decapentaplegic

EVOLUTIONARY HOMOLOGS

Table of contents

Insect Dpp homologs

The Tribolium decapentaplegic gene is similar in sequence, structure, and expression to the Drosophila dpp gene. In addition, the position of an intron within the protein-coding region is conserved in the two genes as well as in the BMP2 and BMP4, mammalian homologs of dpp. Consensus binding sites for the Dorsal transcription factor are found within this intron in the two insect species. Of particular interest is the expression of Tc dpp during embryonic appendage development. In an early stage of germ band extension Tc dpp is detected along the lateral edges of the embryo in ectodermal cells that will give rise to the dorsalmost cells of the embryo. The region-specific expansions of Tc dpp expression within these regions may correspond to the beginnings of appendage formation, but in the case of Tribolium no imaginal discs are formed. Tc dpp is also expressed in what appears to be posterior mesoderm, indicating a possible role for dpp in maintenance of the growth zone of short germ insects such as Tribolium (see Tribolium early embryonic development). When the germ band is fully extended, Tc dpp is detected in the labrum, in the distal tips of the developing antennal, gnathal, and thoracic appendages, and in the putative dorsal ectoderm (Sanchez-Salazar, 1996).

All insect legs are structurally similar, characterized by five primary segments. However, this final form is achieved in different ways. Primitively, the legs developed as direct outgrowths of the body wall, a condition retained in most insect species. In some groups, including the lineage containing the genus Drosophila, legs develop indirectly from imaginal discs. The current understanding of the molecular mechanisms regulating leg development is based largely on analysis of this derived mode of leg development in the species D. melanogaster. The current model for Drosophila leg development is divided into two phases, embryonic allocation and imaginal disc patterning, which are distinguished by interactions among the genes wingless, decapentaplegic and distalless. In the allocation phase, dll is activated by wg but repressed by dpp. During imaginal disc patterning, dpp and wg cooperatively activate dll and also indirectly inhibit the nuclear localization of Extradenticle (Exd), which divide the leg into distal and proximal domains. In the grasshopper Schistocerca americana, the early expression pattern of dpp differs radically from the Drosophila pattern, suggesting that the genetic interactions that allocate the leg differ between the two species (Jockusch, 2000).

At the earliest accessible stages in grasshopper development (approx. 15% to 20% of development), dpp is expressed in two partial stripes in each hemisegment, paralleling the D/V axis. One stripe lies roughly in the middle of the segment and the other lies near the presumed intersegmental boundary. This pattern does not resemble any dpp expression pattern seen in the early Drosophila embryo. dpp is also expressed along the periphery of the germ anlage, a domain that corresponds to the most dorsal longitudinal stripe of dpp expression in the Drosophila embryo. This dorsal stripe persists throughout the stages examined in this study. There are no additional longitudinal stripes or modulation of expression along the D/V axis of the embryo, as seen in Drosophila embryos. Thus, early grasshopper dpp expression does not parallel Drosophila dpp allocation phase expression. Despite early differences in dpp expression, Wg, Dll and Exd are expressed in similar patterns throughout the development of grasshopper and fly legs, suggesting that some aspects of proximodistal (P/D) patterning are evolutionarily conserved. Differences in later dpp expression, which suggests that dpp likely plays a role in limb segmentation in Schistocerca, but not in Drosophila, are also observed. Specifically, a series of intrasegmental rings of dpp expression appear prior to leg segmentation in grasshoppers. The divergence in dpp expression is surprising given that all other comparative data on gene expression during insect leg development indicate that the molecular pathways regulating this process are conserved. However, it is consistent with the early divergence in developmental mode between fly and grasshopper limbs (Jockusch, 2000).

Insects can be grouped into two main categories, holometabolous and hemimetabolous, according to the extent of their morphological change during metamorphosis. The three thoracic legs, for example, are known to develop through two overtly different pathways: holometabolous insects make legs through their imaginal discs, while hemimetabolous legs develop from their leg buds. Thus, how the molecular mechanisms of leg development differ from each other is an intriguing question. In the holometabolous long-germ insect, these mechanisms have been extensively studied using Drosophila melanogaster. However, little is known about the mechanism in the hemimetabolous insect. Leg development of the hemimetabolous short-germ insect, Gryllus bimaculatus (cricket), has been studied focusing on expression patterns of the three key signaling molecules, hedgehog, wingless and decapentaplegic, which are essential during leg development in Drosophila. In Gryllus embryos, expression of hh is restricted in the posterior half of each leg bud, while dpp and wg are expressed in the dorsal and ventral sides of its anterior/posterior (A/P) boundary, respectively. Their expression patterns are essentially comparable with those of the three genes in Drosophila leg imaginal discs, suggesting the existence of the common mechanism for leg pattern formation. However, expression pattern of dpp is significantly divergent among Gryllus, Schistocerca (grasshopper) and Drosophila embryos, while expression patterns of hh and wg are conserved. Furthermore, the divergence is found between the pro/mesothoracic and metathoracic Gryllus leg buds. These observations imply that the divergence in the dpp expression pattern may correlate with diversity of leg morphology (Niwa, 2000).

