Lab Anim Res.  2015 Jun;31(2):93-98. 10.5625/lar.2015.31.2.93.

Expression of pancreatic and duodenal homeobox1 (PDX1) protein in the interior and exterior regions of the intestine, revealed by development and analysis of Pdx1 knockout mice

• Hashimoto
• Kamisako T
• Kagawa T
• Haraguchi
• Yagoto M
• Takahashi
• Kawai K
• Suemizu H

• KMID: 2053683
• DOI: http://doi.org/10.5625/lar.2015.31.2.93

Abstract

We developed pancreatic and duodenal homeobox1 (Pdx1) knockout mice to improve a compensatory hyperinsulinemia, which was induced by hyperplasia in the beta cells or Langerhans' islands, as the diabetic model mice. For targeting of Pdx1 gene by homologous recombination, ES cells derived from a 129(+Ter)/SvJclxC57BL/6JJcl hybrid mouse were electroporated and subjected to positive-negative selection with hygromycin B and ganciclovir. As these results, one of the three chimeric mice succeeded to produce the next or F1 generation. Then, the mouse fetuses were extracted from the mother's uterus and analyzed immunohistologically for the existence of a pancreas. The fetuses were analyzed at embryonic day 14.5 (E14.5) because Pdx1 knockout could not alive after birth in this study. Immunohistochemical staining revealed that 10 fetuses out of 26 did not have any PDX1 positive primordium of the pancreas and that the PDX1 expresses in both the interior and exterior regions of intestine. In particular, one the exterior of the intestine PDX1 was expressed in glands that would be expected to form the pancreas. The result of PCR genotyping with extracted DNA from the paraffin sections showed existence of 10 Pdx1-knockout mice and corresponded to results of immunostaining. Thus, we succeeded to establish a Pdx1-knockout (Pdx1-/-) mice.

Keyword

Pdx1 gene; knockout mice; pancreas

MeSH Terms

Animals
DNA
Fetus
Ganciclovir
Homologous Recombination
Hygromycin B
Hyperinsulinism
Hyperplasia
Intestines*
Islands
Mice
Mice, Knockout*
Pancreas
Paraffin
Parturition
Polymerase Chain Reaction
Uterus
DNA
Ganciclovir
Hygromycin B
Paraffin

Figure

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  Figure 1 Targeting of Pdx1 gene. (A) The Pdx1 gene and targeting starategy. The gray bold bars indicate the position of the probe for genomic Southern blot analysis using ClaI- and EcoRV-digested samples (mutated allele, 7,604 bp; wild-type allele, uncut (Probe 1) or none (Probe 2)). Genomic DNA from ES cells was digested with Cla I and Eco RV, and subjected to hybridization with the probe-1 (B) and probe-2 (C). Lane 1: DIG size marker, lane 2: ES-No.1, lane 3: ES-No. 9, lane 4: ES-No.17, lane 5: ES-No.53, lane 6: ES-No.59, lane 7: ES-No.87, lane 8: ES-No.88, lane 9: ES-No.2, lane 10: ES-No.4, lane 11: ES-No.159. Each number indicates ES clone No. M: size marker. (D) Chromosome banding of ES-No. 53. (E) Chimeric mice generated from ES-No. 53. (F) Genomic DNA from F1 mice was digested with Cla I and Eco RV, and subjected to hybridization with the probe-1. Lane 1: DIG size marker, lane 2: female Pdx1+/+ mouse , lane 3: female Pdx1+/- mouse, lane 4: male Pdx1+/+ mouse, lane 5: male Pdx1+/- mouse.

• 

  Figure 2 Analysis of Pdx1 gene and PDX1 protein expression at E14.5 embryos.(A) Genotyping of E14.5 embryos by PCR. DNA was extracted from paraffin-embedded tissues. (B-O) Immunohistochemical and HE staining of pancreatic primordium region at E14.5 embryos. Paraffin-embedded sections of Pdx1+/-, Pdx1+/+, and Pdx1-/- embryos were stained with anti-PDX1. D, E and N, O were expanded from B and L, respectively. Black bars represent 100 µm (B, C, L and M), 200 µm (D, E, N and O), and 400 µm(F, G, H, I, J, and K).

Reference

1. Wright CV, Schnegelsberg P, De Robertis EM. XlHbox 8: a novel Xenopus homeo protein restricted to a narrow band of endoderm. Development. 1989; 105(4):787–794. PMID: 2574662.

2. Ohlsson H, Karlsson K, Edlund T. IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J. 1993; 12(11):4251–4259. PMID: 7901001.

3. Leonard J, Peers B, Johnson T, Ferreri K, Lee S, Montminy MR. Characterization of somatostatin transactivating factor-1, a novel homeobox factor that stimulates somatostatin expression in pancreatic islet cells. Mol Endocrinol. 1993; 7(10):1275–1283. PMID: 7505393.

4. Miller CP, McGehee RE Jr, Habener JF. IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene. EMBO J. 1994; 13(5):1145–1156. PMID: 7907546.

5. Guz Y, Montminy MR, Stein R, Leonard J, Gamer LW, Wright CV, Teitelman G. Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny. Development. 1995; 121(1):11–18. PMID: 7867492.

