1. Neuroscience

Deleting Mecp2 from the cerebellum rather than its neuronal subtypes causes a delay in motor learning in mice

  1. Nathan P Achilly
  2. Ling-jie He
  3. Olivia A Kim
  4. Shogo Ohmae
  5. Gregory J Wojaczynski
  6. Tao Lin
  7. Roy V Sillitoe
  8. Javier F Medina
  9. Huda Y Zoghbi  Is a corresponding author
  1. Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, United States
  2. Program in Developmental Biology, Baylor College of Medicine, United States
  3. Medical Scientist Training Program, Baylor College of Medicine, United States
  4. Department of Human and Molecular Genetics, Baylor College of Medicine, United States
  5. Howard Hughes Medical Institute, Baylor College of Medicine, United States
  6. Department of Neuroscience, Baylor College of Medicine, United States
  7. Department of Pathology and Immunology, Baylor College of Medicine, United States
  8. Department of Neurology, Baylor College of Medicine, United States
  9. Department of Pediatrics, Baylor College of Medicine, United States
https://doi.org/10.7554/eLife.64833
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Cite this article
  1. Nathan P Achilly
  2. Ling-jie He
  3. Olivia A Kim
  4. Shogo Ohmae
  5. Gregory J Wojaczynski
  6. Tao Lin
  7. Roy V Sillitoe
  8. Javier F Medina
  9. Huda Y Zoghbi
(2021)
Deleting Mecp2 from the cerebellum rather than its neuronal subtypes causes a delay in motor learning in mice
eLife 10:e64833.
https://doi.org/10.7554/eLife.64833
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4 figures, 1 table and 2 additional files

Figures

Figure 1
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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+ neurons (yellow) in the molecular layer (C: solid cyan circle). Scale bar, 25 µm. (D) Quantification of the percentage of NeuN+, Calbindin+, and Parvalbumin+ neurons that express MeCP2. N = 4 biologically independent mice per group. Data are presented as mean ± s.e.m.

Figure 1—source data 1

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Figure 2 with 3 supplements
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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 in mice lacking Mecp2 in the granule cells (Atoh1Cre), Purkinje cells (Pcp2Cre), and Purkinje cells and molecular layer interneurons (Ptf1aCre). (D) Schematic of eyeblink conditioning that pairs an LED light (conditioned stimulus, cs) with an air puff (unconditioned stimulus, us) to generate an anticipatory eyelid closure (conditioned response) before the air puff. (E) Response probability and amplitude of eyelid closure over 12 training days in Flox and KO mice. N = 8–17 biologically independent mice per group. Data are presented as mean ± s.e.m. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. ns (p>0.05), *(p<0.05), **(p<0.01), ****(p<0.0001).

Figure 2—source data 1

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Figure 2—figure supplement 1
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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) with Parvalbumin (yellow), a marker of Purkinje cells (solid cyan circle), and molecular layer interneurons (dashed cyan circle). Scale bar, 25 µm. (C) Immunostaining showing co-expression of tdTomato (magenta) with NeuN (yellow), a marker of granule cells (solid cyan circle). Scale bar, 25 µm. (D) Quantification of MeCP2 protein levels normalized to Histone H3 in the cerebellum and hippocampus of Flox and KO mice. (E) Immunostaining of MeCP2 (magenta) in the cerebellum and hippocampus of Flox and KO mice. Scale bar, 50 µm. N = 3 biologically independent mice per group. Data are presented as mean ± s.e.m. Statistical significance was determined by two-tailed, unpaired student’s t-test. ns (p>0.05), ****(p<0.0001).

Figure 2—figure supplement 1—source data 1

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Figure 2—figure supplement 2
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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 mice showing co-expression of tdTomato (magenta) with NeuN (yellow), a marker of granule cells (solid cyan circle), but not in cells of the Purkinje layer (dashed cyan square) or molecular layer (dashed cyan circle). Scale bar, 25 µm. (C) Immunostaining of MeCP2 (magenta) and NeuN (yellow) in the cerebellum showing the absence of MeCP2 in granule cells of KO mice (solid cyan circle), but not in cells of the Purkinje layer (dashed cyan circle). Scale bar, 25 µm. (D) Pcp2Cre expression was determined by the pattern of tdTomato (magenta) in Pcp2Cre;Rosa26lsl-tdTomato reporter mice. Scale bar, 1 mm. (E) Immunostaining in Pcp2Cre;Rosa26lsl-tdTomato reporter mice showing co-expression of tdTomato (magenta) with Parvalbumin (yellow) in the Purkinje cell layer (solid cyan circle), but not the molecular layer (dashed cyan circle). Scale bar, 25 µm. (F) Immunostaining of MeCP2 (magenta) and Calbindin (yellow) in the cerebellum showing the absence of MeCP2 in Purkinje cells of KO mice (solid cyan circle), but not in molecular layer interneurons (dashed cyan circle). Scale bar, 25 µm. (G) Ptf1aCre expression was determined by the pattern of tdTomato (magenta) in Ptf1aCre;Rosa26lsl-tdTomato reporter mice. Scale bar, 1 mm. (H) Immunostaining in Ptf1aCre;Rosa26lsl-tdTomato reporter mice showing co-expression of tdTomato (magenta) with Parvalbumin (yellow) in the Purkinje layer (solid cyan circle) and molecular layer (dashed cyan circle). Scale bar, 25 µm. (I) Immunostaining of MeCP2 (magenta) and Parvalbumin (yellow) in the cerebellum showing the absence of MeCP2 in Purkinje cells (solid cyan circle) and molecular layer interneurons of KO mice (dashed cyan circle). Scale bar, 25 µm.

