Vick P , Kreis J , Schneider I , Tingler M , Getwan M , Thumberger T , Beyer T , Schweickert A , Blum M .

	Graphical Abstract
	Figure 1. Expression of pkd2 mRNA during Xenopus Embryogenesis. (A–C) Maternally deposited mRNA localized to the animal hemisphere of the zygote (A, A′) and four-cell stage embryos (B). Note animal-vegetal shift of expression as indicated in the zygote. (C) Lack of a specific signal in specimen hybridized with a sense probe. (D–F) pkd2 transcripts in deep mesodermal layers of the early gastrula (D, D′, D′; black arrowheads; plane of section indicated in D by dotted line), in dorsal notochordal mesoderm at mid gastrula (E), and in tail organizer (white arrow) and posterior notochord at late gastrula stages (F, F′). Please note the lack of transcripts in the dorsal lip and the SM (white arrowheads). Border of inner and outer layer of marginal zone indicated by dotted line. Yolk plug and archenteron roof indicated by dashed lines. (G) Deep neuroectodermal, tail organizer, and posterior notochordal expression at neurula stages; no expression of GRP detected (G4). a, anterior; an, animal; ar, archenteron; bf, blastocoel floor; cbc, circumblastoporal collar; dl, dorsal lip; e, endoderm; el, epithelial layer (of cbc); l, left; n, notochord; ne, neuroectoderm; psm, presomitic mesoderm; r, right; s, somites; v, ventral; veg, vegetal; yp, yolk plug.
	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.
	Figure 3. Leftward Flow and GRP Morphogenesis Are Compromised in pkd2 Morphants (A–D) Unilateral injection of Pkd2-MO (A) but not co-MO (B) caused disruption of leftward flow at targeted area (red fluorescence: lineage tracer), as revealed by gradient time trail (GTT); see Transparent Methods section and Schweickert et al., (2007) analysis. Quantification of directionality and velocity of bead transport on morphant (C) and control (D) sides of dorsal explants. Rho (r) represents a value for robustness of directionality of leftward flow. Scale bars represent 50 micrometers. (E–G) Stage 17 GRP ciliation (E) was lost in pkd2 morphants (F) and regained in specimens co-injected with pkd2-MO and a full-length pkd2 mRNA. IF with acetylated α-tubulin antibodies (red) and Alexa 488-phalloidin (green) to highlight GRP cilia and cell borders. (H–K) tekt2 expression at the GRP of stage 17 dorsal explants (H) was lost in pkd2 morphants (I) and rescued upon co-injection of a full-length pkd2 mRNA (J). (K) Quantification of results. (L and M) nodal1 expression in lateral GRP cells (L) was lost in morphants (M). See also Figure S3 and Video S1
	Figure 4. Polycystin-2 and Xnr3 Synergize to Induce the LRO Precursor Tissue of the Superficial Mesoderm (A–F) Increased xnr3 (A–C) and reduced foxj1 expression (D–F) in stage 10.5 pkd2 morphants (B and E) as compared with control specimens (A and D). Dotted lines in A and B indicate plane of histological sections shown in A′, and B′, respectively. Note that foxj1 expression was rescued upon co-injection of a full-length pkd2 mRNA (F). (G–I) Reduced foxj1 expression in early gastrula stages (st. 10.5) following Tg treatment (G, DMSO control embryo; H, Tg treated specimen; I, quantification of results). (J–S) xnr3 and pkd2 synergize SM foxj1 induction. Loss of xnr3 resulted in attenuated foxj1 expression (K and O) compared with control uninjected specimens (J and O), which was highly efficiently rescued by co-injection of full-length xnr3 mRNA (L and O). Both dorsal (M) and ventral (N) overexpression of xnr3 mRNA caused an increase of endogenous or induction of ectopic foxj1, respectively (M–O). Injections of reduced MO doses of Xnr3-MO (O, R; 2 × 0.6pmol) or Pkd2-MO (O, Q; 2 × 0.75pmol) caused mild reductions of foxj1 expression. Co-injection of low doses of MOs resulted in strong inhibitory effects (O and S). (T) Schematic depiction of known (black) and proposed (green) interactions required for SM specification. For details refer to main text. Increase of expression highlighted with white arrowheads and decrease with black arrowheads. Numbers in parentheses indicate the number of embryos analyzed for each condition. See also Figures S4 and S5.
