Fig. 4. (A) Western blot of whole embryo lysates prepared from progressively older embryos that were injected at the two-cell stage with mRNA encoding Id3 isoforms with epitope tags appended to either their N or C terminus. N-terminally tagged Id3 protein persists for a substantially longer period of development. (B) In situ hybridization of embryos injected with mRNA encoding the stabilized N-terminally tagged Id3 protein. Forced Id3 expression of Id3 leads to a modest increase in the expression of Slug in at the neural plate border on the injected side of the embryo (arrowhead). Cyan staining is lineage tracer ß-gal. (C) Siblings of the embryos shown in B show substantially increased expression of Slug at migratory neural crest stages on the injected side of the embryo (left panel, arrows). At these stages expression of Slug has normally been downregulated in migrating neural crest cells, as seen on the control side of the embryo (right panel). (D) Phosphohistone H3 immunocytochemistry of Id3-injected embryos. No difference is seen in the number of actively cycling cells on the control versus Id3 injected (arrowhead) side of the embryo, indicating that the increase in Slug expression is unlikely to be secondary to an increase in cell proliferation. (E) Whole-mount TUNEL staining of Id3-injected embryos. No change in the numbers of apoptotic cells is noted on the Id3-depleted side of the embryo (arrowhead). Red staining is lineage tracer ß-gal.