In the allocation phase of Drosophila 5h embryos, wg and hh are expressed in a stripe along the A/P compartment boundary and in the posterior region of each segment, respectively. However, dpp is expressed throughout the dorsal region and then in the dorsal side of the wg stripe. Later, the expression changes to give two thin stripes running anteroposteriorly along the length of the embryo. Wg, but not Dpp, is responsible for initial specification of the limb primordia with a distal identity and for induction of Dll. A model for the allocation of the limb primordium (the G-H model) is presented. A stripe of Wg induces the limb primordium expressing Dll. Repression of Dll by Dpp from the dorsal side and by Spitz (Drosophila EGF) from the ventral side limits the limb formation only in the lateral position. Then, Dpp specifies proximal cell identity in the primordium in a concentration-dependent manner. In Gryllus and Schistocerca embryos, expression of wg is detected in a stripe along the A/P compartment boundary of the body segment. In Gryllus embryos, expression of dpp is first detected along the periphery of the germ band. Similar expression patterns have been observed in Tribolium. Although the cricket and grasshopper belong to the same Orthoptera, the expression patterns of Sadpp are more complicated than those of Gbdpp. In Schistocerca embryos at early stages, Sadpp is expressed in two partial stripes in each hemisegment, intrasegmentally and intersegmentally, paralleling the D/V axis. The intrasegmental stripes extend along both dorsal and ventral sides of the presumptive leg field. Early expression patterns of Gbdpp resemble those of Dmdpp or Tcdpp more closely than those of Sadpp. Thus, the wg expression pattern appears conserved in the allocation phase, while early expression patterns of dpp seems divergent even in the Orthoptera. Thus, more data are necessary to judge whether the G-H model is also applicable as a model for initiation of limb formation in other insects (Niwa, 2000 and references therein).

In Phase 2, in the Drosophila leg imaginal disc, hh is expressed in the posterior compartment of the disc, determining the A/P pattern, and induces dpp and wg expression in the dorsal and ventral side of the A/P boundary, respectively. They act cooperatively in a concentration-dependent manner to organize the P/D axis and induce expression of Dll at the center of the disc. In Gryllus and Schistocerca limb buds, since hh and wg are expressed in the posterior and the ventral side of the A/P boundary, respectively, their functions during limb development should be conserved among the fly, cricket, beetle and grasshopper. However, expression patterns of Gbdpp are considerably different from those of Drosophila dpp: Gbdpp expression is limited to a dorsal stripe, transiently around the time of limb bud emerging, at stage 6-7. At this time, expression of Dll was found in the distal tip of the limb bud. This transient expression pattern also occurs in Schistocerca embryos. In Drosophila, removal of Dpp signaling prior to the second larval instar results in loss of Dll expression, while later removal of Dpp does not affect Dll expression, indicating that Dpp is required for the initiation but not maintenance of Dll transcription. Thus, it is reasonable to consider that transient dpp expression is enough to induce expression of Dll, which is required for the P/D leg pattern formation (Niwa, 2000 and references therein).

In the Drosophila leg imaginal disc, dpp expression is restricted to a stripe in the dorsal side of the anterior compartment. Hh induces expression of dpp in the dorsal side along the A/P boundary, and is repressed in the posterior leg compartment by the En protein. In the Gryllus leg buds, however, Gbdpp is expressed as four spots in the dorsal side of the A/P boundary at stage 9, then at stages 10-11, expression domains transform into a nearly circumferential ring that is located roughly in the middle of the primary leg segments, as observed in Schistocerca. Since GbEn and Gbhh are always expressed throughout the posterior compartment, the relation between the expression domain of Drosophila dpp with that of hh/en does not hold true in the Gryllus leg bud, indicating divergent roles of dpp in leg patterning. Sadpp may play roles in establishment of boundaries between the leg segments, because the appearance of segmentally reiterated rings of Sadpp precedes morphological segmentation of leg buds. This idea may be applicable to Gryllus leg buds (Niwa, 2000 and references therein).

