Ex vivo cultures combined with vivo-morpholino induced gene knockdown provide a system to assess the role of WT1 and GATA4 during gonad differentiation

Abstract

Gonad morphogenesis relies on the correct spatiotemporal expression of a number of genes that together fulfill the differentiation of the bipotential gonad into testes or ovaries. As such, the transcription factors WT1 and GATA4 are pivotal for proper gonadal development. Here we address the contributions of GATA4 and WT1 to the sex differentiation phase in testes and ovaries. We applied an ex vivo technique for cultivating gonads in hanging droplets of media that were supplemented with vivo-morpholinos to knockdown WT1 and GATA4 either alone or in combination at the same developmental stage. We show that WT1 is equally important for both, the initial establishment and the maintenance of the sex-specific gene expression signature in testes and ovaries. We further identified Foxl2 as a novel putative downstream target gene of WT1. Moreover, knockdown of WT1 reduced mRNA levels of several molecular components of the hedgehog signaling pathway in XY gonads, whereas Gata4 vivo-morpholino treatment increased transcripts of Dhh and Ptch1 in embryonic testes. The data suggest that for its proper function, WT1 relies on the correct expression of the GATA4 protein. Furthermore, GATA4 down-regulates several ovarian promoting genes in testes, such as Ctnnb1, Fst, and Bmp2, suggesting that this repression is required for maintaining the male phenotype. In conclusion, this study provides novel insights into the role of WT1 and GATA4 during the sex differentiation phase and represents an approach that can be applied to assess other proteins with as yet unknown functions during gonadal development.

Fig 1. Deletion of Wt1 results in the disruption of a sex-specific signature in XX and XY gonads.

(A, B) Transcripts were measured by qRT-PCR in wild-type (Wt1^+/+) and Wt1-deficient (Wt1^-/-) XX and XY gonads. Genes are classified as being expressed at similar levels in both sexes or as exhibiting a clear predominance in either XX or XY gonads. Loss of Wt1 showed a significant change in expression of (A) Dax1, Nr5a1(Sf1), Amhr2, Star, and Gata4 in both sexes. Expression of (B) Fst, Foxl2, Amh2, Sox9, and Ctnnb1 was changed sex-specifically. (C) Representative morphology of Wt1^+/+ and Wt1^-/- XX and XY gonads (marked by dashed lines) with attached mesonephroi. Tissues were obtained from embryos at 13.5 dpc. Note the disrupted morphology (dashed lines) in the Wt1^-/- XY and XX gonads. Scale bars indicate 500 μm. For qRT-PCR data (A, B) relative transcript levels were normalized to Gapdh (2^-ΔCt) and shown in percent. Error bars indicate S.E.M. calculated from independent biological replicates (n ≥ 5). Statistical significances are marked by brackets (ANOVA with Tukey’s post hoc test) and asterisk (*p<0.05, t-test). n.d. = not detectable.

Fig 2. Co-expression of WT1 and GATA4 in XY and XX gonads.

(A, B) Dissected XY and XX gonads (13.5 dpc) were co-immunostained as whole-mounts using antibodies against WT1 and GATA4. Bound primary antibodies were visualized with Cy3- (GATA4) and 488 dye (WT1) conjugates. Nuclear co- localization of WT1 and GATA4 is shown in the merged images. Cell nuclei were stained with Dapi. Scale bars are 200 μm (low-power magnification) and 100 μm (high-power magnification), respectively. G = gonad. M = mesonephros.

Fig 3. Experimental workflow and effect on cell proliferation upon WT1 or GATA4 knockdown.

(A) Dissected XX and XY gonad/ mesonephroi regions (12.5 dpc) were incubated for 72 h in a droplet of medium supplemented with either Wt1 or Gata4 vivo-morpholino. Transfection with the corresponding mismatch vivo-morpholinos was performed as control. Efficient gene silencing was assessed by immunostaining of WT1 (B) and GATA4 (C) in XX and XY gonad/ mesonephroi explants. Cell proliferation was determined by means of BrdU incorporation, and nuclei were visualized with Dapi. Proliferating cells were reduced in both XX and XY gonad/ mesonephroi explants upon WT1 knockdown (B), and knockdown of GATA4 only showed an effect on XY explants (C). BrdU-positive cells were counted in at least 5 tissue sections obtained from 3 different embryos and normalized to Dapi-stained nuclei. Scale bars indicate 100 μm. Error bars represent S.E.M., *p<0.05, **p<0.01, ***p<0.001, t-test. mo = vivo-morpholino. mism = vivo-morpholino-mismatch. G = gonad. M = mesonephros.

