Genetic and mechanical analysis of tube morphogenesis in the living Drosophila embryo

Morphogenesis requires simultaneous force generation and cell deformation. Although the transcriptional control of force generation has been studied, the concurrent modulation of cellular material properties to enable deformation has remained purely speculative. In this work, we develop high-resolution live imaging of deep tissue morphogenesis in the Drosophila embryo and combine live imaging with genetic and mechanical analysis to better understand the role of the Ribbon BTB domain transcription factor in salivary gland and tracheal morphogenesis. These studies indicate a novel role for Ribbon-mediated transcription in the modulation of apical membrane material properties during epithelial morphogenesis.;Despite its many advantages as a model organism, the Drosophila embryo has proven difficult to image live, predominantly due to extensive light scattering from tissue pigmentation and refractive index variation. Consequently, live imaging in the Drosophila embryo has been limited to surface epithelia or to bright and shallow structures such as the late trachea. To achieve high-resolution imaging of deep tissue morphogenesis, we utilize microscopy theory and experimentation to troubleshoot poor image quality in live salivary glands expressing alpha-catenin-GFP and nuclear DsRed, ultimately optimizing a highly sensitive custom two-photon microscope to achieve high quality images while preserving cell viability.;Using fixed samples, we find that Ribbon functions with another BTB domain protein, Lola Like, to upregulate crumbs expression. We also find that Ribbon is required to downregulate apical Moesin activity subsequent to invagination and that constitutively active Moesin can mimic the ribbon mutant phenotype. TEM indicates that ribbon mutants exhibit decreased numbers of apical vesicles and increased microvillar structure, defects consistent with Crumbs and Moesin function. Based on studies in other systems, we reasoned that the decreased Crumbs and increased Moesin activity observed in ribbon mutants could affect the material properties of the apical membrane. Indeed, high-resolution live imaging of ribbon mutants reveals slowed and incomplete lumenal morphogenesis, and mechanical models suggest that ribbon mutant epithelia fail primarily in cell extension due to approximately 2-fold increased apical stiffness and 1.6-fold increased apical viscosity. We conclude that Ribbon and Lola Like function as a novel transcriptional cassette regulating material properties of the apical membrane during morphogenesis.