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The relative contribution of proximal 5' flanking sequence and microsatellite variation on brain vasopressin 1a receptor (Avpr1a) gene expression and behavior.

Donaldson ZR, Young LJ - PLoS Genet. (2013)

Bottom Line: Previous work has suggested that both the proximal 5' flanking region and a polymorphic microsatellite element within that region of the vole Avpr1a gene are associated with variation in V1a receptor (V1aR) distribution and behavior, but neither has been causally linked.This is the first direct evidence that polymorphic microsatellite elements near behaviorally relevant genes can contribute to diversity in brain gene expression profiles, providing a mechanism for generating behavioral diversity both at the individual and species level.However, our results suggest that many features of species-specific expression patterns are mediated by elements outside of the immediate 5' flanking region of the gene.

View Article: PubMed Central - PubMed

Affiliation: Division of Integrative Neuroscience, Department of Psychiatry, Columbia University, New York, New York, United States of America. zoe.donaldson@gmail.com

ABSTRACT
Certain genes exhibit notable diversity in their expression patterns both within and between species. One such gene is the vasopressin receptor 1a gene (Avpr1a), which exhibits striking differences in neural expression patterns that are responsible for mediating differences in vasopressin-mediated social behaviors. The genomic mechanisms that contribute to these remarkable differences in expression are not well understood. Previous work has suggested that both the proximal 5' flanking region and a polymorphic microsatellite element within that region of the vole Avpr1a gene are associated with variation in V1a receptor (V1aR) distribution and behavior, but neither has been causally linked. Using homologous recombination in mice, we reveal the modest contribution of proximal 5' flanking sequences to species differences in V1aR distribution, and confirm that variation in V1aR distribution impacts stress-coping in the forced swim test. We also demonstrate that the vole Avpr1a microsatellite structure contributes to Avpr1a expression in the amygdala, thalamus, and hippocampus, mirroring a subset of the inter- and intra-species differences observed in central V1aR patterns in voles. This is the first direct evidence that polymorphic microsatellite elements near behaviorally relevant genes can contribute to diversity in brain gene expression profiles, providing a mechanism for generating behavioral diversity both at the individual and species level. However, our results suggest that many features of species-specific expression patterns are mediated by elements outside of the immediate 5' flanking region of the gene.

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The 5′ flanking region of Avpr1a contributes to neural V1aR expression patterns.In order to determine whether the replaced 5′ flanking region influences V1a receptor patterns, we compared brains of pvKI-long mice (B) with those of WT mice (C). pvKI-long mice display V1a patterns that contain elements of both mice and prairie vole patterns (A). Specifically, mice carrying the prairie vole promoter region displayed increased levels of V1a receptor in the dentate gyrus (DG), paraventricular nucleus of the thalamus (PVthal), and the central amygdala (CeA), but not in the ventral pallidum (VP), cingulate cortex (Cing), laterodorsal thalamus (LDthal), or medial amygdala (MeA).
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pgen-1003729-g003: The 5′ flanking region of Avpr1a contributes to neural V1aR expression patterns.In order to determine whether the replaced 5′ flanking region influences V1a receptor patterns, we compared brains of pvKI-long mice (B) with those of WT mice (C). pvKI-long mice display V1a patterns that contain elements of both mice and prairie vole patterns (A). Specifically, mice carrying the prairie vole promoter region displayed increased levels of V1a receptor in the dentate gyrus (DG), paraventricular nucleus of the thalamus (PVthal), and the central amygdala (CeA), but not in the ventral pallidum (VP), cingulate cortex (Cing), laterodorsal thalamus (LDthal), or medial amygdala (MeA).

