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Figure 2. pkd2 Is Required for LR Asymmetry in Xenopus, Independently of dand5
(A) Putative sensory function of Polycystin-2 during symmetry breakage downstream of GRP-flow.
(B–D) Dose-dependent loss of left-sided pitx2c expression following Pkd2-MO targeting to the GRP. Black arrowhead indicates approximate location of present or absent pitx2c expression on both sides of the embryo. White arrow highlights expression of pitx2c around the eye as proof of working ISH.
(E) Model of hierarchy of the flow sensing module and possible role of pkd2 on the lateral side of the LRO downstream of flow and upstream of Dand5.
(F) Epistatic single and double knockdown experiments, performed on the left or right side, as indicated. Differences in expected and experimental outcome highlighted by red significance. See text for details.
See also Figure S2.
**p < 0.01, ***p < 0.001 in all Figures.
Numbers in parentheses indicate the number of embryos analyzed for each condition. |
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Figure S4: Related to Figure 4. Calcium manipulating agents Thapsigargin or BAPTA-AM impacted differently on LR development
(A-C) foxj1 expression (A) was reduced in pkd2 morphants (B) and rescued upon co-injection of Pkd2-
MO and a full-length pkd2 rescue mRNA not targeted by the MO (C).
(D-F) A 20-minute thapsigargin treatment before gastrulation resulted in loss of pitx2c expression in
>50% of embryos without concomitant gastrulation defects, reminiscent of Pkd2-MO injected specimens
(cf. Figure 2D).
(G-J) Treatment of embryos between blastula (st. 9) and early gastrula stages (st. 10.5) with different
concentrations of the calcium chelator BAPTA-AM did not reduce foxj1 expression (H, I), in comparison
to 0.05-1.00% DMSO treatment (G, I). (J) Embryos analyzed for pitx2c expression at tailbud stages after
short (left side, until stage 11) or long (right side, until stage 12.5) treatment with BAPTA-AM. Longer
(st. 9-12.5), but not short treatment (st. 9-11) resulted in significant LR defects. Please note the high proportion of embryos with general axis malformations when treated until late gastrulation (right side) as compared to shorter treatment (left side). Significances in (J) were calculated for LR expression patterns of pitx2c only, not including malformed embryos, which are also shown in the graph. *p<0.05, **p<0.01, **p<0.001 for all panels |