For the patterning along the proximodistal (P/D) axis in the Drosophila limb disc, two models have been proposed: the gradient model and the intercalation model. In the gradient model, it is proposed that since Wg and Dpp are secreted from the respective ventral and dorsal halves of the cells at the A/P compartment boundary, forming a gradient along the P/D axis, differential response to this gradient leads to circular patterns corresponding to the leg segments. However, in the intercalation model, it has been proposed that after formation of extreme proximal and distal structures by Dpp and Wg, the intermediate leg segments are intercalated by a signal from the proximal domain. In the case of Gryllus limb buds, the intercalation model appears favorable, because the discrete expression of Gbdpp can not maintain the Dpp gradient in the limb bud necessary for circular patterns corresponding to the leg segments. Furthermore, intercalation of the leg segments occurs during development of Gryllus leg buds: the most distal part is formed at first, then limb segmentation occurs at the coxopodite/telopodite boundary, followed by the femur/tibia, trochanter/femur and tibia/tarsus boundaries, judged from the expression patterns of Dll, Gryllus homothorax, dachshund and aristaless. These results are consistent with those observed in the Schistocerca leg buds, using an Annulin antibody for detection of the limb segment boundary. The intercalation model may be supported also by the fact that regeneration of amputated cricket legs occurs through intercalation of the missing segments as observed in the cockroach leg (Niwa, 2000 and references therein).

For establishment of the dorsoventral pattern of the Drosophila leg disc, it has been suggested that the two signals of Wg and Dpp act antagonistically to repress each otherís expression and to specify dorsal and ventral expression patterns. However, in later stages, in early third instar to early pupal leg discs of Drosophila, dpp is expressed in both dorsal and ventral sides. This faint ventral expression in the wg expression domain may correspond to the ventral expression of Gbdpp in the region where Gbwg is expressed intensely. These observations indicate that expression of the two genes may be regulated independently in later stages. It is interesting to note that the Gbdpp expression pattern changes from rings to the intense dorsal and ventral stripes, when each leg segment begins to change from a circular shape to an elliptical shape extended dorsoventrally at stage 11. Thus, the dorsal and ventral expressions of dpp are likely to correlate with the dorsoventral extension of the leg bud. Furthermore, the Gbdpp expression patterns are different between the metathoracic (T3) leg bud and pro/mesothoracic (T1/T2) leg buds in stage 11-12: the circumferential band of Gbdpp expression in the femur and tibia of the T3 leg segment becomes more intense and wider than that in the femur and tibia of the T1/T2 leg segments. This may be related to the fact that the Gryllus adult T3 leg, which has large muscles in the dorsal and ventral side of the femur and tibia for jumping, is several times larger than the T1 and T2 legs. Since the appearance of these segmentally reiterated rings of Gbdpp precedes elongation of leg segments, Gbdpp may be involved in outgrowth of the leg segment (Niwa, 2000 and references therein).

It is speculated that the similar difference in expression patterns of Sadpp among the three legs should be observed in the Schistocerca embryo. However, since expression patterns of Sadpp in the corresponding stages have not been reported so far, this speculation cannot be verified. In Drosophila, in which there is little difference in morphology among the Drosophila thoracic legs, no difference is reported in expression patterns of dpp among the three leg imaginal discs. Thus, the expression patterns of dpp are likely to correlate with leg morphology, implying that temporal and spatial regulations of the dpp gene expression are closely correlated with morphological diversity. In conclusion, although leg developmental pathways are different among insect species, the principal mechanism of leg development is likely to be conserved, judged from expression patterns of hh, wg and dpp. Divergence in dpp expression patterns during leg development is likely to correlate with morphological diversity of the leg (Niwa, 2000).

To understand the mechanism of regeneration, many experiments have been carried out with hemimetabolous insects, since their nymphs possess the ability to regenerate amputated legs. Patterns of hedgehog, wingless, and decapentaplegic expression were examined during leg regeneration of the cricket Gryllus bimaculatus. The observed expression patterns are essentially consistent with the predictions derived from the boundary model modified by Campbell and Tomlinson (CTBM). Thus, it is concluded that the formation of the proximodistal axis of a regenerating leg is triggered at a site where ventral wg-expressing cells abut dorsal dpp-expressing cells in the anteroposterior (A/P) boundary, as postulated in the CTBM (Mito, 2002).

In the cricket leg, the single layer of surface epidermal cells forms precise patterns of structures, including bristles and spines, in the overlying cuticle. The regional specialization of the leg epidermal cells is evident along the three major axes of the leg, which include the anteroposterior (A/P), dorsoventral (D/V), and proximodistal (P/D) axes. The P/D axis relates to the distance from the body trunk, while the A/P and D/V axes unite to form the single circumferential axis. When a metathoracic leg of a Gryllus nymph in the third instar is amputated at the tibia, the distal missing part is completely recovered after about 30-35 days through four molts subsequent to the amputation. Just after the amputation, a trachea running along the P/D axis, reticulate fat bodies, and muscles are observed in sagittal sections. By 6 h after amputation, wounded muscles already start to degenerate, while hemocytes aggregate in the wound to form a scab. By day 2, epidermal cells migrate over the wound surface, and epidermal continuity is restored underneath the scab. Cell proliferation can be detected in epidermis lining the scab during this process. By day 5, the wound epidermis thickens to form a regeneration bud, or blastema, and cell proliferation is greatly activated in the blastema. Cells in the blastema lose their differentiated character and start to grow. By day 7, the blastema becomes the primordia of the tibia and tarsus concomitant with muscle recovery. By day 10, the boundary of the tibia-tarsus is visible in the blastema. Finally, all of the structures that normally lie distal to the point of amputation are restored (Mito, 2002).

