Mechanism of X-TSK function: signal analysis.

<p>(A) Whole mount in situ hybridization of germ layer markers in embryos injected with 500 pg <i>β-Gal</i> with 250 pg <i>truncated BMP receptor (tBR)</i> or 125 pg <i>Chordin (Chd),</i> with percentage occurance of demonstrated phenotype and ‘n’ numbers. <i>Xbra</i> and <i>Sox17α</i> phenotypes differ in comparison to <i>X-TSK</i> overexpression, whereas <i>Gsc</i> expression is commonly expanded. (B) Whole mount in situ hybridization of <i>Gsc</i> in embryos injected with 500 pg <i>β-Gal</i> with 1 ng <i>X-TSK</i>, 500 pg <i>caALK3</i> and <i>X-TSK</i> with <i>caALK3</i>, dorsal orientation. caALK3 blocks X-TSK mediated expansion of <i>Gsc</i> expression. (C) Western blotting of MAPK and Smad1 phosphorylation in animal caps and Smad2 phosphorylation in DMZ explants, with total MAPK, Smad2 and Smad1 controls in explants injected with <i>X-TSK</i> (125 pg-1 ng) (D) 125 pg <i>Chd</i> or 250 pg <i>tBR</i>. X-TSK inhibits MAPK and BMP phosphorylation in animal caps whilst activating Smad2 phosphorylation in DMZ. Chd and tBR similarly inhibit BMP phosphorylation, but contrast with X-TSK in MAPK and Smad2 phosphorylation status.</p

X-TSK requires intact Xnr signaling for endoderm induction; X-TSK binds to and enhances Xnr2 Signaling.

<p>(A) Whole mount in situ hybridization of <i>Sox17α</i> in embryos injected with 500 pg <i>β-Gal</i> with 1 ng <i>X-TSK,</i> 500 pg <i>CerS</i> and 500 pg <i>CerS</i> with 1 ng <i>X-TSK,</i> lateral orientation. (B) Introduction of CerS blocks X-TSK expansion of <i>Sox17α</i> in 100% of embryos analyzed (p = <0.001). (C) Western blotting of nickel bead pulldown of Xnr2-Myc in complex with X-TSK-Myc-His. Top panel: detection of Xnr2-Myc in complex with X-TSK-Myc-His (third lane). Second panel: detection of X-TSK-Myc-His pulled down. Third and bottom panels: detection of Xnr2-Myc and X-TSK-Myc-His input into the pulldown reaction. (D) Western blotting of Smad2 phosphorylation in animal caps injected with 1 ng <i>X-TSK</i>, 5 pg and 50 pg <i>Xnr2</i>. X-TSK enhances Smad2 phosphorylation, particularly evident with 5 pg Xnr2.</p

Transcriptional regulation of <i>X-TSK</i> and model of X-TSK function in germ layer formation and patterning.

<p>(A) Semi-quantitative RT-PCR of <i>X-TSK</i> expression in animal caps injected with 1 ng <i>XFD,</i> 50 pg <i>V-ras</i> or 50 pg <i>caFGFR.</i> WE = Whole embryo, WOC = Water only control. Inhibition of FGF signals with XFD enhances <i>TSK</i> expression, whereas activation of FGF signals with V-ras or caFGFR reduces <i>TSK</i> expression levels. (B) Model of TSK function in <i>Xenopus</i> germ layer formation and patterning: dorsal-ventral mesoderm patterning. X-TSK in dorsal mesoderm (red) inhibits BMP signaling to promote dorsal mesoderm formation, as marked by <i>Gsc</i> expression. This is possibly also enhanced through activation of Xnr2 signals by TSK. MAPK activation inhibits <i>X-TSK</i> expression in ventrolateral mesoderm, where X-TSK inhibits expression of ventrolateral mesoderm markers such as <i>Xbra</i>, through inhibition of FGF signaling. This network of signaling may contribute to clear patterning of the mesoderm. (C) Model of TSK function in endoderm formation. X-TSK coordinates inhibition of FGF and BMP signals with activation of Xnr2 signaling to induce endoderm formation (green), as marked by <i>Sox17α.</i> Again, X-TSK inhibits expression of ventrolateral mesoderm (blue) markers such as <i>Xbra</i>, through inhibition of FGF signaling and may contribute to the distinction between endoderm and mesoderm specific gene expression.</p

