Endogenous OptoRhoGEFs reveal biophysical principles of epithelial tissue furrowing.

During development, epithelia function as malleable substrates that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate the mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in tool expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tag Drosophila RhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities. Using quantitative optogenetic perturbations, we uncover a dose-dependence of tissue furrow depth and bending behavior on RhoGEF recruitment, revealing mechanisms by which developing embryos can shape tissues into particular morphologies. We show that at the onset of gastrulation, furrows formed by cell lateral contraction are oriented and size-constrained by a stiff basal actomyosin layer. Our findings demonstrate the use of quantitative, 3D-patterned perturbations of cell contractility to precisely shape tissue structures and interrogate developmental mechanics.

Bidirectional communication in oogenesis: a dynamic conversation in mice and Drosophila.

In most animals, the oocyte is the largest cell by volume. The oocyte undergoes a period of large-scale growth during its development, prior to fertilization. At first glance, tissues that support the development of the oocyte in different organisms have diverse cellular characteristics that would seem to prohibit functional comparisons. However, these tissues often act with a common goal of establishing dynamic forms of two-way communication with the oocyte. We propose that this bidirectional communication between oocytes and support cells is a universal phenomenon that can be directly compared across species. Specifically, we highlight fruit fly and mouse oogenesis to demonstrate that similarities and differences in these systems should be used to inform and design future experiments in both models.

Size scaling in collective cell growth.

Size is a fundamental feature of living entities and is intimately tied to their function. Scaling laws, which can be traced to D'Arcy Thompson and Julian Huxley, have emerged as a powerful tool for studying regulation of the growth dynamics of organisms and their constituent parts. Yet, throughout the 20th century, as scaling laws were established for single cells, quantitative studies of the coordinated growth of multicellular structures have lagged, largely owing to technical challenges associated with imaging and image processing. Here, we present a supervised learning approach for quantifying the growth dynamics of germline cysts during oogenesis. Our analysis uncovers growth patterns induced by the groupwise developmental dynamics among connected cells, and differential growth rates of their organelles. We also identify inter-organelle volumetric scaling laws, finding that nurse cell growth is linear over several orders of magnitude. Our approach leverages the ever-increasing quantity and quality of imaging data, and is readily amenable for studies of collective cell growth in other developmental contexts, including early mammalian embryogenesis and germline development.

Coupled oscillators coordinate collective germline growth.

Developing oocytes need large supplies of macromolecules and organelles. A conserved strategy for accumulating these products is to pool resources of oocyte-associated germline nurse cells. In Drosophila, these cells grow more than 100-fold to boost their biosynthetic capacity. No previously known mechanism explains how nurse cells coordinate growth collectively. Here, we report a cell cycle-regulating mechanism that depends on bidirectional communication between the oocyte and nurse cells, revealing the oocyte as a critical regulator of germline cyst growth. Transcripts encoding the cyclin-dependent kinase inhibitor, Dacapo, are synthesized by the nurse cells and actively localized to the oocyte. Retrograde movement of the oocyte-synthesized Dacapo protein to the nurse cells generates a network of coupled oscillators that controls the cell cycle of the nurse cells to regulate cyst growth. We propose that bidirectional nurse cell-oocyte communication establishes a growth-sensing feedback mechanism that regulates the quantity of maternal resources loaded into the oocyte.

The essential Drosophila CLAMP protein differentially regulates non-coding roX RNAs in male and females.

Heterogametic species require chromosome-wide gene regulation to compensate for differences in sex chromosome gene dosage. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the male-specific lethal (MSL) complex, which increases transcript levels of active genes 2-fold. The MSL complex contains several protein components and two non-coding RNA on the X ( roX) RNAs that are transcriptionally activated by the MSL complex. We previously discovered that targeting of the MSL complex to the X-chromosome is dependent on the chromatin-linked adapter for MSL proteins (CLAMP) zinc finger protein. To better understand CLAMP function, we used the CRISPR/Cas9 genome editing system to generate a frameshift mutation in the clamp gene that eliminates expression of the CLAMP protein. We found that clamp null females die at the third instar larval stage, while almost all clamp null males die at earlier developmental stages. Moreover, we found that in clamp null females roX gene expression is activated, whereas in clamp null males roX gene expression is reduced. Therefore, CLAMP regulates roX abundance in a sex-specific manner. Our results provide new insights into sex-specific gene regulation by an essential transcription factor.