In normally developing cricket leg buds, hh i expressed in the posterior (P) compartment, while wg and dpp are expressed in the ventral (V) side and dorsal (D) side of the anteroposterior (A/P) boundary, respectively. In a normal leg at the stage corresponding to the regeneration samples, hybridization signals for hh are weakly detected in epidermal cells located in the posterior region, whereas the expressions of wg and dpp are not observed. In contrast, the induced expressions of hh, wg, and dpp are observed in the blastemata of regenerating legs. The expression signals of hh are localized on the posterior side of the leg epidermis. The localization of the En protein was examined in cryosections with the monoclonal antibody mAb4D9. Signals were detected in both sagittal and transverse sections, indicating that En is localized in the posterior half of epidermis and supporting the results for hh. In the transverse sections, the En expression domain looks slightly broader than that of hh (Mito, 2002).

The expression pattern of wg is clearly observed in the ventral region of the blastema with a distal-to-proximal gradient in the signal intensity. The signals of the dpp expression are much weaker than the wg signals. Furthermore, there was variation in the expression patterns. Since such variation was not observed in the wg expression pattern, it is considered that the expression pattern of dpp is dynamically changed, as observed during leg development. The observed expression patterns of dpp were classified mainly into three types: Type I, with signals restricted in dorsodistal epithelial cells of the blastema, where intense non-specific signals appear in the trachea due to longer staining reactions; Type II, with signals observed in dorsal and distal epithelial cells, and weakly in ventral cells; and Type III, with signals so weak that no pattern is discernible (n=24). Type I expression patterns are observed in the early stages, while Type II patterns are observed even in the later stages (~4 days). Therefore, it is reasonable to consider that the expression pattern of dpp changes from Type I to Type II as the regeneration proceeds (Mito, 2002).

The expression patterns of wg and dpp in the blastema are comparable to those in the leg bud of the cricket embryo. In particular, the discrete expression of dpp (Type I) observed in the blastema is also observed in the dorsal side along the A/P boundary in the cricket leg bud, which differs from the expression of dpp in the leg imaginal disc. However, a major difference between the leg bud and blastemata is the size of the wg/dpp expression boundary: the boundary becomes a line in the blastema, similar to the apical ectodermal ridge of vertebrate limb buds, rather than a point in the insect leg bud. After wound healing, the restoration of the epidermal continuity results in the formation of a D/V boundary where dpp-expressing epidermal cells abut wg-expressing cells, which possibly initiates the formation of the P/D axis in the regeneration blastema (Mito, 2002).

The Drosophila genes wingless and decapentaplegic comprise the top level of a hierarchical gene cascade involved in proximal-distal (PD) patterning of the legs. It remains unclear, whether this cascade is common to the appendages of all arthropods. Here, wg and dpp are studied in the millipede Glomeris marginata, a representative of the Myriapoda. Glomeris wg (Gm-wg) is expressed along the ventral side of the appendages compatible with functioning during the patterning of both the PD and dorsal-ventral (DV) axes. Gm-wg may also be involved in sensory organ formation in the gnathal appendages by inducing the expression of Distal-less (Dll) and H15 in the organ primordia. Expression of Glomeris dpp (Gm-dpp) is found at the tip of the trunk legs as well as weakly along the dorsal side of the legs in early stages. Taking data from other arthropods into account, these results may be interpreted in favor of a conserved mode of WG/DPP signaling. Apart from the main PD axis, many arthropod appendages have additional branches (e.g., endites). It is debated whether these extra branches develop their PD axis via the same mechanism as the main PD axis, or whether branch-specific mechanisms exist. Gene expression in possible endite homologs in Glomeris argues for the latter alternative. All available data argue in favor of a conserved role of WG/DPP morphogen gradients in guiding the development of the main PD axis. Additional branches in multibranched (multiramous) appendage types apparently do not utilize the WG/DPP signaling system for their PD development. This further supports recent work on crustaceans and insects, that lead to similar conclusions (Prpic, 2004).

