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LIM-homeodomain (LIM-hd) proteins form a multifunctional family of transcription factors that plays critical roles in the development of progenitor and post-mitotic cells. Considerable work has focused on what regulates their expression post-mitotically in the spinal cord. However, little is known about what regulates LIM-hd genes at earlier developmental stages. To address this question, we explored the role of fibroblast growth factor (FGF) signalling in regulating the expression of a Xenopus laevis Lhx9 orthologue (XLhx9). XLhx9 is first expressed in the eye field and hindbrain, and when FGF receptor (FGFR) activation was inhibited prior to its onset, both brain and eye field expression was diminished. However, when FGFRs were inhibited after XLhx9 onset, retinal expression remained strong and brain expression was again diminished. These data suggest that while FGF signalling initiates and maintains brainXLhx9 expression, in the eye primordium the requirement of FGFs for expression is rapidly lost.
Figure 2. Developmental expression pattern of XLhx9 compared to XPax6 and XLhx2 as determined by whole-mount in situ hybridization. A, B: XLhx9 expression is apparent in the future hindbrain (hb) and retina (ret) at stages 18 (A) and 25 (B). C, D: At stages 30 (C) and 33/34 (D), XLhx9 is expressed in the telencephalon (tel), dorsal diencephalon (di), mesencephalon (mes), and hindbrain. Inset in C: Enlarged view of diencephalon and mesencephalon; relative size of expression domains indicated with brackets. E: XPax6 expression in a stage-25 embryo for comparison to XLhx9. F-H: Vibratome retinal sections after XLhx9 in situ. At stage 25 (F), XLhx9 is expressed throughout the presumptive retinal pigmented epithelium (PRPE) and presumptive neural retina (PNR), but reduced or absent in the optic stalk (OS). By stage 30 (G), XLhx9 expression is confined to the proliferative ciliary margin zone (CMZ) of the retina (arrows). At stage 33/34 (H), XLhx9 is expressed throughout the inner nuclear layer (INL, arrowheads). I-L: In situ hybridizations against XLhx2 on stage matched siblings of embryos in A-D. At stage 18 (I) and 25 (J), XLhx2 is expressed strongly within the eye field, but has reduced or absent expression in the hindbrain as compared to XLhx9. Strong telencephalic expression is also obvious at stage 25 (J). K, L: XLhx2 is expressed within the telencephalon, diencephalon, pineal gland (pi), mesencephalon, and hindbrain at stages 30 (K) and 33/34 (L).
Figure 4. SU5402 treatment effectively knocks down FGF signalling in stage-12 and -16 Xenopus laevis embryos as evidenced by decreased XSpry1 expression. A-D: Stage-12 (A, C) and stage-16 (B, D) Xenopus laevis embryos were incubated overnight (18-22 hr) at 16 in a control dimethyl sulfoxide (CNT; A,B) or a 200- mu M SU5402 (SU5402; C, D) solution and then processed in parallel for XSpry1 expression by in situ hybridization. Note: The stage indicated at the bottom right of each image refers to the age of the embryos at the beginning of their treatment not the stage of the embryos in the photo (embryos that started treatment at stage 12 developed until stage 21, and those that began at stage 16, reached stage 25). E, F: Embryos were scored from 1 to 3 with 1 indicating the lightest or no staining and 3 indicating the darkest level of staining, and the results are graphed as a percentage of embryos that received each score. The number of embryos in each group are as follows: (E) stage-12 CNT, n = 20, stage-12 SU5402 treated, n = 19, (F) stage-16 CNT, n = 20, stage-16 SU5402, n = 19.
Figure 5. XLhx9 requires FGFR activation for initiation of expression in the hindbrain and retina and maintenance of expression in the hindbrain. A-D: Embryos were incubated in a control DMSO (CNT; A, B) or a 200- mu M FGFR inhibitor (SU5402; C, D) solution overnight (18-22 hr) at 16 and then processed in parallel in an in situ hybridization reaction using a probe targeted to XLhx9. A, C: To test the role of FGFR activation in initiation of XLhx9 expression, incubations began at stage 12. B, D: To test the role of FGFRs in the maintenance of XLhx9 expression, incubations began at stage 16. Embryos were scored from 1 to 3 for both the hindbrain (hb) and the retinal (eye) expression in control and SU5402 inhibitor-treated groups, with 1 being the lightest and 3 being the darkest level of staining. E, F: Results are graphed as a percentage of embryos that received each score in either region. The number of embryos in each group are as follows: (E) stage-12 CNT, n = 31; stage-12 SU5402 treated, n = 27; (F) stage-16 CNT, n = 27; stage-16 SU5402, n = 25.
Figure 6. FGF signalling is not required for XPax6 expression in developing Xenopus laevis. A, B: Stage-12 Xenopus laevis embryos were placed in a 200- mu M solution of SU5402 (SU5402; B) or a control solution (CNT; A) with an equivalent amount of DMSO. They were incubated for 18-22 hr at 16 and then fixed and processed in parallel for XPax6 expression by in situ hybridization. C: As in Figures 4 and 5, embryos were scored from 1 to 3 with 1 indicating the lightest or no staining and 3 indicating the darkest level of staining, and the results are graphed as a percentage of embryos that received each score. The number of embryos in each group are as follows: stage-12 CNT, n = 17; stage-12 SU5402 treated, n = 20.
lhx2 (LIM homeobox 2 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, head region only, lateral view, anteriorleft, dorsal up. Inset shows higher magnification of staining in midbrain (mes) pineal gland (pi) and diencephalon (di).
