In vivo imaging of emerging endocrine cells reveals a requirement for PI3K-regulated motility in pancreatic islet morphogenesis.

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.

Cell type and tissue specific function of islet genes in zebrafish pancreas development.

Isl1 is a LIM homeobox transcription factor showing conserved expression in the developing and mature vertebrate pancreas. So far, functions of pancreatic Isl1 have mainly been studied in the mouse, where Isl1 has independent functions during formation of exocrine and endocrine tissues. Here, we take advantage of a recently described isl1 mutation in zebrafish to address pancreatic isl1 functions in a non-mammalian system. Isl1 in zebrafish, as in mouse, shows transient expression in mesenchyme flanking the pancreatic endoderm, and continuous expression in all endocrine cells. In isl1 mutants, endocrine cells are specified in normal numbers but more than half of these cells fail to establish expression of endocrine hormones. By using a lineage tracking approach that highlights cells leaving cell cycle early in development, we show that isl1 functions are different in first and second wave endocrine cells. In isl1 mutants, early forming first wave cells show virtually no glucagon expression and a reduced number of cells expressing insulin and somatostatin, while in the later born second wave cells somatostatin expressing cells are strongly reduced and insulin and glucagon positive cells form in normal numbers. Isl1 mutant zebrafish also display a smaller exocrine pancreas. We find that isl1 expression in the pancreatic mesenchyme overlaps with that of the related genes isl2a and isl2b and that pancreatic expression of isl-genes is independent of each other. As a combined block of two or three isl1/2 genes results in a dose-dependent reduction of exocrine tissue, our data suggest that all three genes cooperatively contribute to non-cell autonomous exocrine pancreas extension. The normal expression of the pancreas mesenchyme markers meis3, fgf10 and fgf24 in isl1/2 depleted embryos suggests that this activity is independent of isl-gene function in pancreatic mesenchyme formation as was found in mouse. This indicates species-specific differences in the requirement for isl-genes in pancreatic mesenchyme formation. Overall, our data reveal a novel interaction of isl1 and isl2 genes in exocrine pancreas expansion and cell type specific requirements during endocrine cell maturation.

Requirement for Pdx1 in specification of latent endocrine progenitors in zebrafish.

BACKGROUND: Insulin-producing beta cells emerge during pancreas development in two sequential waves. Recently described later-forming beta cells in zebrafish show high similarity to second wave mammalian beta cells in developmental capacity. Loss-of-function studies in mouse and zebrafish demonstrated that the homeobox transcription factors Pdx1 and Hb9 are both critical for pancreas and beta cell development and discrete stage-specific requirements for these genes have been uncovered. Previously, exocrine and endocrine cell recovery was shown to follow loss of pdx1 in zebrafish, but the progenitor cells and molecular mechanisms responsible have not been clearly defined. In addition, interactions of pdx1 and hb9 in beta cell formation have not been addressed. RESULTS: To learn more about endocrine progenitor specification, we examined beta cell formation following morpholino-mediated depletion of pdx1 and hb9. We find that after early beta cell reduction, recovery occurs following loss of either pdx1 or hb9 function. Unexpectedly, simultaneous knockdown of both hb9 and pdx1 leads to virtually complete and persistent beta cell deficiency. We used a NeuroD:EGFP transgenic line to examine endocrine cell behavior in vivo and developed a novel live-imaging technique to document emergence and migration of late-forming endocrine precursors in real time. Our data show that Notch-responsive progenitors for late-arising endocrine cells are predominantly post mitotic and depend on pdx1. By contrast, early-arising endocrine cells are specified and differentiate independent of pdx1. CONCLUSIONS: The nearly complete beta cell deficiency after combined loss of hb9 and pdx1 suggests functional cooperation, which we clarify as distinct roles in early and late endocrine cell formation. A novel imaging approach permitted visualization of the emergence of late endocrine cells within developing embryos for the first time. We demonstrate a pdx1-dependent progenitor population essential for the formation of duct-associated, second wave endocrine cells. We further reveal an unexpectedly low mitotic activity in these progenitor cells, indicating that they are set aside early in development.