BIOLOGICAL OVERVIEW

Decapentaplegic is responsible for induction of dorsal-ventral polarity in the fly. Imagine the effects of its removal: the back of the fly fails to develop and becomes instead a neurogenic ectoderm resembling tissue usually found in the ventral portion of the trunk. The resulting fly would have two fronts and no back.

Saxophone, along with Thick veins and Punt, constitute the Drosophila receptor for decapentaplegic. These receptors mediate the transduction of DPP signals into the cell. Mutations in these genes have the same effect as mutation in dpp, since without DPP's receptors, its signals fail to be communicated to the cells that should receive them.

Saxophone has an intracellular kinase domain and a conserved extracellular cysteine reside structure. Kinases add phosphate resides to target proteins. This phosphorylation event is the main process by which receptors pass signals between proteins; the transfer of signals to downstream effectors. Along with Thickveins, SAX is homologous to type I TGF-beta receptors.

sax appears to be expressed more ubiquitously but required less ubiquitously than Thick veins, and SAX requires the function of both Thick veins and Punt (Wharton, 1995, and Ruberte, 1995). Since sax is only necessary for the specification of the dorsalmost cell fate (amnioserosa) which requires the highest levels of DPP activity, SAX might serve to interpret peak levels of DPP. Thus additional type I receptors for DPP, such as SAX, probably function to augment or modify a response to DPP mediated by the primary receptors encoded by Punt and TKV (Ruberte, 1995).

In Drosophila wing discs, a morphogen gradient of Dpp has been proposed to be a determinant of the transcriptional response thresholds of the downstream genes spalt (sal) and optomotor blind (omb). Evidence is presented that the concentration of the type I receptor Tkv must be low to allow long-range Dpp diffusion. However, low Tkv receptor concentrations result in low signaling activity. To enhance signaling at low Dpp concentrations, a second ligand, Tgf-beta-60A, has been found to augment Dpp/Tkv activity. Tgf-beta-60A signals primarily through the type I receptor Sax, which synergistically enhances Tkv signaling and is required for proper Omb expression. Omb expression in wing discs is found to require synergistic signaling by multiple ligands and receptors to overcome the limitations imposed on Dpp morphogen function by receptor concentration levels (Haerry, 1998).

The phenotypic consequences of overexpressing constitutively active forms of Tkv and Sax receptors in the developing wing was investigated using the GAL4-UAS system. The A9-Gal4 line was used: this drives high-level expression of Gal4 in the entire wing disc before it is restricted to the dorsal pouch at late third instar stage. In wild-type discs, the Sal and Omb products are symmetrically expressed along the anterior/posterior (A/P) boundary in response to Dpp. Normally, the Sal domain is restricted to cells in the wing pouch that are in close proximity to the Dpp-expressing cells, while Omb responds to lower levels of Dpp and is expressed in cells further away from the A/P boundary. The anterior boundary of Sal has been shown to specify the location where the longitudinal vein 2 (L2) is formed, while the formation of L5 coincides approximately with the posterior boundary of the Omb domain, but a causal relationship has not yet been established. When Dpp is ubiquitously expressed in wing discs, they become overgrown and the expression of both Sal and Omb is expanded. Like Dpp, overexpression of constitutively active Tkv (TkvA) also leads to disc overgrowth and ectopic induction of Sal and Omb. All cells in wings derived from animals expressing either Dpp or activated Tkv appear to differentiate into vein tissue, as exemplified by production of vein-specific morphological markers such as dark pigment and longer bristles. In contrast to TkvA, expression of either one or two copies of SaxA or development at 30ƒC, which results in an approximately twofold increase of Gal4 activity, is not sufficient to expand either Sal or Omb and produces only weak adult phenotypes consisting primarily of ectopic and thickened veins with a small amount of wing blistering in the region of the posterior cross vein. This phenotype is similar to that seen in animals raised at 18ƒC, which express low levels of TkvA. Although these findings suggest that Sax function may be qualitatively similar to that of Tkv but simply weaker, higher levels of activated Sax (four copies) still cannot mimic the effects of activated Tkv, such as the expansion of Sal and Omb (Haerry, 1998).

When high levels of activated Sax activity are combined with low levels of activated Tkv, the result is more than additive. The combination of one copy of saxA and low levels of Tkv leads to overgrowth, with the expansion of Omb (but not Sal), and results in a strong wing phenotype. The interaction of Sax and Tkv is synergistic. Taken together, these data suggest that Sax and Tkv synergistically interact and control the expression of a common target gene, omb. Activation of omb expression requires a level of signaling that can be activated by either high levels of Tkv activity alone or by a synergistic interaction between low levels of Tkv and high levels of Sax activity. In contrast, Sal activation requires a higher level of signaling, which can only be achieved by high levels of Tkv activity (Haerry, 1998).

