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Figure 4. Ectopic and early induction of beta cells by Ngn3-GR. Insulin expression in tadpoles that have been sectioned through the dorsal pancreas, with the head removed. (a) Control injected embryos. Endogenous insulin expression is first detected at Stage 32 in a small dorsal domain. Endogenous insulin expression in dorsal pancreas (dp) is labeled by arrow in panel (c). (e) Ngn3-GR injected embryos treated with Dex for 4 h at Stage 12 showed increased insulin expression at Stage 28 (n = 14/18), Stage 30 (n = 24/30), and Stage 32 (n = 46/56). (h) Side view of Stage 34 tadpole treated with Dex showing the extent of ectopic insulin expression (line). |
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Figure 9. Tbx2, Mtg8, Mtg16, and Mtgr1 function is required for ectopic and endogenous beta cell development at Stage 32. Tbx2 or Mtg morpholinos (40 ng each) were injected alone or with Ngn3-GR mRNA (1,800 pg) and activated with Dex for 4 h at Stage 12 and the embryos grown to Stage 32 and analyzed for insulin expression. (a) Injection of Mtg8 morpholino blocked ectopic insulin expression by Ngn3-GR (n = 20/27). Mtg8 morpholino alone inhibited endogenous insulin expression (n = 40/77). (e) Mtg16 morpholino blocked ectopic insulin expression by Ngn3-GR (n = 16/20). Mtg16 morpholino alone inhibited endogenous insulin expression (n = 27/52). (i) Mtgr1 morpholino blocked ectopic insulin expression by Ngn3-GR (n = 28/28). Mtgr1 morpholino alone did not inhibit endogenous insulin expression (n = 46/59). (m) Tbx2 morpholino blocked ectopic insulin expression by Ngn3-GR (n = 27/27). Tbx2 morpholino alone inhibited endogenous insulin expression (n = 20/37). |
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Figure 10. Knockdown of Tm4sf3 does not affect Ngn3 promotion of ectopic insulin expression. Ngn3-GR was injected alone or in combination with Tm4sf3 morpholino. Insulin expression at Stage 32 in (a) control tadpoles; (b) +dex treated tadpoles, and (c) in tadpoles injected with Tm4sf3 morpholino. (d) Insulin expression at Stage 44. |
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Figure 1. Differential effects of Ngn3-GR temporal activation. Whole mount in situ hybridization of Stage 44 whole guts from embryos injected with 1,800 pg of XenopusNgn3-GR mRNA at the eight-cell stage. (a,d,g) Control embryos injected with Ngn3-GR mRNA but not treated with dex. (b,e,h) Dex treatment from Stages 12 to 44. Increased expression of glucagon (n = 14/20), insulin (n = 23/23), and somatostatin (n = 10/10). Arrowheads indicate ectopic insulin expression in the liver, stomach, and duodenum. (c,f,i) Dex treatment for 4 h from Stages 12 to 15. Increased insulin (n = 55/55) and somatostatin (n = 22/22) but not glucagon (n = 24/24). (j,k) Schematics illustrating organs in the whole gut of panels a and b (blue-insulin). (l) Insulin expression was detected in rare instances (n = 2/50) in posterior areas of the intestine. Pa: pancreas. |
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Figure 3. Transient activation of mouse Ngn3-GR and human Ngn3-GR also promoted increased insulin expression. (a) Embryos injected with 15 pg of mouse Ngn3-GR and activated with dexamethasone for 4 h at Stage 12 showed increased insulin (n = 10/10) and somatostatin (n = 7/7) expression. (e,f) Injection of 1,800 pg of human Ngn3-GR also caused increased insulin expression (n = 8/8). Pancreas is outlined in panels (a) and (c). |
