Kreis J , Wielath FM , Vick P .

             gastrulation
             Wnt signaling pathway

Xla Wt + rab7a MO (Fig. 1 M)
Xla Wt + rab7a MO (Fig. 1 O U)
Xla Wt + rab7a MO (Fig. 2B B'B'' E E'E'')
Xla Wt + rab7a MO (Fig. 2 BDD'F)
Xla Wt + rab7a MO (Fig. 2 I J J')
Xla Wt + rab7a MO (Fig. 4 H K Q R T)
Xla Wt + rab7a MO (Fig. 5 B)
Xla Wt + rab7a MO (Fig. 7 KM OQ)
Xla Wt + rab7a MO (Fig. S2 J J')
Xla Wt + rab7a MO (Fig S2 L L'l L'')
Xla Wt + rab7a MO (Fig. S2. NPR)
Xla Wt + wnt3a (Fig. 7 F)
Xla Wt + wnt8a (Fig. 7 BD)

	Fig. 1. rab7 shows dynamic expression and inhibition resulted in gastrulation defects. (A,B) Expression of rab7 mRNA in animal hemisphere at stage 3 and sagittal section of late blastula stage. (C) Upon gastrulation transcripts were enriched in deep mesodermal ring, (C′) sagittal section as indicated in (C), (D) dorsal area close-up view of a vibratome-sectioned specimen. (E) At stage 12.5 rab7 accumulates in neural plate ectoderm, (E′) sagittal section of E. (F) During neurulation transcripts get restricted to notochord, neural tube and brain tissue, (G) sagittal and (H) transversal section indicated in F. (I) In tailbud stages transcripts were detected in the notochord and pronephric and head tissues, (J) transversal section indicated in I, (J′) enlargement of J. (K,N) Control specimen at stages 13 and 28. (L,O) Injection of rab7 TBMO in dorsal lineage caused gastrulation defects, resulting in severe dorsal phenotypes. (M,P) Co-injection of ca-rab7 mRNA rescued loss of function phenotype of morphant embryos. (Q) Quantification of results in K–P. (R,T) Tailbud control embryos, (S,U) siblings treated with rab7 CRNP (S+L) or rab7 SBMO showing dorsal phenotypes. (V,W) Quantification of results in (R,S and T,U). a, anterior; an, animal; ca, constitutive active; co, control; CRNP, Cas9 Ribonucleoprotein; d, dorsal; GD, gastrulation defect; p, posterior; SBMO, splice blocking Morpholino Oligonucleotide; st., stage; TBMO, translation blocking Morpholino Oligonucleotide; v, ventral; veg, vegetal; wt, wild type. Scale bars: 250 µm.
	Fig. 2. rab7 morphants show impaired dorsal lip formation. (A) Untreated embryos at the beginning of gastrulation forming a dorsal lip, (B) which can also be observed in rab7 deficient siblings (black arrowheads). (C) In stage 10.5, sagittal sectioned control embryos revealed early involution movements. (C′) Enlargement of the area in the dashed black box in C, showing formation of Brachet's cleft was visible (white outlined arrowheads), (D) dorsal rab7 knockdown specimen rarely showed involution in sagittal sections or Brachet's cleft formation, (D′) enlargement of the area in the dashed black box in D, (black arrowheads). (E) Older control embryo (mid-gastrula) with evenly shaped lip, (E′) sagittal section with Hoechst stained nuclei revealed proceeded dorsal involution and archenteron formation. (F) Same-age morphant siblings developed irregularly shaped lips around the blastopore (black arrowheads), and (F′) did not show any involuting tissue or archenteron formation in Hoechst stained sagittal section (white arrowhead). (G) Wild-type nog expression of control embryos and (I) reduced expression of nog and concomitant failed notochord elongation of dorsal rab7 morphants. (H) Sagittal section and (H′, enlargement of the area in the dashed black box in H) exhibited proceeded notochord elongation marked by nog in untreated embryos. (J) Sagittal section and (J′) enlargement of the area in the dashed black box in J clearly depicted impaired nog expression (black arrowhead). (K) Schematic figure of sagittally bisected mid- to late gastrula embryo indicating view of dorsal lips shown in M-O″. (L) Quantification of overall embryonic phenotypes exemplified in M-O″. (M,N,O) bisected embryos stained for β1-Integrin (white). (M′,N′,O′) Histon 2B (H2B) GFP signal used as lineage tracer (green), (M″,N″,O″) merged channels. (M,N) Normal distributed β1-Integrin in wild-type dorsal lips or h2b mRNA-injected specimen, indicating target site. (O) Axial rab7 morphant cells revealed altered shapes and lack of involution behavior and archenteron formation. Scale bars: A-J′, 250 µm; M-O″, 100 µm.
