miR-9 regulates basal ganglia-dependent developmental vocal learning and adult vocal performance in songbirds
- Zoe Piccus
- Xiaofang Zhang
- Huidi Yang
- Hannah Jarrell
- Yan Ding
- Zhaoqian Teng
- Ofer Tchernichovski
- XiaoChing Li
- Louisiana State University School of Medicine, United States
- Hunter College, United States
Figures
Figure 1 with 2 supplements
A lentiviral approach to manipulate miR-9 expression in Area X of the zebra finch brain.
(A) Schematic drawing of the song control circuit in the zebra finch brain. The motor pathway (green), which connects HVC (used as a proper name) to RA (robust nucleus of the arcopallium) and …
Figure 1—figure supplement 1
A lentiviral approach to manipulate miR-9 expression.
(A) 293T cells infected with the lenti-control or the lenti-miR-9 virus. Note the virally expressed mCherry fluorescence. (B) Western blot analysis of FOXP1 and FOXP2 protein expression in 293T …
Figure 1—figure supplement 2
Impacts of viral injection on Area X volume and neuron number.
(A) Area X volumes in animals receiving no injection or receiving lenti-control virus injection or receiving lenti-miR-9 virus injection. p = 0.73, F(2,12) = 0.3255, one-way ANOVA; n = 3 for the …
Figure 2
Syllable omission in pupils with miR-9 overexpression in juvenile Area X.
(A) Experimental timeline for viral injection, song learning, and song recording. (B) Representative sonograms of a tutor, a control pupil, and an miR-9 pupil. Syllables are labeled alphabetically, …
-
Figure 2—source data 1
- https://doi.org/10.7554/eLife.29087.006
Figure 3 with 1 supplement
miR-9 overexpression in juvenile Area X impairs song learning.
(A) Motif similarity scores of uninjected pupils, pupils injected with control virus, and pupils injected with miR-9 virus. p = 0.002, U = 0, two-tailed Mann-Whitney U Test; n = 6 for control and …
-
Figure 3—source data 1
- https://doi.org/10.7554/eLife.29087.009
Figure 3—figure supplement 1
Maximum motif similarity of control and miR-9 pupils at 100 d.
For each pupil, 20 pupil motif renditions were compared to 10 tutor motif renditions. Then, the 10 highest values among the 200 pairwise measurements were averaged for each pupil. p = 0.002, U = 2, …
Figure 4
Overexpression of miR-9 in juvenile Area X impairs song performance in adulthood.
(A) Representative sonograms of miR-9 pupil Yellow 33 showing an example of variation in syllable order. (B) Representative sonograms of miR-9 pupil Yellow 23 showing an example of syllable …
-
Figure 4—source data 1
- https://doi.org/10.7554/eLife.29087.011
Figure 5
Overexpression of miR-9 in juvenile Area X abolishes social-context-dependent modulation of vocal performance.
(A) An example syllable of a directed song. The lowest constant fundamental frequency (cFF) of a syllable segment between the red and the blue lines was measured with SAP. (B) Variations in cFF of …
-
Figure 5—source data 1
- https://doi.org/10.7554/eLife.29087.013
Figure 6 with 1 supplement
Developmental trajectory of vocal learning in virus-injected pupils.
(A) Motif similarity scores of the control and miR-9 pupils during developmental learning. p = 0.002, U = 0 at 65 d, 80 d, and 100 d, and p = 0.008, U = 0 at 150 d, two-tailed Mann-Whitney U test; n …
Figure 6—figure supplement 1
Amount of song production by control and miR-9 pupils at 65 d and 100 d.
The number of syllables produced from 8:00 a.m. to 12:00 p.m. during one day are plotted. p = 0.485, U = 13 for the 65 d groups; p = 0.132, U = 8.00 for the 100 d groups, two-tailed Mann-Whitney U …
Figure 7 with 1 supplement
miR-9 overexpression in Area X downregulates the expression of FoxP1 and FoxP2 and disrupts dopamine signaling.
(A) Expression of FoxP1 and FoxP2 mRNAs in Area X measured 4 weeks after injection with the lenti-miR-9 virus by qRT-PCR. p = 0.006, t(12) = 4.608 for FoxP1; p < 0.0001, t(12) = 7.062 for FoxP2, …
Figure 7—figure supplement 1
Western blots showing specificity of detection of FoxP2 and DARPP-32 proteins in Area X tissue indicated by inhibition of detection of(A) FoxP2 and (B) DARPP-32 after primary antibodies were pre-incubated with 10-fold molar excess of competing peptides.
https://doi.org/10.7554/eLife.29087.017
Figure 8 with 1 supplement
miR-9 overexpression alters expression levels of numerous FoxP1 and FoxP2 downstream target genes in Area X.
Blue bars, upregulated genes; red bars, downregulated genes. Genes are grouped into five functional groups: (A) neurotransmitter receptors and channels; (B) proteins involved in dendritic growth and …
Figure 8—figure supplement 1
Downstream target genes of FoxP1 and FoxP2 that were unaffected by miR-9 overexpression in Area X.
Reverse transcription and qRT-PCR were performed twice, and qRT-PCR was performed in triplicate. All p values > 0.05, unpaired t-test. n = 7 for all genes except for AUTS2, CDH8, CNTN4, DLGAP2, n = 4…
Additional files
-
Supplementary file 1
Symbols, names, and functional descriptions of genes shown in Figure 8.
Functional descriptions are based on NCBI (National Center for Biotechnology Information) database. For associations between genes and neural developmental and mental disorders, see the online database SFARI Genes (Simons Foundation Autism Research Initiative) and references therein. For genes not included in the SFARI Gene list (e.g., KCNA4 and SRPX2), see references cited in the main text.
- https://doi.org/10.7554/eLife.29087.020
-
Supplementary file 2
Sequences of primers used in all qRT-PCR experiments.
- https://doi.org/10.7554/eLife.29087.021
-
Audio file 1
A song file of tutor Blue 63 (the tutor for Gray 180 and Gray 139).
- https://doi.org/10.7554/eLife.29087.022
-
Audio file 2
A song file of control pupil Gray 180.
- https://doi.org/10.7554/eLife.29087.023
-
Audio file 3
A song file of miR-9 pupil Gray 139.
- https://doi.org/10.7554/eLife.29087.024
-
Audio file 4
A song file of tutor Black 45 (the tutor for Yellow 30).
- https://doi.org/10.7554/eLife.29087.025
-
Audio file 5
A song file of miR-9 pupil Yellow 30.
- https://doi.org/10.7554/eLife.29087.026
-
Transparent reporting form
- https://doi.org/10.7554/eLife.29087.027
