelial monolayers in an automated and unbiased fashion. • ALAn requires two inputs: the spatial distributions of nuclei and actin in cultured cells obtained using confocal fluorescence microscopy. • ALAn code is written in Python3 using the Jupyter Notebook interactive format. • Optimized for use in Marbin-Darby Canine Kidney (MDCK) cells and successfully applied to characterize human MCF-7 mammary gland-derived and Caco-2 colon carcinoma cells. • This protocol utilizes Imaris software to segment nuclei but may be adapted for an alternative method. ALAn requires the centroid coordinates and volume of nuclei.

Fas2EB112: a tale of two chromosomes.

The cell-cell adhesion molecule Fasciclin II (Fas2) has long been studied for its evolutionarily conserved role in axon guidance. It is also expressed in the follicular epithelium, where together with a similar protein, Neuroglian (Nrg), it helps to drive the reintegration of cells born out of the tissue plane. Remarkably, one Fas2 protein null allele, Fas2G0336, demonstrates a mild reintegration phenotype, whereas work with the classic null allele Fas2EB112 showed more severe epithelial disorganization. These observations raise the question of which allele (if either) causes a bona fide loss of Fas2 protein function. The problem is not only relevant to reintegration but fundamentally important to understanding what this protein does and how it works: Fas2EB112 has been used in at least 37 research articles, and Fas2G0336 in at least three. An obvious solution is that one of the two chromosomes carries a modifier that either suppresses (Fas2G0336) or enhances (Fas2EB112) phenotypic severity. We find not only the latter to be the case, but identify the enhancing mutation as Nrg14, also a classic null allele.

Fas2EB112: A Tale of Two Chromosomes.

The cell-cell adhesion molecule Fasciclin II (Fas2) has long been studied for its evolutionarily-conserved role in axon guidance. It is also expressed in the follicular epithelium, where together with a similar protein, Neuroglian (Nrg), it helps to drive the reintegration of cells born out of the tissue plane. Remarkably, one Fas2 protein null allele, Fas2G0336, demonstrates a mild reintegration phenotype, whereas work with the classic null allele Fas2EB112 showed more severe epithelial disorganization. These observations raise the question of which allele (if either) causes a bona fide loss of Fas2 protein function. The problem is not only relevant to reintegration but fundamentally important to understanding what this protein does and how it works: Fas2EB112 has been used in at least 37 research articles, and Fas2G0336 in at least three. An obvious solution is that one of the two chromosomes carries a modifier that either suppresses (Fas2G0336) or enhances (Fas2EB112) phenotypic severity. We find not only the latter to be the case, but identify the enhancing mutation as Nrg14, also a classic null allele.

New insights into the genome and transmission of the microsporidian pathogen Nosema muscidifuracis.

Introduction: Nosema is a diverse genus of unicellular microsporidian parasites of insects and other arthropods. Nosema muscidifuracis infects parasitoid wasp species of Muscidifurax zaraptor and M. raptor (Hymenoptera: Pteromalidae), causing ~50% reduction in longevity and ~90% reduction in fecundity. Methods and Results: Here, we report the first assembly of the N. muscidifuracis genome (14,397,169 bp in 28 contigs) of high continuity (contig N50 544.3 Kb) and completeness (BUSCO score 97.0%). A total of 2,782 protein-coding genes were annotated, with 66.2% of the genes having two copies and 24.0% of genes having three copies. These duplicated genes are highly similar, with a sequence identity of 99.3%. The complex pattern suggests extensive gene duplications and rearrangements across the genome. We annotated 57 rDNA loci, which are highly GC-rich (37%) in a GC-poor genome (25% genome average). Nosema-specific qPCR primer sets were designed based on 18S rDNA annotation as a diagnostic tool to determine its titer in host samples. We discovered high Nosema titers in Nosema-cured M. raptor and M. zaraptor using heat treatment in 2017 and 2019, suggesting that the remedy did not completely eliminate the Nosema infection. Cytogenetic analyses revealed heavy infections of N. muscidifuracis within the ovaries of M. raptor and M. zaraptor, consistent with the titer determined by qPCR and suggesting a heritable component of infection and per ovum vertical transmission. Discussion: The parasitoids-Nosema system is laboratory tractable and, therefore, can serve as a model to inform future genome manipulations of Nosema-host system for investigations of Nosemosis.

The Mechanical Influence of Densification on Initial Epithelial Architecture.

