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We report the characterization of two vertebrate homologs of Drosophila mothers against dpp (Mad) isolated from the mouse and the Xenopus embryo, named MusMLP (mad-like protein) and XenMLP, respectively, together with a summary of their expression patterns in the embryo. Overexpression of XenMLP causes ventralization of Xenopus embryos and we demonstrate that the C-terminal domain is necessary and sufficient to confer this biological effect. This domain also has the potential for transcriptional activation, as shown in one-hybrid assays in mammalian cells. We further demonstrate that MLPs are multidomain proteins by showing a cis-negative effect of the N-terminal domain on the transactivation by the C-terminal domain and that the proline-rich, middle domain maximizes the activity of the C-terminal domain. We also mapped the MusMLP gene to a region on mouse chromosome 13 that corresponds to a region on human chromosome 5q that contains cancer-related genes.
Fig. 2. A. Northern blot analysis of poly-A RNA isolated from multiple tissues from the adult mouse (left panel) and mouse embryo (middle panel) and
Xenopus stage 11 embryo (right panel). Blots were hybridized with the respective species-specific MUprobe. Left panel (each lane contains 2 pg RNA)
shows testis (lane I), kidney (2), skeletal muscle (3). liver (4), lung (51, spleen (6). brain (7) and heart (8); middle panel. 17 (lane l), 15 (2). 11 (3) and 7
days (4) old mouse embryo; right panel, Xenopus stage 11 embryo. (B) In situ hybridization analysis of MLP expression in sections of the mouse embryo
(left panel) and whole-mount Xenopus embryos (right panel). The left panel shows sagittal sections of a 9.5 days p.c. embryo (upper part of this panel)
and two sections of a 12.5 days p.c. embryo (middle and lower part of this panel, which are transversal and sagittal sections, respectively). Note the
uniform expression pattern in the E9.5 and the strongly elevated MusMLP expression in gonads and in mesenchymal cells lining the lungepithelium of
the El25 embryo. For a descriptive summary, see text: ba, branchial arches: du, Mulletian or Wolffian duct; go, gonads: lu. lung; so, somites; sp, spleen;
vb, vertebrae. Right panel: (A) stage 10 (early gastrula) Xenopus embryo shown from the vegetal side, dorsal side up; (B) is a stage 30 (tadpole) embryo
in lateral view: ba, branchial arches: br. brain: sn, spinal nerves.
Fig. 5. The C-terminal domain of XenMLP is required and sufficient for biological activity. A. Phenotype of embryos microinjected at the 4-cell stage
into all four blastomeres with 1.2 ng RNA per blastomere with RNA encoding full-length XenMLP, or deletion constructs lacking aa 1-145 (the Nterminal
domain) or lacking 424466 (part of C-terminal domain), as indicated on the right. Control, uninjected embryo. The frequency of ventralized
(microcephalic, Bauchsttick-type) embryos was 74% (flXenMLP, n = 43), 67% (Dl-145, II = 33), 13% (D424-466, n = 38) and 66% (aa263-466.
n = 29; not shown). (B). Modulation of endogenous levels of Xvenl-1 RNA, a ventral mesodermal marker in isolated dorsal marginal zones (DMZ),
confirms ventralizing activity of XenMLP and the essential function of the C-terminal domain of XenMLP. Embryos were microinjected with 1.2 ng
RNA per blastomere with RNA encoding full-length XenMLP or deletion constructs lacking the N-terminal domain (Dl-145) or lacking the C-terminal
domain (D263-466). DM zones were explanted at the early gastrula stage and incubated until sibling embryos reached stage 19. Total RNA was isolated
and analyzed by RT-PCR assays for expression of Xvent-1 (Gawantka et al., 1995) and histone H4 mRNA as indicated. DMZ and VMZ (ventral marginal
zone) on the left were uninjected controls; -RT, PCR of uninjected control DMZ sample without prior reverse transcription.
