Elongation of a tissue along one body axis is a conserved morphogenetic process that happens repeatedly during embryo and organ morphogenesis. While the mechanisms of tissue elongation in 2D sheet-like tissues are increasingly well understood, the molecular and cellular mechanisms that elongate topologically distinct tubular and acinar organs may be distinct. In this dissertation, I utilize Drosophila egg chamber (follicle) morphogenesis in the adult female ovary as a model system to understand tissue elongation in the unbounded tube-like organs. The follicle grows spherically during early oogenesis and then elongates during mid-oogenesis along the anterior-posterior axis to become an ellipsoidal organ. Whole-tissue rotation of the follicle during the elongation phase is thought to provide Planar Cell Polarity (PCP) information that orients basal actin bundles and basement membrane Collagen IV fibrils around the circumference, through a process requiring the atypical cadherin Fat2. Despite its essential role in the process, how Fat2 regulates PCP symmetry-breaking to specify rotation in one chirality was unresolved.

With long-term live imaging, I showed that the follicle initiates rotation immediately after it buds off from the germarium. To trace the earliest sign of chiral symmetry-breaking, I developed an in toto 3D image analysis platform to comprehensively chart the cytoskeletal PCP in curved structures. In collaboration with my labmate Katherine Lipari, we found that microtubule growth polarity exhibits a bias within the germarium, prior to follicle budding and rotation. This germarial microtubule bias is dependent on Fat2, and provides chiral information that breaks the PCP symmetry prior to rotation. We propose a PCP relay mechanism that initiates from germarial microtubule chirality, to rotational polarity and actin bundle alignment, to finally a polarized basement membrane organization that elongates the organ.

The limitations of current ex vivo cultures for long-term morphogenetic analyses compelled me to develop a tool that can track cell motility over extended periods. I developed M-TRAIL, which utilizes cell-ECM interactions to retrospectively trace cell movements in fixed tissues. By making clones overexpressing GFP-tagged ECM proteins, cell motility trajectories are revealed based on the long-lasting traces of fluorescently-tagged ECM proteins deposited onto basement membrane. I applied M-TRAIL to compare ex vivo versus in vivo rotation and between WT and fat2 to validate M-TRAIL’s validity. Strikingly, a previously reported protein-truncating fat2 mutant allele, which does not rotate ex vivo yet produces elongated follicles, actually rotates in vivo at half the speed than WT. These results revalidate the model that tissue rotation is required to build a proper basement membrane that elongate the follicle. The M-TRAIL approach also demonstrates how reliance solely on ex vivo imaging can mislead.

By combining our previous work that chart the BM stiffness profile and by measuring BM stiffness in the slow rotating Fat2-truncated follicle, we show that rather than the absolute BM stiffness, an anterior-posteriorly patterned BM stiffness gradient elongate the follicle. By further refining the 3D image analysis platform I developed and use it to chart tissue and cellular scale morphometrics, I found three major cell behaviors that can contribute to follicle elongation, which are oriented cell division, polarized cell shape changes, and cell rearrangement. With analyses of either mutants that fail to elongate or by genetic manipulations that disrupt one particular cell behavior, I found cell shape changes and cell rearrangement play a major role in elongating the follicle. The tools I developed and my findings provide a platform and new insights in understanding the molecular, cellular, and mechanical mechanisms in organ morphogenesis.