Drosophila gene Dsrc28C (now termed Btk family kinase at 29A or Tec29), can be immunoprecipitated by anti-v-src antisera. RNA blot hybridization reveals that the gene is expressed predominantly during embryogenesis, in imaginal discs of third-instar larvae, and in adult females. In situ hybridization shows that expression in adult females is largely confined to nurse cells and developing oocytes (Gregory, 1987).
In situ hybridization was used to study the developmental RNA expression of dsrc29A (now termed Btk family kinase at 29A or Tec29). This gene encodes two proteins differing at their amino termini. Both gene products contain a protein-tyrosine kinase domain and resemble the protein encoded by vertebrate src. An examination was carried out of most stages of development in the Drosophila life cycle: embryos, third instar larvae, pupae and adults. Tec29 expression is specialized throughout development, being prominent at various times in neural tissue, phagocytic cells, dorsal vessel, ovaries, gut, developing salivary glands, imaginal discs and disc derivatives. These findings confirm and extend previous results for the distribution of Tec29 protein, indicating that the regulation of this gene is primarily at the level of transcription. In some tissues expression is transient, whereas in others, it is continuous, and expression occurs in proliferative, differentiating and differentiated tissue. These patterns of expression demonstrate how a single protein-tyrosine kinase might play diverse roles at different times during development. Comparison of the expression of Tec29 and other members of the protein-tyrosine kinase gene superfamily reveals that the genes are expressed in distinctive but sometimes overlapping patterns (Katzen, 1990).
Expression of the Drosophila src-related gene, Dsrc28C (now termed Btk family kinase at 29A or Tec29) has been investigated at the protein level using monoclonal antibodies. This analysis has revealed that the Tec29 gene encodes two protein forms: a 66-kD doublet predicted from the sequence of a cDNA clone and an additional 55-kD form. The 66-kD protein doublet is observed first at the cellular blastoderm stage and is not detectable in embryos after 12 hr of development. Expression of the 55-kD protein lags behind that of the 66-kD doublet and then persists throughout embryogenesis. Tec29 protein is localized to the cell periphery during cellular blastoderm and gastrulation. The cell periphery-associated staining is then resolved into ectodermal stripes along the fully extended germ band. After the stripes fade, cytoplasmic staining of the majority of cells within the central and peripheral nervous system is observed. The 66-kD protein was shown to represents the cell periphery-associated form of the protein through antibody staining of larval salivary glands expressing a heat shock promoter-driven, full-length Tec29 cDNA. The 55-kD protein is the nervous system form. Thus, the 66- and 55-kD proteins are products of the Tec29 gene, which exhibit different temporal and spatial patterns of expression in the embryo (Vincent, 1989).
The Dsrc28C gene encodes two major proteins, p66 and p55, each of which contains a tyrosine kinase domain. A detailed investigation was carried out of the spatial expression of Dsrc28C proteins during embryonic and larval development. Differentiation of a number of embryonic tissues is accompanied by the induction of Tec29 expression. With the exception of the developing salivary glands, which express high levels of p66, developing tissues express the p55 form of Tec29. Notable examples are cells of the peripheral nervous systems which express p55 from the early stages of neurogenesis through the remainder of embryogenesis and pole cells, which transiently express p55 during portions of embryonic stages 10 and 11. Nervous system expression includes the cell bodies and neuronal fibers of the central nervous system, the anterior sensory organs, and the peripheral sensory neurons. During larval development, p55 levels within the central nervous system remain high but substantial changes in the pattern of expression take place. p55 gradually disappears from the neuronal fibers of the central nervous system and from embryonic cell bodies. During the third larval instar, the birth of immature neuroblasts within the ventral and midbrain ganglia, but not within the optic ganglia, is marked by a transient high level of p55 expression. All imaginal cells that have been observed within the larva express the p66 protein. The patterns of expression that have been noted suggest that expression of the p55 form of Tec29 protein is an early event in the differentiation of neuronal cells, while expression of the p66 form is characteristic of cells committed to ectodermal cell differentiation (Wadsworth, 1990)
Tec29 RNA and protein expression has been described in embryos (Katzen, 1990; Vincent, 1989; Wadsworth, 1990), but the details of salivary gland expression were not reported. Tec29 RNA expression begins at stage 11 (5.5-7.5 hours) in the dorsal posterior part of the salivary placodes, colocalizing with the site of initial invagination. By stage 12, all salivary placode cells express Tec29. Tec29 continues to be expressed in the invaginating salivary glands at stage 12 and later during embryogenesis. Then as the placodes invaginate, Tec29 expression expands into the more ventral cells that will become the salivary ducts and is weakly expressed in the salivary ducts of older embryos. A similar expression pattern was observed for TEC29 protein. TEC29 is localized to the apical membrane in the salivary gland cells. The shorter isoform of TEC29 is expressed in the epidermis and at high levels in the salivary glands. Thus, TEC29 function in the salivary glands apparently does not require a PH domain (Chandrasekaran, 2005).
