Loss of Satb2 in the Cortex and Hippocampus Leads to Abnormal Behaviors in Mice

Abstract

Satb2-associated syndrome (SAS) is a genetic disorder that results from the deletion or mutation of one allele within the Satb2 locus. Patients with SAS show behavioral abnormalities, including developmental delay/intellectual disability, hyperactivity, and symptoms of autism. To address the role of Satb2 in SAS-related behaviors and generate an SAS mouse model, Satb2 was deleted in the cortex and hippocampus of Emx1-Cre; Satb2^flox/flox [Satb2 conditional knockout (CKO)] mice. Satb2 CKO mice showed hyperactivity, increased impulsivity, abnormal social novelty, and impaired spatial learning and memory. Furthermore, we also found that the development of neurons in cortical layer IV was defective in Satb2 CKO mice, as shown by the loss of layer-specific gene expression and abnormal thalamocortical projections. In summary, the abnormal behaviors revealed in Satb2 CKO mice may reflect the SAS symptoms associated with Satb2 mutation in human patients, possibly due to defective development of cortical neurons in multiple layers including alterations of their inputs/outputs.

Keywords: Satb2; Satb2-associated syndrome; cerebral cortex; hippocampus; mouse behavior.

FIGURE 1

Reduced body weight and survival rate in adult Satb2 CKO mice. (A,B) Satb2 immunostaining verified that Satb2 was deleted in the cerebral cortex in Satb2 CKO mice. (C,D) The growth curve of male (C) and female (D) control, Satb2 heterozygotes, and Satb2 CKO mice. Both male and female Satb2 heterozygotes (Emx1-Cre; Satb2^flox/+) and CKO mice had a normal body weight until postnatal day 15 (P15). The one-way ANOVA test measured the significant differences between these groups appeared at P15 in male mice {F[2,32] = 14.55, P < 0.0001; Tukey’s multiple comparisons test showed P = 0.7673 (controls vs. Satb2 heterozygotes); P < 0.0001 (controls vs. Satb2 CKO mice); P = 0.0008 (Satb2 heterozygotes vs. Satb2 CKO mice)}, and P20 in female mice {F[2,31] = 4.697, P = 0.0165; Tukey’s multiple comparisons test showed P = 0.9858 (controls vs. Satb2 heterozygotes); P = 0.0225 (controls vs. Satb2 CKO mice); P = 0.0406 (Satb2 heterozygotes vs. Satb2 CKO mice)}. (E,F) The survival rate of male (E) and female (F) wild-type, Satb2 heterozygotes, and Satb2 CKO mice. The survival rate of female Satb2 CKO mice was lower than male CKO mice. In (C,D), data are presented as the mean ± SEM. Scale bar = 200 μm (B).

FIGURE 2

Hyperactivity and increased impulsivity in Satb2 CKO mice. (A) The traveling trace in the open field of the control and Satb2 CKO mice. (B) The total distance traveled by Satb2 CKO mice in the open field was much higher than that of control mice (t[37] = 4.553, P < 0.001). (C) The ambulatory time of Satb2 CKO mice was longer than that of the control mice in the open field (t[37] = 4.951, P < 0.001). (D,E) The distance traveled was analyzed every 5 min; it gradually reduced in control mice over time, whereas this was not observed in Satb2 CKO mice as shown by persistent moving in the field (D). The traveled distance in block 6 was significantly lower than that in block 1 in control mice and comparable with that in block 1 in Satb2 CKO mice (E, t_control[36] = 2.687, P = 0.0108; t_CKO[38] = 0.1505, P = 0.8812). (F) The average velocity of Satb2 CKO mice was lower than that of control mice (t[37] = 2.196, P = 0.0345). (G,H) The CAR test was performed to test impulsivity. During the 30-min test, more Satb2 CKO mice fell from the platform than did control mice, none of which fell (G). In total, about 80% of Satb2 CKO mice fell from the platform (F). In (B–F), data are presented as the mean ± SEM; each dot represents a mouse in (B,C,F). ^∗P < 0.05, ^∗∗∗P < 0.001 via Student’s t-tests in (B,C,E,F). Data were analyzed using a repeated measures ANOVA in (D).

FIGURE 3

Reduced anxiety-like behaviors in Satb2 CKO mice. (A,B) The dark-light choice test showed that Satb2 CKO mice spent more time in the light box than did control mice (A, t[29] = 3.362, P = 0.0022). The transition number of Satb2 CKO mice was comparable with that of control mice (B, t[29] = 1.361, P = 0.1841), probably due to more time spent in the light box. (C,D) The elevated plus maze test showed that the time Satb2 CKO mice spent in the open arms was significantly longer than that of control mice (C, t[28] = 6.597, P < 0.0001). Satb2 CKO mice exhibited a higher transition number than control mice (D, t[28] = 2.699, P = 0.0116). In (A–D), data are presented as the mean ± SEM and each dot represents a mouse. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 via Student’s t-tests.

