Oropeza D , Horb M .

DK077197 NIDDK NIH HHS , R01 DK077197 NIDDK NIH HHS , R01 DK077197-05 NIDDK NIH HHS , P40 OD010997 NIH HHS

Xla Wt + Mmu.neurog3-GR + DEX (Fig 3b)
Xla Wt + Mmu.neurog3-GR + DEX (Fig 3d)
Xla Wt + neurog3-GR + DEX (Fig 1. b)
Xla Wt + neurog3-GR + DEX (Fig 1. c)
Xla Wt + neurog3-GR + DEX (Fig 1. e)
Xla Wt + neurog3-GR + DEX (Fig 1. h)
Xla Wt + neurog3-GR + DEX (Fig 2 b)
Xla Wt + neurog3-GR + DEX (Fig 2 e)
Xla Wt + neurog3-GR + DEX (Fig 2 h)
Xla Wt + neurog3-GR + DEX (Fig 4 e)
Xla Wt + neurog3-GR + DEX (Fig 4 f)
Xla Wt + neurog3-GR + DEX (Fig 4 g)
Xla Wt + neurog3-GR + DEX (Fig 4 h)
Xla Wt + neurog3-GR + DEX (Fig 5 c)
Xla Wt + neurog3-GR + DEX (Fig 5 e)
Xla Wt + neurog3-GR + DEX (Fig 5 g)
Xla Wt + neurog3-GR + insm1 MO + DEX (Fig 6d)
Xla Wt + neurog3-GR + rfx6 MO + DEX (Fig 6h)

	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.
	Figure 2. Ngn3-GR activation affects anterior gut markers at Stage 44. (a) Exocrine pancreatic markers PDI and elastase are slightly decreased (n = 16/16 and n = 14/15, respectively). (e,f) Stomach marker cathepsin E is significantly reduced (n = 14/14). (g,h) Liver and duodenum marker hex is slightly decreased (n = 7/7). Pancreas is outlined in panels (c) and (d).
	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).
	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).
	Figure 6. Insm1 and Rfx6 are necessary for ectopic beta cell promotion by Ngn3-GR. In situ hybridizations for insulin expression in all panels. Ngn3-GR mRNA (1,800 pg) was coinjected with a control mismatch morpholino (mMO) or morpholinos targeting insm1 or rfx6 mRNA, activated with Dex for 4 h and fixed at Stage 32. (a,b) Insm1 mMO (20 ng) had no effect on endogenous insulin expression in either ex (n = 8/8) or +Dex (n = 12/17) treated embryos. (c,d) Injection of Insm1 MOs (2 morpholinos, 20 ng each) abolished endogenous insulin expression in ex embryos (n = 7/7) as well as ectopic insulin expression in +Dex embryos (n = 15/15). (e,f) Rfx6 mismatch morpholino (25 ng) did not affect endogenous or ectopic insulin expression in ex (n = 9/9) or +Dex embryos (n = 19/41). (g,h) Antisense Rfx6 morpholino (25 ng) inhibited endogenous insulin expression in −Dex embryos (n = 33/33) as well as ectopic insulin expression in +Dex embryos (n = 46/46).
	Figure 7. Summary of results and schematic diagram of microarray experiment. (A) Summary of ectopic expression of endocrine markers. Red boxes indicate time of Dex treatment. Activation of Ngn3-GR for 1 or 4 h beginning at Stage 12 resulted in increased insulin and somatostatin expression, whereas continuous activation beginning at Stage 12 or a 4-h activation beginning at Stage 15 resulted in increased expression of insulin, somatostatin, and glucagon. (b,c) Diagram of microarray experiment. Ngn3-GR mRNA was injected in the two dorsal vegetal blastomeres at the eight cell stage. Embryos were grown until Stage 12 activated with dexamethasone for 4 h and confirmed targeting to the anterior endoderm at Stage 15 with GFP fluorescence. All dorsal structures were removed and RNA was extracted immediately after. Four replicates of ten embryos were used to hybridize the Affymetrix Xenopus laevis GeneChip 2.0.
	Figure 8. Validation of microarray data. Following 4 h of Ngn3-GR activation at Stage 12 candidate genes from the microarray analysis were increased in the endoderm at Stage 15: (a,b) tbx2 (n = 24/24). (c,d) mtg8 (n = 21/21). (e,f) hes3 (n = 14/42). (g,h) geminin (n = 13/44). (i,j) oct25 (n = 13/13). And at Stage 20: (k,l) mtgr1 (n = 9/13). Dashed circles and arrows indicate ectopic expression in the anterior endoderm. Dashed line indicates ectopic expression in the dorsal endoderm.
	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).
	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.
	ctse (cathepsin E) gene expression in dissectd Xenopus laevis foregut, NF stage 44, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	runx1t1 (runt-related transcription factor 1; translocated to, 1 (cyclin D-related) ) gene expression in Xenopus laevis embryos, NF stage 15, as assayed by in situ hybridization. Bisected sagitally, lateral view: anterior left, dorsal up.
	Figure 5. Ngn3-GR promotes beta cell differentiation. (A) RT-PCR for insulin of whole embryos at Stages 24 and 26. (b,c) Increased pax4 expression detected in Dex treated embryos at Stage 32 (n = 15/17). (d,e) Increase in the mature beta cell marker, sur1, at Stage 32 (n = 19/31) in Dex treated embryos. In panel (d), arrow indicates nonendodermal expression of sur1. In panel (e), bracket denotes ectopic expression in endoderm. (f,g) Increase in somatostatin at Stage 32 (n = 12/27) in Dex treated embryos. In panel (F), arrow indicates expression of som outside the endoderm. In panel G, bracket denotes ectopic expression in endoderm. (h) RT-PCR at Stage 26 of whole endoderm (WE) and whole endoderm plus mesoderm explants (WEM). Explants were dissected at Stage 15 following 4 h of Ngn3-GR activation, cultured until Stage 26 and collected for RT-PCR. Twist and FoxF1 are mesoderm markers, which are detected only in the WEM explants.

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