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The GTF2IRD1 general transcription factor is a candidate for involvement in the varied cognitive and neurobehavioral symptoms of the microdeletion disorder, Williams–Beuren syndrome (WBS). We show that mice with heterozygous or homozygous disruption of Gtf2ird1 exhibit decreased fear and aggression and increased social behaviors. These findings are reminiscent of the hypersociability and diminished fear of strangers that are hallmarks of WBS. Other core features of WBS, such as increased anxiety and problems with spatial learning were not present in the targeted mice. Investigation of a possible neurochemical basis for the altered behaviors in these mice using high-performance liquid chromatography analysis showed increased levels of serotonin metabolites in several brain regions, including the amygdala, frontal cortex and parietal cortex. Serotonin levels have previously been implicated in fear and aggression, through modulation of the neural pathway connecting the prefrontal cortex and amygdala. These results suggest that hemizygosity for GTF2IRD1 may play a role in the complex behavioral phenotype seen in patients with WBS, either individually, or in combination with other genes, and that the GTF2I transcription factors may influence fear and social behavior through the alteration of neurochemical pathways.
Williams–Beuren syndrome (WBS; OMIM 194050) is an autosomal dominant disorder, with a frequency of between in 1/7500 and 1/20 000 live births, that presents with a unique spectrum of physical and behavioral features (Greenberg 1990; Pober & Dykens 1996; Stromme et al. 2002). WBS is associated with mild to moderate mental retardation, but the uneven neurocognitive profile is characterized by significant deficits in visuospatial constructive cognition alongside relative strengths in language, auditory rote memory and facial recognition (Mervis & Klein-Tasman 2000; Mervis et al. 2000). The most striking aspect of the WBS phenotype is the distinctive behavioral profile, which is a unique combination of both friendliness and anxiety (Mervis & Klein-Tasman 2000; Pober & Dykens 1996).
Both Gtf2i and Gtf2ird1 are widely expressed during the embryonic stages of mouse development (Enkhmandakh et al. 2004; Palmer et al. 2006). In adult mice, GTF2I is present exclusively in neurons, with the greatest expression levels observed in cerebellar Purkinje cells, hippocampal interneurons and the large neurons of the cerebral cortex, whereas GTF2IRD1 showed greatest expression in granular cell layer of the olfactory bulb, the Purkinje cells of the cerebellum and the neurons in the piriform cortex (Danoff et al. 2004; Palmer et al. 2006). These results suggest that the two proteins play nonredundant, differentially regulated roles, despite their similar structure.
A Gtf2ird1 insertional mutant was generated previously, but only limited cognitive testing was performed (Durkin et al. 2001; van Hagen et al. 2006; Tassabehji et al. 2005). To better understand the contribution GTF2IRD1 haploinsufficiency makes to the cognitive and behavioral phenotype of WBS, we generated a gene-targeted mouse model and subjected heterozygous and homozygous Gtf2ird1 mutant mice to a variety of neurobehavioral paradigms that evaluate different domains of central nervous system functioning. In addition, to further probe the mechanism by which haploinsufficiency for this gene might translate into altered behavior, we analyzed neurotransmitter levels in a variety of different brain areas.
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- Materials and methods
Individuals with WBS display a spectrum of clinical, neurobehavioral and cognitive abnormalities, but to date only a single gene (elastin) has been unequivocally implicated in any aspect of the disorder, namely the cardiovascular abnormalities (Curran et al. 1993). This is, at least in part, because of the paucity and phenotypic heterogeneity of individuals with smaller deletions of the region, making genotype–phenotype correlation difficult. In addition, it is possible, perhaps likely, that there are combinatorial consequences of multiple gene deletion. As a result of these limitations, mouse models have become a logical route to understanding the role of specific genes in the complex WBS phenotype. Here, we have shown that mice either heterozygously or homozygously disrupted for the Gtf2ird1 transcription factor exhibit some behavioral features of WBS that have not been reported in previous mouse models (Crackower et al. 2003; Fujiwara et al. 2006; van Hagen et al. 2006; Hoogenraad et al. 2002; Li et al. 1998; Meng et al. 2002; Tassabehji et al. 2005; Zhao et al. 2005). The distinctive behavioral profile seen in people with WBS is one of the defining features of WBS, and insight into the genetic basis of aspects of this unique phenotype will be important not only for understanding the molecular basis of WBS, but of normal human behavior.