Tec29 encodes the only known Drosophila member of the Tec tyrosine kinases. By identifying the first mutations in Tec29 (formerly Src29A), it has been shown that Tec29 is essential for head involution during embryogenesis and for ring canal development during oogenesis. Tec29 mutant egg chambers are defective in the transfer of cytoplasm from the accessory nurse cells through the ring canals into the oocyte. Growth of the mutant ring canals is arrested, and they lack the strong phosphotyrosine localization seen in wild-type ring canals. Mutants lacking the Drosophila Src homolog Src64 show the same phenotype, and Src64 is required for the localization of Tec29 to the ring canals. This interaction is similar to that between vertebrate Src and Tec kinases and suggests that Tec29 is an effector of Src64 that modifies ring canal components required for growth (Roulier, 1998).
Mutation of the Src64 gene of Drosophila results in ovarian ring canal defects and reduced female fertility. A dosage-sensitive modifier screen was used to search for downstream components of the SRC64 signaling pathway. Mutations affecting Tec29, an essential gene encoding a member of the Tec family of protein tyrosine kinases, dominantly enhance the Src64 ring canal phenotype. Loss of Tec29 function in the female germline results in a phenotype strikingly similar to that caused by the loss of Src64 function. In each case, the ring canals are reduced in size and phosphotyrosine content. Tec29 is shown to localize to the ring canal, and this subcellular localization requires Src64 function. These data suggest that Tec29 is a downstream target of SRC64, and that regulating Tec29 localization during ring canal growth may be a crucial Src64 function (Guarnieri, 1998).
Drosophila type 2 Btk29A reveals the highest homology to Btk among mammalian Tec kinases. In Btk29A(ficP) mutant males, the apodeme holding the penis splits into two pieces. Human Btk rescues this phenotype in 39% of Btk29A(ficP) males, while the Drosophila transgenes do so in 90%-100% of mutants. The Btk29A(ficP) mutation reduces adult longevity to 11% that of wild-type. This effect is counteracted by Drosophila type 2, yielding 76% of the wild-type lifespan. Human Btk extends the lifespan of Btk29A(ficP) mutants to only 20% that of wild-type. Thus human Btk can partially replace Drosophila Btk29A+ in male genital development and survival (Hamada, 2005).
Src family tyrosine kinases respond to a variety of signals by regulating the organization of the actin cytoskeleton. During early oogenesis Src64 mutations lead to uneven accumulation of cortical actin, defects in fusome formation, mislocalization of septins, defective transport of Orb protein into the oocyte, and possible defects in cell division. Similar mutant phenotypes suggest that Src64, the Tec29 tyrosine kinase, and the actin crosslinking protein Kelch act together to regulate actin crosslinking, much as they do later during ring canal growth. Condensation of the oocyte chromatin into a compact karyosome is also defective in Src64, Tec29, and kelch mutants and in mutants for spire and chickadee (profilin), genes that regulate actin polymerization. These data, along with changes in G-actin accumulation in the oocyte nucleus, suggest that Src64 is involved in a nuclear actin function during karyosome condensation. These results indicate that Src64 regulates actin dynamics at multiple stages of oogenesis (Djagaeva, 2005).
Expression in the salivary glands suggests that Tec29 might be important for salivary gland morphogenesis. Embryos mutant for a P-element allele of Tec29, Tec29K00206 have abnormally long salivary glands that do not fully invaginate and are still connected to the surface. The phenotype is fully penetrant, and an identical phenotype is observed in embryos mutant for at least two other Tec29 alleles, Tec29K05610 and Tec29e482, as well as Tec29K00206/Tec29K05610 heterozygotes (Chandrasekaran, 2005).
