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Fig. 1. Fgf signalling is necessary for proper LPM pattern. Foxf1, hand1, sall3 and Xbra were assayed for a response in expression domain when Fgf signalling was inhibited with SU5402. The anterior domain, marked by foxf1 expression (A–D), was expanded toward the ventral side of the embryo (arrowheads in A and B), and was upregulated at the posterior end of the LPM just ventral to the blastopore (arrowheads in C and D). The posterior border of hand1 (E–H) is displaced further posterior, particularly at the dorsal edges of the domain (arrowheads in E–H). The LPM domain of sall3 (I–L) is displaced posterior with a loss of Fgf signalling (white arrowheads in K and L) while the posterior neural tube domain is undetectable (black arrowheads in K and L). Expression of Xbra in the tailbud domain is completely lost in the absence of Fgf signalling (arrowheads in M and N). llv: left lateral view, embryos oriented with the anterior pole toward the left of the image, dorsal at top. pos: posterior view, embryos oriented with dorsal at top of image. The total number of embryos examined for each panel is indicated in the lower left hand corner.
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Fig. 2. Fgf is required for the patterning of late LPM markers. Hand1 (A and B) and foxf1 (C and D) are normally restricted in the anterior and middle LPM with a clear domain free of expression in both cases. When embryos are treated with SU5402, both hand1 (B) and foxf1 (D) are expressed along the entire anterior–posterior axis. Conversely, the LPM expression domains of both hoxc10 (E and F; normally expressed in the posterior half of the LPM) and tnni3 (G and H; marker of cardiac differentiation) are completely undetectable when Fgf signalling is inhibited. (A–F): lateral view of the embryos is shown, with anterior toward the left, dorsal at top. (G–H); ventral view of the heart region is shown, with anterior toward left. The total number of embryos examined for each panel is indicated in the lower left hand corner.
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Fig. 3. Fgf signalling is required for the initiation of heart specification. Expression of both nkx2.5 and isl1 is readily detectable in the cardiac crescent of stage 20 embryos (A and C). Embryos treated with SU5402 to inhibit fgfr1 activity show a loss of nkx2.5 expression (B), and a down regulation of isl1 expression (D). In both cases, the anterior pole of the embryo is viewed, with dorsal folds visible toward the top of the image. The total number of embryos examined for each panel is indicated in the lower left hand corner.
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Fig. 4. Fgf signalling is necessary for heart patterning and morphogenesis. Embryos were treated with SU5402 during different developmental windows to determine when Fgf signalling is necessary for heart development and assayed for expression of nkx2.5 (A–D and I–L) or tnni3 (E–H and M–P). Fgf signalling was inhibited at successively later stages (top panel: A–H) and compared to control embryos (A and E). Embryos treated with SU5402 at either stage 12.5 (B and F) or stage 20 (C and G) demonstrate a complete loss of heart marker expression by stage 32, while embryos treated at stage 24 (D and H) show expression of both nkx2.5 and tnni3 but no discernable heart tube. Fgf signalling was also inhibited at stage 12.5 and restored at later stages by removing the inhibitor (bottom panel: I–P) and compared to control embryos (I and M). When Fgf signalling is restored by either stage 20 (J and N) or stage 22 (K and O), both heart markers are expressed however a normal heart tube is not formed. If signalling is not restored until stage 26 (L and P), neither nkx2.5 or tnni3 are detectable. White arrows mark the heart region. The total number of embryos examined for each panel is indicated in the lower left hand corner.
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Fig. 5. Fgf and RA signalling are required for proper patterning of the trunk vascular system. Embryos were treated with DMSO or SU5402 at stage 12 (top panel: A–H) and allowed to develop until stage 32 at which point vascular pattern was assessed by expression of etv2 (A–D) and aplnr (E–H). Both etv2 (A and B) and aplnr (E and F) are normally restricted from the posterior end of the LPM. However, when Fgf signalling is lost, the expression domains of both etv2 (C and D) and aplnr (G and H) are extended posterior to the end of the trunk. Also, the anterior gap in vasculature between the rostral lymph sac and trunk vasculature, corresponding to the location of developing heart, is absent in SU5402 treated embryos. Embryos treated at stage 14 with a synthetic RA antagonist (RAA), all-trans RA or a DMSO control (bottom panel: I–T) were assayed for etv2 (A–F) or aplnr (G–L) expression. While no obvious changes are present after treating embryos with RAA (I,J and O,P), the posterior limit of the LPM vasculature (white arrowheads) is extended further posterior in RA treated embryos (M,N and S,T) when compared with control embryos (K,L and Q,R). The staining seen in the rostral lymph sac is also absent in embryos treated with RA. White arrows: posterior limit of trunk vascular pattern. Red arrows: rostral lymph sac. Black arrows: gap between rostral lymph sac and trunk vasculature. The total number of embryos examined for each panel is indicated in the lower left hand corner.