	Fig. 3. rab7 is required for axial elongation and notochord morphogenesis. (A,D) Early wild-type neurula embryos showing elongated notochords marked by the expression of not or shh. (A′,D′) Transversal sections indicated in (A,D) revealed not and shh expression in the axial neural plate and shh additionally throughout the whole notochord. (A″,D″) enlargement of the area in the dashed black boxes in A′ and D′, respectively. (B,E) Dorsal rab7 TBMO injection resulted in embryos failing to elongate their notochord, although not expression intensity (black outlined arrowheads) was not reduced in comparison to shh (black arrowheads). (B′) Transversal section of morphant embryos showed lateral expanded expression of not above the dorsal lip and (E′) severely reduced shh expression. (B″,E″) enlargement of the area in the dashed black boxes in B′ and E′, respectively. (C,F) Quantification of results in A,B and D,E, respectively. Scale bars: 250 µm.
	Fig. 4. Dorsal mesoderm specification requires Rab7 independent of the organizer. (A,D,G,J,M,P,S) Untreated control specimen, or β-gal mRNA control injections confirming dorsal targeting (white outlined arrowheads in A,M) with wild-type gsc, ventx1, nodal3, foxj1, myod1, myf5 and tbxt expression, respectively. (B,E) rab7 loss of function in dorsal or ventral lineage did not alter gsc or ventx1 expression, respectively. (H) nodal3 expression was slightly reduced in some rab7 morphant embryos. (K,N,Q,T) expression of foxj1, myod1, myf5 and tbxt were severely affected in specimen with rab7 deficiency (black arrowheads). (C,F,I,L,O,R,U) Quantification of results in A,B,D,E,G,H,J,K,M,N,P,Q,S,T. Scale bars: 250 µm.
	Fig. 5. Specification of the ventral mesoderm requires Rab7. (A) Untreated early tadpoles with normal tail development, which was impaired by rab7 loss of function in ventral lineage resulting in posterior truncations (B, black arrowhead). (C) Quantification of results in A,B. (D) Mid-gastrula control embryo depicting wild-type myod1 expression, (E) injected β-gal mRNA marks ventrally targeted area (outlined white arrowheads). (F) Co-injection of β-gal mRNA and rab7 TBMO revealed reduced or absent ventral myod1 expression in most specimen at corresponding target site (white outlined black arrowheads). (G) Quantification of results in D-F. (H) Ventro-lateral mesoderm shown by wild-type myod1 expression. (I) Ventral injection of suboptimal doses of ctnnb1 MO resulted in minor reduction of myod1 (black outlined arrowheads), (J) whereas optimal doses caused complete loss of ventro-lateral expression (black arrowheads). (K) Parallel injection of rab7 TBMO and ctnnb1 MO, both in suboptimal doses, lead to absence of myod1 (black arrowheads). (L) Quantification of epistatic function of Rab7 and Ctnnb1 in ventro-lateral mesoderm, different manifestations of myod1 expression exemplified below. Scale bars: 250 µm.
