A second tolloid in Drosophila

tolloid-related-1 (tlr-1), also known as tolkin, maps immediately proximal totolloid. Sequence analysis indicates that tlr-1 has a large N-terminal extension relative to tld, but otherwise shows the same general organization of sequence motifs found in tld and other BMP-1 family members. tlr-1expression shows unique transcriptional patterns in late stage embryos that are not seen with tld. In larval stages, both genes are expressed in identical patterns in imaginal discs and in the optic lobes of the brain, but tlr-1 is more abundant than tld. Deletions that eliminate tlr-1 expression cause lethality during larval and pupal stages of development. It would seem that tld and tlr-1 arose by gene duplication; each has evolved independently to acquire distinct tissue specific roles in Drosophila development (Nguyen, 1994).

Expression patterns of tlr-1 overlap those of tolloid and dpp in early embryos and diverge in later stages. In larval tissues, both tolloid and tlr-1 are expressed uniformly in the imaginal disks. In the brain, both tolloid and tlr-1 are expressed in the outer proliferation center, whereas tlr1 has another stripe of expression near the outer proliferation center. Analysis of lethal mutations in tlr-1 indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp (Finelli, 1994).

Evolution of the dorsal-ventral patterning network in the mosquito: Altered expression of sog and tld correlates with a broader domain of Dpp signaling, when compared with Drosophila.

The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. This study addressed how changes in this network produce evolutionary diversity in insect gastrulation. Focus was placed on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene (Goltsev, 2007).

A variety of dorsal patterning genes were examined in A. gambiae embryos in an effort to determine the basis for the formation of distinct ectodermal derivatives. For example hindsight (hnt; also known as peb - Flybase) is expressed along the dorsal midline of D. melanogaster embryos, while tailup (tup) is expressed in a broader pattern that encompasses both the presumptive amnioserosa and dorsolateral ectoderm. The hnt expression pattern seen in A. gambiae is similar to that detected in D. melanogaster, although there is a marked expansion in the dorsal-ventral limits of the presumptive extra-embryonic territory. By contrast, the tup pattern in A. gambiae is dramatically different from that seen in D. melanogaster -- it is excluded from the prospective serosa and restricted to the future amnion (Goltsev, 2007).

The T-box genes Dorsocross1 (Doc1) and Doc2 are involved in amnioserosa development and expressed along the dorsal midline and in a transverse stripe near the cephalic furrow of gastrulating D. melanogaster embryos. The Doc1 and Doc2 orthologues in A. gambiae exhibit restricted expression in the presumptive amnion, similar to the tup pattern. The expression patterns of the two genes are identical but only Doc1 is shown. They are initially expressed in a broad dorsal domain but come to be repressed in the serosa. There is also a head stripe of expression comparable to the D. melanogaster pattern. Additional dorsal-ventral patterning genes are also expressed in a restricted pattern within the developing amnion. Overall, the early expression patterns of tup, Doc1 and Doc2 (and additional patterning genes) foreshadow the subdivision of the dorsal ectoderm into separate serosa and amnion lineages in Anopheles (Goltsev, 2007).

In D. melanogaster, the patterning of the dorsal ectoderm depends on Dpp and Zen, along with a variety of genes encoding Dpp signaling components, such as the Thickveins (Tkv) receptor. Most of the corresponding genes are expressed in divergent patterns in A. gambiae embryos. For example, dpp and tkv are initially expressed throughout the dorsal ectoderm, but become excluded from the presumptive serosa and restricted to the amnion. By contrast, both genes have broad, nearly uniform expression patterns in the dorsal ectoderm of D. melanogaster embryos (Goltsev, 2007).

There is an equally dramatic change in the zen expression pattern. In A. gambiae, expression is restricted to the presumptive serosa territory, even at the earliest stages of development. By contrast, zen is initially expressed throughout the dorsal ectoderm of cellularizing embryos in D. melanogaster, and becomes restricted to the dorsal midline by the onset of gastrulation. Thus, the dpp/tkv and zen expression patterns are essentially complementary in A. gambiae embryos, but extensively overlap in Drosophila (Goltsev, 2007).

The loss of dpp, tkv, Doc1, Doc2 and tup expression in the presumptive serosa of A. gambiae embryos raises the possibility that zen activates the expression of one or more repressors in the serosa. It is unlikely that Zen itself is such a repressor since the expression of the A. gambiae zen gene in transgenic Drosophila embryos does not alter the normal development of the amnioserosa (Goltsev, 2007).

Different segmentation genes were examined in an effort to identify putative serosa-specific repressors. For example, the gap gene hunchback (hb) is initially expressed in the anterior regions of A. gambiae embryos, in a similar pattern to that seen in D. melanogaster, but by the onset of gastrulation a novel pattern arises within the presumptive serosa. hb expression has also been seen in the developing serosa of other insects, including a primitive fly (Clogmia) and the flour beetle, Tribolium (Goltsev, 2007).

Two additional segmentation genes behave like hb, empty spiracles (ems) and tramtrack (ttk). ems is involved in head patterning in D. melanogaster. Its expression is limited to a single stripe in anterior regions of cellularizing D. melanogaster embryos. Staining is seen in a comparable anterior region of A. gambiae embryos, but a second site of expression (not seen in Drosophila) is also detected in the presumptive serosa (Goltsev, 2007).

Ttk is a maternal repressor that helps establish the expression limits of several pair-rule stripes. It is ubiquitously expressed throughout the early D. melanogaster embryo, but has a tightly localized expression pattern within the presumptive serosa of A. gambiae embryos. Thus, novel patterns of ems and ttk expression are consistent with the possibility that serosa-specific repressors help subdivide the dorsal ectoderm into separate serosa and amnion lineages in A. gambiae embryos (Goltsev, 2007).

The analysis of dorsal-ventral patterning genes identified two critical differences between the pre-gastrular fly and mosquito embryos. First, there are separate serosa and amnion lineages in A. gambiae, but just a single amnioserosa in D. melanogaster. Second, there is an expansion in the limits of the dorsal ectoderm in A. gambiae as compared with the D. melanogaster embryo. Localized repressors might help explain the former observation of separate lineages, but do not provide a basis for the expansion of the dorsal ectoderm (Goltsev, 2007).

In D. melanogaster, the limits of Dpp signaling are established by the repressor Brinker and the inhibitor Sog. Genetic studies suggest that Sog is the more critical determinant in early embryos. It is related to Chordin, which inhibits BMP signaling in vertebrates, and is expressed in broad lateral stripes encompassing the entire neurogenic ectoderm. The secreted Sog protein directly binds Dpp, and blocks its ability to interact with the Tkv receptor. However, Sog-Dpp complexes are proteolytically processed by the Tolloid (Tld) metalloprotease, which is expressed throughout the dorsal ectoderm of early Drosophila embryos. Tld helps ensure that high levels of the Dpp signal are released at the dorsal midline located far from the restricted source of the inhibitor Sog (Goltsev, 2007).

The expression patterns of the sog and tld genes in A. gambiae are very different from those seen in D. melanogaster. sog expression is primarily detected in the ventral mesoderm, although low levels of sog transcripts might extend into the ventral-most regions of the neurogenic ectoderm. This pattern is more restricted across the dorsal-ventral axis than the D. melanogaster sog pattern. tld expression is restricted to lateral regions of A. gambiae embryos and is excluded from the dorsal ectoderm, which is the principal site of expression in Drosophila. These significant changes in the sog and tld expression patterns might account, at least in part, for the expanded limits of Dpp signaling in the dorsal ectoderm of A. gambiae embryos (Goltsev, 2007).

