Dissertations and Theses
Collective Behavior of Drosophila Melanogaster Neural Progenitor and Imaginal Disc Cells within Controlled Microenvironments
Date of Award
Microfluidics, Drosophila, Transport, Chemotaxis, Microfabrication, Retina
Regenerative therapies for the damaged visual system have introduced stem-derived cells to recapitulate developmental processes and initiate functional regeneration in different components of the eye. The developing visual system in Drosophila Melanogaster offers a model in which to analyze the associated processes in retinogenesis. The optic nerve is critical to vision and is developmentally preceded in Drosophila by a structure called the Optic Stalk (OS). Collective migration of neural and retinal progenitor cells (RPCs) from the developing brain lobes (DBL) to the Imaginal Disc (ID), through the OS, is a fundamental part of regenerative strategies in retina. Developmental signals governing retinal cell fate and migration have been well-studied using Drosophila Melanogaster. While conserved signaling pathways are known to drive retinogenesis across invertebrate and vertebrate species, the role(s) of diffusible signaling molecules in the collective migratory processes critical to eye development remain incompletely understood. Invertebrate models remain largely underutilized for in vitro study of cell response to controlled stimuli.
In this thesis, the collective behavior and migration of primary Drosophila-derived neural progenitor cells (NPC) and Drosophila imaginal disc cells were analyzed on different extracellular matrices and coatings, poly-L-lysine (PLL), laminin (LM) and Concanavalin- A (Con-A), when exposed to exogenous gradients of growth factors and within microfluidic systems in order to propose an animal model for retinogenesis. The formation of single, small clusters and large clusters of NPCs were observed on all matrices and within the μLane, a bridged microchannel system. Furthermore, small and large clusters were demonstrated chemotaxis, directed cell migration, to gradients of fibroblast growth factor 8 (FGF8), while single cells demonstrated chemokinesis, non- directed cell migration within the μLane. A microfluidic system called the Micro Optic Stalk (μOS), that recapitulated in vivo geometric constraints seen during Drosophila retinal development, was designed, fabricated and validated. When NPCs were cultured and exposed to FGF8 within the μOS, the formation of single cells, small clusters and large clusters was observed. Furthermore, clusters demonstrated chemotaxis and gradient-dependent migration patterns. Imaginal disc cells were studied in order to look at the behavior of the secondary structure involved in retinogenesis. Dm-D17-c3 (D17) cells were examined as they have been utilized for motility studies in literature. D17 cells demonstrated two cell populations, rounded and elongated cells, on PLL and Con- A, while they did not adhere and grew in suspension on LM. Utilizing Boyden Chamber Assays, D17 cells showed significant migration toward brain-derived neurotrophic factor (BDNF), concentration-dependent migration toward Insulin (In), and no significant migration toward FGF8. Furthermore, D17 cells showed high viability when cultured within the μLane when seeded at higher cell densities (7.5 ́105 cells and 1 ́106 cells). Although D17 cells were shown to not be suitable for examination in a retinogenesis model centering on the role of FGF8, they show promise for use in other developmental models and microfluidic systems. Future work will utilize FGFR receptor knock outs in Drosophila within the μOS, in order to further understand FGF8’s role in retinogenesis.
Pena, Caroline D., "Collective Behavior of Drosophila Melanogaster Neural Progenitor and Imaginal Disc Cells within Controlled Microenvironments" (2019). CUNY Academic Works.