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Figure 1: FGF signaling is required for LR axis development during gastrulation. (A) Timeline of events relevant for LR axis development (top), and SU5402 incubation periods (bottom; marked as 1-5). (B) Representative specimens encountered following co-staining for pitx2c and myod1 mRNA expression. Top: DMSO-treated embryo; WT morphology and expression patterns (left-sided expression of pitx2c, marked by green arrowhead, and myod1 in somites). Middle: specimen treated with SU5402 at early gastrulation (stage 10.5) displaying lack of pitx2c expression (red arrowhead). Bottom: specimen treated with SU5402 at late gastrula (stage 12). Note the strongly decreased expression of myod1 upon early, but not late, SU5402 treatments. (C) Quantification of pitx2c expression patterns in specimens treated during indicated time periods (1-5). n.s., not significant; st., stage; wt, wild type; LR, left-right; LRO, left-right organizer; LPM, lateral plate mesoderm; SM, superficial mesoderm. n, number of analyzed embryos.
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Figure 2: Inhibition of FGF signaling at early gastrulation acts upstream of foxj1. (A,B) Expression patterns of the SM marker gene nodal3. Expression was unaltered following SU5402 treatment from stage 9 to 10.25 (N=3). (C,D) Representative foxj1 expression patterns at stage 11.5 in embryos treated with DMSO (C) or SU5402 up to gastrula stage 10.5 (D) demonstrating downregulation of the SM marker gene foxj1 in embryos treated with SU5402 (N=3). (E,F) Representative wnt11b expression patterns at stage 11 in embryos treated with DMSO (E) or SU5402 starting before stage 10.5 (F) demonstrating downregulation of wnt11b in the SM of embryos treated with SU5402 (N=2). (E′,F′) Mid-sagittal view of bisected specimens shown in E and F. (G) Quantification of results following early treatments. (H-K) Representative examples of expression patterns of foxj1 (H,I), and tekt2 (J,K) in embryos treated with DMSO (H,J) or 80 µM SU5402 from mid-gastrula stages onward (I,K) demonstrating unaltered expression of foxj1 (in the SM) and tekt2 (in the GRP) of embryos treated with SU5402. Embryos in J and K show ventral views of dorsal explants displaying archenteron roof and embedded GRP. Gray arrowheads highlight WT expression, blue arrowheads highlight reduced expression. a, anterior; l, left; p, posterior; r, right; st., stage; wt, wild type. N, number of experiments; n, number of embryos analyzed.
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Figure 3: FGF signaling regulates somitic GRP specification from mid to late gastrulation. (A-F) Analysis of nodal1 (A-C) and dand5 (D-F) mRNA expression in stage 17 embryos treated with DMSO (A,D) or 50 µM SU5402 (B,C,E,F) from stage 12 (A,B,D,E) or 13 (C,F) onwards. Lateral GRP-specific expression patterns shown in ventral view of dorsal explants. (G) Quantification of results (N=3). (H-K) Analysis of nodal1 (H,I) and dand5 (J,K) in stage 17 control specimens (H,J) or embryos unilaterally injected with fgf8b DNA (5-8 pg; I,K), specifically targeting the right expression domain of nodal1/dand5. Injected embryos showed a lateral increase of expression domains on the targeted side. (L) Quantification of results (N=3). a, anterior; co, control; l, left; n.s., not significant; p, posterior; r, right; st., stage; wt, wild type. N, number of experiments; n are numbers of embryos analyzed.
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Figure 4: FGF inhibition during late gastrulation blocks myod1 expression in the lateral GRP. (A) Schematic of explant preparation for myod1 analyses. Neurula-stage embryos were bisected transversally and the posterior part bisected into dorsal and ventral halves (top). Wild-type myod1-expression in the somitic GRP is highlighted in blue. Dorsal halves were analyzed for myod1 expression (B-F′; N=4). (B-D) ISH analysis of myod1 expression in embryos treated with DMSO (B) or 50 µM SU5402 (C,D) from stage 12 (B,C) or stage 13 (D) onwards. Ventral views of representative dorsal explants. Position of GRP is indicated by the curved dashed line. (B′-D′) Histological analysis of myod1 in transversal sections, as indicated by dashed white lines in B-D. Blue bars mark widths of myod1-positive lateral GRP, which was strongly (C′; stage 12) or slightly (D′; stage 13) reduced by SU5402 treatment. Black bars indicate unaltered central part of GRP. (E) Quantification of relative widths of central (top) and lateral somitic (bottom) GRP in DMSO- and SU5402-treated embryos, as described in the Materials and methods section. (F) Analysis of myod1 in stage 17 dorsal explants (ventral view) injected unilaterally with dnfgfr1 mRNA (8-12 pg) targeted to the left lateral GRP. (F′) Histological analysis in transversal section (at the level indicated in F). Contribution of somitic myod1-positve cells to the GRP (highlighted by dotted line on uninjected side) was lost on the injected side (black arrowheads). a, anterior; ar, archenteron; ec, ectoderm; en, endoderm; l, left; no, notochord; n.s., not significant; p, posterior; psm, presomitic mesoderm; r, right; s, somite; st., stage; wt, wild type. N, number of experiments; n are numbers of embryos analyzed.
