Injection of AAV-PHP.eB-hSyn1-Cre into the retro-orbital sinus yields widespread biodistribution and rapid recombination in the brain.

(A) Schematic depicting injections of AAV-PHP.eB-hSyn1-Cre into the retro-orbital sinus (RO) of P14 Ai9 reporter mice and experimental time course of biodistribution assessment. (B-E) Representative Cre recombinase immunofluorescence and resulting tdTomato immunofluorescence in grayscale at the indicated ages. Scale bars, 2 mm (low-magnification) or 0.5 mm (high-magnification).

Representative biodistribution of AAV-hSyn1-mCherry and AAV-hSyn1-Cre in adult Tcf4-LSL mice.

(A) Schematic depicting study paradigm including retro-orbital injection of AAV-PHP.eB-hSyn1-mCherry and AAV-PHP.eB-hSyn1-Cre in Tcf4-LSL mice, assessment of mCherry (native) or Cre (antibody-enhanced) immunofluorescence (IF) at postnatal day 60 and behavioral assessments and postmortem measures in mice separate from immunofluorescence. (B,C) Representative immunofluorescence for mCherry and Cre at postnatal day 60. Scale bars, 2 mm (low-magnification) or 0.5 mm (high-magnification).

Behavioral recovery following juvenile reinstatement of TCF4 in Tcf4-LSL mice is limited to spatial memory.

(A) Schematic of behavioral study timeline and behavioral assays in WT and Tcf4-LSL mice, following injection with AAV-hSyn1-mCherry or AAV-hSyn1-Cre. (B-G) Behavioral assessments, n = 25-30 mice per group. (B) Total distance traveled in the open field assay. Main effect of group by one-way ANOVA (p < 0.0001). (C) Distance traveled over time in the open field assay. Main effect of genotype (p < 0.0001) and time (p = 0.0085) by two-way ANOVA. (D) Time spent in and (E) entries to the open arms of the elevated plus maze. Main effect of group by one-way ANOVA (p < 0.0001, p < 0.0001, respectively). (F) Time spent in the center of the elevated plus maze. No effect by one-way ANOVA. (G) Relative novel object exploration time in the object location memory assay. Main effect of group by one-way ANOVA (p = 0.0024). (H) Percentage of nesting material used over time in the nest building assay, n = 23-26 mice per group. Main effect of genotype (p < 0.0001), trial (p < 0.0001), and interaction (p < 0.0001) by two-way ANOVA. Comparisons between WT Cherry - LSL Cherry (gray) and LSL Cre-LSL Cherry (red) are plotted. Data are represented as means ± SEM. Tukey’s multiple comparisons post hoc testing. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Juvenile reinstatement of TCF4 in Tcf4-LSL mice does not improve body weight or brain weight phenotypes.

(A-B) Body weights of (A) male and (B) female WT and Tcf4-LSL mice following injection with AAV-hSyn1-mCherry or AAV-hSyn1-Cre, n = 12-16 mice per group. (C) Brain weights of WT and Tcf4-LSL mice following injection with AAV-hSyn1-mCherry or AAV-hSyn1-Cre, n = 10-12 mice per group. Comparisons between WT Cherry, LSL Cherry, and LSL Cre groups are plotted. Main effect of group by one-way ANOVA (p < 0.0001). Data are represented as means ± SEM. Tukey’s multiple comparisons post hoc testing. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Juvenile reinstatement of TCF4 in Tcf4-LSL mice partially improves transcriptional dysregulation.

(A-D) Relative expression of (A) Tcf4, (B) Mal, (C) Glra3, and (D) Tcerg1l in the hippocampus of WT and Tcf4-LSL mice following injection with AAV-hSyn1-mCherry or AAV-hSyn1-Cre, normalized to average of WT groups. Comparisons between WT Cherry, LSL Cherry, and LSL Cre groups are plotted, n = 13-17 mice per group. Main effect of group by one-way ANOVA (p < 0.0001, p = 0.0002, p = 0.0033, p = 0.0002 for A-D, respectively). Data represent means ± SEM. Tukey’s multiple comparisons post hoc testing. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Summary of mouse behavioral and anatomical phenotypes with demonstrated recovery (R) or no recovery (NR) following TCF4 reinstatement at various stages of development from Kim et al., 2022 and the present study.

Primers used throughout study.