The mechanism by which Decapentaplegic (Dpp) and its antagonist Short gastrulation (Sog) specify the dorsoventral pattern in Drosophila embryos has been proposed to have a common origin with the mechanism that organizes the body axis in the vertebrate embryo. However, Drosophila Sog makes only minor contributions to the development of ventral structures that hypothetically correspond to the vertebrate dorsum where the axial notochord forms. In this study, a homologue of the Drosophila sog gene was isolated in the spider Achaearanea tepidariorum, and its expression and function were characterized. Expression of sog mRNA initially appears in a radially symmetrical pattern and later becomes confined to the ventral midline area, which runs axially through the germ band. RNA interference-mediated depletion of the spider sog gene leads to a nearly complete loss of ventral structures, including the axial ventral midline and the central nervous system. This defect appeared to be the consequence of dorsalization of the ventral region of the germ band. By contrast, the extra-embryonic area forms normally. Furthermore, embryos depleted for a spider homologue of dpp failed to break the radial symmetry, displaying evenly high levels of sog expression except in the posterior terminal area. These results suggest that dpp is required for radial-to-axial symmetry transformation of the spider embryo and sog is required for ventral patterning. It is proposed that the mechanism of spider ventral specification largely differs from that of the fly. Interestingly, ventral specification in the spider is similar to the process in vertebrates in which the antagonism of Dpp/BMP signaling plays a central role in dorsal specification (Akiyama-Oda, 2006).

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).

BMP processing

Bone morphogenetic protein-4 (BMP-4) is a multifunctional developmental regulator. BMP-4 is synthesized as an inactive precursor that is proteolytically activated by cleavage following the amino acid motif -Arg-Ser-Lys-Arg-. Very little is known about processing and secretion of BMPs. The proprotein convertases (PCs) are members of a family of seven structurally related serine endoproteases, at least one of which, furin, cleaves after the amino acid motif -Arg-X-Arg/Lys-Arg-. To examine the potential roles of PCs during embryonic development, a potent protein inhibitor of furin, alpha1-antitrypsin Portland (alpha1-PDX), was expressed in early Xenopus embryos. Ectopic expression of alpha1-PDX phenocopies the effect of blocking endogenous BMP activity, leading to dorsalization of mesoderm and direct neural induction. alpha1-PDX-mediated neural induction can be reversed by co-expression of downstream components of the BMP-4 signaling pathway. Thus, alpha1-PDX can block BMP activity upstream of receptor binding, suggesting that it inhibits an endogenous BMP-4 convertase(s). Consistent with this hypothesis, alpha1-PDX prevents cleavage of BMP-4 in an oocyte translation assay. Using an in vitro digestion assay, it is demonstrated that four members of the PC family have the ability to cleave BMP-4, but of these, only furin and PC6B are sensitive to alpha1-PDX. These studies provide the first in vivo evidence that furin and/or PC6 proteolytically activate BMP-4 during vertebrate embryogenesis (Cui, 1998).

The Xolloid secreted metalloprotease, a Tolloid-related protein, cleaves and inactivates Chordin and cleaves Chordin/BMP-4 complexes at two specific sites in biochemical experiments. Xolloid mRNA blocks secondary axes caused by chordin, but not by noggin, follistatin, or dominant-negative BMP receptor, mRNA injection. Xolloid-treated Chordin protein is unable to antagonize BMP activity. 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 the 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: that is, XOLL inhibits Chordin, which in turn inhibits the BMPs. BMPs function to activate BMP receptors. Consequently Xolloid indirectly regulates BMP function by its inactivation of Chordin (Piccolo, 1997).

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, 1999).

Bone morphogenetic proteins (BMPs) are derived from inactive precursor proteins by endoproteolytic cleavage. Processing of Nodal and Myc-tagged BMP4 has been shown to be significantly enhanced by SPC1/Furin or SPC4/PACE4, thus providing direct evidence that regulation of BMP signaling is likely to be controlled by subtilisin-like proprotein convertase (SPC) activities. Nodal processing is dramatically enhanced if two residues adjacent to the precursor cleavage site are substituted with amino acids found at the equivalent positions of Activin, demonstrating that structural constraints at the precursor cleavage site limit the processing efficiency. However, in transfection assays, mature Nodal is undetectable either in culture supernatants or in cell lysates, despite efficient cleavage of the precursor protein, suggesting that mature Nodal is highly unstable. Domain swap experiments support this conclusion since mature BMP4 or Dorsalin are also destabilized when expressed in conjunction with the Nodal pro domain. By contrast, mature Nodal is stabilized by the Dorsalin pro domain, which mediates the formation of stable complexes. Collectively, these data show that the half-life of mature BMPs is greatly influenced by the identity of their pro regions (Constam, 1999).

