May 1, 2008;
The role of FGF signaling in the establishment and maintenance of mesodermal gene expression in Xenopus.
FGF signaling is important for the formation of mesoderm
in vertebrates, and when it is perturbed in Xenopus, most trunk
and tail mesoderm
fails to form. Here we have further dissected the activities of FGF in patterning the embryo
by addressing its inductive and maintenance roles. We show that FGF signaling is necessary for the establishment of xbra
expression in addition to its well-characterized role in maintaining xbra
expression. The role of FGF signaling in organizer
formation is not clear in Xenopus. We find that FGF signaling is essential for the initial specification of paraxial mesoderm
but not for activation of several pan-mesodermal and most organizer
genes; however, early FGF signaling is necessary for the maintenance of organizer
gene expression into the neurula stage
. Inhibition of FGF signaling prevents VegT
activation of specific mesodermal transcripts. These findings illuminate how FGF signaling contributes to the establishment of distinct types of mesoderm
[+] show captions
Figure 1. FGF signaling is necessary for initiation of xbra expression. All embryos were processed by RNA in situ hybridization for expression of xbra. Embryos were treated with SU5402 (90-100 mu M) (A-D, K-P) or DMSO alone (E-J). A-D: The bar represents time from fertilization until 11 hr post-fertilization with each hour represented by hash marks; the black region represents the time the embryos were in carrier (DMSO) only, and the blue region represents the time frame when embryos were treated with the inhibitor SU5402. Lateral views. A: DMSO only. B,C: Initially treated with DMSO, then SU5402 was added at 10 and 9 hr post-fertilization, respectively. D: Embryos were first treated with SU5402 then the inhibitor was washed out after 4 hr. E-P: Embryos were treated with either DMSO alone or SU5402 from the 8-16-cell stage until the indicated time point post-fertilization and xbra expression was analyzed in these blastula stage embryos. Embryos were cleared in BB:BA (2:1). The two views in each panel are of the same embryo with an animal pole view above and a lateral view below.
Figure 2. FGF signaling effects on endoderm and mesoderm. These embryos have been processed by in situ hybridization for expression of the indicated transcript above each column. Embryos were treated with DMSO or SU5402 from the 8- to 16-cell stage as indicated on the side. A,B: Lateral views; embryos were collected at the blastula stage, 7.5 hr post-fertilization. C-S: Gastrula stage. C-N: Blastoporal views. O,P: Lateral views with vegetal to the bottom. Q-S: Embryos were injected into one cell at the two-cell stage with the dominant-negative FGFR1 construct XFD (500 pg); the red staining is the lineage tracer n beta gal. T: Whole embryos were treated with either DMSO or SU5402, collected at the time indicated above the lanes, and analyzed by RT-PCR for expression of the indicated markers along the side. ODC was used as a loading control.
Figure 3. The involvement of FGF signaling in organizer and paraxial mesoderm development. Embryos were treated with either DMSO or SU5402 from the 8- to 16-cell stage and analyzed at the gastrula stage for expression of the indicated transcripts. A,B,E-H,M-P: Embryos are displayed in a lateral blastoporal view. C,D,I-L,Q,R: Embryos are displayed from a lateral view with blastopore toward the bottom. Axial/organizer genes are present, but expression of paraxial mesodermal transcripts is absent in inhibitor-treated embryos.
Figure 4. Early FGF signaling is necessary to maintain organizer gene expression. Embryos were treated with either DMSO or SU5402 and analyzed at the early neurula stage for expression of a range of mesodermal, posterior and anterior, and endodermal genes, except for Q-V, which were analyzed at stage 28. A-H,W-ZD: Dorsal views with anterior to the left. I-M: Anterior views. O: Posterior view. J,L,N,P: Dorsal blastoporal views. Q-U: Lateral views, anterior to the left. V: Dorsal view. A-P: Embryos were treated from the 8- to 16-cell stage until the early neurula stage. R: Injected with XFD RNA at 250 pg into each blastomere at the 4-cell stage. S,U,V: Treated from the 8- to 16-cell stage until stage 28. W-ZD: Embryos were treated from mid-gastrulation until the early neurula stage. Y',Z': Embryos demonstrating the range of effects from inhibitor treatment.
Figure 5. VegT activity in the absence of FGF signaling. VegT has dose-specific effects in the whole embryo. A,B: Control uninjected embryos at the mid-gastrula stage and processed by in situ hybridization for expression of sox17 or xbra. C-L: Embryos were injected with different doses of VegT mRNA into one cell at the two-cell stage marked by the lineage tracer nuclear beta -gal in red and analyzed at the mid-gastrula stage. All embryos are displayed in lateral views with the blastopore toward the bottom. At a very low dose, VegT mRNA has no detectable effect (C,D). At the 10-pg dose, VegT induces ectopic xbra expression but not sox17 while higher doses induce ectopic sox17 at the focal point of injection and xbra at the periphery (E-L). M-X: FGF signaling is necessary for VegT-mediated expansion of xbra but not sox17. Embryos were either uninjected, or injected with VegT mRNA (250 pg) or FGF8b mRNA (10 pg) and treated with either DMSO or SU5402 as indicated. At the early gastrula stage, embryos were analyzed for xbra and sox17 expression. Y: An RTPCR experiment on whole embryos treated as indicated along the top and analyzed for a range of markers. EF1 alpha and ODC were used as loading controls.
Crx-a (Otx5) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 10, lateral blastoporal view.