Pleiotropic functions for transcription factor zscan10

Abstract

The transcription factor Zscan10 had been attributed a role as a pluripotency factor in embryonic stem cells based on its interaction with Oct4 and Sox2 in in vitro assays. Here we suggest a potential role of Zscan10 in controlling progenitor cell populations in vivo. Mice homozygous for a Zscan10 mutation exhibit reduced weight, mild hypoplasia in the spleen, heart and long bones and phenocopy an eye malformation previously described for Sox2 hypomorphs. Phenotypic abnormalities are supported by the nature of Zscan10 expression in midgestation embryos and adults suggesting a role for Zscan10 in either maintaining progenitor cell subpopulation or impacting on fate choice decisions thereof.

Figure 1. Zscan10 mutant females show decreased body weight.

(A) Body mass time curve of mutant and control cohorts, n = 60. (B) Body weight boxplot with strip chart, split by sex and genotype. Bodyweight is significantly reduced in female mutant mice (p = 0.042). (C) Bodyweight means, standard deviation and p-values calculated by a linear model (D) Body composition analysis by NMR: means, standard deviation and p-values of a linear model. (E) DXA analysis: Group means, standard derivation and ANOVA (Tukey multiple comparisons of means). (F) Tibia length box plot with strip chart. Split by sex and genotype. Tibia length is significantly reduced in female mutant mice. (G) Indirect calorimetry: Average body weight at indirect calorimetry boxplot with strip chart, split by sex and genotype. Points are individual animals; circles represent females (f); diamonds represent males (m) of Zscan10 homozygous mutants (mut) and wild type littermates (con). Line within the boxplot represents median. Box represents the 25% and 75% quartile. Asterix represents the mean. Circled points: Values outside the upper whisker (min(max(x), 75% quantile +1.5*IQR) and outside the lower whisker (max(min(x), 25% quantile+1.5*IQR)  =  outliers. IQR  =  interquartile range (75% quantile–25% quantile).

Figure 2. Hematological and clinical chemistry analysis.

(A) Plasma electrolyte, protein, creatinine and urea concentrations of ad libitum fed mice. (B) Lipid and glucose levels as well as selected enzyme activities in plasma of ad libitum fed mice. (C) Plasma concentrations of minerals, iron and ALP activity of ad libitum fed mice. (D) Values obtained from EDTA-blood samples. Means, standard deviations and p-values for genotype, sex and genotype x sex interaction effects calculated by a linear model (A-D). Platelet (E) and mean platelet volume (F) boxplot strip chart, split by sex and genotype. (G) Concentration of steroid hormones in plasma: Medians, first and third quartile and p-values calculated by a Wilcoxon rank-sum test (n = 57). Points are individual animals; circles represent females (f); diamonds represent males (m) of Zscan10 homozygous mutants (mut) and wild type littermates (con). Line within the boxplot represents median. Box represents the 25% and 75% quantile. Asterix represents the mean. Circled points: Values outside the upper whisker (min(max(x), 75% quantile +1.5*IQR) and outside the lower whisker (max(min(x), 25% quantile+1.5*IQR)  =  outliers. IQR  =  interquartile range (75% quantile–25% quantile).

Figure 3. Open Field Testing.

Distance traveled (A), percent distance in the center (B) and percent time spent in the center (C). Total boxplot with strip chart, split by sex and genotype. Points are individual animals; circles represent females (f); diamonds represent males (m) of Zscan10 homozygous mutants (mut) and wild type littermates (con). Line within the boxplot represents median. Box represents the 25% and 75% quantile. Asterix represents the mean. Circled points: Values outside the upper whisker (min(max(x), 75% quantile +1.5*IQR) and outside the lower whisker (max(min(x), 25% quantile+1.5*IQR)  =  outliers. IQR  =  interquartile range (75% quantile–25% quantile).

Figure 4. Small eye phenotype in Zscan10 homozygous mutant mice.

Lateral (A) and frontal (B) view of adult mutant (left) and wild type (right) mice. Lateral view of right eye of a wild type (C) and mutant (D) adult female. (E) Eye size measurements by laser interference biometry revealed significantly reduced axial eye length in mutants of both sexes (p<0.001). Anterior eye investigation by Scheimpflug imaging indicated significantly increased mean lens density between controls and mutants for left eyes in males (p = 0.002) (F) and right eyes in females (p = 0.016) (G). Virtual drum vision testing showed a reduced response with borderline significance in female mutants (p = 0.034) (H). (E) to (H) are displayed as boxplot split by sex and genotype. Outliers are indicated by open circles.

Figure 5. Organ weight determined in pathology screen.

(A) Normalized liver weight is significantly increased in male mutant mice (P = 0.008). (B) Absolute spleen weight is significantly reduced in mutant mice of both genders. (C) Normalized spleen weight is significantly reduced in female mutant mice (P = 0.002). (D) Absolute heart weight is significantly reduced in female mutant mice (p = 0.003). (E) Normalized heart weight is significantly reduced in female mutant mice (p = 0.016). All results demonstrated as boxplot with strip chart, split by sex and genotype. For tibia length refer to Figure 1F. Points are individual animals; circles represent females (f); diamonds represent males (m) of Zscan10 homozygous mutants (mut) and wild type littermates (con). Line within the boxplot represents median. Box represents the 25% and 75% quantile. Asterix represents the mean. Circled points: Values outside the upper whisker (min(max(x), 75% quantile +1.5*IQR) and outside the lower whisker (max(min(x), 25% quantile+1.5*IQR)  =  outliers. IQR  =  interquartile range (75% quantile–25% quantile).

Figure 6. Punctuated Zscan10 expression in E12.5 wild type embryos as determined by SISH.

Sagittal sections through E12.5 wild type embryos for reference as Mallory tetrachrome staining (A), with T7 antisense probe (B) and T3 sense probe (C). T7 antisense probe on transverse sections reflecting Zscan10 expression in the lining of the stomach (D), kidney and lining of the gut (E) at 4x; on sagittal sections through the eye (F), lower abdomen (G), heart (H), humerus (I) and vertebral body (K), as well as transverse sections through the neural tube (J) at 10x. All pictures were taken on a Motic compound microscope with a Motic Moticam 2.0MP digital camera. Brain (br), liver (li), dorsal root ganglia (drg), stomach (sto), kidney (ki), gut (gt), lens (le), retina (re), heart (he), bone (bn), neutral tube (nt), notochord (nc) and vertebral body (vb).

Figure 7. Punctuated Zscan10 expression persists in adult tissue.

SISH on sections through heart (A,D), kidney (B,E) and eye (C,F) probed with the T7 Zscan10 antisense probe taken at 0.63× (A,B) and 2.5× (C) on a Leica S6 dissecting scope and magnifications thereof (D,E,F) taken on a Motic compound microscope at 10× all documented with a Motic Moticam 2.0MP digital camera.