Proteolytic maturation of proBMP-4 is required to generate an active signaling molecule. ProBMP-4 is cleaved by furin in a sequential manner. Cleavage at a consensus furin site (-R-X-R/K-R-; the S1 site) adjacent to the mature ligand domain allows for subsequent cleavage at an upstream nonconsensus furin site (-R-X-X-R-; the S2 site) within the prodomain. Consistent with the possibility that both of these sites are utilized in vivo, primary and upstream furin cleavage motifs are conserved in all known vertebrate BMP-2 and BMP-4 precursor proteins and in the Drosophila ortholog, Decapentaplegic (DPP), but not in other BMP superfamily members, such as BMP-7. BMP-4 synthesized from precursor in which the upstream site is noncleavable is less active, signals at a shorter range, and accumulates at lower levels than does BMP-4 cleaved from native precursor. Conversely, BMP-4 cleaved from precursor in which both sites are rapidly cleaved is more active and signals over a greater range. Differential use of the upstream cleavage site could provide for tissue-specific regulation of BMP-4 activity and signaling range (Cui, 2001).

Twisted gastrulation (TSG) is an extracellular modulator of bone morphogenetic protein (BMP) activity and regulates dorsoventral axis formation in early Drosophila and Xenopus development. Studies on tsg-deficient mice also indicate a role of this protein in skeletal growth, but the mechanism of TSG activity in this process has not yet been investigated. This study shows by in situ hybridization and immunohistochemistry that TSG is strongly expressed in bovine and mouse growth plate cartilage as well as in fetal ribs, vertebral cartilage, and cartilage anlagen of the skull. Furthermore, evidence is provided that TSG is directly involved in BMP-regulated chondrocyte differentiation and maturation. In vitro, TSG impairs the dose-dependent BMP-2 stimulation of collagen II and X expression in cultures of MC615 chondrocytes and primary mouse chondrocytes. In the presence of chordin, a BMP antagonist, the inhibitory effect of TSG was further enhanced. TSG also inhibits BMP-2-stimulated phosphorylation of Smad factors in chondrocytes, confirming the role of TSG as a modulator of BMP signaling. For analysis of TSG functions in cartilage development in vivo, the gene was overexpressed in transgenic mice under the control of the cartilage-specific Col2a1 promoter. As a result, Col10a1 expression was significantly reduced in the growth plates of transgenic embryos and newborns in comparison with wild type littermates as shown by in situ hybridization and by real time PCR analysis. The data suggest that TSG is an important modulator of BMP-regulated cartilage development and chondrocyte differentiation (Schmidl, 2006).

BMP interactions other TGFß family members and with its receptors

Nodal ligands are essential for the patterning of chordate embryos. Genetic studies have revealed that Nodal and its orthologs require EGF-CFC factors for their biological effects. EGF-CFC factors are membrane proteins that attach to the extracellular surface via glycosylphosphatidylinositol (GPI) linkages. They are named for two conserved motifs common to all EGF-CFC family members: (1) a region with homology to epidermal growth factor (the EGF-like motif), and (2) a region with homology specific to the EGF-CFC family (the CFC motif). To date, seven EGF-CFC factors are known: human Cripto and human Cryptic, mouse Cripto and mouse Cryptic, Xenopus FRL-1, zebrafish one-eyed pinhead (Oep), and chick Cripto. The role of Cripto in Nodal signaling has been investigated. Cripto interacts with the type I receptor ALK4 via the conserved CFC motif in Cripto. Cripto interaction with ALK4 is necessary both for Nodal binding to the ALK4/ActR-IIB receptor complex and for Smad2 activation by Nodal. Nodal can inhibit BMP signaling by a Cripto-independent mechanism. Inhibition appears to be mediated by heterodimerization between Nodal and BMPs, indicating that antagonism between Nodal and BMPs can occur at the level of dimeric ligand production (Yeo, 2001).

While Nodal-BMP7 heterodimers have not yet been identified in vivo, the finding that Nodal's affinity for BMP7 in heterodimerization is similar to Nodal's affinity for itself, strongly suggests that this heteromeric interaction can occur in vivo in tissues in which Nodal and BMPs are expressed at similar levels. This heterodimerization provides a novel, intracellular mechanism for the patterned inhibition of BMP signals. This intracellular inhibitory mechanism is significant in that in this case a BMP-antagonizing signal would be restricted to Nodal-expressing cells and not propagated into adjacent cell layers. In this model, Nodal antagonism of BMP signaling would complement diffusible BMP antagonists to permit the generation of spatially complex patterns of BMP antagonism (Yeo, 2001).

