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内容記述 |
During mouse embryogenesis, many morphogenetic events occur sequentially <br />according to the scheduled time, indicating that these sequential events are linked <br />with the precise temporal regulation. Such regulations must exist throughout <br />embryogenesis to coordinate many developmental processes, although the molecular <br />nature coordinating such temporal regulation is largely unknown. <br /> The vertebrate body is subdivided into repeating segments along the <br />anterior-posterior (AP) axis. This segmental or metameric pattern is established early <br />in embryogenesis by the process of somitogenesis. Somites are blocks of paraxial <br />mesoderm cells that give rise to the axial skeleton and their associated muscles and <br />tendons, which retain a metameric pattern. During development, somitogenesis is <br />tightly coupled with axis elongation. Precursors of the somites, called presomitic <br />mesoderm (PSM), arise from the posterior end of embryo, called tail bud. Somites are <br />aligned along the neural tube, and budding off from the anterior-most end of the <br />unsegmented presomitic mesoderm at the regular time. Therefore, somitogenesis is an <br />event that occurs according to the scheduled time, and it is believed that somitogenesis <br />is under the precise control of temporal information. <br /> The timing of somitogenesis is regulated by the so-called `segmentation clock', <br />which is associated with a periodic activation of Notch signal pathway in PSM cells. <br />Notch signal .activates the target genes, Hes7 and L-fng. The transcription factor Hes7 <br />(hairy and enhancer of split 7) in turn represses own transcription as well as that of <br />L-fng, making negative feedback loops. L-fng encodes a glycosyltransferase that acts <br />as a negative regulator of Notch activity, which generates the oscillation of Notch <br />signal activity within the PSM. However, the oscillation itself does not make a <br />segmental boundary, as exemplified by a pendulum clock in which the correct time is <br />not provided by the rhythm of pendulum. This temporal information has therefore to <br />be accurately translated into a spatial pattern during somitogenesis. <br /> The basic helix-loop-helix (bHLH) protein Mesp2 is a crucial factor in this process. <br />Mesp2 expression is periodically observed only in the anterior PSM, and the anterior <br />border of the Mesp2 expression domain determines the next somite segmental border. <br />To understand dynamic expression of Mesp2, the enhancer sequence, which is required <br />for the expression in the PSM, has been mapped within 185bp upstream region in the 5' <br />flanking region of Mesp2 gene, and it has been shown that a T-box transcriptional <br />factor, Tbx6, directly binds to the enhancer elements, and is essential for the <br />activation of Mesp2. Furthermore, it is shown that Notch signaling synergistically <br />works with Tbx6 and enhances Mesp2 activation when these factors coexist. However, <br />since the enhancer analysis was mainly conducted using the cultured cell system, <br />mechanisms involved in the spatial restriction and periodic regulation of Mesp2 <br />expression remain elusive. <br /> In this study, I have employed high resolution fluorescent in situ hybridization in <br />conjunction with immunohistochemical methods to analyze sections derived from <br />single specimens. These methods have enabled me to determine the spatio-temporal <br />relationship among several factors involved in mouse somitogenesis. Initially I show <br />that the timing of Mesp2 expression is determined by the periodic waves of Notch <br />activity, indicating the temporal link between Notch signal oscillation and Mesp2 <br />transcription cycle. Next, I find that Tbx6 defines the anterior limit of Mesp2 <br />expression domain by serving as an important transcription activator. Intriguingly, <br />Mesp2 mRNA initially shares an identical anterior border, but that once translated, <br />the Mesp2 protein is found to suppress Tbx6 expression post-translationally. This was <br />strongly supported by the fact that Tbx6 protein expression was expanded to the <br />anterior somitic region in the Mesp2-null embryo without altering expression pattern <br />of the transcript. The negative regulation of the Tbx6 by Mesp2 is critically important <br />to set up the next anterior border of Mesp2 expression domain. These results indicate <br />that interactions of three factors, Mesp2, Tbx6 and Notch activity are critically <br />important to translate temporal information to the spatial patterning. I also find that <br />onset of Mesp2 transcription is intimately linked with the initiation of Notch signal <br />oscillation, indicating that the relationship of three factors appears to be established <br />in the early stage embryo via initial Notch oscillation. I further show that the lack of <br />FGF signaling results in the posterior shift of Mesp2 expression domain, indicating <br />that FGF signaling provides a spatial cue to position the posterior border of Mesp2 <br />expresslon. <br /> Furthermore, to reveal the mechanism of post-translational Tbx6 suppression <br />downstream of Mesp2, I tried to determine the domain of Tbx6 protein that was <br />required for the suppression process. I generated transgenic mice harboring several <br />types of Tbx6 protein that had truncation in several domains, under the control of <br />endogenous promoter and enhancers of Tbx6 using a BAC-base transgenic mouse <br />technology. These results indicate that the T-box domain containing a DNA-binding <br />motif, is essential and sufficient for the suppression of Tbx6 expression. In good <br />agreement with these results, I find that Mesp2 also suppresses the expression of <br />Brachyury, the other T-box factor protein, by the post-translational mechanism. <br /> Taken together, I conclude that Mesp2 is the final output signal by which the <br />temporal information from the segmentation clock is translated to the segmental <br />patterning, and reciprocal regulation between Mesp2 and Tbx6 creates the periodic <br />pattern during somitogenesis. <br /> |
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