The development of the pancreas is a complex process comprising of regionalization, cell differentiation, and morphogenesis. Using zebrafish as a model can contribute to helping visualize its development in which signaling molecules allow the ultimate differentiation of pancreatic cell types as well as its use in pancreatic regeneration studies and diabetes. More interestingly, zebrafish have now been used as a model to demonstrate how endodermal cells can be influenced to differentiate into either liver cells or pancreatic cells. This is the topic we’ll be focusing on.
Zebrafish share the same basic structure and cellular makeup with mammals, implying that the developmental data from zebrafish will be broadly applicable (Kinkel et al, 2009). The basic organization of the digestive tract is conserved from mammals to fish. The pancreas develops from the endoderm germ layer. It consists of an exocrine compartment (acinar cells, which are secreted into the ductal system and produce and transport digestive enzymes to the digestive tract), and the endocrine compartment (the islets), which is critical for blood sugar homeostasis.
Bmp2b Signaling in Hepatopancreatic Fate Decision
Research has shown in vivo evidence that some endodermal cells can give rise to both the liver and ventral pancreas (Chung et al, 2008). In one study, the Bmp receptor gene Alk8 was identified as playing a positive role in hepatic induction, and data from these analyses indicated that Bmp signaling was essential for hepatic specification. This particular set of work centered around identifying the relevant expression pattern of this inducer.
Lineage Tracing Reveals the Existence of Bipotential Hepatopancreatic Progenitors
Lineage tracing is the identification of all progeny of a single cell (Kretzschmar et al, 2012). For this particular scenario involving zebrafish, single endodermal cells are labeled in the following medio-lateral (M-L) fashion: the most medial cells (medial), cells immediately adjacent to the medial cells (lateral 1), and cells one cell away from the medial cells (lateral 2). They are also co-labeled the cells somites 1, 2, 3, and 4 (this region comprises the liver and pancreas progenitors). What was discovered was that medial endodermal cells at 6-8 somite stage gave rise mostly to pancreatic endocrine cells at 50hpf. In constrast, the fate of lateral 1 cells was variable; these cells could give rise to both pancreatic exocrine cells and intestinal cells as well as a small number of pancreatic endocrine cells. Most of the descendants of the lateral 1 cells populate the pdx1-positive organs (i.e. the pancreas and intestine). Most interestingly, single endodermal cells in the lateral 2 position between somites 1 and 3 gave rise not only to pancreatic exocrine cells and intestinal cells, but also to liver cells. These data show that endodermal cells, although at the same A-P level, can contribute to different organs depending on their M-L position.
What happens in the decrease of Bmp signaling?
In testing the role of bmp2b in directing cells toward the liver or to the ventral pancreas, bmp2b morpholinos were injected into zebrafish embryos. Injection of 210 pg or more of bmp2b MO led to dorsalization of the embryos. When injected with lower doses (100–150 pg) of bmp2b MO, hhex expression was slightly reduced in the liver region [hhex is known to regulate liver development (Wallace et al, 2001)]. This reduction in hhex expression was more pronounced when injecting the same amount of bmp2b MO into alk8 heterozygous embryos. These data, together with the analysis of alk8 mutants, indicate that the Alk8 signaling of bmp2b regulates hepatic specification and that reducing bmp2b signaling leads to an expansion of the pdx1 expression domain.
What happens in the overexpression of Bmp2b ?
The next step was to see what happens in the liver and pdx1 domains with an increase in bmp2b expression. The Tg(hsp70l:bmp2b)f13 line of zebrafish can be manipulated to overexpress bmp2b by induced heat shock treatment (see also heat shock proteins). The embryos were heat shocked at the 8 and 14 somite stages, which is before and after the initiation of endogenous bmp2b expression.
At this stage in wild type, hhex is expressed in the liver and dorsal pancreatic bud while pdx1 is expressed in a broad region of the foregut endoderm (which includes the dorsal and ventral pancreas and the intestine, but not the liver). When bmp2b expression was induced at the 8 somite stage, hhex expression at 44 hpf was greatly expanded while pdx1 expression was severely reduced (although its expression in pancreatic endocrine cells appeared unaffected). However, when bmp2b expression was induced at the 14 somite stage, hhex expression was only slightly expanded and pdx1 expression appeared unaffected, although the morphology of the gut tube was disrupted.
To see the effects of bmp2b in the ventral pancreas, the expression of Tg(ptf1a:GFP)jh1 was examined (which marks the development of the ventral pancreas) along with Prox1 and Islet1 [to mark the liver (Burke et al, 2002) and pancreatic endocrine cells (Schmitt et al, 2008), respectively]. Compared to control embryos, in embryos where bmp2b expression was induced at the 8 somite stage, the domain of Prox1 expression was dramatically expanded in the endoderm anterior to the pancreatic endocrine cells. In these embryos, Tg(ptf1a:GFP)jh1 expression was completely absent, indicating that all the Prox1-expressing cells are liver cells. This complete absence of the ventral pancreas is likely due to the severe reduction of pdx1 expression in the developing gut. Altogether, these data show that increased Bmp2b signaling caused ventral pancreas and intestine progenitors to become liver cells.
As such, this study arrived at the following conclusions:
1. Endodermal cells can contribute to different organs depending on their M-L position.
2. Reducing bmp2b signaling leads to an expansion of the pdx1 expression domain.
3. Increasing bmp2b signaling causes ventral pancreas and intestine progenitors to become liver cells.
In an ideal situation, diseased pancreatic islets of diabetic patients would be replaced by healthy functional cells. Early studies using the Edmonton Protocol, which involves cadaver-harvested islets transplanted into diabetic patients, shows that this is a feasible option. The protocol, however, is limited by the availability of harvested islets. This encourages efforts to either derive functional islets from stem cells or to induce regeneration of pancreatic cells.
Previous studies have used the mouse as a model system. The mouse is an advantageous model because it shares many characteristics with humans. But because the accessibility and optical clarity of zebrafish embryos allows sophisticated in vivo imaging as well as cell manipulation, an endeavor much more difficult to do with mammals, it has allowed the zebrafish to become a model for pancreatic studies.
Discussion of Paper
The authors of the paper did a great job in outlining specifically the experiments done and the rationale behind each one. Experimental protocol was also explained sufficiently. This is also a great discovery in developmental biology. The discovery of bipotential hepatopancreatic progenitors could have a large impact in studies involving diabetes. Also, this finding has paved the way in future studies involving pancreas and liver specification; ablation experiments can now involve the redetermination the fate of cells rather than having to regenerate them. I found no obvious weaknesses in this paper.
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