Direct evidence for broader Dpp signaling was obtained using an antibody that detects phosphorylated Mad (pMad), the activated form of Mad obtained upon induction of the Tkv receptor. In D. melanogaster pMad expression is restricted to the dorsal midline. This is the domain where Sog-Dpp complexes are processed and peak levels of Dpp interact with the receptor Tkv. The spatial limits of the sog expression pattern are decisive for this restricted domain of pMad activity. Just a twofold reduction in the levels of Sog (sog/+ heterozygotes) causes a significant expansion in pMad expression (Goltsev, 2007).

There is a marked expansion of the pMad expression domain in A. gambiae embryos as compared with Drosophila. The domain encompasses the entire presumptive serosa and extends into portions of the presumptive amnion. The dpp and tkv expression patterns are downregulated in the presumptive serosa, nonetheless, the pMad staining pattern clearly indicates that this is the site of peak Dpp signaling activity. The early expression of both dpp and tkv encompasses the entire dorsal ectoderm. It would appear that peak Dpp signaling is somehow maintained in the developing serosa even after the downregulation of dpp and tkv expression in this tissue. A similar scenario is seen in the Drosophila embryo, in that there is downregulation of both dpp and tkv expression along the dorsal midline of gastrulating embryos (Goltsev, 2007).

To determine the basis for expanded Dpp signaling a sog enhancer was identified and characterized in A. gambiae. The D. melanogaster enhancer is located in the first intron of the sog transcription unit. It is ~300 bp in length and contains four evenly spaced, optimal Dorsal binding sites. These sites permit activation of sog expression by low levels of the Dorsal gradient; however, closely linked Snail repressor sites inactivate the enhancer in the ventral mesoderm. A putative A. gambiae enhancer was identified by scanning the sog locus for potential clusters of Dorsal binding sites. The recently developed cluster-draw program was used for this purpose since it successfully identified a sim enhancer in the honeybee, Apis mellifera, which is even more divergent than Anopheles. The best putative Dorsal binding cluster was identified within the first intron of the A. gambiae sog locus. Several genomic DNA fragments were tested for enhancer activity, but only this cluster was found to activate gene expression in transgenic Drosophila embryos (Goltsev, 2007).

Two different genomic DNA fragments, 3.7 kb and 1.1 kb, that encompass the intronic binding cluster were tested in transgenic embryos. Both fragments were attached to a lacZ reporter gene containing the core eve promoter from D. melanogaster, and both direct lacZ expression in the presumptive mesoderm. They exhibit the same restricted dorsal-ventral limits of expression as that seen for the endogenous sog gene in A. gambiae, although the smaller fragment produces ventral stripes whereas the larger fragment directs a more uniform pattern. The change in the dorsal-ventral limits -- broad expression in D. melanogaster and restricted expression in A. gambiae -- might be due to the quality of individual Dorsal binding sites in the two enhancers (Goltsev, 2007).

Therefore, s comprehensive analysis of dorsal-ventral patterning genes in the A. gambiae embryo reveals elements of conservation and divergence in the gastrulation network of D. melanogaster. There is broad conservation in the expression of regulatory genes responsible for the patterning of the mesoderm and neurogenic ectoderm, including sequential expression of sim, vnd and ind in the developing nerve cord. By contrast, there are extensive changes in the expression of regulatory genes that pattern the dorsal ectoderm. These changes foreshadow the subdivision of the dorsal ectoderm into separate serosa and amnion lineages in A. gambiae (Goltsev, 2007).

The major difference in the early patterning of the mesoderm in flies and mosquitoes concerns the manner in which mesoderm cells enter the blastocoel of gastrulating embryos. In D. melanogaster, there is a coherent invagination of the mesoderm through the ventral furrow, much like the movement of bottle cells through the blastocoel of Xenopus embryos. By contrast, there is no invagination of the mesoderm in A. gambiae. Instead, the mesoderm undergoes progressive ingression during germband elongation. This type of ingression is seen in D. melanogaster mutants lacking fog signaling. The A. gambiae genome lacks a clear homologue of fog, and it is therefore conceivable that fog represents an innovation of the higher Diptera that was only recently incorporated into the D. melanogaster dorsal-ventral patterning network (Goltsev, 2007).

D. melanogaster is somewhat unusual in having an amnioserosa, rather than separate serosa and amnion tissues as seen in most insects. In certain mosquitoes the serosa secretes an additional proteinaceous membrane that provides extra protection against desiccation. The changes in gene expression in the D. melanogaster and A. gambiae dorsal ectoderm provide a basis for understanding the evolutionary transition of two dorsal tissues in A. gambiae into a novel single tissue in higher dipterans (Goltsev, 2007).

The D. melanogaster amnioserosa expresses a variety of regulatory genes, including Doc1/2 and tup. The expression of most of these genes is restricted in the presumptive amnion of the A. gambiae embryo. zen is the only dorsal patterning gene, among those tested, that exhibits restricted expression in the serosa. Several segmentation genes have a similar pattern, and one of these, ttk, encodes a known repressor. Ectopic expression of Ttk causes a variety of patterning defects in Drosophila embryos, including disruptions in head involution and germband elongation that might arise from alterations in the amnioserosa. It is proposed that zen activates ttk in the serosa of A. gambiae embryos. The encoded repressor might subdivide the dorsal ectoderm into separate serosa and amnion tissues by inhibiting the expression of Doc1/2 and tup in the serosa. The loss of this putative zen-ttk regulatory linkage might be sufficient to allow Dpp signaling to activate tup and Doc1/2 throughout the dorsal ectoderm, thereby transforming separate serosa and amnion tissues into a single amnioserosa. According to this scenario, the loss of zen binding sites in ttk regulatory sequences might be responsible for the evolutionary transition of the amnioserosa (Goltsev, 2007).

The formation of separate amnion and serosa tissues is not the only distinguishing feature of A. gambiae embryos when compared with D. melanogaster. There is also a significant expansion in the overall limits of the dorsal ectoderm. This can be explained, in part, by distinct patterns of sog expression. The broad expression limits of the Sog inhibitor are responsible for restricting Dpp/pMad signaling to the dorsal midline of the D. melanogaster embryo. This pattern depends on a highly sensitive response of the sog intronic enhancer to the lowest levels of the Dorsal gradient. The Dorsal binding sites in the sog enhancer are optimal sites, possessing perfect matches to the idealized position weighted matrix of Dorsal recognition sequences. By contrast, the A. gambiae intronic sog enhancer contains low-quality Dorsal binding sites, similar to those seen in the regulatory sequences of genes activated by peak levels of the Dorsal gradient, such as twist. The binding sites in the D. melanogaster sog enhancer have an average score of ~10. By contrast, the best sites in the A. gambiae sog enhancer have scores in the 6.5-7 range, typical of enhancers that mediate expression in the mesoderm in response to high levels of the Dorsal gradient. Although every potential regulatory sequence in the A. gambiae sog locus was not explicitly tested, none of the putative Dorsal binding clusters in the vicinity of the gene possess the quality required for activation by low levels of the Dorsal gradient in the neurogenic ectoderm. Thus, the narrow limits of sog expression in A. gambiae embryos can be explained by the occurrence of low-quality Dorsal binding sites, along with the loss of Snail repressor sites (Goltsev, 2007).