Epithelial tissues are the most abundant tissue type in animals, lining body cavities and generating compartment barriers. The function of a monolayer epithelium - whether protective, secretory, absorptive, or filtrative -relies on regular tissue architecture with respect to the apical-basal axis. Using an unbiased 3D analysis pipeline developed in our lab, we previously showed that epithelial tissue architectures in culture can be divided into distinct developmental categories, and that these are intimately connected to cell density: at sparse densities, cultured epithelial cell layers have a squamous morphology (Immature); at intermediate densities, these layers develop lateral cell-cell borders and rounded cell apices (Intermediate); cells at the highest densities reach their full height and demonstrate flattened apices (Mature). These observations prompted us to ask whether epithelial architecture emerges from the mechanical constraints of densification, and to what extent a hallmark feature of epithelial cells, namely cell-cell adhesion, contributes. In other words, to what extent is the shape of cells in an epithelial layer a simple matter of sticky, deformable objects squeezing together? We addressed this problem using a combination of computational modeling and experimental manipulations. Our results show that the first morphological transition, from Immature to Intermediate, can be explained simply by cell crowding. Additionally, we identify a new division (and thus transition) within the Intermediate category, and find that this second morphology relies on cell-cell adhesion.

The Drosophila mitotic spindle orientation machinery requires activation, not just localization.

The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins - called Pins, Gαi, Mud, and Dynein in flies - that exerts a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. In this study we revisit the canonical model and find that it is incomplete. Spindle orientation in the Drosophila follicular epithelium and embryonic ectoderm requires not only Pins localization but also direct interaction between Pins and the multifunctional protein Discs large. This requirement can be over-ridden by interaction with another Pins interacting protein, Inscuteable.

A novel tool for the unbiased characterization of epithelial monolayer development in culture.

The function of an epithelial tissue is intertwined with its architecture. Epithelial tissues are often described as pseudo-two-dimensional, but this view may be partly attributed to experimental bias: many model epithelia, including cultured cell lines, are easiest to image from the "top-down." We measured the three-dimensional architecture of epithelial cells in culture and found that it varies dramatically across cultured regions, presenting a challenge for reproducibility and cross-study comparisons. We therefore developed a novel tool (Automated Layer Analysis, "ALAn") to characterize architecture in an unbiased manner. Using ALAn, we find that cultured epithelial cells can organize into four distinct architectures and that architecture correlates with cell density. Cells exhibit distinct biological properties in each architecture. Organization in the apical-basal axis is determined early in monolayer development by substrate availability, while disorganization in the apical-basal axis arises from an inability to form substrate connections. Our work highlights the need to carefully control for three-dimensional architecture when using cell culture as a model system for epithelial cell biology and introduces a novel tool, built on a set of rules that can be widely applied to epithelial cell culture.

Emerging Cnidarian Models for the Study of Epithelial Polarity.

Epithelial tissues are vital to the function of most organs, providing critical functions such as secretion, protection, and absorption. Cells within an epithelial layer must coordinate to create functionally distinct apical, lateral, and basal surfaces in order to maintain proper organ function and organism viability. This is accomplished through the careful targeting of polarity factors to their respective locations within the cell, as well as the strategic placement of post-mitotic cells within the epithelium during tissue morphogenesis. The process of establishing and maintaining epithelial tissue integrity is conserved across many species, as important polarity factors and spindle orientation mechanisms can be found in many phyla. However, most of the information gathered about these processes and players has been investigated in bilaterian organisms such as C. elegans, Drosophila, and vertebrate species. This review discusses the advances made in the field of epithelial polarity establishment from more basal organisms, and the advantages to utilizing these simpler models. An increasing number of cnidarian model organisms have been sequenced in recent years, such as Hydra vulgaris and Nematostella vectensis. It is now feasible to investigate how polarity is established and maintained in basal organisms to gain an understanding of the most basal requirements for epithelial tissue morphogenesis.

Interaction between Discs large and Pins/LGN/GPSM2: a comparison across species.

