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Determining the functional attributes of pancreatic transcription factors is essential to understand how the pancreas is specified distinct from other endodermal organs, such as liver, stomach and duodenum, and to direct the differentiation of other cell types into pancreas. Previously, we demonstrated that Pdx1-VP16 was sufficient to convert liver to pancreas. In this paper, we characterize the functional ability of another pancreatic transcription factor, Ptf1a, in promoting ectopic pancreatic fates at early stages throughout the endoderm and later during organogenesis. Using the transthyretin promoter to drive expression in the early liver region/bud of transgenic Xenopus tadpoles, we find that Ptf1a-VP16 is able to convert liver to pancreas. Overexpression of the unmodified Ptf1a on the other hand has no effect in liver but is able to convert stomach and duodenum to pancreas. When overexpressed at earlier embryonic stages throughout the endoderm, Ptf1a activity is similarly limited, whereas Ptf1a-VP16 has increased activity. Interestingly, in all instances we find that Ptf1a-VP16 is only capable of promoting acinar cell fates, whereas Ptf1a promotes both acinar and endocrine fates. Lastly, we demonstrate that, similar to mouse and zebrafish, Xenopus Ptf1a is essential for the initial specification of both endocrine and exocrine cells during normal pancreas development.
Fig. 1. Transgenic overexpression of Ptf1a and Ptf1a-VP16 promotes ectopic pancreas formation. (A) Control St.44 Elas-GFP transgenic tadpole showing GFP expression only in the pancreas (p). No expression is seen in the stomach and duodenum (st + d). (B) St.45 TTR-Ptf1a:Elas-GFP transgenic tadpole. No stomach or duodenum is evident and instead ectopic GFP is detected throughout this region (p + ep). The liver (l) is normal. (C) St.45 TTR-Ptf1a-VP16:Elas-GFP transgenic tadpole. Ectopic expression (ep) of Elas-GFP is seen opposite the pancreas in the liver. Stomach and duodenum are normal. (The punctate GFP expression in between the ectopic and normal pancreas does not represent epithelial cells in the stomach or duodenum but are cells released from the ectopic pancreas due to mechanical abrasion during processing of samples for photography.) (D) Amylase RNA expression in stage 46/47 whole guts. (D) In control whole guts amylase is only detected in the pancreas. (E) In TTR-Ptf1a transgenics ectopic amylase expression is now seen in the stomach and duodenum that is fused with the normal pancreas (p + ep) and not in the liver. (F) Ectopic expression (ep) is seen in the livers of TTR-Ptf1a-VP16 tadpoles. (G) Insulin RNA expression in stage 46/47 whole guts. (G) In controls, insulin is found expressed in a punctate fashion within the pancreas. (H) In TTR-Ptf1a transgenics insulin expression is detected in the ectopic pancreatic tissue encompassing the stomach, duodenum and pancreas (p + ep). The normal pancreas is seen bulging out to the left, while the ectopic pancreas is fused with it just to the right. The shape of this ectopic pancreatic tissue resembles the normal stomach. The liver (l) is normal. (I) Expression of insulin is only seen in the pancreas of TTR-Ptf1a-VP16 transgenics. No expression is seen in the ectopic pancreas (ep). (J) Glucagon RNA expression in stage 46/47 whole guts. (J) In controls, glucagon is expressed in a punctate fashion much like insulin. (K) In TTR-Ptf1a transgenics, ectopic glucagon expression is detected in the stomach and duodenum (p+ep). (L) In TTR-Ptf1a-VP16 transgenics, no ectopic glucagon expression is detected outside the pancreas.
Fig. 2. Effects of transgenic overexpression of Ptf1a-VP16 and Ptf1a on liver, stomach and duodenum. (A) Liver differentiation marker, transthyretin expression in stage 46/47 whole guts. (A) Control showing normal domain of TTR expression. Notice the size of the liver. (B) In Ptf1a transgenics transthyretin expression is normal. (C) Ptf1a-VP16 transgenic whole gut. Only a small domain of transthyretin expression is evident, located adjacent to the ectopic pancreas. (D) Expression of the stomach differentiation marker frp5 in stage 46/47 whole guts. (D) Frp5 expression in control whole guts extends from the duodenum through the stomach region (st + d), (E) Ptf1a transgenic whole gut. Almost no frp5 expression is detected. A small region at the posterior end of the pancreas and ectopic pancreas does express frp5 and may be marking a small remnant of the duodenum (. (F) Ptf1a-VP16 transgenic whole gut-frp5 expression is normal.
Fig. 3. Histological analysis of Ptf1a and Ptf1a-VP16 transgenic whole guts. (A) Control whole gut section stained for amylase showing normal stomach (st), pancreas (p) and liver (l). (B) Single whole gut from TTR-Ptf1a:Elas-GFP transgenic tadpole was processed for histology after staining for amylase expression. 12-μm serial sections numbers correlate to specific section, with number 1 corresponding to the first section obtained from the isolated whole gut. The posteriorsmall intestine (si) is evident in every section on the left side for orientation. (B) Section 5sophagus (e), pancreas (p) and liver (l) are all present. (C) Section 11he pancreas has almost completely replaced the stomach and duodenum (p + e). A small portion of remaining stomach tube is evident but contains no recognizable stomach cells and the tube is open. Part of the liver (l) can still be seen. (D) Section 18o gut tube is evident, and a large ectopic pancreas (p + e) is found in place of the stomach and duodenum. (E) Section 24he small intestine (si) first reappears, with pancreatic tissue and liver still present. We measured the lack of stomach and duodenum from sections 10 to 24 to be 168 μm in total. (F) Section from Ptf1a-VP16 transgenic whole gut stained with amylase. The ectopic pancreas (ep) is seen fused with the liver, and the normal pancreas adjacent but separate.
