Figure 5.

Dll4 acts as a Notch ligand in Xenopus embryos. One blastomere of 2-cell albino embryos was injected with either (a) nLacZ RNA and 0.1–0.25 ng of Dll4 RNA, or (b) nLacZ RNA only. At stage 14, the embryos were fixed, stained in whole mount with X-gal (light blue reaction product), and then labeled by in situ hybridization for the expression of N-tubulin (dark blue). Embryos are presented anterior to the left with the injected side on the top. Note that N-tubulin expression is suppressed in the area of Dll4 injection (as visualized with lacZ). (c) Results of RNase protection assay to measure the levels of ESR-1 and ESR-7, targets of Notch signaling, in neural ectoderm isolated from embryos injected with the indicated RNAs. Ectoderm was neuralized by injection of Noggin. RNAs were extracted at stage 13 and the ability of Dll4 to activate Notch targets were examined. For quantification, specific band intensities in each lane were normalized to the amount of EF1-α RNA to control for sample recovery, and the gene expression results are represented relative to the uninjected controls. Note that Dll4 weakly activates ESR-1 and ESR-7 expression via endogenous X-Notch activation (column 3), whereas ESR gene activation by Dll4 is strongly potentiated by coinjection of murine Notch1 (column 4) to reach the levels induced by X-Delta1 (column 5). In a separate experiment (columns 7–10), the ability of Dll4 to signal through the murine Notch4 receptor was tested. The activation of ESR-1 and ESR-7 expression by Dll4 (column 9) is strongly potentiated by coinjection with murine Notch4 (column 10). In both experiments, activation of targeted genes was increased >3.5-fold upon coinjection of Dll4 with either murine Notch1 or Notch4.