Figure 2—figure supplement 3
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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) Footslip count on the parallel rod assay normalized to total distance traveled. (D) Total distance traveled in the open-field assay. (E) Grip strength normalized to body weight. (F) Hang time on an inverted wire grid. (G) Maximum acoustic startle response to a 120 dB stimulus. (H) Pre-pulse inhibition to 74, 78, and 82 dB pre-pulses. (I) Interaction time in the three-chamber social interaction assay between a novel mouse or object. (J) Time spent freezing during contextual memory recall. (K) Time spent freezing during cued memory recall. N = 10–17 biologically independent mice per group. For (C–K), mice are 6 months old. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way (C–G, J–K) or two-way ANOVA (A–B, H–I) with Tukey’s multiple comparisons test. ns (p>0.05).

Figure 2—figure supplement 3—source data 1

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Figure 3
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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 cell firing in Flox and KO mice displaying simple spikes (ss) and complex spikes (cs). (D) Simple spike firing rate, complex spike firing rate, coefficient of variation (CV), and coefficient of variation 2 (CV2). Simple and complex spikes were differentiated by their characteristic waveforms during offline analysis. (E) Golgi stain of Purkinje cells in Flox and KO mice. Scale bar, 25 µm. Inner panel demonstrates dendritic spines on Purkinje cells. Scale bar, 5 µm. Sholl analysis and spine density quantification in Flox and KO mice. (F) Staining and quantification of Vglut1 (cyan), Vglut2 (magenta), and Vgat (gray) puncta density in the cerebellum of Flox and KO mice. Scale bar, 25 µm. For (D), 23–27 neurons were analyzed from three biologically independent mice per group. For (E), 10–15 neurons were analyzed from three biologically independent mice per group. For (F), N = 4 biologically independent mice per group. Data are presented as mean ± s.e.m. Statistical significance was determined by two-tailed, unpaired student’s t-test (D, F) and two-way ANOVA with Tukey’s multiple comparisons test (E). ns (p>0.05), **(p<0.01).

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
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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) Normalized DAPI intensity in heterochromatic foci. (B) Normalized H3K4me3 intensity in heterochromatic foci. (C) Normalized H3K9me3 intensity in heterochromatic foci. (D) Normalized H3K27me3 intensity in heterochromatic foci. 15–20 neurons were analyzed per mouse. Data were normalized to the values of Flox mice. N = 4–5 biologically independent mice per group. Data are presented as mean ± s.e.m. Statistical significance was determined by two-tailed, unpaired student’s t-test. ns (p>0.05), *(p<0.05), **(p<0.01).

Figure 4—source data 1

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Figure 4—figure supplement 1
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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 H3K9me3. Scale bar, 5 µm. (C) Representative images of cerebellar neurons in Flox and KO mice showing DAPI and H3K27me3. Scale bar, 5 µm.

