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???displayArticle.abstract??? Endoderm development is an area of intense interest in developmental biology, but progress has been hampered by the lack of specific markers for differentiated endodermal cells. In an unbiased secretion cloning screen of Xenopus gastrula embryos we isolated a novel gene, designated Darmin. Darmin encodes a secreted protein of 56 kDa containing a peptidase M20 domain characteristic of the glutamate carboxypeptidase group of zinc metalloproteases. We also identified homologous Darmin genes in other eukaryotes and in prokaryotes suggesting that Darmin is the founding member of a family of evolutionarily conserved proteins. Xenopus Darmin showed zygotic expression in the early endoderm and later became restricted to the midgut. By secretion cloning of Xenopus cleavage-stage embryos we isolated another novel protein, designated Darmin-related (Darmin-r) due to its sequence similarity with Darmin. Darmin-r was maternally expressed and showed at later stages expression in the lens and pronephric glomus. The endoderm-specific expression of Darmin makes this gene a useful marker for the study of endoderm development.
Fig. 2. Darmin is an endoderm-specific marker. Whole-mount in situ hybridization was performed on intact (A, F, G, J, K) or hemisectioned Xenopus embryos (B–E, H, I). (A) Animal view of 4-cell stage embryo; note the lack of maternal transcripts. (B) Internal view of a hemisectioned early gastrulaembryo (stage 10); Darmin expression starts in the dorsal vegetal cell mass; the arrowhead indicates the dorsal blastopore lip in this and subsequent panels. (C) Close-up of embryo in (B); note nuclear staining in the yolky endoderm adjacent to invaginating mesoderm of Spemann's organizer. (D) At mid-gastrula (stage 11), Darmin-labeled cells are found throughout the endoderm. (E) Internal view of late gastrulaembryo (stage 13) showing increased transcript levels in the endoderm excluding the anterior region; the arrow marks the liver diverticulum, a derivative of the foregut. (F, G) Lateral and dorsal views of late neurula (stage 20) displaying Darmin expression in the midgut. (H) Internal view showing signals in the ventral and lateral walls of the midgut. (I) At early tail bud stage (stage 25), staining in the midgut becomes very abundant. (J, K) Lateral views of advanced tail bud embryos (stages 32 and 40); Darmin expression remains strong in the midgut.
Fig. 3. Expression of Endodermin, Darmin and Darmin-related. Embryos are shown in lateral (A, B, D) and animal views (C). (A) Tail bud embryo (stage 28); note expression of Endodermin (Edd) in the fore-, mid-, and hindgut. (B) Sibling embryo showing Darmin expression restricted to the midgut. (C) Abundant maternal transcripts of Darmin-related (Darmin-r) in 4-cell stage embryo. (D) Advanced tail bud embryo (stage 32) displaying weak expression of Darmin-r in the lens (ls) and pronephric glomus (gl). Transversal section in inset shows transcripts in the glomera.
darmin (darmin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, midsagittal section, dorsal right.
darmin (darmin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anteriorleft.
a2m (alpha-2-macroglobulin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
cndp2 (CNDP dipeptidase 2 (metallopeptidase M20 family)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3 (4-cell), dorsal view.
cndp2 (CNDP dipeptidase 2 (metallopeptidase M20 family)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anteriorleft, dorsal up.