The altered sog expression pattern is probably not the sole basis for the expansion of the dorsal ectoderm. A. gambiae embryos also exhibit a significant change in the tld expression pattern. tld is expressed throughout the dorsal ectoderm in D. melanogaster, but restricted to the neurogenic ectoderm of A. gambiae. Tld cleaves inactive Tsg-Sog-Dpp complexes to produce peak Dpp signaling along the dorsal midline of Drosophila embryos. It is proposed that the altered tld pattern in combination with altered sog leads to two dorsolateral sources of the active Dpp ligand in mosquito embryos. The sum of these sources might produce a step-like distribution of pMad across dorsal regions of mosquito embryos. This broad plateau of pMad activity might be responsible for the observed expansion of the dorsal ectoderm territory, and the specification of the serosa (Goltsev, 2007).

In Drosophila, tld is regulated by a 5' silencer element that prevents the gene from being expressed in ventral and lateral regions in response to high and low levels of the Dorsal gradient. This silencing activity is due to close linkage of Dorsal binding sites and recognition sequences for 'co-repressor' proteins. Preliminary studies suggest that Dorsal activates the A. gambiae tld gene, possibly by the loss of co-repressor binding sites in the 5' enhancer (Goltsev, 2007).

It is proposed that there are at least two distinct threshold readouts of Dpp signaling in the dorsal ectoderm of A. gambiae embryos. Type 1 target genes, such as hb, ems, ttk and zen, are activated by high levels and thereby restricted to the presumptive serosa. Type 2 target genes, such as tup and Doc1/2, can be activated - in principle - by both high and low levels of Dpp signaling in the presumptive serosa and amnion. However, these target enhancers contain binding sites for one or more type 1 repressors expressed in the serosa. The favorite candidate repressor is Ttk. Perhaps the type 2 tup enhancer contains optimal pMad activator sites as well as binding sites for the localized repressor Ttk, which keeps tup expression off in the serosa and restricted to the amnion. As discussed earlier, the simple loss of ttk regulation by the Dpp signaling network might be sufficient to account for the evolutionary conversion of separate serosa and amnion tissues into a single amnioserosa. Localization of this single tissue within a restricted domain along the dorsal midline would arise from concomitant dorsal shifts in the sog and tld expression patterns (Goltsev, 2007).

Evolution of extracellular Dpp modulators in insects: The roles of tolloid and twisted-gastrulation in dorsoventral patterning of the Tribolium embryo

The formation of the BMP gradient which patterns the DV axis in flies and vertebrates requires several extracellular modulators like the inhibitory protein Sog/Chordin, the metalloprotease Tolloid (Tld), which cleaves Sog/Chordin, and the CR domain protein Twisted gastrulation (Tsg). While flies and vertebrates have only one sog/chordin gene they possess several paralogues of tld and tsg. A simpler and probably ancestral situation is observed in the short-germ beetle Tribolium castaneum (Tc), which possesses only one tld and one tsg gene. This study shows that in T. castaneum tld is required for early BMP signalling except in the head region and Tc-tld function is, as expected, dependent on Tc-sog. In contrast, Tc-tsg is required for all aspects of early BMP signalling and acts in a Tc-sog-independent manner. For comparison with Drosophila melanogaster fly embryos were constructed lacking all early Tsg activity (tsg;;srw double mutants); they were shown to still establish a BMP signalling gradient. Thus, these results suggest that the role of Tsg proteins for BMP gradient formation has changed during insect evolution (Nunes da Fonseca, 2010).

Tolloid homologs in C. elegans

The hch-1 gene of C. elegans encodes a tolloid/BMP-1 family protein. The phenotype of hch-1 mutants shows that it is required for normal hatching and normal migration of a post-embryonic neuroblast. Drosophila Tolloid is not involved in either of these functions. In spite of its expression in embryogenesis, it is not required for viability of embryos. The possession of only one CUB domain distinguishes HCH-1 from TLD which has five. There are at least eight proteins in C. elegans that resemble HCH-1 in amino acid sequence and domain arrangement (Hishida, 1996).

BMP signaling regulates the dorsal planarian midline and is needed for asymmetric regeneration

Planarians can be cut into irregularly shaped fragments capable of regenerating new and complete organisms. Such regenerative capacities involve a robust ability to restore bilateral symmetry. Three genes needed for bilaterally asymmetric fragments to regenerate missing body parts have been identified. These genes are candidate components of a signaling pathway that controls the dorsal-ventral patterning of many animal embryos: a BMP1/Tolloid-like gene (smedolloid-1), a SMAD4-like gene (smedsmad4-1), and a BMP2/4/DPP-like gene (smedbmp4-1). BMP signaling is involved in the formation of new tissues at the midline of regeneration, the dorsal-ventral patterning of new tissues, and the maintenance of the dorsal-ventral pattern of existing adult tissue in homeostasis. smedbmp4-1 is normally expressed at the dorsal midline. Asymmetric fragments lacking a midline display new smedbmp4-1 expression prior to formation of a regenerative outgrowth (blastema). Asymmetric fragments containing the midline display expanded smedbmp4-1 expression towards the wound. It is suggested injured animals that lack left-right symmetry reset their midline through modulation of BMP activity as an early and necessary event in regeneration (Reddien, 2007; full text of article).

Tolloid in Zebrafish and Frogs

Dorsoventral patterning of vertebrate and Drosophila embryos requires bone morphogenetic proteins (BMPs) and antagonists of BMP activity. The Drosophila gene tolloid encodes a metalloprotease similar to BMP-1 that interacts genetically with decapentaplegic, the Drosophila homolog of vertebrate BMP-2/4. Zebrafish embryos overexpressing a zebrafish homolog of tolloid resemble loss-of-function mutations in chordino, the zebrafish homolog of the Xenopus BMP-4 antagonist Chordin. Zebrafish tld transcripts are detected throughout the early gastrula stage embryo. Toward the end of gastrulation expression becomes restricted, accumulating both dorsally and ventrally along the closing blastopore. Expression is also detected in the ectoderm flanking the anterior neural plate at this stage. At the 10-somite stage tld mRNA is expressed in the developing tailbud and in cells flanking the midbrain and hindbrain; these cells presumably correspond to migrating cranial neural crest. Chordin is degraded by COS cells expressing Tolloid. These data suggest that Tolloid antagonizes Chordin activity by proteolytically cleaving Chordin. A conserved function for zebrafish and Drosophila Tolloid during embryogenesis is proposed (Blader, 1997).

BMP signaling is modulated by a number of extracellular proteins, including the inhibitor Chordin, Tolloid-related enzymes (Tld), and the interacting protein Twisted Gastrulation (Tsg). Although in vitro studies have demonstrated Chordin cleavage by Tld enzymes, its significance as a regulatory mechanism in vivo has not been established in vertebrates. In addition, Tsg has been reported in different contexts to either enhance or inhibit BMP signaling through its interactions with Chordin. The zebrafish gastrula has been used to carry out structure/function studies on Chordin, by making versions of Chordin partially or wholly resistant to Tld cleavage and introducing them into chordin-deficient embryos. The cleavage products generated in vivo from wild-type and altered Chordins were examined, and their efficacy as BMP inhibitors was tested in the embryo. Tld cleavage is shown to be crucial in restricting Chordin function in vivo, and is carried out by redundant enzymes in the zebrafish gastrula. Evidence is presented that partially cleaved Chordin is a stronger BMP inhibitor than the full-length protein, suggesting a positive role for Tld in regulating Chordin. Depletion of embryonic Tsg leads to decreased BMP signaling, and to increased levels of Chordin. Finally, it was shown that Tsg also enhances BMP signaling in the absence of Chordin, and its depletion can partially rescue the chordin mutant phenotype, demonstrating that important components of the BMP signaling pathway remain unidentified (Xie, 2005).

Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. The zebrafish BMP1 gene bmp1 was identified and mapped. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. It is concluded that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis (Jasuja, 2006).

The Xolloid secreted metalloprotease, a tolloid-related protein, was found to cleave Chordin and Chordin/BMP-4 complexes at two specific sites. In biochemical experiments Xolloid mRNA blocks secondary axes caused by chordin, but not by noggin, or follistatin, and not by injection of mRNA coding for dominant-negative BMP receptor. Xolloid-treated Chordin protein is unable to antagonize BMP activity. Furthermore, Xolloid digestion releases biologically active BMPs from Chordin/BMP inactive complexes. Injection of dominant-negative Xolloid mRNA indicates that the in vivo function of Xolloid is to limit the extent of Spemann's organizer field. It is proposed that Xolloid regulates organizer function by a novel proteolytic mechanism involving a double inhibition pathway required to pattern the dorsoventral axis (Piccolo, 1997).

Bone morphogenetic protein 1 (BMP1) is a metalloproteinase closely related to Drosophila Tolloid (Tld). Tld regulates dorsoventral patterning in early Drosophila embryos by enhancing the activity of Dpp, a member of the TGF-beta family most closely related to BMP2 and BMP4. In Xenopus BMP4 appears to play an essential role in dorsoventral patterning, promoting the development of ventral fates during gastrula stages. To determine if BMP1 has a role in regulating the activity of BMP4, cDNAs were isolated for Xenopus BMP1 and a novel closely related gene that has been called xolloid (xld). Whereas xbmp1 is uniformly expressed at all stages tested, the initial uniform expression of xld becomes localized to two posterior ectodermal patches flanking the neural plate and later to the inner ectoderm of the developing tailbud. xld is also expressed in dorsal regions of the brain during tailbud stages and is especially abundant in the ventricular layer of the dorsal hindbrain caudal to the otic vesicle. Overexpression of either gene inhibits the development of dorsoanterior structures in whole embryos and ventralizes activin-induced dorsal mesoderm in animal caps. Since ventralization of activin-induced animal caps can be blocked by coinjecting a dominant-inhibitory receptor for BMP2 and BMP4, a role is suggested for BMP1 and Xld in regulating the ventralizing activity of these molecules (Goodman, 1998).

Bone morphogenetic protein 1 (BMP1) is a metalloprotease that ventralizes dorsal mesoderm when overexpressed in early Xenopus embryos. Xenopus BMP1 blocks the dorsalising activity of chordin, but not noggin or DeltaxBMPR, when coexpressed in the ventral marginal zone and degrades chordin protein in vitro. A dominant-negative mutation for XBMP1 (dnBMP1) dorsalizes ventral mesoderm in vivo, and blocks degradation of chordin by both XBMP1 and Xolloid, a closely related Xenopus metalloprotease, in vitro. dnBMP1 does not dorsalize ventral mesoderm in UV-irradiated embryos, demonstrating that this activity is dependent upon a functional organizer -- the natural source of chordin in Xenopus gastrulae. These results suggest that XBMP1 may regulate the availability of chordin during vertebrate embryogenesis (Wardle, 1999b).

A number of genetic and molecular studies have implicated Chordin in the regulation of dorsoventral patterning during gastrulation. Chordin, a BMP antagonist of 120 kDa, contains four small (about 70 amino acids each) cysteine-rich domains (CRs) of unknown function. The Chordin CRs define a novel protein module for the binding and regulation of BMPs. The biological activity of Chordin resides in the CRs, especially in CR1 and CR3, which have dorsalizing activity in Xenopus embryo assays and bind BMP4 with dissociation constants in the nanomolar range. The activity of individual CRs, however, is 5- to 10-fold lower than that of full-length Chordin. These results shed light on the molecular mechanism by which Chordin/BMP complexes are regulated by the metalloprotease Xolloid, which cleaves in the vicinity of CR1 and CR3 and would release CR/BMP complexes with lower anti-BMP activity than intact Chordin. CR domains are found in other extracellular proteins such as procollagens. Full-length Xenopus procollagen IIA mRNA has dorsalizing activity in embryo microinjection assays and the CR domain is required for this activity. Similarly, a C. elegans cDNA containing five CR domains induces secondary axes in injected Xenopus embryos. These results suggest that CR modules may function in a number of extracellular proteins to regulate growth factor signaling (Larrain, 2000).

The Chordin/BMP pathway is regulated by the zinc metalloprotease Xolloid, a homolog of Drosophila Tolloid that regulates the activity of Sog. The observations made in this study begin to provide a molecular explanation for how Xolloid may regulate Chordin. Xolloid cleaves Chordin at two sites, which had been roughly mapped close to a region downstream of CR1 and CR3. Recently, the cleavage sites have been sequenced and found to correspond to conserved aspartic residues. The CR1 protein used in this study is very similar in length (only 8 amino acids shorter) to the fragment generated by metalloprotease cleavage in the N-terminal site of Chordin. CR1 binds BMP4 with a lower affinity (8-fold lower), is less efficient in competing BMP4 binding to BMPR (10 times lower), and has less biological activity (5- to 10-fold lower) than full-length Chordin. It is conceivable that the Xolloid protease inactivates Chordin by the generation of smaller fragments that can still bind BMP and perhaps transport it. However, each of these binding modules alone would not have high enough affinity to compete with the higher affinity of BMP for its cognate receptors, which is in the same range as that of full-length Chordin for BMP4 (Larrain, 2000 and references therein).

In Drosophila, Sog not only inhibits Dpp signaling but is also able to enhance it at a distance. This enhancement of BMP signals requires Sog diffusion (presumably carrying bound Dpp or Screw) and the activity of the Tolloid protease. It has been suggested that the cleavage products of Sog, or Sog fragments complexed with Dpp, could augment the binding of Dpp/Screw to its receptors. None of the Chordin constructs used in the current study, including a series of carboxy-terminal protein truncations, display ventralizing effects as would be expected if there were increased binding to receptors. Rather, the observation that Chordin fragments are either weakly dorsalizing or inactive in Xenopus assays tends to support the proposal that diffusion of Chd/Sog complexed with BMP/Dpp contributes to the formation of morphogen gradients in which maximal levels of signaling are achieved by cleavage of the inhibitor and release of the active BMP signal (Larrain, 2000 and references therein).

A novel Tolloid-like metalloprotease, Xolloid-related (Xlr), has been identified that is expressed during early Xenopus development. Transcripts for xlr are localized to the marginal zone of mid-gastrulae and are most abundant in ventral and lateral sectors. At neurula stages xlr is strongly expressed around the blastopore and in the pharyngeal endoderm, and more weakly expressed throughout the ventral half of the embryo. Transcripts are detected in the nervous system, particularly the hindbrain and spinal cord, and tailbud of tailbud stage embryos, with weaker expression in the anterior nervous system, otic vesicle, heart, and pronephric duct. Transcription of xlr is increased by BMP4 and decreased by Noggin and tBR (dominant-negative bmprI mRNA), indicating that xlr is regulated by BMP signaling. Injection of xlr mRNA inhibits dorsoanterior development and the dorsal axis-inducing ability of coinjected chordin, but not noggin or tBR, mRNA. Xlr conditioned media cleaves Chordin in vitro, indicating that this protease may regulate the availability of Chordin in vivo (Dale, 2002).

