Le Bouffant R et al. (2025), Comprehensive analysis of Ephrin ligand and rec...

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Int J Dev Biol 2025 Oct 13;693:133-142. doi: 10.1387/ijdb.250065jr.

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Comprehensive analysis of Ephrin ligand and receptor expression reveals exclusive domains during nephrogenesis for epha4/epha7 and efna3.

Le Bouffant R , Bello V , Buisson I , Umbhauer M , Riou JF .

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Interactions between ephrins and their Eph receptors regulate a broad range of cellular processes, including attraction, repulsion, adhesion and migration, all of which play crucial roles in tissue remodeling and homeostasis. While several ephrin ligands and Eph receptors are known to be expressed in the developing kidney, their specific roles, particularly during nephrogenesis, remain poorly understood. The development of the Xenopus pronephros provides an accessible and relatively simple model for studying vertebrate nephrogenesis. Through a comprehensive gene expression analysis of all ephrin ligands and Eph receptors present in Xenopus genomes, we have identified members of the Eph-ephrin signaling pathway that may contribute to pronephric development. Among them, efna3, which encodes an ephrin ligand, is strongly expressed in the ventral region of the pronephric anlage and later in the intermediate and distal segments of the developing tubule. This expression pattern is strikingly complementary to that of epha4 and epha7, which are expressed in the region forming the proximal tubule. This suggests a potential role for efna3-epha4/epha7 signaling in establishing a boundary between these domains during pronephros development.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: efna1 efna2 efna3 efna4 efna5 efnb1 efnb2 efnb3 epha10 epha2 epha3 epha4 epha5 epha6 epha7 epha8 ephb1 ephb2 ephb3 ephb4

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	Fig. 1. Expression of genes encoding the members of the Eph-ephrin signaling components in the pronephros at early tadpole stage 35/36. The enlarged region corresponding to the pronephric region is shown with a general view of the tadpole. (A) Genes encoding ephrin ligands. Expression of efna3 and b2 is detected in the developing pronephros; efna3 is expressed in the intermediate and distal segments of the tubule (It, Dt). Expression of efnb2 is observed in the glomus region (G). Expression of efna5 and b1 is detected at the level of the vessels associated with the pronephros: in the pronephric sinus (Ps), the posterior cardinal vein (Pcv), the common cardinal vein (Ccv) and the intersomitic veins (Iv). Efna5 expression is also observed in the heart (H). Efna1 seems to be weakly expressed in the pronephric sinus and intersomitic vein. Scale bars: 0.8 mm for whole embryo and 0.3 mm for embryo magnification. (B) Genes encoding Eph receptors. Expression of epha4, a7, b3 and b2 are clearly detected in the pronephros. Epha4 and EphA7 are expressed in the PT1, PT2 and PT3 segments of the proximal tubule (Pt1, three lines, Pt2, line and Pt3 arrowhead) while ephb3 is detected only in PT3 (arrowhead). Ephb2 is detected in the proximal, intermediate and the first distal segment of the tubule. No pronephric expression of epha2, a3, a5, a6, a8, a10 and b1 is observed. Expression of ephb4 is detected in the vessels associated with the pronephros. Pt, It, Dt: proximal, intermediate and distal tubule. Scale bars: 1.7 mm for whole embryo and 0.5 mm for embryo magnification.
	Fig. 2. Conservation of Xenopus efna3 in vertebrates. (A) Multiple alignment of vertebrate efna3 protein sequences. (B) Phylogenetic tree. List of accession numbers used for sequences analysis is shown in the Materials and Methods section.
