Volumetric analysis of epithelial morphogenesis with high spatiotemporal resolution

Project Summary
How epithelial sheets remodel themselves to adopt new tissue conformations through changes in neighbor
relationships and cell shape dynamics has been a key question in development and disease. Interestingly,
many of the pioneering studies performed in model systems have largely been confined to 2D analysis, and
have often been challenged to image cell behaviors that occur in basal regions that lie deeper into the tissue.
The intercalation movements that occur during tissue elongation in the Drosophila gastrula have been a classic
system for understanding epithelial remodeling, and have been fundamental to informing the developmental
paradigms that describe how cells can change position in an adherent epithelium. Nearly all of the studies in
this system have been confined to 2D analysis of apical events in the early fly embryo, and no studies to date
have systematically analyzed the full 3D behaviors that drive epithelial remodeling and tissue extension in the
Drosophila embryonic epithelium. Thus, one of the biggest remaining questions in the field is how the
volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface along the entire
apical-basal axis. Fundamental questions on where forces originate from as well as how far and fast forces
propagate across different apical-basal layers have remained unanswered. In our preliminary analysis, we
have been successful in completing the first full 4D segmentation of the intercalating Drosophila epithelium
through the use of Lattice Light Sheet Microscopy (LLSM). We find that intercalation can be initiated at any
position we have surveyed along the apical-basal axis. This is striking as previous studies have largely
implicated apical force generation, and a single study has suggested that contractile forces can also originate
from the basal surface of the epithelium. In the proposed project, we are developing the tools to perform the
first comprehensive, quantitative 3D analysis of cell intercalation in the early Drosophila embryo. We will then
determine the molecular mechanisms driving 3D force generation, and whether different mechanical regimes
exist across the apical-basal axis. Preliminary data suggests highly novel dynamic Myosin II and F-actin
populations that show rapid axial propagation in lateral and basal regions. The 3D distributions of these
populations are being mapped and the relevant actin nucleating and Myosin regulatory networks will be
determined. These results will provide the first comprehensive understanding of the cortical and contractile
networks that determine the mechanical environment of a gastrulating epithelium. We will also use 3D data
sets in wild-type and functionally compromised backgrounds to examine how epithelial forces propagate along
apical-basal and planar dimensions using topological mapping metrics. We will determine how far, and at what
velocities, contractile forces spread in an intact, developing epithelium. These results will give fundamental
answers into how the viscous cytoplasm and elastic cell cortex respond to force-driven displacements, and
how these displacements spread within individual cells and across tissues to drive new tissue topologies.

Grant Number: 5R01GM144506-03
NIH Institute/Center: NIH
Principal Investigator: James Blankenship