Cell polarity determinant Dlg1 facilitates epithelial invagination by promoting tissue-scale mechanical coordination

Epithelial folding mediated by apical constriction serves as a fundamental mechanism to convert flat epithelial sheets into multilayered structures. It remains elusive whether additional mechanical inputs are required for folding mediated by apical constriction. Using Drosophila mesoderm invagination as a model, we identified an important role for the non-constricting, lateral mesodermal cells adjacent to the constriction domain ("flanking cells") in facilitating epithelial folding. We found that depletion of the basolateral determinant, Dlg1, disrupts the transition between apical constriction and invagination without affecting the rate of apical constriction. Strikingly, the observed delay in invagination is associated with ineffective apical myosin contractions in the flanking cells that lead to overstretching of their apical domain. The defects in the flanking cells impede ventral-directed movement of the lateral ectoderm, suggesting reduced mechanical coupling between tissues. Specifically disrupting the flanking cells in wildtype embryos by laser ablation or optogenetic depletion of cortical actin is sufficient to delay the apical constriction-to-invagination transition. Our findings indicate that effective mesoderm invagination requires intact flanking cells and suggest a role for tissue-scale mechanical coupling during epithelial folding.

An Analysis of the Progression of Conjunctivalisation after Transplantation of Cultivated Corneal Epithelium

Purpose. To analyse the recurrence of superficial neovascularisation after previous corneal surface reconstruction with cultivated corneal epithelial cells. Materials and Methods. Forty-eight eyes underwent autologous transplantation of cultivated corneal epithelium to treat partial or total limbal stem cell deficiency caused by chemical or thermal injury. The carrier for the epithelial sheets was a denuded amniotic membrane. Follow-up was conducted for up to 120 months. Recurrent revascularisation (measured in terms of clock hours affected) was evaluated with slit-lamp examination and the support of confocal microscopy. Results. During the long-term observation, only 7 eyes had stable epithelia with no neovascularisation from the conjunctiva. Nineteen eyes developed pathologic vessels in 1 quadrant, with additional 4 eyes developing them in 2 quadrants. Twelve patients developed subtotal or total conjunctivalisation of the corneal surface. They were referred for second cultivated epithelium transplantation (3 patients), allogenic keratolimbal transplantation (7 patients), or keratoprosthesis (2 patients). Six patients withdrew consent. The use of confocal scans of up to 100 µm in resolution enabled the detection of pathologic microvasculature originating from the conjunctiva and the exclusion of stromal vascular ingrowth. Conclusions. Local ingrowth of the conjunctiva is a common complication after the transplantation of cultivated epithelial cells. Severe and progressive vascularisation inevitably leads to graft failure. However, if local ingrowth stops before reaching the central cornea, the treatment even with this complication can be considered a success.

Origami: Single-cell 3D shape dynamics oriented along the apico-basal axis of folding epithelia from fluorescence microscopy data

A common feature of morphogenesis is the formation of three-dimensional structures from the folding of two-dimensional epithelial sheets, aided by cell shape changes at the cellular-level. Changes in cell shape must be studied in the context of cell-polarised biomechanical processes within the epithelial sheet. In epithelia with highly curved surfaces, finding single-cell alignment along a biological axis can be difficult to automate in silico. We present ‘Origami’, a MATLAB-based image analysis pipeline to compute direction-variant cell shape features along the epithelial apico-basal axis. Our automated method accurately computed direction vectors denoting the apico-basal axis in regions with opposing curvature in synthetic epithelia and fluorescence images of zebrafish embryos. As proof of concept, we identified different cell shape signatures in the developing zebrafish inner ear, where the epithelium deforms in opposite orientations to form different structures. Origami is designed to be user-friendly and is generally applicable to fluorescence images of curved epithelia.

3D Cell neighbour dynamics in growing pseudostratified epithelia

During morphogenesis, epithelial sheets remodel into complex geometries. How cells dynamically organize their contact with neighbouring cells in these tightly packed tissues is poorly understood. We have used light-sheet microscopy of growing mouse embryonic lung explants, three-dimensional cell segmentation, and physical theory to unravel the principles behind 3D cell organization in growing pseudostratified epithelia. We find that cells have highly irregular 3D shapes and exhibit numerous neighbour intercalations along the apical-basal axis as well as over time. Despite the fluidic nature, the cell packing configurations follow fundamental relationships previously described for apical epithelial layers, i.e., Euler's formula, Lewis' law, and Aboav-Weaire's law, at all times and across the entire tissue thickness. This arrangement minimizes the lateral cell-cell surface energy for a given cross-sectional area variability, generated primarily by the distribution and movement of nuclei. We conclude that the complex 3D cell organization in growing epithelia emerges from simple physical principles.

Reconstruction of distinct vertebrate gastrulation modes via modulation of key cell behaviours in the chick embryo

The morphology of gastrulation driving the internalisation of the mesoderm and endoderm differs dramatically among vertebrate species. It ranges from involution of epithelial sheets of cells through a circular blastopore in amphibians to ingression of mesenchymal cells through a primitive streak in amniotes. By targeting signalling pathways controlling critical cell behaviours in the chick embryo, we generated crescent- and ring-shaped mesendoderm territories in which cells can or cannot ingress. These alterations subvert the formation of the chick primitive streak into the gastrulation modes seen in amphibians, reptiles and teleost fish. Our experimental manipulations are supported by a theoretical framework linking cellular behaviors to self-organized multi-cellular flows in the accompanying paper. All together, this suggests that the evolution of gastrulation movements are largely determined by the shape of and cell behaviours in the mesendoderm territory across different species, and controlled by a relatively small number of signalling pathways.