The orientation of the mitotic spindle determines the direction of cell division, and therefore contributes to tissue shape and cell fate. Interaction between the multifunctional scaffolding protein Discs large (Dlg) and the canonical spindle orienting factor GPSM2 (called Pins in Drosophila and LGN in vertebrates) has been established in bilaterian models, but its function remains unclear. We used a phylogenetic approach to test whether the interaction is obligate in animals, and in particular whether Pins/LGN/GPSM2 evolved in multicellular organisms as a Dlg-binding protein. We show that Dlg diverged in C. elegans and the syncytial sponge Opsacas minuta and propose that this divergence may correspond with differences in spindle orientation requirements between these organisms and the canonical pathways described in bilaterians. We also demonstrate that Pins/LGN/GPSM2 is present in basal animals, but the established Dlg-interaction site cannot be found in either Placozoa or Porifera. Our results suggest that the interaction between Pins/LGN/GPSM2 and Dlg appeared in Cnidaria, and we therefore speculate that it may have evolved to promote accurate division orientation in the nervous system. This work reveals the evolutionary history of the Pins/LGN/GPSM2-Dlg interaction and suggests new possibilities for its importance in spindle orientation during epithelial and neural tissue development.

Gametogenesis: Germ Cells Aren't Just Along for the Ride.

A new study explores the mechanical basis of germline encapsulation in Drosophila gametogenesis, reporting that it is not driven solely by somatic tissue, as previously assumed, but instead relies on actomyosin-generated force in the germline cells.

An Axon-Pathfinding Mechanism Preserves Epithelial Tissue Integrity.

Epithelial tissues form the boundaries of organs, where they perform a range of functions, including secretion, absorption, and protection. These tissues are commonly composed of discrete cell layers-sheets of cells that are one-cell thick. In multiple systems examined, epithelial cells round up and move in the apical direction before dividing, likely in response to neighbor-cell crowding [1-6]. Because of this movement, daughter cells may be born displaced from the tissue layer. Reintegration of these displaced cells supports tissue growth and maintains tissue architecture [4]. Two conserved IgCAMs (immunoglobulin superfamily cell adhesion molecules), neuroglian (Nrg) and fasciclin 2 (Fas2), participate in cell reintegration in the Drosophila follicular epithelium [4]. Like their vertebrate orthologs L1CAM and NCAM1/2, respectively, Nrg and Fas2 are cell adhesion molecules primarily studied in the context of nervous system development [7-10]. Consistent with this, we identify another neural IgCAM, Fasciclin 3 (Fas3), as a reintegration factor. Nrg, Fas2, and Fas3 are components of the insect septate junction, the functional equivalent of the vertebrate tight junction, but proliferating follicle cells do not have mature septate junctions, and we find that the septate junction protein neurexin IV does not participate in reintegration [11, 12]. Here, we show that epithelial reintegration works in the same way as IgCAM-mediated axon growth and pathfinding; it relies not only on extracellular adhesion but also mechanical coupling between IgCAMs and the lateral spectrin-based membrane skeleton. Our work indicates that reintegration is mediated by a distinct epithelial adhesion assembly that is compositionally and functionally equivalent to junctions made between axons.

Neuronal immunoglobulin superfamily cell adhesion molecules in epithelial morphogenesis: insights from Drosophila.

In this review, we address the function of immunoglobulin superfamily cell adhesion molecules (IgCAMs) in epithelia. Work in the Drosophila model system in particular has revealed novel roles for calcium-independent adhesion molecules in the morphogenesis of epithelial tissues. We review the molecular composition of lateral junctions with a focus on their IgCAM components and reconsider the functional roles of epithelial lateral junctions. The epithelial IgCAMs discussed in this review have well-defined roles in the nervous system, particularly in the process of axon guidance, suggesting functional overlap and conservation in mechanism between that process and epithelial remodelling. We expand on the hypothesis that epithelial occluding junctions and synaptic junctions are compositionally equivalent and present a novel hypothesis that the mechanism of epithelial cell (re)integration and synaptic junction formation are shared. We highlight the importance of considering non-cadherin-based adhesion in our understanding of the mechanics of epithelial tissues and raise questions to direct future work. This article is part of the discussion meeting issue 'Contemporary morphogenesis'.

The role of integrins in Drosophila egg chamber morphogenesis.

The Drosophila egg chamber comprises a germline cyst surrounded by a tightly organised epithelial monolayer, the follicular epithelium (FE). Loss of integrin function from the FE disrupts epithelial organisation at egg chamber termini, but the cause of this phenotype remains unclear. Here, we show that the ß-integrin Myospheroid (Mys) is only required during early oogenesis when the pre-follicle cells form the FE. Mutation of mys disrupts both the formation of a monolayered epithelium at egg chamber termini and the morphogenesis of the stalk between adjacent egg chambers, which develops through the intercalation of two rows of cells into a single-cell-wide stalk. Secondary epithelia, like the FE, have been proposed to require adhesion to the basement membrane to polarise. However, Mys is not required for pre-follicle cell polarisation, as both follicle and stalk cells localise polarity factors correctly, despite being mispositioned. Instead, loss of integrins causes pre-follicle cells to constrict basally, detach from the basement membrane and become internalised. Thus, integrin function is dispensable for pre-follicle cell polarity but is required to maintain cellular organisation and cell shape during morphogenesis.