6. Peshavaria M, Gamer L, Henderson E, Teitelman G, Wright CV, Stein R. XIHbox 8, an endoderm-specific Xenopus homeodomain protein, is closely related to a mammalian insulin gene transcription factor. Mol Endocrinol. 1994; 8(6):806–816. PMID: 7935494.

7. Hashimoto H, Arai T, Takeguchi A, Hioki K, Ohnishi Y, Kawai K, Ito M, Suzuki R, Yamauchi T, Ohsugi M, Saito M, Ueyama Y, Tobe K, Kadowaki T, Tamaoki N, Kosaka K. Ontogenetic characteristics of enzyme activities and plasma metabolites in C57BL/6J:Jcl mice deficient in insulin receptor substrate 2. Comp Med. 2006; 56(3):176–187. PMID: 16774126.

8. Hashimoto H. Study on establishment of congenic strains and screening of characteristics in IRS-2 deficient mice to support translational research on type 2 diabetes. Exp Anim. 2011; 60(1):21–32. PMID: 21325749.

9. Hashimoto H, Arai T, Mori A, Kawai K, Hikishima K, Ohnishi Y, Eto T, Ito M, Hioki K, Suzuki R, Ohsugi M, Saito M, Ueyama Y, Okano H, Yamauchi T, Kubota N, Ueki K, Tobe K, Tamaoki N, Kadowaki T, Kosaka K. Reconsideration of insulin signals induced by improved laboratory animal diets, Japanese and American diets, in IRS-2 deficient mice. Exp Clin Endocrinol Diabetes. 2009; 117(10):577–586. PMID: 19629935.

10. Terauchi Y, Takamoto I, Kubota N, Matsui J, Suzuki R, Komeda K, Hara A, Toyoda Y, Miwa I, Aizawa S, Tsutsumi S, Tsubamoto Y, Hashimoto S, Eto K, Nakamura A, Noda M, Tobe K, Aburatani H, Nagai R, Kadowaki T. Glucokinase and IRS-2 are required for compensatory beta cell hyperplasia in response to high-fat diet-induced insulin resistance. J Clin Invest. 2007; 117(1):246–257. PMID: 17200721.

11. Fukushima M, Suzuki H, Seino Y. Insulin secretion capacity in the development from normal glucose tolerance to type 2 diabetes. Diabetes Res Clin Pract. 2004; 66(Suppl 1):S37–S43. PMID: 15563978.

12. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes. 2003; 52(1):102–110. PMID: 12502499.

13. Brissova M, Shiota M, Nicholson WE, Gannon M, Knobel SM, Piston DW, Wright CV, Powers AC. Reduction in pancreatic transcription factor PDX-1 impairs glucose-stimulated insulin secretion. J Biol Chem. 2002; 277(13):11225–11232. PMID: 11781323.

14. Kobayashi T, Yamaguchi T, Hamanaka S, Kato-Itoh M, Yamazaki Y, Ibata M, Sato H, Lee YS, Usui J, Knisely AS, Hirabayashi M, Nakauchi H. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell. 2010; 142(5):787–799. PMID: 20813264.

15. Jonsson J, Carlsson L, Edlund T, Edlund H. Insulin-promoter-factor 1 is required for pancreas development in mice. Nature. 1994; 371(6498):606–609. PMID: 7935793.

16. Sugawara A, Goto K, Sotomaru Y, Sofuni T, Ito T. Current status of chromosomal abnormalities in mouse embryonic stem cell lines used in Japan. Comp Med. 2006; 56(1):31–34. PMID: 16521857.

17. Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan BL, Wright CV. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development. 1996; 122(3):983–995. PMID: 8631275.

18. Ahlgren U, Jonsson J, Edlund H. The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development. 1996; 122(5):1409–1416. PMID: 8625829.

19. Kanai-Azuma M, Kanai Y, Gad JM, Tajima Y, Taya C, Kurohmaru M, Sanai Y, Yonekawa H, Yazaki K, Tam PP, Hayashi Y. Depletion of definitive gut endoderm in Sox17-null mutant mice. Development. 2002; 129(10):2367–2379. PMID: 11973269.

20. Takahashi M, Ikemoto S, Ezaki O. Effect of the fat/carbohydrate ratio in the diet on obesity and oral glucose tolerance in C57BL/6J mice. J Nutr Sci Vitaminol (Tokyo). 1999; 45(5):583–593. PMID: 10683810.

21. Matschinsky FM. Banting Lecture 1995. A lesson in metabolic regulation inspired by the glucokinase glucose sensor paradigm. Diabetes. 1996; 45(2):223–241. PMID: 8549869.

22. Kulkarni RN, Jhala US, Winnay JN, Krajewski S, Montminy M, Kahn CR. PDX-1 haploinsufficiency limits the compensatory islet hyperplasia that occurs in response to insulin resistance. J Clin Invest. 2004; 114(6):828–836. PMID: 15372107.

23. Okada T, Liew CW, Hu J, Hinault C, Michael MD, Krtzfeldt J, Yin C, Holzenberger M, Stoffel M, Kulkarni RN. Insulin receptors in beta-cells are critical for islet compensatory growth response to insulin resistance. Proc Natl Acad Sci U S A. 2007; 104(21):8977–8982. PMID: 17416680.