Since both Tkv and Sax, as well as the type II receptor Put, have been implicated in mediating Dpp signaling, whether the loss in signal activity of these receptors would cause similar patterning defects in the wing was investigated. If these three receptors all bind the same ligand and signal to the same sets of downstream genes, it would be expected that a reduction in the activity of any individual receptor should result in qualitatively similar phenotypes that differ in severity only. Increasing levels of dominant negative receptors were expressed in different regions of the developing wing disc. Similar to using an allelic series of hypomorphic mutations, it was expected that expression of increasing copy numbers of dominant negative receptors should result in progressively more severe phenotypes. Ubiquitous expression of 3-4 copies of either form of two dominant negative Tkv1 constructs results in small wings with partial loss of L4 and both cross veins. In addition, L2 and L3 are closer together and the triple-row margin bristles are shifted more distally/posteriorly, as expected if the level of Dpp signal is reduced by titration of Dpp into nonproductive complexes. At higher levels (6-8 copies) of dominant negative Tkv1, very small adult wings are produced that show fusion of L2 and L3 as well as L4 and L5. Similar phenotypes are produced by expressing dominant negative versions of the alternative Tkv isoform that have an N-terminal extended extracellular domain, and also by expression of dominant negative Put. Both the Sal and the Omb domains are strongly reduced: the adult wings show fusion of L2 with L3 and L4 with L5. The wing phenotypes obtained with increasing levels of dominant negative Tkv and Put resemble those of certain combinations of dpp loss-of-function alleles, which is consistent with the notion that Dpp is primarily signaling through the combination of the Tkv and Put receptors. In contrast to these observations, dominant negative Sax constructs produce different results. When increasing copy numbers (1-8 copies) of dominant negative Sax are expressed, the discs become smaller and the Omb domain is reduced to the size of the normal Sal domain. But unlike expressing dominant negative Tkv, the Sal domain is not affected. In the adult wing, L5 and the posterior cross vein are lost compared to losing L3 and L4 after expression of dominant negative Tkv or Put. In addition, L2 is shifted more proximally and the proximal triple-row bristles that expand more distally/posteriorly in dominant negative Tkv wings are replaced by more proximal costa bristles. While the distance between L3 and L4 is normal, the overall shape of the wing becomes more ëstrap-likeí, suggesting loss of peripheral tissue rather than the central tissue that is deleted in animals expressing Tkv or Put dominant negative receptors. These results suggest that dominant negative Sax acts in a qualitatively different manner from dominant negative Tkv (Haerry, 1998).

These results indicate that while the reduction of Tkv and Put activity affects the whole disc (Sal, Omb and growth), the expression of dominant negative Sax only affects the peripheral region of the disc (Omb and peripheral growth). If the dominant negative receptors function primarily by titrating Dpp, then it is curious why the overexpression phenotypes of dominant negative Sax are different. One possibility is that these receptors do not simply signal in response to Dpp but also in response to the binding of other ligands as well. Of the other two BMP-type ligands that have been described in Drosophila, scw shows no detectable expression at this stage. However, Tgf-beta-60A is expressed broadly in wing discs, and mutant analyses indicate that Tgf-beta-60A is required for normal wing development. Given its role in wing patterning, the effects of heteroallelic Tgf-beta-60A mutations were examined on Sal and Omb expression. Similar to discs expressing dominant negative Sax, Sal expression in Tgf-beta-60A mutant discs is normal while the Omb domain is reduced, particularly in the dorsal compartment. These observations are consistent with the notion that a second BMP-type ligand, Tgf-beta-60A, is required in addition to Dpp for proper Omb expression. Furthermore, the similarity of the Tgf-beta-60A loss-of-function and the dominant negative Sax phenotypes is consistent with recently described genetic interactions between Tgf-beta-60A and sax mutations and suggests that Tgf-beta-60A could signal in part through Sax (Haerry, 1998).

Ubiquitous overexpression of moderate levels of Tgf-beta-60A does not result in excessive disc overgrowth and does not alter the distribution of Sal and Omb. The resulting wings are slightly larger and exhibit minor venation defects along L2 and L5. However, similar to Dpp or TkvA, higher levels of Tgf-beta-60A overexpression expands both Sal and Omb and results in blistered and pigmented adult wings. Since only activated Tkv but not Sax is able to expand Sal and Omb expression, these findings are consistent with the notion that expression of moderate levels of Tgf-beta-60A leads to signaling preferentially through Sax, producing relative mild phenotypes, while higher concentrations of Tgf-beta-60A may also result in signaling through Tkv, producing phenotypes similar to activated Tkv (Haerry, 1998).