	Figure S1: Related to Figure 1. Late expression pattern of pkd2. Expression in the neuroectoderm (A-C), intermediate mesoderm (B, C), pronephric system (D), branchial arches (D’) and tailbud (E) in early to late tailbud stages. Key: a, anterior; ba, branchial arches; d, dorsal; e, endoderm; fp, floorplate; im, intermediate mesoderm; l,left; lpm, lateral plate mesoderm; mhb, mid hindbrain boundary; n, notochord; ne, neuroectoderm; pd, pronephric duct; pn, pronephros; r, right; s, somites; v, ventral
	pkd2 (polycystin 2, transient receptor potential cation channel) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, lateral view, anterior left, dorsal up. Key: ne= neuroectoderm.
	pkd2 (polycystin 2, transient receptor potential cation channel) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, lateral view, anterior left, dorsal up. Key: im, intermediate mesoderm.
	pkd2 (polycystin 2, transient receptor potential cation channel) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, lateral view, anterior left, dorsal up. Key: pd, pronephric duct.
	pkd2 (polycystin 2, transient receptor potential cation channel) gene expression in Xenopus laevis embryo, head region, assayed via in situ hybridization, NF stage 36, and tail tip at NF stage 34, lateral view, anterior left, dorsal up. Key: pn, pronephric nephrostomes; ba, pharyngeal/branchial arches; mhb, mid hindbrain boundary.
	Figure S2: Related to Figure 2. Lineage-specific knockdown of pkd2 reveals a left-sided dorsal requirement of Polycystin-2 in the LRO (A) Schematic depiction of the differentially targeted areas of the gastrocoel roof in a dorsal explant of a stage 17 neurula embryo with the central, flow-generating population of the LRO (1. red), the lateral, sensory population of the LRO (2. green), and the surrounding endodermal cells (3. yellow) that cover the lateral plate mesoderm, which will express nodal and pitx2c on the left side after flow-dependent symmetry breakage to govern asymmetric organogenesis. (B) Unilateral left-sided pkd2 knockdown experiment showing dose-dependent loss of pitx2c when injected into the left dorsal marginal zone (DMZ) to target the central, flow-generating GRP. Injection into the dorso-lateral marginal zone (D-LMZ) to target the sensory part of the GRP (i.e. the lateral LRO cells) caused fewer, yet significant LR defects. Injecting the ventral marginal zone (VMZ) to target the left lateral plate mesoderm did not result in miss-expression of pitx2c. *p<0.05, **p<0.01, **p<0.001 for all panels.
	Figure S3: Related to Figure 3. GRP defects in pkd2 morphants (A, B) SEM analysis of co-MO (A) and Pkd2-MO (B) injected specimens reveal loss of ciliation and altered GRP cell morphology in morphants. Examples are representative for 6 specimens analyzed each. Scale bars represent 20 micrometers. (C-H) Unilateral absence of dnah9 (D), nodal1 (F) and dand5 (H) in left-injected embryos in comparison to internal control sides at stage 17. Control embryos (C, E, G) show wildtype expression levels. (I) SEM analysis of ciliation and cell morphology in dorsal explant of stage 17 specimen unilaterally injected with Pkd2-MO targeted exclusively to the lateral, sensory part of the GRP. Shown is a ventral view of the GRP broken transversally and revealing the deep tissue arrangements in the top half of the picture. Right (I’) and left (I’’) magnifications show both the sensory areas of the GRP. Note that the lateral GRP cells were absent on the MO-injected side, such that central GRP cells, which are characterized by posteriorly polarized cilia, directly bordered non-ciliated endodermal cells. Sensory GRP cilia highlighted with red arrowheads, central GRP cilia with green arrowheads. (J-L) Expression of nodal1 in lateral GRP cells of flow stage (st. 17) specimens following Tp (K, L) or 1% DMSO (J) treatment during mid to late gastrula stage (st. 11.5). nodal1 was lost (K’) or strongly reduced (L) after Tp treatment, without causing gastrulation defects. (M-P) Expression of nodal1 in lateral GRP cells of flow stage (st. 16-19) specimens following 25-200µM BAPTA-AM (N-P) or 0.05-1% DMSO (M, P) treatment during mid and late gastrula stages (st. 11.5- 13/14). nodal1 was lost (O) or reduced (N) after BAPTA-AM treatment. *p<0.05, **p<0.01, **p<0.001 Black arrowheads indicate reduction or lack of expression. a, anterior; e, endoderm; l, left; n, notochord; p, posterior; r, right; s, somites
	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
	Figure S5: Related to Figure 4. Polycystin-2 function in LR axis formation is independent of canonical Wnt signaling (A-C) Wnt-dependent double axis formation. Injection of wnt8a mRNA resulted in >75% of conjoined twinning (A, C). Co-injection of Pkd2-MO resulted in a very moderate drop of twinning rates to approx. 70% (B, C). Quantification of results (C). Arrowhead indicates induced secondary axis. (D-F) Wildtype expression of brachyury during gastrulation in control specimens (D) was reduced (E) or lost (F) after injection of Xnr3-MO into the dorsal marginal zone (DMZ), demonstrating that Xnr3 was required for dorsal bra expression. Bisected embryos in D’, E’ and F’ highlight these effects.
