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Combined Ectopic Expression of Pdx1 and Ptf1a/P48 Results in the Stable Conversion of Posterior Endoderm Into Endocrine and Exocrine Pancreatic Tissue

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Combined Ectopic Expression of Pdx1 and Ptf1a/P48 Results in the Stable Conversion of Posterior Endoderm Into Endocrine and Exocrine Pancreatic Tissue Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION results reported in this communication, ectopic expres- Combined ectopic expression sion of Pdx1 in nonpancreatic chicken endoderm re- of Pdx1 and Ptf1a/p48 results sulted in the initiation of pancreatic budding, but it was not sufficient to promote differentiation of either exo- in the stable conversion crine or endocrine cells (Grapin-Botton et al. 2001). of posterior endoderm into Ptf1a/p48 is a bHLH transcription factor, which was originally identified as a part of a heterotrimeric pan- endocrine and exocrine creas transcription factor complex, referred to as PTF1 that activates transcription of exocrine specific pancre- pancreatic tissue atic genes in the mature pancreas (Cockell et al. 1989; Beres et al. 2006). Mice bearing a null mutation of Ptf1a/ Solomon Afelik, Yonglong Chen, p48 are completely devoid of exocrine pancreas, while 1 and Tomas Pieler endocrine pancreatic cells still form, but are found to be translocated to the spleen (Krapp et al. 1998). More re- Georg-August-Universität Göttingen, Zentrum Biochemie und cent studies have revealed that Ptf1a/p48 is already ex- Molekular Zellbiologie, Abteilung Entwicklungsbiochemie, pressed in pancreatic precursor cells which contribute to 37077 Göttingen, Germany all pancreatic cell types, and that, in the absence of Patterning of the embryonic endoderm into distinct sets Ptf1a/p48, pancreatic precursor cells adopt a duodenal fate (Kawaguchi et al. 2002). These findings suggest a of precursor cells involves the precisely regulated activi- role for Ptf1a/p48 that is not solely in exocrine differen- ties of key transcription regulators. Ectopic, pan-endo- tiation, but that is also relevant for cells of the endocrine dermal activation of XPtf1a/p48 during pancreas precur- lineages. sor cell stages of Xenopus embryogenesis results in an Here, we report on observations that are in strong sup- expansion of the pancreatic territory, precisely within port of the concept that a combination of these two tran- the borders of XlHbox8 expression. A combination of scription factors, XlHbox8 and XPtf1a/p48, is sufficient both activities is sufficient to expand the pancreatic pre- for the commitment of endodermal cells to a pancreatic cursor cell population also into more posterior portions fate in Xenopus embryos. of the endoderm. Both treatments result in the forma- tion of a giant pancreas that persists up to late tadpole Results and Discussion stages of development and carries both supernumerary Within the endoderm, expression of XPtf1a/p48 is spe- endocrine and exocrine cells. A combination of XPtf1a/ cifically restricted to the entire dorsal and ventral pan- p48 and XlHbox8 is thus sufficient to convert nonpan- creatic anlagen as early as at stage 27, while expression creatic endodermal cells into pancreatic precursor cells. of XlHbox8 also includes adjacent regions of the pre- sumptive stomach and duodenum (Fig. 1). To test for the Supplemental material is available at http://www.genesdev.org. function of XPtf1a/p48 in the context of early pancreas development, mRNA encoding a dexamethasone-induc- Received January 4, 2006; revised version accepted March 9, ible variant of XPtf1a/p48, referred to as XPtf1a/p48GR, 2006. was injected into all four vegetal blastomeres of eight- cell-stage Xenopus embryos. Following the temporal ex- The vertebrate pancreas develops from one dorsal and pression profile of the endogenous gene, dexametha- two ventral evaginations in the endodermal epithelium sone treatment was performed at embryonic stage 27. (Slack 1995). Early pancreatic precursor cells express Coinjection of ␤-galactosidase-encoding mRNA indi- Pdx1, a homeodomain protein that is also expressed cates that the injected RNA is evenly distributed in the adjacent presumptive stomach and duodenum throughout the entire endoderm at tadpole stages (Sup- (Ohlsson et al. 1993; Jonsson et al. 1994), as well as plementary Fig. 1). As a result, we observed ectopic ex- Ptf1a/p48, a basic helix–loop–helix (bHLH) transcription pression of exocrine pancreatic differentiation markers, factor (Kawaguchi et al. 2002). Lineage tracing studies such as pancreatic protein disulphide isomerase (XPDIp) have indicated that the Pdx1-expressing cells represent and elastase (Fig. 2A, panels 3, 6); other exocrine marker precursor cells that contribute to the formation of both genes like amylase, carboxypeptidase, and trypsinogen endocrine and exocrine lineages of the mature pancreas showed similar