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In order to study anteroposterior neural patterning in Xenopus embryos, we have developed a novel assay using explants and tissue recombinants of early neural plate. We show, by using region-specific neural markers and lineage tracing, that posterior axial tissue induces midbrain and hindbrain fates from prospective forebrain. The growth factor bFGF mimics the effect of the posterior dorsal explant in that it (i) induces forebrain to express hindbrain markers, (ii) induces prospective hindbrain explants to make spinal cord, but not forebrain and midbrain, and (iii) induces posterior neural fate in ectodermal explants neuralized by the dominant negative activin receptor and follistatin without mesoderm induction. The competence of forebrain explants to respond to both posterior axial explants and bFGF is lost by neural groove stages. These findings demonstrate that posterior neural fate can be derived from anterior neural tissue, and identify a novel activity for the growth factor bFGF in neural patterning. Our observations suggest that full anteroposterior neural patterning may be achieved by caudalization of prospective anterior neural fate in the vertebrate embryo.
Fig. 1. RT-PCR detection of regionally expressed neural markers in
neural plate explants. (A) Schematic diagram of an early neurula
stage embryo (stage 14; red=forebrain, green=midbrain,
yellow=hindbrain, blue=spinal cord) based on the fate map of
Eagleson and Harris (1989). At stage 14, the neural plate was
removed (including the underlying axial mesoderm) and divided into
three pieces containing, prospective forebrain (A), hindbrain (M),
and spinal cord (P) regions. (B) The explants were cultured to tailbud
stage and then assayed by RT-PCR for the expression of the
forebrain marker OtxA, the hindbrain marker Krox-20, and the spinal
cord marker Xlhbox6 (HoxB9). Lane 1: whole embryo (early tailbud
stage). Lane 2: negative control containing all the reagents used in
lane 1 except the reverse transcriptase. Lanes 3, 4 and 5 contain
RNA derived from explants of presumptive forebrain, hindbrain, and
spinal cord respectively. Detection of EF1-a shows that comparable
amounts of total RNA were assayed in all of the reactions.
Fig. 2. Recombination of prospective forebrain and spinal cord
induces expression of the hindbrain marker Krox-20. (A) Schematic
diagram of the recombinant strategy used in these assays,
prospective forebrain (F) is shown in red and spinal cord (S) in blue.
(B) RT-PCR assay for the expression of region specific neural
markers in explants and recombinants. Krox-20 expression was not
detected in the control forebrain (lane 3) or spinal cord (lane 4)
explants but was expressed in the forebrain-spinal cord recombinants
(lane 5). The negative control (lane 2) contained all of the RT-PCR
ingredients except the reverse transcriptase. (C) Hindbrain marker
induction requires the forebrain-spinal cord interaction. Explants of
prospective forebrain and spinal cord regions of the neural plate were
recombined forebrain-forebrain (FF), forebrain-spinal cord (FS), and
spinal cord-spinal cord (SS) and cultured to tailbud stage for RTPCR
detection of OtxA, Krox-20 and Xlhbox6. Krox-20 is only
induced in the forebrain-spinal cord recombinants. Controls are as
described in Fig. 1.
Fig. 3. Prospective forebrain is caudalized by forebrain-spinal cord recombination. Forebrain-spinal cord recombinants were made at the early
neural plate stage using lineage-labeled prospective spinal cord explants and unlabeled prospective forebrain explants. The recombinants,
control explants and whole sibling embryos were assayed at the early tailbud stage for En-2 expression by whole-mount in situ hybridization.
Forebrain and spinal cord explants alone, as well as middle piece explants, from which the prospective forebrain and spinal cord regions were
removed, served as controls for dissections. (A) Whole embryo. (B) Prospective forebrain explants alone. (C) Middle pieces. (D) Spinal cord
explants alone. No En-2 expression was detected in the forebrain or spinal cord explants alone whereas whole embryos and the middle pieces
showed the characteristic band in the midbrain-hindbrain border. Forebrain-spinal cord recombinants (E) displayed a band or patch of En-2
expression. F shows a typical recombinant and the detection of the lineage label is shown in G and H. The patch of En-2 staining (arrow) lies
outside of the lineage label (red fluorescence, left side, G and H). A chip of cover glass, seen in the upper left, was necessary to position the
explant. The En-2 staining was always localized to the unlabeled forebrain component of the recombinant.