<i>X-TSK</i> expression in <i>Xenopus.</i>

<p>(A) Whole mount in situ hybridization of <i>X-TSK</i> in <i>Xenopus</i> gastrula stage embryos, including sense control. Purple staining indicates <i>X-TSK</i> expression. Orientations and stages as indicated. <i>X-TSK</i> is expressed in dorsal marginal zone (DMZ) and ectoderm from stage 10, and endoderm from stage 10.5. (B) In situ hybridization of <i>X-TSK</i> in sectioned <i>Xenopus</i> embryos, including sense control. Orientation: animal top, vegetal bottom, dorsal right, stages as indicated. <i>X-TSK</i> is expressed maternally (stage 7) in the animal region, with light staining in the vegetal region. From stage 10.5, <i>X-TSK</i> expression is detected throughout the endoderm. (C) Expression levels of <i>X-TSK</i> (upper panel) measured by RT-PCR from egg to stage 41, including <i>ODC</i> expression (middle panel) and -RT control (lower panel). WOC = Water Only Control. <i>X-TSK</i> is expressed at highest levels during germ layer formation and gastrulation. (D) Comparative expression of <i>Sox17α</i> (marking endoderm), <i>Gsc</i> (dorsal mesoderm), and <i>Xbra</i> (pan-mesoderm) in sectioned stage 10 embryos. (E) Schematic of <i>X-TSK</i> expression (grey) in ectoderm, dorsal mesoderm and endoderm.</p

Signaling involved in <i>Xenopus</i> germ layer formation.

<p>(A) Selected signaling pathways involved in <i>Xenopus</i> mesoderm and endoderm formation. Activation of pathways indicated by ‘+++’, inhibition of pathways indicated by ‘−−−‘. Ectoderm = red, mesoderm = green, endoderm = blue. FGF signal activity is required for mesoderm formation in addition to activity of activin-like signaling (represented here by Xnr2). FGF and BMP signal inhibition with Xnr2 signal activation is involved in endoderm induction mechanisms. (B) Selected signaling pathways involved in <i>Xenopus</i> mesoderm patterning. Active BMP signaling produces mesoderm with ventral character, whereas inhibition of BMP signaling produces mesoderm of dorsal character. Also, Xnr2 expressed in the dorsal region has activity to induce dorsal mesoderm.</p

Loss of X-TSK function.

<p>(A) In situ hybridization of endoderm markers, <i>Sox17α</i> (upper row), and <i>GATA4</i> (lower row) in sectioned early gastrula (stage 10) embryos, purple staining indicates expression. Orientation: animal top, vegetal bottom. All embryos injected with 500 pg <i>β-Galactosidase (β-Gal)</i> to identify targeted area (blue staining), with 20 ng control morpholino (CMO) or 20 ng X-TSK morpholino (XMO). Endoderm marker staining is reduced in XMO injected embryos, as indicated by general loss of purple staining (<i>Sox17α</i>) and loss of punctate staining (<i>GATA4</i>), detailed in the zoomed panel. Rescues were performed with 1 ng <i>H-TSK</i>, or 50 pg <i>Xnr2</i>, restoring endoderm marker expression. Detailed analysis of <i>GATA4</i> staining in sectioned embryos. Numbers of <i>GATA4</i> foci were counted, as represented graphically, relative to uninjected control. XMO injection reduces <i>GATA4</i> foci by 50% (p = <0.001), partially rescued by 1 ng <i>H-TSK</i> and 50 pg <i>Xnr2</i> to over 80% relative to control (p = <0.001). (B) Whole mount in situ hybridization of dorsal mesoderm marker, <i>Gsc</i> in stage 10.5 embryos (dorsal orientation) and <i>MyoD</i> in stage 16 (anterior top, posterior bottom) in embryos injected with 500 pg <i>β-Gal,</i> with 20 ng CMO or 20 ng XMO. <i>Gsc</i> expression is reduced in XMO injected embryos, whereas <i>MyoD</i> expression is expanded by 30% (relative to control, p = <0.001) on the injected side, as identified by blue <i>β-Gal</i> staining. (C) Gut morphology in stage 40 embryos injected with 20 ng CMO or 20 ng XMO. Gut width is reduced by 21% in XMO injected embryos, relative to uninjected embryos (p = <0.001).</p

BMP and FGF signal activation blocks X-TSK mediated endoderm induction: triple signal regulation.

