Fig 1: Ptgds transcription modulated by the Auts2-Ctip2-NuRD complex in postmitotic GCs is involved in NSC/IPC migration from SPZ to SGZ.(A and B) Detection of Ptgds mRNA expression in DG by RNAscope [(A) scale bar, 100 µm] and quantitative real-time polymerase chain reaction (PCR) [(B) four mice per genotype). (C) Detection of Ptgds protein expression in DG; five mice per genotype. (D to F) Nex-Cre mice were injected with AAV2/9-DIO-Ptgds-2A-EGFP or AAV2/9-DIO-EGFP in DG at P3 for assessment of IPC migration [(D) scale bar, 100 µm], the area of DG [(E) scale bar, 200 µm], and SGZ proliferation [(F) scale bar, 100 µm]; 15 to 18 slices from four mice per genotype. (G). Three-chamber SI test. Nex-Cre mice with Ptgds overexpression showed a decreased preference for S2 over S1; 11 to 12 mice per group. (H to K) Pairwise interactions among AUTS2, CTIP2, and MTA1. The lysates of HEK293T cells were immunoprecipitated, and the precipitates were analyzed with antibodies against HA, FLAG, and Myc. (L) DG lysates from Auts2fl/fl mice and Auts2fl/fl; Emx1-Cre mice at P4 were immunoprecipitated with antibody against Ctip2. The precipitates were analyzed with antibodies against Auts2, Mta1, and Ctip2; four mice per genotype. (M) Transcriptional activity was detected by using dual luciferase assay from five independent tests. (N to P) Chromatin immunoprecipitation (ChIP) assay. DG extracts at P4 were immunoprecipitated with antibodies against Ctip2 and Hdac2 to obtain the precipitates. The segments of Ptgds promoter were detected by PCR and agarose gel electrophoresis with Gapdh as control (N). The fold change of Ptgds promoter segment 2 immunoprecipitated by Hdac2 (O) and Ctip2 (P) antibodies was analyzed by quantitative real-time PCR; three mice per genotype. (Q) Proposed model for Ptgds transcription modulated by the Auts2-Ctip2-NuRD complex in postmitotic GCs. cko, conditional knockout. Data were shown as means ± SEM. Two-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 2: Co-expression of AUTS2 and GALNT17 in different cell types in CB. Expression of AUTS2 (red; top) and GALNT17 (red; bottom) in various cell types was determined by co-staining with antibodies to different markers, and by cell shape and location in CB. Both proteins are expressed in Bergmann glia (A and E; arrowheads) as marked by GFAP in green at P1, and in PCs (B and F; stars) as marked by Calbindin (CalB) in green at P60. Both proteins were also co-expressed in cerebellar GCs (B and F; asterisks), stellate cells (B and F; arrowheads), large neurons in the deep cerebellar nuclei (C and G; arrowheads), and choroid plexus (D and H). scale bar = 10 µm. PCL = Purkinje cell layer; EGL = external granule layer; ML = molecular layer; GCL = granule cell layer.
Fig 3: Cerebellar and hippocampal expression of Auts2 transcripts in 16GsoT/T compared to WT controls at P14 and P35. Auts2 isoforms (T1, T4, T5 and T6) expression in 16GsoT/T HC and CB at P14 and P35 was normalized to WT levels, which were set at 1. The expression of Auts2 isoforms T1, T4 and T6 was decreased in P14 16GsoT/T HC, while in CB, only Auts2 isoform T6 was significantly reduced. At P35, Auts2 isoform T6 was significantly decreased in HC, and no Auts2 isoform is dysregulated in CB. n = 3 for each genotype and sample. ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001. CB = cerebellum; HC = hippocampus.
Fig 4: Deletion of Auts2 in forebrain excitatory neural progenitors leads to DG hypoplasia and autism-related behaviors.(A and B) The mRNA expression pattern of Auts2 in wild-type HIP (A) and FC (B) at different postnatal days. Scale bars, 200 µm. (C) Auts2 mRNA expression in HIP from Auts2fl/fl mice and Auts2fl/fl;Emx1-Cre mice at P2 and P10. Scale bars, 200 µm. (D) Auts2 protein expression in FC and HIP of Auts2fl/fl mice and Auts2fl/fl;Emx1-Cre mice at P4. (E) P14 coronal sections revealed abnormal morphology of DG in Auts2fl/fl;Emx1-Cre mice. Scale bar, 500 µm. (F to H) Morphological changes in postnatal Auts2-deleted HIP at P4 (F), P14 (G), and P60 (H). Scale bars, 200 µm. (I to M) Quantification of the suprapyramidal blade (I) and infrapyramidal blade (J) length, the CA length (L), and the area of DG (K) and CA (M) at P4, P14, and P60; three mice per genotype at each time point. (N to P) Three-chamber SI test. Representative heatmaps for the different genotypes (N). In trial 1 (S1 versus EM), mice of both genotypes spent more time in close interaction with S1 than EM (O). In trial 2 (S1 versus S2), Auts2fl/fl;Emx1-Cre mice displayed no preference for the novel social partner S2 [(P) 13 to 14 male mice per genotype]. (Q) Social recognition memory test. Male Auts2fl/fl mice, but not Auts2fl/fl;Emx1-Cre mice, habituated to the same mouse (trials 1 to 3) and dishabituated to a novel mouse (trial 4); 12 male mice per genotype. (R) Numbers of ultrasonic vocalizations (USVs) emitted by pups during maternal separation at P3, P6, and P9; 23 to 24 pups per genotype. (S) Increased stereotyped grooming and digging behaviors in male Auts2fl/fl;Emx1-Cre mice; 10 to 11 male mice per genotype. Data were shown as means ± SEM. Two-tailed Student’s t test and two-way analysis of variance (ANOVA). **P < 0.01 and ***P < 0.001; n.s., no significance.
Fig 5: Sequence and genomic map location of the 16Gso translocation breakpoints. (A) Sequences obtained by PCR across the 16Gso breakpoint aligned with the WT sequence at the same genomic sites. Sequences that align between WT chr5 and chr8 and the mutant DNA sequences are shown in caps, while lower case letters represent WT sequences beyond the breakpoint site. The alignment revealed the deletions on both translocated chromosomes (underlined), involving one copy of a homologous triplet (GCT) shared by the two WT chromosomes in the translocated chr5;8, and deletion of a single C at the chr8;5 breakpoint site. (B) The breakpoint-flanking sequences mapped between Galnt17 and Auts2 genes on chr5, and upstream of Sox1 on chr8 (red arrows on each chromosome map). Genes are drawn to scale within each map; arrows indicate direction of transcription for each gene. For Auts2, transcripts T1, T4, T5, and T6 are labeled at the locations of their promoters. Above both maps, vertical lines demark a genomic distance of 500 kb.
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