During development, newborn neurons frequently migrate before assembling into neural networks that control complex motor and cognitive functions. In addition, neuronal growth cones must mediate precise axon pathfinding to generate functional neural circuits. Cell adhesion molecules play important roles during development, including in neuronal migration and axon guidance. Contactin2 (Cntn2)/Transiently-expressed Glycoprotein 1 (Tag1), a glycosylphosphatidylinositol anchored cell adhesion molecule of the immunoglobulin superfamily, is expressed in specific neuronal types during vertebrate nervous system development. In zebrafish, the facial branchiomotor (FBM) neurons, a subset of the hindbrain motor neurons, express cntn2 while migrating caudally from rhombomere 4 to rhombomeres 6 and 7. Our previous studies using morpholinos suggested that cntn2 is necessary for FBM neuron migration, and that it genetically interacts with the planar cell polarity gene vangl2 during FBM neuron migration. To validate these data, we generated loss-of-function mutations in cntn2 using CRISPR/Cas9, and identified three alleles including two frameshift mutations generating premature stop codons (zou20 and zou22) that exhibited identical phenotypes. cntn2 expression was reduced in cntn2zou20 and cntn2zou22 homozygotes, and Cntn2 protein was undetectable in both cases, suggesting these identified alleles are null. In contrast to the morphant phenotype, zygotic and maternal-zygotic (MZ) cntn2-/- mutants exhibited normal FBM neuron migration, suggesting either that the morphant phenotype may be non-specific or that there is genetic compensation from related genes in MZ mutants. Consistent with the latter, FBM neurons failed to migrate caudally in a significant fraction of cntn2+/-; vangl2+/- embryos obtained from cntn2-/- mothers, supporting the genetic interaction observed using morpholinos.
Previous studies showed that morpholino-mediated cntn2 knockdown generated defasiculation defects in midbrain nucMLF and spinal cord Rohon-Beard (RB) axons. However, MZ cntn2-/- mutants exhibited no nucMLF defects. To assay for defects in the sensorimotor circuits of RB neurons in cntn2 mutants, touch-evoked escape responses was evaluated. Wild type and mutants larvae exhibited normal escape responses when touched on the head. In contrast, mutants were significantly less responsive when touched in the trunk consistent with putative defects in RB circuits. Together, these data support distinct roles for Cntn2 in the development and function of neural circuits in zebrafish.