Fig. 7

Pcdh1α splice-blocking morpholinos result in apoptosis throughout the CNS. (A) Shown here are 24 hpf embryos processed for TUNEL to assess levels of apoptosis. Wild type and 5 basepair mismatch control morpholinos exhibited relatively little cell death at 24 hpf, as indicated by the lack of darkly stained nuclei. In contrast, pcdh1α-morpholino-injected embryos (2.5 ng) showed a dramatic increase in labeled nuclei, which was not prevented by co-injection of 5 ng p53MO. Thus, the reduction in full-length Pcdh1α protein results in neuronal death. The insets show magnified views of the boxed portion of the spinal cord. (B) Dorsal view of the hindbrain region in a pcdh1α-morpholino-injected embryo at 24 hpf. Bilateral clusters of apoptotic cells can be observed that exhibit a rhombomeric pattern (arrows) that mimics the expression pattern determined by in situ hybridization and immunocytochemistry. (C) Acridine orange staining was performed to score embryos for the cell death phenotype. We designed two different morpholinos against pcdh1α (MO1.1 and MO1.2), as well as 5 bp mismatch controls for each (MO1.1mis and MO1.2mis). Injection of 2.5 ng of either splice-blocking morpholino yielded extensive cell death in > 90% of injected embryos. Injection of control morpholinos failed to cause cell death. Co-injection of 5 ng of a p53 morpholino with 2.5 ng of either splice-blocking morpholino failed to inhibit apoptosis, demonstrating that the cell death is not due to off-target activation of the p53 pathway. Injection of 1 ng of either MO1.1 or MO1.2 failed to cause a significant increase in apoptosis, while a combined dose of 1 ng of each morpholino resulted in a large increase in cell death.