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First published online October 10, 2008
doi: 10.1242/10.1242/dev.027144

Development 135, 3511-3519 (2008)
Published by The Company of Biologists 2008
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Functional importance of evolutionally conserved Tbx6 binding sites in the presomitic mesoderm-specific enhancer of Mesp2

Yukuto Yasuhiko1,*, Satoshi Kitajima1, Yu Takahashi1, Masayuki Oginuma2, Harumi Kagiwada3, Jun Kanno1 and Yumiko Saga2,*

1 Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
2 Division of Mammalian Development, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
3 Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974 Japan.


Figure 1
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  		Fig. 1. Disruption of Tbx6 binding sites eliminates Mesp2 expression. (A) Targeting strategy to generate the Mesp2 enhancer knockout mouse (P2EmB1D). A DNA fragment containing mutated Tbx6 binding sites (black ovals with X) was substituted for the wild-type sequence (white ovals) by homologous recombination. The PGK-neoR selection marker was removed by the Cre-loxP system to obtain a {Delta}neo allele. (B) PCR detection of homozygotes in the P2EmB1D intercross. (C) Impaired skeletal segmentation in the Mesp2 enhancer knockout mouse. The P2EmB1D/P2EmB1D mouse exhibits severe skeletal malformation at E17.5 (centre) identical to that of the Mesp2-null mouse (P2MCM/P2MCM, right). Note the shortened spine with incompletely segmented vertebrae (upper panels) and fused ribs (bracket in lower panels). (D) Expression of Mesp2 and the somite-specific genes Mesp1, Epha4 and Tbx18 in P2EmB1D/+ (left column) and P2EmB1D/P2EmB1D (right column) embryos. Mesp2 mRNA expression is eliminated in the P2EmB1D/P2EmB1D homozygotes. Wild-type (+/+) and heterozygote (P2EmB1D/+) embryos showed varying Mesp2 expression patterns owing to its cyclic expression. Mesp1 is upregulated and Epha4 is not affected, whereas Tbx18 is completely abolished in P2EmB1D/P2EmB1D.

 

Figure 2
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  		Fig. 2. Tbx6 binds to P2PSME in the PSM and tailbud. (A,B) Characterization of the anti-Tbx6 antibody produced in this study. (A) Whole-mount immunohistochemistry demonstrating the localization of Tbx6 protein in the mouse PSM and tailbud. (B) Western blot analysis showing that the anti-Tbx6 antibody detected a protein of expected molecular weight (58 kDa) in the PSM and tailbud (p) but not in formed somites (s). The asterisk indicates non-specific binding. (C) Double staining of Mesp2 mRNA (purple) and Tbx6 protein (green) demonstrating the coexistence of both signals in the anterior-most part of the Tbx6-positive region (white in merged image). (D) Design of an in vivo technique for detecting Tbx6 binding to the P2PSME by ChIP. Arrows represent primers for the ChIP assay for the Mesp2 and Dll1 genes. Dll1 is known to be downstream of Tbx6 and was therefore used as a positive control. Gray and black boxes represent the P2PSME and Dll1 mesoderm (msd) enhancers, respectively. White ovals indicate Tbx6 binding sites. P2Em and P2Ewt, mutated and wild-type P2PSME regions, respectively; NC, unrelated sequence as negative control. (E) Tbx6 associates with P2PSME in the anterior and posterior PSM. (F) The association of Tbx6 with mutated P2PSME as detected by ChIP assay. Mutated and wild-type P2PSME regions were differentially detected by PCR with different sets of primers in the tails of E10.5 embryos obtained from the crossing of P2EmB1D/+ and ICR mice.