A possible explanation for this phenotype is that the mutant salivary placodes have more cells than wild-type placodes, resulting in long glands. However, cell counts did not support this idea. Tec29 salivary placodes had 113±36 cells compared with 117±39 in wild type. In addition, location of the placodes is normal. As shown by co-staining for Engrailed, the placodes respect both the normal AP boundaries at the edges of parasegment 2 and the DV boundaries that separate them from the dorsal epidermis and the more ventral salivary duct cells. In addition, staining for the mitotic marker phosphohistone H3 produced no evidence of extra mitoses, either in the placodes or at later stages. Thus, the long glands do not result from the recruitment of extra cells into the primordium or from the production of extra cells during development (Chandrasekaran, 2005).
The salivary ducts are defective in Tec29 mutant embryos. As evidenced by staining for duct markers such as dead ringer, duct cell fate appears to be normally specified in these embryos, but the duct cells do not undergo normal morphogenesis. Staining for a lumenal marker, Crumbs, shows that tubular ducts are not formed in these embryos (Chandrasekaran, 2005).
Since the long salivary glands in Tec29 mutants are not due to extra cells and there are uninvaginated cells in the salivary glands of Tec29 embryos, it was reasoned that the detailed analysis of salivary placode invagination in Tec29 embryos might explain the salivary gland phenotype. In wild-type embryos, the progress of salivary invagination can be monitored relative to germ band retraction, since both processes occur during stage 12 of embryogenesis (Chandrasekaran, 2005).
In wild-type embryos, there is an inverse relationship between the area of the placodes left on the surface and the length of the invaginated gland, such that by the end of stage 12 there are no placodes left on the surface. The length of the glands increases as the embryos progress through stage 12, but then decreases at later stages. Since germ band retraction appears to be normal in Tec29 embryos, similar analyses of salivary glands were performed in these embryos. The salivary placodes begin invagination on schedule and at early stage 12 look similar to wild type. During mid stage 12, when there is a linear increase in the length of the invaginated gland in wild type, Tec29 embryos stall; the glands do not increase in length and the placode area do not decrease. By late stage 12, the length of the invaginated salivary glands in Tec29 embryos is comparable with wild type, but by stage 15 the salivary glands in Tec29 embryos are significantly longer than wild type. Despite this, there are still many placode cells remaining on the surface at late stage 12 and even at stage 13-14. Thus, in Tec29 embryos there is an arrest in the salivary gland invagination process at mid stage 12, followed by a late increase in gland length. These results indicate that the delay in invagination precedes the lengthening of the gland in Tec29 mutants and is the main cause of the Tec29 salivary gland phenotype (Chandrasekaran, 2005).
There is a paradox in understanding the Tec29 phenotype. Although fewer cells invaginate, the mutant glands eventually become much longer than in wild-type embryos. Since the increase in the salivary gland length occurs late in Tec29 embryos, it was guessed that this increase might come from stretching them during head involution in older embryos. To test this possibility, Tec29 embryos were immunostained either for Crumbs, which outlines the apical ends of epithelial cells or for Scribble, a septate junction protein that is localized to the lateral margins of epithelial cells, more basal to Crumbs. Both markers highlight the salivary gland cells, producing nearly isodiametric outlines in wild-type embryos at stage 15. In Tec29 embryos stained for either Scribble or Crumbs, the distal region of the salivary glands looks normal, but the cells in the proximal region of the glands are elongated in the AP direction. These results show that the arrest of invagination coupled with the anterior movement of the epidermis during head involution causes stretching of the mutant salivary glands, leading to the observed phenotype of long salivary glands (Chandrasekaran, 2005).
Stretching of the glands in Tec29 mutants suggests that they are tethered at both ends. The anterior tether probably results from the invagination defect. As the placodes do not completely invaginate in Tec29 embryos, the anterior part of the gland appears to remain anchored to the moving ectoderm. To permit stretching, the posterior part of the gland must also be tethered, probably by attachment to tissues close to the distal tip of the gland, maybe the anterior midgut. This posterior attachment may be part of normal salivary morphogenesis since the posterior part of the Tec29-mutant glands are located normally and have the same apical outlines as wild-type glands (Chandrasekaran, 2005).
