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The floor plate, a cell group that develops at the midline of the neural plate in response to inductive signals from the notochord, has been implicated in the control of dorsoventral neural pattern. The frog Pintallavis gene, encoding a member of the HNF-3/fork head transcription factor family, is expressed in the notochord and in midline neural plate cells that give rise to the floor plate. To examine whether Pintallavis might be involved in regulating the differentiation of the floor plate, we ectopically expressed Pintallavis by injection of synthetic mRNA into two-cell frog embryos. Injection of Pintallavis mRNA resulted in the ectopic expression of F-spondin, a gene encoding a floor plate-specific adhesion molecule, at the dorsal midline of the neural tube. The expression of Pintallavis in midline cells may therefore contribute to the establishment of the floor plate fate.
FIG. 1. Comparison of the frog and rat
F-spondin proteins. (Upper) Schematic representation
of the domain structure of F-spondin
protein. The black box represents the signal
sequence. The gray boxes represent the six
thrombospondin type I repeats (TSRs). (Lower) Amino acid alignment of the frog and rat F-spondin
proteins. Underlined NH2-terminal sequences correspond to the putative signal sequences.
Bars above the sequence delineate the
TSRs, numbered 1-6 as in the schematic. The
overall similarity of the frog and rat F-spondin
proteins is 84%. The NH2-terminal domain of
the proteins, which does not contain the TSRs,
exhibits 86% identity, indicating that this do-
main may have a role in the structure or function
of the protein. Apart from the signal sequence,
the only region of extensive sequence divergence
is the highly charged segment between
TSRs 5 and 6. Numbers at
FIG. 2. Localization of F-spondin mRNA in frog embryos. (A) Whole-mount in situ hybridization of a X. laevis tadpole, stage 34-36, showing
F-spondin RNA in the floor plate at the ventral midline of the neural tube and in cells of the hypochord. Note the higher F-spondin expression
in hindbrain compared with spinal cord. (B) Whole-mount in situ hybridization of an exogastrulated embryo at a stage equivalent to that in A,
showing that F-spondin RNA is not present in the ectoderm (ec). In contrast, F-spondin RNA is detected in cells of the hypochord found adjacent
to the notochord (n). en, Endoderm; me, mesoderm. (C and D) High magnification of midbrain-hindbrain region of tadpole (stage 36) labeled in
whole-mount, showing F-spondin RNA in the floor plate (fp) of the hindbrain and in the hypochord (h), located under the notochord (n). There
is also F-spondin RNA in the lateral region of the midbrain. In contrast to the expression in rat embryos (17), in frogs F-spondin is not expressed
by the most anterior floor plate cells located in the midbrain and there is a clear boundary of expression in the floor plate at the boundary of the
midbrain (mb) and the hindbrain (hb) (arrowhead in D). F-spondin is expressed at low levels in the posterior part of the notochord and in branchial
arches (data not shown). (E and F) Cross sections of the hindbrain of tadpoles (stage 36) after whole-mount in situ hybridization. F-spondin RNA
is restricted to the floor plate (fp) at the ventral midline. In F, F-spondin RNA is detected in the hypochord (h), an embryonic endodermal structure
located ventrally to the notochord (n). s, Somites. (G) Dorsal view of the anteriorhindbrain of a tailbud-stage (stage 22) embryo after whole-mount
in situ labeling, showing early expression of F-spondin by small groups of cells at the ventral midline of the neural tube. (H and I) Whole-mount
in situ hybridization oflate blastula (stage 9) (H) and late gastula (stage 12.5) (I) embryos. (H) Vegetal view showing normal expression ofPintallavis
in the organizer region, located dorsally, and the injected RNA (arrowhead). d, Dorsal; v, ventral. (I) Dorsal view of an embryo injected with
Pintallavis RNA, showing normal expression ofPintallavis in the midline. a, Anterior; p, posterior. (J) Cross section of a tadpole (stage 36) injected
with RLDx into one cell at the two-cell stage, showing unilateral restriction of the injected tracer. Note the labeling of decussated axons in the
ventral funiculus of the unlabeled half. [Bar = 600 ,m (A and B), 150 ,um (C), 30 pm (D), 40 ,pm (E and J), 20 ,um (E and G), or 200 pm (H and I).]
FIG. 3. Localization of F-spondin mRNA in Pintallavis
mRNA-injected tadpoles. (A-G) Cross sections
of the hindbrain of tadpole stage embryos injected with
Pintallavis RNA at the two-cell stage, showing the
localization of F-spondin RNA by whole-mount in situ
hybridization. F-spondin RNA is expressed in floor
plate (fp) cells at the ventral midline of the neural tube.
Arrowheads depict the sites of ectopic F-spondin RNA
expression. Embryos shown in A, B, D, and F show
ectopic F-spondin RNA in cells adjacent to the dorsal
midline, which form part of the roof plate. In B and D
the dorsal cells were displaced slightly during the
labeling procedure. The embryo shown in B was pigmented.
Embryos shown in C, E, and G show an
expanded area of F-spondin expression adjacent to the
floor plate. C shows F-spondin RNA expression in
hypochord cells under the notochord (n). As previously
shown (8), injection of Pintallavis RNA leads to an
increase in the amount of posterior neural tissue in
affected embryos. C and E show enlarged neural tubes
(arrows) and (in C) a secondary neurocoel. (H and I)
Lateral view of tadpoles (stage 36) injected with Pinn
tallavis RNA after whole-mount in situ localization of
F-spondin RNA. Arrowheads point to the sites of
ectopic F-spondin expression. The embryo in H faces
right and that in I faces left. In all cases, dorsal side is
up. [Bar = 10 ,um (A-G) or 200 Zm (H and I).]
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