Functional importance of evolutionally conserved Tbx6 binding sites in the presomitic mesoderm-specific enhancer of Mesp2.
The T-box transcription factor Tbx6 controls the expression of Mesp2, which encodes a basic helix-loop-helix transcription factor that has crucial roles in somitogenesis. In cultured cells, Tbx6 binding to the Mesp2 enhancer region is essential for the activation of Mesp2 by Notch signaling. However, it is not known whether this binding is required in vivo. Here we report that an Mesp2 enhancer knockout mouse bearing mutations in two crucial Tbx6 binding sites does not express Mesp2 in the presomitic mesoderm. This absence leads to impaired skeletal segmentation identical to that reported for Mesp2-null mice, indicating that these Tbx6 binding sites are indispensable for Mesp2 expression. T-box binding to the consensus sequences in the Mesp2 upstream region was confirmed by chromatin immunoprecipitation assays. Further enhancer analyses indicated that the number and spatial organization of the T-box binding sites are critical for initiating Mesp2 transcription via Notch signaling. We also generated a knock-in mouse in which the endogenous Mesp2 enhancer was replaced by the core enhancer of medaka mespb, an ortholog of mouse Mesp2. The homozygous enhancer knock-in mouse was viable and showed normal skeletal segmentation, indicating that the medaka mespb enhancer functionally replaced the mouse Mesp2 enhancer. These results demonstrate that there is significant evolutionary conservation of Mesp regulatory mechanisms between fish and mice.
Yasuhiko
Yukuto
Y
Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. yasuhiko@nihs.go.jp
Kitajima
Satoshi
S
Takahashi
Yu
Y
Oginuma
Masayuki
M
Kagiwada
Harumi
H
Kanno
Jun
J
Saga
Yumiko
Y
D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D017124:Conserved Sequence; D004622:Embryo, Mammalian / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D019143:Evolution, Molecular; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting; D008648:Mesoderm / Q000196:embryology / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009928:Organ Specificity; D009990:Oryzias / Q000235:genetics; D051880:Receptors, Notch / Q000378:metabolism; D015398:Signal Transduction; D019170:Somites / Q000378:metabolism*; D020825:T-Box Domain Proteins; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D051792:Basic Helix-Loop-Helix Proteins; D051880:Receptors, Notch; D020825:T-Box Domain Proteins; D014157:Transcription Factors; C107407:Mesp2 protein, mouse; C102932:Tbx6 protein, mouse