Fig. 2. RBs but not PMNs are eliminated by ngn1 morpholinos. (A-D) Lateral views of 18 hpf wild-type embryos at somites 15-20, processed to reveal isl2 RNA expression. In this and subsequent figures, the portrayed phenotype was observed in at least 80% of injected embryos. (A) Uninjected embryo. Expression of isl2 can be seen in RBs in dorsal neural tube and in a subset of primary motoneurons (PMN) in ventral neural tube. (B) Embryo injected at the two-cell stage with approximately 1.5 ng of ngn1 MO that straddles the start AUG of the ngn1 transcript. As revealed by isl2 expression, RBs were absent or highly reduced, whereas PMNs were normal (40 embryos scored). Note: in embryos injected with 7.5 ng of ngn1 MO, PMNs in the tail were reduced in some embryos; however, it is unclear whether this phenotype resulted from targeting of the ngn1 transcript, because target specificity may not be maintained at this concentration. (C) Embryo injected at two-cell stage with approximately 3.0 ng of ngn1^mismatch MO, which is identical to ngn1 MO except at four base positions (28 embryos scored). RBs and PMNs are normal, indicating that sequence complementarity is essential for protein knockdown at this morpholino concentration. At 6.0 ng of injected ngn1^mismatch MO, embryos resembled ngn1 MO-injected embryos (20 embryos scored), suggesting that at this high dose, the ngn1 mismatch MO targets the ngn1 transcript, despite the lack of complementarity at four out of 25 positions. We therefore ignored effects of morpholinos at this high dose, because of the uncertainty of the transcript being targeted. (D) Embryo injected at two-cell stage with approximately 1.2 ng of ngn1^5'UTR MO, that complements an upstream region of the 5'UTR. The phenotype appeared identical to that in ngn1 MO-injected embryos at this dose (25 embryos scored). At 3.6 ng and higher, embryos showed patchy necrosis in the CNS. This phenotype was presumed to result from toxicity of the morpholino at this dose, as opposed to further reduction of Ngn1 function. Scale bar: 50 µm.

Return to article