We created dual morphants by injecting embryos with both the MO-E3 and MO-E2 morpholinos for the lgi1a and lgi1b genes respectively. Since the compound morphants were likely to experience more severe phenotypes, reflecting the importance of both genes for normal development, we created morphants using high and low concentrations of the morpholinos and compared them to morphants created using the equivalent concentrations of the mismatch morpholinos. Compared with the single morphants, the compound morphants showed a much higher incidence of premature death with,50% dying after 24 hfp and.75% mortality after 48 hpf. The high dose morphants showed slightly greater mortality compared with the low dose. The single morphants, over the 72 hpf period, only showed mortality of,20%. As we have reported throughout this series of experiments the mismatch morphants do not show any developmental abnormalities even at high dose of MO. At a low dose lgi1a and lgi1b morphants do not show abnormalities of the tail, which is correlated with a proportional knockdown of the respective mRNAs in the MK-4827 1038915-60-4 enbryos. In contrast, the compound morphants show significant tail deformity in addition to smaller eyes and head size. The highdose, compound morphants show ever more dramatic developmental abnormalities than the low dose morphants. One of the striking observations in the development of the compound morphants was their inability to escape from the chorion, which precluded behavioral analysis as described in figure 5. For those rare embryos that survived beyond 48 hours, when we manually removed the chorion, once freed, these embryos appeared to show a hyperactivity not seen in mismatch morphants, which suggests a similar phenotype to that seen in lgi1a morphants. In addition, the compound morphants at low or high dose demonstrated the hydrocephalus seen in the lgi1b morphants. These data suggest firstly, that specific phenotypes related to knockdown of the individual lgi1 genes are retained in the compound morphants and that lower doses of the individual MOs can produce the more extreme phenotypes seen only with high doses of the individual MOs. The high mortality rate and early onset of death in the embryos precluded many of the behavioral studies but supports an important role for the lgi1 genes in development. Duplication of the zebrafish genome allows subfunctionalization of the paralogs. This appears to be the case for the lgi1 genes. Lgi1a morphants demonstrate seizure-like behavior within the 3– 4 ng range, whereas the lgi1b morphants do not. Inactivation of either paralog, however, predisposes to PTZ induced hyperactivity using low dose MO treatments, which indicates a potentially common response to this epilepsy inducing drug. It has also been shown that mice with heterozygous inactivation of Lgi1 are hyper sensitive to PTZ induced seizures. The lgi1b morphants show a pronounced enlargement of the ventricles, which was not seen in the lgi1a morphants. Interestingly, the Lgi1 gene was shown to be highly expressed in the choroid plexus in mice, which is also seen at early stages of embryonic brain development.