Diabetes mellitus is characterized by disrupted glucose homeostasis due to loss or dysfunction of insulin-producing beta cells. In this work, we characterize pancreatic islet development and function in zebrafish mutant for pdx1, a gene which in humans is linked to genetic forms of diabetes and is associated with increased susceptibility to Type 2 diabetes. Pdx1 mutant zebrafish have the key diabetic features of reduced beta cells, decreased insulin and elevated glucose. The hyperglycemia responds to pharmacologic anti-diabetic treatment and, as often seen in mammalian diabetes models, beta cells of pdx1 mutants show sensitivity to nutrient overload. This unique genetic model of diabetes provides a new tool for elucidating the mechanisms behind hyperglycemic pathologies and will allow the testing of novel therapeutic interventions in a model organism that is amenable to high-throughput approaches.

The three-dimensional architecture of the pancreatic islet is integral to beta cell function, but the process of islet formation remains poorly understood due to the difficulties of imaging internal organs with cellular resolution. Within transparent zebrafish larvae, the developing pancreas is relatively superficial and thus amenable to live imaging approaches. We performed in vivo time-lapse and longitudinal imaging studies to follow islet development, visualizing both naturally occurring islet cells and cells arising with an accelerated timecourse following an induction approach. These studies revealed previously unappreciated fine dynamic protrusions projecting between neighboring and distant endocrine cells. Using pharmacological compound and toxin interference approaches, and single-cell analysis of morphology and cell dynamics, we determined that endocrine cell motility is regulated by phosphoinositide 3-kinase (PI3K) and G-protein-coupled receptor (GPCR) signaling. Linking cell dynamics to islet formation, perturbation of protrusion formation disrupted endocrine cell coalescence, and correlated with decreased islet cell differentiation. These studies identified novel cell behaviors contributing to islet morphogenesis, and suggest a model in which dynamic exploratory filopodia establish cell-cell contacts that subsequently promote cell clustering.

Pancreatic islets, discrete microorgans embedded within the exocrine pancreas, contain beta cells which are critical for glucose homeostasis. Loss or dysfunction of beta cells leads to diabetes, a disease with expanding global prevalence, and for which regenerative therapies are actively being pursued. Recent efforts have focused on producing mature beta cells in vitro, but it is increasingly recognized that achieving a faithful three-dimensional islet structure is crucial for generating fully functional beta cells. Our current understanding of islet morphogenesis is far from complete, due to the deep internal location of the pancreas in mammalian models, which hampers direct visualization. Zebrafish is a model system well suited for studies of pancreas morphogenesis due to its transparency and the accessible location of the larval pancreas. In order to further clarify the cellular mechanisms of islet formation, we have developed new tools for in vivo visualization of single-cell dynamics. Our results show that clustering islet cells make contact and interconnect through dynamic actin-rich processes, move together while remaining in close proximity to the duct, and maintain high protrusive motility after forming clusters. Quantitative analyses of cell morphology and motility in 3-dimensions lays the groundwork to define therapeutically applicable factors responsible for orchestrating the morphogenic behaviors of coalescing endocrine cells.

Mitochondria, organelles critical for energy production, modify their shape and location in response to developmental state and metabolic demands. Mitochondria are altered in diabetes, but the mechanistic basis is poorly defined, due to difficulties in assessing mitochondria within an intact organism. Here, we use in vivo imaging in transparent zebrafish larvae to demonstrate filamentous, interconnected mitochondrial networks within islet cells. Mitochondrial movements highly resemble what has been reported for human islet cells in vitro, showing conservation in behaviour across species and cellular context. During islet development, mitochondrial content increases with emergence of cell motility, and mitochondria disperse within fine protrusions. Overall, this work presents quantitative analysis of mitochondria within their native environment and provides insights into mitochondrial behaviour during organogenesis.