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???displayArticle.abstract???
We have reviewed the evidence supporting the notion that the fibrillar extracellular matrix on the basal surface of the blastocoel roof in amphibian embryos directs and guides mesodermal cell migration during gastrulation. Based on extensive experimental evidence in several different systems, we conclude the following: (i) the fibrillar extracellular matrix contains fibronectin (FN) and laminin. (ii) The fibrils are oriented in such a way as to promote directional migration of mesodermal cells during migration. (iii) We have used several different probes to disrupt the interaction between migrating mesodermal cells and the fibrillar extracellular matrix. These probes include: (a) nucleocytoplasmic and interspecific hybridization. Such embryos have defects in FN synthesis and gastrulation. (b) Fab' fragments of anti-FN and anti-integrin VLA-5 IgGs prohibit mesodermal cell adhesion both in vitro and in vivo and gastrulation is arrested. (c) Peptides containing the RGDS sequence specifically inhibit interactions between migrating mesodermal cells and the FN-fibrillar matrix. (d) Tenascin blocks cell adhesion to FN in vitro and gastrulation in vivo. (e) Antibodies against the cytoplasmic domain of beta 1 integrin, when injected into blastomeres, prevent FN-fibrillogenesis in progeny of injected blastomeres and delay mesodermal cell migration selectively in the progeny of injected blastomeres but not in the uninjected blastomere progeny.
Fig. 1. Scanning electron micrographs. Gastrulation of Pleurodeles waltl embryos from rhe vegetal aspect. !A) Blastopore 15a pigmented area just
beginning to Invagmate (81 Blastopore nearlV a complere Circle ICI Yolk plug disappears from surface and blastopore closes. Bar: 500,um.
Fig. 2. Scanning electron micrographs
of migrating mesodermal
cells on the inner surface of the
blastocoel roof in Ambystoma maculatum
gastrulae. (A) Two migrating mesodermal cells.
The animal pole is to the left and the dorsal lip of
the blastopore is to the right. Notice that the cells have large lameillpodia on the leading edge of the cells directed toward the animal pole and a uropodlike
arrachment on the Iraillng edge of the cells. Bar 50 pm. (B) Filopodia on the leading edge of a lamelilpodium associated with the fibrillar ECM on
the basal surface of the blastocoel roof. Bar: 1um
Fig. 3. Fluorescent micrograph of ECM fibrils transferred from the basal suriace ofthe blastocoel roof of an Ambystoma maculatum gastrula to a plastic dish. DeposIted flbnls were stamed with a polyclonal antibody directed against A mexicanum plasma flbronectm, The dorsal lip region of the conditioning explant was on the left and the ammal pole region was on the right Notice that the fibrils are highly aligned parallel to the blastopore-animal pole aXIs (parallel to the long aXIs of the photograph). Migrating mesodermal cells or explanrs of the DMZ adhere to such substrata and migrate preferentially toward the animal pole region of such conditioned areas. Bar: 50 um.
Fig. 4. Orientation of DMZ outgrowth toward the animal pole (AP) on ECM-conditioned substratum.(AI Diagram of explants containing DMZ and adjacent ectoderm was deposited in the center of a substratum conditioned by ECM deposition from the basal surface of the blastocoel roof The original animal pole to blastopore axis (thick arrow) was perpendicular to that of the fragment used to condition the substratum.
(8) After 24 h of culture. DMZ
outgrowth has fully spread toward the animal pole, a cohesive cel/sheet developed on this sIde. The front of migratIon reached the edge of animal pole
and stopped there because ECM-conditloned substratum was no longer available. AP, animal pole, BP, blastopore. Bar: 500lJm
Fig. 5. Scanning electron micrograph of the in vivo effect of Fab' fragments of anti-FN during gastrulation. Pleurodeles waltl embryos were
mJcrolnjected at the early gastrula stage (Stage Bb) and observed 24 h later. (A) Injection of Fab' fragmenrs of antl-FN. This result IS typical of the more
severely arrested embryos.A complete inhibition of gastrulation was observed. Note the highly convoluted roof of the blastocoel, Circular blastopore, and
smooth exposed endoderma! mass. (B) Control embryo Injected with Fab' preimmune IgG The embryo undergoes normal gastrulation. An early neural
plate has formed in this control embryo indicating that primary embryonic induction has taken place. Bar. 250 11m.
Fig. 6. Migratory behavior of mesodermal cells in lIitrofrom Pleurodeles walt/gastrulae on a substrate conditioned by FN and TN. (A) An explant
contammg the DMZ and adjacent ectoderm was placed at the edge of one track conditioned by FN (left) and another conditioned by FNand TN{right}.
The migrating mesodermal sheet fails to spread on FN-and TN-coated substratum. The vertica/lines through the micrographs represent the boundaries
between each rype of substratum. (B) Control where both tracks were conditioned by FN alone. The cell sheer spread equally in both tracks. Bar: 500pm.
Fig. 7. Inhibition of FN.fibril formation
in selected blastomeres
by Fab' to cytoplasmic domain
of integrin B, subunit. (A-B)
Monovalent antibodies against
the cytoplasmic domain of integrin
61 (50 ng/embryo) were Injected
into each uncleaved Pleurodeles
waltl embryo. Embryos weremamrained
at 1Ef'C, then dissected after
the indicated times of incubation.
Immunodetection of FN was done
on whole mounts of rhe blastocoel
roof. (A) Twenry-four hours
afrer injection, the embryo has
reached rhe late blastula stage
(stage 7), bur no staining for FN
can be detected. IS) The first FN
fibrils appear at the early gastrula
stage (stage 8a). They are distributed
around the periphery of
cells. Their pattern is comparable to normal embryos observed at the early blastula stage (17 h earlier) Bar 5.um. !C-DJ Embryos Injected with antibodies
to the cytoplasmic domain of integrin 6, subunit. (C) Embryo at the two-cell stage was Injected into the leftblastomere with 100 ng of anti-I3, COGH
Fab'. Ar rht! rime ofobservarion 72 h farer, rhe left neural fold IS defectIVe. (D) Comrol expenment. Antl-B1 COOH Fab' was premcubated with amphibian
inregrin 13,and injected into the leftblastomere at the two-cell stage. Neurulation occurs normally. Bar: 0_3 mm.
