A short loop on the ALK-2 and ALK-4 activin receptors regulates signaling specificity but cannot account for all their effects on early Xenopus development.

Activin, a member of the transforming growth factor beta (TGF-beta) superfamily, signals through a heteromeric complex of type I and type II serine-threonine kinase receptors. The two activin type I receptors previously identified, ALK-2 (ActR-I) and ALK-4 (ActR-IB), have distinct effects on gene expression, differentiation and morphogenesis in the Xenopus animal cap assay. ALK-4 reproduces the effects of activin treatment including the dose-dependent induction of progressively more dorso-anterior mesodermal and endodermal markers, whereas ALK-2 induces only ventral mesodermal markers and counteracts the effects of ALK-4. To identify regions of the receptors that determine signaling specificity we have generated chimeras of the constitutively active ALK-2 and ALK-4 receptors (termed ALK-2* and ALK-4*). The effects of these chimeric receptors on gene expression and morphogenetic movements implicate the loop between kinase subdomains IV and V in mediating the strong dorsal gene-inducing properties of ALK-4*; when the seven amino acids comprising this loop are transferred from ALK-4* to ALK-2*, the resulting chimeric receptor is capable of inducing the expression of dorsal-specific genes. In contrast, when the equivalent region of ALK-2* is transferred to the ALK-4* backbone it cannot effectively counteract the dorsalizing effects of ALK-4*, suggesting that other regions of type I receptors are also involved in determining signal specificity.

The ALK-2 and ALK-4 activin receptors transduce distinct mesoderm-inducing signals during early Xenopus development but do not co-operate to establish thresholds.

The TGFbeta family member activin induces different mesodermal cell types in a dose-dependent fashion in the Xenopus animal cap assay. High concentrations of activin induce dorsal and anterior cell types such as notochord and muscle, while low concentrations induce ventral and posterior tissues such as mesenchyme and mesothelium. In this paper we investigate whether this threshold phenomenon involves the differential effects of the two type I activin receptors ALK-2 and ALK-4. Injection of RNA encoding constitutively active forms of the receptors (here designated ALK-2* and ALK-4*) reveals that ALK-4* strongly induces the more posterior mesodermal marker Xbra and the dorsoanterior marker goosecoid in animal cap explants. Maximal levels of Xbra expression are attained using lower concentrations of RNA than are required for the strongest activation of goosecoid, and at the highest doses of ALK-4*, levels of Xbra transcription decrease, as is seen with high concentrations of activin. By contrast, the ALK-2* receptor activates Xbra but fails to induce goosecoid to significant levels. Analysis at later stages reveals that ALK-4* signalling induces the formation of a variety of mesodermal derivatives, including dorsal cell types, in a dose-dependent fashion, and that high levels also induce endoderm. By contrast, the ALK-2* receptor induces only ventral mesodermal markers. Consistent with these observations, ALK-4* is capable of inducing a secondary axis when injected into the ventral side of 32-cell stage embryos whilst ALK-2* cannot. Co-injection of RNAs encoding constitutively active forms of both receptors reveals that ventralising signals from ALK-2* antagonise the dorsal mesoderm-inducing signal derived from ALK-4*, suggesting that the two receptors use distinct and interfering signalling pathways. Together, these results show that although ALK-2* and ALK-4* transduce distinct signals, the threshold responses characteristic of activin cannot be due to interactions between these two pathways; rather, thresholds can be established by ALK-4* alone. Furthermore, the effects of ALK-2* signalling are at odds with it behaving as an activin receptor in the early Xenopus embryo.

The comparative genomic structure and sequence of the surfeit gene homologs in the puffer fish Fugu rubripes and their association with CpG-rich islands.