Fig 4. Single- and double-knockdown of WT1 and GATA4 in XX and XY gonads.

(A-D) XX and XY gonads were dissected from murine embryos (12.5 dpc) and incubated for 72 h with Wt1 and/ or Gata4 vivo-morpholinos. Transfection with appropriate mismatch vivo-morpholinos served as a negative control. For double-knockdown of WT1 and GATA4, vivo-morpholinos were applied simultaneously. (A) Efficient knockdown of WT1 and/or GATA4 was assessed by immunoblot analysis. (B-D) Relative transcript levels were determined by qRT-PCR and normalized to Sdha transcripts according to the 2^ΔΔCt method [34]. Results are shown as fold differences between cultures treated with mismatch vs. Wt1- (B) and mismatch vs. Gata4-morpholino (C), respectively. Panel D represents the effect of the double-knockdown of WT1 and GATA4. Error bars represent S.E.M. from independent biological replicates (n ≥ 5). scale = log2. Statistical differences are indicated by asterisks: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0005, paired t-test. n.d., not detectable.

Fig 5. Sex-specific effect of WT1 and GATA4 knockdown on Gata4 E1a and Gata4 E1b isoforms expression.

(A, B) Knockdown of GATA4 showed a strong significant increase in Gata4 E1b mRNA transcripts in XY but not in XX gonads. WT1 knockdown resulted in a significant decrease of Gata4 E1b and Gata4 E1a transcripts in XY gonads but not in XX gonads. Transcripts were measured by qRT-PCR and normalized to Sdha [34]. Results are shown as fold differences between cultures treated with mismatch vs. Gata4- (A) and mismatch vs. Wt1-morpholino (B). Error bars represent S.E.M. from independent biological replicates (n ≥ 5). Scale = log2. Statistical differences are indicated by asterisks: *p<0.05, **p<0.01, paired t-test.

Fig 6. Single- and double-knockdown of WT1 and GATA4 affect mRNA levels of hedgehog pathway components in XY and XX gonads.

XY (A-C) and XX (D-F) gonads, dissected from murine embryos (12.5 dpc), were incubated for 72 h with Wt1 and/ or Gata4 vivo-morpholinos as described in Fig 4. Relative transcript levels were determined by qRT-PCR and normalized to Sdha transcripts according to the 2^ΔΔCT method [34]. Results are shown as fold differences between cultures treated with mismatch vs. Wt1- (A, D) and mismatch vs. Gata4-morpholino (B, E), respectively. Panels C and F represents the effect of double-knockdown of WT1 and GATA4. Error bars represent S.E.M. from independent biological replicates (n ≥ 8). scale = log2. Statistical differences are indicated by asterisks: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, paired t-test.

Fig 7. WT1 and GATA4 in the regulation of sex-specific gonadal gene expression.

During testis development, WT1 is required for activating various testis promoting genes, i.e. Amh, Amhr2, Nr5a1(Sf1), Star, Sox9, Gli1, Gli2, Smo, Dhh and Ptch1. GATA4 represses ovarian promoting transcripts such as Ctnnb1, Bmp2, and Fst, in the testis, and thereby may be essential for the maintenance of the testis phenotype. GATA4 is also involved in repressing Dhh and Ptch1 transcript levels in embryonic testis, which may contribute to the fine-tuning of their threshold levels. Given such a pivotal role of GATA4, it is likely that sufficient GATA4 levels are ensured by a back-up mechanism provided by the GATA4 E1b isoform in the testis. During ovary development, i.e. in the absence of SRY, WNT4 and RSPO-1 levels are stabilized and promote canonical Wnt signaling. In the ovary, WT1 is required for Dax1 expression. GATA4 acts as a repressor of Ctnnb1 and serves as an activator for the transcripts Gli2, Gli3, and Smo, the latter of which may be regulated synergistically by both WT1 and GATA4. WT1 and GATA4 may also synergize on Foxl2 transcription, which contributes to an ovarian specific signature. Suggested stimulatory (→) and inhibitory (⊥) pathways identified herein are marked in red color.