Wang JH , Deimling SJ , D'Alessandro NE , Zhao L , Possmayer F , Drysdale TA .

MOP-74663 Canadian Institutes of Health Research

	Figure 1. Temporal expression of lung and thyroid markers. Whole mount in situ hybridization was used to compare the temporal expression of key early genes in the development of the thyroid and lung. Expression of nkx2.1 is first detectable in the thyroid (red arrows) at stage 30 and is subsequently expressed in the lung (green arrows) at stage 34. Expression of pax2 begins at stage 32 in the thyroid. Expression of the differentiation markers sftpb and sftpc is first detectable at stage 38.
	Figure 2. Retinoic acid is required for lung differentiation but excess retinoic acid causes expression of lung differentiation markers in the thyroid. When embryos were treated with a retinoic acid antagonist (RAA) at stage 14 of development, the lung failed to differentiate as judged by the lack of sftpb expression at stage 38/40. If the retinoic acid antagonist is not added until stage 26, the expression of sftpb in the lung was essentially normal. If embryos were exposed to exogenous retinoic acid (RA) starting between stages 20 to 34, expression of sftpb was detectable in both the lung (green arrows) and in the thyroid (red arrow). Addition of retinoic acid at earlier (stage 14) or later stages (stage 36) did not result in expression of sftpb in the presumptive thyroid. Carrier control (DMSO) embryos had normal expression of sftpb. Treatment times are indicated at the top of each column (eg. T-st14 indicates that the treatment was initiated at embryonic stage 14).
	Figure 3. Fgf signalling is required for lung and elements of thyroid differentiation in Xenopus. When embryos were treated with SU5402 at either stage 12, 20 or 26, expression of nkx2.1 was lost in the lung (green arrows) but not the thyroid (red arrows) or anterior neural tissue. Although treatment with SU5402 at stage 20 caused clear deformations in the lens ectoderm expression (yellow arrows) of foxe4, there was no clear effect on the thyroid expression of foxe4. Expression of pax2 in the thyroid was lost when SU5402 was applied at stage 12 but expression of pax2 was observed in most embryos at stage 20 and all embryos when treated at stage 26. Note the severe tail defects in embryos treated with SU5402 at stage 12 demonstrating the effectiveness of the block to Fgf signalling. (side - side view, ventral - ventral view).
	Additional file 1. Treatment with SU5402 causes a loss of both sftpb and sprouty2 expression. Embryos treated with SU5402, in order to block Fgf signalling, do not express sftpb indicating that there is a loss of differentiated lung. The location of the differentiated lung (green arrow) can be seen in the control embryo (DMSO treated). Expression of sprouty2 (spry2) was used to demonstrate the effectiveness of the Fgf signalling block. Sprouty2 is a target of Fgf signalling and strong expression can normally be seen at the midbrain-hindbrain border (yellow arrow) and in the pharynx (purple arrow). The expression of sprouty2 in these regions is effectively eliminated by addition of SU5402 at all times tested. Treatment times are indicated at the top of each column (eg. T-st12 indicates that the treatment was initiated at embryonic stage 12).
	Figure 4. Exogenous retinoic acid causes ectopic expression of sftpc in the presumptive thyroid. Whole mount in situ hybridization for sftpc demonstrated that embryos treated at stage 26 with exogenous retinoic acid showed ectopic expression of sftpc in the developing thyroid (red arrows) as well as the normal expression in the lung (green arrows). (side - side view, ventral - ventral view).
	Figure 5. Exogenous retinoic acid signalling results in the loss of early thryroid gene expression. When embryos are treated with retinoic acid (RA) at stage 24/26, expression of sftpb was detected in the thyroid (red arrows) and in the lung (green arrows) but it is only expressed in the lung in control (DMSO) embryos. Pax2 and foxe4 are normally expressed in the thyroid although both have distinct expression domains in other tissues. Embryos treated with RA still expressed both pax2 and foxe4 but the expression in the thyroid was eliminated. Expression of foxe1 could be observed in all embryos, particularly in the pharynx, but expression in the thyroid was undetectable. Treatment with the retinoic acid antagonist at stage 26 did not eliminate the expression of any tested markers in either the thyroid or lung.
	Figure 6. Treatment with retinoic acid eliminated expression of hhex in the thyroid but not liver. When embryos were treated at stage 24/26 and cultured to stage 36, expression could be detected near the thyroid even in RA treated embryos but in ventral view it can be clearly seen that midline expression of hhex (red arrow) is eliminated by RA but there is an additional region of expression lateral to the thyroid (blue arrows) that is not lost when embryos were treated with RA. There is also no obvious change in the expression of the large liver expression domain found caudal to the thyroid expression.
	Figure 7. Transplantation of the presumptive thyroid explants into the embryo flank is sufficient to cause expression of sftpb in the explant. When explants of the presumptive thyroid region are cultured alone, they do not express sftpb (A) but do express pax2 (B - black arrows). Note that the small blue flecks are seen on the surface of many embryos due to the stickiness of the cement gland that is also usually in part of the explant. When those same explants are placed on to the flank region immediately after they are removed from donor embryos, sftpb expression can be seen in the explant (blue arrows) as well as the endogenous lung (green arrows). When explants are transplanted, expression of pax2 (red arrows) can be seen in the explants. Embryos in C and D are normal views and embryos in E and F have been cleared to better visualize staining although cavity staining is also seen when embryos are cleared.
	sftpb (surfactant, pulmonary-associated protein B ) gene expression in Xenopus laevis embryo, via in situ hybridization, NF stage 29 & 30, lateral view, anterior left, dorsal up.
	spry2 (sprouty homolog 2) gene expression in Xenopus laevis embryo, via in situ hybridization, NF stage 29&30, lateral view, anterior left, dorsal up.
	nkx2-1 (NK2 homeobox 1) gene expression in Xenopus laevis embryo, via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.
	foxe3 (forkhead box E3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 37 & 38, lateral view, anterior left, dorsal up. Red arrow: thyroid primordium; yewllow arrow: lens.
	foxe3 (forkhead box E3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 37 & 38, ventral view, anterior left.
	sftpc (surfactant, pulmonary-associated protein C ) gene expression in Xenopus laevis embryo, via in situ hybridization, NF stage 39, lateral view, anterior left, dorsal up.

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