Previous studies have shown that Tec29 can reorganize the actin cytoskeleton in the ovary and during the cellularization of Drosophila embryos (Djagaeva, 2005; Roulier, 1998; Thomas, 2004). Therefore, whether the actin distribution is normal in the placodes of Tec29 embryos was examined. Staining with phalloidin to visualize F actin or with an alpha-spectrin antibody did not reveal any gross abnormalities. However, use of an actin monoclonal antibody that detects both F and G actin shows that in Tec29 embryos, actin is disorganized at the apical end of the placode cells, but looks similar to wild type on the basolateral surface. The disorganization of actin occurs early in stage 12 in the ventral cells of the placodes and precedes the delay in invagination observed in these cells. In addition, there are genetic interactions between Tec29 and the actin-binding proteins, profilin and cofilin. The Drosophila profilin homolog, chickadee (chic), is important for promoting actin polymerization, thereby increasing F-actin in the ovary, embryo and imaginal discs, whereas the Drosophila cofilin homolog twinstar (tsr), promotes depolymerization, thus limiting actin filament growth and increasing G-actin. Embryos mutant for either chic or tsr alone have normal salivary glands. However, Tec29 chic double mutants show an enhancement of the Tec29 salivary gland phenotype. The salivary glands in 80% of Tec29 chic double mutants showed more severe invagination defects with large placodes left on the surface. The remaining 20% of the embryos had salivary glands similar to Tec29. By contrast, 30% of the Tec29 tsr double mutants have salivary glands that invaginated normally or nearly so, indicating a partial suppression of the Tec29 mutant phenotype. These genetic interactions show that in Tec29 mutants there is shift from F-actin to G-actin on the apical surface of the salivary placode cells. They also suggest that the apparent disorganization seen with the actin antibody results from increases in G-relative to F-actin. The partial rescue of the Tec29 salivary gland phenotype by tsr indicates that the shift in the balance between G-actin and F-actin in the salivary placodes causes the invagination delay in Tec29 mutant salivary placodes. Therefore, Tec29 is necessary to facilitate the formation or maintenance of F-actin at the apical surface of the salivary placodes cells, and this localization of actin is crucial for normal invagination (Chandrasekaran, 2005).
The Src superfamily of non-receptor-type tyrosine kinases is composed of four families (the Src family, the Csk family, the Abl family, and the Btk family), each of which is represented by multiple family members. These kinases have been suggested to play diverse roles in cell proliferation, differentiation, survival, and death. Among these four families, the Bruton's tyrosine kinase (Btk) family is unique in that the kinases in this family have extended N-terminal sequences that are called collectively the pleckstrin homology (PH) domain. This domain is known to bind the gamma subunits of heterotrimeric G proteins as well as membrane lipids such as phosphatidylinositol-4,5-bisphosphate. Another feature unique to the Btk family resides in the restricted localization of its members in mammalian tissues. For example, Btk is present only in B cells whereas the expression of Itk, another Tek protein-tyrosine kinase family member, is confined strictly to T cells. Similarly, one of the two forms of the Tec kinase is liver specific whereas the other is hepatocyte specific. In Drosophila, Dsrc29A (Btk family kinase at 29A) is the sole kinase that represents the Btk family, and it is most similar to Btk itself in terms of overall homology. However, the reported N-terminal sequence of Dsrc29A has no similarity to the PH domain. This fact raises the possibility that Dsrc29A is not an ortholog of Btk but represents a distinct member of the Btk family. Deficits in Btk function are responsible for X chromosome-linked agammaglobulinemia in humans and X chromosome-linked immunodeficiency in mice, where B-cell maturation is blocked. The defense mechanism involving immunoglobulins secreted from B lymphocytes exists only in higher vertebrates, and the machinery for antibody production is expected to have its origin in an apparently unrelated cellular function in lower animals. Thus, functional analysis of Dsrc29A would provide insights into the evolution of Btk-like kinases (Baba, 1999a and references).