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Figure 5: FGF inhibition during late gastrulation impairs somitic GRP morphogenesis. (A) Schematic of explant preparation for morphological analysis. Neurula-stage embryos were bisected transversally to isolate posterior-dorsal halves, which were bisected transversally to enable visualization of GRPs by SEM. Lateral somitic GRP and somites are indicated in red, central GRP and notochord in green, and endoderm in yellow. (B-D) SEM of stage 17 dorsal explants (ventral view) treated with DMSO (B) or 60 µM SU5402 (C) from stage 12 onwards, highlighting GRP subpopulations and flanking LECs. Higher magnification of DMSO- (B′,B″) and SU5402-treated (C′) specimens reveal the presence (B′,B″) or absence (C′) of lateral, somitic GRP areas, which are characterized by non-polarized central cilia (indicated by black arrowheads). Cells are outlined by dashed lines and colored in red (lateral somitic GRP), green (central GRP) and yellow (LECs). White arrowheads indicate polarized cilia of central GRP cells, which are localized to the posterior part of the cell. Note the lack of non-polarized, somitic GRP after late SU5402 treatments, as illustrated in D. (E) Quantification of cilia polarization (N=2). (F) Quantification of GRP widths (mean widths of DMSO-treated GRPs set to 1.0; N=2). (G,H) Representative SEM photographs of stage 17 dorsal explants (ventral view) injected unilaterally on the left with dnfgfr1 mRNA (G; 8-12 pg; N=3; n=25) or with fgf8b DNA on the right (H; 5 pg). (G′,H′) Higher magnifications of the boxed areas in G,H reveal reduction (G′; dnfgfr1-injections on left indicated by asterisk) or expansion (H′; fgf8b-injection on right indicated by asterisk) of lateral somitic GRP areas. a, anterior; l, left; no, notochord; n.s., not significant; p, posterior; r, right; s, somite; st., stage; wt, wild type. N, number of experiments; n, number of embryos analyzed.
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Figure 6: Sprouty1-mediated FGF inhibition blocks specification of somitic GRP. (A-L) Marker gene analyses of control and spry1-injected embryos at stage 11 (A-C), and dorsal explants at stage 17 (D-L; N=3). (C,F,I,L) Quantification of results. (A-C) Wild-type foxj1 expression in control specimens (A,C) and embryos bilaterally injected with spry1 mRNA (B,C; N=3). (D-F) Narrowed tekt2 expression in spry1-injected specimens (E; light blue arrowheads), compared with control embryos (D; gray arrowheads; N=3). (G-L) nodal1 (G-I) and dand5 (J-L) were reduced or absent on the spry-injected sides following unilateral mRNA injection (dark blue arrowheads; N=3). spry1 mRNA was injected at concentrations ranging from 320 to 800 pg. (A,B) Vegetal views, dorsal side up. (D,E,G,H,J,K) Ventral views of dorsal explants. bi, bilateral; n.s., not significant; wt, wild type. N, number of experiments; n, numbers of embryos analyzed.
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Figure 7: spry1 and pkd2 cooperate in somitic GRP specification. (A-F) Epistatic analysis of FGF/Ca2+ and Polycystin-2 function by simultaneous overexpression of spry1 and knockdown of pkd2, using sub-phenotypic doses. ISH analysis of dand5 (A-E; N=3) or nodal1 (F; N=3) in stage 17 embryos showed wild-type expression in control specimens (A,E,F). No or minor reductions were observed following unilateral injection of low doses of spry1 mRNA (50-160 pg) in combination with a control-MO (4 ng; C,E,F), or by injection of a Pkd2-MO (4 ng) in combination with a control mRNA (50-160 pg; B,E,F). Combinations of spry1 mRNA (50-160 pg) and Pkd2-MO (4 ng) resulted in a high proportion (>50%) of embryos with reduced or absent gene expression levels (D-F). GFP mRNA was used as control mRNA, control (co) bar represents uninjected specimens. (G-J) Rescue of nodal1 expression (G) in spry1-injected specimens (H; 320-800 pg) by co-injection of pkd2 mRNA (I). (J) Quantification of results (N=4). (G-I). Ventral views of dorsal explants. wt, wild type. N, number of experiments; n, numbers of embryos analyzed.
