Inducible Mosaic Cell Labeling Provides Insights Into Pancreatic Islet Morphogenesis

Abstract

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.

Mitochondrial network expansion and dynamic redistribution during islet morphogenesis in zebrafish larvae

“…Although the above studies suggest that the overall mitochondrial network characteristics remained relatively stable during islet assembly, there was generation of new mitochondria in extended cells. We know from previous studies that these cells are highly motile [23,35], and in still images we observed a striking distribution of discrete mitochondrial units at proximal to distal locations in long cellular protrusions (Fig. S6A,B).…”

“…Although considered ‘ubiquitous’, the EF1a promoter is variably downregulated during development [ 32 , 33 ] and the EF1a:MLS‐EGFP transgenic line appeared not optimal for studies of islet mitochondria. As we previously characterised the robust pancreatic islet expression driven from a 5 kb neurod promoter [ 23 ], we now generated a neurod:mitoGFP transgenic line to label mitochondria in pancreatic islet cells. In the newly established transgenic line, the neurod promoter directed robust expression of a mitochondrial targeted GFP within islet cells (Fig.…”

“…This is problematic for analysing cell clusters, as the peripherally dispersed islet cell mitochondria network cannot be easily assigned to the corresponding cell. Therefore, we used the membrane labelling of the neurod:memKate transgenic line [ 23 ] to distinguish boundaries of cells in clusters and to generate masks for cell segmentation (Fig. 3A,B ).…”

“…Mitochondrial topology adapts to cell state and metabolic needs, with a progression from fragmented to elongated forms characterising the transition from progenitor to a more differentiated state [ 1 ]. Previous studies showed that the endocrine cells are very dynamic during islet morphogenesis, as progenitors differentiate and coalesce into clusters [ 23 , 35 , 36 ], but mitochondria have not been examined during this process. To investigate mitochondria morphology and distribution in relation to islet assembly, we analysed larvae expressing neurod:memKate , in addition to neurod:mitoGFP .…”

“…Three independent F1 lines showing robust and consistent pancreatic islet expression, and 50% transgene transmission, were maintained for further experiments. Previously published lines used in this study were: ins:mCherry [ 21 ], pax6b:dsRed [ 22 ], neurod:memKate [ 23 ].…”