 

Figure 3
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  		Fig. 3. Multiple Tbx6 binding is required for Mesp2 activation. (A) The sequence of the mouse Mesp2 enhancer region that contains four presumptive Tbx6 binding sites (Sites B, D, F and G). Sites A and C are presumptive RBPJ-{kappa} binding sites (Yasuhiko et al., 2006Go). The Tbx6 consensus binding site was originally reported by White and Chapman (White and Chapman, 2005Go). (B) Site G, but not Site F, binds to Tbx6 in an electromobility shift assay (EMSA). Site G produced a bandshift indicating a single bound Tbx6 molecule (lane 16), whereas Site B produced two bands (lane 1). Mutated oligonucleotide probes mB1, mD and mG produced no shifted bands (lanes 5, 11 and 20, respectively). The mB2 probe showed a single shifted band, implying the loss of one Tbx6 binding site in Site B (lane 6). (C,D) Luciferase reporter assays were conducted using several mutated enhancer elements. Luciferase activity was measured after transfection of reporter constructs along with an empty vector (None), Tbx6 expression vector (+Tbx6), or both Tbx6 and Notch intracellular domain expression vectors (+Tbx6 +NICD). Each reporter construct is presented schematically to the left of each graph. Black oval, mutated Tbx6 binding site; white oval, wild-type Tbx6 binding site; arrow, transcription start site. The number of wild-type Tbx6 binding sites is also indicated (number of Tbx6 binding: C, 4 to 2; D, 2 to 0). To the left are representative images of lacZ staining in transgenic embryos with P2PSME-lacZ reporters bearing the indicated enhancers. The number of lacZ-positive/transgene-positive embryos is indicated. The results of a consecutive series of reporter assays, as described in C, are shown in D, but on a different scale owing to the steep declines in activity. The P2EmDG lane represents the same data in both C and D. Each luciferase assay was performed in triplicate in at least three independent experiments. Error bars represent s.d.

 

Figure 4
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  		Fig. 4. The medaka mespb PSM enhancer is functionally equivalent to its counterpart in the mouse. (A) A comparison of the medaka mespb and mouse Mesp2 PSME regions. Black and gray boxes represent presumptive T-box binding sites. The numbers above the boxes represent the nucleotide positions from the first ATG. The nucleotide sequences of the putative T-box binding sequences are shown beneath. Consensus Tbx6 binding sequences and their directions are indicated by arrows. The dashed arrow in Site B of the Mesp2 PSME depicts an incomplete Tbx6 binding sequence that only binds to Tbx6 if an adjoining complete Tbx6 binding sequence is present. The T-box proteins that might bind to these sequences are indicated. (B) EMSA analysis of the T-box binding sites in the medaka mespb PSME. T-box binding site T1 associates with a single Tbx24 molecule and T2 and T3 with two Tbx24 molecules, which is consistent with their nucleotide sequences as shown in A. (C) The targeting strategy used to generate the medaka mespb PSME knock-in mouse (medakaP2). A 2.8-kb fragment of mespb genomic DNA that is required for PSM-specific mespb expression was substituted for Mesp2 PSME by homologous recombination. The neoR selection marker was removed by recombination using the Cre-loxP system. (D) medakaP2 homozygotes are viable and have normal external features. (E) Homozygotes are indistinguishable from heterozygotes and wild-type littermates in skeletal preparations.

 

Figure 5
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  		Fig. 5. Putative mechanism for the Tbx6-mediated regulation of Mesp genes. (A) Tbx6 activates Mesp2 expression through multiple Tbx6 binding sites. Notch signaling (NICD, red ovals) activates Mesp2 expression via factor X (gray ovals), which recognizes two neighboring Tbx6 binding sites in P2PSME. (Left) Schematic description of the Tbx6-dependent activation of Mesp2. (Middle column) lacZ expression in the P2Ewt transgenic embryo. (Right) Normal skeletal formation in the heterozygous fetus of a P2EmB1D mouse. (B) Mutation in Site B results in decreased expression of Mesp2. (C) A single Tbx6 binding site is unable to activate Mesp2 expression, presumably owing to an inability to respond to Notch signaling.

 

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© The Company of Biologists Ltd 2008