Matthew C. Gibson

The emergence of geometric order in proliferating metazoan epithelia

The predominantly hexagonal cell pattern of simple epithelia was noted in the earliest microscopic analyses of animal tissues, a topology commonly thought to reflect cell sorting into optimally packed honeycomb arrays. Here we use a discrete Markov model validated by time-lapse microscopy and clonal analysis to demonstrate that the distribution of polygonal cell types in epithelia is not a result of cell packing, but rather a direct mathematical consequence of cell proliferation. On the basis of in vivo analysis of mitotic cell junction dynamics in Drosophila imaginal discs, we mathematically predict the convergence of epithelial topology to a fixed equilibrium distribution of cellular polygons. This distribution is empirically confirmed in tissue samples from vertebrate, arthropod and cnidarian organisms, suggesting that a similar proliferation-dependent cell pattern underlies pattern formation and morphogenesis throughout the metazoa.

Control of the Mitotic Cleavage Plane by Local Epithelial Topology

For nearly 150 years, it has been recognized that cell shape strongly influences the orientation of the mitotic cleavage plane (e.g., Hofmeister, 1863). However, we still understand little about the complex interplay between cell shape and cleavage-plane orientation in epithelia, where polygonal cell geometries emerge from multiple factors, including cell packing, cell growth, and cell division itself. Here, using mechanical simulations, we show that the polygonal shapes of individual cells can systematically bias the long-axis orientations of their adjacent mitotic neighbors. Strikingly, analyses of both animal epithelia and plant epidermis confirm a robust and nearly identical correlation between local cell topology and cleavage-plane orientation in vivo. Using simple mathematics, we show that this effect derives from fundamental packing constraints. Our results suggest that local epithelial topology is a key determinant of cleavage-plane orientation, and that cleavage-plane bias may be a widespread property of polygonal cell sheets in plants and animals.

Epithelial junctions maintain tissue architecture by directing planar spindle orientation

During epithelial cell proliferation, planar alignment of the mitotic spindle coordinates the local process of symmetric cell cleavage with the global maintenance of polarized tissue architecture. Although the disruption of planar spindle alignment is proposed to cause epithelial to mesenchymal transition and cancer, the in vivo mechanisms regulating mitotic spindle orientation remain elusive. Here we demonstrate that the actomyosin cortex and the junction-localized neoplastic tumour suppressors Scribbled and Discs large 1 have essential roles in planar spindle alignment and thus the control of epithelial integrity in the Drosophila imaginal disc. We show that defective alignment of the mitotic spindle correlates with cell delamination and apoptotic death, and that blocking the death of misaligned cells is sufficient to drive the formation of basally localized tumour-like masses. These findings indicate a key role for junction-mediated spindle alignment in the maintenance of epithelial integrity, and also reveal a previously unknown cell-death-mediated tumour-suppressor function inherent in the polarized architecture of epithelia.

Interkinetic Nuclear Migration Is a Broadly Conserved Feature of Cell Division in Pseudostratified Epithelia

Animal development requires tight integration between the processes of proliferative growth and epithelial morphogenesis, both of which play out at the level of individual cells. In this respect, not only must polarized epithelial cells assume complex morphologies, these distinct forms must be radically and repeatedly transformed to permit mitosis. A dramatic illustration of this integration between epithelial morphogenesis and cell proliferation is interkinetic nuclear migration (IKNM), wherein the nuclei of pseudostratified epithelial cells translocate to the apical epithelial surface to execute cell division. IKNM is widely considered a hallmark of pseudostratified vertebrate neuroepithelia, and prior investigations have proposed both actomyosin- and microtubule-dependent mechanisms for apical localization of the mitotic nucleus. Here, using comparative functional analysis in arthropod and cnidarian systems (Drosophila melanogaster and Nematostella vectensis), we show that actomyosin-dependent IKNM is likely to be a general feature of mitosis in pseudostratified epithelia throughout Eumetazoa. Furthermore, our studies suggest a mechanistic link between IKNM and the fundamental process of mitotic cell rounding.

Lumenal transmission of decapentaplegic in Drosophila imaginal discs.

Drosophila imaginal discs are sac-like appendage primordia comprising apposed peripodial and columnar cell layers. Cell survival in disc columnar epithelia requires the secreted signal Decapentaplegic (DPP), which also acts as a gradient morphogen during pattern formation. The distribution mechanism by which secreted DPP mediates global cell survival and graded patterning is poorly understood. Here we report detection of DPP in the lumenal cavity between apposed peripodial and columnar cell layers of both wing and eye discs. We show that peripodial cell survival hinges upon DPP signal reception and implicate DPP-dependent viability of the peripodial epithelium in growth of the entire disc. These results are consistent with lumenal transmission of the DPP survival signal during imaginal disc development.

TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis

Non-bilaterian phyla represent key lineages for exploring the evolutionary history of early animals. However, despite an increasing number of sequenced genomes from early-branching metazoans, efficient and reproducible methodologies for analysis of gene function remain a major challenge. Here we report the utilization of the TALEN and CRISPR/Cas9 systems to induce targeted mutations and homologous recombination-mediated transgenesis in the sea anemone Nematostella vectensis. We also present a new method to isolate genetically modified animals using engineered selection cassettes introduced by homologous recombination. Taken together, these methods will permit sophisticated gain- and loss-of-function analyses in Nematostella and perhaps other early metazoan species that allow for zygotic injection.

Decapentaplegic and growth control in the developing Drosophila wing

As a central model for morphogen action during animal development, the bone morphogenetic protein 2/4 (BMP2/4)-like ligand Decapentaplegic (Dpp) is proposed to form a long-range signalling gradient that directs both growth and pattern formation during Drosophila wing disc development. While the patterning role of Dpp secreted from a stripe of cells along the anterior–posterior compartmental boundary is well established, the mechanism by which a Dpp gradient directs uniform cell proliferation remains controversial and poorly understood. Here, to determine the precise spatiotemporal requirements for Dpp during wing disc development, we use CRISPR–Cas9-mediated genome editing to generate a flippase recognition target (FRT)-dependent conditional null allele. By genetically removing Dpp from its endogenous stripe domain, we confirm the requirement of Dpp for the activation of a downstream phospho-Mothers against dpp (p-Mad) gradient and the regulation of the patterning targets spalt (sal), optomotor blind (omb; also known as bifid) and brinker (brk). Surprisingly, however, third-instar wing blade primordia devoid of compartmental dpp expression maintain relatively normal rates of cell proliferation and exhibit only mild defects in growth. These results indicate that during the latter half of larval development, the Dpp morphogen gradient emanating from the anterior–posterior compartment boundary is not directly required for wing disc growth.

Cell division and the maintenance of epithelial order

Epithelia are polarized layers of adherent cells that are the building blocks for organ and appendage structures throughout animals. To preserve tissue architecture and barrier function during both homeostasis and rapid growth, individual epithelial cells divide in a highly constrained manner. Building on decades of research focused on single cells, recent work is probing the mechanisms by which the dynamic process of mitosis is reconciled with the global maintenance of epithelial order during development. These studies reveal how symmetrically dividing cells both exploit and conform to tissue organization to orient their mitotic spindles during division and establish new adhesive junctions during cytokinesis.

Chapter 4 Cell Topology, Geometry, and Morphogenesis in Proliferating Epithelia

Epithelia are sheets of tightly adherent cells that line both internal and external surfaces in a vast array of metazoans. During development, an intrinsic consequence of coupling tight adhesion with cellular proliferation is the emergence of an epithelial form characterized by a stereotyped distribution of polygonal cell shapes. Despite the near universality of this constraint on cell shape and tissue organization, very little is known about the possible implications of cell pattern geometry for mechanical properties of tissues or key biological processes, such as planar polarization, tissue remodeling, and cell division. In this chapter, through an examination of increasingly complex models, we highlight what is known about the role of mitotic proliferation in the emergence of epithelial cell geometry, and examine some possible implications for tissue morphogenesis. Ideally, continued progress in this area will address a major conceptual challenge in biology, which is to understand aspects of morphogenesis that are not explicitly directed by genetic control, but instead emerge from the complex interactions between geometric and biomechanical properties of epithelial tissues.

Modeling and Inferring Cleavage Patterns in Proliferating Epithelia

The regulation of cleavage plane orientation is one of the key mechanisms driving epithelial morphogenesis. Still, many aspects of the relationship between local cleavage patterns and tissue-level properties remain poorly understood. Here we develop a topological model that simulates the dynamics of a 2D proliferating epithelium from generation to generation, enabling the exploration of a wide variety of biologically plausible cleavage patterns. We investigate a spectrum of models that incorporate the spatial impact of neighboring cells and the temporal influence of parent cells on the choice of cleavage plane. Our findings show that cleavage patterns generate “signature” equilibrium distributions of polygonal cell shapes. These signatures enable the inference of local cleavage parameters such as neighbor impact, maternal influence, and division symmetry from global observations of the distribution of cell shape. Applying these insights to the proliferating epithelia of five diverse organisms, we find that strong division symmetry and moderate neighbor/maternal influence are required to reproduce the predominance of hexagonal cells and low variability in cell shape seen empirically. Furthermore, we present two distinct cleavage pattern models, one stochastic and one deterministic, that can reproduce the empirical distribution of cell shapes. Although the proliferating epithelia of the five diverse organisms show a highly conserved cell shape distribution, there are multiple plausible cleavage patterns that can generate this distribution, and experimental evidence suggests that indeed plants and fruitflies use distinct division mechanisms.