Btk family kinase at 29A/Dsrc29A is required for adult survival and male genital formation in Drosophila. fickleP (ficP), which removes the Btk homolog in Drosophila, was isolated in a screening of P-element mutants with defects in mating behavior. The mating behavior of Drosophila is made up of several discrete elementary steps. First, the male finds and tracks the female. While tracking, the male approaches the female to tap her abdomen with his forelegs. The male then performs courtship songs by using unilateral wing vibration. Provided that she is sexually receptive, the female frequently stops moving when exposed to the courtship songs, offering the male a chance to lick her genitalia and to attempt copulation. If the male is successful, he mounts on and copulates with the female for 10 to 17 min. Upon termination of copulation, the male releases his genitalia from the female's and dismounts. These behavioral acts are considered to be fixed-action patterns, i.e., instinctive behavior (Baba, 1999a and references).
There are mutations that are known to affect the unique aspects of mating behavior in D. melanogaster. One class of mutations affect copulation and postcopulatory behavior. These include stuck (sk) and coitus interruptus (coi). The sk males often fail to withdraw their genitalia after copulation, with the result that the male and female pair tug at each other, pulling in opposite directions. The coi mutation affects males, causing copulation to terminate prematurely, even before completion of the sperm transfer from the male to the female. Phenotypically, ficP seemed to belong to this class of mutations. The ficP flies exhibit an extremely variable copulatory duration ranging from 1 to 15 min, in sharp contrast to the wild-type flies. Unlike wild-type flies, ficP mutant flies tended to mate repeatedly within a short period (minutes). These behavioral phenotypes of ficP are probably a consequence of malformation of male genitalia. Another conspicuous phenotype of ficP is a reduced life span after adult emergence. The functional rescue experiments of the ficP mutant demonstrate that Drosophila Btk is required in the pupal stage for normal adult longevity and male genital formation. The longevity phenotype is believed to be linked to Btk expression in the developing brain, while the genital phenotype is associated with its expression in the developing genital disc (Baba, 1999a).
Interestingly, the Btk homolog is generated by an alternative exon usage of the transcription unit for the Btk family kinase at 29A/Dsrc29A. Both the Btk-coding transcript (referred to here as type 2) and the Dsrc29A kinase transcript (type 1) are expressed in the central nervous system (CNS) and imaginal discs; the domains and/or timing of expression in these tissues are distinct from each other. Complete loss of function of the gene (i.e., loss of both types 1 and 2) causes oocyte undergrowth and embryonic death accompanied by defective head involution, while selective loss of the type 2 transcript spares life but reduces the life span in the adult and leads to malformation of the male genitalia. Thus, the single Btk/Dsrc29A kinase gene exerts pleiotropic functions in different developmental contexts in different tissues through the generation of distinct forms of protein products by means of alternative splicing (Baba, 1999a, and references).
Two types of cDNA for this gene were found by library screenings. The type 1 clone is about 2.9 kb long whereas the type 2 clone is about 3.7 kb long. Although the type 1 and 2 clones had identical 3' coding sequences, they differed from each other in the 5' half. This difference appears to have resulted from alternative exon usage. Sequencing of the type 1 cDNA clone reveals a long open reading frame that encodes a protein of 603 amino acids (if the first ATG codon is chosen as the translation initiation site). A database search for similar sequences revealed that an amino acid sequence very similar to the protein has been described as Dsrc29A. An important difference between the sequence found in this study and the reported Dsrc29A sequence (Gregory, 1987) is found in their N termini; the open reading frame for the cDNA clones isolated in this study is open for an additional 45 bp upstream of the methionine start codon chosen in the previous stydy. Furthermore, there are 34 amino acid differences in the deduced protein sequences. Dsrc29A belongs to the Src superfamily, having highly conserved sequence motifs including the SH2 (Src-homology 2), the SH3 (Src-homology 3), and the catalytic (kinase) domains. Dsrc29A differs from Src in that it has a long, basic N-terminal region upstream of the SH3 domain (Baba, 1999a).
Analysis of the type 2 cDNA reveals that the protein encoded by the second form of the transcript has an amino acid sequence identical to that of the type 1 product in the C-terminal two-thirds, including the SH3, SH2, and kinase domains, but has a unique N-terminal stretch of 231 amino acids. This isoform has not been reported previously. Although the type 1 product does not contain any discernible conserved motifs in its N-terminal extension, the newly identified type 2 isoform bears typical PH and TH domains, which are regarded as hallmarks of the Btk family kinases, such as Atk, Itk, Tec, and Btk. A striking difference between the type 2 product and Btk is the presence of a polyglycine stretch insertion in the proline-rich region. Overall, the percent similarity between the Drosophila type 2 protein and mammalian Btk is 67.7%. When compared for each domain, the value is 56.9% (PH), 33.8% (TH), 78.8% (SH3), 80.4% (SH2), and 85.3% (kinase). The low value for the TH domain is ascribable to the polyglycine stretch present in the Drosophila sequence. The similarity increases to 76.9% if only the Btk motif in the TH domain is considered for comparison. Thus, the type 2 product is very likely to be the Drosophila ortholog of Btk (Baba, 1999a).
