Fig 2: Wnt7a/b replenishment rescues the astrocytic network abnormality in the parental isolation model. A) Demonstration of micro‐injection procedure. The observation site was highlighted by the magenta square. B) Immunostaining of GFAP and active (nonphosphorylated) β‐catenin. Arrowheads highlight GFAP+ cells. Scale bar: 10 µm. GFAP+ astrocytes in Wnt7a and Wnt7b injected mice had a significantly higher level of active β‐catenin compared to 0.9% NaCl injected control (Saline). C) Immunostaining of BLBP showed that the number of BLBP+ cells was not altered by Wnt7a and Wnt7b injection, while the branch number was increased. The injection site was highlighted by the dashed line. Scale bar: 1000 µm. D) Wnt7a and Wnt7b injected mouse displayed increased GFAP+ cell branches. E,F) Immunostaining of Cx43, Cx30, and GFAP showed increased expression of Cx43, Cx30 in Wnt7a and Wnt7b injected mouse hippocampus. Scale bar: 50 µm. Data presented as mean ± SD. N = 3 mice for immunostaining experiments. p‐values are calculated using unpaired t‐test. *p < 0.05, **p < 0.01, n.s. not significant.

Fig 3: Identification of prognostic signatures. (A) Univariate Cox analysis of 28 KRAS-related genes with p < 0.05. (B) Kaplan–Meier survival analysis of NTNG1 and GJB6 in TCGA cohort (p < 0.05) (C) Multivariate Cox regression analysis of 5 prognostic genes determined by Kaplan–Meier survival analysis. (D) RT-PCR validation of GJB6 in different COAD cell lines. HT-29 was KRAS-wild while HCT-116 (G13) and SW620 (G12) were KRAS-mutated (E) Representive IHC results of GJB6 in CRC patients with KRAS mutated or not. Scale, 200x. Percent of GJB6 positive samples in KRAS-mutated and wild patients. (F) Kaplan–Meier survival analysis of KRGPS in the TCGA cohort (Log-rank test) (G) Validation of KRGPS in the GEO cohort by Kaplan–Meier survival analysis (Log-rank test).

Fig 4: Improved parental isolation‐induced neuropsychiatric behaviors and astrocyte maldevelopment. A) To set up the isolated group (Isolated), mouse pups were separated from their parents and housed individually for 8 h daily (9:00–17:00) from P2 to P12. In the control group (Father‐only), mouse pups were separated from their mothers but stayed with their fathers. Mouse pups were subjected to behavioral tests and IHC analysis on P23. B) Cliff avoidance test: Left: representative diagram of the cliff avoidance test. Black dots represent the mouse position at the experiment endpoint. Middle: reduced cliff avoidance reaction (CAR) was observed in the isolated mice compared to the Father‐only control, suggesting an impulsive behavioral trait. Right: the ratio of travel distance at the edge to total distance was not significantly different between the two groups. The log‐rank (Mantel‐Cox) test was used to compare survival curves. C) Tail suspension test: Isolated mice displayed prolonged immobility times, suggesting depressive behavior. D) Elevated plus maze test: Isolated mice showed reduced entries into the open arm in the elevated plus maze test, suggesting anxiety‐like behavior. E) Open field test: Left: representative diagram of the open field test. The isolated mice exhibited a preference for staying at the edges. Middle and right: the open field test showed that isolated mice traveled less distance and spent less time in the central area, while they showed no statistical differences in mobility, as suggested by travel distance measurements. F) Novel object recognition test: no statistical differences in cognitive function between the Father‐only control and isolated mice. G) BLBP staining showed no difference in astrocyte numbers, but instead displayed reduced astrocyte processes per cell in isolated mice. Scale bar: 100 µm. H) 3D reconstruction of GFAP staining. GFAP+ branch number was reduced in isolated mice. Scale bar: 10 µm. I) Immunostaining of GFAP and astrocytic network proteins Cx43. Reduced Cx43+ area was found in isolated mice. Scale bar: 50 µm. J) Immunostaining of GFAP and astrocytic network proteins Cx30. Reduced Cx30+ area was found in isolated mice. Scale bar: 50 µm. K) gap‐FRAP: Left: representative images of the gap‐FRAP experiment at three different time points. Right: gap‐FRAP experiments showed that the fluorescence recovery rate and amplitude were decreased in the astrocytes of the isolated group, suggesting reduced gap junction channel function. Scale bar: 20 µm. Data presented as mean ± SEM, n = 10. L) Calcium Imaging on acute brain slices stained with Rhod‐4 showed that Isolated mice displayed reduced astrocytic Ca2+ waves. Data presented as mean ± SEM, n = 27. M) The isolated mice demonstrated fewer c‐Fos+ neurons. Scale bar: 50 µm. Data presented as mean ± SD unless stated otherwise. N ≥ 7 mice for behavioral tests, N = 3 mice for immunostaining experiments. p‐values are calculated using unpaired t‐test. *p < 0.05, **p < 0.01, n.s. not significant.

Fig 5: Expression profile of astrocyte‐enriched proteins in the upper dorsal horn. Immunolabeling for 10 astrocyte‐enriched proteins (green). IB4 and PKCγ staining (both in blue) served as orientation markers for L2. Confocal images of the spinal cord dorsal horn (1) and the respective magnification of L1, L2, and L3 (2) are shown for the following proteins: GFAP (a1, a2), S100B (b1, b2), GS (c1, c2), NDRG2 (d1, d2), Cx43 (e1, e2), Cx30 (f1, f2), GLAST (g1, g2), GLT1 (h1, h2), AQP4 (i1, i2), and Kir4.1 (j1, j2). Scale bar: 100 μm (1) and 50 μm (2). (k) Analysis of the mean intensity (au) in L1, L2, and L3. N = 5–6 mice (mean of 4–6 slices/mouse). GFAP (p < .01), S100B (p = .522), GS (p = .543), NDRG2 (p = .073), Cx43 (p = .009), Cx30 (p = .004), GLAST (p < .001), GLT1 (p = 0.114), AQP4 (p = .003), and Kir4.1 (p = .009). Data are presented as raw data with mean ± SEM. One‐way ANOVA with Holm–Sidak post hoc correction was performed. Values for GFAP, S100B and GS were further corrected for three parameters tested (density, mean intensity, co‐localization). In case normality failed, a Kruskal–Wallis one‐way ANOVA with Tukey post hoc correction was performed (GS, NDRG2, Cx43, GLT1, AQP4, and Kir4.1). n.s. not significant, *p < .05, **p < .01, ***p < .001 [Color figure can be viewed at wileyonlinelibrary.com]