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A new bHLH gene from mouse that we call pMesogenin1 (referring to paraxial mesoderm-specific expression and regulatory capacities) and its candidate ortholog from Xenopus were isolated and studied comparatively. In both organisms the gene is specifically expressed in unsegmented paraxial mesoderm and its immediate progenitors. A striking feature of pMesogenin1 expression is that it terminates abruptly in presumptive somites (somitomeres). Somitomeres rostral to the pMesogenin1 domain strongly upregulate expression of pMesogenin's closest known paralogs, MesP1 and MesP2 (Thylacine1/2 in Xenopus). Subsequently, the most rostral somitomere becomes a new somite and expression of MesP1/2 is sharply downregulated before this transition. Thus, expression patterns of these bHLH genes, together with that of an additional bHLH gene in the mouse, Paraxis, collectively define discrete but highly dynamic prepatterned subdomains of the paraxial mesoderm. In functional assays, we show that pMesogenin1 from either mouse or frog can efficiently drive nonmesodermal cells to assume a phenotype with molecular and cellular characteristics of early paraxial mesoderm. Among genes induced by added pMesogenin1 is Xwnt-8, a signaling factor that induces a similar repertoire of marker genes and a similar cellular phenotype. Additional target genes induced by pMesogenin1 are ESR4/5, regulators known to play a significant role in segmentation of paraxial mesoderm (W. C. Jen et al., 1999, Genes Dev. 13, 1486-1499). pMesogenin1 differs from other known mesoderm-inducing transcription factors because it does not also activate a dorsal (future axial) mesoderm phenotype, suggesting that pMesogenin1 is involved in specifying paraxial mesoderm. In the context of the intact frog embryo, ectopic pMesogenin1 also actively suppressed axial mesoderm markers and disrupted normal formation of notochord. In addition, we found evidence for cross-regulatory interactions between pMesogenin1 and T-box transcription factors, a family of genes normally expressed in a broader pattern and known to induce multiple types of mesoderm. Based on our results and results from prior studies of related bHLH genes, we propose that pMesogenin1 and its closest known relatives, MesP1/2 (in mouse) and Thylacine1/2 (in Xenopus), comprise a bHLH subfamily devoted to formation and segmentation of paraxial mesoderm.
FIG. 3. pMesogenin1 is zygotically expressed in presumptive mesoderm of gastrulae and tailbud region in Xenopus embryos. (A)RT-PCR analysis of Mesogenin1 expression shows that pMesogenin1 is zygotically expressed and developmentally regulated during
Xenopus embryogenesis. T-box transcription factor VegT, on the other hand, is expressed both maternally and zygotically (Zhang and King, 1996). X-max2 gene expression (King et al., 1993) was also monitored as a control for RT-PCR. (B) Whole-mount in situ analysis of pMesogenin1 expression during Xenopus embryogenesis. During gastrulation, pMesogenin1 expression was detected in ventrolateral mesoderm. After gastrulation, pMesogenin1 is expressed in the tailbud (presomitic mesoderm) region until early tadpole stage. Abbreviations: D, dorsal; V, ventral; A, anterior; and P, posterior.
FIG. 6. Comparative analysis of expression of pMesogenin1 and T-box transcription factor genes in mouse and Xenopus embryos. (A) pMesogenin1 (A and B), Brachyury T (C and D), and Tbx6 (E and F)expression was examined by whole-mount in situ hybridization of mouse embryos at 7.5 (A, C, and E) and 9.0 dpc (B, D, and F). (G) pMesogenin1 (G), Xbra (H), and VegT (I) expression was monitored in Xenopus
embryos at gastrulation stages.
FIG. 1. pMesogenin1 encodes a novel bHLH transcription factor. (A) Deduced amino acid sequences of pMesogenin1 from both mouse and
Xenopus. The sequences were aligned using the BESTFIT program in a GCG package. Identical amino acids are indicated as red letters. The
bHLH domain is blocked. (B) Comparison of bHLH domain of pMesogenin1 to other closely related bHLH proteins in vertebrates. The
peptide sequence of the bHLH domain of mouse pMesogenin1 was used to perform a BLAST search. The six top matched sequences were
selected and aligned to each other using the CLUSTALW program. Sequences of Neurogenin1, M-Twist, and MyoD were also included in
the alignment. Identical amino acids are indicated as a hyphen and gaps are indicated as a period. (C) Schematic diagram showing
similarities among pMesogenin1 and its related bHLH genes. The bHLH domain is colored black. Numbers on the boxed areas indicate the
percentage of same amino acids in the domain compared to mouse pMesogenin1. (D) Phylogenetic dendrogram of pMesogenin1 and its
related bHLH proteins. The peptide sequences of the bHLH domains were analyzed by the Kimura method for distance calculation and the
resulting distance matrix data were processed by the UGPMA method in the NEIGHBOR program of the PHYLIP sequence analysis
software package to obtain a tree dendrogram.
FIG. 4. pMesogenin1 induces presomitic paraxial mesoderm markers in animal cap explants. (A and B) RT-PCR analysis of mesoderm marker
gene expression in animal cap explants injected with either mouse or Xenopus pMesogenin1 RNA. Water (Mock) or Xenopus (0.5 and 2 ng per
embryo) or mouse pMesogenin1 RNA (2 ng per embryo) was injected into the animal pole of both blastomeres of two-cell stage embryos, which
were explanted at stage 8 and cultured until stage 10.5 (A) or stage 12 (B). Expression of a number of marker genes was analyzed by RT-PCR. At
least three independent experiments were done and produced the same results. Data from one experiment are shown here. (C–E) Overexpressed
pMesogenin1 induced ventrolateral phenotypes in animal cap explants cultured until the time equivalent of stage 35 to 36. The culture explants
were paraffin embedded, sectioned, and stained with hematoxylin and eosin. Explants injected with pMesogenin1 (2 ng per embryo) develop a
cavity filled with mesenchymal cells, which is a typical phenotype for lateral ventral mesoderm induction.
FIG. 5. Ectopically expressed pMesogenin1 suppresses dorsal mesoderm formation. pMesogenin1 RNA (4 ng per embryo) was injected into
either the dorsal (DMZ) or the ventral (VMZ) marginal zone of four-cell stage blastomeres, and expression of the dorsal marker Chordin (A,
B, and C) and ventrolateral mesodermal marker Xwnt-8 (D, E, and F) was analyzed by whole-mount in situ hybridization. (G) Morphological
defects in the tadpoles injected in the DMZ of four-cell stage embryos with pMesogenin1. Tadpoles showing more severe defects are
arranged from top to bottom. pMesogenin1 caused a failure of axis extension, probably due to the defect in dorsal structure formation.
FIG. 7. Interactions between pMesogenin1 and T-box genes. (A)
RT-PCR analysis of T-box gene expression in animal cap explants
injected with Xenopus pMesogenin1 RNA (0.5 and 2 ng per
embryo). The animal cap explants were harvested at stage 11
(midgastrulation). (A) pMesogenin1 induced Xbra and VegT expression
readily and Eomesodermin expression weakly. pMesogenin1,
however, failed to induce accumulation of endogenous pMesogenin1
RNA. (B) VegT failed to induce pMesogenin1. RT-PCR analysis
of pMesogenin1 expression in the stage 11 animal cap explants
injected with VegT RNA (1 and 2 ng per embryo) showed no
induction of pMesogenin1 expression, but efficient induction of
VegT itself and Xwnt-8. The data shown are from one of three
independent experiments which produced essentially identical
results.
