Biben C et al. (1998), Murine cerberus homologue mCer-1: a candidate a...

XB-ART-15315

Dev Biol 1998 Feb 15;1942:135-51. doi: 10.1006/dbio.1997.8812.

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Murine cerberus homologue mCer-1: a candidate anterior patterning molecule.

Biben C , Stanley E , Fabri L , Kotecha S , Rhinn M , Drinkwater C , Lah M , Wang CC , Nash A , Hilton D , Ang SL , Mohun T , Harvey RP .

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Xenopus cerberus (Xcer) is a cytokine expressed in anterior mesendoderm overlapping and surrounding Spemann's gastrula organiser. When misexpressed in blastomeres, Xcer can induce ectopic heads with well-defined brain, cement gland, olfactory placodes, cyclopic eye, and occasionally liver and heart. We report here the identification of mCer-1, a murine gene related to cerberus. Both mCer-1 and Xcer appear to belong to the cystine knot superfamily, which includes TGF beta s and BMPs. In Xenopus animal cap assays, mCer-1 and Xcer induced cement glands and markers of anterior neural tissue and endoderm, characteristic of BMP inhibition. Furthermore, both antagonised the ventrolateral mesoderm-inducing activity of coexpressed BMP4. In mouse embryos, mCer-1 was expressed at early gastrulation in a stripe of primitive endoderm along the future anterior side of the egg cylinder, a region essential for anterior patterning. A second phase of expression was detected in anterior embryonic mesendoderm, and by late-streak stages most of the anterior half of the embryo was positive, except for the node and cardiac progenitors. Expression was later seen in the cranial portion of the two most-recently formed somites and in two stripes within presomitic mesoderm. In embryos lacking Otx2, a homeogene with a demonstrated role in anterior patterning, mCer-1 was still expressed in an anterior zone, although often abnormally. The data suggest that mCer-1 shares structural, functional, and expression characteristics with Xcer and may participate in patterning the anterior of the embryo and nascent somite region, in part, through a BMP-inhibitory mechanism.

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Species referenced: Xenopus
Genes referenced: a2m bmp4 cer1 eef1a2 egr2 hba4 ncam1 ndp nkx2-3 nkx2-5 otx2 tbx2 tgfb2

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FIG. 1. (A) Alignment of the predicted amino acid sequences of mCer-1 and Xenopus Xcer. Amino acid identities are shaded and similarities (see Materials and Methods) shown within open boxes. The putative cystine knot domains of mCer-1 and Xcer are outlined. The potential hydrophobic signal sequence of mCer-1 is overlined. The arrowhead indicates the N-terminus of secreted CFLAG– mCer-1 produced from CHO and 293T cells, as determined by N-terminal amino acid sequence analysis. (B) Alignment of the putative cystine knot regions of mCer-1, Xcer, mouse Norrie disease protein (NDP), and rat mucin with the equivalent domain of human TGFb2, known to form a cystine knot from crystallographic analysis. Cysteines are numbered according to Meitinger et al. (1993), with 1–6 (red) forming the conserved core of the cystine knot (see C), whilst a–d (yellow) are proposed to form additional stabilising disulphide bridges in some members. Note that mCer-1 and Xcer have additional cysteines a–c, but not d. Another cysteine (blue), used for dimerisation in TGFbs, is absent in mCer-1 and Xcer. The star indicates the glycine conserved in all cytokine members of the cystine knot superfamily. (C) Schematic representation of a cystine knot protein. The disulphide bonds that configure the cystine knot are indicated by red vertical lines, while the proposed additional disulphide bridges in NDP (Meitinger et al., 1993) are represented by blue lines. The Genbank Accession No. for mCer-1 is AF035579.

FIG. 3. Comparison of mCer-1 and Xcer activities in Xenopus animal cap assays. (A) Formation of cement glands in individualised animal caps (stage 35) after mock injection (a) or injection of mRNAs encoding mCer-1 (b), CFLAG–mCer-1 (c), or Xcer (d). A single darkly pigmented cement gland is induced by injected cerberus mRNAs in some cases (see B), but not by mock injection. Note in b the secretion of sticky exudate. (B) Histogram depicting frequency of cement gland induction in animal caps after injection of mRNAs encoding Xcer, mCer-1, or CFLAG–mCer-1, compared to uninjected controls. Data were compiled for each mRNA after examination of 100–120 injected caps. (C) RT-PCR analysis of markers induced in animal caps at stage 28 after injection of mRNAs encoding Xcer (XCer; lane 1), mCer-1 (MCer; lane 2), and BMP4 (lane 5) or after coinjection of 1:1 Xcer:BMP4 (lane 3) and 1:1 mCer-1:BMP4 (lane 4), compared to uninjected control caps (lane 7) and whole stage 25 embryos (lane 6). Nkx2.3 and Nkx2.5 are expressed in cardiac progenitors and anterior pharyngeal endoderm (Tonissen et al., 1994; Evans et al., 1995). T4 globin is specific to blood, a ventral mesodermal derivative (Banville and Williams, 1985). XeHAND marks cardiac and vascular smooth muscle progenitors in lateral mesoderm (D. B. Sparrow, S. Kotecha, N. Towers, and T. J. Mohun, submitted for publication). N-CAM is a panneural marker (Kintner and Melton, 1987). Otx2 is a marker of anterior tissues, expressed in midbrain, forebrain, placodes, cement gland, and anterior mesoderm (Pannese et al., 1995; Gammill and Sive, 1997). Krox20 is expressed in rhombomeres 3 and 5 in the hindbrain (Bradley et al., 1993). CG13 is specific to cement gland (Jamrich and Sato, 1989; Sive et al., 1989). Edd expression is ubiquitous at low levels (data not shown), but enriched in endoderm (Sasai et al., 1996). Equal mRNA input was assessed by expression of EF-1 a (EF1a) (Krieg et al., 1989).