	Fig. 3. Expression of efna3 at stages of pronephric development. WISH analysis at stages ranging from early neurula stage 14 (appearance of the kidney field) to early tadpole stage 35/36 (functional pronephros). Anterior, dorsal and lateral views are shown at neurula stages (st14 and 20). Lateral and dorsal views are shown at tailbud stage 25. Only lateral views are shown for tailbud stages 28 and 32, and early tadpole (st35/36). At stage 14, 20 and 25, images of sectioned embryos are shown with red dotted lines, indicating the corresponding levels on whole mounts. For convenience, these levels are just indicated with arrows 1, 2, and 3 at stage 28, 32 and 35/36 (note that the sections were not made from the whole mount specimen shown). Efna3 starts to be detected in the developing tubule from the stage 28. It is detected prior to stage 28 in the developing nervous system and migrating cephalic neural crest cells. Efna3 is expressed at stage 14 in the primary neuronal precursors (Np), and weakly in the anterior neural folds (Nf). Neural expression expands at st20 to the closing neural tube (Nt). It is strong in the emerging first (n1) and third/fourth (n3, n4) branchial arches neural crest streams. Cg: cement gland. At stage 25, efna3 expression is strong in branchial neural crest streams populating the first arch around the eye rudiment (n1), as well as the third and fourth arches (n3, n4). It is also detected in the dorsal neural tube (Nt). Efna3 is also expressed in the proctodeal region (Pr). At stage 28, efna3 is now expressed in the ventral region of the pronephric anlage (red arrowhead). It is detected in the dorsal region of the midbrain (Mb) and in the hindbrain (Hb), as well as in the developing branchial arches (Ba). In addition to the first branchial arch around the eye (n1), and the third and fourth branchial arches (n3, n4), efna3 is now also detected in the second branchial arch (n2). Expression of efna3 is observed in developing neural placodes, in the epidermis overlying the optic vesicle that contains the lens placode (Lp), in the ventral region of the otic vesicle (Ov) and in the developing posterior lateral line placode (Pp, blue arrowhead). Efna3 is also detected in the proctodeal region (Pr). Pronephric expression at stage 32 is very strong in a region of the tubule at the origin of the intermediate and distal segments (red arrowhead). Efna3 continues to be detected in the anterior midbrain (Mb) and in a hindbrain (Hb) region likely corresponding to rhombomere 4. It continues to be detected in the four branchial arches (n1, n2, n3 and n4), in the posterior lateral line placode and in the otic vesicle (Ov). It is now expressed in the developing lens (L). Expression at stage 35/36 is similar to stage 32 in the intermediate and distal segments of the developing tubule (It, Dt). Expression is diminishing in the branchial arches (Ba) and the hindbrain (Hb) but is very strong in dorsal midbrain (Mb), otic vesicle (Ov), and lens (L). Scale bar: 1.7 mm for whole embryo, 0.5 mm for embryonic magnification and 280 μm for section.
	Fig. 4. Expression of Eph receptors epha4, a7, b2 and b3 at stages of pronephric development. WISH analysis at stages ranging from neurula stage 14-18 (kidney field) to early tadpole stage 35/36 (functional pronephros). Anterior, dorsal and lateral views are shown at neurula stages (st14-18 and 20). Only lateral views are shown for tailbud stages 25, 28 and 32, and early tadpole (st35/36). Images of sectioned embryos at stage 35/36 are shown, with red dotted lines, indicating the corresponding levels on whole mounts (note that the sections were not made from the whole mount specimen shown). Pronephric expression of epha4, a7 and b2 is detected from stage 25 onward, while ephb3 is only detected in the pronephros at stage 32. Epha4 and a7 are mostly co-expressed in the dorsal region of the pronephric anlage at stages 25 and 28. Their expression is maintained in the emerging PT1 segments at stage 32 (blue arrowheads) and expands at stage 35/36 in PT2 (line) and PT3 (arrowhead). EphA4 is also expressed in connecting tubule (Ct). Expression of ephb2 is observed in the whole anlage at stages 25 and 28, and later in the developing tubule especially in intermediate region (It), while ephb3 transcripts are detected in the PT3 segment at stages 32 and 35/36. Expression of epha4 in the developing central nervous system (Fb, Mb, r3, 5: rhombomeres 3 and 5; cephalic neural crest n3: neural crest destined for third arch), developing heart (h) and tailbud (Bc) confirms previously published observations (Barnett et al., 2012; Rothe et al., 2017; Smith et al., 1997; Winning and Sargent, 1994; Xu et al., 1995), as well as epha7 expression in forebrain (Fb) and hindbrain (Hb), Cement gland (Cg), otic vesicle (Ov) and developing heart (H) (Wang et al., 2016). Ephb2 expression at neurula stage is observed in the anterior neural plate. At stage 20, it is observed in the whole neural tube and in the optic placode (Op), and at lower levels in the cement gland (Cg). It is detected at tailbud stage in the developing eye and migrating neural crest cells populating every branchial arch. Expression of ephb3 at neurula stage is principally observed in the cement gland (Cg) and also at low levels of expression in the neural plate. At tailbud stages 25 and 28, it is detected in the developing eye, midbrain and with a segmented pattern in the hindbrain, and at lower levels in migrating cephalic neural crest. Strong ephb3 expression is detected in the fourth branchial arch at stages 32 and 35/36 (n4). The red arrowheads indicate the pronephric anlage at stages 25 and 28, and the pronephros at stage 32 and 35/36. Scale bar: 1.7 mm for whole embryo, 0.5 mm for embryonic magnification and 280 m for section.