	Fig. 6. Small GTPase Rab7 acts epistatically with the LE regulator Vps4. (A) Untreated specimen; (B) Injection of dn-vps4 mRNA or (C) rab7 TBMO in suboptimal dose led to minor reduction of myod1 expression on ventral side (black outlined arrowheads in C). (D) Parallel suboptimal injection of rab7 TBMO and dnvps4 mRNA resulted in absent myod1 expression (black arrowheads). (E) Quantification of results, different manifestations of myod1 expression in analysis are exemplified below quantification. Scale bar: 250 µm.
	Fig. 7. Rab7 is required for canonical Wnt pathway activation. (A) Wild-type dorsal expression of chd in comparison to specimen with (B) radial injected wnt8a mRNA showing an extended chd expression domain around the blastopore. (C) Co-injection of rab7 TBMO restricted additional chd expression to normal wild-type state. (D) Quantification of results in A-C. (E) Untreated control specimen, (F) in comparison to induced double axis (white arrowhead) after ventral wnt3a mRNA injection, (G) parallel injection of rab7 TBMO inhibited secondary axis formation (black arrowhead). (H) Quantification of results in E-G. (I) Luciferase-based Wnt reporter assay analysis at stage 10.5 illustrated that wnt8a induced reporter activity was blocked by rab7 loss of function in animal caps. (J,N) Wild-type expression of myod1 and myf5 in stage 11 control embryos. (K,O) Absent dorsal myod1 expression and lost domains upon rab7 loss of function (black arrowheads). (L,P) Treatment with LiCl partially rescues dorsal expression of myod1 and myf5 in rab7 morphant embryos (white arrowheads). Please note that LiCl-induced ectopic expression in the axial mesoderm was not inhibited by the rab7 TBMO. (M,Q) Quantification of results in J-L and N-P, respectively. Scale bars: 250 µm.
	Fig. S1. rab7 exhibits dynamic expression pattern. (A) rab7 expression in animal hemisphere of st. 1 embryo, (A') sagittal section of (A). (B,C) Early and late gastrula stages displayed transcripts in deep mesoderm, (C') sagittal section of (C). (D) Onset of neurulation with rab7 restricted to neural plate and notochord, (D') sagittal section of (D), (E) transversal section as indicated in (D). (F,G) Tailbud stage expression in the notochord, trunk neural crest, eyes, pronephric and head tissues and in dorsal fin mesenchyme. a, anterior; an, animal; d, dorsal; p, posterior; st., stage; v, ventral; veg, vegetal.
	Fig. S2. Loss of rab7 caused gastrulation and axis elongation defects. (A-C) Synthego ICE CRISPR analysis of rab7 sgRNAs targeting both S and L alleles or L alone. (D) Wildtype control embryo compared to lateral view of rab7 CRNP (E) treated specimen with severe gastrulation defects, (E') dorsal view. (F) Quantification of results in (D-E). (G) Scheme of binding sites of rab7 SBMO targeting both homeologs. (H) RT-PCR of knockdown by rab7 SBMO demonstrated successful inhibition of splicing.
	Fig. S2. Loss of rab7 caused gastrulation and axis elongation defects.( continued) (I,K) Wildtype neurula and tailbud control specimen with stage specific rab7 transcripts, (I',K') transversal sections of (I,K), (K'') blow-up of (K'). (J,L) Radial knockdown of rab7 by SBMO caused significant reduction of rab7 expression in neurula and tailbud stages, respectively, (J',L') transversal sections of (J,L), (L'') blow-up of (L'). (M,O,Q) Untreated control tadpoles and (N,P,R) milder affected morphant embryos, suffering from AP-axis defects, knockdown was induced using (N) rab7 TBMO, (P) rab7 SBMO and (R) rab7 CRNP (S+L). Scale bars: 250μm a, anterior; an, animal; co, control; CRNP, Cas9 Ribonucleoprotein; d, dorsal; EF1 α, Elongation factor 1 alpha; FP, forward primer; G, genomic DNA; GD, gastrulation defect; p, posterior; RP, reverse primer; RT, reverse transcriptase; st., stage; v, ventral; veg, vegetal; wt, wildtype.
