| |
Fig. 4. Knockdown of XTcf-4 results in reduced proliferation of dorsal brain tissue. (A) XTcf-4 morpholino, but not control morpholino, blocks the expression of NCAM protein in the dorsal midbrain. Ten picomoles of the morpholino were injected together with 100 pg of myc-tagged EGFP mRNA into one blastomere of two-cell stage embryos. Transverse sections of stage 30 embryos were incubated with α-NCAM and visualized with a Cy3-coupled goat anti-mouse antibody. The injected side is traced by EGFP. Note: At the XTcf-4 morpholino-injected side, the NCAM signal is missing in the dorsal half of the midbrain (arrow). Consistently, the dorsal part appears to consist of less tissue (arrow, brightfield). (B) The pan-neural marker gene nrp-1 is still expressed in the Tcf-4-depleted, size-reduced dorsal midbrain. Fifty-micrometer transversal sections through the midbrain region of stage 30 embryos stained for nrp-1 reveal that the dorsal neural tissue (arrow) is less thick at the injected side (asterisk). (C) XTcf-4 morpholino blocks cell proliferation in the dorsal brain. Transversal sections of stage 25 embryos were stained with the M-phase marker phosphoH3 and visualized with Cy-3-coupled secondary antibody. The injected side is marked by α-myc staining for the coinjected GFPmyc. The white line illustrates the subdivision of the midbrain into ventral (v) and dorsal (d) part for the quantification in C. (D) Quantification of the phosphoH3-positive nuclei in the brain reveals that on the injected side (filled bars), the number of proliferating cells is decreased, while at the ventral midbrain cell proliferation was not altered. The phosphoH3-positive nuclei at the uninjected side (open bars) were set as 100%. |