<p>(A) Whole mount in situ hybridization of <i>Sox17α</i> in embryos injected with 500 pg <i>β-Gal</i> with 1 ng <i>X-TSK,</i> 500 pg <i>caALK3,</i> 50 pg <i>V-ras</i> and 500 pg <i>caALK3,</i> 50 pg <i>V-ras</i> with 1 ng <i>X-TSK,</i> lateral orientation. (B) Graphic representation of quantity of embryos demonstrating expanded <i>Sox17α</i> expression. Introduction of caALK3 and V-ras partially blocks X-TSK expansion of <i>Sox17α</i> (p = 0.01 and 0.05 respectively). (C) Whole mount in situ hybridization of <i>GATA4</i> in embryos injected with 500 pg <i>β-Gal</i> with combinations of 1 ng <i>XFD,</i> 500 pg <i>tBR,</i> and 50 pg <i>Xnr2,</i> lateral orientation. Demonstrated phenotype frequencies with n-numbers in white text. A triple combination of 1 ng <i>XFD,</i> 500 pg <i>tBR,</i> and 50 pg <i>Xnr2</i> produces the strongest expansion of <i>GATA4</i> expression.</p

X-TSK Loss-of-Function

<p>X-TSK Loss-of-Function</p

X-TSK inhibition and binding of FGF8b.

<p>(A) Western blotting of MAPK phosphorylation in animal caps injected with 20–40 ng CMO and 20–40 ng XMO. Depletion of X-TSK with XMO activates MAPK phosphorylation. (B) Semi-quantitative RT-PCR of <i>Xbra</i> expression in DMZ injected with 20 ng CMO, 20 ng XMO, 500 pg <i>XFD</i> and 500 pg <i>XFD</i> with 20 ng XMO. WE = Whole embryo, WOC = Water only control. Inhibition of FGF signals with XFD blocks <i>Xbra</i> expression activated upon depletion of X-TSK with XMO. (C) Whole mount in situ hybridization of <i>Xbra</i> in stage 10.5 embryos, lateral orientation. Embryos injected with 500 pg <i>β-Gal</i> with 1 ng <i>X-TSK</i>, 50 pg <i>V-ras</i> and <i>X-TSK</i> with <i>V-ras</i>. V-ras blocks X-TSK mediated inhibition of <i>Xbra</i> expression in 100% of embryos analyzed (p = <0.01), represented graphically in (D). (E) Western blotting of MAPK phosphorylation in animal caps injected with <i>X-TSK</i> and <i>V-ras</i>. V-ras blocks X-TSK mediated inhibition of MAPK phosphorylation. (F) Western blotting of MAPK phosphorylation in animal caps injected with <i>X-TSK</i> and <i>iFGFR,</i> in the presence or absence of chemical dimerisation agent, AP20187. Induced dimerisation blocks the activity of X-TSK to inhibit MAPK phosphorylation. (G) Western blotting of MAPK phosphorylation in animal caps injected with <i>X-TSK</i> and <i>FGF8b</i>. X-TSK inhibits MAPK phosphorylation activated by FGF8b. (H) Western blotting of nickel bead pulldown of FGF8b-FLAG in complex with X-TSK-Myc-His. Top panel: detection of FGF8b-FLAG in complex with X-TSK-Myc-His (third lane). Second panel: detection of X-TSK-Myc-His pulled down. Third and bottom panels: detection of FGF8b-FLAG and X-TSK-Myc-His input into the pulldown reaction.</p

<i>Brca2^{(p.T1942fs/+)}</i> dissipates ovarian reserve in rats through oxidative stress in follicular granulosa cells

Pathogenic variants of BRCA1/2 constitute hereditary breast and ovarian cancer (HBOC) syndrome, and BRCA1/2 mutant is a risk for various cancers. Whereas the clinical guideline for HBOC patients has been organized for the therapy and prevention of cancer, there is no recommendation on the female reproductive discipline. Indeed, the role of BRCA1/2 pathogenic variants in ovarian reserve has not been established due to the deficiency of appropriate animal models. Here, we used a rat model of Brca2(p.T1942fs/+) mutant of Sprague-Dawley strain with CRISPR-Cas9 editing to evaluate ovarian reserve in females. Fertility and ovarian follicles were evaluated and anti-Müllerian hormone (AMH) was measured at 8–32 weeks of age with a comparison between the wild-type and the mutant rats (MUT). MUT revealed a significantly smaller number of deliveries with fewer total pups. Furthermore, MUT showed a significant decrease in primordial follicles at 20 weeks and a low AMH level at 28 weeks. RNA-sequencing of the ovary at 10 weeks detected acceleration of the DNA damage repair pathway, which was accompanied by oxidative stress-induced DNA double-strand breaks, a decrease in PTEN, and an increase in mTOR in follicular granulosa cells. In conclusion, Brca2(p.T1942fs/+) dissipates primordial follicles via early activation of granulosa cells through oxidative stress, leading to earlier termination of fertility.</p