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Since both RA and FGF are necessary to pattern separate areas of the LPM, and have been previously shown to form mutually antagonistic gradients in other tissues, I wished to test if the two pathways interacted in patterning the LPM. To determine if the RA signalling pathway directly impacts FGF signalling, embryos were treated with 1 µM

Figure 17. FGF is required for the patterning of late LPM markers. Hand1 (A, B) and

foxf1 (C, D) are normally restricted in the anterior and middle LPM with a clear posterior domain free of expression in both cases. When embryos were treated with SU5402 at stage 12.5 both hand1 (B) and foxf1 (D) were observed along the entire anterior-posterior axis by stage 32. Conversely, the LPM expression domains of both hoxc10 (E-F;

normally expressed in the posterior half of the LPM) and cTnI (G-H; marker of cardiac differentiation) were completely undetectable when FGF signalling is inhibited, although expression of hoxc10 in the somites and neural tube is still observed. 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.

RA, RAA or a DMSO control and assayed for both fgf4 and fgf8 expression at the end of neurulation. When RA signalling was antagonized, the anterior trunk domain of fgf8, located just posterior to the cement gland, was decreased in intensity in comparison to staining in the pituitary anlagen (Figure 18D-E). However, the posterior domains of both

fgf4 and fgf8 remained unaffected. Increasing RA signalling by treatment with all-trans

RA resulted in an increase in the fgf8 expression domain in anterior trunk (Figure 18E-F), becoming much thicker and surrounding the entire cement gland rather than residing posterior to it. Fgf8 expression in the posterior neural tube was also extended much further anterior along the neural folds (Figure 18H-I), which is in agreement with previous results (Moreno and Kintner, 2004). Conversely, the expression domain of fgf4

was decreased in the posterior neural tube to essentially background levels (Figure 18B- C).

Finally, I used sprouty2 expression as an assay of active FGF signalling. Sprouty2

is normally expressed in both anterior and posterior domains overlapping with the fgf8

expression domains (Figure 18K, N). When RA signalling is increased the anterior domain of sprouty2 becomes much broader in both the heart region and the pituitary anlagen (Figure 18L). Expression of sprouty2 is also increased in the neural tube (Figure 18O), as it is normally present in the posterior half of the neural folds, but is displaced much further anterior with increased RA signalling.

To test if FGF signalling impacted the RA signalling system, I treated embryos at stage 12.5-13 with 10 µM SU5402 and assayed at the end of neurulation for both raldh2, the enzyme predominantly responsible for synthesizing all trans RA in vivo, and cyp26, the enzyme responsible for RA catabolism. Raldh2 is normally expressed in the anterior

Figure 18. 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) at stage 12.5 and compared to a DMSO control (center column). Embryos were assayed for fgf4 (A-C) and fgf8 (D-I) expression at stage 20. 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 however is expanded both anteriorly (E-F) and posteriorly (H-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 at stage 12.5 and assayed for expression of raldh2 (P- Q) or cyp26 (R-S) at stage 20 to determine the effect of a loss of FGF signalling on the RA signalling pathway. The expression domain of raldh2 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.

of the LPM. Inhibiting FGF signalling caused a posterior expansion of the dorsal raldh2

domain much further posterior in the somites and LPM than in control embryos (Figure 18 P-Q). Cyp26 is also normally expressed in the posterior neural tube and tail bud in a domain reminiscent of the fgf4 and fgf8 expression domains, and in the tail bud region that corresponds to the normal bra domain. Inhibiting FGF signalling caused a complete loss of the posterior cyp26 domain (Figure 18R-S), which was no longer detectable in the posterior neural folds or the adjacent tail bud domain, consistent with previous studies (Moreno and Kintner, 2004).

To confirm that RA signalling was necessary for expression of the Fgf ligands, I also treated embryos with inhibitors of Raldh2 (ketoconazole) and Cyp26 (DEAB), the endogenous enzymes primarily responsible for RA synthesis and catabolism respectively. A reduction in RA levels by treatment with DEAB caused a similar reduction in fgf8

staining in the anterior domain to that seen with RAA treatments (Figure 19 H-I), although to a lesser degree. However, increasing endogenous RA signalling by treating with ketoconazole did not seem to have an obvious effect on fgf8 expression (Figure 19G- H). Neither an endogenous increase or decrease in RA signalling led to obvious changes of the sprouty2 domain (M-R).