The ultrastructural organization of the rat exocrine pancreas: II. Centroacinar cells, intercalary and intralobular ducts

Centroacinar cells (CACs) are ductal Notch-responsive progenitors that in the larval zebrafish pancreas differentiate to form new islets and ultimately contribute to the majority of the adult endocrine mass. Uncovering the mechanisms regulating CAC differentiation will facilitate understanding how insulin-producing β cells are formed. Previously we reported retinoic acid (RA) signaling and Notch signaling both regulate larval CAC differentiation, suggesting a shared downstream intermediate. Sox9b is a transcription factor important for islet formation whose expression is upregulated by Notch signaling in larval CACs. Here we report that sox9b expression in larval CACs is also regulated by RA signaling. Therefore, we hypothesized that Sox9b is an intermediate between both RA- and Notch-signaling pathways. In order to study the role of Sox9b in larval CACs, we generated two cre/lox based transgenic tools, which allowed us to express full-length or truncated Sox9b in larval CACs. In this way we were able to perform spatiotemporal-controlled Sox9b gain- and loss-of-function studies and observe the subsequent effect on progenitor differentiation. Our results are consistent with Sox9b regulating CAC differentiation by being a downstream intermediate of both RA- and Notch-signaling pathways. We also demonstrate that adult zebrafish with only one functional allele of sox9b undergo accelerated β-cell regeneration, an observation consistent with sox9b regulating CAC differentiation in adults.

The Notch signaling pathway regulates embryonic development of the pancreas, inhibiting progenitor differentiation into exocrine acinar and endocrine islet cells. The adult pancreas appears to lack progenitor cells, and its mature cell types are maintained by the proliferation of pre-existing differentiated cells. Nonetheless, Notch remains active in adult duct and terminal duct/centroacinar cells (CACs), in which its function is unknown. We previously developed mice in which cells expressing the Notch target gene Hes1 can be labeled and manipulated, by expression of Cre recombinase, and demonstrated that Hes1⁺ CACs do not behave as acinar or islet progenitors in the uninjured pancreas, or as islet progenitors after pancreatic duct ligation. In the current study, we assessed the function of Notch signaling in the adult pancreas by deleting the transcription factor partner of Notch, Rbpj, specifically in Hes1⁺ cells. We find that loss of Rbpj depletes the pancreas of Hes1-expressing CACs, abrogating their ongoing contribution to growth and homeostasis of more proximal duct structures. Upon Rbpj deletion, CACs undergo a rapid transformation into acinar cells, suggesting that constitutive Notch activity suppresses the acinar differentiation potential of CACs. Together, our data provide direct evidence of an endogenous genetic program to control interconversion of cell fates in the adult pancreas.

Zebrafish provide a highly versatile model in which to study vertebrate development. Many recent studies have elucidated early events in the organogenesis of the zebrafish pancreas; however, several aspects of early endocrine pancreas formation in the zebrafish are not homologous to the mammalian system. To better identify mechanisms of islet formation in the zebrafish, with true homology to those observed in mammals, we have temporally and spatially characterized zebrafish secondary islet formation. As is the case in the mouse, we show that Notch inhibition leads to precocious differentiation of endocrine tissues. Furthermore, we have used transgenic fish expressing fluorescent markers under the control of a Notch-responsive element to observe the precursors of these induced endocrine cells. These pancreatic Notch-responsive cells represent a novel population of putative progenitors that are associated with larval pancreatic ductal epithelium, suggesting functional homology between secondary islet formation in zebrafish and the secondary transition in mammals. We also show that Notch-responsive cells persist in the adult pancreas and possess the classical characteristics of centroacinar cells, a cell type believed to be a multipotent progenitor cell in adult mammalian pancreas.

To determine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in pancreas development, we generated a pancreas-specific knockout of Pten, a negative regulator of PI3-K signaling. Knockout mice display progressive replacement of the acinar pancreas with highly proliferative ductal structures that contain abundant mucins and express Pdx1 and Hes1, two markers of pancreatic progenitor cells. Moreover, a fraction of these mice develop ductal malignancy. We provide evidence that ductal metaplasia results from the expansion of centroacinar cells rather than transdifferentiation of acinar cells. These results indicate that Pten actively maintains the balance between different cell types in the adult pancreas and that misregulation of the PI3-K pathway in centroacinar cells may contribute to the initiation of pancreatic carcinoma in vivo.

In the rat pancreas oval cells were shown to contain albumin transcripts and are considered to be precursors of pancreatic hepatocytes. Pancreatic oval cells have been maintained in our laboratory as a cell line. In the present study we have examined the differentiation potential of rat pancreatic epithelial oval cells under different culture conditions using light and electron microscopy and in situ hybridization techniques. Oval cells cultured on collagen gels formed a monolayer. However, when oval cells were embedded in collagen gels and cultured with or without fibroblasts, they readily organized into duet-like structures within 1 week. Over the next 3 weeks there was a gradual increase in the number and size of ductules. Morphological examination of duct-like structures showed true lumen formation. Ultrastructurally, cells showed features of ductal cells characterized by well-formed cell junctions, interdigitation of plasma membranes, well-developed cytoplasmic organelles, and apical microvilli. Light microscopic autoradiography showed proliferation of cells lining the ductules throughout the 4 week culture period. Albumin mRNA expression was demonstrable by in situ hybridization with albumin probe in oval cells cultured on collagen gels and embedded in collagen gels. Results of these studies indicate that under these in vitro conditions, oval cells appear to express albumin transcripts but exhibit duct-like morphogenetic features.

Ultrastructural study showed the effects of dietary conditions on the exocrine pancreas of the sea bass Dicentrarchus labrax L. The characteristics of this organ were first determined in feral sea bass, used as controls for comparing changes caused by fasting or intensive artificial feeding. In fasted sea bass, collagen developed at the periphery of exocrine tissue and within it. The lumina of excretory ducts became narrower, sometimes even disappearing, and only carried minute quantities of pancreatic juice. This condition corresponded to reduced cellular activity, as indicated by the scarcity of zymogen granules and the small number of apical microvilli. Other changes occurred in pancreatic cells, i.e. mitochondria lengthened and pseudomyelin structures and lysosomes appeared. On the other hand, in intensively fed sea bass, zymogen granules were more numerous and larger, and a greater quantity of material was carried by the ducts, indicating increased cellular activity compared to control and fasted fish. The observations are discussed in terms of their functional significance.