Jones CM et al. (1995), Nodal-related signals induce axial mesoderm and...

XB-ART-19067

Development 1995 Nov 01;12111:3651-62.

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Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation.

Jones CM , Kuehn MR , Hogan BL , Smith JC , Wright CV .

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Mouse embryos homozygous for a null mutation in nodal arrest development at early gastrulation and contain little or no embryonic mesoderm. Here, two Xenopus nodal-related genes (Xnr-1 and Xnr-2) are identified and shown to be expressed transiently during embryogenesis, first within the vegetal region of late blastulae and later in the marginal zone during gastrulation, with enrichment in the dorsal lip. Xnrs and mouse nodal function as dose-dependent dorsoanterior and ventral mesoderm inducers in whole embryos and explanted animal caps. Using a plasmid vector to produce Xnr proteins during gastrulation, we show that, in contrast to activin and other TGF beta-like molecules, Xnr-1 and Xnr-2 can dorsalize ventral marginal zone explants and induce muscle differentiation. Xnr signalling also rescues a complete embryonic axis in UV-ventralized embryos. The patterns of Xnr expression, the activities of the proteins and the phenotype of mouse nodal mutants, all argue strongly that a signaling pathway involving nodal, or nodal-related peptides, is an essential conserved element in mesoderm differentiation associated with vertebrate gastrulation and axial patterning.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a gsc ifn2 (provisional) mt-tr nodal nodal1 nodal2 nog odc1 tbxt trna

