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???displayArticle.abstract??? Fibroblast growth factor (FGF) has been implicated in a variety of developmental processes including posteriormesoderm and neural patterning. Previous work has led to contradictory roles for FGF in neural induction and anteroposterior neural patterning. Launay et al. (Development 122, 869-880, 1996) suggested a requirement for FGF in anterior neural induction. In contrast, Kroll and Amaya (Development 122, 3173-3183, 1996) and Bang et al. (Development 124, 2075-2085, 1997) proposed that FGF is not required for early neural patterning. Here we use a loss-of-function assay to examine whether FGF is required for neural patterning in three experimental situations: (i) in Xenopus early embryos, (ii) in embryonic explants consisting of presumptive dorsal mesoderm and neurectoderm (Keller explants), and (iii) in explants of dorsal ectoderm and posteriormesoderm in which FGF signaling is specifically blocked in the ectoderm. When cultured until tailbud stages, Keller explants develop neural tissue with normal anteroposterior pattern. Overexpression of the dominant-negative FGF receptor (XFD) in Keller explants inhibited the posterior neural markers En-2, Krox-20, and HoxB9, but not the panneural marker nrp-1 and the anterior neurectodermal markers XAG-1 and Xotx-2. Similar results were seen in whole embryos, but only when XFD RNA was targeted to both the dorsal and lateral regions. In contrast, addition of FGF to Keller explants resulted in a shift of the midbrain-hindbrain boundary marker En-2 to a more anterior position normally fated to become cement gland. We also determined whether FGF is required specifically by the neurectoderm for anteroposterior neural patterning. Recombinants of dorsal ectoderm and posteriormesoderm were made in which FGF was specifically blocked in the ectoderm. Spinal cord and hindbrain markers were inhibited in these recombinants, whereas anterior markers and cement gland development were enhanced. Our results demonstrate that FGF is important for posterior development in both mesoderm and neurectoderm and that neural induction and posteriorization represent separable developmental events.
FIG. 1. Anterior neural markers occupy the same position in explants as in intact embryos. (A) Fate of ectodermal cells in the stage
10-minus gastrula. A point in the dorsal midline, at the midpoint between the bottle cells and the animal pole, was stained with Di-I
(asterisk). Blue lines indicate outline of representative Keller explant. (B) In tailbud embryos (face view, stage 27), the marked point (arrow,
yellow cells) occupies the face plate, between the eyes and posterior to the cement gland. e, eyes; cg, cement gland; ncz, nonciliated zone.
(C) Marking the same region in explants using a glass bead (gb). (D) Explant at tailbud stage (stage 23) stained for cement gland (XAG-1),
and the midbrain–hindbrain boundary (En-2). The glass bead left a hole in the explant (gb) posterior to cement gland. (E) Expression of En-2
and XAG-1 in stage 23 embryo (face view).
FIG. 2. Keller explants express neural and mesodermal markers with correct anteriorâposterior patterning. (AâH) Neural markers (green
arrows: Krox-20 for hindbrain; HoxB9 for spinal cord; nrp-1 for panneural; Xotx-2 for forebrain) and cement gland marker (red arrows,
XAG-1) in explants (AâD) and whole embryos (EâH). All explants and embryos stained at stage 22 except for D and H which were stained
at stage 15. bp, blastopore. (D) Note both mesodermal (m) and neural expression (n) of Xotx-2 and location of glass bead (gb). Occasionally,
in situ hybridization resulted in a brown-staining product (D,H) but the reason for this variation is not understood. (I,J) Expression of early
mesodermal markers in stage 10-minus explants (I, red arrows) and stage 10.5 gastrulae (J). I. Anterior mesoderm (gsc) occupies the lower
edge of the explant, while more posterior markers (Xbra, Xnot) are found in the interior. Dots in J indicate dorsal lip.
FIG. 3. The organizer region is required for neural pattern in Keller explants and is itself specified to form posterior neural tissue. de, dorsal
ectoderm. dm, dorsal mesodermal portion (A) Experimental scheme. (BâE) Explants with dorsal mesoderm removed from the ectodermal
portion of Keller explants. Distribution of XAG-1 (BâD), En-2 (B), HoxB9 (C), nrp-1 (D), and anterior mesodermal Xotx-2 (E). Neural markers
were not expressed in the ectodermal portions, while only spinal cord and panneural (C,D) and anterior mesoderm markers (E) were detected
in the fragment containing dorsal mesoderm.