Dorsoventral patterning is regulated by a system of interacting secreted proteins involving BMP, Chordin, Xolloid and Twisted gastrulation (Tsg). The molecular mechanism by which Tsg regulates BMP signaling has been analyzed. Overexpression of Tsg mRNA in Xenopus embryos has ventralizing effects similar to Xolloid, a metalloprotease that cleaves Chordin. In embryos dorsalized by LiCl treatment, microinjection of Xolloid or Tsg mRNA restores the formation of trunk-tail structures, indicating an increase in BMP signaling. Microinjection of Tsg mRNA leads to the degradation of endogenous Chordin fragments generated by Xolloid. The ventralizing activities of Tsg require an endogenous Xolloid-like activity, since they can be blocked by a dominant-negative Xolloid mutant. A BMP-receptor binding assay has revealed that Tsg has two distinct and sequential activities on BMP signaling. (1) Tsg makes Chordin a better BMP antagonist by forming a ternary complex that prevents binding of BMP to its cognate receptor. (2) After cleavage of Chordin by Xolloid, Tsg competes the residual anti-BMP activity of Chordin fragments and facilitates their degradation. This molecular pathway, in which Xolloid switches the activity of Tsg from a BMP antagonist to a pro-BMP signal once all endogenous full-length Chordin is degraded, may help explain how sharp borders between embryonic territories are generated (Larraín, 2001).

The opposing activities of Tsg on BMP binding to its receptor suggest a sequential molecular mechanism that may help reconcile disparate observations in the literature. (1) Tsg forms a ternary complex with Chordin and BMP, which is a potent inhibitor of BMP signaling. This antagonist function must be the predominant one in zebrafish, because loss-of-function of Tsg and Chordin using antisense morpholinos ventralizes the embryo. (2) After cleavage of Chordin by Xolloid, Tsg competes the residual activity of Chordin fragments, providing a permissive signal that promotes BMP binding to its cognate receptor. This function is consistent with injection experiments in Xenopus embryos, in which reduction of endogenous Xenopus Tsg activity enhances the anti-BMP activity of CR1 fragments. (3) Overexpression of Tsg facilitates the degradation of endogenous Chordin in Xenopus. This activity may help explain why Tsg can ventralize the embryo and inhibit axis duplication by Chordin in a Xolloid-dependent manner. It is proposed that in overexpression experiments, an excess of Tsg protein displaces the equilibrium in the reaction, so that after cleavage of Chordin by Xolloid, Tsg dislodges BMP from the proteolytic products and facilitates their degradation in vivo. The Tsg/BMP binary complex acts as a permissive signal, because at physiological concentrations Tsg does not interfere with BMP binding to its receptor. Finally, at high concentrations, Tsg can also act as a BMP antagonist in the absence of Chordin, inducing in animal cap explants the cement gland marker XAG-1, but not the neural marker NCAM, by partially inhibiting BMP activity (Larraín, 2001).

The present results provide mechanistic insights into how sharp borders may be generated in embryos. In Drosophila, Tsg is required for the peak BMP signaling that induces a sharp band of Mad phosphorylation in the dorsal-most tissue. In lateral regions of the Xenopus embryo, where free full-length Chordin is still present, Tsg/BMP binary complexes released by Xolloid will have a higher affinity for Chordin than for the BMP receptor promoting the re-formation of inhibitory ternary complexes that can diffuse further. However, once all Chd is proteolytically cleaved by Xolloid, the function of Tsg switches from an inhibitory to a permissive signal that increases binding of BMPs to their cognate receptors. This switch in activity would facilitate the formation of sharp boundary differences. In lateral regions, where ternary complexes are constantly re-formed and re-cleaved as diffusion takes place, the situation is conceptually analogous to that occurring in an organic chemistry fractional distillation column. Although much remains to be learned about this interesting patterning system, the opposing functions of Tsg suggest a novel molecular mechanism for the establishment of cell differentiation territories in the embryo (Larraín, 2001).

An unexpected role is reported for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl -| Xlr -| Chd -| BMP --> P-Smad1 --> Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10-8 M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibits Xlr, suggesting a wider role for this molecular mechanism (Lee, 2006).

Enzymatic regulation of pattern: BMP4 binds CUB domains of Tolloids and inhibits proteinase activity

In Xenopus embryos, a dorsal-ventral patterning gradient is generated by diffusing Chordin/bone morphogenetic protein (BMP) complexes cleaved by BMP1/Tolloid metalloproteinases in the ventral side. A new BMP1/Tolloid assay was developed using a fluorogenic Chordin peptide substrate, and an unexpected negative feedback loop was identified for BMP4, in which BMP4 inhibits Tolloid enzyme activity noncompetitively. BMP4 binds directly to the CUB (Complement 1r/s, Uegf [a sea urchin embryonic protein] and BMP1) domains of BMP1 and Drosophila Tolloid with high affinity. Binding to CUB domains inhibits BMP4 signaling. These findings provide a molecular explanation for a long-standing genetical puzzle in which antimorphic Drosophila tolloid mutant alleles displayed anti-BMP effects. The extensive Drosophila genetics available supports the relevance of the interaction described in this study at endogenous physiological levels. Many extracellular proteins contain CUB domains; the binding of CUB domains to BMP4 suggests a possible general function in binding transforming growth factor-beta (TGF-beta) superfamily members. Mathematical modeling indicates that feedback inhibition by BMP ligands acts on the ventral side, while on the dorsal side the main regulator of BMP1/Tolloid enzymatic activity is the binding to its substrate, Chordin (Lee, 2009).

The differentiation of cell types along the vertebrate D-V axis is regulated by an extracellular network of BMPs and their regulators, such as Chordin, BMP1/Tolloid, Tsg, and Crossveinless-2, in animals as diverse as Xenopus, Drosophila, zebrafish, amphioxus, hemichordates, and spiders. In addition, in the vertebrates, additional extracellular BMP antagonists such as Noggin and Follistatin cooperate with the anti-BMP activity of Chordin. The complexity of this biochemical pathway raises the question of why so many components and regulatory interactions are required to establish a simple gradient of BMP signaling through the transcription factors Smad1/5/8. One reason is that a stable gradient must be robustly maintained through many hours of development (from blastula until the end of gastrulation) at a time during which the three embryonic germ layers are undergoing massive morphogenetic movements. In addition, the frog embryo must have the ability to adapt to changes in temperature in its environment (Lee, 2009)

The patterning system must be resilient, given the self-regulating nature of development. When Xenopus embryos are cut in half, they will attempt to regenerate an embryo as perfect as possible, producing in some cases identical twins. This implies that cells in the dorsal and ventral poles of the early embryo communicate with each other, forming a self-regulating embryonic field. At a molecular level, these cell-cell communications can be explained by a pathway in which dorsal BMPs (ADMP and BMP2) and their antagonist, Chordin, are repressed at the transcriptional level by BMP signaling, while on the ventral side, BMP4/7 and CV2 are activated by the same signal, providing a self-regulating system. The key controlling element in this D-V conversation is provided by BMP1/Tolloid enzymes that degrade Chordin/BMP complexes releasing active BMP that are regulated by the Sizzled/Ogon-secreted competitive inhibitor. In this study, a novel regulatory node in the D-V patterning pathway, in which BMP4 serves as a feedback inhibitor of the BMP1 and Tolloid-related enzymes, was introduced (Lee, 2009).

