Placental Igf1 Overexpression Sex-Specifically Impacts Mouse Placenta Structure, Altering Offspring Striatal Development and Behavior

Abstract

Insulin-like growth factor 1 (IGF1) is produced primarily in the placenta in utero and is an essential hormone for neurodevelopment. Specifically, how placental IGF1 production persistently influences the brain is unclear. This study evaluated the effects of placental Igf1 overexpression on embryonic and postnatal brain development, particularly for striatum, a region highly linked to neurodevelopmental disorders. Placental Igf1 was overexpressed via placental-targeted CRISPR manipulation. This overexpression altered placenta structure and function distinctly in females and males. Early differences in placental function altered the trajectory of striatal development, as adult females showed persistent changes in striatal cell composition and striatal dependent behavior while males were less affected in brain and behavior outcomes. Overall, these results demonstrate that placental Igf1 expression alters striatal development and behavior in ways relevant to neurodevelopmental disorders. These findings expand our understanding of placental influence on neurodevelopment and will aid in identifying placental-targeted preventive interventions.

Keywords: CRISPR; Ganglionic Eminence; Insulin-like Growth Factor 1; Neurodevelopment; Neurodevelopmental Disorders; Neuroplacentology; Placenta; Striatum.

Figure 1.. Placental-targeted CRISPR mediated overexpression resulted in sex-specific phenotypes

(A) Schematic of placental-targeted manipulation followed by embryonic and postnatal experiment timepoints. Expression of placental Igf1 analyzed by qPCR relative to 18s at E13 in females (B) and males (C) (n=5-7 per group). Placental protein levels of IGF1 by ELISA in E14 females (D) and males (E) (n=3-10 per group). Body levels of IGF1 protein in E14 females (F) and males (G) (n=5-12 per group). Placental Igf2 expression in females (H) and males (I) (n=4-10 per group). All graphs show mean and SEM. ns=nonsignificant, #p<0.1, *p < 0.05, and **p < 0.01 by linear mixed effects model with litter as a covariate.

Figure 2.. Placental Igf1 overexpression promoted growth in female placentas

Placental expression of angiogenic factors Plgf (A), Flt1 (B), and Hif-1α (C) analyzed by qPCR relative to 18s in E14 female samples (n=4-11 per group). (D) E18 labyrinth zone area in females (n=13 per group). (E) E18 total placental area in Igf1-OE females (n=13 per group). Representative H&E images of E18 control (F) and Igf1-OE (G) female placental sections. Black line borders the junctional zone, D=decidua, JZ=junctional zone, and LZ=labyrinth zone. E18 placental expression of Plgf (H), Flt1 (I), and Hif-1α in female placentas (n=4-10 per group). (K) E18 Igf1 placental expression (n=7-9 per group). (L) E18 placental IGF1 protein levels (n=7-10 per group). (M) Schematic representing the changes in placental function in Igf1-OE female placentas after placental-targeted Igf1 manipulation compared to control female placentas. All graphs show mean and SEM. Scale bar represents 1mm. ns=nonsignificant, #p<0.1, *p < 0.05, **p < 0.01, and ****p<0.00001 by linear mixed effects model with litter as a covariate.

Figure 3.. Male placenta downregulated growth after Igf1 overexpression manipulation

E14 male placental expression by qPCR relative to 18s for Igfbp3 (A), Igbp5 (B), and Plgf (C) (n=8-12 per group). (D) E18 total placental area (n=9 per group). (E) E18 junctional zone thickness (n=9 per group). (F) E18 male junctional zone area (n=9 per group). E18 labyrinth thickness (G) and area (H) (n=9 per group). (I) E18 placental Plac1 expression in males (n=8-10 per group). Representative H&E images of E18 control (J) and Igf1-OE (K) male placental sections. Black line borders the junctional zone, D=decidua, JZ=junctional zone, and LZ=labyrinth zone. E18 male placental expression of Igfbp3 (L), Igbp5 (M) and Plgf (N) (n=7-10 per group). (O) Placental Igf1 expression at E18 in males (n=6-8 per group). (P) E18 male placental IGF1 protein levels (n=7-9 per group). (Q) Schematic representing the changes in placental function in Igf1-OE male placentas after placental-targeted Igf1 manipulation compared to control male placentas. All graphs show mean and SEM. Scale bar represents 1mm. ns=nonsignificant, *p < 0.05 and **p < 0.01 by linear mixed effects model with litter as a covariate.

