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The developing vertebrate retina produces appropriate ratios of seven phenotypically and functionally distinct cell types. Retinal progenitors remain multipotent up until the last cell division, favoring the idea that extrinsic cues direct cell fate. We demonstrated previously that fibroblast growth factor (FGF) receptors are necessary for transduction of signals in the developing Xenopus retina that bias cell fate decisions (S. McFarlane et al., 1998, Development 125, 3967-3975). However, the precise identity of the signal remains unknown. To test whether an FGF signal is sufficient to influence cell fate choices in the developing retina, FGF-2 was overexpressed in Xenopus retinal precursors by injecting, at the embryonic 16-cell stage, a cDNA plasmid encoding FGF-2 into cells fated to form the retina. We found that FGF-2 overexpression in retinal precursors altered the relative numbers of transgene-expressing retinal ganglion cells (RGC) and Müller glia; RGCs were increased by 35% and Müller glia decreased by 50%. In contrast, the proportion of retinal precursors that became photoreceptors was unchanged. Within the photoreceptor population, however, we found a twofold increase in rod photoreceptors at the expense of cone photoreceptors. These data are consistent with an endogenous FGF signal influencing cell fate decisions in the developing vertebrate retina.
FIG. 2. Overexpressing FGF-2 in retinal precursors alters the
distribution of retinal cell fates. Graph of the distribution of GFP,
FGI:-2, and GFP/FGE-2 transgene-expressing cells in stage 40
retinas. FGF-2-expressing cells were identified by immunolabeling
with an antibody against FGF-2 (R&D Systems). Transgene~
expressing ceils were identified on the basis of laminar position and
morphology. Error bars are SEM (n, number of retina, GFP = 3848
cells; GFP/FGF-2 = 2096 cells; FGF-2 = 1694 cells) (*P < 0.01;
* *P < 0,001 ANOVA, Dunnett's post hoc test),
FIG. 3. Overexpression of FGF-2 in retinal precursors favors a rod
photoreceptor fate. Graph of the percentage of transgene-expressing
photoreceptors that are rods in GFP and GFP/FGF-2 blastomere
injected embryos. Stage 40 transgene-expressing retina were la~
beled with either anti-rhodopsin to label rods or anti-calbindin to
label cones. Transgene-expressing rod photoreceptors, photoreceptots
that were either rhodopsin-positive or calbindin-negative, are
represented as a percentage of the total number of transgene~
expressing photoreceptors (rods + cones}. Numbers above bars are
the numbers of retinas, and the numbers in parentheses are the
numbers of transgene*expressing photoreceptors. Error bars are
SEM ( * P < 0.01, Kruskal-Wallis nonparametric ANOVA, Dunn's
post hoc test).
FIG. 4. Overexpression of FGF-2 may promote rod photoreceptor
cell fate in the developing Xenopus retina. Twelvemicrometer
transverse sections through stage 40 retinas of GFPand
GFP/FGF-2-expressing embryos. (A and B) Retina expressing
GFP (A) or GFP/FGF~2 (B) immunolabeled with anti-rhodopsin
(red) to identify rod photoreceptors (yellow). Cells labeled
with stars are rods (rhodopsin positive), and circles are cones
(rhodopsin negative). (C) High-power view of the outer nuclear
layer (ONL) of a retina containing GFP/FGF-2-expressing cells
(green) iramunostained with anti~rhodopsin (red). (D) Retina
expressing GFP/FGF-2 (green) labeled with antbcalbindin (red)
to identify cone photoreceptors (yellow). (E and F) Apoptotic
cells (green, arrows) in a control retina (E) and a GFP/FGF-2-
expressing retina (F). (G and H) GFP- (G) and GFP/FGF-2- (H)
expressing :retinas (green) ilmnunolabeled with an~i-BrdU (red)
to identify mitotic cells. With the exception of a few BrdUlabeled
cells in the central retina the only actively dividing ceils
in transgene-expressing retina are in the CMZ. Bar in B is 50/xm
for A and B, 20 b~m for C, and 100 ~m for D-H. L, lens; PE,
pigment epithelium; ONL, outer nuclear layer; D, dorsal; V,
ventral. Orientation in A is for A, B, and D. Orientation in C is
for C and E-H.
FIG. 5. Overexpression of FGF-2 does not affect the survival or
proliferation of transgene-expressing cells. At different developmental
stages, retinas were assayed for proliferation (BrdU
incorporation) and apoptosis and no significant difference was
observed between the control and the experimental data sets. (A)
Graph showing the average number of apoptotic cells/12-~ln
retinal section that either were nonexpressing controls or expressed
the myc or FG£-2 transgenes. The same mean nmnber of
apoptotic cells was observed in all three sets of retinas. Numbers
are the numbers of retinas analyzed (>1400 transgene-positive
cells were counted for each data point). Statistical significance
was assayed using an ANOVA. (B) Graph showing the mean
percentage of transgene-positive cells that were apoptotic. Nmnbets
are the numbers of retinas analyzed. Statistical significance
was assayed using a two-tailed Student t test. (C) Graph showing
the percentage of transgene-expressing cells/retina that were
BrdU positive, Numbers represent the numbers of retina, while
numbers in parentheses represent the numbers of transgeneexpressing
cells. Statistical significance was analyzed using a
two-tailed Student t test.
