Figure 1.
Juvenile social interactions in B6, BTBR, and F2 mice.
(A) As compared to B6, BTBR exhibited significantly lower levels of nose-to-nose sniff, (E) follow, (G) push-crawl, (C) and a trend toward lower levels of front-approach. (B, F, H, D) In the present study, F2 subjects exhibited broad ranges of these behaviors. A, C, E, G display Mean ± SEM; bold red line in B, D, F & H indicates mean value. B6 and BTBR data from A, E and G are adapted from Yang et al., 2009, European Journal of Neuroscience, Copyright (c) 2009, Blackwell Publishing, Ltd., with reuse and reprint permission via the Copyright Clearance Center (CCC).
Figure 2.
Adult social approach in B6, BTBR, and F2 mice.
(A) B6 exhibited high sociability, spending significantly more time in the chamber containing the novel mouse than in the chamber containing the novel object, and (C) more time sniffing the novel mouse than the novel object. BTBR exhibited low sociability and did not show preference for the novel mouse. (E) As compared to B6, BTBR mice exhibited significantly higher levels of self-grooming in an empty cage. In the present study, F2 mice exhibited broad ranges of time spent in the novel mouse chamber (B), time sniffing the novel mouse (D), and self-grooming in an empty cage (F). The parental strains and F2 generation were tested using identical methods, by the same NIMH investigators in the Bethesda laboratory. The F2 scores shown in panel B and panel D represent the time spent in the chamber with the novel mouse Panel A and the time spent sniffing the novel mouse and time spent sniffing the novel object in Panel C, respectively. (A, C)*p<.05 for the comparison between novel mouse and novel object; (E)*p<.01 vs. B6. A, C, E display Mean ± SEM; bold red line in B, D, F indicates mean value. B6 and BTBR data from A, C, and are adapted from Yang et al., 2009, European Journal of Neuroscience, Copyright (c) 2009, Blackwell Publishing, Ltd., with reuse and reprint permission via the Copyright Clearance Center (CCC).
Figure 3.
Callosal phenotype of the BTBR T+ tf/J mouse.
(A) As compared to the C57BL6/J mouse strain, a midsaggital section of the BTBR T+ tf/J mouse brain displays deficits in the formation of commissural structures (CC – red, HC – green; AC – blue); (B, C) a complete loss of the corpus callosum (CC), a reduction in the (D) length and (E) area of the hippocampal commissure (HC), and (F) a slightly enlarged anterior commissure (AC). (B,–F) Graph of the distribution of (B) corpus callosum, and (D) hippocampal commissure lengths, and (F) anterior commissure areas of F2 intercrossed progeny, indicates a widespread distribution.
Figure 4.
QTL for autism-relevant behaviors.
For the related juvenile reciprocal social interaction trait parameters “novel mouse sniff” (the cumulative time spent sniffing the novel mouse) and “diff sniff” (the difference between seconds spent sniffing the novel mouse and seconds spent sniffing the novel object) (A, B), a significant QTL was found on chromosome 3 (LOD = 4.52). (B) An additional QTL was found on the X chromosome (LOD = 4.12) for the “novel mouse sniff” trait, but did not meet genome-wide significance for the trait “diff sniff.” (C) The trait “Juvenile Nose to Nose” displayed a QTL on chromosome 1 (LOD = 4.43) and chromosome 9 (LOD = 3.80). (D) The trait “Juvenile Push Crawl” displayed QTL on chromosome 10 (LOD = 4.77) and chromosome 12 (LOD = 4.09), respectively. Red arrows indicate statistically significant QTL; red hatched arrows indicate transgressive loci.
Table 1.
List of QTL for Autism-Relevant Behaviors and Callosal Phenotypes.
Figure 5.
QTL for commissural morphology.
For the trait “corpus callosum length” (A), a significant QTL was found on chromosome 4 (LOD = 20.21). (B) Interestingly, a different QTL, also located on chromosome 4, was found for the “corpus callosum area” trait (LOD = 10.9), and (C) was also noted as a secondary locus for the trait “hippocampal commissure length” (LOD = 5.27), whereas the primary locus for this trait was found on chromosome 6 (LOD = 5.36). (D) The trait “anterior commissure area” displayed a novel QTL on chromosome 12 (LOD = 6.11), and additionally, shared the same locus on chromosome 4 (LOD = 4.48) as the trait “corpus callosum length.” Red arrows indicate statistically significant QTL; red hatched arrows indicate transgressive loci.
Figure 6.
Gene candidate analysis flowchart.
Following fine mapping, QTL meeting genome-wide significance of 5% were further analyzed to identify a list of gene candidates for future study. Sequencing data from the BTBR T+tf/J strain was compared to known SNPs in the C57BL/6J and LP/J strains, and potential pathogenicity of BTBR-specific SNPs were determined (using Polyphen-2). Genes containing potentially pathogenic SNPs were filtered to only those involved in neurological behavior or within the nervous system, and then filtered again to limit genes to only those with cortical expression to develop the final list of gene candidates for AgCC and autism-relevant behaviors in the BTBR T+tf/J strain of mouse.
Table 2.
List of Candidate Genes located within QTL Significant for Social Deficits and Callosal Phenotypes.
Figure 7.
Categories of candidate genes for AgCC and autism-relevant phenotypes in the BTBR mouse.
Candidate genes identified in the present study fall into four major protein categories: (1) Developmental proteins, involved in processes of cell cycle regulation, cell adhesion, axon growth, guidance, migration, actin-binding and cytoskeletal organization, (2) Synaptic and receptor proteins, (3) Kinases, and (4) Immune and heat shock proteins. *Indicates gene has been previously implicated in ASD (SFARIgene; gene.sfari.org) #Indicates gene family member has been previously implicated in ASD (SFARIgene; gene.sfari.org).