Perhaps the most intriguing finding in mice with disruption of Gtf2ird1 was the decrease in aggressive behavior toward, and increased social interest in, unfamiliar mice. This was paired with a significantly blunted natural fear response. People with WBS almost universally exhibit overfriendliness with inappropriate social boundaries and lack of normal risk assessment, and frequently approach and/or initiate social interactions with strangers (Doyle et al. 2004; Klein-Tasman & Mervis 2003). The altered behaviors seen in the Gtf2ird1 mutants are intriguingly reminiscent of these hallmark features of WBS.
In an apparent direct contrast to people with WBS, the majority of whom have either generalized anxiety disorder or simple non-social phobias (Dykens 2003; Mervis & Klein-Tasman 2000), Gtf2ird1−/− mice displayed decreased anxiety when tested in the elevated plus maze and open field (Fig. 4). Although this was somewhat unexpected, it could be that alteration in the expression of GTF2IRD1 affects the anxiety state of both humans and Gtf2ird1-targeted mice, albeit in different ways. Alternatively, it is possible that these tests do not examine the same behavioral response as that seen in people with WBS. The presentation of a novel object normally elicits fear response, or neophobia, in animals. However, Gtf2ird1-targeted mice showed an enhanced interest in the home cage cube exploration test (Fig. 4d), in part confirming their less fearful state observed in the plus maze and open field. However, it is also possible that the apparent lack of fear (approach/avoidance behavior) exhibited by these mice is masking any potential anxiety that would normally be elicited by the elevated plus maze or open field. Additional testing, including the administration of anxiogenic drugs before testing in the elevated plus maze may help distinguish between lack of fear response and reduced anxiety-observed behavior of Gtf2ird1-targeted mice.
The neural mechanisms regulating social behavior and aggression are still being elucidated, but models of human aggression specifically implicate the amygdala and paralimbic prefrontal regions (Davidson et al. 2000), with lesions of the amygdala in non-human primates resulting in impaired or inappropriate social function (Amaral 2002; Prather et al. 2001). The molecular mechanisms governing the perception of, and reaction to, danger and threatening situations have also been linked to the amygdala, with the orbitofrontal cortex hypothesized to play a key role in modulating limbic reactivity to threat (Davidson et al. 2000; Izquierdo et al. 2005). Recent functional neuroimaging studies of people with WBS showed reduced activation of the amygdala when processing images of threatening faces, suggesting an underlying dysfunction (Meyer-Lindenberg et al. 2005). Amygdala function is often measured using associative fear conditioned learning and memory, specifically with a cue, such as a tone. Impaired cued fear conditioning was noted in both the Gtf2ird1+/− and Gtf2ird1−/− mice although no difference was detected in contextual fear conditioning. Biochemically, oxytocin has been firmly established as central mediator of social behavior through its action in the amygdala, and stathmin, a molecule highly expressed in this region, has also been implicated in both innate and learned fear (Shumyatsky et al. 2005; Winslow & Insel 2002). Mice lacking GDI1, which encodes a protein controlling the activity of the small guanosine triphosphatase of the Rab family in vesicle fusion and intracellular trafficking, also exhibit decreased aggression and altered social behavior, but the biological basis for this remains unknown (D’Adamo et al. 2002). It will be interesting to investigate these and other molecules and genes that have been implicated in fear and social response, in the Gtf2ird1 mutant mice.
It is known that 5-HT plays an important role in emotional disorders with decreased 5-HT levels shown to cause an increase in aggressive behavior in rodents (Vergnes et al. 1986) as well as depression in humans (Ogilvie et al. 1996), while administration of a 5-HT(1B) receptor agonist reduced aggression in rats (De Almeida et al. 2006). Alteration of 5-HT(1A) and 5-HT(2A) receptor density and binding have also been linked to changes in aggression in rodents (Caramaschi et al. 2007; Schiller et al. 2006). Gtf2ird1−\− mice showed a significant increase of the 5-HT metabolite 5-HIAA in the frontal and parietal cortices and the amygdala, although the tissue 5-HT content was not significantly increased in any of the regions tested. These observations suggest an alteration in postsynaptic 5-HT turnover rather than an overall increase in 5-HT production. Further experiments, including studies of 5-HT receptor density and binding, are needed to determine the mechanism by which serotonergic pathways are altered in the Gtf2ird1-targeted mice.