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Fig. 6. The extent of the vascular plexus is related to the size of the hand1 domain. To more directly assess the relationship between the forming vasculature and hand1 expression double in situ hybridizations were done with hand1 being visualized in light blue and etv2, marking the forming vasculature, in purple. The forming vascular plexus (emphasized in the lower panels of each example) along the side of the embryo does not extend to the end of the embryo in control (DMSO, ATP) embryos nor does the hand1 staining. When the extent of the vascular plexus is shifted towards the anterior end by treatment with RAA (A and D) this is reflected in the changes to the hand1 expression domain. When the vascular plexus is shifted towards the end of the embryo by addition of RA (C and F) or SU5402 (H and J) this is again reflected in the changes to the extent of hand1 expression domain along the anterior–posterior axis. Note that the close correspondence appears to be lost in the dorsal ventral axis in the SU5402-treated embryos (H and J).
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Fig. 7. The RA and Fgf pathways regulate each other. The levels of RA signalling (A–I) were altered by addition of a synthetic RA antagonist (left column) or all-trans RA (right column) and compared to a DMSO control (centre column). Embryos were assayed for fgf4 (A–C) and fgf8 (D–I) expression. The posterior domain of fgf4 (a) is lost in RA treated embryos (C) when compared to the control (B), but unaffected in embryos treated with RAA. Expression of fgf8 is expanded both anteriorly (E and F) and posteriorly (H and I; compare distance between arrowheads (d) marking the anterior limits of domain, and (e) marking posterior limits of domain) under treatment with RA. Decreasing RA signalling also reduces the anterior domain of fgf8 underlying the heart region (compare ratio of staining intensity between (b) marking the pituitary anlagen to (c)). A similar effect is seen with sprouty2 expression (J–O), as its domain is increased with RA in both the anterior heart region (L; arrowhead f) and it extends further anterior (g) in the dorsal neural tube (O) when compared to controls (K–N). Conversely, embryos were treated with SU5402 and assayed for expression of aldh1a2 (P and Q) or cyp26 (R and S) to determine the effect of a loss of Fgf signalling on the RA signalling pathway. The expression domain of aldh1a2 was expanded posterior (Q) (arrowhead: h – marking posterior limit of expression domain) as compared to control embryos (P). Cyp26, normally present in the posterior LPM tailbud domain (R; arrowhead i) is undetectable when Fgf signalling is inhibited (S). Ant: anterior view with dorsal at top of image. Dor: dorsal view with anterior at top. Llv: left lateral view with anterior toward left, dorsal at top of image. Pos: posterior view with dorsal at top. The total number of embryos examined for each panel is indicated in the lower left hand corner.
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Fig. 8. Retinoic acid and fgf are opposing signalling molecules in patterning the LPM. Embryos were treated at stage 12 with either RA, SU5402 or both RA and SU5402 and assayed by whole mount in situ hybridization for expression of nkx2.5 (arrowhead a), isl1 (arrowhead b) and foxf1. Treating embryos with RA reduces the expression domain of both nkx2.5 (B) and isl1 (F) compared with controls (A and E). Significantly reduced domains of nkx2.5 and isl1 were also present in SU5402 treated embryos (C), however when embryos are treated with both RA and SU5402 neither marker is detectable (D and H). The expression domain of foxf1 was expanded in both the RA (J) and SU5402 (K) treatments when compared to controls (I), however when embryos were treated with both RA and SU5402, foxf1 expression was detectible across the entire LPM although expression was still graded. Ant: anterior view with dorsal at top of image. Pos: posterior view with dorsal at top of image. Llv is left lateral view. The total number of embryos examined for each panel is indicated in the lower left hand corner.
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Fig. 9. Loss of retinoic acid signalling partially rescues effects of loss of Fgf signalling on the domains of isl1 and Xbra. Decreasing RA signalling in SU5402 treated embryos had little effect on the nkx2.5 domain (D) as it was still present in a highly restricted domain similar to the SU5402 treatment (C). However, a loss of Fgf signalling leads to a loss of isl1 (H) and Xbra (T), while neither domain is changed when RA signalling is lost (F and R). However, when Fgf signalling is decreased in conjunction with reduced RA signalling both isl1 and Xbra expression is detectable in their normal domains (H and T). However, the restriction of the hand1 expression domain under reduced RA condition (J and N) seems to be dominant to the extended domain seen in the SU5402 treated embryos (K and O) as losing both RA and Fgf signalling (L) leads to a restricted domain similar to the RA antagonist alone.