Figure 4.. Placental Igf1-OE led to sex-specific cell cycle changes in the embryonic ganglionic eminence

(A) Representative image of a coronal hemi-section of E13 forebrain stained with DAPI; ganglionic eminence (GE) traced in white. Cell cycle exit in the E13 female GE (B) and the E13 male GE (C) (n=5-8 per group). Total cell (DAPI) population in the E13 GE for females (D) and males (E) (n=5-9 per group). (F) E14 female body mass (n=13-19 per group). Representative images of DAPI (blue) and Ki67 (red) staining in the GE: control females (G) and Igf1-OE females (H). The same representative images are shown with Ki67 only in black and white (G’ and H’). White arrows show normal Ki67 diffuse staining and white arrowheads show S phase puncta Ki67 staining. (I) E14 female S phase cell accumulation in the GE (n=4-9 per group). (J) E14 female Sox2+ cell density in the GE (n=4-6 per group). (K) Total cell (DAPI) population in the female GE (n=5-10 per group). (L) E14 male body mass (n=16-19 per group). Representative images of DAPI (blue) and Ki67 (red) staining in the GE: control males (M) and Igf1-OE males (N). The same representative images are shown with Ki67 only in black and white (M’ and N’). (O) S phase cell accumulation in the GE of E14 males (n=5-8 per group). (P) E14 male Sox2+ cell density in the GE (n=3-9 per group). (Q) Total cell (DAPI) population in the male GE (n=5-9 per group). All graphs show mean and SEM. Scale bar represents 500μm in panel A and 20μm in panel N’. ns=nonsignificant, #p<0.1, *p < 0.05, and **p < 0.01 by linear mixed effects model with litter as a covariate.

Figure 5.. Placental Igf1-OE affected E18 female and male brain and body growth

(A) Representative image of a coronal hemi-section of E18 forebrain stained with DAPI; striatum traced in white. E18 striatal volume for females (B) and males (C) (n=8 per group). Total cell (DAPI) population of the E18 striatum of females (D) and males (E) (n=7-8 per group). E18 forebrain expression of Igf1R females (F) and males (G) (n=8-10 per group). E18 forebrain expression of Igf1 in females (H) and males (I) (n=8-10 per group). (J) Representative image of an E18 embryo body. IGF1 protein levels in the bodies of E18 female embryos (K) and male embryos (L) (n=8 per group). (M) Female body mass at E18 (n=28-35 per group). (N) Male body mass at E18 (n=25-37 per group). All graphs show mean and SEM. Scale bar represents 1mm in panel A and 1cm in panel J. ns=nonsignificant, *p < 0.05 and **p < 0.01 by linear mixed effects model with litter as a covariate.

Figure 6.. Altered juvenile growth and striatal-dependent behavior in Igf1-OE female and male mice

P14 female (A) and male (B) body length (n=9-15 per group). P26 female (C) and male (D) body mass (n=13-14 per group). Representative images of OFT (E) and rotarod testing (F). (G) Time spent in the center during OFT in P26 females (G) and males (H) (n=13-14 per group). Distance traveled during OFT in P26 females (I) and males (J) (n=13-14 per group). (K) Juvenile female learning coefficient score for rotarod testing (n=13-14 per group). (L) Juvenile male learning coefficient score for rotarod testing (n=13-14 per group). Frequency distribution of juvenile behavior composite score representative of all juvenile behavior tests for females (M) and males (N) versus same sex controls (n=13-14 per group). Juvenile behavior composite scores for females (O) and males (P) (n=13-14). All graphs show mean and SEM. ns=nonsignificant, *p < 0.05 by Welch’s t-test.

Figure 7.. Persistent changes in adult Igf1-OE female striatal dependent behavior

(A) Schematic of adult behavior testing timeline. (B) Adult female Y-maze unique trip score (n=13-14 per group). (C) Adult female time in the open arms during EPM testing (n=12-14 per group). (D) Adult female errors made during water T maze reversal testing (n=13-14 per group). (E) Grooming incidences seen during stereotypy in adult females (n=9 per group). Adult male Y-maze unique trip score (F), time spent in open arm during EPM testing (G), and errors made during water T reversal testing (H) (n=12-14 per group). (I) Adult male grooming incidences during stereotypy testing (n=8-10 per group). (J) Frequency distribution of adult behavior composite score representative of adult behavior tests for Igf1-OE females versus same sex controls (n=13-14 per group). (K) Adult behavior composite scores for females (n=13-14). (L) Frequency distribution of adult behavior composite score representative of adult behavior tests for Igf1-OE males versus same sex controls (n=13-14 per group). (M) Adult behavior composite scores for males (n=13-14). All graphs show mean and SEM. ns=nonsignificant, #p<0.1, *p < 0.05, and **p < 0.01 by Welch’s t-test.

Figure 8.. Altered striatal cell composition only in adult Igf1-OE female brain

(A) Representative image of a coronal hemi-section of adult forebrain stained with DAPI (blue) and NeuN (red); striatum traced in white. (B) Representative image of NeuN+ (arrowhead) and NeuN− (arrow) stained cells in the adult striatum. Adult striatal volume for females (C) and males (D) (n=9-11 per group). Total cell (DAPI) population in the adult female (E) and male (F) striatum (n=8-11 per group). Neuron percentage of total cells in the adult female (G) and male (H) striatum (n=8-11 per group). All graphs show mean and SEM. Scale bar represents 2mm in panel A and 50μm in panel B. ns=nonsignificant, *p < 0.05 by Welch’s t-test.