The increased sociability seen in the Gtf2ird1 mutant mice suggests that this gene plays an important role in the regulation of normal social interaction in rodents, possibly in pathways that influence transcription of the molecules mentioned above. Interestingly, although almost all individuals with WBS exhibit the same cognitive profile, three children with smaller than normal deletions of the WBS region, leaving genes at the distal end intact, did not exhibit hypersociability (Doyle et al. 2004; van Hagen et al. 2006; Tassabehji et al. 2005). Recently, an individual was identified with a unique deletion that extends out of the WBS region toward the telomere, resulting in hemizygosity for GTF2IRD1 and GTF2I, but no other genes from the common WBS deletion region (Edelmann et al. 2006). This patient exhibited inappropriate friendliness toward strangers, even though her phenotype was compounded by a diagnosis of autism. It seems likely, therefore, that in humans, the WBS behavioral profile may be the product of the combinatorial effect of hemizygosity for both GTF2IRD1 and GTF2I. Mouse models with multiple gene deletions will go some way to elucidate this and will be very helpful in studying the interplay between different genes within the WBS deletion.
Even homozygous disruption of Gtf2ird1 was not sufficient to produce deficits in learning tasks that rely heavily on the hippocampus, such as the Morris water maze test of spatial learning and memory and contextual fear conditioning. This was supported by electrophysiological recordings of the CA1 region of the hippocampus, which showed normal basal synaptic activity and long-term potentiation (data not shown). Reports of two patients with unusual deletions of 7q11.23 support the role of other genes in spatial learning. One individual with a deletion that removed GTF2IRD1, but left GTF2I intact, did not exhibit as severe visual spatial impairment as people with WBS (Tassabehji et al. 2005), whereas a second individual with a deletion that removed both GTF2IRD1 and GTF2I, but none of the other commonly deleted genes (Edelmann et al. 2006), showed weakness in visuospatial skills equivalent to that seen in people with WBS. Our findings, together with the published reports, suggest that GTF2IRD1 and GTF2I may both play an important role in proper visuospatial cognition, but that their effect may only be evident when both are in the heterozygous state.
Growth deficiency has long been associated with WBS, with mean adult height corresponding to the third percentile in both sexes (Pankau et al. 1992). Consistent with this, mild growth deficiencies were observed in Gtf2ird1-deficient mice, as was recently reported for homozygous Gtf2ird1 mutants (Tassabehji et al. 2005). Similar results were seen in a mouse deficient for another gene from the WBS deletion, Cyln2 (Hoogenraad et al. 2002), raising the possibility that the growth deficiency seen in individuals with WBS results from additive hemizygosity for Cyln2 and Gtf2ird1.
Craniofacial abnormalities were also reported in Gtf2ird1 mutants, including a proportion of homozygous mice with severely misaligned jaws (Tassabehji et al. 2005). We did not see any obvious craniofacial abnormalities in our homozygous mice although a careful quantitative analysis may be required to identify subtle abnormalities. A recent report of a second Gtf2ird1 null mouse generated using fusion of a LacZ cassette into exon 2 of the gene, also failed to detect any overt craniofacial abnormalities (Palmer et al. 2006). The difference in phenotype penetrance may be because of the influence of other genes involved in the pathways regulating craniofacial development. Our mice are maintained on a mixed, predominantly outbred genetic background, whereas the insertional mutants were on a mixed inbred background (C57BL/6 × CBA/J). The penetrance of craniofacial anomalies in mouse models of Smith–Magenis syndrome was recently shown to be highly dependent on genetic background (Yan et al. 2007).
Disruption of only a single copy of Gtf2ird1 in mice results in decreased aggression and natural fear response, and increased social interaction combined with impaired amygdala-based learning. These alterations closely resemble phenotypes observed in WBS and suggest that the haploinsufficiency of GTF2IRD1 contributes to the physical and behavioral deficits associated with this disorder. The Gtf2ird1 mutant mice present an opportunity to identify downstream genes and pathways that are essential for proper development and maintenance of certain aspects of human behavior. These mice provide the basis for manipulations not possible in humans, for example, the global analysis of gene expression in the amygdala before and after behavioral testing, and should prove a valuable model for an intriguing human disorder.