Mentions: We used V1aR autoradiography as a proxy for Avpr1a gene expression since this technique is much more quantitative and sensitive than in situ hybridization, provides greater anatomical resolution than qPCR, and accurately reflects Avpr1a mRNA patterns (Young 1997). Furthermore, since the replaced region lies upstream of the transcription start site, variation in that sequence should not affect post-transcriptional processing. The greater signal to noise ratio of this technique allows us to detect relatively subtle differences in V1aR protein binding. Replacement of the 5′ flanking region of the murine Avpr1a locus with the same region from prairie voles yielded qualitative patterns of V1aR with elements of both mouse and prairie vole expression (Figure 3). To initially explore the effects of our manipulation on V1aR levels, we performed an overall ANOVA with three factors: genotype (WT, mvKI, pvKI-short, and pvKI-long), brain region, and sex. We identified main effects of genotype (F(3, 137) = 35.7, p<0.001), brain region (F(4, 137) = 1035.1, p<0.001), and sex (F(1, 137) = 10748.9, p<0.001). In addition, there were genotype×brain region (F(12, 137) = 8.902, p<0.001) and genotype×sex interactions (F(3, 137) = 3.451, p = 0.02), but no brain region×sex (F(4, 137) = 2.0, p = 0.09) or genotype×brain region×sex (F(12, 137) = 1.425, p = 0.16) interactions. Therefore, we did not analyze sex differences for each brain region in each of the lines. The main effect of sex appeared to be driven by the fact that females tend to have slightly higher levels of V1aR binding in some brain regions. However, since there are equal numbers of males and females across groups, and our focus was on the impact of promoter elements on expression, we collapsed males and females into a single group. We then performed three separate ANOVAs to test the a priori hypotheses regarding 1) the role of the 5′ flanking region, 2) species differences in the microsatellite, and 3) intraspecies differences in the microsatellite. To address the first of these, we compared pvKI-long mice homozygous for this region with WT mice. pvKI-long mice showed an increase in V1aR binding in the ventral pallidum (VP), central amygdala (CeA), paraventricular nucleus of the thalamus (PVthal), and dentate gyrus (DG) of the hippocampus, consistent with the distribution in the prairie vole. Specifically, we compared V1aR binding in pvKI-long mice to WT mice, and identified a significant effect of both brain region (F(4, 100) = 511.6, p<0.001) and genotype (F(4, 100) = 79.2, p<0.001) on V1aR levels. In addition, there was a significant interaction between brain region and genotype (F(4, 100) = 10.6, p<0.001), and simple main effects with Sidak-adjusted α showed that pvKI-long animals had significantly higher levels of V1aR than WT mice in the VP (p<0.001), CeA (p<0.001), PVThal (p<0.001), and DG (p<0.001), but not in the lateral septum (LS; p = 0.24; Figure 3). While it is intriguing that there is a significant difference in binding in the VP of these two lines, the actual difference is quite modest (on average, 1.15 fold higher). In addition, it should be noted that the binding in the dentate gyrus was distributed differently between prairie voles compared to pvKI-long mice, potentially due to different effects of the mouse versus vole coding sequences on receptor trafficking. However, the pvKI-long mice did not show the prairie vole specific binding in the laterodorsal thalamus (LDthal) or medial amygdala (MeA) (Figure 3). The overall similarities in binding pattern between the pvKI-long and WT mice demonstrate that elements outside of the replaced element (e.g. distal 5′ flanking regions, introns, and other surrounding elements) contribute significantly to species-typical expression, perhaps more so than the sequences in the 3.4 kb regions that we tested. However, the quantitative differences between these two lines of mice demonstrate conclusively that the proximal 3.4 kb of the 5′ flanking region also contributes to species-specific patterns of V1aR distribution in the brain.


The relative contribution of proximal 5' flanking sequence and microsatellite variation on brain vasopressin 1a receptor (Avpr1a) gene expression and behavior.

Donaldson ZR, Young LJ - PLoS Genet. (2013)

The 5′ flanking region of Avpr1a contributes to neural V1aR expression patterns.In order to determine whether the replaced 5′ flanking region influences V1a receptor patterns, we compared brains of pvKI-long mice (B) with those of WT mice (C). pvKI-long mice display V1a patterns that contain elements of both mice and prairie vole patterns (A). Specifically, mice carrying the prairie vole promoter region displayed increased levels of V1a receptor in the dentate gyrus (DG), paraventricular nucleus of the thalamus (PVthal), and the central amygdala (CeA), but not in the ventral pallidum (VP), cingulate cortex (Cing), laterodorsal thalamus (LDthal), or medial amygdala (MeA).
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Related In: Results  -  Collection