Fig. 4. Posterior dorsal mesoderm alone caudalizes prospective
forebrain. Tissue explants are designated F for prospective forebrain,
PM for posterior dorsal mesoderm, and FPM for the forebrainposterior
dorsal mesoderm recombinants. RT-PCR examination
showed that Krox-20 is induced in the recombinants (lane 5) but not
in either tissue alone (lanes 3 and 4). Muscle actin is a marker of
dorsal mesoderm whereas N-CAM is a pan-neural marker. Detection
of EF1-a shows that comparable amounts of total RNA were assayed
in all of the reactions. The negative control (lane 2) contained all of
the RT-PCR ingredients except the reverse transcriptase.
Fig. 5. Forebrain explants express Krox-20 following treatment with
bFGF but do not respond to activin. Early neurula prospective
forebrain and spinal cord explants were treated with (A) bFGF
(0.005-5 ng/ml) or (B) activin (0.2-8 ng/ml), cultured to tailbud
stage, and then processed for RT-PCR detection of OtxA, Krox-20,
Xlhbox6 and EF1-a. (A) Treatment with 5 ng/ml bFGF induced
Krox-20 expression in forebrain explants (lane 7) while lower doses
(lanes 3-6) did not. Posterior explants, treated and untreated, did not
express Krox-20 (lanes 8-12). (B) Activin treatment did not induce
Krox-20 in explants of prospective forebrain (lanes 3-7) or spinal
cord (lanes 8-12). Detection of EF1-a shows that comparable
amounts of total RNA were assayed in all of the reactions. The
negative control (lane 2) contained all of the RT-PCR ingredients
except the reverse transcriptase.
Fig. 6. Treatment of prospective hindbrain explants with bFGF
induces spinal cord but not forebrain-midbrain marker expression.
Explants of the prospective hindbrain were treated with bFGF (5, 50,
150 ng/ml) and at early tailbud stage assessed for anteriorization and
posteriorization by detection of the forebrain and midbrain markers
OtxA and En-2 and the spinal cord marker Xlhbox6. Treated explants
(lanes 4, 5, 6) expressed Xlhbox6 but not OtxA or En-2. Untreated
explants (lane 3) did not express OtxA, En-2, or Xlhbox6. Detection
of EF1-a shows that comparable amounts of total RNA were assayed
in all of the reactions. The negative control (lane 2) contained all of
the RT-PCR ingredients except the reverse transcriptase.
Fig. 7. Neural tissue induced by follistatin and dominant negative
activin receptor is caudalized by bFGF. Gastrula stage (stage 10.5-
11) ectodermal explants from uninjected embryos or those injected
with 2 ng of either follistatin or dominant negative activin receptor
(D1XAR1) RNA were treated with 0, 5, or 50 ng/ml bFGF, cultured
to tailbud stage and then assayed by RT-PCR for the expression of
A-P neural markers. Mesoderm induction was assayed by muscle
actin expression. Levels of EF1-a shows that comparable amounts of
total RNA were assayed in all of the reactions. The negative control
(lane 2) contained all of the RT-PCR ingredients except the reverse
transcriptase.
Fig. 8. The competence of forebrain explants to express Krox-20 in
response to prospective spinal cord explant recombination and bFGF.
14, 18, 20, 25, 30 and 35 are embryonic stages representing: open
neural plate, neural groove, neural tube, tailbud and tadpole stages.
(A) Competence of forebrain to respond to the prospective spinal
cord in recombinants. s, spinal cord alone; +, recombinants; -,
forebrain explants alone. (B) Competence of forebrain explants to
respond to bFGF. +, forebrain cultured in the presence of bFGF at 5
ng/ml; -, forebrain explants cultured in buffer alone. Detection of
EF1-a shows that comparable amounts of total RNA were assayed in
all of the reactions. The negative control (lane 2) contained all of the
RT-PCR ingredients except the reverse transcriptase. The
competence of prospective forebrain explants to respond to either the
posterior piece or to bFGF ends by neural groove stage (stage 18).
