Human-specific transcriptional regulation of CNS development genes by FOXP2.

The signalling pathways controlling both the evolution and development of language in the human brain remain unknown. So far, the transcription factor FOXP2 (forkhead box P2) is the only gene implicated in Mendelian forms of human speech and language dysfunction. It has been proposed that the amino acid composition in the human variant of FOXP2 has undergone accelerated evolution, and this two-amino-acid change occurred around the time of language emergence in humans. However, this remains controversial, and whether the acquisition of these amino acids in human FOXP2 has any functional consequence in human neurons remains untested. Here we demonstrate that these two human-specific amino acids alter FOXP2 function by conferring differential transcriptional regulation in vitro. We extend these observations in vivo to human and chimpanzee brain, and use network analysis to identify novel relationships among the differentially expressed genes. These data provide experimental support for the functional relevance of changes in FOXP2 that occur on the human lineage, highlighting specific pathways with direct consequences for human brain development and disease in the central nervous system (CNS). Because FOXP2 has an important role in speech and language in humans, the identified targets may have a critical function in the development and evolution of language circuitry in humans.

Mutations in a novel gene encoding a CRAL-TRIO domain cause human Cayman ataxia and ataxia/dystonia in the jittery mouse.

Cayman ataxia is a recessive congenital ataxia restricted to one area of Grand Cayman Island. Comparative mapping suggested that the locus on 19p13.3 associated with Cayman ataxia might be homologous to the locus on mouse chromosome 10 associated with the recessive ataxic mouse mutant jittery. Screening genes in the region of overlap identified mutations in a novel predicted gene in three mouse jittery alleles, including the first mouse mutation caused by an Alu-related (B1 element) insertion. We found two mutations exclusively in all individuals with Cayman ataxia. The gene ATCAY or Atcay encodes a neuron-restricted protein called caytaxin. Caytaxin contains a CRAL-TRIO motif common to proteins that bind small lipophilic molecules. Mutations in another protein containing a CRAL-TRIO domain, alpha-tocopherol transfer protein (TTPA), cause a vitamin E-responsive ataxia. Three-dimensional protein structural modeling predicts that the caytaxin ligand is more polar than vitamin E. Identification of the caytaxin ligand may help develop a therapy for Cayman ataxia.

Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene.

Autism is a genetically complex neurodevelopmental syndrome in which language deficits are a core feature. We describe results from two complimentary approaches used to identify risk variants on chromosome 7 that likely contribute to the etiology of autism. A two-stage association study tested 2758 SNPs across a 10 Mb 7q35 language-related autism QTL in AGRE (Autism Genetic Resource Exchange) trios and found significant association with Contactin Associated Protein-Like 2 (CNTNAP2), a strong a priori candidate. Male-only containing families were identified as primarily responsible for this association signal, consistent with the strong male affection bias in ASD and other language-based disorders. Gene-expression analyses in developing human brain further identified CNTNAP2 as enriched in circuits important for language development. Together, these results provide convergent evidence for involvement of CNTNAP2, a Neurexin family member, in autism, and demonstrate a connection between genetic risk for autism and specific brain structures.

Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain.

Mutations in FOXP2, a member of the forkhead family of transcription factor genes, are the only known cause of developmental speech and language disorders in humans. To date, there are no known targets of human FOXP2 in the nervous system. The identification of FOXP2 targets in the developing human brain, therefore, provides a unique tool with which to explore the development of human language and speech. Here, we define FOXP2 targets in human basal ganglia (BG) and inferior frontal cortex (IFC) by use of chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) and validate the functional regulation of targets in vitro. ChIP-chip identified 285 FOXP2 targets in fetal human brain; statistically significant overlap of targets in BG and IFC indicates a core set of 34 transcriptional targets of FOXP2. We identified targets specific to IFC or BG that were not observed in lung, suggesting important regional and tissue differences in FOXP2 activity. Many target genes are known to play critical roles in specific aspects of central nervous system patterning or development, such as neurite outgrowth, as well as plasticity. Subsets of the FOXP2 transcriptional targets are either under positive selection in humans or differentially expressed between human and chimpanzee brain. This is the first ChIP-chip study to use human brain tissue, making the FOXP2-target genes identified in these studies important to understanding the pathways regulating speech and language in the developing human brain. These data provide the first insight into the functional network of genes directly regulated by FOXP2 in human brain and by evolutionary comparisons, highlighting genes likely to be involved in the development of human higher-order cognitive processes.

Characterization of a mutagenic B1 retrotransposon insertion in the jittery mouse.

B1 elements are an abundant class of short interspersed elements (SINEs) in the mouse genome and mobilize by a process known as retrotransposition. Here, we report the characterization of a mutagenic B1 insertion into exon 4 of the Atcay gene, which was previously shown to be responsible for the jittery mouse. Mutations in the human ortholog of this gene, ATCAY, are responsible for Cayman ataxia. The B1 insertion is approximately 150-bp long, ends in a 45-50-bp polyadenylic acid (poly A) tail, is flanked by a perfect 13-bp target-site duplication, and is inserted into a sequence that resembles a LINE-1 endonuclease consensus cleavage site. Computational analysis indicates that the mutagenic insertion is most closely related to elements of the B1-C subfamily, and we have identified two possible progenitor B1 sequences on mouse chromosome 19. Together, these data demonstrate that B1 retrotransposition is ongoing in the mouse genome and is consistent with the hypothesis that the reverse transcriptase and endonuclease encoded by LINE-1 elements mediate B1 mobility.

Fate of cajal-retzius neurons in the postnatal mouse neocortex.

Cajal-Retzius (CR) neurons play a critical role in cortical neuronal migration, but their exact fate after the completion of neocortical lamination remains a mystery. Histological evidence has been unable to unequivocally determine whether these cells die or undergo a phenotypic transformation to become resident interneurons of Layer 1 in the adult neocortex. To determine their ultimate fate, we performed chronic in vivo two-photon imaging of identified CR neurons during postnatal development in mice that express the green fluorescent protein (GFP) under the control of the early B-cell factor 2 (Ebf2) promoter. We find that, after birth, virtually all CR neurons in mouse neocortex express Ebf2. Although postnatal CR neurons undergo dramatic morphological transformations, they do not migrate to deeper layers. Instead, their gradual disappearance from the cortex is due to apoptotic death during the second postnatal week. A small fraction of CR neurons present at birth survive into adulthood. We conclude that, in addition to orchestrating cortical layering, a subset of CR neurons must play other roles beyond the third postnatal week.

X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment.

Epilepsy and mental retardation limited to females (EFMR) is a disorder with an X-linked mode of inheritance and an unusual expression pattern. Disorders arising from mutations on the X chromosome are typically characterized by affected males and unaffected carrier females. In contrast, EFMR spares transmitting males and affects only carrier females. Aided by systematic resequencing of 737 X chromosome genes, we identified different protocadherin 19 (PCDH19) gene mutations in seven families with EFMR. Five mutations resulted in the introduction of a premature termination codon. Study of two of these demonstrated nonsense-mediated decay of PCDH19 mRNA. The two missense mutations were predicted to affect adhesiveness of PCDH19 through impaired calcium binding. PCDH19 is expressed in developing brains of human and mouse and is the first member of the cadherin superfamily to be directly implicated in epilepsy or mental retardation.