Figure 4—figure supplement 2
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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). Scale bar, 5 µm. (B) Neurons stained for H3K9me3 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). Scale bar, 5 µm. (C) Neurons stained for H3K27me3 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). Scale bar, 5 µm.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional
information
AntibodyRabbit polyclonal anti-Histone H3AbcamRRID:AB_302613
Cat# ab1791		1:20,000
AntibodyRabbit monoclonal anti-MeCP2Cell Signaling TechnologiesRRID:AB_2143849
Cat# 3456		1:1000
AntibodyMouse monoclonal anti-MeCP2AbcamRRID:AB_881466
Cat# ab50005		1:500
AntibodyMouse monoclonal anti-NeuNMillipore SigmaRRID:AB_2298772
Cat# MAB377		1:250
AntibodyMouse monoclonal anti-Calbinin-D28KSwantRRID:AB_10000347
Cat# 300		1:10,000
AntibodyRabbit polyclonal anti-ParvalbuminSwantRRID:AB_2631173
Cat# PV27		1:1000
AntibodyRabbit polyclonal anti-Vglut1Synaptic SystemsRRID:AB_887877
Cat# 135 302		1:1000
AntibodyGuinea pig polyclonal anti-Vglut2Synaptic SystemsRRID:AB_887884
Cat# 135 404		1:1000
AntibodyGuinea pig polyclonal anti-VgatSynaptic SystemsRRID:AB_1106810
Cat# 131 005		1:1000
AntibodyRabbit polyclonal anti-histone H3 (tri methyl K4)Cell Signaling TechnologiesRRID:AB_2616028
Cat# 9751		1:500
AntibodyRabbit polyclonal anti-histone H3 (tri methyl K9)AbcamRRID:AB_306848
Cat# ab8898		1:500
AntibodyRabbit polyclonal anti-histone H3 (tri methyl K27)Millipore SigmaRRID:AB_310624
Cat# 07–449		1:500
AntibodyGoat polyclonal anti-RORαSanta Cruz BiotechnologyRRID:AB_655755
Cat# sc-6062		1:250
AntibodyGoat anti-mouse IgG Alexa Fluor 488Thermo FischerRRID:AB_2534069
Cat# A-11001		1:500
AntibodyGoat anti-guinea pig IgG Alexa Fluor 555Thermo FischerRRID:AB_2535856
Cat# A-21435		1:500
AntibodyGoat anti-rabbit IgG Alexa Fluor 647Thermo FischerRRID:AB_2535812
Cat# A-21244		1:500
AntibodyDonkey anti-rabbit IgG Alexa Fluor 488Thermo FischerRRID:AB_2535792
Cat# A-21206		1:500
AntibodyDonkey anti-goat IgG Alexa Fluor 555Thermo FischerRRID:AB_2535853
Cat# A-21432		1:500
AntibodyDonkey anti-mouse IgG Alexa Fluor 647Thermo FischerRRID:AB_162542
Cat# A-31571		1:500
Commercial assay, kitParaformaldehydeMillipore SigmaCat# 158127
Commercial assay, kitPierce BCA Protein AssayThermo FischerCat# 23225
Commercial assay, kitFD Rapid Golgi Stain KitFD NeurotechnologiesCat# PK401
Strain, strain background (Mus musculus)(C57BL/6J)
Rosa26lsl-tdTomato		The Jackson LaboratoryRRID: IMSR_JAX:007914
Strain, strain background (Mus musculus)(C57BL/6J)
En1Cre		The Jackson LaboratoryRRID: IMSR_JAX:007916
Strain, strain background (Mus musculus)(C57BL/6J)
Atoh1Cre		The Jackson LaboratoryRRID:IMSR_JAX:011104
Strain, strain background (Mus musculus)(C57BL/6J)
Pcp2Cre		The Jackson LaboratoryRRID:IMSR_JAX:004146
Strain, strain background (Mus musculus)(C57BL/6J)
Ptf1aCre		The Jackson LaboratoryRRID:IMSR_JAX:007909
Strain, strain background (Mus musculus)(C57BL/6J)
Mecp2flox/+ and Mecp2flox/flox		The Jackson LaboratoryRRID:IMSR_JAX:007177
OtherDAPI stainThermo FischerRRID:AB_2629482
Cat# D-1306
OtherTissue-Tek Optimum Cutting Temperature CompoundSakuraCat# 4583
OtherSuperfrost Plus microscope slidesThermo FischerCat# 12-550-15
OtherProLong Gold Antifade mounting mediumThermo FischerCat# P10144
OtherNuPAGE LDS sample bufferThermo FischerCat# NP0007
OtherNuPAGE Sample reducing agentThermo FischerCat# NP0004
Other15-well NuPAGE 4–12% Bis–Tris GelThermo FischerCat# NP0336BOX
Other15-well NuPAGE 4–12% Bis–Tris GelThermo FischerCat# NP0336BOX
OtherPVDF blotting membraneGE Healthcare Life SciencesCat# 10600021
OtherOdyssey TBS Blocking BufferLI-COR BiosciencesCat# 927–50000
Software, algorithmSpike2Cambridge Electronic DesignRRID:SCR_000903
Software, algorithmMATLABMathworksRRID: SCR_001622
Software, algorithmImage Studio LiteLI-COR BiosciencesRRID:SCR_013715
Software, algorithmImageJ-FijiOtherRRID:SCR_002285
Software, algorithmNeurolucida 360MBF BiosciencesRRID:SCR_016788
Software, algorithmNeurolucida ExplorerMBF BiosciencesRRID:SCR_017348
Software, algorithmImarisBitplaneRRID:SCR_007370
Software, algorithmPrismGraphPad SoftwareRRID: SCR_002798

Additional files

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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https://cdn.elifesciences.org/articles/64833/elife-64833-transrepform-v1.docx

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  1. Nathan P Achilly
  2. Ling-jie He
  3. Olivia A Kim
  4. Shogo Ohmae
  5. Gregory J Wojaczynski
  6. Tao Lin
  7. Roy V Sillitoe
  8. Javier F Medina
  9. Huda Y Zoghbi
(2021)
Deleting Mecp2 from the cerebellum rather than its neuronal subtypes causes a delay in motor learning in mice
eLife 10:e64833.
https://doi.org/10.7554/eLife.64833