Loss-of-function nerfin-1 mutations were generated by the 'ends-in' homologous recombination gene knockout technique. The targeted gene knockout resulted in the isolation of five independent embryonic recessive lethal alleles. Although late-stage homozygous mutant embryos appeared normal, with no detectable gross morphological or segmentation defects, they failed to hatch from their egg chambers. To confirm that the lethality and cellular phenotype observed in the mutant embryos was due to the loss of nerfin-1, independent P-element insertions that contained an 11,154 bp nerfin-1 genomic DNA fragment were used to rescue the viability and cellular phenotype and to restore the nerfin-1 expression level.

Given the absence of any detectable alteration in NB-lineage development in nerfin-1 null embryos, we sought to determine if Nerfin-1 exhibited axon guidance phenotypes. Immunostains of nerfin-1 null embryos revealed significant alterations in axon projections within the embryonic CNS but not in the PNS. For example, within the ventral nerve cord of stage 13 and older nerfin-1 null embryos, the longitudinal connective axon fascicles were disrupted between segments, and both the anterior and posterior commissures of each ventral cord ganglia were malformed (see accompanying figure). Axons that normally project through fascicles that make up the intersegmental longitudinal connectives appeared to either stall or randomly turn at or near segmental boundaries, creating disorganized tangles. In addition, the organization of longitudinal connectives within each of the segments was abnormal with misrouted axons projecting laterally away from the longitudinal tracks. In stage 15 and older mutants, the medial, intermediate, and lateral longitudinal tracks were disrupted along the entire length of the ventral cord.

In contrast to the axon fascicle organization defects observed in the ventral cord and brain, no significant patterning defects were detected in the motoneuron nerve tracts that exit the CNS. In addition, the axon patterning of PNS neurons, outside the CNS, also appeared normal in nerfin-1 null embryos.