	Fig. S3. Lineage-specific knockdown of rab7 in the dorsal or ventral mesoderm. (A,F) Lineage tracing scheme of injection site (dorsal or ventral) in st. 3 embryos; tested st. 10-10.5 illustrates observed localization of injected material at correct target side (dorsal or ventral), and approximately one stage later before fixation (for late gastrula analyses). mGFP mRNA lineage tracer was co-injected with coMO (B,D,G,I) or rab7 TBMO (C,E,H,J), correct targeting and corresponding phenotypes visualized via fluorescent mGFP (B'-E',G'-J'), brightfield (B-E,G-J) and merged channels (B''-E'',G''-J''). coMO injected specimen depicted normal gastrulation at st. 10- 10.5 and 11-11.5, both, when injected dorsally (B,D) or ventrally (G,I). (C,E) Impaired dorsal lip formation of rab7 morphant embryos at onset of gastrulation, with increasing severeness during gastrulation. (H) Ventral injected specimen did not show altered tissue at st. 10-10.5, (J) impaired lip formation at later gastrula stages with diffuse appearance of ventral lip. coMO, control Morpholino Oligonucleotide; d, dorsal; p, posterior; st., stage; TBMO, translation blocking Morpholino Oligonucleotide; v, ventral; veg, vegetal.
	Fig. S4. rab7-deficient embryos develop axial elongation defects and impaired notochord morphogenesis. (A) Elongated notochords of control embryos highlighted by not expression (B) in comparison to dorsal rab7 SBMO injection, which resulted in reduced notochord elongation (arrowhead). (C) Quantification of results in (A-B). (D) Tailbud stage embryos revealed not expression throughout the notochord, (D') also shown in sagittal and (E) transversal section and (E') blow-up of (E). (F) rab7 knockdown in dorsal lineage did not reduce not expression, (F') but affected notochord morphogenesis depicted in thicker notochord as shown in sagittal section. (G) Transversal sections revealed some morphant specimen also developed split notochords, (G') blow-up highlighting open dorsal tissue. (H-K) Transversal sections of st. 24/25 embryos stained for F-Actin (blue) and MZ- 15 (green). (H,J) control specimen displayed wildtype outer sheets of notochords (green), (I) MZ- 15 staining was absent in rab7 TBMO injected or (K) reduced in rab7 CRNP treated embryos. Scale bars: (A-G) 250μm; (H-K) 50μm a, anterior; CE, convergent extension; co, control; CRNP, Cas9 Ribonucleoprotein; d, dorsal; n.s., not significant; p, posterior; st., stage; v, ventral; wt, wildtype.
	Fig. S5. Loss of Rab7 does not impact organizer gene expression or DV patterning. (A,C) St. 10.5 untreated controls showing wildtype gsc expression (B,D) comparable to embryos either injected with rab7 SBMO or rab7 CRNP (S+L). (E) Quantification of results in (C-D). (F) Wildtype chd expression in control embryos were unaffected in embryos injected with (G) rab7 CRNP (S+L). (H) Quantification of results in (F-G). (I) DV range of chd expression of control embryos was unaltered in embryos injected with rab7 TBMO in (J) dorsal or (K) ventral lineage. Scale bars: 250μm co, control; CRNP, Cas9 Ribonucleoprotein; d, dorsal; n.s., not significant; v, ventral; wt, wildtype.
