The Mesp2 transcription factor establishessegmental borders by suppressing Notch activityMitsuru Morimoto1, Yu Takahashi3, Maho Endo1& Yumiko Saga1,2The serially segmented (metameric) structures of vertebrates arebased on somites that are periodically formed during embryo-genesis. A ‘clock and wavefront’ model has been proposed toexplain the underly ing mechanism of somite form ation1,inwhich the periodicity is generated by oscillation of Notch com-ponents (the clock) in the posterior pre-somitic mesoderm(PSM)2–6. This temporal periodicity is then translated into thesegmental units in the ‘wavefront’7,8. The wavefront is thought toexist in the anterior PSM and progress backwards at a constantrate; however, there has been no direct evidence as to whether thelevels of Notch activity really oscillate and how such oscillation istranslated into a segmental pattern in the anterior PSM. Here, wehave visualized endogenous levels of Notch1 activ ity in mice,showing that it oscillates in the posterior PSM but is arrested inthe anterior PSM. Somite boundaries formed at the interfacebetween Notch1-activated and -repressed domains. Genetic andbiochemical studies indicate that this interface is generated bysuppression of Notch activity by mesoderm posterior 2 (Mesp2)through induction of the lunatic fringe gene (Lfng). We proposethat the oscillation of Notch activity is arrested and translated inthe wavefront by Mesp2.Mesp2—a basic helix–loop–helix-type transcription factor—andits related proteins have an essential role in both segmentation androstro-caudal patterning of somites in the anterior PSM9–12.Tounderstand further the involvement of Mesp2 in boundary for-mation, we generated a Mesp2–venus knock-in mouse (Supplemen-tary Fig. 1) in which Mesp2 localization is detected in live embryosusing confocal microscopy (Fig. 1a–g). As expected, the Mesp2–venus fusion protein was detected in the nuclei of cells (Fig. 1d).Furthermore, in embryos at 8.75 days post coitus (d.p.c.), two tothree segmental stripes are detectable, showing an anterior toposterior gradient for each segment (Fig. 1a–d). The signal in themost anterior segment is very weak but a second stripe has a clearanterior boundary that perfectly matches the segmental border;indeed, it lines the entire border. The strongest third stripe also hasa clear anterior border in the PSM in which no sign of a morpho-logical boundary can be detected. This band may therefore demarcatethe next segmental border. In later-stage embryos, one clear stripewas detected, although we occasionally observed an additional signalin the newly formed somite (Fig. 1e– g).We have previously shown that the expression domain of Mesp2transcript changes during somitogenesis13, appearing at S21orS22as a single band of one somite in length, and then graduallyshrinking, leaving the rostral half of its expression domain intact.To elucidate further the dynamic behaviour of Mesp2, we haverecorded its expression pattern using time-lapse microscopy. Anew Mesp2 expression domain is evident as a single band of aboutone somite in length, whereas the expression domain of the pre-existing band undergoes changes such that the rostral part of theband is maintained longer before finally disappearing (Supplemen-tary Movie 1 and Supplementary Fig. 2). This dynamic change isconsistent with the expression profile of Mesp2 messenger RNA,previously obtained using explant cultures13. However, becauseMesp2–venus is a fusion protein, it may not faithfully reproducethe pattern of endogenous protein distribution. This prompted usto generate antibodies against the Mesp2 protein. The antibody-staining pattern reveals that the distribution of endogenous Mesp2 issimilar to that of Mesp2–venus (Fig. 1h–j). The neighbouring sectionwas subjected to in situ hybridization analysis. The mRNA localiza-tion pattern was found to be consistent with the immunohistochemi-cal results, indicating that message is translated immediately and thatthe protein is rapidly degraded but the transcript disappears earlier,as expected (Fig. 1j). We occasionally observed two bands of Mesp2protein: an anterior band located in the segmented border and anposterior one in the next presumptive border. In this case, transcriptwas only observed in the posterior region (Fig. 1h). Taken together,we conclude that the anterior border of the expression domain of theMesp2 protein coincides with the next segmental border in the PSM.The expression of Notch components, such as Hes7 (hairy andenhancer of split 7) and Lfng, oscillates in the PSM3,4,6, whereasNotch1 is expressed throughout the entire PSM and at higher levels inthe anterior PSM9. We attempted to visualize the activation of Notchsignalling by detecting a processed NICD (Notch intracellulardomain) using a specific antibody. Unlike its RNA expressionpattern, Notch1 activity exhibits a dynamic pattern in the PSMduring somitogenesis. We analysed 52 embryos, which exhibited fourdistinct patterns; the positive signals were detected in the tailbud(Fig. 1k), the middle of the PSM (Fig. 1l, m) or more anteriorlywithin the PSM (Fig. 1n). Notably, in most of the embryos, Notchactivation is in a similar phase to the transcript of Lfng, whichencodes a glycosyltransferase that can modify Notch activity14,15(Fig. 1k–n). The expression domain of Lfng appears slightly laterthan Notch activation, supporting the idea that Lfng transcription isactivated by Notch signalling, as previously indicated16. These resultsindicate that the activity of Notch oscillates in the posterior PSM. In aprevious report, Hes7 was shown to function as a suppressor of Hes7and Lfng transcription17, forming a feedback loop in the coreoscillator. We thus tested the pattern of Notch activation in micedefective in Notch pathway components (Fig. 2a–e). In Dll1-nullembryos18, no Notch1 activity is found anywhere in the PSM(Fig. 2c), whereas in Lfng-null embryos19, Notch1 activity is detectedthroughout the entire PSM but does not seem to oscillate (Fig. 2d).These observations indicate that the Dll1 ligand is required forNotch1 activation, and that Lfng functions as a suppressor ofNotch activity, thus generating the oscillatory activation of Notch1.In the anterior PSM, Notch oscillatory activity is arrested andlocalizes in the caudal portion of the S0 somite, whereas Lfngtranscripts are detectable in the rostral part of S 2 1 and theirLETTERS1Division of Mammalian Development, National