FIGURE 4

Impaired sensorimotor gating and social novelty in Satb2 CKO mice. (A) The pre-pulse inhibition (PPI) test shows that Satb2 CKO mice had a significant PPI deficit compared with control mice at 73 and 82 dB pre-pulse intensities. The repeated measures ANOVA (2 genotypes × 3 pre-pulse intensities with repeated measures on pre-pulse intensities) showed that both the genotypes and pre-pulse intensities affected response reactivity {genotypes effect: F[1,42] = 17.76, P = 0.0001; pre-pulse intensities effect: F[2,42] = 33.52, P < 0.0001; interaction: F[2,42] = 1.246, P = 0.298; P = 0.0074 (73dB); P = 0.0165 (82 dB); P = 0.5856 (65 dB)}. (B,C) The three-chamber social interaction test was performed. Satb2 CKO mice showed a preference for the animate stranger mouse (S1) over the inanimate ball, with no difference compared with control mice (B, t_control[14] = 2.755, P = 0.0155; t_CKO[14] = 9.409, P < 0.0001). Satb2 CKO mice showed a similar preference to the S2 mouse and S1 mouse, while the control mice showed a preference for the S2 mouse (C, t_control[14] = 2.673, P = 0.0182; t_CKO[14] = 0.3878, P = 0.7040). (D) In the direct social interaction test, the interaction time of Satb2 CKO mice was longer compared with control mice (t[15] = 3.057, P = 0.0080). In (A–D), data are presented as the mean ± SEM and each dot represents a mouse in (D). ^∗P < 0.05 via Student’s t-tests in (B–D), and repeated measures ANOVA in (A). ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 5

Impaired spatial learning and memory in Satb2 CKO mice. The Morris water maze test was performed. (A) During the learning phase, the latency to the platform was significantly longer in Satb2 CKO mice than in the control mice. The analysis of the latency using a repeated measures ANOVA (2 genotypes × 7 days with repeated measures on days) showed that both the genotypes and training days affected learning ability (genotype effect: F[1,18] = 38.53, P < 0.0001; days effect: F[6,108] = 11.28, P < 0.0001; and interaction: F[6,108] = 1.59, P = 0.1571). (B) During the memory trial, the latency to the platform was longer in Satb2 CKO mice compared with the control mice (t[18] = 2.872, P = 0.0101). (C) During the memory trial, the mean distance to the platform was higher in the Satb2 CKO mice compared with the control mice (t[18] = 3.944, P = 0.0010). (D) The duration in the target quadrant was shorter in Satb2 CKO mice compared to control mice (t[18] = 3.493, P = 0.0026). (E) The frequency of platform crossings was lower in Satb2 CKO mice compared to control mice (t[18] = 3.633, P = 0.0019). (F) Satb2 CKO mice showed a similar swimming velocity to control mice (t[18] = 0.6638, P = 0.5153). In (A), data are presented as the mean and ^∗P < 0.05 by a repeated measures ANOVA. In (B–F), data are presented as the mean ± SEM, and each dot represents a mouse. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 via Student’s t-tests.

FIGURE 6

Altered corpus callosum and gene expression in the cortex of Satb2 CKO mice. (A–B^′) A few of AuCl₃ labeled axons cross the midline at anterior section (A^′) but not posterior section (B^′) in Satb2 CKO mice compared with control mice (A,B). (C,C^′) Cux2 mRNA was dramatically decreased in layers II–IV at P6 in Satb2 CKO mice (C^′) compared with control mice (C). (D,D^′) Ctip2 mRNA was increased in Satb2 CKO cortex (D^′) compared with the control mice (D). (E,E^′) Tle4 mRNA was increased in VI of Satb2 CKO mice (E^′) when compared with control mice (E). Scale bar = 500 μm (C^′), 200 μm (A^′,B^′,D^′,E^′).

FIGURE 7

Loss of “barrels” in layer IV in Satb2 CKO mice. (A,A^′) RORβ mRNA was dramatically reduced in the cortex at P0 in Satb2 CKO mice (A^′) compared with that in control mice (A). (B–C^′) The expression of RORβ mRNA was hardly detected in Satb2 CKO mice at P6 (B^′) and totally absent at P15 (C^′). (D,D^′) 5-HTT-positive fibers reached the deep layer of cortex in Satb2 CKO mice (D^′) at P0 as control mice did so (D). (E,E^′) Septal regions among the “barrels” can be clearly seen in a control mouse, as shown by the presence of densely packed Hoechst-stained cells (E, arrows), but were not observed in Satb2 CKO mice, as shown by homogenous distribution of stained cells (E^′, arrows). (F–G^′) Thalamocortical axons labeled by 5-HTT in layer IV were clustered within “barrels” in control mice (F,G), while they were sparsely and homogenously distributed in layer IV of Satb2 CKO mice (F^′,G^′). Scale bar = 500 μm (A^′–C^′,F^′) and 100 μm (D^′–E^′,G^′).