A synthetic Chordin octapeptide spanning the C-terminal cleavage site that fluoresces when cleaved by Tolloids provided a quantitative enzymatic assay. This new assay was essential to the work, because both Chordin and Sog become better substrates for Tolloids when bound to BMP. It is therefore not possible to conduct a biochemical study on the digestion of full-length Chordin/Sog plus or minus BMP, because BMP affects both the substrate and the enzyme. The conformational change in the Chordin/Sog substrate would have precluded the discovery of the inhibition of enzyme activity by BMP4 (Lee, 2009).

Inhibition of BMP1/tolloids by BMP4 was specific, because it was not observed with other proteins such as Activin A, Tsg, Follistatin, and Noggin. The kinetics followed those of a Michaelis-Menten noncompetitive inhibition. This meant that BMP4 affected the activity of the enzyme by binding to a site distinct from the catalytic center. BMP4 was found to bind directly to CUB domains with high affinity. The Ki or inhibition constant (concentration at which half of the enzyme is bound to the inhibitor) for BMP1 was in the 40 nM range, and in the 14-20 nM range when measured by direct binding. This is within physiological levels, since the Km (Michaelis constant or affinity of the enzyme for its substrate) of BMP/Tolloids for Chordin substrate was between 17 and 25 nM, and of 96 nM for its BMP1/PCP activity (Lee, 2009).

The ventral center of the Xenopus gastrula expresses a chordin-like protein called CV-2 that strongly binds Chordin/BMP complexes transported from more dorsal regions of the embryo and facilitates BMP signaling through its cognate receptors after cleavage of Chordin by BMP1/tolloids. This suggests that in vivo free BMP is locally concentrated at sites of high CV2 and chordinase activity; it is in these regions that the negative feedback loop should be most effective. Not only will the BMP levels be highest, but also the Chordin levels will be lowest. The affinities of the interaction between BMP4/Tsg/Chordin and Tolloid may also be enhanced by the recently described Olfactomedin-related adaptor protein Ont-1, which brings together Chordin and tolloids (Lee, 2009).

The importance of the interaction between Tolloid and BMPs for developmental patterning in vivo is suggested by Drosophila genetics. A very large allelic series of tolloid mutants has been obtained that display a graded series of patterning defects along the D-V axis in Drosophila. This suggests that Tolloid provides a rate-limiting step during patterning. Therefore, any decrease in its activity caused by binding of BMPs would be expected to regulate the signaling gradient. The antimorphic tolloid mutations, which are proteolytically inactive but display anti-BMP effects, demonstrate that endogenous Tolloid enzyme is expressed at high enough levels to function antagonistically toward Dpp in vivo. Thus, at least in Drosophila, the interactions between Tolloid and BMPs discovered in this study function at physiological concentrations of D-V pathway components (Lee, 2009).

There previously had been isolated reports showing that DN-BMP1/tolloids dorsalized Xenopus ventral mesoderm, which should lack Chordin. One possible interpretation for these results was the presence of a Chordin counterpart, such as CV2, expressed in the high-BMP regions of the embryo. However, it was later found that CV2 is resistant to degradation by tolloids/BMP1. Instead, it was found that the anti-BMP effect of DN-tolloids, which can take place in Chordin-depleted embryos, are due to the sequestration of BMP ligands through direct binding to CUB domains (Lee, 2009).

It was initially hoped that the second site mutations described in Drosophila Tolloid CUB domains would point to amino acid residues critical for Tolloid binding of BMP4. Instead, all second site mutations affected Tolloid/BMP1 protein secretion. These second site antimorphic revertants behave essentially as null mutations of tolloid because they are not secreted. It is likely that the original antimorphic mutants displayed anti-Dpp effects because they bound BMPs in the Drosophila embryo (Lee, 2009).

CUB domains are also required for enzymatic activity. In the case of BMP1/PCP, it has been shown that the procollagen substrate is not efficiently recognized when CUB2 of BMP1 is deleted. However, the protease domain plus CUB1 is sufficient for BMP1 chordinase activity. In the case of Drosophila Tolloid, CUB4 and CUB5 are required to cleave Sog, and for Xolloid, CUB1 and CUB2 are required for recognition and cleavage of Chordin. Thus, CUB domains in Tolloid/BMP1 have specific functions in substrate recognition. CUB domains are also required for secretion, in addition to serving as inhibitory BMP-binding sites. As an interaction between the BMP1 prodomain and BMP4 has also been reported, it should be noted that the prodomain was lacking in all the CUB domain constructs used in the present study (Lee, 2009).

CUB domains are present in many secreted or transmembrane proteins, but their biochemical function remains unknown. The human genome contains 56 different loci encoding CUB domain-containing proteins. The finding that the CUB domains of BMP1 and Tolloid bind BMP4 suggests the exciting possibility that CUB domains may serve as binding modules for TGF-β superfamily ligands in other extracellular proteins as well. In the future it will be interesting to investigate, for example, the binding properties of the CUB domains found in Complement components C1r and C1s, which function in the opsonization of antigens. Another interesting protein is CUB domain-binding protein 1 (CDCP-1), a transmembrane receptor with three CUB domains that activates the Src tyrosine protein kinase and promotes metastases in human cancers; TGF-β also promotes metastases. Other CUB domain-containing proteins include membrane frizzled-related protein (MFRP), in which mutations in CUB domains cause nanophthalmos; procollagen C-peptidase enhancer (PCPE), known as a potent enhancer of BMP1/PCP activity in procollagen processing; the WNT transmembrane coreceptor Kremen; and many other extracellular or transmembrane proteins (Lee, 2009).

The effects of enzymatic inhibition -- in this case, noncompetitive inhibition by BMP4 -- were integrated into a reaction-diffusion model to understand its effect on the BMP morphogen gradient of the early Xenopus embryo. This mathematical modeling predicted that Tld activity will be inhibited in ventral regions in which BMPs are present in high concentrations. An unexpected finding was that Chordin itself is a major regulator of BMP/Tolloid activity. At high concentrations, such as in the dorsal side of the frog gastrula and likely in the fly ventral blastoderm, Chd/Sog complexed with Tld is predicted to decrease the availability of free (active) BMP/Tolloid. This will inhibit degradation of Chordin-BMP complexes, preventing local BMP release and signaling, enabling the complex to diffuse further (Lee, 2009).

These observations suggest that the Tolloid inhibition by BMP also takes place in fruit flies, which provide a system much more amenable to the visualization of gradients, and for which sophisticated mathematical modeling already exists. In the future, it will be interesting to investigate whether CUB domains generally serve as BMP or TGF-β superfamily-binding modules. This approach has been productive in the case of the CR/vWFc domains of Chordin, which function as BMP-binding modules in many proteins (Lee, 2009).

The present study suggests that the antimorphic revertant mutations, were based on direct Dpp-Tolloid associations and were indicators of a crucial step in the formation or maintenance of the self-adjusting D-V morphogen gradient. The findings in Drosophila and Xenopus also suggest that this extracellular negative feedback regulation was already present in the patterning system of Urbilateria, the last common ancestor of all bilateral animals that lived more than 535 million years ago. Finally, the direct binding of BMP4 to BMP1 explains why highly purified bone-inducing protein preparations contained BMP1/Tolloid in addition to BMP2-7. It may be worthwhile to explore the value of BMP1 or its CUB domains as a delivery system for BMPs in therapeutic interventions, such as the repair of bone fractures (Lee, 2009).

