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FIGURE 1. Rab7 is necessary for correct LR axis development. (A) Experimental setup for the shown experiments: Injections at four-cell stage (left) targeted the dorsal mesoderm to analyze mesoderm specification (tbxt; middle-left) at gastrula stages, a prerequisite for the formation of the dorsal mesoderm derived LRO (middle-right) in wild type situation during neurula stages, which in turn induces LR asymmetric features (here pitx2 expression; right) at tailbud stages. (B) Wild type circular tbxt expression of st. 10.5 control specimen, (C) injection of reduced amounts of rab7 TBMO did not alter tbxt expression, (D) whereas higher doses reduced transcripts (black outlined arrowheads) or (E) abolished them in the targeted dorsal lineage (black arrowheads). (F) Quantification of tbxt expression. (G) Uninjected st. 32 tadpole, or specimen injected with (H) coMO depicted wild type axis elongation compared to (I) shortened axis of rab7 TBMO treated specimens. (G′–I′) Please note in frontal sections through somitic lineage that all specimen showed myod1 expression in somites along AP-axis. (G′′–I′′) Magnification of myod1 expressing somites as indicated in dashed boxes, respectively. Please note altered somite shapes in morphants. (J) Quantification of axis phenotype. (K) Left-sided pitx2 expression in untreated st. 32 tadpoles and (L) coMO injected ones. (M) Lost left-sided pitx2 expression upon rab7 knockdown (black arrowhead). (N) Quantification of pitx2 expression. a, anterior; cLRO, central LRO; co, control; coMO, control Morpholino oligomere; d, dorsal; l, left; LRO, left-right organizer; N, number of experiments; n, number of evaluated embryos; n.s., not significant; p, posterior; pmol, picomole; r, right; sLRO, somitic LRO; st., stage; TBMO, translation blocking Morpholino oligomere; v, ventral; wt, wild type.
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FIGURE 2. Rab7 knockdown resulted in loss LRO sensor tissue. (A) Experimental setup for the shown experiments: Injections at four-cell stage (left) targeted the dorsal/dorsolateral mesoderm to analyze superficial mesoderm induction (foxj1; middle) or somitic mesoderm specification (myf5; middle) at gastrula stages, following analyses of general LRO formation (tekt2; right) or sLRO specification (dand5/nodal1; right) during neurulation. (B) Wild type crescentic foxj1 transcripts were (C) unaltered or (D) mildly affected by low dose rab7 TBMO injections (black outlined arrowheads). (E) Quantification of foxj1 expression. (F) Expression domain of tekt2 in the LRO of st. 17 embryos, (G) depicted slightly stronger posterior and reduced anterior signals upon low dose rab7 TBMO treatment. (H) Wild type dorsolateral myf5 expression (I) was significantly reduced in rab7-morphant embryos (black outlined arrowheads). (J) Quantification of myf5 expression. (K,O) Expression of dand5 and nodal1 in the somitic domains of the LRO of control and (L,P) coMO injected specimen, respectively. (M) Transcripts of dand5 or (Q) nodal1 were lost (black arrowheads) or severely reduced (black outlined arrowheads) in rab7-deficient embryos (low dose TBMO). (N,R) Quantification of dand5 and nodal1 expression, respectively. (S) Left-sided unilateral knockdown (marked by asterisk) of low dose rab7 TBMO in myod1 expressing LRO, (S′) section revealed lost superficial expression of myod1 at injection side (black arrowhead). cLRO, central LRO; co, control; coMO, control Morpholino oligomere; d, dorsal; l, left; LRO, left-right organizer; N, number of experiments; n, number of evaluated embryos; n.s., not significant; r, right; sLRO, somitic LRO; st., stage; TBMO, translation blocking Morpholino oligomere; v, ventral; wt, wild type.
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FIGURE 3. Loss of Rab7 blocks dorsal mesoderm specification upstream of Mapk activation. (A) Uniform circular appearance of tbxt expression of st. 10 Xenopus embryos (B) faded due to dorsal knockdown of rab7 (black arrowheads). (C) Rescue injection of ets2 mRNA restored expression almost back to wild type level (outlined black arrowheads). (A′–C′) Panels beneath depict corresponding sagittal sections. (D) Quantification of tbxt expression rescued by ets2 mRNA. (E) Circular tbxt expression of st. 11 specimen (F) faded upon unilateral dorsal rab7 LOF (black arrowhead). (G) Coinjection of mapk1D324N mRNA in morphant embryos partially restored lost dorsal tbxt expression (black outlined arrowhead). (H) Quantification of tbxt expression rescued by mapk1D324N mRNA. (I) myod1 horseshoe-shaped expression of st. 11 specimen (J) displayed downregulated paraxial transcripts after dorsal loss of rab7 (black arrowheads). (K) ets2 mRNA injected dorsally rescued the paraxial mesoderm identity (outlined black arrowheads); please note ectopic expression found in animal areas above the dorsal gap (white arrowhead). (L) Quantification of myod1 expression rescued by ets2 mRNA. (M) Horseshoe myod1 expression of st. 11 specimen (N) faded upon dorsal rab7 LOF (black arrowheads). (O) Coinjection of mapk1D324N mRNA in morphant embryos partially restored lost dorsal myod1 expression (black outlined arrowheads). (P) Quantification of myod1 expression rescued by mapk1D324N mRNA. (Q) Injection scheme and localization of imaged animal tissue of st. 12.5 embryos (R,R′′) Nucleic and cytosolic accumulation of pMapk1 (magenta) at posterior neural plate of wild type specimen. (S,S′′) Downregulation of pMapk1 signal intensity in rab7-morphant neural plates. (R′,S′) For lineage tracing and nuclear staining of h2b mRNA (green) was used, cell borders are highlighted by F-actin (blue). (T) Intensity quantification of pMapk1 signal in panels (R,S). All rab7 TBMO injections in this figure were done with high dose approach. a, anterior; co, control; d, dorsal; N, number of experiments; n, number of evaluated embryos; p, posterior; pmol, picomole; st., stage; TBMO, translation blocking Morpholino oligomere; v, ventral; wt, wild type; µm, micrometer.
