DEV00629 Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Expression of the rostral genes Cer1 and Epha4 reflects expression of Mesp2. Throughout our genetic analysis, expression levels and patterns of the rostral genes Cer1 and Epha4 were correlated with those of Mesp2. Expression of Cer1 and Epha4 is observed as stripes at the anterior PSM and is finally localized to the rostral half of each somite in the wild-type embryo (A,E). In the Dll3^pu/pu embryo, Cer1 and Epha4 are expressed as a stripe but their expression appears blurred and the rostrally localized thin stripes are never observed (B,F). Stripes of these genes are almost lost in the Mesp2-null embryo (C,G) as well as in the Dll3/Mesp2 double-null embryo (D,H), indicating that Mesp2 is more directly required for expression of the rostral genes, whereas Dll3 normally localizes the effect of Mesp2 to activate the rostral genes. Expression level of Mesp2 is moderately decreased and its expression pattern is blurred in the Dll3-null, Psen1-null and Dll3/Psen1 double-null embryos (Fig. 5). Likewise, expression levels of Cer1 (I-L) and Epha4 (M-P) are comparable among the three genotypes and rostral localization is disrupted.

• Supplemental Figure 2 - Proximal rib phenotypes also show that Dll3-Notch signaling counteracts Psen1-dependent Dll1-Notch signaling. The proximal rib element is derived from the caudal half of the somite as well as the pedicle of neural arch, and its formation is specified by Uncx4.1 (Mansouri et al., 2000; Leitges et al., 2000). The caudalization of somites in Mesp2-null embryo results in complete fusion of the proximal rib (Saga et al., 1997), while the loss of the caudal half property in the Psen1-null embryo results in the absence of the proximal rib and separation of the distal rib from the vertebral body (Koizumi et al., 2001). We examined proximal rib morphology and quantified it by counting number of separate ribs and ribs fused at the proximal region in the Dll3/Psen1 intercross. In the wild-type fetus at 17.5 dpc, a normal arrangement of ribs is observed (A). In the Dll3^pu/pu fetus, most of the ribs are fused at the proximal region and separation is relatively rare (B,F; arrows in B point to the fused region). The Dll3^+/+Psen1^–/– fetus shows typical separate ribs and virtually no fusion (C, arrows; F). In the Dll3^+/puPsen1^–/– fetus, the number of separate ribs is decreased, and thus the caudal half property is rescued (D,F). In the Dll3^pu/puPsen1^–/– fetus, the number of separate ribs is further decreased and fused ribs are observed (E, arrows; F), showing this double-null phenotype is intermediate between the Dll3-null and Psen1-null phenotypes. The graph in F indicates the average numbers of separate and fused ribs, with standard deviation, for each genotype. The number of fetuses counted is indicated in parentheses. Thus, Dll3-Notch signaling can counteract Psen1-dependent Dll1-Notch signaling.

  References

  Koizumi, K., Nakajima, M., Yuasa, S., Saga, Y., Sakai, T., Kuriyama, T., Shirasawa, T. and Koseki, H. (2001). The role of presenilin 1 during somite segmentation. Development 128, 1391-1402.

  Leitges, M., Neidhardt, L., Haenig, B., Herrmann, B. G. and Kispert, A. (2000). The paired homeobox gene Uncx4.1 specifies pedicles, transverse processes and proximal ribs of the vertebral column. Development 127, 2259-2267.

  Mansouri, A., Voss, A. K., Thomas, T., Yokota, Y. and Gruss, P. (2000). Uncx4.1 is required for the formation of the pedicles and proximal ribs and acts upstream of Pax9. Development 127, 2251-2258.

  Saga, Y., Hata, N., Koseki, H. and Taketo, M. M. (1997). Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev. 11, 1827-1839.