Fig. 4. Overexpression of Ptf1a and Ptf1a-VP16 mRNA promotes ectopic and enlarged pancreas formation. Stages 424 dissected whole guts are shown in each image. (A) Control F2 Elas-GFP transgenic tadpole whole gut. (B) Ptf1a-injected embryos showing expanded GFP fluorescence. Stomach (st), duodenum (d) and liver (l) are normal, but the pancreas (p) is enlarged. (C) Ectopic pancreas formation in Ptf1a-injected embryo. The pancreas, stomach and duodenum (p + ep) form a large ectopic pancreas expressing Elas-GFP. (D) Ptf1a-VP16-injected embryo showing ectopic GFP fluorescence throughout the anteriorendoderm. (E) In situ hybridization for elastase expression on isolated whole guts from (E) control, (F, G) Ptf1a-injected embryos and (H) Ptf1a-VP16-injected embryos. Notice expanded and ectopic expression of elastase in Ptf1a and Ptf1a-VP16-injected embryos. (I) In situ hybridization for insulin in (I) control, (J, K) Ptf1a-injected embryos and (L) Ptf1a-VP16-injected embryos. More insulin-expressing cells are detected in Ptf1a-injected whole guts, while there is a large decrease in insulin-expressing cells in Ptf1a-VP16-injected whole guts (arrowheads). (M) Real-time PCR analysis of endocrine and exocrine markers in Ptf1a and Ptf1a-VP16-injected embryos for amylase and insulin expression in control, Ptf1a-injected and Ptf1a-VP16-injected whole embryos at stage 35. Each bar is an average of 4 individual whole tadpoles. Amylase expression is increased in both, while insulin expression is decreased in Ptf1a-VP16 and increased in Ptf1a-injected embryos. Purpleontrol tadpoles, redtf1a mRNA-injected tadpoles, yellowtf1a-VP16-injected tadpoles.
Fig. 5. Development of glucagon and somatostatin expressing cells in Ptf1a and Ptf1a-VP16-injected embryos. (A) In situ hybridization for glucagon expression in control, Ptf1a and Ptf1a-VP16-injected embryos at stage 45. (B) Stomach and duodenal expression of glucagon is absent, while pancreatic glucagon expression appears slightly reduced in Ptf1a-injected ectopic pancreas phenotype. (C) No glucagon expression is detected in Ptf1a-VP16-injected embryos. (D) Somatostatin expression. (E) Overall somatostatin expression is reduced in Ptf1a-injected embryos. Pancreatic expression appears normal, while stomach and duodenal expression is completely absent. (F) Somatostatin expression is completely absent in Ptf1a-VP16-injected embryos.
Fig. 6. Effects of Ptf1a and Ptf1a-VP16 mRNA overexpression on organogenesis of liver, stomach and duodenum. (A) In situ hybridization for frp5 expression in control, Ptf1a-injected embryos and Ptf1a-VP16-injected embryos. (B) Normal expression is seen in the enlarged pancreas phenotype from Ptf1a-injected embryos, (C) whereas reduced expression is seen in the ectopic pancreas phenotype from Ptf1a-injected embryos. (D) No frp5 expression is detected in whole guts from Ptf1a- VP16-injected embryos. (E) In situ hybridization for Hex expression in Control, Ptf1a-injected embryos and Ptf1a-VP16-injected embryos. (F, G) Normal expression of Hex is detected in both enlarged and ectopic phenotypes from Ptf1a-injected embryos. (H) No Hex expression is detected in whole guts from Ptf1a- VP16-injected embryos.
Fig. 7. Elastase expression is absent in Ptf1a morphants, while insulin expression is reduced at late stages, but absent at early stages. (A) Whole mount in situ hybridization for elastase RNA expression in control and Ptf1a-MO dissected whole guts at stage 42. Elastase expression is not detected in either single or double morpholino-injected guts. (E) Whole mount in situ hybridization for insulin RNA expression in control and Ptf1a-MO whole guts. (F, G) Insulin expression is decreased in both single morpholino injections. (H) In embryos injected with both morpholinos together insulin expression is also reduced, but still present. (I) Initial insulin expression is lacking in Ptf1a morphants. Insulin RNA expression by whole mount in situ hybridization in control and Ptf1a-MO-injected embryos at stage 35. Inset in each panel is the low power view. (I) Control tadpole showing normal punctate insulin expression in the dorsal pancreas (arrow). (J, K) Insulin expression is reduced in single morpholino-injected embryos but (L) completely lacking in double morpholino-injected embryos. (M) Expression of the liver marker Hex is normal in both single and double Ptf1a morpholino-injected embryos. (Q) Real-time PCR analysis of insulin expression in control and Ptf1a-MO- injected embryos at stage 35 confirms insulin reduction in single and double morpholino-injected embryos. Each bar is an average of four individual tadpoles. There is a 500% reduction in single morpholino tadpoles (40 ng), but an almost complete absence in the double morpholino tadpoles (20 ng each). (R) RT-PCR analysis of splicing in control and MO2-injected embryos showing the inhibition of splicing by MO2. Loading control was established with EF1α (not shown). Primers were designed flanking the single Ptf1a intron.
Fig. 8. Mouse Ptf1a rescues the Ptf1a-morpholino induced phenotype. (A) Control whole gut showing elastase expression in the pancreas. (B) In Ptf1a-MO whole guts elastase is absent. (C, D) Pancreatic tissue and elastase expression is restored in Ptf1a-MO embryos co-injected with mouse Ptf1a
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