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. 2020 Mar 11;9:e52981. doi: 10.7554/eLife.52981

Figure 1. Dnmt3a cKO and Mecp2 cKO mice show overlapping as well as distinct neurological deficits.

(A) Mice that lack Dnmt3a or MeCP2 in inhibitory neurons present with hindlimb spasticity. (B) Obsessive grooming is increased in both cKO models. (C) Nest building, (D) grip strength, (E) open field, (F) fear conditioning tests revealed impairments in both cKO lines. (G) Self-injury in Dnmt3a cKO and Mecp2 cKO mice necessitated humane euthanasia. (H) Example traces of miniature inhibitory postsynaptic currents (mIPSCs) recorded in the dorsal striatum. Both cKO models show similar alterations in (I) amplitude. (J) Weekly body weight records for Dnmt3a cKO and Mecp2 cKO mice showed only Dnmt3a cKO mice (here separated by sex- see Materials and methods) were runted. (K) Dnmt3a cKO and Mecp2 cKO mice showed opposite alterations in acoustic startle response. (L) Only Mecp2 cKO mice displayed impairment on the parallel rod. M) Dnmt3a cKO mice had to undergo earlier euthanasia than Mecp2 cKO mice due to the severity of their self-lesioning. n = 11–52 (behavior), n = 5–9 mice per genotype with 24–50 neurons total (electrophysiology). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Supplementary file 1 for full statistics.

Figure 1.

Figure 1—figure supplement 1. Protein levels of Dnmt3a and MeCP2 are reduced in inhibitory neurons of conditional knockout mice.

Figure 1—figure supplement 1.

(A) Western blot of half brain hemisphere from Dnmt3a or Mecp2 cKO mice demonstrating loss of each protein. (B) Immunofluorescence (IF) images of WT, Dnmt3a cKO or Mecp2 cKO mice probing for Dnmt3a or MeCP2 (red), the Sun1-sfGFP-myc-PA fusion protein that marks the nuclear envelope and is dependent on Cre expression (green), and DAPI to mark genomic DNA (blue). n = 3 mice per genotype (western), n = 3 mice (IF) representative images for two brain regions shown.
Figure 1—figure supplement 2. Supplemental behavioral and physiological data for Dnmt3a cKO and Mecp2 cKO mice.

Figure 1—figure supplement 2.

(A) Both mouse lines showed decreased rearing in the open field test. (B) Dnmt3a cKO mice showed impaired fear learning, whereas Mecp2 cKO mice did not differ from control mice. (C) Cue memory was normal in both cKO mice. (D–F) Tests for anxiety-like behaviors (open field, light dark or elevated plus maze, respectively). (G) Hot plate and (H) tail flick testing for nociceptive pain in both cKO mice. (I) Rotarod test for motor learning and coordination and (J) the partition test for social interaction. (K–N) Frequency, charge, rise and decay measures from mIPSCs from the striatum. (O) Only Mecp2 cKO had a trend for increased pre-pulse inhibition. n = 11–50 per genotype (behavior), n = 5–9 mice per genotype with 24–50 neurons total (electrophysiology), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Supplementary file 1 for full numbers and statistics.
Figure 1—figure supplement 3. X-gal staining of LacZ+/-;Slc32a1-Cre+/- and control E14.5 embryos shows widespread expression of Cre transgene in the nervous system and select expression in peripheral tissues.

Figure 1—figure supplement 3.

(A) X-gal staining of LacZ+/- control and (B) LacZ+/-;Slc32a1-Cre+/- reporter mice demonstrating specific staining of tissues with expression of Cre recombinase under the Slc32a1 promoter. (C–H) Images showing positive X-gal staining in nervous system tissues consistent across three biological replicates: (C) brain (D) spinal cord (E) dorsal root ganglia (F) inner ear (G) olfactory epithelium (H) and eye. (I–L) Images showing positive X-gal staining in non-neural tissues consistent across three biological replicates: (I) kidney (J) heart (K) skin (only in select regions: surrounding genital tubercle and tail and area on the face/nose) (L) ureter and bladder. Additional positive X-gal staining was noted in the (M) testis in the only embryo where the testis were visible, as well as rare, scattered cells of the intestine and pancreas (not shown). The observation of expression of Slc32a1-Cre in the testis is consistent with our noted germline recombination of floxed genes if F1, Dnmt3aflox/+;Slc32a1-Cre+/- males are used to generate F2 offspring; a breeding scheme we deliberately avoided to generate our experimental mice (see Materials and methods for more details).
Figure 1—video 1. Forepaw stereotypies are apparent in Dnmt3a cKO mice.
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Video shows an example of forepaw stereotypies in a Dnmt3a cKO male mouse at 8 week of age.