	Fig. 5. Summary of gene expression domains for Ephrin ligands and Eph receptors in pronephros segments at stage 35/36. Schematic representation of the pronephros and table (inspired by Raciti el al., 2008); Ephrin ligands are outlined in green and Eph receptors in light orange. Dark blue: strong expression. Turquoise blue: middle expression. Efnb2 expression is detected in the glomus at stage 35/36 and not in the tubule and is therefore not included in the table.
	efna2 (ephrin A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efna3 (ephrin A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efna1 (ephrin A1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efna5 (ephrin A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efnb1 (ephrin B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efnb2 (ephrin B2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efnb3 (ephrin B3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	epha2 (EPH receptor A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	epha3 (EPH receptor A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	epha4 (EPH receptor A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	epha5 (EPH receptor A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	epha6 (EPH receptor A6) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	epha7 (EPH receptor A7) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	epha8 (EPH receptor A8) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	epha10 (EPH receptor A10) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efna3 (ephrin A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 14 anterior and dorsal views, illustrating expression in the primary neuronal precursors (Np), and in the anterior neural folds (Nf).
	efna3 (ephrin A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior view, dorsal up. showing expression in the neural tube, and in the emerging first (n1) and third/fourth (n3, n4) pharyngeal arches of the cranial neural crest.
	efna3 (ephrin A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, dorsal view, anterior left, showing expression in craniial neural crest streams of the first pharygeal arch around the eye, dorsal aspects of neural tube and as well as the third and fourth pharyngeal arches .
	efna3 (ephrin A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view, anterior left, dorsal up. Expression in midbrain, hindbrain, pharyngeal arches, otic vesicle, eye, proctodeum and the pronephric /intermediate mesoderm.
	efna3 (ephrin A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view, anterior left, dorsal up. Expression in midbrain, hindbrain (R4), pharyngeal arches, otic vesicle, eye lens, proctodeum and the pronephric ducts, as well posterior lateral line placode.
	efna3 (ephrin A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35 & 36, ateral view, anterior left, dorsal up. Expression in brain (forebrain, midbrain, hindbrain ), pharyngeal arches, otic vesicle, lens, the intermediate and distal segments of the developing pronephric tubule
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 16, anterior, lateral and dorsal views.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view, anterior left, dorsal up.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view, anterior left, dorsal up.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view, anterior left, dorsal up.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35 & 36, left image: lateral view, anterior left, dorsal up. right image, in transverse section at leel indicted by red dashed line.
	epha7 (EPH receptor A7) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 14, anterior, lateral and dorsal views.
	epha7 (EPH receptor A7) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	epha7 (EPH receptor A7) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view, anterior left, dorsal up.
	epha7 (EPH receptor A7) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view, anterior left, dorsal up.
	epha7 (EPH receptor A7) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view, anterior left, dorsal up.
	epha7 (EPH receptor A7) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35 & 36, left image: lateral view, anterior left, dorsal up; right image: transverse section through trunk as level of red line.
	ephb2 (EPH receptor B2) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 18, anterior, lateral and dorsal views,
	ephb2 (EPH receptor B2) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views,
	ephb2 (EPH receptor B2) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view, anterior left, dorsal up.
	ephb2 (EPH receptor B2) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view, anterior left, dorsal up.
	ephb2 (EPH receptor B2) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view, anterior left, dorsal up.
	phb2 (EPH receptor B2) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35 & 36, transverse section through trunk, at level of the pronephric kidney, anterior up.
	ephb3 (EPH receptor B3) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 14, anterior, lateral and dorsal views.
	ephb3 (EPH receptor B3) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	ephb3 (EPH receptor B3) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view, anterior left, dorsal up.
	ephb3 (EPH receptor B3) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view, anterior left, dorsal up.
	ephb3 (EPH receptor B3) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35 & 36, lateral view, anterior left, dorsal up.
	ephb3 (EPH receptor B3) gene expression in X. laevisembryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35 & 36, transverse section through trunk, at level of the pronephros, dorsal up.