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Fig. 10. Model of signalling input into the LPM of the neurula stage Xenopus embryo. A high level of RA signalling input is suggested by the expression domain of raldh2 in the anterior and dorsal LPM (A). Conversely, Fgf signalling is proposed in the anterior-ventral and posterior-ventral LPM, suggested by the expression domains of fgf8 in the anterior, and fgf4 and fgf8 in the posterior pole of the embryo. Dark blue and yellow bars represent the expression of raldh2 and fgf ligands respectively, while blue and yellow arrows depict the proposed area which RA (blue) and fgf (yellow) are required for normal patterning. The model depicts a left lateral view of the LPM. Ant: anterior, Dor: dorsal, Lat: lateral view, Pos: posterior, Ven: ventral. (B) Diagrammatical representation of LPM expression domain response to decrease in either RA (left) or Fgf (right) signalling when compared with the DMSO control (centre). The anterior-dorsal and middle LPM domains require RA signalling for their full expression domain. When embryos are treated with the RA antagonist the anterior-dorsal domain is restricted (a) and the middle LPM domain is contracted (b). When Fgf signalling is inhibited the anterior-dorsal domain is expanded ventrally (c) and the posterior domains are severely restricted (d), indicating that the anterior-ventral and posterior LPM domains are dependant on Fgf signalling. Red: nkx2.5, blue: foxf1, yellow: hand1, green: sall3, purple: Xbra.
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Supplementary Fig. 2. Altering the bio-availability of endogenous RA ligand by inhibiting members of the RA signalling pathway leads to similar effects as exogenous RA treatments. Expression of cyp26, a biomarker of RA signalling, is slightly increased under treatment with ketoconazole (B and E), and decreased with DEAB (C and F) as compared to control embryos (A and D). The anterior fgf8 expression domain (arrowhead a) was unchanged with a moderate increase in RA signalling when treated with ketoconazole (H) was also reduced in comparison to expression in the pituitary anlagen (arrowhead b) under treatment with DEAB (I) when compared to controls (G). However consistent changes were seen with either ketoconazole or DEAB on the posterior domain of fgf8 expression (J–L). Also, no obvious and consistent changes were seen on the size and position of the sprouty2 domain (M–R).
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Supplementary Fig. 3. ATP promotes the efficacy of SU5402. (A) Embryos were treated with DMSO + ATP, 10 μM SU5402 or 10 μM SU5402 in conjunction with 0.1 mM ATP. Embryos treated with SU5402 alone elongated similar to DMSO controls, while embryos treated with SU5402 in conjunction with ATP demonstrate the truncated phenotype characteristic of a loss of FGF signalling. (B) Similar results are obtained when using cardiac differentiation, as assayed by expression of tnni3, although lower concentrations of SU5402 were effective. When embryos were exposed to 1 μM SU5402 alone cardiac differentiation was similar to controls but when embryos were exposed to 1 μM SU5402 in conjunction with 0.1 mM ATP, cardiac differentiation was blocked.
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Supplementary Fig. 4. Sprouty2 expression demonstrates the efficacy of SU5402. Treatment of embryos at stage 12.5 with 10 μM SU5402 with 0.1 mM ATP is sufficient to largely eliminate sprouty2 expression based on whole mount in situ hybridization when assayed at either stage 20 (A–D) or stage 32 (E F).
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Supplementary Fig. 1. Inhibiting Vegf signalling with KRN633, a vegfr specific inhibitor does not recapitulate the SU5402 phenotype. Treating embryos with either 10 μM, or 25 μM KRN633 leads to a loss of angiogenesis when embryos are assayed for either etv2 (G–L) or aplnr (M–R) as demonstrated by a loss of vascular budding in the ventral trunk, and a loss of intersomitic vessels (white arrow heads). However, no concentration tested was able to block expression of tnni3 (A–F), or cause significant truncations of the tail.
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hand1 (heart and neural crest derivatives expressed 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
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isl1 (ISL LIM homeobox 1) gene expression in a X. laevis embryo, NF stage 20, assayed via in situ hybridization, anterior view, dorsal up.
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