The puffer fish Fugu rubripes (Fugu) has a compact genome approximately one-seventh the size of man, mainly owing to small intron size and the presence of few dispersed repetitive DNA elements, which greatly facilitates the study of its genes at the genomic level. It has been shown previously that, whereas the Surfeit genes are tightly clustered at a single locus in mammals and birds, the genes are found at three separate loci in the Fugu genome. Here, Fugu gene homologs of all six Surfeit genes (Surf-1 to Surf-6) have been cloned and sequenced, and their gene structure has been compared with that of their mammalian and avian homologs. The predicted protein products of each gene are well conserved between vertebrate species, and in most cases their gene structures are identical to their mammalian and avian homologs except for the Fugu Surf-6 gene, which was found to lack an intron present in the mouse gene. In addition, we have identified conserved regulatory elements at the 5' and 3' ends of the Surf-3/rpL7a gene by comparison with the mammalian and chicken Surf-3/rpL7a gene homologs, including the presence of a polypyrimidine tract at the extreme 5' end of this ribosomal protein gene. The Fugu Surfeit gene homologs appear to be associated with CpG-rich islands, like the Surfeit genes in higher vertebrates, but these Fugu CpG islands are similar to the nonclassical islands characteristic of other fish species. Our observations support the use of the Fugu genome to study vertebrate gene structure, to predict the structure of mammalian genes, and to identify vertebrate regulatory elements. [The sequence data described in this paper have been submitted to the data library under accession nos. Y15170 (Surf-2, Surf-4), Y15171 (Surf-3, Surf-1, Surf-6), and Y15172 (Surf-5.)]

Signalling by TGF-beta family members: short-range effects of Xnr-2 and BMP-4 contrast with the long-range effects of activin.

BACKGROUND: One way of establishing a morphogen gradient in a developing embryo involves the localized synthesis of an inducing molecule followed by its diffusion into surrounding tissues. The morphogen-like effects of the mesoderm-inducing factor activin provide support for this idea in amphibian development. The questions remain, however, of how activin exerts its long-range effects, and whether long-range signalling is a property of all transforming growth factor beta (TGF-beta) family members. RESULTS: We compare the signalling ranges of activin and two other TGF-beta family members, Xnr-2 and BMP-4. Unlike activin, Xnr-2 and BMP-4 act over short distances. Furthermore, the effects of constitutively active activin receptors are strictly cell-autonomous. These observations suggest that the long-range effects of activin occur through protein diffusion and that "relay' mechanisms are not initiated by any of these TGF-beta family members. Mechanisms limiting the signalling range of Xnr-2 were addressed by studying Xnr-2 processing and secretion. An activin-Xnr-2 fusion protein signals over many cell diameters, suggesting that regulated processing or secretion is one limiting factor. Disaggregation and reaggregation of Xnr-2-producing tissues also extends the range of Xnr-2, suggesting that components of intact tissue restrict spread of the protein. CONCLUSIONS: The long-range effects of activin are likely to occur through the diffusion of activin protein. The short-range effects of Xnr-2 and BMP-4 emphasize that long-range diffusion is not a general property of TGF-beta-related molecules. Finally, signalling ranges may be regulated by constraints on processing or secretion and by interactions with extracellular components of embryonic tissues.

Surfeit locus gene homologs are widely distributed in invertebrate genomes.

The mouse Surfeit locus contains six sequence-unrelated genes (Surf-1 to -6) arranged in the tightest gene cluster so far described for mammals. The organization and juxtaposition of five of the Surfeit genes (Surf-1 to -5) are conserved between mammals and birds, and this may reflect a functional or regulatory requirement for the gene clustering. We have undertaken an evolutionary study to determine whether the Surfeit genes are conserved and clustered in invertebrate genomes. Drosophila melanogaster and Caenorhabditis elegans homologs of the mouse Surf-4 gene, which encodes an integral membrane protein associated with the endoplasmic reticulum, have been isolated. The amino acid sequences of the Drosophila and C. elegans homologs are highly conserved in comparison with the mouse Surf-4 protein. In particular, a dilysine motif implicated in endoplasmic reticulum localization of the mouse protein is conserved in the invertebrate homologs. We show that the Drosophila Surf-4 gene, which is transcribed from a TATA-less promoter, is not closely associated with other Drosophila Surfeit gene homologs but rather is located upstream from sequences encoding a homolog of a yeast seryl-tRNA synthetase protein. There are at least two closely linked Surf-3/rpL7a genes or highly polymorphic alleles of a single Surf-3/rpL7a gene in the C. elegans genome. The chromosomal locations of the C. elegans Surf-1, Surf-3/rpL7a, and Surf-4 genes have been determined. In D. melanogaster the Surf-3/rpL7a, Surf-4, and Surf-5 gene homologs and in C. elegans the Surf-1, Surf-3/rpL7a, Surf-4, and Surf-5 gene homologs are located on completely different chromosomes, suggesting that any requirement for the tight clustering of the genes in the Surfeit locus is restricted to vertebrate lineages.