	Supplemental Fig. 1. [efna1 and efna2] Expression of genes encoding ephrin-a ligands at stages of pronephric development from kidney field (neurula stages 17-18) to functional pronephros (early tadpole stage 35/36), except for efna3 that is shown in detail in Fig.3. Anterior, dorsal and lateral views are shown at neurula stages (stages 17-18, 20). Only lateral views are shown for tailbud stages 25, 28 and 32. A lateral view with the enlarged pronephric region is shown at early tadpole stage 35/36. From stage 25 onward, low levels of efna1 [and efna4] expression are detected at the level of the pronephric primordium, possibly in the associated developing vasculature. At the early tadpole stage (stage 35/36), efna1 is expressed in the vasculature associated to the pronephric region. .... Expression of efna2 is detected in the Hindbrain (Hb) at stage 35/36. Scale bars: 1.7 mm for whole embryo, 0.5 mm for embryonic magnification.
	Supplemental Fig. 1. [efna4 and efna5] Expression of genes encoding ephrin-a ligands at stages of pronephric development from kidney field (neurula stages 17-18) to functional pronephros (early tadpole stage 35/36), except for efna3 that is shown in detail in Fig.3. Anterior, dorsal and lateral views are shown at neurula stages (stages 17-18, 20). Only lateral views are shown for tailbud stages 25, 28 and 32. A lateral view with the enlarged pronephric region is shown at early tadpole stage 35/36. From stage 25 onward, low levels of efna1 and a4 expression are detected at the level of the pronephric primordium, possibly in the associated developing vasculature. .... Expression of efna5 in the pronephric region is observed in the pronephric sinus (Ps). It is also observed in the vascular system, the intersomitic veins (Iv), the posterior cardinal vein (Pcv), the common cardinal vein (Ccv) and the heart (H). .....Scale bars: 1.7 mm for whole embryo, 0.5 mm for embryonic magnification.
	efna1 (ephrin A1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 17, anterior, lateral and dorsal views.
	efna1 (ephrin A1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	efna1 (ephrin A1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view, anterior left, dorsal up.
	efna1 (ephrin A1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view, anterior left, dorsal up.
	efna1 (ephrin A1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view, anterior left, dorsal up.
	efna1 (ephrin A1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35 & 36, lateral view, anterior left, dorsal up.
	efna2 (ephrin A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 18, anterior, lateral and dorsal views.
	efna2 (ephrin A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	efna2 (ephrin A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	efna2 (ephrin A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	efna2 (ephrin A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 18, anterior, lateral and dorsal views.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	efna4 (ephrin A4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efna5 (ephrin A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 18, anterior, lateral and dorsal views.
	efna5 (ephrin A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	efna5 (ephrin A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	efna5 (ephrin A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	efna5 (ephrin A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	efna5 (ephrin A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	Supplemental Fig. 2. [efnb1 and efnb2] Expression of genes encoding ephrin-b ligands at stages of pronephric development from kidney field (neurula stages 14-18) to functional pronephros (early tadpole stage 35/36). Anterior, dorsal and lateral views are shown at neurula stages (st14-18 and 20). Only lateral views are shown for tailbud stages 25, 28 and 32. A lateral view with the enlarged pronephric region is shown at early tadpole stage 35/36. Images of sectioned st 35/36 embryos are shown for efnb1 and efnb2 with red dotted lines indicating the corresponding levels on whole mounts (note that the sections were not made from the whole mount specimen shown). Only efnb2 appears to be expressed in the developing pronephros. Its pronephric expression is detected from tailbud stage 28 in the dorsal region of the pronephric anlage. At tadpole stage 35/36, it appears to be expressed in the glomus (G) at the level of differentiating podocytes (arrowhead on section) (White et al., 2010). Efnb1 is expressed in the pronephric region, but rather in the pronephric sinus (Ps). It is also detected in other vascular components, the intersomitic veins (Iv) and the com- mon cardinal vein (Ccv). ....The red arrowheads indicate the pronephric anlage at stage 28 and the pronephros at stage 32 and 35/36. Scale bars: 1.7 mm for whole embryo, 0.5 mm for embryonic magnification and 280 μm for section.