Tolloid in Sea Urchin

The Xenopus embryo was used as a test system for analyzing the activity of SpAN, a sea urchin metalloprotease in the astacin family containing BMP1 and tolloid. Embryos expressing SpAN initiate gastrulation on a time scale indistinguishable from controls, but invagination of the vegetal pole is subsequently delayed by several hours. At tailbud stages the most severely affected embryos are completely ventralized, lacking all dorsal structures. Molecular analysis of injected embryos, using probes for both dorsal (xgsc and xnot) and ventral (xhox3 and xwnt8) mesoderm, indicates that SpAN ventralizes dorsal mesoderm during gastrula stages. These results mirror those previously obtained with BMP4, suggesting that SpAN may enhance the activity of this ventralizing factor. Consistent with this suggestion, SpAN is shown to block the dorsalizing activity of noggin and chordin, two inhibitory binding proteins for BMP4, but not that of a dominant-negative receptor for BMP4. In contrast, a dominant-negative SpAN, in which the metalloprotease domain has been deleted, dorsalizes ventral mesoderm, a phenotype that can be rescued by coexpressing either SpAN or XBMP1. This suggests that SpAN is mimicking a Xenopus metalloprotease responsible for regulating the activity of Xenopus BMPs during gastrulation. Moreover, these results raise the possibility that SpAN may function to facilitate BMP signaling in early sea urchin embryos (Wardle, 1999a).

Tollid in birds

Expression and regulation of the mRNAs for the type I BMP receptors, BMPR-IA and BMPR-IB, were examined in quail embryos in vivo and in neural crest cultures in vitro. BMPR-IB mRNA is expressed in the primordial sympathetic ganglia at stage 17, soon after the first expression of Cash-1 mRNA, the avian homolog of the Drosophila transcription factor achaete-scute. BMP-4 mRNA is detected in the dorsal aorta at stage 17, coincident with BMPR-IB mRNA expression in the sympathetic ganglia. BMPR-IA mRNA is first expressed in the sympathetic ganglia at stage 18. Moreover, BMP-4 ligand mRNA is detected in the sympathetic ganglia starting at stage 18. BMPR-IA and BMPR-IB are differentially regulated in cultured neural crest cells. BMPR-IB is expressed in primary outgrowths of neural crest cells but is downregulated after primary outgrowths are harvested and replated in secondary cultures. In secondary cultures of neural crest cells, exogenous BMP-2 and BMP-4 increase the expression of BMPR-IA but decrease the expression of BMPR-IB. The expression of both type I BMP receptors is inhibited by exogenous TGF-beta1. These results suggest distinct roles for BMPR-IA and BMPR-IB in the development of the sympathoadrenal phenotype from cells of the neural crest (McPherson, 2000).

Mammalian Tolloids

Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal-ventral patterning by releasing TGFbeta-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of this family of developmental proteases, novel family member mammalian Tolloid-like 2 (mTLL-2) is identified and enzymatic activities and expression domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2] are compared. Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site, while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and counteract the dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and post-natal skeletogenesis (Scott, 1999).

In humans and mice, alternatively spliced transcripts encode BMP-1 and a longer protein, designated mammalian Tolloid (mTld), with a domain structure identical to that of Drosophila Tld. Low levels of transcripts for mTld are found in all adult human tissues surveyed, while BMP-1 transcripts are detectable in all adult tissues except brain. This differential expression is mirrored in embryonic mouse tissues where high levels of mTld transcripts are found in the floor plate of the neural tube of the developing central nervous system, but no BMP-1 transcripts (Takahara, 1994).

The organization is described of the 46-kb, 22-exon human BMP1/mTld gene that encodes the alternatively spliced BMP1 forms. Exons corresponding to each of the alternatively spliced transcripts are identified, and comparison with the Drosophila tld gene reveals alignment of introns at only three positions (Takahara, 1995).

The mouse bone morphogenetic protein1 (Bmp1) gene encodes a secreted astacin metalloprotease that cleaves the COOH-propeptide of procollagen I, II and III. BMP-1 is also related to the product of the Drosophila patterning gene, tolloid. The mouse Bmp1 gene was disrupted by deleting DNA sequences encoding the active site of the astacin-like protease domain common to all splice variants. Homozygous mutant embryos appear to have a normal skeleton, apart from reduced ossification of certain skull bones. However, they have a persistent herniation of the gut in the umbilical region and do not survive beyond birth. Analysis of the amnion of homozygous mutant embryos reveals the absence of the fold that normally tightly encloses the physiological hernia of the gut. At the electron microscopic level, the extracellular matrix of the amnion contains collagen fibrils with an abnormal morphology, consistent with the incorporation of partially processed procollagen molecules. Metabolical labelling and immunofluorescence studies also reveal abnormal processing and deposition of procollagen by homozygous mutant fibroblasts in culture (Suzuki, 1996).

BMPs are bone-derived factors capable of inducing ectopic bone formation. Unlike other BMPs, BMP-1 is not like transforming growth factor-beta (TGF-beta), but it is the prototype of a family of putative proteases implicated in pattern formation during development in diverse organisms. Although some members of this group, such as Drosophila Tolloid, are postulated to activate TGF-beta-like proteins, actual substrates are unknown. Procollagen C-proteinase (PCP), identical to BMP-1, cleaves the COOH-propeptides of procollagens I, II, and III to yield the major fibrous components of vertebrate extracellular matrix (Kessler, 1996).

Transforming growth factor-beta1 (TGF-beta1) induces increased extracellular matrix deposition. Bone morphogenetic protein-1 (BMP-1) also plays key roles in regulating vertebrate matrix deposition; it is the procollagen C-proteinase (PCP) that processes procollagen types I-III, and it may also mediate biosynthetic processing of lysyl oxidase and laminin 5. BMP-1 is itself up-regulated by TGF-beta1; secreted BMP-1, induced by TGF-beta1, is either processed to an active form or remains as unprocessed proenzyme, in a cell type-dependent manner. In MG-63 osteosacrcoma cells, TGF-beta1 elevates levels of BMP-1 mRNA approximately 7-fold and, to a lesser extent, elevates levels of mRNA for mammalian tolloid (mTld), an alternatively spliced product of the BMP1 gene. Induction of RNA is dose- and time-dependent and cycloheximide-inhibitable. Secreted BMP-1 and mTld, induced by TGF-beta1 in MG-63 and other fibrogenic cell cultures, are predominantly in forms in which proregions have been removed to yield activated enzyme. TGF-beta1 treatment also induces procollagen N-proteinase activity in fibrogenic cultures, while expression of the procollagen C-proteinase enhancer (PCPE), a glycoprotein that stimulates PCP activity, is unaffected. In contrast to fibrogenic cells, keratinocytes lack detectable PCPE under any culture conditions and are induced by TGF-beta1 to secrete BMP-1 and mTld predominantly as unprocessed proenzymes (Lee, 1997).