An investigation was carried out to determine if Tgf-beta-60A contributes to wing development primarily in the form of homodimers or Tgf-beta-60A/Dpp heterodimers. Results: (1) the level of Tgf-beta-60A mRNA appears to be significantly less than that of DPP, based on RNA in situ hybridization, indicating that heterodimers are not likely to be very abundant assuming similar translational efficiencies. (2) Localized overexpression of Tgf-beta-60A in the dpp-expressing cells does not result in any mutant phenotypes. (3) Expression of Tgf-beta-60A in the posterior compartment results in overgrowth, an expansion of the Sal and Omb domains, and restriction all adult wing defects exclusively to the posterior compartment. Since Tgf-beta-60A expression in this experiment does not overlap with Dpp-secreting cells, no Dpp/Tgf-beta-60A heterodimers should form, since heterodimer formation requires expression of both proteins in the same cell. Therefore, Tgf-beta-60A functions most likely as a homodimer. This finding is consistent with recent genetic analysis showing that clones of Tgf-beta-60A mutant cells that do not include dpp-expressing cells nevertheless produce patterning defects. It has been shown that dominant negative Tkv is more potent than Sax for inhibiting Dpp signaling, while dominant negative Sax is a stronger suppressor than Tkv of Tgf-beta-60A signaling. High levels of Tkv receptor limit Dpp diffusion and restrict Omb expression (Haerry, 1998).

PROTEIN STRUCTURE

Amino Acids - 570

Structural Domains

saxophone was cloned on the basis of its containing homology to mammalian serine-threonine kinases. Degenerate primers were designed in order to isolate candidate receptors by polymerase chain reaction. The primers corresponded to the conserved cytoplasmic domains of the serine-threonine kinase receptors of activin and TGF-beta (Xie, 1994).

The serine-threonine kinase domains of the Drosophila and vertebrate receptors are 78% homologous, the two fly genes (sax and tkv) being no more closely related to one another than they are to their vertebrate homologs. The extracellular domains show resemblence only in the spacing of the cysteine residues (Nellen, 1994 and Xie, 1994).

EVOLUTIONARY HOMOLOGS

Two structural elements, the L45 loop on the kinase domain of the transforming growth factor-beta (TGF-beta) family type I receptors and the L3 loop on the MH2 domain of Smad proteins, determine the specificity of the interactions between these receptors and Smad proteins. The L45 sequence of the TGF-beta type I receptor (TbetaR-I) specifies Smad2 interaction, whereas the related L45 sequence of the bone morphogenetic protein (BMP) type I receptor (BMPR-I) specifies Smad1 interactions. Members of a third receptor group, which includes ALK1 and ALK2 from vertebrates and Saxophone from Drosophila, specifically phosphorylate and activate Smad1 even though the L45 sequence of this group is very divergent from that of BMPR-I. The structural elements that determine the specific recognition of Smad1 by ALK1 and ALK2 have been investigated. In addition to the receptor L45 loop and the Smad1 L3 loop, the specificity of this recognition requires the alpha-helix 1 of Smad1. The alpha-helix 1 is a conserved structural element located in the vicinity of the L3 loop on the surface of the Smad MH2 domain. Thus, Smad1 recognizes two distinct groups of receptors (the BMPR-I group and the ALK1 group) through different L45 sequences on the receptor kinase domain and a differential use of two surface structures on the Smad1 MH2 domain (Chen, 1999).