Bone morphogenetic protein-2 (BMP-2) induces bone formation and regeneration in adult vertebrates and regulates important developmental processes in all animals. BMP-2 is a homodimeric cysteine knot protein that, as a member of the transforming growth factor-ß superfamily, signals by oligomerizing type I and type II receptor serine-kinases in the cell membrane. The binding epitopes of BMP-2 for BMPR-IA (type I) and BMPR-II or ActR-II (type II) were characterized using BMP-2 mutant proteins for analysis of interactions with receptor ectodomains. A large epitope 1 for high-affinity BMPR-IA binding was detected spanning the interface of the BMP-2 dimer. A smaller epitope 2 for the low-affinity binding of BMPR-II was found to be assembled by determinants of a single monomer. Symmetry-related pairs of the two juxtaposed epitopes occur near the BMP-2 poles. Mutations in both epitopes yield variants with reduced biological activity in C2C12 cells; however, only epitope 2 variants behave as antagonists, partially or completely inhibiting BMP-2 activity. These findings provide a framework for the molecular description of receptor recognition and activation in the BMP/TGF-ß superfamily (Kirsch, 2000).

The identification and characterization of two distinct binding epitopes in human BMP-2 as well as the detection of antagonistic BMP-2 variants, provides new insights into the primary steps and mechanism of BMP receptor activation. Receptor-binding epitopes have not been described before for any of the closely related members of the TGF-ß superfamily that signal via type I and type II receptor serine-kinases. All TGF-ß-like proteins are dimers, usually homodimers, where the monomers have been compared with an open hand, with the central alpha-helix (alpha3) being the wrist or heel and the two aligned two-stranded ß-sheets representing the four fingers, with loop 1 and loop 2 at the tips of each pair of fingers. The N-terminal segment exists at the position of the thumb. Consequently, the epitope 1 assembled around the central alpha-helix is called in the following the 'wrist epitope' and epitope 2 located at the back of the hand near the outer finger segments is called the 'knuckle epitope' (Kirsch, 2000 and references therein).

The wrist epitope has dimensions of ~20 x 25-30 Å (500-600 Ų). This large area would be compatible with the function as a high-affinity interaction site. The knuckle epitope seems to be smaller with dimensions of 10 x 20-25 Å (200-250 Ų) in accordance with the lower affinity of the interaction with BMPR-II at this site. The wrist epitope is highly discontinuous and it comprises different elements of both monomers. In heterodimers, e.g. of BMP-4 and BMP-7 or of the inhibin/activin-type factors, this has the interesting consequence that the two symmetry-related epitopes are no longer equivalent and may therefore exhibit different receptor-binding properties. In the knuckle epitope, the binding residues are provided by one monomer only and are located in sheets ß3, ß4, ß7 and ß8 and possibly also in ß9 (E109)(Kirsch, 2000 and references therein).

The juxtaposed knuckle and wrist epitopes are only 10-15 Å apart. The distances between the two type I (~40 Å) or the two type II chains (~55 Å), that possibly are assembled at BMP-2, are much larger. This appears to be especially relevant for the receptor serine-kinases. Their small ectodomain is connected to the membrane-spanning segment by a short linker of <12 residues. In the receptor complex with BMP-2, this short distance between epitopes 1 and 2 might facilitate the interaction of the type I and type II receptor serine-kinases (Kirsch, 2000).

The occurrence of the BMP-2 antagonists detected in this study is most likely to be a consequence of an ordered sequential binding mechanism operating during receptor activation. The antagonist blocks the high-affinity type I receptor chain via its intact wrist epitope, and the disrupted knuckle epitope prevents the subsequent interaction with the low-affinity type II chain(s). The similarly low IC₅₀ of the antagonists as well as their efficient competition with BMP-2 for receptor binding could indicate that it is predominantly the type I chain(s) that adjust(s) the binding affinity of BMP-2 for the whole receptor complex. Interestingly, the velocity of complex formation and dissociation with BMPR-IA is equally critical, as revealed by the complete loss of biological activity of the respective double mutants. An intriguing finding is the dramatic loss of biological activity in variants of the knuckle epitope, considering that binding to the ectodomains of the type II receptor chain(s) is reduced only 10- to 15-fold. It is possible that the simultaneous binding of two type II chains is necessary for an efficient receptor activation, and therefore a decrease in binding affinity becomes aggravated (Kirsch, 2000 and references therein).

Activins and bone morphogenetic proteins (BMPs) elicit diverse biological responses by signaling through two pairs of structurally related type I and type II receptors. The crystal structure is reported of BMP7 in complex with the extracellular domain (ECD) of the activin type II receptor. The structure produces a compelling four-receptor model, revealing that the types I and II receptor ECDs make no direct contacts. Nevertheless, truncated receptors lacking their cytoplasmic domain retain the ability to cooperatively assemble in the cell membrane. Also, the affinity of BMP7 for its low-affinity type I receptor ECD increases 5-fold in the presence of its type II receptor ECD. Taken together, these results provide a view of the ligand-mediated cooperative assembly of BMP and activin receptors that does not rely on receptor-receptor contacts (Greenwald, 2003).

