Functional and pharmacological analyses of visual habituation learning in larval zebrafish.

Habituation allows animals to learn to ignore persistent but inconsequential stimuli. Despite being the most basic form of learning, a consensus model on the underlying mechanisms has yet to emerge. To probe relevant mechanisms, we took advantage of a visual habituation paradigm in larval zebrafish, where larvae reduce their reactions to abrupt global dimming (a dark flash). We used Ca2+ imaging during repeated dark flashes and identified 12 functional classes of neurons that differ based on their rate of adaptation, stimulus response shape, and anatomical location. While most classes of neurons depressed their responses to repeated stimuli, we identified populations that did not adapt or that potentiated their response. These neurons were distributed across brain areas, consistent with a distributed learning process. Using a small-molecule screening approach, we confirmed that habituation manifests from multiple distinct molecular mechanisms, and we have implicated molecular pathways in habituation, including melatonin, oestrogen, and GABA signalling. However, by combining anatomical analyses and pharmacological manipulations with Ca2+ imaging, we failed to identify a simple relationship between pharmacology, altered activity patterns, and habituation behaviour. Collectively, our work indicates that habituation occurs via a complex and distributed plasticity processes that cannot be captured by a simple model. Therefore, untangling the mechanisms of habituation will likely require dedicated approaches aimed at sub-component mechanisms underlying this multidimensional learning process.

Gradient in cytoplasmic pressure in germline cells controls overlying epithelial cell morphogenesis.

It is unknown how growth in one tissue impacts morphogenesis in a neighboring tissue. To address this, we used the Drosophila ovarian follicle, in which a cluster of 15 nurse cells and a posteriorly located oocyte are surrounded by a layer of epithelial cells. It is known that as the nurse cells grow, the overlying epithelial cells flatten in a wave that begins in the anterior. Here, we demonstrate that an anterior to posterior gradient of decreasing cytoplasmic pressure is present across the nurse cells and that this gradient acts through TGFß to control both the triggering and the progression of the wave of epithelial cell flattening. Our data indicate that intrinsic nurse cell growth is important to control proper nurse cell pressure. Finally, we reveal that nurse cell pressure and subsequent TGFß activity in the stretched cells combine to increase follicle elongation in the anterior, which is crucial for allowing nurse cell growth and pressure control. More generally, our results reveal that during development, inner cytoplasmic pressure in individual cells has an important role in shaping their neighbors.

Variations in basement membrane mechanics are linked to epithelial morphogenesis.

The regulation of morphogenesis by the basement membrane (BM) may rely on changes in its mechanical properties. To test this, we developed an atomic force microscopy-based method to measure BM mechanical stiffness during two key processes in Drosophila ovarian follicle development. First, follicle elongation depends on epithelial cells that collectively migrate, secreting BM fibrils perpendicularly to the anteroposterior axis. Our data show that BM stiffness increases during this migration and that fibril incorporation enhances BM stiffness. In addition, stiffness heterogeneity, due to oriented fibrils, is important for egg elongation. Second, epithelial cells change their shape from cuboidal to either squamous or columnar. We prove that BM softens around the squamous cells and that this softening depends on the TGFß pathway. We also demonstrate that interactions between BM constituents are necessary for cell flattening. Altogether, these results show that BM mechanical properties are modified during development and that, in turn, such mechanical modifications influence both cell and tissue shapes.