Northern blot analysis using a sequence common to both type 1and 2 clones as a probe revealed that a major 3-kb transcript and a minor 4-kb transcript are expressed at constant levels throughout development. The 4-kb transcript is detectable in poly(A)+ mRNA extracted from the heads but not from the bodies of adult flies. This transcript may represent an isoform specific to neural tissue that occupies most of the head. In the wild-type Drosophila, a 4-kb transcript is detected in the adult head poly(A)+ RNA when probed with the type 2-specific sequence. In addition, the type 2-specific probe hybridized with a transcript, of about 3 kb, in both the head and body parts. On the other hand, the type 1-specific probe detects a 3-kb transcript in the head and body poly(A)+ RNA. Thus, the type 1 cDNA corresponds to a 3-kb transcript whereas the type 2 cDNA corresponds to a different 3-kb transcript and a 4-kb transcript. The 3-kb type 1 and 3-kb type 2 transcripts are expressed in both the head and body, while the 4-kb type 2 transcript is head specific (Baba, 1999a).
The difference between wild-type and ficP strains was evident when a type 2-specific probe was used for Northern blot analysis; neither of the 4-kb and 3-kb transcripts are detected in ficP flies. The absence of these transcripts is associated with the fic mutation, since ficR, a phenotypic revertant obtained by excision of the inserted P element, has both the transcripts. The sole transcript detectable in ficP flies with the type 2-specific probe is distinct from any of the transcripts found in the wild type. The mutant transcript is slightly shorter than the wild-type 3-kb type 2 transcript, implying that it is a truncated version of the type 2 transcript. (Baba, 1999a).
To examine whether the ficP phenotypes are causally linked to malfunction of the cloned gene, ficP mutant lines were created carrying the full-length type 1 or 2 wild-type cDNA driven by the heat shock promoter (hs-cDNA). Although the phenotypes of ficP results from the selective loss of the type 2 transcript, ubiquitous overexpression of either type 1 or 2 similarly rescues these phenotypes. This fact suggests that the specific phenotype of the ficP mutant reflects disruption of specific temporal and/or spatial expression of the type 2 transcript rather than functional specificity of the type 2 product (Baba, 1999a).
Mutant flies carrying hs-cDNA, reared at 25°C, retain the ficP phenotype: the apodeme of the male genitalia remains torn. Consistent with this observation, the histogram for copulatory duration reveals a pattern similar to that for ficP flies without hs-cDNA. Subjecting the ficP;hs-cDNA males to heat shock throughout the pupal stage restores normal apodeme structure and is accompanied by a dramatic alteration in mating behavior after emergence. The pattern of distribution of copulatory duration for the ficP;hs-cDNA flies after heat shock resembles that for wild-type flies. Thus, expression of hs-cDNA in the pupal stage rescues the ficP defect in the genitalia and restores normal copulation behavior, establishing the causal relationship between the mutant phenotype and the gene. Heat shock induction of hs-cDNA on the first or second day of pupal life is sufficient for rescue of both the behavioral and genital phenotypes. Repeated remating is not observed between pairs of ficP;hs-cDNA males and wild-type females, when the males had been exposed to heat shock after the third-instar larval stage until emergence (Baba, 1999a).
Apart from the defects in the male genitalia and mating behavior, it was noted that the life span after eclosion is decreased in ficP flies, when compared to wild-type flies. The longevity phenotype of ficP adult flies is also rescued by ubiquitous overexpression of either type 1 or 2 cDNA, indicating that the type 1 and 2 transcripts are functionally equivalent to each other in terms of life span regulation. This implies that a specific site at which type 2 mRNA, but not type 1 mRNA, is expressed plays a critical role for adult survival. The most effective period of hs-cDNA induction for rescue of the longevity phenotype is between the last day of the third larval instar stage and the first day of pupation. Thus, the results of the heat shock experiments suggest a requirement for Btk/Dsrc29A in the prepupal to early pupal stage for normal copulation and genital formation and in the third larval instar to early pupal stage for normal adult life span (Baba, 1999a).