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	Fig. 1. Expression of dorsoanterior mesoderm markers in nodalinjected embryos. (A) goosecoid transcript levels are elevated during early gastrula stages and remain higher than controls at least into sibling neurula stages. ODC signal assesses RNA loading in the samples. (B) Muscle-specific (ms) actin expression is upregulated in nodal-injected embryos. Higher levels of ms-actin mRNA are detected precociously in embryos expressing nodal. Cytoskeletal actin serves as a control for RNA integrity in the lanes. C, control; N, mouse nodal RNA-injected.
	Fig. 2. Characterization of animal caps induced by injection of mouse nodal RNA. Morphological (A,B), and histological (C,D) analysis of control and nodal-loaded animal caps. (A,C) Control caps form atypical epidermis, but caps expressing mouse nodal (B,D) elongate and differentiate dorsal mesodermal tissues, often consisting almost entirely of notochord and small patches of muscle. Abbreviations: epi, atypical epidermis; ms, muscle; no, notochord. (E) Analysis of gene expression induced in animal explants by different nodal RNA doses. High doses (1 ng RNA/embryo) induce goosecoid (a dorsoanterior mesoderm marker), muscle-specific actin and Xbra. Decreased doses (100, 10, or 1 pg/embryo) do not induce goosecoid, but intermediate concentrations induce muscle-specific actin and Xbra. All samples are from the same injection experiment. goosecoid and Xbra were assayed at sibling stage 10. 5, and muscle actin at stage 20. ODC and cytoskeletal actin serve as controls for RNA integrity and loading in samples.
	Fig. 3. Alignment of amino acid sequences of mouse nodal with Xnr-1, Xnr-2 and a chicken nodal-related peptide. (A) Deduced amino acid sequences of Xnr-1 and Xnr-2. A hydrophobic region (underlined) at the N terminus of each Xnr resembles a secretory signal sequence, with cleavage predicted according to the algorithm of von Heijne (1986). Four potential N-linked glycosylation sites (consensus N-X-T/S) are present in each protein (centered on residues 72, 137, 174, and 345 for Xnr-1, and residues 72, 160, 174, and 344 for Xnr-2). Three are positionally conserved between Xnr-1/Xnr-2. Putative basic proteolytic processing sites (RRxRR, underlined) begin at residues 277 (Xnr-1) and 278 (Xnr-2). Asterisks indicate identities, double dots represent conservative changes. (B) Alignment of C-terminal mature regions of Xnr-1, Xnr-2, mouse nodal and a newly isolated chick nodal-related sequence. Alignments begin at the putative basic processing site of each molecule. The region of cysteine spacing unique to the Xnr factors (C-X-X-C) is underlined. Vertical lines represent identities in all four proteins. A consensus sequence is presented below the alignment. Dashes represent spaces introduced to optimize alignments.
	Fig. 4. Temporal and spatial expression of Xnr-1 and Xnr-2 during Xenopus development. (A) RNAse protection analysis of Xnr-1 and Xnr-2 expression during Xenopus development. Transcripts are detected during late blastula (stage 9) and gastrula (stage 10 and 10. 5) stages. A very low level of Xnr-1 RNA is detected during neurula stages (stage 17), but no expression is detected for Xnr-2 after gastrulation (stage 13). Stage 8 represents an RNA pool before zygotic transcription begins, and transcripts for Xnr-1 and Xnr-2 are not detected. ODC is a loading control, and the tRNA lane demonstrates specificity of signal to embryo RNA. (B-H) Whole-mount in situ hybridization analysis of Xnr-1 and Xnr-2 expression. All embryos are cleared albino embryos, viewed from the vegetal surface with dorsal oriented upward. The dorsal lip is indicated by the black arrowhead. (B) Stage 9 embryos show punctate perinuclear Xnr-1 signal over the entire vegetal region. Xnr-2 shows the same pattern (data not shown). (C) Xnr-1 signal at stage 10.25 is restricted to the dorsal marginal zone (dark arc at bottom left is a background artefact). (D) Xnr-2 signal in stage 10 pregastrula is primarily located in the dorsal marginal zone, but also in adjacent dorsovegetal cells. (E) Xnr-2 signal in the stage 10.5 gastrula is highly concentrated just above the dorsal lip, with a gradual decrease laterally and ventrally. (F) Whole-mount stained stage 10.25 embryo, split open along the dorsal/ventral plane and viewed internally to show Xnr-2 expression at the dorsal lip. Superficial and slightly deeper staining is observed. Some out-of-focus vegetal cells below the lip express Xnr-2 (white arrowhead). (G) noggin mRNA hybridization in a stage 10.5 embryo shows deeper mesodermal expression extending anteriorly along the dorsal midline. (H) Xnr-2 sense strand control, stage 10.25 embryo. (I) RNAse protection analysis of Xnr-1 and Xnr-2 distribution in dissected gastrulae. Lanes 1-3: at stage 10.25, Xnr-1 and Xnr-2 transcripts are detected in the marginal zone, at greatly reduced levels in vegetal tissue, but are undetectable in animal tissue. Lanes 4 and 5: in stage 10 embryos, Xnr-1 RNA and, to a lesser extent, Xnr-2 RNA, is enriched in dorsal halves of embryos compared to ventral halves. EF1-a assesses RNA integrity and loading.
	Fig. 5. RNAse protection analysis of Xnr-1 and Xnr-2 expression in animal caps treated with growth factors and LiCl- and UV-treated embryos. (A) Xnr-1 and Xnr-2 expression is induced in animal caps by activin, but not FGF, indicating activation by dorsal mesoderminducing signals. Control protections show Xnr-1 and Xnr-2 RNA in stage 10.5 sibling embryos. EF1-a assesses RNA loading in the samples. (B) Expression of Xnr-1 and Xnr-2 at stage 10 in UVventralized or LiCl-dorsalized embryos. UV-ventralization greatly reduces Xnr-1 and Xnr-2 transcript levels. In contrast, LiCl dorsalization results in a 2-4 fold increase (evaluated densitometrically) in Xnr-1 and Xnr-2 RNA compared to untreated siblings. EF1-a is used as a loading control. DAI, dorso-anterior index score of sibling embryos (Kao and Elinson, 1988).
	Fig. 6. Effects of Xnr-1 and Xnr-2 on animal cap explants. Morphological (A-C) and corresponding histological analysis (D-F) of caps explanted from Xnr-1 or Xnr-2 RNA-injected embryos. (A,D) Control explants remain rounded, differentiating into atypical epidermis. (B,E) Most Xnr-1-injected caps extend slightly compared to controls, and primarily differentiate blocks of striated muscle (ms). Notochord differentiation is observed in Xnr-1 injected caps, but at lower frequency than Xnr-2. (C,F) Xnr-2 expressing explants elongate extensively, and consistently form dorsal mesodermal tissue including notochord (no) and striated muscle. (G) Induction of dorsal mesodermal markers in animal caps by Xnr-2 mRNA. Explants from embryos injected with high Xnr-2 RNA doses (100 pg) differentiate dorsoanterior mesoderm as marked by goosecoid and muscle actin expression. Lower concentrations do not induce goosecoid, but still induce actin and the pan-mesodermal marker, Xbra. As little as 1 pg of Xnr-2 RNA injected into 1-cell embryos induces actin and Xbra expression in animal caps (barely visible in this exposure). goosecoid and Xbra were assayed at sibling stage 10.5, and actin at sibling stage 20. Sibling embryo RNAs provide positive controls, and EF1-a and cytoskeletal actin assess RNA integrity and loading. (H) While high Xnr-2 RNA doses induce dorsal mesoderm markers (compare to G), intermediate doses of Xnr-2 RNA induce the ventrolateral mesodermal markers Xhox-3 and globin. The highest levels are seen at 10 pg/embryo. Low levels of Xhox-3 are indicated by the arrowheads.
	Fig. 7. Muscle-specific actin expression in ventral marginal zone explants dorsalized by Xnr-1 and Xnr-2 expressed zygotically. (A) Ventral marginal zones (VMZs) isolated from stage 10 gastrulae previously injected at the 4-cell stage with pCSKA:Xnr-1 or pCSKA:Xnr-2 express muscle-specific actin (ms-actin), indicating dorsalization compared to control VMZ explants, which do not express ms-actin. In four separate experiments (two separate examples are shown), pCSKA:activin never induced detectable levels of ms-actin. Dorsal marginal zone (DMZ) explants express high levels of msactin. (B-E) Histological analysis of explanted marginal zones. (B) Control VMZs differentiate ventral-type tissues, including mesothelium (mt) and loose mesenchyme. ym, yolk mass. (C) Control DMZs form notochord (no), striated muscle (ms), and neural tissue (nt). (D,E) VMZ explants preinjected with pCSKA:Xnr-1 or pCSKA:Xnr-2, respectively, are dorsalized and differentiate large blocks of striated muscle.
	Fig. 8. Complete axial rescue of UV-ventralized embryos by localized Xnr-1 RNA injection. (A) Normal tadpole, (B) UV-treated embryo (DAI=0; Kao and Elinson, 1988) at the same age of development, (C) Embryo resulting from injection of Xnr-1 RNA into one cell of a 4-cell stage UV-irradiated embryo. Except for a small injection artefact in the belly, Xnr-1 rescued embryos are indistinguishable from sibling normal embryos. In a representative experiment, 13 of 21 injected embryos (61%) were rescued to a DAI of 3-5, and 8 of these 13 had a DAI of 4 or 5, representing complete rescue. A DAI score of 3 was recorded if definitive melanized eye tissue was seen, while a DAI of 5 represents a normal tadpole.