Fig. 7. MAK functions in regional brain patterning, but does not affect organizer markers. (A-O) MAK regulates midbrain-hindbrain boundary. Dorsal-animal region of four-cell embryos was injected with 50 ng of MOs or 2 ng of MAK RNAs, as indicated, together with nßgal RNA as a lineage tracer. At stage 20, Otx2, En2 and Gbx2 were assessed by whole-mount in situ hybridization. MAK MO inhibited Otx2 and En2 (D,E), but upregulated Gbx2 (F). MAK RNA expanded and posteriorly shifted Otx2 and En2 on the injected side (J,K), and inhibited Gbx2 (L). Red arrows indicate altered expression; black arrows indicate marker expression on the uninjected side. (P) MAK depletion or overexpression does not affect organizer marker expression. Two to four-cell embryos were injected twice in the dorsal margin with MAK MO or COMO (100 ng), ß-catenin MO (30 ng) or indicated RNA (4 ng). RT-PCR was carried out with total RNA from stage 10 embryos. DNA fragments were separated in 6% SDS-polyacrylamide gel. EF-1 is a loading control. Uninj, uninjected embryos. RT-, no reverse transcriptase.