Gut specific expression using mammalian promoters in transgenic Xenopus laevis.
The recent development of transgenic methods for the frog Xenopus laevis provides the opportunity to study later developmental events, such as organogenesis, at the molecular level. Our studies have focused on the development of the tadpole gut, where tissue specific promoters have yet to be identified. We have used mammalian promoters, for the genes elastase, pancreatic duodenal homeobox-1, transthyretin, and intestinal fatty acid binding protein to drive green fluorescent protein expression in live tadpoles. All of these were shown to drive appropriate tissue specific expression, suggesting that the molecular mechanisms organising the gut are similar in amphibians and mammals. Furthermore, expression from the elastase promoter is initiated in the pancreatic buds before morphological definition becomes possible, making it a powerful tool for the study of pancreatic determination.
PubMed ID: 10534620
Article link: Mech Dev.
Grant support: Wellcome Trust
Genes referenced: fabp2 pdx1 ttr unnamed
Article Images: [+] show captions
|Fig. 1. Xenopus tadpoles transgenic for elastase-GFP. (a,b) GFP reporter activity in live tadpoles. Green fluorescence shows where the reporter is active, and yellow colour is due to yolk autofluorescence. (a) Four-day-old tadpole from right hand side, showing GFP limited to the pancreas (arrows). Anterior is to the right. (b) Ventral view of 7-day tadpole with GFP in the pancreas (arrow), anterior is to the right. (c,d) In situ hybridisations to show distribution of GFP mRNA (dark blue) in transgenic embryos, anterior is to the left. (c) Stage 31 embryo, cleared, to show onset of expression in the future dorsal pancreatic bud (d). (d) Stage 33 embryo, cleared, to show onset of expression in the future ventral pancreatic bud (v). Note the close contact between the dorsal bud (d) and the overlying notochord (nc). (e,f), In situ hybridisation to show distribution of GFP mRNA (dark blue) in dissected gut whole mounts. Anterior is to the left. (e) Stage 37 gut, viewed from the left side, to show expression is restricted to the dorsal (d) and ventral (v) pancreatic buds. (f) Expression in the pancreas after fusion of the buds, at 7 days of development, to show persistence of elastase promoter driven expression in all cells of the pancreas (pa). (g,h) Schematic diagrams showing the development and fusion of the pancreatic buds in Xenopus embryos based on elastase promoter studies. (g) At stage 37 the dorsal and ventral buds begin to move from the midline, the dorsal pancreas moves to the left and the ventral pancreas to the right. Both buds also begin to extend towards each other, pushing inwards between the stomach and duodenal endoderm. (h) At 3 days of development (stage 41) the pancreatic buds have fused. Subsequent gut coiling then moves the pancreas first to the left side of the embryo, and then to the right side, where it remains from 4 days onwards (not shown). Abbreviations: pa, pancreas; d, dorsal pancreatic bud; v, ventral pancreatic bud; li, liver; st, stomach; si, small intestine; nc, notochord; i, intestine; ph, pharynx.|
|Fig. 2. Xenopus tadpoles transgenic for PDX-1-GFP. (a,b) GFP reporter activity in live tadpoles. Green fluorescence shows where the reporter is active, and yellow colour is due to yolk autofluorescence. (a) Three-day-old tadpole from left hand side showing GFP in the pancreatic and duodenal endoderm (black arrow) and lens of the eye (white arrow). Some of the melanocytes, which partially obscure the gut at this stage, have been removed. Anterior is to the left. (b) Four-day-old tadpole viewed from the right hand side to show GFP in the pancreas (pa) but not in the surrounding gut coils. Anterior is to the right. (c) In situ hybridisation to show GFP mRNA in pancreatic buds and duodenum of 3-day-old transgenics (dark blue). View from the left, melanocytes covering the gut have been removed to improve visibility. Anterior is to the left. (d) In situ hybridisation to detect GFP mRNA in whole guts taken from transgenic tadpoles. By 6 days, GFP expression is no longer visible in the pancreas (pa), but low levels of expression are maintained in the duodenum (du). Anterior is to the left. Abbreviations: pa, pancreas; li, liver; du, duodenum.|
|Fig. 3. Xenopus tadpoles transgenic for transthyretin-nGFP or IFABP-GFP. (a-c) Transthyretin nGFP transgenics. (a,b) GFP reporter activity (green) in live tadpoles, yolk autofluorescence is yellow, anterior is to the right. (a) Four-day tadpole, ventral view, GFP is present in the liver (arrow) but absent from the rest of the gut, which autofluoresces yellow. (b) Six-day tadpole, ventral/left view to show GFP in the liver (li). The gall bladder (gb), to the left of the liver, fluoresces bright yellow at this stage, as does the small intestine (si). (c) In situ hybridisation to GFP mRNA in whole mount guts from transgenic tadpoles. (c) GFP is expressed exclusively in the liver (dark blue) at 5 days, anterior view. (d±f) Xenopus tadpoles transgenic for IFABP-GFP. (d,e) GFP reporter activity (green) in the coils of the small intestine of live 5-day (d) and 7-day (e) tadpoles, both ventral views, anterior to the left. Gall bladder (gb) autofluoresces yellow. (f) In situ hybridisation to GFP mRNA in 7 day whole mount gut. Expression of the reporter is seen in the small intestine (si, dark blue) but absent from the stomach (st), pancreas (pa), liver (li), gall bladder (gb) and colon (co). Abbreviations: gb, gall bladder; li, liver; si, small intestine; pa, pancreas; st, stomach; co, colon.|