Lissencephaly is a pediatric neurological disorder that causes mental retardation and epilepsy. Haploinsufficiency is thought to cause a neuronal migration defect, whereas homozygous patients are not seen and are thought to be miscarriages. Similarly, in a mouse model, weak knockout mutations disrupt brain development, while strong knockout mutations produce embryonic arrest. The full-length worm
lis-1 cDNA was cloned by PCR from a worm cDNA library and by sequencing of several ESTs from the Kohara EST database. A 5' RACE experiment showed that the 5' UTR is preceded by an SL1 trans-spliced leader sequence. The cDNA cloning and 5' RACE data agree with those of a Northern blot which showed a single band in the range of 2.1KB in mixed-stage wild-type worms. Taken together, these results show that the worm LIS-1 protein is 58% identical to the human LIS1 protein. Both genes contain an amino-terminal coiled-coil domain followed by seven WD40 repeats. Like the human LIS1 protein, the worm homolog appears to be broadly expressed. A GFP reporter driven by ~ 3 KB from the worm
lis-1 promoter region is expressed in a variety a tissues, including the seam cells, body wall and pharyngeal muscle, vulval cells, spermatheca, the tail, and many neurons including the hermaphrodite-specific neurons. Polyclonal anti-LIS-1 antisera is also being used for western blot analysis and whole-mount immunofluorescence studies of LIS1 expression. To study the loss-of-function phenotypes associated with the worm homolog, I used a PCR-based deletion screening approach and obtained two knockout mutations within
lis-1 , a 2 kb out-of-frame deletion removing exons 4-6 and a 1.5 kb in-frame deletion removing exons 4-5. Worms heterozygous for either deletion produce homozygous progeny that exhibit a backing defect and release some but not all eggs. These
lis-1 homozygotes also show fully penetrant maternal-effect lethality and lay eggs that arrest early in embryogenesis. Injection of cosmid T03F6 encompassing the
lis-1 genomic sequence rescues these defects. Thus, the worm loss-of-function mutants display a phenotypic pattern similar to mammals. As in human and mouse, weak loss-of-function mutants display neurological abnormalities, and strong loss-of-function mutants display embryonic abnormalities. I performed immunohistochemical studies using DAPI, anti-tubulin antibody, and an antibody to a centrosomal marker, polo-like kinase, to triple-stain one-cell stage embryos for DNA, tubulin, and centrosomes, respectively. In
lis-1 embryos centrosomal migration is impaired, and in many embryos centrosomes are physically dissociated from pronuclei altogether. These experiments confirmed the absence of bipolar spindles in
lis-1 embryos. Instead, microtubules are disordered and shortened, perhaps secondary to centrosomal dysfunction, centrosomes normally acting as the microtubule-organizing centers. Nuclei become asymmetrically distributed as a result of failed chromosomal segregation. Time-lapse Nomarski analysis of a series of
lis-1 embryos aso revealed defects occurring in the following sequence: delayed or failed pronuclear migration, misplaced pronuclear meeting, failed rotation and centering of the centrosomes and pronuclei, endoreduplication, and failed cytokinesis. In conclusion, immunohistochemical and Nomarski time-lapse studies show that centrosomal and nuclear migration defects are the earliest abnormalities seen at the one cell-stage, consistent with the notion that
lis-1 plays a role in microtubule-based intracellular motility.