TGFbeta signaling pathways of the bone morphogenetic protein (BMP) subclass are essential for dorsoventral pattern formation of both vertebrate and invertebrate embryos. It has been determined, by chromosomal mapping, linkage analysis, cDNA sequencing and mRNA rescue, that the dorsalized zebrafish mutant lost-a-fin (laf) is defective in the gene activin receptor-like kinase 8 (alk8), which encodes a novel type I TGFbeta receptor. The alk8 mRNA is expressed both maternally and zygotically. Embyros that lack zygotic, but retain maternal Laf/Alk8 activity, display a weak dorsalization restricted to the tail and die by 3 days postfertilization. The laf dorsalized mutant phenotype was rescued by alk8 mRNA injection. Homozygous laf/alk8 mothers were generated to investigate the maternal role of Laf/Alk8 activity. Adult fish lacking Laf/Alk8 activity are fertile, exhibit a growth defect and are significantly smaller than their siblings. Embryos derived from homozygous females, which lack both maternal and zygotic Laf/Alk8 activity, display a strongly dorsalized mutant phenotype, no longer limited to the tail. These mutant embryos lack almost all gastrula ventral cell fates, with a concomitant expansion of dorsal cell types. During later stages, most of the somitic mesoderm and neural tissue circumscribe the dorsoventral axis of the embryo. Zygotic laf/alk8 mutants can be rescued by overexpression of the BMP signal transducer Smad5, but not the Bmp2b or Bmp7 ligands, consistent with the Laf/Alk8 receptor acting within a BMP signaling pathway, downstream of a Bmp2b/Bmp7 signal. Antibodies specific for the phosphorylated, activated form of Smad1/5, show that BMP signaling is nearly absent in gastrula lacking both maternal and zygotic Laf/Alk8 activity, providing further evidence that Laf/Alk8 transduces a BMP signal. In total, this work strongly supports the role of Laf/Alk8 as a type I BMP receptor required for the specification of ventral cell fates (Mintzer, 2001).

In Drosophila, two BMP type I receptors, Thickveins and Saxophone, act in the transduction of the Dpp and Screw signals, respectively. However, Dpp and Screw together with their respective receptors play non-equivalent roles in dorsoventral pattern formation, unlike the Bmp2b and Bmp7 ligands in the zebrafish. Thickveins and Dpp act in the establishment of all dorsal cell fates, while Saxophone and Screw specify only a subset. The Laf/Alk8 receptor, which is more similar to Saxophone phlyogenetically than Thickveins, plays an extensive role in dorsoventral pattern formation, more similar to Thickveins in the fly. It is possible that all Bmp2b and/or Bmp7 signaling acts through the Laf/Alk8 type I receptor in dorsoventral patterning; however, this does not exclude the role of additional type I receptors in this process. In the fly, Thickveins, Saxophone, and two Punt type II receptor subunits are proposed to form a tetrameric complex together with one Dpp and one Screw homodimer. Hence, additional type I receptors may function together with the Alk8/Laf receptor. Further studies are required to determine the roles of other BMP type I receptors in dorsoventral patterning in vertebrates (Mintzer, 2001).

Neural crest cells (NCCs) are pluripotent migratory cells that contribute to the development of various craniofacial structures. Many signaling molecules have been implicated in the formation, migration and differentiation of NCCs, including bone morphogenetic proteins (BMPs). BMPs signal through a receptor complex composed of type I and type II receptors. Type I receptors (Alk2, Alk3 and Alk6) are the primary determinants of signaling specificity and therefore understanding their function is important in revealing the developmental roles of molecular pathways regulated by BMPs. A Cre/loxP system has been used for neural crest specific deletion of Alk2. The results show that mice lacking Alk2 in the neural crest display multiple craniofacial defects, including cleft palate and a hypotrophic mandible. It is concluded that signaling via Alk2 receptors is non-redundant and regulates normal development of a restricted set of structures derived from the cranial neural crest (Dudas, 2004).

Cardiac neural crest cells are multipotent migratory cells that contribute to the formation of the cardiac outflow tract and pharyngeal arch arteries. Neural crest-related developmental defects account for a large proportion of congenital heart disorders. Recently, the genetic bases for some of these disorders have been elucidated, and signaling pathways required for induction, migration and differentiation of cardiac neural crest have emerged. Bone morphogenetic proteins comprise a family of secreted ligands implicated in numerous aspects of organogenesis, including heart and neural crest development. However, it has remained generally unclear whether BMP ligands act directly on neural crest or cardiac myocytes during cardiac morphogenesis, or function indirectly by activating other cell types. Studies on BMP receptor signaling during organogenesis have been hampered by the fact that receptor knockouts often lead to early embryonic lethality. A Cre/loxP system was used for neural crest-specific deletion of the type I receptor, ALK2, in mouse embryos. Mutant mice display cardiovascular defects, including persistent truncus arteriosus, and abnormal maturation of the aortic arch reminiscent of common forms of human congenital heart disease. Migration of mutant neural crest cells to the outflow tract is impaired, and differentiation to smooth muscle around aortic arch arteries is deficient. Moreover, in Alk2 mutants, the distal outflow tract fails to express Msx1, one of the major effectors of BMP signaling. Thus, the type I BMP receptor ALK2 plays an essential cell-autonomous role in the development of the cardiac outflow tract and aortic arch derivatives (Kaartinen, 2004).

date revised: 30 October 98

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