Cellular Morphodynamics Reveals Divergent Mechanisms of Gastrulation in Insects

During embryonic development, dynamic cellular rearrangements collectively result in tissue morphogenesis. One fundamental morphogenetic process is tissue internalisation, of which the earliest occurrence drives mesoderm gastrulation in a wide variety of organisms. In both the fly Drosophila melanogaster and the beetle Tribolium castaneum, the mesoderm is specified ventrally by conserved genetic factors as an epithelial sheet that buckles inwards, forming a tube that subsequently collapses. While the overall morphogenesis of the mesoderm appears analogous between the two species at the tissue level, the level of conservation of the cellular behaviours driving this process remains elusive. To address this gap, I developed a morphodynamics pipeline to segment and analyse 3D cell shapes in an unbiased and quantitative framework. This enabled me to mathematically describe the coordinated cell shape changes responsible for mesoderm morphogenesis in Drosophila. These transformations are linked to known cellular processes that take place sequentially: apical constriction of tall columnar epithelial cells, cell shortening, mitosis and cell spreading, together contributing to a collective epithelial-mesenchymal transition. In contrast, the application of my pipeline in Tribolium embryos revealed asynchronicity in cellular behaviours driving mesoderm invagination and epithelial-mesenchymal transition. By complementing my morphometric analysis with high-resolution live imaging and staining of cytoskeletal components, I identified two distinct phases of mesoderm internalisation in Tribolium. First, the mesoderm is specified as a cuboidal monolayer which undergoes a wave of semisynchronous cell divisions. Strikingly, at this stage a significant portion of mesodermal cells individually ingress via out of plane divisions and, in small part, cell extrusion. Internalised cells adopt a mesenchymal phenotype, whilst cells that have remained in the epithelium plane apically constrict and collectively fold inside. The tube then collapses and cells adopt a spread mesenchymal phenotype, which coincides in shape distribution with Drosophila mesenchymal cells. The differences between these species in mesodermal cell shape trajectories leading to the mesenchymal state can be ultimately attributed to two factors: the epithelium sheet's initial height, and the timing of cell division, either prior or subsequent to tissue folding. My study provides a generalised framework for extracting, analysing and comparing three-dimensional cell shapes from microscopy images of tissues, further offering adaptability to other systems. In the context of mesoderm gastrulation in Drosophila and Tribolium, it brings new insights to our understanding of conserved and divergent mechanisms underlying tissue internalisation. While apical constriction appears to orchestrate collective cell invagination via tissue folding in both insects, in Tribolium cells can individually ingress via out of plane mitosis and undergo a sudden transition from an epithelial to a mesenchymal phenotype. Out of plane divisions are a relatively underexplored phenomenon in developmental biology, whilst spindle misorientation in epithelia has been correlated with pathologies such as cancer and microcephaly. My study opens the door for exploring the mechanisms triggering out of plane divisions and their mechanical effects on tissue morphogenesis in an accessible organism.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Battistara, Margherita

Advisor dc:contributor.advisor

• Paluch, Ewa

• morphogenesis
• cell shape
• gastrulation
• insects embryogenesis
• out of plane divisions

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a28616be-c94e-4f69-b712-65fad1bb5f16/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.108827

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/368713