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Molecular analysis of vertebrate eye development has been hampered by the availability of sequences that can selectively direct gene expression in the developing eye. We report the characterization of the regulatory sequences of the Xenopus laevis Rx1A gene that can direct gene expression in the retinal progenitor cells. We have used these sequences to investigate the role of Fibroblast Growth Factor (FGF) signaling in the development of retinal cell types. FGFs are signaling molecules that are crucial for correct patterning of the embryo and that play important roles in the development of several embryonic tissues. FGFs and their receptors are expressed in the developing retina, and FGF receptor-mediated signaling has been implicated to have a role in the specification and survival of retinal cell types. We investigated the role of FGF signaling mediated by FGF receptor 4a in the development of retinal cell types in Xenopus laevis. For this purpose, we have made transgenic Xenopus tadpoles in which the dominant-negative FGFR4a (Delta FGFR4a) coding region was linked to the newly characterized regulatory sequences of the Xrx1A gene. We found that the expression of Delta FGFR4a in retinal progenitor cells results in abnormal retinal development. The retinas of transgenic animals expressing Delta FGFR4a show disorganized cell layering and specifically lack photoreceptor cells. These experiments show that FGFR4a-mediated FGF signaling is necessary for the correct specification of retinal cell types. Furthermore, they demonstrate that constructs using Xrx1A regulatory sequences are excellent tools with which to study the developmental processes involved in retinal formation.
Fig. 1. Transgenic Xenopus laevis embryos at different stages carrying Xrx1A-GFP construct 1 (A,D,G,J; see Fig. 3), displaying GFP fluorescence (B,E,H,K). (C,F,I,L) In situ hybridization of Xrx1A probe to non-transgenic embryos of the same developmental stage to demonstrate the normal expression pattern of the Xrx1A gene. A-C, stage 15; D-F, stage 21 (frontal view); G-I, stage 21 (side view); and J-L, stage 28.
Fig. 5. (A-C,N-R) Immunostaining of sections of tadpoleeyes with antibodies against rhodopsin and calbindin. (D-M) Whole-mount staining of tadpoles with antibodies against rhodopsin. (A) Section of a stage 39 tadpole that does not carry the Xrx1A-δFGFR4a construct stained with antibodies against rhodopsin, demonstrating the presence of photoreceptor cells. (B) Section of a stage 39 tadpole that carries the Xrx1A-δFGFR4a construct stained with antibodies against rhodopsin. Note the lack of photoreceptor cells. (C) Section of a tadpole carrying the Xrx1A-δFGFR4a construct stained with rhodopsin antibodies that shows some photoreceptor cells in ectopic position. (D-G) Whole-mount staining of Xenopus tadpoles that do not carry the Xrx1A-δFGFR4a construct with rhodopsin antibodies at different stages, demonstrating the normal accumulation of photoreceptor cells during development. (H-M) Whole-mount staining of transgenic Xenopus tadpoles expressing the Xrx1A-δFGFR4a construct with rhodopsin antibodies at different stages, demonstrating lower numbers of photoreceptor cells in these embryos at all stages. D,H, stage 33; E,K, stage 35; F,L, stage 36; G,M, stage 38. (N) Staining of a section from a stage 46 embryo that does not carry the Xrx1A-δFGFR4a construct with antibodies against rhodopsin. (O) Staining of a section of stage 45 embryo that does not carry the Xrx1A-δFGFR4a construct with antibodies against cone-specific calbindin. (P) An eye section from a stage 45 tadpole expressing the Xrx1A-δFGFR4a construct stained with rhodopsin antibodies. Note the lack of rhodopsin-positive rods. (R) An eye section from a stage 45 tadpole expressing the Xrx1A-δFGFR4a construct stained with calbindin antibodies. Only few cones are present (arrowheads), some of them in ectopic locations.
Fig. 7. Comparison of retinal cell distribution in tadpoles carrying and lacking the Xrx1A-δFGFR4a construct. (A) Immunostaining of an eye section from a stage 45 non-transgenic tadpole with antibodies against Islet1, which recognizes the ganglion and amacrine cells. (B) Immunostaining of an eye section from a stage 45 tadpole that carries the Xrx1A-δFGFR-4a construct with antibodies against Islet1, demonstrating disturbed layering of retinal cells. (C) Hoechst staining of the section from B. (D) Immunostaining of an eye section from a stage 45 tadpole that does not carry the Xrx1A-δFGFR4a construct with antibodies against glutamine synthetase, which recognizes Müller cells. (E) Immunostaining of an eye section from a stage 45 tadpole that carries the Xrx1A-δFGFR4a construct with antibodies against glutamine synthetase demonstrates irregular distribution of Müller cells in the retina of these tadpoles. (F) Hoechst staining of the section from E. (G) Histogram showing the percentage of Müller glial cells and retinal ganglion cells/amacrine cells in the retina of transgenic tadpoles. Müller glial cells and retinal ganglion cells/amacrine cells are identified by immunostaining with antibodies against glutamine synthetase and Islet1, respectively. MGC, Müller glial cells; RGC, retinal ganglion cells; AC, amacrine cells; Single Tsg, car-GFP transgenic (MGC, n=8 retinas; RGC/AC, n=6 retinas); Double Tsg, car-GFP/Xrx1A-δFGFR4a transgenic (MGC, n=10 retinas; RGC/AC, n=11 retinas).
