As outlined above, the majority of research into Xenopus pancreas development has been focused on the early patterning and specification events that occur prior to NF30. The fact that Xenopus can be used to investigate the function of “late-acting” pancreas-specific genes has only recently become appreciated. From these studies, it is becoming clear that Xenopus is a valuable model system for elucidating the molecular mechanisms underlying pancreatic cell fate specification; including identifying new genes, determining their function, defining transcriptional regulatory networks, and more specifically identifying candidate genes for diabetes.
Ptf1a and Pdx1
Two important genes in pancreas development that have been studied in Xenopus include Pdx1 and Ptf1a. Both genes are expressed in pancreatic progenitors, and are necessary and sufficient for pancreas development (Horb et al.,2003; Afelik et al.,2006; Jarikji et al.,2007). In fact, ectopic expression of both simultaneously converts posterior endoderm into pancreatic tissue (Afelik et al.,2006). The bHLH pancreas-specific transcription factor 1a (PTF1a) is one of the earliest pancreas-specific markers. Loss of Ptf1a function in mice and humans results in the complete absence of acinar cells and a large reduction in insulin expression (Krapp et al.,1996; Kawaguchi et al.,2002; Sellick et al.,2004), whereas in zebrafish it is required mainly for exocrine pancreas development (Zecchin et al.,2004; Lin et al.,2004). In Xenopus, ptf1a is detected as early as NF27/30 in the developing dorsal and ventral pancreatic anlagen (Fig. 2A) (Afelik et al.,2006). It is expressed in the tadpole acinar cells until the climax of metamorphosis when the PTF1a gene is shut off. Its expression returns at the end of metamorphic climax and remains acinar-specific (Mukhi et al.,2008). This expression is identical to that seen in mice, where Ptf1a is expressed in all pancreatic progenitors at the earliest stages of pancreas development, but becomes localized to acinar cells shortly thereafter (Krapp et al.,1996; Kawaguchi et al.,2002).
Two studies examined the function of Xenopus Ptf1a in early pancreas development and found that Ptf1a was both necessary and sufficient for pancreas development. Both studies found that knockdown of Ptf1a resulted in a complete loss of acinar cells; however, phenotypic differences regarding endocrine cell development were found (Afelik et al.,2006; Jarikji et al.,2007). In the first study, a single morpholino was used to knockdown Ptf1a, and early expression of insulin at NF35 was unaffected, while the late expression of both insulin and glucagon was lost at NF48 (Afelik et al.,2006). The second study, using two morpholinos to target distinct regions of the Ptf1a transcript, found the exact opposite: knockdown of Ptf1a resulted in an absence of early insulin expression, while low levels of insulin were detected at later stages (Jarikji et al.,2007). The reason for this discrepancy is currently unclear. Nevertheless, these studies show that the function of Ptf1a in Xenopus pancreas development is quite similar to that observed in other vertebrates where Ptf1a is required for development of all acinar cells and a subset of endocrine cells.
The homeobox gene Pdx1 was originally cloned in Xenopus 20 years ago and called XlHbox8 (Wright et al.,1989). It is expressed in a broad domain in the anterior endoderm beginning at NF27, encompassing the developing dorsal and ventral pancreatic buds as well as parts of the stomach and duodenum (Wright et al.,1989; Afelik et al.,2006). Low levels of Pdx1 are detected by RNase protection analysis as early as NF12.5, and by RT-PCR at NF19/21 (Gamer and Wright,1995; Horb and Slack,2001). Morpholino knockdown of Pdx1 in Xenopus resulted in the complete loss of exocrine markers, but had little effect on insulin expression (Afelik et al.,2006). These results are in contrast to the defects observed in mice and humans, where lack of Pdx1 resulted in pancreas agenesis (Ohlsson et al.,1993; Jonsson et al.,1994; Stoffers et al.,1997; Schwitzgebel et al.,2003). The defect seen in Xenopus is similar to that observed in mice where although loss of Pdx1 leads to pancreatic agenesis, there is a small dorsal bud present that produces insulin and glucagon (Ahlgren et al.,1996).
One of the benefits of Xenopus is the relative ease in which gain-of-function studies can be performed. In addition to these loss-of-function studies showing that Ptf1a and Pdx1 are necessary for pancreas development, ectopic overexpression experiments revealed that both were also sufficient to promote ectopic pancreatic cell fates. Overexpression of Pdx1 in naïve endoderm or transgenic overexpression of Pdx1 in the liver after NF44 did not result in the activation of pancreatic differentiation markers (Horb et al.,2003; Afelik et al.,2006). However, it should be noted that some studies in mammals have found that Pdx1 alone is sufficient to promote ectopic pancreatic cell fates (Ferber et al.,2000; Ber et al.,2003; Sapir et al.,2005; Shternhall-Ron et al.,2007). In contrast, overexpression of Pdx1-VP16, an activated form of Pdx1, was sufficient to promote ectopic endocrine and acinar cell fates in the developing liver; no effect was seen in the intestine (Horb et al.,2003). The relevance to mammals was shown by the fact that this same transgene was able to convert mammalian HepG2 cells to pancreas (Horb et al.,2003; Li et al.,2005). Subsequent studies have confirmed these results. In particular, Pdx1-VP16 was found sufficient to convert rat liver cells into immature beta cells that were able to restore euglycemia in diabetic mice (Cao et al.,2004; Tang et al.,2006). The reasons for this difference between Pdx1 and Pdx1-VP16 are unclear, although it is reasonable to assume that the addition of the VP16 activation domain allows for interactions with other proteins creating a favorable environment in which Pdx1 can activate transcription of its downstream targets.
Overexpression of Ptf1a, on the other hand, was found sufficient to promote ectopic pancreatic cell fates. Injection of Ptf1a mRNA into the two dorso-vegetal blastomeres at the eight-cell stage (targeting the anterior endoderm) resulted in the conversion of stomach/duodenum to pancreas (Afelik et al.,2006; Jarikji et al.,2007). Using a hormone-inducible version (Ptf1a-GR), it was found that when Ptf1a was activated at NF27 or earlier, it was able to promote ectopic pancreatic cell fates, whereas when activated after NF36, it had no effect (Afelik et al.,2006). Activation of Ptf1a between NF30 and 35 did not convert stomach/duodenum to pancreas, but did result in an enlarged pancreas. In contrast, Ptf1a-VP16 was found to have greater activity than the unmodified Ptf1a in naive endoderm. In addition to being able to convert prospective stomach/duodenum to pancreas as seen with unmodified Ptf1a, Ptf1a-VP16 was also able to convert prospective liver to pancreas. However, only the acinar cell fate was promoted (Jarikji et al.,2007). No effect was seen with either Ptf1a or Ptf1a-VP16 when overexpressed in posterior endoderm. Similar results were also found when Ptf1a or Ptf1a-VP16 was ectopically expressed at later stages after the organs had formed. Transgenic overexpression of Ptf1a-VP16 in Xenopus tadpoles was sufficient to convert liver to acinar cells (but had no effect in the stomach or duodenum), while Ptf1a converted stomach/duodenum to endocrine and acinar cell fates (but had no effect in liver) (Jarikji et al.,2007). These results confirm earlier data in mice where it was shown that loss of Hes1 induces ectopic expression of Ptf1a in the stomach, duodenum, and common bile duct resulting in the production of ectopic patches of pancreatic tissue (Fukuda et al.,2006).