Abstract
foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA) of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2nd coding exon: a 17 base-pair (bp) deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development.
Highlights
Language impairment is central to autism and the autistic spectrum disorders, and is a major component in many neurodevelopmental disorders including Angelman syndrome and Fragile X syndrome [1]
We cloned and sequenced the PCR product from exon 1–2 amplification, and found that the mutation led to the predicted shift in codon reading frame. These results suggest that the ZFNinduced foxP2 mutants will lead to expression of truncated, mutant protein products lacking the zinc-finger, leucine zipper, and forkhead domains
Since in humans language ability depends on connections between different language areas [48], we had hypothesized a role for FOXP2 in axon guidance
Summary
Language impairment is central to autism and the autistic spectrum disorders, and is a major component in many neurodevelopmental disorders including Angelman syndrome and Fragile X syndrome [1]. Unraveling the genetic pathways and neural circuitry involved in language development is important for understanding these different disorders. Only one gene, FOXP2, has been linked with the normal development of language [2]. This gene, a forkhead-box transcription factor, was originally identified in a family with a severe speech and language disorder, and has subsequently been identified in other patients as well [2,3,4,5,6,7,8]. Haploinsufficiency for FOXP2 in humans leads to defects in grammatical language construction, as well as in the sequencing of orofacial movements required for speech articulation [9]. Studies on the human FOXP2 mutant pedigrees, using functional and volumetric magnetic resonance imaging (MRI), show abnormalities of the basal ganglia, cerebellum, and prefrontal cortex [10,11,12]
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