	Fig. S6. A Specification of the ventro-lateral mesoderm requires proper Rab7 function and hrs knockdown in the ventral mesoderm phenocopies loss of rab7. (A) Untreated specimens with normal tail development. (B) Impaired posterior development after loss of rab7 in ventral lineage (black arrowhead). (C) Control embryo depicting wildtype tbx6 expression, (D) ventral rab7 knockdown inhibited tbx6 expression (black arrowheads). (E) Quantification of results in (C-D). (F) Wildtype myod1 expression in control specimen, (F') sagittal section of (F) showing ventral expression. (G) Ventral injection of hrs MO (0,8-1 pmol) caused loss of myod1 (black arrowheads), (G') sagittal section as indicated in (G) with absent ventral expression (black arrowhead). (H) Quantification of results. Scale bars: 250μm a, anterior; co, control; d, dorsal; MO, morpholino oligonucleotide; p, posterior; st., stage; v, ventral; wt, wildtype.
	Fig. S7. Rab7 is required for exogenous activation of canonical Wnt pathway. (A) Wildtype dorsal expression of nog in st. 10 embryos in comparison to specimen with (B) radial injected wnt8a mRNA showing complete dorsalization by extended nog expression around the blastopore. (C) Co-injection of rab7 TBMO restricted nog expression to normal wildtype area again. (D) Quantification of results in (A-C). (E) Wildtype ventral expression of ventx1 in st. 10 embryos, (E') sagittal section as indicated in (E) showing ventx1 expression domain. (F) Loss of ventx1 expression upon radial wnt8a mRNA injection, (F') highlighted in bisected embryo. (G) Co-injection of rab7 TBMO partially restored lost ventx1 expression, (G') which in the sagittal section remained smaller and weaker on ventral side. (H) Quantification of results in (E-G). Please note premature (st. 10) appearance of fully radial lips in (B,F) but not in (A,C,E,G) in same age specimens. (I) Late gastrula Luciferase-based BAR-reporter assay (bottom) demonstrating tissue-specific inhibition of endogenously induced Wnt reporter activity in the ventral mesoderm (middle) after co-injection of rab7 TBMO into the ventral mesodermal lineage at 4-cell stage (top). (J) Wildtype AP axis in control specimen. (K) ctnnb1 induced right-sided secondary axis (white arrowhead), (L) which was not inhibited by rab7 knockdown (white arrowhead). (J-L) Axes highlighted by sox3 expression. (M) Quantification of results in (J-L). (N) Control embryos showing normal dorsal gsc expression, (N') confirmed in sagittal section indicated in (N). (O) Ventral wnt3a mRNA injection induced second organizer marked by gsc (white arrowhead), (O') sagittal section revealed additional ventral gsc positive domain (white arrowhead). (P) Lost second gsc expression upon parallel rab7 TBMO injection (black arrowhead), (P') highlighted in sagittal section (black arrowhead). Scale bars: 250μm a, anterior; co, control; d, dorsal; n.s., not significant; p, posterior; v, ventral; wt, wildtype.
	rab7a (RAB7A, member RAS oncogene family) gene expression in X. laevis embryo NF stage 3 (4 cell), assayed via in situ hybridization, animal hemisphere up.
	rab7a (RAB7A, member RAS oncogene family) gene expression in X. laevis embryo NF stage 10.5, assayed via in situ hybridization, dorsal up (C), and in sagittal section(C') with dorsal right.
	rab7a (RAB7A, member RAS oncogene family) gene expression in X. laevis embryo NF stage 12.5, assayed via in situ hybridization, dorsal up (E) , and in sagittal section (E') dorsal right.
	rab7a (RAB7A, member RAS oncogene family) gene expression in X. laevis embryo NF stage 15, assayed via in situ hybridization, anterior view, dorsal up (F) , and in sagittal section (G) with posterior right, and in transverse section (H) with dorsal up.
	rab7a (RAB7A, member RAS oncogene family) gene expression in X. laevis embryo NF stage 32, assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	rab7a (RAB7A, member RAS oncogene family) gene expression in X. laevis embryo NF stage 32, assayed via in situ hybridization, in transverse section through the trunk region, dorsal up.

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