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The DG42 gene is expressed during a short window during embryogenesis of Xenopus laevis. The mRNA for this gene can be first detected just after midblastula, peaks at late gastrula, and decays by the end of neurulation. The sequence of the DG42 cDNA and genomic DNA predicts a 70,000-Da protein that is not related to any other known protein. Antibodies prepared against portions of the DG42 open reading frame that had been expressed in bacteria detected a 70,000-Da protein in the embryo with a temporal course of appearance and decay that follows that of the RNA by several hours. Localization of the mRNA in dissected embryos and immunohistochemical detection of the protein showed that DG42 expression moves as a wave or gradient through the embryo. The RNA is first detected in the animal region of the blastula, and by early gastrula is found everywhere except in the outer layer of the dorsal blastopore lip. By midgastrula DG42 protein is present in the inner ectodermal layer and the endoderm; it disappears from dorsal ectoderm as the neural plate is induced and later decays in a dorsoventral direction. The last remnants of DG42 protein are seen in ventral regions of the gut at the tailbud stage.
FIG. 1. DG42 mRNA sequence. The composite nucleotide sequence of full-length DG42 mRNA was obtained from the original cDNA clone
DG42 and the longer version of this cDNA, pC4202; the 5â-terminal eight nucleotides were derived from the genomic clone G42. The positions of
the four introns are indicated by arrows (see also Fig. 2). Differences between the cDNA sequence shown in the figure and genomic sequences
occur at the following locations; cDNA residue is given first. Position 661, A to G (both Glu); 832, A to G (both Glu); 1057, the genomic sequence
was unreadable; 1720, C to T (both Tyr). The following differences are located in the Sâuntranslated region: 1866, G to A; 1923, A to G; 1941, A to
G; 1942, G to A, 2003, T to A; positions 2098-2101, TGTT, are absent from the genomic DNA, 2164, T to C; 2195, T to C; 2287, C to T.
FIG. 2. DG42 gene structure and upstream sequences. (A) The structure of the DG42 gene, which includes five exons (black rectangles) and
four introns (lines), was deduced by comparison of genomic and cDNA sequence. The numbers refer to the mRNA sequence (above) and the
genomic sequence (below). (B) The DNA sequence of the region located upstream from the transcriptional start site. Sequences of interest (see
text) are underlined.
FIG. 3. Immunochemical characterization and developmental pattern of DG42 protein. (A) %-labeled embryonic protein was extracted and
precipitated with anti-DC42 protein antibody, as described under Materials and Methods. Lane 1, precipitation with anti-DG42 antibody; lane
2, precipitation in the presence of excess unlabeled, bacterially synthesized DG42 protein antigen; lane 3, precipitation in the presence of excess
bacterially synthesized protein from a region of DG42 not used to generate the antibody. (B) The accumulation of DG42 mRNA sequences is
revealed by dot blot analysis (above, reprinted from Jamrich et al, 1985), and the accumulation of DG42 protein as visualized by Western
blotting (below). The stages are according to Nieuwkoop and Faber (1967), and equivalent stages in the two series are connected
FIG. 4. Localization of DG42 mRNA in dissected embryos. (A) Embryos
between stages 9 and 10 were cut into three segments as shown,
and RNA was extracted from animal and vegetal thirds. Total RNA
from approximately 1000 cells per sample was analyzed by dot blot
hybridization to DG42 probe. (B) Inner and outer ectodermal tissue
was dissected from stage 9 through stage 114 embryos, and for each
sample, total RNA from 1000 cells was analyzed by gel blotting. i,
Inner ectoderm, o, outer ectoderm. (C) Stage 104 embryos were dissected
as indicated by the drawing into outer ectoderm (oe), inner
ectoderm (ie), outer blastopore lip (ob), inner blastopore lip (ib),
dorsal endoderm (de), ventralendoderm (ve), and marginal zone
(mz). Lane numbers are indicated on the drawing, with lane 8 containing
whole embryo RNA. Equal amounts of total RNA (2 pg) were
loaded per lane.
FIG. 5. Immunohistochemical localization of DG42 protein in gastrula.
Sections were cut along the dorsal-ventral axis as drawn at the
top and stained with anti-ppAC10 antibodies(only the dorsal region
of the embryo is shown). The antigen is visualized by silver (dark)
deposits. Note that the staining is peripheral to the cells. At this
stage, staining was found in the endoderm and the inner layer of the
ectoderm. In the animal or anterior region( A), the inner layer of the
ectoderm is labeled throughout.In the more posterior section( B), the
region of the ectoderm becoming neural plate is not stained.
FIG. 6. Immunohistochemical localization of DG42 protein in neurula and tailbud stages. Early (stage 15 (A)) and middle (stage 18 (B);
stage 20 (C)) neurula and beginning tailbud (stage 24 (D, E)) embryos were sectioned and stained as in Fig. 5. Staining was found in endoderm,
inner layer of the ectoderm, and, at later stages, ventralmesoderm (lateral plate, see (C) and (E)). Note that staining disappears gradually
from the dorsal side as development proceeds.
