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Abstract
In mammals, it has been well established that gastrointestinal and pancreatic endocrine cells are specified by a cascade of different transcription factors, but whether these same pathways (or linear relationships) operate in Xenopus is currently unknown. We recently identified the endocrine-specific zinc finger transcription factor insulinoma associated protein 1 (insm1) as a dorsal-enriched gene. We found that insm1 is expressed in the dorsal pancreas as early as NF28, making it one of the earliest markers to be localized to the dorsal pancreas. Through morpholino-mediated knockdown, we demonstrate that insm1 is essential for proper specification of both gastrointestinal and pancreatic endocrine cells. In addition, we place insm1 downstream of ngn3 and upstream of pax6 and neuroD in the endocrine cell transcription factor cascade. These are the first results showing that the endodermal endocrine cell development in Xenopus uses the same transcriptional cascade as in mammals.
Figure 1. Expression pattern of insm1 in endoderm. A-D: Expression of insm1 in the dorsal endoderm at NF28, NF34, NF38, and NF39, respectively. Notice expression begins as a small point in the dorsal endoderm (arrowhead) and then is found in bilateral domains at NF34 (two arrows); by NF38 expression is now present in the dorsal midline in scattered cells (outlined). Shortly thereafter expression is now found displaced to the left side of the tadpole (outlined). E,F: Isolated liver/pancreastissue. Insm1 is detected at NF40 in a punctate manner throughout the gastrointestinal tract and in the dorsal pancreas, similar to that seen in the early endoderm. (The dotted line demarcates dorsal and ventral portions of the fused pancreas.) At later stages, expression was found throughout the entire pancreas, but still in a punctate manner. G,H: Whole guts from NF40 and NF44 tadpole. The pancreas is outlined. Expression is found in a punctate manner throughout the entire gastrointestinal tract. Dp, dorsal pancreas; vp, ventralpancreas.
Figure 2. Insm1 is required for endocrine cell differentiation. A-D: Insulin expression at both early (NF35; 90%, n = 15) and late stages (NF40/42; 79%, n = 52) is almost completely abolished. E-H: glucagon expression. I-L: somatostatin expression. At early stages expression of both gcg and som is reduced in the stomach/duodenum of insm1 knockdown animals (100%; n = 33 and n = 12, respectively), but begins to reappear at later stages (100%; n = 8 for both). The initial expression of these markers in the pancreas is also reduced at later stages (100% for gcg and 80% for som). M,N: Expression of the exocrine pancreas marker XPDIp is normal in insm1 morpholino injected tadpoles (100%, n = 10). O: In vitro transcription and translation of insm1 either alone (insm1), in the presence of the 5-bp mismatch morpholino (insm1+mMO) or in the presence of the morpholino (insm1+MO). Pictures presented in control are of uninjected embryos, but in all cases mismatch morpholino embryos (n ge 8 for each marker) gave identical results as control, with normal expression of these markers. The pancreas is outlined.
Figure 3. Expression of endocrine-specific transcription factors in insm1 knockdown whole guts. A,B: Ngn3 expression. Ngn3 is expressed in endocrine progenitors, and its expression is normal in insm1 morpholino injected samples (70%, n = 24). C,D: NeuroD expression. E,F: Pax6 expression. Expression of neuroD and pax6 is absent both in the pancreas and the gut (100%, n = 16 for both). Pictures presented in control column are of uninjected embryos, but in all cases mismatch morpholino embryos (n ge 8 for each marker) gave identical results as control, with normal expression of these markers. The pancreas is outlined.
Figure 4. Development of anterior endodermal organs is unaffected. A,B: Hex gene expression is normal in insm1 morpholino injected samples (88%, n = 8). C,D: Frp5 expression is slightly reduced, but overall the stomach appears normal (66%, n = 25). E,F: Hnf6 expression. Hnf6 is expressed in the posteriorduodenum and the gall bladder. In insm1 knockdown tadpoles, expression in the gall bladder is normal, while its expression in the gut is absent (72%, n = 18). Pa, pancreas; Li, liver; GB, gall bladder. Pictures presented in control column are of uninjected embryos, but in all cases mismatch morpholino embryos (n ge 8 for each marker) gave identical results as control, with normal expression of these markers. The pancreas is outlined.
Figure 5. Initial specification of the anteriorendoderm is normal. A,B: Expression of ptf1a in the developing dorsal and ventral pancreatic buds was normal in insm1 knockdown tadpoles at NF35 (100%, n = 10). C,D: Expression of pdx1 in the pancreas and duodenum was unaffected (95%, n = 20). E,F: Initial expression of hnf6 in the anteriorendoderm was also unaffected by the knockdown on insm1 (100%, n = 8). Pictures presented in control column are of uninjected embryos, but in all cases mismatch morpholino embryos (n = 15 for each marker) gave identical results as control, with normal expression of these markers.
onecut1 (one cut homeobox 1 )gene expression in Xenopus tropicalis embryos, NF stage 36, as assayed by in situ hybridization. Lateral view: anteriorleft, dorsal up.
sst (somatostatin)gene expression in dissectd Xenopus tropicalis foregut, NF stage 36, as assayed by in situ hybridization. Lateral view: anteriorleft, dorsal up.
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