August 1, 1986;
Induction of neural cell adhesion molecule (NCAM) in Xenopus embryos.
Using a classical neural induction protocol (H. Spemann and H. Mangold (1924). Roux'' Arch. Entwicklungsmech. Org. 123, 389-517), it has been demonstrated that the sustained presence of NCAM
in Xenopus embryos, as detected by immunohistochemistry, was confined to the experimentally induced nervous system
and the primary host nervous system
. Furthermore, in vitro NCAM
expression by dorsal blastopore lip
and animal pole tissue
was detected only when the two tissues were cultured in contact. These and other results show that readily detected and sustained levels of NCAM
expression in Xenopus can be used as a marker for neural tissue
and an early positive indicator that neural induction has occurred. They suggest that the observed levels of NCAM
are a consequence of and not a prerequisite for induction. Using NCAM
expression in vitro to determine the minimum time necessary for this induction to occur in vivo, it was found that NCAM
was first detected in cultured animal pole
that had been removed at stage 10
.75 or later. Thus, an inductive step necessary and sufficient for stimulation of NCAM
expression in animal pole
tissues had not occurred or was reversible prior to the first 2 to 2.5 hr of gastrulation.
[+] show captions
FIG. 1. Characterization of antibodies against NCAM by immunoblots
of tissue membrane extracts after fractionation by SDS-PAGE.
Staining using monoclonal antibody 4D with adult liver (lane l), adult
frog brain (lane 2), and adult frog brain after treatment with endoneuraminidase
N (lane 3). Staining using polyclonal rabbit anti-frog
NCAM with adult frog brain (lane 4), adult frog brain after treatment
with endoneuraminidase N (lane 5), and stage 28 frog embryo (lane
6). The predominant polypeptide form of NCAM in each case is the
180 kilodalton chain. The polyclonal antibody also detects a trace of
140 kDa NCAM and an apparent fragment of 90 kDa (Rutishauser et
FIG. 2. Experimental design and summary of the results of the induction experiments in (A) the intact embryo (Fig. 4) and (B-F) in tissue
culture (Fig: 5; Table 1).
FIG. 3. Experimental design and summary of the results of isolating
the animal pole in vitro (Table 2; Fig. 6).
FIG. 4. Tissue sections stained immunohistochemically for NCAM. Wild-type Xenopus embryos at stage 30 that had received a graft of dorsal
blastopore lip in the ventral marginal zone at stage 10. (A) Section cut coronally at the level of the eyes. (B) Section cut in the parasaggital
plane. Greatly enhanced NCAM reactivity is seen in the primary (P) or host nervous system, and secondary (S) or experimentally induced
central nervous system. The dark pigment in the epidermis is melanin. Magnification bar 100 pm.
FIG. 5. NCAM immunohistochemistry of tissue sections excised at stage 10 from albino Xenopu.s embryos (A-C) and wild-type Xenopz~s (D)
and cultured for 48 hr before processing. (A) Dorsal blastopore lip culture alone and (B) animal pole cultured alone, both of which in color
photographs display no detectable immunoperoxidase reaction product. (C and D) Combination of animal pole with dorsal blastopore lip with
strong NCAM immunoreactivity (N). Bar is 50 pm for all photographs.
FIG. 6. Immunohistological sections stained for NCAM. Animal pole from wild-type Xenopus isolated from a stage 10.75 embryo (A), and a
stage 12 embryo (B), and cultured for 48 hr, N. NCAM reaction product. The dark pigment is melanin. Note that although both (A) and (B)
have detectable staining for NCAM, the degree of recognizable neural structure is greater in (B). These two pictures represent extreme
examples of this difference in ability to differentiate. Bar is 50 pm.