3D Image-based morphometric analysis of embryonic epithelia

Abstract

Epithelial cells are the cornerstones of metazoan development. Nonetheless, their three-dimensional (3D) shape, organization, and morphogenetic deformations remain largely unexplored in higher mammals. A plethora of studies has characterized apical epithelial morphology in invertebrates by accounting for cell surface geometric patterns, inspiring a number of models based on topology, geometry, and curvature. However, a lot of the architectural complexity observed in live mammalian rudiments remains unexplained. The projects in this thesis contribute to this field by shedding light on cell morphology, neighbor dynamics, and organizational principles in 3D, as well as epithelial tube elongation and anatomical dysmorphologies during early mouse Mus musculus organogenesis. In the first part of this thesis (chapter 2), the dynamic re-organization of cell-cell contacts and 3D morphology of epithelial cells in tubular and spherical layers is analyzed. To this end, light-sheet fluorescence microscopy of growing mouse lung rudiments and 3D cell segmentation were used to visualize, map, and quantify cell areas and topological changes throughout the apical-basal axis. The results reveal that 3D epithelial shapes are highly irregular, dynamic, and exhibit numerous neighbor intercalations along the apical-basal axis, with the majority localizing at the nucleus's boundaries. Ultimately, this expands the pool of 3D geometrical possibilities for columnar epithelia in developing tissues. Additionally, growing epithelia were found to adhere to fundamental relationships only previously described for apical layers, i.e., Euler’s formula, Lewis’ law, and Aboav-Weaire’s law. These findings strongly suggest that while complex and fluid, the 3D epithelial architecture follows simple physical principles. In the second part of the presented thesis (chapter 3), epithelial tube morphology and outgrowth in the embryonic bronchial system were characterized. To this extent, live fluorescence microscopy, optical clearing, and morphometric analyses in 2D and 3D were used to allow for the morphological quantification of the left primary bronchus during early organogenesis. This work provides insight into the precise extent of elongation between developmental stages and reveals a striking correspondence between 2D and 3D explant cultures. What is more, these datasets provide experimental evidence of closed epithelial tubes with opposing apical sides and narrow luminal spaces. Similar configurations were identified irrespective of the sample preparation, isolation, or clearing duration used for embryonic lungs and kidneys. This study also discusses avenues to query the nature of the observed non-uniform luminal curvature and a number of likely biomechanical queues for anisotropic tube growth. The last part of this thesis (chapter 4) involves the identification of embryonic neuroepithelial alterations in a genetically defined mouse model of autism spectrum disorder (ASD). This project incorporated light-sheet microscopy, optical tissue clearing, and multi-scale morphometric segmentations to reveal developmental aberrations in corticoventricular shape and epithelial architecture. Despite a low sample size, initial results show deviations in the intraocular distance and ventricular sphericity. Moreover, quantifications of apical epithelial geometry show no differences in cortical organization, suggesting that neither cellular nor tissue mechanics abnormalities are likely to precede characteristic ASD-associated postnatal remodeling.