To study the tissue localization of the Btk/Dsrc29A transcript during the critical period, whole-mount in situ RNA hybridization was performed. Both the type 1 and 2 transcripts are detected in the male genital disc, although their expression patterns are distinctly different from each other. The expression of the type 1 transcript is confined to the middle portion of the anterior bulbus, which is known to form the internal genitalia. However, the type 2 transcript is strongly expressed along the edge of the male genital primordium facing the lumen. Fate map studies have indicated that this part of the male genital primordium contributes to the main body of the male genitalia including the penis apparatus, to which the apodeme is attached. This finding supports the notion that the specific phenotype of the ficP mutant correlates with the spatial restriction of the type 2 transcript (Baba, 1999a).
The other tissue that is rich in Btk/Dsrc29A mRNA is the CNS: particularly strong expression is detected in the cells of the inner optic anlage. Several hours after puparium formation, strong Btk/Dsrc29A mRNA expression is noted in the cells above the mushroom bodies. Hybridization with type-specific probes demonstrate that the transcript expressed in the mushroom body region consists exclusively of the type 2 transcript. In other areas of the brain, type 1 transcript predominates. These cells are thought to contribute to the development of mushroom bodies. The expression is detected in many mushroom body ganglion cells but not in neuroblasts. Soon thereafter, weak expression of type 1 is noted in many scattered cells in the central brain and ventral ganglion while expression in the optic lobe ceases. In the ventral ganglia, weak Btk/Dsrc29A expression is detected in many scattered cells in a manner similar to that in the central brain. Strong expression is observed in the cells on the midline of the ventral ganglion. Since these are irregularly shaped cells that enfold the anterior and posterior comissures in each segment, they are likely to be midline glia. The expression in the ventral ganglia consisted exclusively of the type 1 transcript. No expression of the type 2 transcript is detected in this region. At around 72 h after puparium formation, nearly half of the cells in the brain express type 1 mRNA. At this stage, expression of the type 2 mRNA is observed only in a cluster of cells just above each antennal lobe and cells near the mushroom body. The level of type 2 mRNA expression in these cells is remarkably high. Most of the cells expressing this mRNA in the antennal lobe region appear to be neurons because the cell bodies are round. The antennal lobe is known to consist of approximately 1,200 cells. Of these, only a very small portion exhibit strong Btk/Dsrc29A expression. Interestingly, there are other clusters of cells above the antennal lobes that do not express Btk/Dsrc29A. Four clusters of cells expressing the mRNA per hemisphere are observed in the mushroom body region. Each cluster apparently corresponds to the progeny of one of four mushroom body neuroblasts. Thus, it is concluded that the type 2 transcript is selectively expressed in the cells of the mushroom body and the antennal lobe whereas the type 1 transcript is expressed in dispersed neurons and the midline glial cells in the CNS during the larval and pupal stages. The localizations of types 1 and 2 mRNA appear to be mutually exclusive in both the CNS and the genital disc. It is emphasized that the temporal correlation between the genital and behavioral phenotypes revealed by the rescue experiments with time-restricted Btk/Dsrc29A expression does not exclude the possibility of a neural origin of the behavioral phenotype (Baba, 1999a).
The Btk/Dsrc29A gene is distinctly different from other
members of the Btk family genes in two respects: (1) it
is alternatively spliced so as to give rise to two different proteins,
one of which has, at its N terminus, a unique domain without sequence
homology to any other known protein, in place of the PH domain in the
sibling protein. (2) The N-terminal segments in both isomers are
separated from the main body of the protein by an intervening
polyglycine stretch. This structural complexity may be necessary for
the support of the pleiotropic functions of the Btk/Dsrc29A
kinase in Drosophila, in which no other members of the Btk
kinase family exist. It is of interest that the PH domain is dispensable for normal
copulation and survival. It should be pointed
out, however, that the adult life span of the rescued mutant flies is
not identical to that of wild-type flies. Such partial rescue may
indicate that the function of the type 1 product only partially
overlaps that of the type 2 product (Baba, 1999a).