Division orientation: disentangling shape and mechanical forces.

Oriented cell divisions are essential for the generation of cell diversity and for tissue shaping during morphogenesis. Cells in tissues are mechanically linked to their neighbors, upon which they impose, and from which they experience, physical force. Recent work in multiple systems has revealed that tissue-level physical forces can influence the orientation of cell division. A long-standing question is whether forces are communicated to the spindle orienting machinery via cell shape or directly via mechanosensing intracellular machinery. In this article, we review the current evidence from diverse model systems that show spindles are oriented by tissue-level physical forces and evaluate current models and molecular mechanisms proposed to explain how the spindle orientation machinery responds to extrinsic force.

Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium.

We investigated the cell behaviors that drive morphogenesis of the Drosophila follicular epithelium during expansion and elongation of early-stage egg chambers. We found that cell division is not required for elongation of the early follicular epithelium, but drives the tissue toward optimal geometric packing. We examined the orientation of cell divisions with respect to the planar tissue axis and found a bias toward the primary direction of tissue expansion. However, interphase cell shapes demonstrate the opposite bias. Hertwig's rule, which holds that cell elongation determines division orientation, is therefore broken in this tissue. This observation cannot be explained by the anisotropic activity of the conserved Pins/Mud spindle-orienting machinery, which controls division orientation in the apical-basal axis and planar division orientation in other epithelial tissues. Rather, cortical tension at the apical surface translates into planar division orientation in a manner dependent on Canoe/Afadin, which links actomyosin to adherens junctions. These findings demonstrate that division orientation in different axes-apical-basal and planar-is controlled by distinct, independent mechanisms in a proliferating epithelium.

Cell reintegration: Stray epithelial cells make their way home.

Ongoing work shows that misplaced epithelial cells have the capacity to reintegrate back into tissue layers. This movement appears to underlie tissue stability and may also control aspects of tissue structure. A recent study reveals that cell reintegration in at least one tissue, the Drosophila follicular epithelium, is based on adhesion molecules that line lateral cell surfaces. In this article we will review these observations, discuss their implications for epithelial tissue development and maintenance, and identify future directions for study.

Spindle orientation: a question of complex positioning.

The direction in which a cell divides is determined by the orientation of its mitotic spindle at metaphase. Spindle orientation is therefore important for a wide range of developmental processes, ranging from germline stem cell division to epithelial tissue homeostasis and regeneration. In multiple cell types in multiple animals, spindle orientation is controlled by a conserved biological machine that mediates a pulling force on astral microtubules. Restricting the localization of this machine to only specific regions of the cortex can thus determine how the mitotic spindle is oriented. As we review here, recent findings based on studies in tunicate, worm, fly and vertebrate cells have revealed that the mechanisms for mediating this restriction are surprisingly diverse.

Pins is not required for spindle orientation in the Drosophila wing disc.

In animal cells, mitotic spindles are oriented by the dynein/dynactin motor complex, which exerts a pulling force on astral microtubules. Dynein/dynactin localization depends on Mud/NUMA, which is typically recruited to the cortex by Pins/LGN. In Drosophila neuroblasts, the Inscuteable/Baz/Par-6/aPKC complex recruits Pins apically to induce vertical spindle orientation, whereas in epithelial cells Dlg recruits Pins laterally to orient the spindle horizontally. Here we investigate division orientation in the Drosophila imaginal wing disc epithelium. Live imaging reveals that spindle angles vary widely during prometaphase and metaphase, and therefore do not reliably predict division orientation. This finding prompted us to re-examine mutants that have been reported to disrupt division orientation in this tissue. Loss of Mud misorients divisions, but Inscuteable expression and aPKC, dlg and pins mutants have no effect. Furthermore, Mud localizes to the apical-lateral cortex of the wing epithelium independently of both Pins and cell cycle stage. Thus, Pins is not required in the wing disc because there are parallel mechanisms for Mud localization and hence spindle orientation, making it a more robust system than in other epithelia.