	Figure 1. Expression of pkd2 mRNA during Xenopus Embryogenesis(A–C) Maternally deposited mRNA localized to the animal hemisphere of the zygote (A, A′) and four-cell stage embryos (B). Note animal-vegetal shift of expression as indicated in the zygote. (C) Lack of a specific signal in specimen hybridized with a sense probe.(D–F) pkd2 transcripts in deep mesodermal layers of the early gastrula (D, D′, D″; black arrowheads; plane of section indicated in D by dotted line), in dorsal notochordal mesoderm at mid gastrula (E), and in tail organizer (white arrow) and posterior notochord at late gastrula stages (F, F′). Please note the lack of transcripts in the dorsal lip and the SM (white arrowheads). Border of inner and outer layer of marginal zone indicated by dotted line. Yolk plug and archenteron roof indicated by dashed lines.(G) Deep neuroectodermal, tail organizer, and posterior notochordal expression at neurula stages; no expression of GRP detected (G4).a, anterior; an, animal; ar, archenteron; bf, blastocoel floor; cbc, circumblastoporal collar; dl, dorsal lip; e, endoderm; el, epithelial layer (of cbc); l, left; n, notochord; ne, neuroectoderm; psm, presomitic mesoderm; r, right; s, somites; v, ventral; veg, vegetal; yp, yolk plug.See also Figure S1.
	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.
	Figure 3. Leftward Flow and GRP Morphogenesis Are Compromised in pkd2 Morphants(A–D) Unilateral injection of Pkd2-MO (A) but not co-MO (B) caused disruption of leftward flow at targeted area (red fluorescence: lineage tracer), as revealed by gradient time trail (GTT); see Transparent Methods section and Schweickert et al., (2007) analysis. Quantification of directionality and velocity of bead transport on morphant (C) and control (D) sides of dorsal explants. Rho (r) represents a value for robustness of directionality of leftward flow. Scale bars represent 50 micrometers.(E–G) Stage 17 GRP ciliation (E) was lost in pkd2 morphants (F) and regained in specimens co-injected with pkd2-MO and a full-length pkd2 mRNA. IF with acetylated α-tubulin antibodies (red) and Alexa 488-phalloidin (green) to highlight GRP cilia and cell borders.(H–K) tekt2 expression at the GRP of stage 17 dorsal explants (H) was lost in pkd2 morphants (I) and rescued upon co-injection of a full-length pkd2 mRNA (J). (K) Quantification of results.(L and M) nodal1 expression in lateral GRP cells (L) was lost in morphants (M).See also Figure S3 and Video S1
	Figure 4. Polycystin-2 and Xnr3 Synergize to Induce the LRO Precursor Tissue of the Superficial Mesoderm(A–F) Increased xnr3 (A–C) and reduced foxj1 expression (D–F) in stage 10.5 pkd2 morphants (B and E) as compared with control specimens (A and D). Dotted lines in A and B indicate plane of histological sections shown in A′, and B′, respectively. Note that foxj1 expression was rescued upon co-injection of a full-length pkd2 mRNA (F).(G–I) Reduced foxj1 expression in early gastrula stages (st. 10.5) following Tg treatment (G, DMSO control embryo; H, Tg treated specimen; I, quantification of results).(J–S) xnr3 and pkd2 synergize SM foxj1 induction. Loss of xnr3 resulted in attenuated foxj1 expression (K and O) compared with control uninjected specimens (J and O), which was highly efficiently rescued by co-injection of full-length xnr3 mRNA (L and O). Both dorsal (M) and ventral (N) overexpression of xnr3 mRNA caused an increase of endogenous or induction of ectopic foxj1, respectively (M–O). Injections of reduced MO doses of Xnr3-MO (O, R; 2 × 0.6pmol) or Pkd2-MO (O, Q; 2 × 0.75pmol) caused mild reductions of foxj1 expression. Co-injection of low doses of MOs resulted in strong inhibitory effects (O and S).(T) Schematic depiction of known (black) and proposed (green) interactions required for SM specification. For details refer to main text. Increase of expression highlighted with white arrowheads and decrease with black arrowheads. Numbers in parentheses indicate the number of embryos analyzed for each condition.See also Figures S4 and S5.

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