EvoprintHD of baboon

BIOLOGICAL OVERVIEW

Although there is compelling proof for the existence of invertebrate BMP-like signaling pathways, the evidence for invertebrate TGF-beta or Activin-like signaling pathways has been scant. Baboon (Babo) is an invertebrate Activin type I receptor: the characterization of Baboon may well be the first evidence for the existence of an Activin-like signaling pathway in Drosophila. Null mutations and germ-line clonal analysis demonstrate that babo is not required during embryogenesis but is essential for proper pupation and adult viability. Loss of babo function results in late larval or early pupal lethality. The major defect in these mutants is a reduction of cell proliferation within the primordia for adult structures, specifically imaginal discs and brain tissue. Activated Babo can signal to vertebrate TGF-beta/Activin, but not to BMP-responsive promoters in cell culture. Activated Babo cannot bind to or interact with Drosophila Mad in tissue culture but can utilize a new Drosophila Smad homolog, dSmad2 (Smad on X), which relates most closely to the vertebrate Smads 2 and 3. Drosophila dSmad2 is highly expressed in tissues that require babo function and can be phosphorylated by either overexpression of activated Babo or by overexpression of wild-type Punt and Babo together. On the basis of these results, it is proposed that an Activin-like signaling pathway exists in Drosophila, which is required for proper cell proliferation in many primordial adult tissues (Brummel, 1999).

Upon cloning of Baboon, it was first necessary to test whether the newly identified receptor acted as a receptor for the known BMP-type ligands in Drosophila. The reduced size of imaginal discs and brain tissue is not unique to babo mutations: this is also a characteristic feature of mutations in Drosophila BMP signaling pathways. In the embryo, all three characterized BMP ligands, Decapentaplegic (Dpp), Screw (Scw), and Glass bottom boat (Gbb), participate in specific developmental processes. Because Scw and Dpp are both critically important for formation of the amnioserosa tissue at the blastoderm stage, a Kruppel-lacZ line was used to follow the fate of amnioserosa tissue in babo mutants: such tissue was found to be unaffected. The role of Babo in midgut development was analyzed. Between stages 14 and 16, three constrictions form in the midgut producing a four-chambered vessel. Dpp is required for formation of the second midgut constriction, whereas gbb mutants lack the first constriction. Mutant embryos derived from babo germ-line clones exhibit a normal four-chambered midgut at stage 16. Furthermore, visceral mesoderm expression of the Gbb target gene Antennapedia, and the Dpp target gene Ubx, are both normal in babo mutants. In addition to these activities, both Dpp and Gbb are required for growth and patterning of imaginal discs and larval brains. Therefore, the expression of several target genes was examined in babo mutant discs. In wild-type discs, the genes optomotor blind (omb) and spalt (sal) respond to Dpp in a dose-dependent fashion. In addition, proper omb expression also requires input from the Gbb ligand. In leg discs, Dpp represses wg in the ventral portion of the disc. No alterations were found in the expression patterns of omb, sal, and wg in any babo mutant disc. These results indicate that babo does not likely function as a receptor for Dpp, Scw, or Gbb during Drosophila development (Brummel, 1999).

Clonal analysis of babo mutants as well as overexpression of constitutively activated Babo receptors was carried out to identify the developmental function of Babo. The results suggest that Babo primarily regulates cell proliferation and has only minimal affects on patterning. The consequences of expressing a constitutively active form of Babo (Babo*, Q302D) in imaginal discs via the UAS-Gal4 system was examined. Ubiquitous expression of the constitutively active receptor leads to tissue overgrowth in the wing, but only limited pattern abnormalities. Surface area measurements indicate an ~30% increase in wing size for a particular UAS-Babo* line. To determine whether this increase in wing surface area results from an increase in cell size or cell number, wing hairs (single apical extensions found on the surface of each wing blade cell) were counted within a fixed area at two locations on the dorsal surface of the wing. An ~20% increase in the density of cells at both positions was found, as compared with control flies. Assuming that there is no decrease in cell death, and taking into account the ~30% increase in wing size, these results suggest that one of every two cells undergoes an extra round of division during wing formation as a result of ectopically activated Babo expression (Brummel, 1999).

Because genetic data indicate that in Drosophila Babo is not a component of Dpp signaling, the specificity of Babo activity was examined in mammalian cell culture assays using several different pathway specific promoters. Using a TGF-beta/Activin responsive promoter, p3TP, fused to luciferase, cultured cells were cotransfected with the reporter alone or with wild-type forms of Babo or the mammalian TGFbeta type-I receptor, TbetaRI. In the absence of TGF-beta, only basal levels of luciferase activity are observed. Addition of TGF-beta or transfection with an activated TbetaRI results in a strong induction of the tagged promoter. A similar induction is observed when the constitutively active form of Babo is cotransfected with the tagged promoter. In contrast, cotransfection with the constitutively active Dpp receptor Tkv does not stimulate the TGF-beta/Activin responsive reporter. Similar results were obtained with a second Activin/TGF-beta responsive element (known as ARE). Cotransfection of cultured cells with the second tagged promoter and activated Babo results in a fivefold induction in luciferase activity. This stimulation is similar to levels of induction produced by the activated forms of mammalian receptors TbetaRI or ActRIB. Tkv fails to modulate induction of the second TGF-beta/Activin responsive promoter. Together these data indicate that Babo specifically activates a TGF/Activin-like pathway in mammalian cells and suggest that this receptor may regulate a Smad2/Smad3-like pathway in Drosophila (Brummel, 1999).