	Supplemental Fig. 2. [efnb3] Expression of genes encoding ephrin-b ligands at stages of pronephric development from kidney field (neurula stages 14-18) to functional pronephros (early tadpole stage 35/36). Anterior, dorsal and lateral views are shown at neurula stages (st14-18 and 20). Only lateral views are shown for tailbud stages 25, 28 and 32. A lateral view with the enlarged pronephric region is shown at early tadpole stage 35/36. ....Efnb3 is not expressed in the developing pronephros. The red arrowheads indicate the pronephric anlage at stage 28 and the pronephros at stage 32 and 35/36. Scale bars: 1.7 mm for whole embryo, 0.5 mm for embryonic magnification and 280 μm for section.
	efnb1 (ephrin B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 14, anterior, lateral and dorsal views.
	efnb1 (ephrin B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, nterior, lateral and dorsal views.
	efnb1 (ephrin B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	efnb1 (ephrin B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	efnb1 (ephrin B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	efnb1 (ephrin B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efnb1 (ephrin B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, transverse section through trunk at the levele of the pronephros.
	efnb2 (ephrin B2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 15, anterior, lateral view and dorsal views.
	efnb2 (ephrin B2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral view and dorsal views.
	efnb2 (ephrin B2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	efnb2 (ephrin B2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	efnb2 (ephrin B2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	efnb2 (ephrin B2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, lateral view anterior left, dorsal up.
	efnb2 (ephrin B2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35/36, transverse section through trunk at level of pronephros.
	efnb3 (ephrin B3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 14, anterior, lateral and dorsal views.
	efnb3 (ephrin B3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	efnb3 (ephrin B3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	efnb3 (ephrin B3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	efnb3 (ephrin B3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	epha2 (EPH receptor A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 16, anterior, lateral and dorsal views.
	epha2 (EPH receptor A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	epha2 (EPH receptor A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	epha2 (EPH receptor A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	epha2 (EPH receptor A2) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	epha3 (EPH receptor A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 14, anterior, lateral and dorsal views.
	epha3 (EPH receptor A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	epha3 (EPH receptor A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	epha3 (EPH receptor A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	epha3 (EPH receptor A3) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	epha5 (EPH receptor A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 18, anterior, lateral and dorsal views.
	epha5 (EPH receptor A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	epha5 (EPH receptor A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	epha5 (EPH receptor A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	epha5 (EPH receptor A5) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	epha6 (EPH receptor A6) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 18, anterior, lateral and dorsal views.
	epha6 (EPH receptor A6) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	epha6 (EPH receptor A6) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	epha6 (EPH receptor A6) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	epha6 (EPH receptor A6) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	Supplemental Fig. 3. [epha2 and epha3] Expression of genes encoding receptors epha2, epha3, ..... at stages of pronephric development from kidney field (neurula stages 14-18) to functional pronephros (early tadpole stage 35/36). Anterior, dorsal and lateral views are shown at neurula stages (st14-18 and 20). Only lateral views are shown for tailbud stages 25, 28 and 32. A lateral view with the enlarged pronephric region is shown at early tadpole stage 35/36. Expression of epha2, a3 and a5 is observed in the region of the developing pronephros from stage 32 onward. .... For epha2, and epha3 .... in situ hybridization with the sense probes was shown at stage 32 as control. Scale bar: 1.7 mm for whole embryo, 0.5 mm for embryo magnification.
	Supplemental Fig. 3.[epha5 and epha6] Expression of genes encoding receptors ..... epha5 and epha6 at stages of pronephric development from kidney field (neurula stages 14-18) to functional pronephros (early tadpole stage 35/36). Anterior, dorsal and lateral views are shown at neurula stages (st14-18 and 20). Only lateral views are shown for tailbud stages 25, 28 and 32. A lateral view with the enlarged pronephric region is shown at early tadpole stage 35/36. Expression of epha2, a3 and a5 is observed in the region of the developing pronephros from stage 32 onward. Epha6 is detected as soon as the tailbud stage 25. At stage 32 it is detected in the forming intersomitic veins (Iv) and pronephric sinus (Ps). For epha5, in situ hybridization with the sense probes was shown at stage 32 as control. Scale bar: 1.7 mm for whole embryo, 0.5 mm for embryo magnification.