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Fig. S1. Inhibition of FGF signaling during early gastrulation. (A-C) Loss of myf5 expression in embryos treated with SU5402 from stage 9-11 (N=1). (A) WT expression pattern in DMSO-treated embryo. (B) Absence of myf5 expression in SU5402-treated specimen. (C) Quantification of results. (D-K) Partial downregulation of myf5 (D-F; N=2), myod1 (GI; N=3) and tbxt (J-L; N=3) following SU5402 treatments from 10-11.5 (D-F) or 10-12 (G-L), as compared to WT control specimens (D,G,J). (F,I,L) Quantification of results. Embryos shown in vegetal view, dorsal at top. N, number of experiments; n, number of embryos analyzed; st., stage; wt, wildtype.
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Fig. S2. Dose-dependent inhibition of foxj1 expression by dnfgfr1. (A-C) foxj1 expression in stage 10 control embryo (A) and specimens injected with high (B) or low (C) doses of dnfgfr1 mRNA (N=2). Embryos are shown in vegetal view, dorsal at top. (D) Quantification of foxj1 (in A-C). (E) pitx2c analysis at st. 31 after unilateral low-dose dnfgfr1-injection (8pg DNA) targeting the left GRP. co, control; dnfgfr1, dominant-negative FGF receptor 1; N, number of experiments; n, number of embryos analyzed; st., stage; wt, wildtype.
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Fig. S3. Late gastrula inhibition of FGF does not impair GRP morphogenesis or flow. (A-E) SEM-analysis of GRP in stage 17 dorsal explants revealed no significant difference in ciliation between DMSO- and SU5402-treated embryos incubated from late gastrula stages onwards (N=2). SEM photographs of dorsal GRP explants of DMSO (A) or SU5402-treated (B) specimens in ventral view. Colorations in (A,B) and in blow-ups (A',B') indicate non-ciliated cells (red), cells with normal posteriorly polarized cilia (green) and cilia with other locations (blue). Quantification of mean cilia lengths (C), mean ciliation rates (D) and distribution of cilia (E) revealed no significant differences. (F) SEM-analysis of GRP in stage 17 dorsal explants revealed no significant difference in cilia length between wildtype and low-dose dnfgfr1-injected (16pg) embryos. (G-I) Analysis of leftward flow in
dorsal explants of stage 17 embryos revealed WT flow in DMSO- and SU5402-treated specimens incubated from late gastrula stages onwards. (G, H) Wind rose diagrams of representative explants showing mean directionality of transported fluorescent beads and robustness of flow, as indicated by rho (p; N=2). Percentage of beads transported in main direction (left in both cases) is indicated.
n, number of beads analyzed per explant. (I) Quantification of flow quality (rho value), as calculated from different time lapse movies of DMSO- (n=9) or SU5402-treated (n=15) specimens. The dimensionless number rho (p) takes on a value between 0 (no directionality) and 1.0 (strict directionality) and describes the quality of flow. Wildtype flow was defined as >0.6 previously (for details see Methods). a, anterior; l, left; p, posterior; r, right; N, number of experiments; n, number of embryos analyzed; n.s., not significant; p, rho; st., stage
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Fig. S4. Late gastrula inhibition of FGF impairs lateral GRP cell formation. (A-F) Wildtype GRP marker gene expression in central and lateral sub-populations in stage 17 dorsal explants. Level of transversal sections in B’, C’, D’, E’ (dorsal to the top) indicated by white dashed lines in B,C,D, and E. Approximate position of myod1-positive part (presomitic mesoderm and lateral somitic GRP; cf. Fig. 4B) is outlined by black dashed lines in sections. (A) a2ml1 expression marking the endoderm flanking the GRP. (B-C) Expression of cilia marker genes tekt2 (B) and dnah9 (C) highlight entire GRP. Transversal sections (B’,C’) show superficial signals in the GRP, but not in the endoderm or somites. (D,D') nodal1 marks lateral GRP. (E,E') shh expression (central GRP plus deep notochordal cells). (F) Double staining of a2ml1 and shh highlights lateral GRP cells by lack of staining. (G-L) Loss of lateral GRP cells upon SU5402 treatment from stage 12-17 (I, J; n=80) but not 13-17 (K,L; n=69), as assessed by nodal1 (G,I,K) and shh/a2ml1 (H,J,L) staining of DMSO (G,H; n=66) and SU5402 (I-L) incubated specimens. (M-R) Analysis of brachyury (tbxt; M-O) and myod1(P-R) at stage 17 to visualize late mesoderm differentiation. (M,P) WT expression in DMSO-treated specimens. (N,Q) Reduced expression in specimens treated with SU5402 from st. 10.5-17. (O,R) WT expression in embryos treated with SU5402 from st. 11.5-17. (S-X) Down-regulation of lateral GRP marker genes nodal1 and dand5 in embryos unilaterally injected with low dose dnfgfr1 mRNA (16pg; N=10). Injected embryos showed strong reduction or absence of nodal1 (T) and dand5 (W) on injected side (blue arrowhead), as compared to WT expression (S,V) in untreated embryos. (U,X) Quantification of results (in S-W). All embryos are dorsal explants shown in ventral view (except for dorsal view in N,P,Q,R). Anterior at top. co, control; GRP, gastrocoel roof plate; N, number of experiments; n, number of embryos analyzed; st., stage; wt, wildtype.