Baboon was shown to function through the newly identified dSmad2, a Drosophila homolog of mammalian Smad2 and Smad3, which function in Activin signaling. Activated Babo induces phosphorylation on the last two serines of dSmad2. These data suggest that dSmad2 is a downstream target of Babo and is phosphorylated on the last two serine residues in the carboxyl terminus. Babo-dependent phosphorylation of dSmad2 also induces association with Medea, a homolog of mammalian Smad4. Phosphorylation of dSmad2 on the last two serines is necessary for receptor-dependent induction of heteromeric complexes of dSmad2 and Medea. Mammalian Activin receptors require dimerization with a type II receptor for their function. Punt, the Drosophila type II receptor was shown to function to activate Babo. dSmad2 interacts transiently and specifically with Punt-Babo receptor complexes. Taken together, these functional and biochemical analyses strongly suggest that dSmad2 is a Drosophila homolog of Smad2/Smad3 and functions as a downstream signaling component that directly interacts with Babo (Brummel, 1999).

The simplicity of the babo loss-of-function phenotype in Drosophila is striking in light of the number of speculated roles for Activin signaling in vertebrates. In particular, the fact that babo loss-of-function mutations primarily affects cell growth and proliferation in late development, but not cell fate specification or patterning processes, stands in marked contrast to the situation in vertebrates in which Activin or Activin-like signaling pathways have been implicated in a wide range of early developmental events, as well as adult functions such as reproductive potential. Similarly, in Drosophila the BMP-like factor Dpp has been implicated in the control of cell fate, cell proliferation, and patterning processes. Thus, the Babo signaling pathway appears to be unique in that it seems to act primarily in only one of these interconnected processes. It should be recognized, however, that Babo signaling may contribute to some patterning processes and/or cell fate decisions. For example, in the brain, it has yet to be determined whether all of the morphological defects can be accounted for by reduced proliferation rates of certain neuroblast populations or whether there are also changes in cell fate specification. Likewise, the cause of the enlarged anal pads, observed in babo mutants, during larval development has not been investigated. As the anal organ is involved in regulating salt homeostasis in insects, the enlarged anal pads might simply represent swelling due to a salt imbalance or the inability to regulate water content. Larvae missing babo function also fail to exhibit a typical behavioral trait, which is to move away from moist food before pupation. Thus, the inability to assess hydration as the result of abnormal brain development may underlie both the enlarged anal pads and the failure to seek a drier climate for pupation (Brummel, 1999).

The Babo pathway appears to act in a positive manner to stimulate cell proliferation. How might Babo signaling couple to cell proliferation? Recent analysis of cell growth and division in the Drosophila wing imaginal discs has led to the conclusion that the cell division rate is normally coupled to increases in cell mass such that a relatively constant ratio between the two processes is maintained (Neufeld, 1998). Because mutations that affect protein synthesis, such as Minutes, retard cell proliferation without changing cell size, Neufeld favored a model in which tissue growth is upstream and dominant to cell cycle control. Consistent with this view, it was found that if the cell cycle is artificially stimulated to give a higher division rate, then the size of the cells decreases. Conversely, if mitosis in the wing is blocked by inactivation of the Drosophila cdc2 kinase, then wings of normal size are produced but they contain smaller numbers of larger cells (Weigmann, 1997). Overexpression of activated Babo in the wing appears to alter the process that couples growth and proliferation rates with the determination of tissue size. The simplest explanation for these results is that Babo signaling stimulates some aspect of the cell cycle resulting in an increased proliferation rate and reduced cell size similar to that described by Neufeld. However, the fact that larger wings are seen despite the smaller cell size suggests that Babo may independently affect tissue growth parameters as well as the cell cycle. Therefore, additional studies on the mechanism of Babo signaling could help reveal how cell growth and proliferation are linked to the determination of final tissue size (Brummel, 1999).

GENE STRUCTURE

Both BABO transcripts are encoded within a 9-kb genomic fragment and contain seven exons with exon 3a/b being differentially spliced to produce the two isoforms (Brummel, 1999)

Number of exons - 7

PROTEIN STRUCTURE

Amino Acids - 601

Structural Domains

A transmembrane protein serine/threonine kinase, Baboon, that is structurally related to receptors for members of the transforming growth factor-beta (TGF-beta) family has been cloned from Drosophila. The spacing of extracellular cysteines and the cytoplasmic kinase domain of Baboon resemble most closely those of the recently described mammalian type I receptors for TGF-beta and activin. The kinase domain of Baboon bears a 60% to 72% amino acid identity to mammalian type I receptors. The Baboon kinase domain is more distantly related (37% to 40% amino acid sequence identity) to those of the type II receptors, including Drosophila Punt. The extracellular domain of Baboon shows little sequence similarity to other receptors and is larger than those of serine/threonine kinase receptors from vertebrates. The spacing of the 10 extracellular cysteines in Baboon resembles the spacing in the other type I receptors, and includes the cysteine box motif near the transmembrane region that is characteristic of the serine/threonine kinase receptor family. Comparison of Baboon with other type I receptors reveals the presence of a characteristic 30-amino-acid domain immediately upstream of the kinase region in all these receptors. This domain, of unknown function, contains a repeated Gly-Ser sequence and is therefore referred to as the GS domain. Two alternative forms of Baboon have been identified that differ in an ectodomain region encompassing the cysteine box motif characteristic of receptors in this family. The second class of cDNA is represented by one clone of 4.9 kb encoding a product in which a 70-amino-acid sequence replaces a 49-amino-acid sequence in the extracellular region near the transmembrane domain. Aside from the cysteine box, which is partially included in this region, there is virtually no similarity between the two alternative sequences. The larger size of Baboon compared with mammalian type I receptor (Baboon contains a 94 amino acid N-terminal region that is not present in the mammalian proteins) correlates with the presence of a large extracellular region and more N-linked glycosylation sites in Baboon (Wrana, 1994).

date revised: 21 January 99

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