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FIGURE 4. Rab7 is required for FGF-dependent mesoderm induction. (A) Experimental setup for the shown animal cap elongation assay: Injection at four-cell stage (left) targeted the animal hemisphere, at st. 9 animal caps were dissected (middle) and incubated in Activin A or buffer solution (right). (B) Quantification of animal cap elongation. (C) Untreated animal caps (D) elongated upon Activin treatment. (E) rab7-deficient, untreated animal caps remained in a roundish shape, (F) treated ones depicted partial or complete inhibition of elongation. (D) tbxt expression was only observed in Activin treated, elongated caps. (G,K) Wild type animal caps of st. 11 embryos. (H) Induction of tbxt expression (white arrowheads) in animal caps by fgf8 or (L) vras mRNA overexpression. (I,M) Loss of rab7 in those animal caps blocked tbxt induction (outlined arrowheads). (J,N) Quantification of tbxt induction via fgf8 or vras mRNA. All rab7 TBMO injections in this figure were done with high dose approach. (O) Proposed model for Rab7-dependent dorsal mesoderm and subsequent sLRO specification triggering LR development: (Top) Rab7-positive LEs could provide the platform for sustained FGF signal transduction (left). FGF signals mediate deep and superficial mesoderm specification (middle-left). Mesoderm development enables LRO morphogenesis (middle-right), the crucial tissue for LR asymmetry (right). (Bottom) Loss of Rab7 would inhibit FGF signal maintenance due to lost signaling platforms (left), causing a lack of deep and superficial mesoderm specification (middle-left). Loss of mesoderm prevents sLRO formation (middle-right) blocking LR axis establishment (right). cLRO, central LRO; co, control; d, dorsal; ind., induction; LRO, left-right organizer; N, number of experiments; n, number of evaluated embryos; sLRO, somitic LRO; st., stage; TBMO, translation blocking Morpholino oligomere; v, ventral; wt, wild type.
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Supplement Figure 1. Loss of Rab7 blocks dorsal mesoderm specification upstream of Mapk activation.
(A) Xenopusst. 32 tadpoles depicted wildtype axis elongation compared to (B) shortened axis of dn-fgfr1 mRNA
treated specimen, somites are highlighted by myod1 expression. (A', B') Frontal sections through somitic
domain, (A'', B'') zoom-ins of myod1 expressing somites as indicated in dashed boxes, respectively. (C)
Quantification of axis phenotype. (D) Dorsal gap of wildtype myod1 expression of st. 10.5 embryos (E) depicted
ectopic myod1 induction upon ets2 mRNA overexpression (white arrowheads), (D', E') clarified in sagittal
sections respectively. (F) Quantification of dorsal myod1 induction.
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Supplement Figure 2. Dorsal mesoderm induction depends on Rab7.
(A) Experimental setup of artificial organizer induction: Injection at four-cell stage (left) targeted animal
hemisphere to analyze artificial organizer induction and surrounding tbxt expression in animal caps of st. 10
specimens. (B) Quantification of induced artificial organizers in animal caps. (C) Wildtype animal caps of st.
10 embryos. (D) nodal1 mRNA injection induced pigment accumulation, indicating organizer induction (white
arrowhead), induction site is sphered by tbxt expression. (E, F) Co-injection of either rab7 TBMO or dn-fgfr1
mRNA resulted in artificial organizer inhibition (outlined white arrowheads) and blocked tbxt expression in
animal caps. (C'-F') Panels beneath show half sections of treated tissues and (C''-F'') zoom-ins of potential
organizer induction sites, respectively. (C-F) For lineage tracing lacZ staining was used.
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dand5 (DAN domain family member 5, BMP antagonist) gene expression in X. laevis embryo, NF stage 17, in the somitic domains of the LRO (Left Right Organizer) of the gastrocoel roof plate.
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nodal1 (nodal homolog 1) gene expression in X. laevis embryo, NF stage 17, in the somitic domains of the LRO (Left Right Organizer) of the gastrocoel roof plate.
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tekt2 (tektin 2) gene expression in X. laevis embryo, NF stage 17, assayed via in situ hybridization, in etc central domains of the LRO ( left right organizer) of the gastrocoel roof plate.
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