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The neural plate in the amphibian embryo is induced in the ectoderm by signals from the dorsal mesoderm. In the extensively studied species Xenopus laevis, such signals are believed to proceed along two alternate pathways, defined as vertical and planar induction. We have studied the relative importance of these pathways in Rana pipiens. In the embryo of this frog, dorsal mesoderm involution can be diverted from its normal course by injection of peptides that inhibit interaction of fibronectin with its receptor. In such embryos, dorsal mesoderm failed to migrate across the blastocoel roof but moved bilaterally along the equator, leading to the formation of two notochords. Neural tissue differentiation occurred in close association with each notochord, but no neural tissue formed along the dorsal midline as might have been predicted by a predominantly planar induction model. While in X. laevis planar induction has been reported to be a major pathway in neuralizing the ecoderm, the results presented here indicate that vertical induction predominates in initiating neural development in R. pipiens embryos.
FIG. 1. Development of embryos injected at the blastula stage
with 200 nl of GRGDS peptide at 50 mg/ml. At the early gastrula
stage both control (A) and injected embryos (B) display an apparently
normal blastopore lip (arrow) as viewed from the vegetal pole. At late
gastrula, blastopore closure in the control embryo (C) is almost
complete (arrow), but the injected embryo (D) shows a large yolk
plug. By stage 20 [control at tailbud stage (E)], the endomesoderm
of the injected embryo (F) protrudes from the blastopore that
remains largely open. In E and F, anterior is to the right and dorsal
is up. (Bar = 1 mm.)
FiG. 2. Expression of differentiation markers visualized by whole-mount immunostaining in intact and GRGDS embryos. Control embryos
at stage 18 (A, D, and G); GRGDS embryos at equivalent stage 18 (B, E, and H), stage 20 (C, F, I, andJ), and stage 21 (K and L). Muscle-specific
12/101 monoclonal antibody (39) stains somites in control (A) and GRGDS embryos (B and C); note two sets of somites in B and C. Tor 70
monoclonal antibody specifically stains the notochord inR. pipiens, as in other amphibians (40) (D). In GRGDS embryos, two distinct notochords
appear at lateral positions (Eand F). 4d monoclonal antibody, raised against chicken NCAM 180 kDa (36, 37), strongly stains the central nervous
system (CNS) of control embryos (G). NCAM staining was detected as early as stage 18 (H) in GRGDS embryos along both sides of the embryo
and intensified at stage 20 (I). B, C, E, F, and Hare dorsal views, I is ventral, anterior is up in all cases. (J) Lateral view of a GRGDS embryo
stained with LINC antibody (38) (anterior to the right) shows very similar pattern to NCAM staining (Hand I). (K) This ventral view ofaGRGDS
embryo doubly stained with LINC (brown) and 12/101 (blue) shows that segmented somites are associated with LINC-positive tubular
structures; anterior is up. (L) GRGDS embryo doubly stained with Tor 70 (brown) and 4d-NCAM (blue) antibodies; anterior is to the left.
NCAM-positive cells are localized in the vicinity of each notochord while the midline of the ectoderm is unstained; note the apparent segmented
spinal nerves. (A-I, bar = 1 mm; J-L, bar = 1 mm.)
FIG. 3. Schematic representation of 32-cell stage blastomeres (Upper)
and their contribution to notochord and CNS in normal (Lower
Left) and GRGDS-injected (Lower Right) embryos. The notochord
(arrowheads, Not) arise from blastomeres B1 andC1 in both normal and
GRGDS embryos. Shading is proportionate to the contribution of each
blastomere to the CNS; whereas the CNS originates from A and B tier
blastomeres in control embryos, it originates exclusively from B and C
tier blastomeres in GRGDS embryos. Asterisks indicate blastomeres
whose fate was not determined.
FIG. 4. Examples of lineage tracing of 32-cell stage blastomeres in control (A, C, and F) and GRGDS-injected embryos (B, D, E, G, H, and
I), sectioned at stage 21. (A and B) Bi and Cl blastomeres were injected with FLDX (green) and TRLDX (red), respectively. Transverse section
of a control embryo (A) shows Bi progeny in the spinal cord (n) and notochord (arrow); in this section Cl progeny does not appear in the
notochord but in somites and mesenchyme. The notochord (arrow) of GRGDS embryo (B) contains Bi- and Cl-derived cells, and neural tissue
(arrowhead) contains Bi-derived cells. Blue staining in the ventolateral aspects of the neural tube (A; also in C) corresponds to immunostaining
with a mix of 4d-NCAM and LINC antibodies visualized with AMCA fluorochrome. (C-E) Both Al blastomeres were injected with FLDX.
Transverse section of normal embryo (C) shows Al progeny in the spinal cord (n) and the epidermis, while in the GRGDS embryo (D,
phase-contrast; E, fluorescence), Al progeny is restricted to the apical epidermal region; no labeled cells can be detected in a lateral position
where neural tissue is located (see I and Fig. 2 H-J and L). Arrows in D point to the dual notochords. (F-I) The progeny of B2 (FLDX) and
C2 (TRLDX) blastomeres in a normal (F) and a GRGDS (G-I) embryo. (F) In this section, B2 progeny is almost exclusively restricted to one
half of the rhombencephalon (n). C2-derived cells form the pharynx and mesenchyme but are virtually absent from the rhombencephalon (n)
and the notochord (arrow). (G) Phase-contrast image of an oblique transverse section of a GRGDS embryo showing only one of the two axes
(arrow). (H and I) Higher magnification of the axis indicated in G. The neural tube-like structure (n) is composed of cells originating from B2
(green) and C2 (red) blastomeres, while the notochord (arrows in Hand I) is not labeled. (D and E, bar = 0.3 mm; A, F, and G, bar = 0.2 mm;
C, B, H, and I, bar = 0.1 mm.)
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