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Fig. S5. Loss of lateral GRP by late inhibition of FGF occurs in the absence of apoptosis or altered proliferation. (A-C) Unaltered proliferation in DMSO (A) and SU5402-treated specimens (B), as assessed by immunohistochemistry using a phospho-histone3-specific antibody (red) in dorsal explants of stage 13/14 embryos. Cell boarders indicated by Alexa 488 phalloidin (green) and nuclei by Hoechst staining (blue; A,B). (C) Quantification of results. (D,E) TUNEL-staining of stage 13/14 dorsal explants in ventral (D,E) and dorsal view (D',E'), showing the GRP and neuroectoderm, respectively. Apoptosis was unaffected in SU5402-treated specimen (E,E') as compared to DMSO-treated control (D,D'). (F-K) Injection of a bcl-XL DNA-expression construct (H,K) did not rescue loss of nodal1 (G,H) or dand5 (J,K) expression in dorsal explants of SU50402-treated embryos (G,J), as compared to WT nodal1 (F) and dand5 (I) expression of DMSO-treated specimens. Data were derived from three independent experiments. Anterior to the top in all explants. a, anterior; l, left; n, number of embryos analyzed; p, posterior; r, right; st., stage.
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Fig. S6. SEM analysis of GRP and vesicle abundance in SU5402-treated specimens. (A-B) Dorsal explants from DMSO (A) and experimental specimen treated with SU5402 from stage 12-17 (B). Boxed areas were used for evaluation of cilia polarization in Fig. 5B,C. Specimens shown at low magnification in side view (tilted angle) reveal the breaking edge, in order to demonstrate continuity of somite and lateral GRP (pink) in WT sample and lack thereof in SU5402-treated
specimen. (C) Tissue morphology of representative WT GRP in ventral view depicting separation between central and lateral parts of GRP (indicated by dashed lines). Zoom-ins on left (C') or right (C'') dividing lines visualize differences in tissue morphology and vesicle distribution across central and lateral aspects of GRP. (D,E) Quantification of GRP vesicles (per µm2) in different areas of GRP
(D; N=2), and between left and right lateral GRP aspects (E; N=2). Note that vesicle abundance did not differ between left and right sides (E). GRP, gastrocoel roof plate; N, number of experiments; n, number of cells quantified; n.s., not significant; µm, micrometer.
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Fig. S7. Loss of somitic GRP upon Sprouty1-induced inhibition of FGF/Ca2+-signaling. (A-F) ISH-analysis of gastrula (A,B; vegetal view) or dorsal explants of neurula stage (C-F; ventral view) embryos injected bilaterally with spry1-mRNA at the 4-cell stage (N=4). Unaffected expression of tbxt (B,D) and myod1 (F) following spry1 overexpression, as compared to uninjected controls (A,C,E). (G-I) SEM-analysis of stage 17 dorsal explants (ventral view) injected unilaterally on the left with spry1-mRNA. spry1-mRNA overexpression resulted in loss (G,G') or reduction of lateral GRP cells (H,H',H'') on the injected side. Blow-ups (G’,H’,H'’, as indicated in G and H) mark somitic GRP part (colored in yellow). (H'') Higher magnification of boxed area in (H'). White arrowheads indicate posteriorly localized WT cilia on central GRP cells, while red arrowheads highlight residual cilia at reduced lateral GRP. (I) Quantification of results. Dividing lines between lateral and central as well as lateral and endodermal cells indicated by yellow dashed lines. co, control; GRP, gastrocoel roof plate; N, number of experiments; n, number of embryos analyzed; st., stage; wt, wildtype.
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Fig. S8. Loss of Pkd2 resulted in inhibition of somitic GRP marker genes. (A-D) Downregulation of lateral GRP markers nodal1 and dand5 in embryos unilaterally injected with Pkd2-MO (1pmol). Injected embryos showed strong reduction or absence of dand5 on targeted side (B) in comparison to bilateral expression (A) in wildtype embryos. Quantification of dand5 (C) and nodal1 expression (D). Data derived from three independent experiments. co, control; n, number of embryos analyzed; wt, wildtype.
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