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Ectopic expression of Ptf1a and downregulation of Cdx2 and its downstream targets.

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posted on 2013-02-22, 11:26 authored by Kei Semba, Kimi Araki, Ken-ichirou Matsumoto, Hiroko Suda, Takashi Ando, Akira Sei, Hiroshi Mizuta, Katsumasa Takagi, Mai Nakahara, Mayumi Muta, Gen Yamada, Naomi Nakagata, Aritoshi Iida, Shiro Ikegawa, Yusuke Nakamura, Masatake Araki, Kuniya Abe, Ken-ichi Yamamura

A. Strategy for generating ETn-Gm13336-Ptf1alacZ mice. The PGK-neo gene was replaced with the lacZ gene in the ETn-Gm13336-Ptf1aneo allele using Cre-mediated recombination. B. Whole-mount X-gal staining of ETn-Gm13336-Ptf1alacZ embryos. Upper panel: wild-type (WT) littermates. Lower panel: ETn-Gm13336Ptf1alacZ embryos. LacZ expression was detected in the notochord and hindgut at E8.5 to E9.5. Then, lacZ expression extended to the cloaca and mesonephros at E10.5 and to the pancreatic bud at E10.5 and E11.5. C. Ectopic lacZ expression. LacZ expression was strongly detected in the notochord (red arrow), mesonephros (blue arrow), and cloaca (yellow arrow) by histological analysis with X-gal staining at E9.5. The areas shown at higher magnification in the lower panels are indicated by boxes. Bars: 500 µm. D. Quantitative RT-PCR analyses in E9.5+/+, Ptf1a/+, and Ptf1a/Ptf1a littermates and in Sd/Sd embryos with a C57BL/6 genetic background. Although the expression of Ptf1a in ETn-Gm13336-Ptf1aPtf1a/+-+ and homozygous ETn-Gm13336-Ptf1aPtf1a/ETn-Gm13336-Ptf1aPtf1a mice was slightly lower than that in Sd/Sd mice, it was much higher than that in WT mice. The expression levels of Cdx2 and T in ETn-Gm13336-Ptf1aPtf1a/ETn-Gm13336-Ptf1aPtf1a mice were similar to those in Sd/Sd mice, but significantly lower than those in WT mice. The expression of Wnt3a and Cyp26a1 was decreased in ETn-Gm13336-Ptf1aPtf1a/ETn-Gm13336-Ptf1aPtf1a mice, but not in Sd/Sd mice. The data represent the mean ± SD of independent whole embryos (+/+: n = 8, Ptf1a/+: n = 4, Ptf1a/Ptf1a: n = 6, Sd/Sd: n = 3–10). *p<0.05; ** p<0.01. E. Model of Ptf1a action. The ETn insertion induces ectopic expression of Ptf1a, resulting in downregulation of Cdx2 and its downstream targets T, Wnt3a, and Cyp26a1. This combined deficiency of gene expression causes the Sd phenotype.

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