Deconstructing Gastrulation at the Single Cell Level

Gastrulation movements in all animal embryos start with regulated deformations of patterned epithelial sheets. Current studies of gastrulation use a wide range of model organisms and emphasize either large-scale tissue processes or dynamics of individual cells and cell groups. Here we take a step towards bridging these complementary strategies and deconstruct early stages of gastrulation in the entire Drosophila embryo, where transcriptional patterns in the blastoderm give rise to region-specific cell behaviors. Our approach relies on an integrated computational framework for cell segmentation and tracking and on efficient algorithms for event detection. Our results reveal how thousands of cell shape changes, divisions, and intercalations drive large-scale deformations of the patterned blastoderm, setting the stage for systems-level dissection of a pivotal step in animal development.

Allogeneic Cultivated Limbal Epithelial Sheet Transplantation in Reconstruction of Conjunctival Sac After Chemical and Thermal Burns

The study aims to evaluate the effect of allogeneic cultivated limbal epithelial cell sheet transplantation (CLET) in reconstructing conjunctival sac for severe symblepharon after chemical and thermal burns. A retrospective, non-comparative case series. Thirty-six eyes (36 patients) underwent CLET for severe symblepharon and conjunctival sac stenosis or atresia. Symblepharon was separated, and pseudopterygium was preserved to replace the palpebral conjunctiva. Allogeneic cultivated limbal epithelial cell sheet using human amniotic membrane as a carrier was transplanted into the recipient's eye to reconstruct the conjunctival sac. The effect of conjunctival sac reconstruction, eye and eyelid movement, ocular surface restitution, and symblepharon recurrence were analyzed after surgery. Symblepharon was completely relieved in 30 of the 36 eyes (83.3%) by a single surgical procedure, with fornix reconstruction, as well as free movement of eye globe and eyelids. Strip-like symblepharon remained in 6 eyes (16.7%) and was completely relieved after the second CLET. Twenty patients without visual function received prostheses 3 months after surgery and the other sixteen patients underwent different corneal transplantation for visual acuity improvement. During the follow-up period, no one had symblepharon recurrence. The transplantation of cultivated allogeneic limbal epithelial sheets offers an effective and safe alternative in the treatment of symblepharon and reconstruction of conjunctival sac in eyes with severe ocular burns, which lays the foundation for subsequent treatments.

Modelling the collective mechanical regulation of the structure and morphology of epithelial cell layers

The morphology and function of epithelial sheets play an important role in healthy tissue development and cancer progression. The maintenance of structure of closely packed epithelial layers requires the coordination of various mechanical forces within the cells and others resulting from interactions with other cells and other tissues or substrates. However, a general model for the combination of mechanical properties which determine the cell shape and the overall structure of epithelial layers remains elusive. Here, we propose a computational model, based on the Cellular Potts Model, to study the interplay between mechanical properties of cells and dynamical transitions in epithelial structures and cell shapes. We map out phase diagrams as functions of cellular properties and the orientation of cell division. Monolayers of squamous, cuboidal, and columnar cells are found when the axis of cell proliferation is perpendicular to the substrate. Monolayer-to-multilayer transition is promoted via cell extrusion, depending on the mechanical properties of cells and the orientation of cell division. The results and model predictions are discussed in the context of experimental observations.

Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity in vitro and in vivo

It has been a long-standing challenge to obtain from cell cultures adequate amounts of mouse corneal epithelial cells (mCEC) to perform transplantation surgery. This limitation is attributable to the passage dependent declines in their proliferative activity. We describe here development of a novel 6C medium that contains six different modulators of different signaling pathways, which control proliferative mCEC activity. Its usage shortens the time and effort required to obtain epithelial sheets for hastening healing of an epithelial wound in an experimental animal model. This serum-free 6C medium contains:Y27632, forskolin, SB431542, DAPT, IWP-2, LDN-193189 and also DermaLife K keratinocyte calcium. Their inclusion inhibits rises in four specific markers of epithelial mesenchymal transdifferentiation:ZEB1/2, Snail, β-catenin and α-SMA. This medium is applied in a feeder-free air-lifted system to obtain sufficient populations of epithelial progenitor cells whose procurement is facilitated due to suppression of progenitor epithelial cell transdifferentiation into epithelial-mesenchymal cells. Diminution of this decline in transdifferentiation was confirmed based on the invariance of P63, K14, Pax6, and K12 gene expression levels. This cell culture technique is expected to facilitate ex vivo characterization of mechanisms underlying cell fate determination. Furthermore, its implementation will improve yields of progenitor mouse corneal epithelial cells, which increases the likelihood of using these cells as a source to generate epithelial sheets for performing transplantation surgery to treat limbal stem cell deficiency in a clinical setting. In addition, the novel insight obtainable from such studies is expected to improve the outcomes of corneal regenerative medicine.

Origami: Single-cell oriented 3D shape dynamics of folding epithelia from fluorescence microscopy images

A common feature of morphogenesis is the formation of three-dimensional structures from the folding of two-dimensional epithelial sheets aided by spatio-temporal cell shape changes at the cellular-level. Studying cell shape dynamics and polarised processes that underpin them, requires orienting cells within the epithelial sheet. In epithelia with highly curved surfaces, assigning cell orientation can be difficult to automate in silico . We present ‘Origami’, a MATLAB-based image analysis pipeline to compute oriented cell shape-features. Our automated method accurately computed cell orientation in regions with opposing curvature in synthetic epithelia and fluorescence images of zebrafish embryos. As proof of concept, we identified different cell shape signatures in the developing zebrafish inner ear, where the epithelium deforms in opposite orientations to form different structures. Origami is designed to be user-friendly and is generally applicable to fluorescence images of curved epithelia.