Deleting Mecp2 from the cerebellum rather than its neuronal subtypes causes a delay in motor learning in mice
- Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, United States
- Program in Developmental Biology, Baylor College of Medicine, United States
- Medical Scientist Training Program, Baylor College of Medicine, United States
- Department of Human and Molecular Genetics, Baylor College of Medicine, United States
- Howard Hughes Medical Institute, Baylor College of Medicine, United States
- Department of Neuroscience, Baylor College of Medicine, United States
- Department of Pathology and Immunology, Baylor College of Medicine, United States
- Department of Neurology, Baylor College of Medicine, United States
- Department of Pediatrics, Baylor College of Medicine, United States
Figures
Figure 1
MeCP2 is expressed in cerebellar neurons of 6-month-old wild-type mice.
(A–C) MeCP2 (magenta) staining in NeuN+ neurons (yellow) in the granular layer (A: solid cyan circle), Calbindin+ neurons (yellow) in the Purkinje cell layer (B: solid cyan circle), and Parvalbumin+ …
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Figure 1—source data 1
Related to Figure 1.
- https://cdn.elifesciences.org/articles/64833/elife-64833-fig1-data1-v1.xlsx
Figure 2 with 3 supplements
Deleting Mecp2 from the cerebellum, but not its neuronal subtypes, causes motor learning deficits in 6-month-old mice.
(A) Breeding scheme to generate WT, Cre, Flox, and KO mice. (B) Latency to fall on the rotarod over four training days in the En1Cre group. (C) Latency to fall on the rotarod over four training days …
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Figure 2—source data 1
Related to Figure 2.
- https://cdn.elifesciences.org/articles/64833/elife-64833-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Deletion of Mecp2 from the cerebellum.
(A) En1Cre expression was determined by the pattern of tdTomato (magenta) in En1Cre;Rosa26lsl-tdTomato reporter mice. Scale bar, 1 mm. (B) Immunostaining showing co-expression of tdTomato (magenta) …
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Figure 2—figure supplement 1—source data 1
Related to Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/64833/elife-64833-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Cre expression and Mecp2 deletion in cerebellar neuron subtypes.
(A) Atoh1Cre expression was determined by the pattern of tdTomato (magenta) in Atoh1Cre;Rosa26lsl-tdTomato reporter mice. Scale bar, 1 mm. (B) Immunostaining in Atoh1Cre;Rosa26lsl-tdTomato reporter …
Figure 2—figure supplement 3
Deleting Mecp2 from the cerebellum does not cause other behavioral abnormalities.
(A) Latency to fall on the rotarod in 2-month-old mice. (B) Latency to fall on the rotarod in 4-month-old mice. A new cohort of WT, Cre, Flox, and KO mice was assessed for each time point. (C) …
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Figure 2—figure supplement 3—source data 1
Related to Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/64833/elife-64833-fig2-figsupp3-data1-v1.xlsx
Figure 3
Purkinje cell firing rate is more irregular in cerebellar KO mice but is independent of overt morphological abnormalities.
(A) Schematic of in vivo extracellular recording of Purkinje cells. (B) Photograph of a recording electrode inside a surgically implanted recording chamber. (C) Representative traces of Purkinje …
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Figure 3—source data 1
Related to Figure 3.
- https://cdn.elifesciences.org/articles/64833/elife-64833-fig3-data1-v1.xlsx
Figure 4 with 2 supplements
The loss of Mecp2 in cerebellar neurons disrupts histone methylation in heterochromatic foci.
The intensity of DAPI and histone methylation marks was measured in the heterochromatic foci of granule cells (GC), Purkinje cells (PC), and molecular layer interneurons (ML) in Flox and KO mice. (A)…
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Figure 4—source data 1
Related to Figure 4.
- https://cdn.elifesciences.org/articles/64833/elife-64833-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Heterochromatin architecture in mice lacking Mecp2 in cerebellar neurons.
(A) Representative images of cerebellar neurons in Flox and KO mice showing DAPI and H3K4me3. Scale bar, 5 µm. (B) Representative images of cerebellar neurons in Flox and KO mice showing DAPI and …
Figure 4—figure supplement 2
Cerebellar neurons were identified by the expression of RORα and NeuN.