Surf5: a gene in the tightly clustered mouse surfeit locus is highly conserved and transcribed divergently from the rpL7A (Surf3) gene.

The four previously characterized genes (Surf1 to 4) of the mouse Surfeit locus do not share any sequence homology, and the transcription of each gene alternates with respect to its neighbor(s). Adjacent Surfeit genes are separated by very small distances, and two of the genes overlap at their 3' ends. In this work we have further defined the Surfeit gene cluster by the isolation of Surf5, a fifth gene of the locus, and determination of its relationship to the other Surfeit genes. Surft5 does not share any sequence homology with the four cloned Surfeit genes. The transcription of Surf5 is divergent with respect to its neighbor the Surf3 gene, and the 5' ends of Surf5 and Surf3 are separated by only 159 bp, suggesting the presence of a second bidirectional promoter in the locus. The 3' end of Surf5 maps only 68 bp away from the processed 3' end of a pseudogene. The human and partial chicken Surf5 coding regions show greater than 95% identity, and a Caenorhabditis elegans homologue shows 38% identity and 56% similarity with the mouse Surf5 amino acid sequence. The 3.5-kb transcript of Surf5 encodes a small hydrophilic protein of 140 amino acid residues, which differs from the ribosomal protein L7a encoded by the Surf3 gene or the integral membrane protein encoded by the Surf4 gene. Subcellular fractionation located the Surf5 protein to the soluble fraction of the cytoplasm. The surfeit locus appears to represent a novel type of gene cluster in which the genes are unrelated by sequence or function; however, their organization may play a role in their gene expression.

The genomic organization of the region containing the Drosophila melanogaster rpL7a (Surf-3) gene differs from those of the mammalian and avian Surfeit loci.

The Surf-3 gene of the unusually tight mouse Surfeit locus gene cluster has been identified as the highly conserved ribosomal protein gene L7a (rpL7a). The topography and juxtaposition of the Surfeit locus genes are conserved for the 600 million years of divergent evolution between mammals and birds. This suggests cis interaction and/or coregulation of the genes and suggests that, within this locus, gene organization plays an important role in gene expression. The further evolutionary conservation of the organization of the Surfeit locus was investigated. A cDNA encoding the Drosophila melanogaster homolog of the Surf-3/rpL7a gene was cloned, was shown to be present as a single copy, and was expressed constitutively at high levels throughout development. Genomic cosmid clones encompassing the gene and its surrounding DNA were isolated. The gene was determined to have five introns, of which two were located in the 5' untranslated region of the gene. The remaining three introns had splice sites at positions equivalent to those found in the Surf-3/rpL7a mammalian homologs. S1 analysis and 5' rapid amplification of cDNA ends both confirmed the start of transcription to occur in a polypyrimidine tract in the absence of a TATA box in the promoter. The genomic region around the Surf-3/rpL7a gene was analyzed by low-stringency hybridization with murine Surfeit gene probes, by partial sequence analysis, and by hybridization of fragments to Northern (RNA) blots. No homologs of other members of the Surfeit gene cluster were detected in close proximity to the D. melanogaster Surf-3/rpL7a gene. However, a gene which was detected directly 3' to the Surf-3/rpL7a gene was shown to encode a homolog of a mammalian serine-pyruvate aminotransferase.