BMP interaction with components of the extracellular matrix

During early development, cells receive positional information from neighboring cells to form tissue patterns in initially uniform germ layers. Ligands of the transforming growth factor (TGF-ß) superfamily are known to participate in this pattern formation. In particular, activin has been shown to act as a long-range dorsalizing signal to establish a concentration gradient in Xenopus. In contrast, BMP-2 and BMP-4, other members of the family, appear to influence and induce ventral fates only where they are expressed. This raises a question as to how the action of BMPs is tightly restricted to the region within and around the cells that produce them. This study demonstrates that a basic core of only three amino acids in the N-terminal region of BMP-4 is required for BMP-4 restriction to the non-neural ectoderm as its expression domain. It is proposed that the basic amino acids in the N-terminal region of BMP-4 and, particularly, the RKK residues play an essential role in conferring on BMP-4 a short-range action in the animal cap. The motif is conserved from flies to mammals. These results also suggest that heparan sulfate proteoglycans bind to this basic core and thus play a role in trapping BMP-4. The present study is the first to identify the critical domain of BMP that is responsible for its interaction with the extracellular environment that restricts its diffusion in vivo (Ohkawara, 2002).

Glypicans represent a family of six cell surface heparan sulfate proteoglycans in vertebrates. Although heparan sulfate is found ubiquitously on cell surfaces, these carbohydrates are carried predominantly by the protein products of only two gene families, syndecans and glypicans. All members of the glypican family share a common mechanism of attachment to the cell membrane through glycosylphosphatidylinositol linkages, and the characterization of amino acid sequence homologies has suggested the existence of subfamilies of structurally related vertebrate glypicans. Although no specific in vivo functions have thus far been described for these proteoglycans, spontaneous mutations in the human and induced deletions in the mouse glypican-3 (Gpc3) gene result in severe malformations and both pre- and postnatal overgrowth, known clinically as the Simpson-Golabi-Behmel syndrome (SGBS). Mice carrying mutant alleles of Gpc3 created by either targeted gene disruption or gene trapping display a wide range of phenotypes associated with SGBS, including renal cystic dysplasia, ventral wall defects, and skeletal abnormalities that are consistent with the pattern of Gpc3 expression in the mouse embryo. Studies in Drosophila have implicated glypicans in the signaling of decapentaplegic, a BMP homolog. Experiments with mice show a significant relationship between vertebrate BMP signaling and glypican function; GPC3-deficient animals were mated with mice haploinsufficient for bone morphogenetic protein-4 (Bmp4) and their offspring displayed a high penetrance of postaxial polydactyly and rib malformations not observed in either parent strain. This previously unknown link between glypican-3 and BMP4 function provides evidence of a role for glypicans in vertebrate limb patterning and skeletal development and suggests a mechanism for the skeletal defects seen in SGBS (Paine-Saunders, 2000).

There are multiple mechanisms by which GPC3 and its Drosophila homolog Dally could function. For example, they could affect a parallel signaling pathway that alters cellular responses to BMPs. Alternatively, they could more directly modulate the BMP signaling pathway. By analogy with the role of another heparan sulfate proteoglycan, betaglycan, which directly potentiates TGF-beta binding to its signaling receptor, it has been suggested that Dally could function as a coreceptor for Dpp: GPC3 could have a similar function with respect to BMP4 in the vertebrate limb. Consistent with this model, Drosophila Dpp and vertebrate BMP2, -3, -4, and -7 all bind to heparin with high affinity in vitro; therefore, they likely bind to heparan sulfate in vivo. However, the consequence of this interaction to BMP function remains unclear. For example, although heparin potentiates the activity of BMP2 in a chick limb bud differentiation model in vitro, as would be expected if it were serving a coreceptor function, so does destruction of the N-terminal heparin binding domain of BMP2 or addition of a peptide corresponding to the N-terminal heparin binding domain of BMP2 in these same assays. These latter results can be interpreted as evidence that at least some heparan sulfate-containing proteins actually sequester BMPs in vivo and serve to suppress rather than enhance BMP functions (Paine-Saunders, 2000).

Another likely mechanism by which GPC3 could directly affect BMP4 activity in the developing limb could be through regulation of the distribution or level of accumulation of a BMP antagonist, several of which are known to bind to heparin with high affinity. In particular, the heparin binding BMP antagonist Gremlin has recently been shown to mediate the SHH/FGF4 feedback loop in vertebrate limb buds through direct BMP antagonism. Loss of the heparan sulfate proteoglycan GPC3 could result in the increased accumulation and/or a broader distribution of Gremlin protein in the developing limb fields, thus resulting in an increase in signaling through this feedback loop and therefore development of redundant postaxial structures (Paine-Saunders, 2000).

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