(A) Neurons stained for H3K4me3 from Figure 4—figure supplement 1 were co-stained for RORα and NeuN to identify molecular layer interneurons (RORα), Purkinje cells (RORα), and granule cells (NeuN). …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Rabbit polyclonal anti-Histone H3 | Abcam | RRID:AB_302613 Cat# ab1791 | 1:20,000 |
| Antibody | Rabbit monoclonal anti-MeCP2 | Cell Signaling Technologies | RRID:AB_2143849 Cat# 3456 | 1:1000 |
| Antibody | Mouse monoclonal anti-MeCP2 | Abcam | RRID:AB_881466 Cat# ab50005 | 1:500 |
| Antibody | Mouse monoclonal anti-NeuN | Millipore Sigma | RRID:AB_2298772 Cat# MAB377 | 1:250 |
| Antibody | Mouse monoclonal anti-Calbinin-D28K | Swant | RRID:AB_10000347 Cat# 300 | 1:10,000 |
| Antibody | Rabbit polyclonal anti-Parvalbumin | Swant | RRID:AB_2631173 Cat# PV27 | 1:1000 |
| Antibody | Rabbit polyclonal anti-Vglut1 | Synaptic Systems | RRID:AB_887877 Cat# 135 302 | 1:1000 |
| Antibody | Guinea pig polyclonal anti-Vglut2 | Synaptic Systems | RRID:AB_887884 Cat# 135 404 | 1:1000 |
| Antibody | Guinea pig polyclonal anti-Vgat | Synaptic Systems | RRID:AB_1106810 Cat# 131 005 | 1:1000 |
| Antibody | Rabbit polyclonal anti-histone H3 (tri methyl K4) | Cell Signaling Technologies | RRID:AB_2616028 Cat# 9751 | 1:500 |
| Antibody | Rabbit polyclonal anti-histone H3 (tri methyl K9) | Abcam | RRID:AB_306848 Cat# ab8898 | 1:500 |
| Antibody | Rabbit polyclonal anti-histone H3 (tri methyl K27) | Millipore Sigma | RRID:AB_310624 Cat# 07–449 | 1:500 |
| Antibody | Goat polyclonal anti-RORα | Santa Cruz Biotechnology | RRID:AB_655755 Cat# sc-6062 | 1:250 |
| Antibody | Goat anti-mouse IgG Alexa Fluor 488 | Thermo Fischer | RRID:AB_2534069 Cat# A-11001 | 1:500 |
| Antibody | Goat anti-guinea pig IgG Alexa Fluor 555 | Thermo Fischer | RRID:AB_2535856 Cat# A-21435 | 1:500 |
| Antibody | Goat anti-rabbit IgG Alexa Fluor 647 | Thermo Fischer | RRID:AB_2535812 Cat# A-21244 | 1:500 |
| Antibody | Donkey anti-rabbit IgG Alexa Fluor 488 | Thermo Fischer | RRID:AB_2535792 Cat# A-21206 | 1:500 |
| Antibody | Donkey anti-goat IgG Alexa Fluor 555 | Thermo Fischer | RRID:AB_2535853 Cat# A-21432 | 1:500 |
| Antibody | Donkey anti-mouse IgG Alexa Fluor 647 | Thermo Fischer | RRID:AB_162542 Cat# A-31571 | 1:500 |
| Commercial assay, kit | Paraformaldehyde | Millipore Sigma | Cat# 158127 | |
| Commercial assay, kit | Pierce BCA Protein Assay | Thermo Fischer | Cat# 23225 | |
| Commercial assay, kit | FD Rapid Golgi Stain Kit | FD Neurotechnologies | Cat# PK401 | |
| Strain, strain background (Mus musculus) | (C57BL/6J) Rosa26lsl-tdTomato | The Jackson Laboratory | RRID: IMSR_JAX:007914 | |
| Strain, strain background (Mus musculus) | (C57BL/6J) En1Cre | The Jackson Laboratory | RRID: IMSR_JAX:007916 | |
| Strain, strain background (Mus musculus) | (C57BL/6J) Atoh1Cre | The Jackson Laboratory | RRID:IMSR_JAX:011104 | |
| Strain, strain background (Mus musculus) | (C57BL/6J) Pcp2Cre | The Jackson Laboratory | RRID:IMSR_JAX:004146 | |
| Strain, strain background (Mus musculus) | (C57BL/6J) Ptf1aCre | The Jackson Laboratory | RRID:IMSR_JAX:007909 | |
| Strain, strain background (Mus musculus) | (C57BL/6J) Mecp2flox/+ and Mecp2flox/flox | The Jackson Laboratory | RRID:IMSR_JAX:007177 | |
| Other | DAPI stain | Thermo Fischer | RRID:AB_2629482 Cat# D-1306 | |
| Other | Tissue-Tek Optimum Cutting Temperature Compound | Sakura | Cat# 4583 | |
| Other | Superfrost Plus microscope slides | Thermo Fischer | Cat# 12-550-15 | |
| Other | ProLong Gold Antifade mounting medium | Thermo Fischer | Cat# P10144 | |
| Other | NuPAGE LDS sample buffer | Thermo Fischer | Cat# NP0007 | |
| Other | NuPAGE Sample reducing agent | Thermo Fischer | Cat# NP0004 | |
| Other | 15-well NuPAGE 4–12% Bis–Tris Gel | Thermo Fischer | Cat# NP0336BOX | |
| Other | 15-well NuPAGE 4–12% Bis–Tris Gel | Thermo Fischer | Cat# NP0336BOX | |
| Other | PVDF blotting membrane | GE Healthcare Life Sciences | Cat# 10600021 | |
| Other | Odyssey TBS Blocking Buffer | LI-COR Biosciences | Cat# 927–50000 | |
| Software, algorithm | Spike2 | Cambridge Electronic Design | RRID:SCR_000903 | |
| Software, algorithm | MATLAB | Mathworks | RRID: SCR_001622 | |
| Software, algorithm | Image Studio Lite | LI-COR Biosciences | RRID:SCR_013715 | |
| Software, algorithm | ImageJ-Fiji | Other | RRID:SCR_002285 | |
| Software, algorithm | Neurolucida 360 | MBF Biosciences | RRID:SCR_016788 | |
| Software, algorithm | Neurolucida Explorer | MBF Biosciences | RRID:SCR_017348 | |
| Software, algorithm | Imaris | Bitplane | RRID:SCR_007370 | |
| Software, algorithm | Prism | GraphPad Software | RRID: SCR_002798 |
Additional files
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Source data 1
Statistical analysis related to Figure 1, Figure 2, Figure 2—figure supplement 1, Figure 2—figure supplement 3, Figure 3, and Figure 4.
- https://cdn.elifesciences.org/articles/64833/elife-64833-data1-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/64833/elife-64833-transrepform-v1.docx
