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A cloned cDNA fragment, homologous to domain II of the mouse laminin B1 chain, was obtained from a Xenopus neurula cDNA library. Using this probe Northern-blot analysis over the course of embryogenesis revealed a first signal of a laminin transcript at midgastrula (stage 11). Affinity-purified polyclonal antibodies directed against Xenopus laminin were obtained via a lacZ fusion protein. Immunohistology demonstrated the appearance of the antigen at about one developmental stage after the detection of the transcript. At stage 12 1/2 a faint immunofluorescence staining surrounded the developing notochord and somitogenic mesodermal cells. Thereafter laminin appeared in distinct locations outlining notochord, somites and neural tube. The epidermal basement membrane seemed to be endowed with laminin only at the relatively late postneurula stage. The observation that laminin could not be detected before stage 12 1/2 is discussed with respect to a proposed role of laminin in the gastrulation process.
Fig. 1. Comparison of nucleic acid sequence between the mouse
and Xenopus laminin fragment. Top, structural model of the laminin
molecule and, in detail, the B1 chain with the different positions
of the domains I-VI [43,40]. The model on the right schematically
shows the cloned 3-kb Xenopus cDNA fragment, its position
within the B1 chain, the 1.8-kb subfragment (marked by UYYUM>-
heah) and the site of the sequenced portion (arrow). Bottom, nucleic
acid sequence of the Xenopus laminin (x) compared to the
corresponding mouse (m) sequence [2]
Fig. 2A, B. Specificity of Xenopus laminin antibodies. A Coomassie-blue-stained 7% sodium dodecyl sulfate (SDS)-electrophoresis gel
(lanes 2-5) and immunoblot (lanes 6-9), showing total protein from induced bacteria containing the unfused trpE gene (lanes 2, 6),
the trpE-LN fusion gene (lanes 4 , S), the unfused lacZ gene (lanes 3, 3, or the IacZ-LN fusion gene (lanes 5 , 9). Molecular-weight
marker proteins are shown in lane 1. For immunoblots (lanes 6-9), the Xenopus laminin antiserum was diluted 1:3000 as described
in Methods. B Coomassie-blue-stained 7% SDS-PAGE of mouse laminin (lane I ) and total Xenopus kidney (lane 2) and the corresponding
immunoblots using mouse laminin antibodies (1:200) for the mouse laminin (lune 3) and Xenopus laminin antibodies (1:300) for the
kidney extract (lane 4 )
Fig. 3A, B. Tissue-specific distribution of laminin in muscle and kidney. Indirect immunofluorescence staining for laminin in 10-pm
frozen sections of Xenopus muscle (A) and kidney (B). The Xenopus antibodies were used undiluted. Bar, 50 pm
Fig.
4A-D. Expression of Xenopus laminin mRNA
during development. A, B Poly (A)' RNA (2 pg per
lane) was denatured and electrophoresed on a 1 YO
agarose formaldehyde gel, blotted onto nitrocellulose
and hybridized with a 3zP-labeled Xenopus laminin
cDNA probe. The lanes contained RNA from
different embryonic stages or from adult Xenopus
kidney ( K ) , respectively. C, D The same nitrocellulose
blots as shown in A, B stained with methylene blue,
showing the 28s ribosomal RNA
Fig. 5A, B. Immunofluorescence localization of laminin and fibronectin on the blastocoel roof. Whole-mount staining of the bkdstocoel
roof of stage embryos for laminin (A) or fibronectin (B). The bovine fibronectin antibodies were diluted 1:2OO as described
In Methods, and the Xenopus laminin antibodies were used undiluted. Bur, 10 pm
Fig. 6 A-D. Distribution of laminin during development. A-D Indirect immunofluorescence staining for laminin on sections of embryos,
stage 121/2 (A), 17 (B), 20 (C) and 23/24 (D). The embryos were staged according to Nieuwkoop and Faber [33]. The Xenopus antibodies
were applied undiluted. Bar, 20 Fm
