XB-ART-44499Genesis March 1, 2012; 50 (3): 271-85.
Transient expression of Ngn3 in Xenopus endoderm promotes early and ectopic development of pancreatic beta and delta cells.
Promoting ectopic development of pancreatic beta cells from other cell types is one of the strategies being pursued for the treatment of diabetes. To achieve this, a detailed outline of the molecular lineage that operates in pancreatic progenitor cells to generate beta cells over other endocrine cell types is necessary. Here, we demonstrate that early transient expression of the endocrine progenitor bHLH protein Neurogenin 3 (Ngn3) favors the promotion of pancreatic beta and delta cell fates over an alpha cell fate, while later transient expression promotes ectopic development of all three endocrine cell fates. We found that short-term activation of Ngn3 in Xenopus laevis endoderm just after gastrulation was sufficient to promote both early and ectopic development of beta and delta cells. By examining gene expression changes 4 h after Ngn3 activation we identified several new downstream targets of Ngn3. We show that several of these are required for the promotion of ectopic beta cells by Ngn3 as well as for normal beta cell development. These results provide new detail regarding the Ngn3 transcriptional network operating in endocrine progenitor cells to specify a beta cell phenotype and should help define new approaches to promote ectopic development of beta cells for diabetes therapy.
PubMed ID: 22121111
PMC ID: PMC3294191
Article link: Genesis
Grant support: DK077197 NIDDK NIH HHS , R01 DK077197 NIDDK NIH HHS , R01 DK077197-05 NIDDK NIH HHS , P40 OD010997 NIH HHS
Genes referenced: abcc8 aif1l ankrd37 arl5b asb5 atoh7 bmp7.1 btg5 cbfa2t2 cbfa2t3 ccnb1 cer1 chn1 chst10 ctse dbn1 dbndd1 dlc dlc1 dll1 egln3 elavl3 esr10 fgf13 foxa4 foxf1 frzb gadd45g gcg gmnn gramd2b hba3 hes3.1 hes3.3 hes5.1 hes9.1 hey1 hhex hoxd1 igf2 ins insm1 irf6 kcnk5 loc100135144 mcoln2 mespb mospd1 mxi1 ndrg4 neurod4 neurog1 neurog3 nudt22 pax4 pdia2 pdk4 pgam1 pik3ip1 pou4f1 pou5f3.2 ppm1k ppp1r14a prag1 ptgs2 rfesd rflnb rfx6 rgs1 ripply2.1 runx1t1 sat1 slc16a3 slc3a2 slc7a8 sst stmn3 tbx2 tob1 tsc22d3 tspan8 twist1 twsg1 unc119b unc93a ventx1.1 zfand2a
Morpholinos referenced: cbfa2t2 MO1 cbfa2t3 MO1 insm1 MO1 insm1 MO2 rfx6 MO1 rfx6 MO2 runx1t1 MO1 tbx2 MO1
Disease Ontology references: diabetes mellitus
External Resources: GO Terms referenced: type B pancreatic cell development
Article Images: [+] show captions
|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.|