Mammalian Tolloid and heart development

Mammalian Tolloid-like 1 (mTLL-1) is an astacin-like metalloprotease, highly similar in domain structure to the morphogenetically important proteases bone morphogenetic protein-1 (BMP-1) and Drosophila Tolloid. BMP-1 is structurally most similar to the Drosophila protein Tolloid (Tld), although Tld contains additional C-terminal EGF and CUB domains. A single mammalian gene is now known to produce alternatively spliced mRNAs for BMP-1 and for a larger protein designated mammalian Tolloid (mTLD), due to a domain structure identical to that of Tld. To investigate possible roles for mTLL-1 in mammalian development, gene targeting in ES cells was used to produce mice with a disrupted allele for the corresponding gene, Tll1. Homozygous mutants are embryonic lethal, with death at mid-gestation from cardiac failure and a unique constellation of developmental defects that are apparently confined solely to the heart. Constant features are incomplete formation of the muscular interventricular septum and an abnormal and novel positioning of the heart and aorta. Consistent with roles in cardiac development, Tll1 expression is specific to precardiac tissue and endocardium in 7.5 and 8.5 days p.c. embryos, respectively. Tll1 expression is also high in the developing interventricular septum, where expression of the BMP-1 gene, Bmp1, is not observed. Cardiac structures that are not affected in Tll1-/- embryos either show no Tll1 expression (atrio-ventricular cushions) or show overlapping expression of Tll1 and Bmp1 (aortico-pulmonary septum), suggesting that products of the Bmp1 gene may be capable of functionally substituting for mTLL-1 at sites in which they are co-expressed. Together, the various data show that mTLL-1 plays multiple roles in formation of the mammalian heart and is essential for formation of the interventricular septum (Clark, 1999).

The effects of mTLL-1 protease activity on formation of muscular septa, such as the MIVS (the muscular interventricular septum), may again involve the potentiation of signaling by BMPs, at least two of which (BMPs 5 and 7) are expressed in developing ventricular myocardium. Thus, it is of interest that the temporal patterns of expression for the Chordin and mTLL-1 genes, Chrd and Tll1, are inversely related in developing heart. In particular, from 11.5 to 13.5 dpc Tll1 expression in heart is highest and Chrd expression is lowest, and it is during this same period that Tll1 -/- septation defects manifest themselves. Therefore, during this period, BMP signaling may normally be particularly important in the formation of the muscular septa and be maximized in these structures through concomitant down-regulation of inhibitory Chordin and up-regulation of potentiating mTLL-1 molecules. Electron microscopic examination found no evidence for collagen fibrils in wild-type MIVS, making it unlikely that mTLL-1 procollagen C-proteinase activity plays a major role in MIVS formation. Nevertheless, some portion of the cardiac defects in Tll1 -/- embryos may be due to the failure to process some, as yet unidentified, nonfibrillar matrix substrate(s) of mTLL-1. If so, mTLL-1 may normally influence heart morphogenesis through coordinated effects on the potentiation of TGFbeta-like molecules and the deposition of matrix. Atrial septal defects and defects of the MIVS constitute the most common cardiac abnormalities in humans. Thus, identification here of the essential roles of the astacin-like protease mTLL-1 in heart morphogenesis, not only provides new insights into the molecular events underlying formation of the mammalian heart, but has the potential to further understanding of human cardiovascular disease as well (Clark, 1999).

Tsg enhances chordin cleavage by vertebrate tolloid-related proteases at a site poorly used in Tsg's absence

Twisted gastrulation gene products have been identified from human, mouse, Xenopus, zebrafish and chick. Expression patterns in mouse and Xenopus embryos are consistent with in vivo interactions between Tsg, BMPs and the vertebrate SOG ortholog, chordin. Tsg binds both the vertebrate Decapentaplegic ortholog BMP4 and chordin, and these interactions have multiple effects. Tsg increases chordin's binding of BMP4, potentiates chordin's ability to induce secondary axes in Xenopus embryos, and enhances chordin cleavage by vertebrate tolloid-related proteases at a site poorly used in Tsg's absence; also, the presence of Tsg enhances the secondary axis-inducing activity of two products of chordin cleavage. It is concluded that Tsg acts as a cofactor in chordin's antagonism of BMP signaling (Scott, 2001).

Tsg is coexpressed with chordin and various BMPs in vertebrate development. In Xenopus, maternal Tsg RNA was detected in eggs by RT (reverse transcription)-PCR, while whole-mount in situ hybridization showed uniform Tsg expression across the entire animal hemisphere and marginal zone of the early gastrula. At tailbud stage, Tsg, chordin and BMP4 expression domains partially overlap in the developing tail, anterior brain, eye and heart. In mouse, Tsg is broadly expressed throughout the 7.5-days-post-coitus (7.5-d.p.c.) gastrula and in extraembryonic tissues. Chordin, Tsg and BMPs 2, 4 and 7 are highly expressed in the digital rays of 15.5- and 17.5-d.p.c. embryo hindlimbs. Strong chordin expression in the interzone of the joint cavity is juxtaposed with strong Tsg expression at the joint articular surfaces and the interzone. Thus, Tsg is properly situated for potential interactions with chordin and BMPs during various stages of vertebrate embryogenesis (Scott, 2001).

In Drosophila, TSG influences cleavage of the chordin ortholog Short gastrulation (SOG) by Tolloid, altering the pattern of SOG cleavage products. There are four mammalian tolloid-related proteases. Two of these, BMP1 and mammalian tolloid-like 1 (mTll1), each cleave chordin at two specific sites, yielding fragments of relative molecular mass (Mr) 15K, 13K and 83K, corresponding to the amino-terminal, carboxy-terminal, and internal portions of chordin, respectively. Murine Tsg appears to enhance cleavage of mouse chordin and to influence the relative abundance of cleavage products, such that fragments of Mr 65K and 29K, minor forms in the absence of Tsg, become major products in the presence of Tsg. A third related protease, mammalian tolloid (mTld), which has little detectable chordin-cleaving activity, has significant activity in the presence of Tsg, also producing the 65K and 29K fragments as major forms. The fourth mammalian tolloid-like protease, mTll2, lacks chordin-processing activity in the presence or absence of Tsg (Scott, 2001).

The 65K and 29K chordin cleavage products preferentially produced in the presence of Tsg are subfragments of the 83K internal chordin fragment, as established by N-terminal amino-acid sequencing, and result from cleavage at a previously unmapped site between Ala 670 and Thr 671. Thus, the 29K form contains chordin cysteine-rich repeats (CRs) 2 and 3, whereas the 65K form contains no CR domains (Scott, 2001).

To determine how Tsg affects chordin cleavage, Tsg's ability to physically interact with tolloid-like proteases was examined. Co-immunoprecipitation of Tsg with BMP1 or mTll1 fails to detect physical interactions. However, co-immunoprecipitations show that Tsg binds chordin. Whether Tsg/chordin interactions might influence chordin's ability to bind BMP4 was examined. Co-immunoprecipitation of chordin and BMP4 is greatly enhanced in the presence of Tsg. It was also found that Tsg binds BMP4. In summary, Tsg's interactions with chordin and/or BMP4 enhance chordin/BMP4 complex formation, suggesting that Tsg might enhance chordin's antagonism of BMP signaling (Scott, 2001).

In vertebrates and invertebrates, the bone morphogenetic protein (BMP) signaling pathway patterns cell fates along the dorsoventral (DV) axis. In vertebrates, BMP signaling specifies ventral cell fates, whereas restriction of BMP signaling by extracellular antagonists allows specification of dorsal fates. In misexpression assays, the conserved extracellular factor Twisted gastrulation (Tsg) is reported to both promote and antagonize BMP signaling in DV patterning. To investigate the role of endogenous Tsg in early DV patterning, morpholino (MO)-based knockdown studies of Tsg1 were performed in zebrafish. Loss of tsg1 results in a moderately strong dorsalization of the embryonic axis, suggesting that Tsg1 promotes ventral fates. Knockdown of tsg1 combined with loss of function of the BMP agonist tolloid (mini fin) or heterozygosity for the ligand bmp2b (swirl) enhance dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppresses the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). These results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning (Little, 2004).

tolloid: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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