effects (data not shown). Embryos that (Gu et al. 2002). In Pdx1-homozygous mutant mice, pan- were injected with the XPtf1a/p48GR mRNA, but not creatic buds seem to be arrested in their development; treated with dexamethasone, showed no such effect, and however, early glucagon- and insulin-expressing cells they were indistinguishable from uninjected embryos can still be detected (Ahlgren et al. 1996; Offield et al. treated with dexamethasone (Fig. 2A, panels 2, 5). Strik- 1996). Such mice exhibit additional malformations in ingly, the ectopic expression of exocrine pancreatic stomach and duodenum, where Pdx1 is normally ex- genes is specifically restricted to the territory of prospec- pressed. Interestingly, and of primary relevance for the tive stomach and duodenum, adjacent to the developing pancreas. Insulin expression is first detected exclusively in the [Keywords: Xenopus laevis; pancreas development; Ptf1a/p48; trans- dorsal pancreas by stage 32, representing the only endo- specification] crine differentiation gene expressed before stage 45 1Corresponding author. E-MAIL [email protected]; FAX 0049-551-3914614. (Kelly and Melton 2000; Horb and Slack 2002). Insulin Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.378706. expression remained unaffected in embryos overexpress- GENES & DEVELOPMENT 20:1441–1446 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org 1441 Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Afelik et al. to convert nonpancreatic into pancreatic endoderm. Coinjection of XPtf1a/p48GR with XlHbox8GR results in an induction of exocrine-specific differentiation genes also outside of stomach and duodenal regions, now ex- panding into the more posterior portions of the endo- derm (Fig. 2B, panel 3). The occurrence of several discrete foci of ectopic pancreatic gene expression provides a fur- ther argument for fate conversion rather than expansion of a primary pancreatic territory by increased prolifera- tion being responsible for the effects observed. The ex- pression of insulin in the dorsal pancreas of early (stage 36) embryos remained again unaffected (Fig. 2B, panel 6). In all cases of ectopic pancreatic gene induction, the on- set of expression in the ectopic domain followed the nor- mal temporal pattern, as observed in uninjected control embryos (data not shown). Figure 1. Whole-mount in situ hybridization analysis of XPtf1a/ p48 expression during Xenopus embryogenesis and in comparison to XlHbox8.(A) Dorsal view of a stage 20 embryo, anterior toward the left. XPtf1a/p48 transcripts (blue) are detected along two parallel longitudinal stripes representing the neural folds. (B) Double-stain- ing in situ hybridization of tailbud-stage (stage 26) embryos using En2 (red) as a midbrain–hindbrain boundary marker (white arrow- head) and Krox20 (red) as a marker for rhombomeres 3 and 5 (black arrowheads). Neural expression of XPtf1a/p48 becomes restricted to the hindbrain, with the anterior end defined by the midbrain–hind- brain boundary and the posterior limit by rhombomere 5. XPtf1a/ p48 transcripts are also becoming detectable in the developing retina. During later phases of development, retinal expression is confined to the proliferating precursor cells of the ciliary marginal zone and expression in the neural tube to dorsal elements (shown in E,F). (C,D) Lateral view of stage 28 and stage 35 embryos stained for XPtf1a/p48 expression. (E–G) Transverse sections (S1, S2, S3) of a stage 35 embryo at the levels indicated in D, dorsal to the top. (H) Lateral view. (I) Ventral view. (J,K) Lateral and ventral view of XlHbox8 expression. (duo) Duodenum; (dp) dorsal pancreatic bud; (st) stomach; (vp) ventral pancreatic buds. ing XPtf1a/p48GR (Fig. 2A, panel 9). Similarly, examina- tion of embryos at stages 41 and 43 showed no obvious effect on insulin expression (data not shown). In normal embryos, the Forkhead transcription factor Foxa1 is expressed in the stomach and duodenum but excluding the pancreas, as well as in the proctodaeum (Fig. 2A, panel 10). Upon ectopic activation of XPtf1a/ p48 in the endoderm, expression of Foxa1 is lost specifi- Figure 2. A combination of XPtf1a/p48 and XlHbox8 induces ec- cally at the site of ectopic pancreatic gene expression, topic pancreatic differentiation. (A) Whole-mount in situ hybridiza- whereas its expression in the proctodaeum remains un- tion analysis of a panel of markers reveals that ectopic expression of affected (Fig. 2A, panel 12). These findings suggest adap- XPtf1a/p48 converts part of the presumptive stomach and duode- num into a pancreatic fate. (Panels 1–3) Lateral view. Ninety-three tation of a pancreatic fate with a concomitant loss of percent of the embryos examined showed effects as in panel 3 duodenal and stomach identities within the XlHbox8 (n = 62). (Panels 4–6) Ventral view. Phenotype in panel 6, 87%, positive territory,
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