	Supplemental Fig. 4. [epha8 and epha10] Expression of genes encoding receptors epha8, epha10, ..... at stages of pronephric development from kidney field (neurula stages 16-18) to functional pronephros (early tadpole stage 35/36). Anterior, dorsal and lateral views are shown at neurula stages (st16-18 and 20). Only lateral views are shown for tailbud stages 25, 28 and 32. A lateral view with the enlarged pronephric region is shown at early tadpole stage 35/36. Images of sectioned st 35/36 embryos are shown for epha10... with red dotted lines indicating the corresponding levels on whole mounts (note that the sections were not made from the whole mount specimen shown). Epha8 expression is observed in the developing pronephros (red arrowhead) from tailbud stage 28, while epha10 is transiently detected in the pronephros (red arrowhead) from tailbud stage 25 to 32. At tadpole stage 35/36 epha10 is not anymore expressed in pronephros, but rather in migrating hypaxial myoblasts (Myo). ..... Scale bar: 1.7 mm for whole embryo, 0.5 mm for embryo magnification and 280 μm for section.
	Supplemental Fig. 4. [ephb1 and ephb4]. Expression of genes encoding receptors ephb1 and ephb4 at stages of pronephric development from kidney field (neurula stages 16-18) to functional pronephros (early tadpole stage 35/36). Anterior, dorsal and lateral views are shown at neurula stages (st16-18 and 20). Only lateral views are shown for tailbud stages 25, 28 and 32. A lateral view with the enlarged pronephric region is shown at early tadpole stage 35/36. Images of sectioned st 35/36 embryos are shown for .... ephb1 and ephb4 with red dotted lines indicating the corresponding levels on whole mounts (note that the sections were not made from the whole mount specimen shown)...... Ephb1 and ephb4 are not detected in the developing pronephros. Vascular ephb4 expression in posterior cardinal vein (Pcv), pronephric sinus (Ps), and intersomitic veins (Iv) has been previously described (Helbling et al., 2000). Scale bar: 1.7 mm for whole embryo, 0.5 mm for embryo magnification and 280 μm for section.
	epha8 (EPH receptor A8) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 16, anterior, lateral and dorsal views.
	epha8 (EPH receptor A8) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	epha8 (EPH receptor A8) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	epha8 (EPH receptor A8) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	epha8 (EPH receptor A8) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	epha10 (EPH receptor A10) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 18 anterior, lateral and orsal views.
	epha10 (EPH receptor A10) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20 anterior, lateral and dorsal views.
	epha10 (EPH receptor A10) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view anterior left, dorsal up.
	epha10 (EPH receptor A10) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view anterior left, dorsal up.
	epha10 (EPH receptor A10) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view anterior left, dorsal up.
	epha10 (EPH receptor A10) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35 & 36,transverse section through trunk at level of the pronephros.
	ephb1 (EPH receptor B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 18, anterior, lateral and dorsal views.
	ephb1 (EPH receptor B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	ephb1 (EPH receptor B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view, anterior left, dorsal up.
	ephb1 (EPH receptor B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view, anterior left, dorsal up.
	ephb1 (EPH receptor B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view, anterior left, dorsal up.
	ephb1 (EPH receptor B1) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35 & 36, lateral view, anterior left, dorsal up. Transverse section at level of pronephros.
	ephb4 (EPH receptor B4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 18, anterior, lateral and dorsal views.
	ephb4 (EPH receptor B4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 20, anterior, lateral and dorsal views.
	ephb4 (EPH receptor B4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 25, lateral view, anterior left, dorsal up.
	ephb4 (EPH receptor B4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 28, lateral view, anterior left, dorsal up.
	ephb4 (EPH receptor B4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 32, lateral view, anterior left, dorsal up.
	ephb4 (EPH receptor B4) gene expression in X. laevis embryo, assayed whole-mount via situ hybridization (WISH) at NF stage 35 & 36, lateral view, anterior left, dorsal up. Transverse section through trunck at level of pronephros as indicated by red dashed line.