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pgen-1003729-g003: The 5′ flanking region of Avpr1a contributes to neural V1aR expression patterns.In order to determine whether the replaced 5′ flanking region influences V1a receptor patterns, we compared brains of pvKI-long mice (B) with those of WT mice (C). pvKI-long mice display V1a patterns that contain elements of both mice and prairie vole patterns (A). Specifically, mice carrying the prairie vole promoter region displayed increased levels of V1a receptor in the dentate gyrus (DG), paraventricular nucleus of the thalamus (PVthal), and the central amygdala (CeA), but not in the ventral pallidum (VP), cingulate cortex (Cing), laterodorsal thalamus (LDthal), or medial amygdala (MeA).
Mentions: We used V1aR autoradiography as a proxy for Avpr1a gene expression since this technique is much more quantitative and sensitive than in situ hybridization, provides greater anatomical resolution than qPCR, and accurately reflects Avpr1a mRNA patterns (Young 1997). Furthermore, since the replaced region lies upstream of the transcription start site, variation in that sequence should not affect post-transcriptional processing. The greater signal to noise ratio of this technique allows us to detect relatively subtle differences in V1aR protein binding. Replacement of the 5′ flanking region of the murine Avpr1a locus with the same region from prairie voles yielded qualitative patterns of V1aR with elements of both mouse and prairie vole expression (Figure 3). To initially explore the effects of our manipulation on V1aR levels, we performed an overall ANOVA with three factors: genotype (WT, mvKI, pvKI-short, and pvKI-long), brain region, and sex. We identified main effects of genotype (F(3, 137) = 35.7, p<0.001), brain region (F(4, 137) = 1035.1, p<0.001), and sex (F(1, 137) = 10748.9, p<0.001). In addition, there were genotype×brain region (F(12, 137) = 8.902, p<0.001) and genotype×sex interactions (F(3, 137) = 3.451, p = 0.02), but no brain region×sex (F(4, 137) = 2.0, p = 0.09) or genotype×brain region×sex (F(12, 137) = 1.425, p = 0.16) interactions. Therefore, we did not analyze sex differences for each brain region in each of the lines. The main effect of sex appeared to be driven by the fact that females tend to have slightly higher levels of V1aR binding in some brain regions. However, since there are equal numbers of males and females across groups, and our focus was on the impact of promoter elements on expression, we collapsed males and females into a single group. We then performed three separate ANOVAs to test the a priori hypotheses regarding 1) the role of the 5′ flanking region, 2) species differences in the microsatellite, and 3) intraspecies differences in the microsatellite. To address the first of these, we compared pvKI-long mice homozygous for this region with WT mice. pvKI-long mice showed an increase in V1aR binding in the ventral pallidum (VP), central amygdala (CeA), paraventricular nucleus of the thalamus (PVthal), and dentate gyrus (DG) of the hippocampus, consistent with the distribution in the prairie vole. Specifically, we compared V1aR binding in pvKI-long mice to WT mice, and identified a significant effect of both brain region (F(4, 100) = 511.6, p<0.001) and genotype (F(4, 100) = 79.2, p<0.001) on V1aR levels. In addition, there was a significant interaction between brain region and genotype (F(4, 100) = 10.6, p<0.001), and simple main effects with Sidak-adjusted α showed that pvKI-long animals had significantly higher levels of V1aR than WT mice in the VP (p<0.001), CeA (p<0.001), PVThal (p<0.001), and DG (p<0.001), but not in the lateral septum (LS; p = 0.24; Figure 3). While it is intriguing that there is a significant difference in binding in the VP of these two lines, the actual difference is quite modest (on average, 1.15 fold higher). In addition, it should be noted that the binding in the dentate gyrus was distributed differently between prairie voles compared to pvKI-long mice, potentially due to different effects of the mouse versus vole coding sequences on receptor trafficking. However, the pvKI-long mice did not show the prairie vole specific binding in the laterodorsal thalamus (LDthal) or medial amygdala (MeA) (Figure 3). The overall similarities in binding pattern between the pvKI-long and WT mice demonstrate that elements outside of the replaced element (e.g. distal 5′ flanking regions, introns, and other surrounding elements) contribute significantly to species-typical expression, perhaps more so than the sequences in the 3.4 kb regions that we tested. However, the quantitative differences between these two lines of mice demonstrate conclusively that the proximal 3.4 kb of the 5′ flanking region also contributes to species-specific patterns of V1aR distribution in the brain.

Bottom Line: Previous work has suggested that both the proximal 5' flanking region and a polymorphic microsatellite element within that region of the vole Avpr1a gene are associated with variation in V1a receptor (V1aR) distribution and behavior, but neither has been causally linked.This is the first direct evidence that polymorphic microsatellite elements near behaviorally relevant genes can contribute to diversity in brain gene expression profiles, providing a mechanism for generating behavioral diversity both at the individual and species level.However, our results suggest that many features of species-specific expression patterns are mediated by elements outside of the immediate 5' flanking region of the gene.

View Article: PubMed Central - PubMed

Affiliation: Division of Integrative Neuroscience, Department of Psychiatry, Columbia University, New York, New York, United States of America. zoe.donaldson@gmail.com

ABSTRACT
Certain genes exhibit notable diversity in their expression patterns both within and between species. One such gene is the vasopressin receptor 1a gene (Avpr1a), which exhibits striking differences in neural expression patterns that are responsible for mediating differences in vasopressin-mediated social behaviors. The genomic mechanisms that contribute to these remarkable differences in expression are not well understood. Previous work has suggested that both the proximal 5' flanking region and a polymorphic microsatellite element within that region of the vole Avpr1a gene are associated with variation in V1a receptor (V1aR) distribution and behavior, but neither has been causally linked. Using homologous recombination in mice, we reveal the modest contribution of proximal 5' flanking sequences to species differences in V1aR distribution, and confirm that variation in V1aR distribution impacts stress-coping in the forced swim test. We also demonstrate that the vole Avpr1a microsatellite structure contributes to Avpr1a expression in the amygdala, thalamus, and hippocampus, mirroring a subset of the inter- and intra-species differences observed in central V1aR patterns in voles. This is the first direct evidence that polymorphic microsatellite elements near behaviorally relevant genes can contribute to diversity in brain gene expression profiles, providing a mechanism for generating behavioral diversity both at the individual and species level. However, our results suggest that many features of species-specific expression patterns are mediated by elements outside of the immediate 5' flanking region of the gene.

Show MeSH