FIG. 4. A dominant-negative FGF receptor (XFD) inhibits posterior neural development in explants. (A) RNA injection in the dorsal
midline at the two-cell stage (4 injection sites, total of 4 ng RNA per embryo). (B) Control d50-injected embryo at the tailbud stage. Black
arrow, En-2. Red arrow, XAG-1. (C) XFD-injected embryo expresses XAG-1 and En-2. Note lack of blastopore closure. (D) Control (d50 RNA)
explants express cement gland and En-2. (E) Explants isolated from XFD embryos lack En-2. Cement gland develops normally. Explants in
F and G were obtained from embryos injected with RNA in the dorsal midline and lateral regions. XFD-expressing explants are marked by
an asterisk. Black arrows indicate glass bead that was placed in the center of the explant. (F) Control explants express XAG-1 (white arrow)
and HoxB9 (green arrows). XFD explants (*) do not express HoxB9, although they do have cement gland. (G) Xotx-2 (white arrows) is
expressed in explants. All explants were stained at stage 22, except ones in G, which were stained at stage 15.
FIG. 5. Lateral and dorsal injections of XFD lead to loss of posterior but not anterior neural markers. XFD-injected embryos are marked
by an asterisk. (A) Injection scheme (4 dorsal and 2 lateral injection sites, total of 8â10 ng of RNA per embryo). (B) Embryo injected with
control d50 RNA (arrow, En-2; light blue stain, XAG-1). (C) XFD-injected embryo lacks En-2 but retains XAG-1. (D) Krox-20 is lost in XFD
embryos on one side (middle embryo) or entirely (right embryo). (E) HoxB9 spinal cord marker is lost entirely (lower left embryo) or on one
side (lower right embryo). (F) nrp-1 (arrows) throughout nervous system (head and spinal cord) in control, and in head region of XFD embryos
(*). (G) Xotx-2 expression in forebrain regions is maintained in XFD embryos (*). In XFD-injected embryos, convergent extension is inhibited
causing condensation of the neural plate. As a result, the expression domains of nrp-1 and Xotx-2 become spatially restricted (F,G).
FIG. 6. Posterior neural markers are lost when XFD is expressed throughout the marginal zone. (A) XFD RNA (lanes 1 and 3) and d50 RNA
(lanes 2 and 4) were injected at the two-cell stage into either the dorsal regions (âD,â lanes 1 and 2; 4 injection sites, total of 6 ng of RNA
per embryo) or into the dorsal and lateral regions (âD 1 L,â lanes 3 and 4; 6 injection sites, total of 8â10 ng of RNA per embryo). cDNA
for RTâPCR was prepared from stage 27 embryos. Although injection of XFD in dorsal regions inhibits muscle actin expression (m-actin,
lane 1), loss of posterior neural markers (En-2; Krox-20, HoxB9) is only seen when injection is targeted to both the dorsal and lateral regions
(lane 3). EF-1a is a loading control. (B) RTâPCR analysis of embryos (lanes 6 and 7) injected with RNA as in lanes 3 and 4. Dorsal marginal
zone (DMZ) explants were prepared from stage 10-minus gastrulae and cultured until stage 27 (lanes 8 and 9). Note loss of posterior neural
markers and up-regulation of XAG-1 and Xotx-2 due to XFD RNA overexpression (lane 8). EF-1a is a loading control. (C,D) Dorsal marginal
zone explants isolated from embryos that had been injected with control d50 RNA (C) or XFD RNA (D) and stained for En-2 (dark blue) and
XAG-1 (light blue).
FIG. 7. Addition of FGF posteriorizes neural cell fate in explants but does not expand the neural field. (A) XAG-1 staining (light blue) is
found next to the location of a glass bead, that was placed in the center of the explant (black arrow). Midbrainâhindbrain boundary marker
(En-2, red arrows, dark blue stain) in more posterior region. (B) In explants treated with 10 ng/ml basic FGF, En-2 (red arrow, dark blue stain)
occupies a region significantly closer to the glass bead. Cement glands are frequently absent. (C) Control (left) and FGF-treated (right)
explants stained for a panneural marker (nrp-1) show that FGF does not alter the expand the border of neural tissue.
FIG. 8. FGF signaling is specifically required in the neurectoderm
for complete anteriorâposterior neural patterning. Explants of dorsal
ectoderm (DE) were prepared from stage 10-minus gastrulae that
had been previously injected with d50 or XFD RNA (4 dorsal
animal injection sites, total of 4 ng RNA per embryo). Posterior
mesoderm was obtained from stage 12 embryos. d50-injected
embryos express neural markers and muscle actin (lane 1). For
RTâPCR, cDNA was prepared from explants and embryos at stage
27. Embryos injected with XFD RNA express all neural markers,
but have a decrease in muscle actin (lane 2; note that XFD injection
was limited to the animal pole and dorsal midline). Uninduced
explants of dorsal ectoderm (XFD and d50) do not express significant
amounts of neural tissue (lanes 3 and 5). Posterior mesoderm
can induce dorsal ectoderm expressing d50 RNA to form neural
tissue of all anteriorâposterior levels (lane 4). Blocking of FGF
signaling specifically in dorsal ectoderm (with XFD RNA) inhibits
the induction of spinal cord and hindbrain by posterior mesoderm
and enhances anterior development (lane 6). Posterior mesoderm,
cultured alone, forms muscle but not neural tissue (lane 7).