Formation of the Drosophila cellular blastoderm involves both
membrane invagination and cytoskeletal regulation. Mutations in src64 and tec29 (Btk29A - FlyBase) reveal a novel role for these genes in controlling contraction of the actin-myosin microfilament ring during this process. Although membrane invagination still proceeds in mutant embryos, its depth is not uniform, and basal closure of the cells does not occur during late cellularization. Double-mutant analysis between scraps (a mutation in anillin that eliminates microfilament rings) and bottleneck suggests that microfilaments can still contract even though they are not organized into
rings. However, the failure of rings to contract in the src64
bottleneck double mutant suggests that src64 is required for
microfilament ring contraction even in the absence of Bottleneck protein. These results suggest that src64-dependent microfilament ring contraction is resisted by Bottleneck to create tension and coordinate membrane invagination during early cellularization. The absence of Bottleneck during late cellularization allows src64-dependent microfilament ring constriction to drive basal closure (Thomas, 2004).
tec29, a non-receptor tyrosine kinase, is, like src64, required for the
morphogenesis of ovarian ring canals and interacts with Src64 protein to
control ovarian ring canal growth. Cellularizing embryos derived from
tec29k00206 germline clones have large and
non-rounded microfilament rings like those of src64 embryos. tec29 microfilament rings have a circularity index of 0.82, similar to that of src64 but very different from that of wild-type microfilament rings. Tec29 protein is expressed at the cellularization front and more weakly along the lateral cellular membrane in both wild-type embryos and src64Delta17 embryos, suggesting that src64 does not act to localize Tec29 protein during cellularization. This is in
contrast to the situation in the ovary where Src64 protein acts to localize Tec29 protein to the ring canal. Phosphotyrosine staining is observed at the cellularization front in both wild-type and src64 mutant embryos. This suggests that
Src64 is not the major source of phosphotyrosine during cellularization as it is in the egg chamber. Thus both src64 and tec29 are required for microfilament ring contraction during cellularization, but tec29 is localized to the cellularization front independent of src64 activity (Thomas, 2004).
To determine whether other actin-binding proteins affect microfilament ring contraction in a manner similar to src64, the
actin-binding protein anillin, which is expressed at the cellularization front in a domain similar that of Src64, was examined. During later development the anillin protein also localizes to contractile rings during cytokinesis. Anillin is encoded by the gene scraps, which is defined by a maternal effect lethal mutation. The phenotype of embryos from mothers trans-heterozygous for two strong reduction-of-function mutations of scraps was examined. The most informative phenotype of scraps mutants is the absence of microfilament rings. This can be readily seen by observing the density and continuity of myosin staining around the basal openings in the cellularization front. The furrow canals are collapsed and lack the early bulb-like or the late flask-like morphology of the wild type. Only some furrow canals show strong myosin staining. Myosin is seen in
dense rod-like clumps lying between some of the basal cellular openings. This myosin distribution is similar to that of F-actin in the septin mutant peanut during cellularization, suggesting
that both anillin and certain septins play a role in the assembly or
maintenance of the microfilament rings (Thomas, 2004).
During early cellularization in scraps mutant embryos, the basal cytoplasmic openings are angular and resemble polygons with relatively straight sides. Because the sides of these polygons are somewhat uniform in length, they approximate circles and have a circularity index of 0.89, differing only slightly from wild-type microfilament rings. Unlike in src64 mutants,
they are not convoluted or wavy, and indentations are not observed. During late cellularization, the scraps phenotype becomes more severe, significantly differing from wild type. This decrease in
circularity reflects an increase in the length of some of the sides of each polygon and a decrease in the length of others so that the basal openings more closely resemble polygons with fewer sides. The sides remain straight and show no waviness that would indicate a lack of tension. Instead, the gradual distortion of the polygons in scraps mutants is consistent with stretching due to microfilament contraction in the absence of an organizing structure. The scraps phenotype is therefore distinct from that of src64 mutants, which appear to lack microfilament tension and maintain similar, but deviant, circularities throughout cellularization. This
interpretation implies that microfilament rings are not required for
microfilament contraction. The low circularity index of later basal openings in scraps mutants suggests that microfilament ring structure provides a stabilizing framework for microfilament contraction during the late phase of cellularization (Thomas, 2004).
Mutations in bottleneck (bnk) have been used to define more clearly the roles that src64 and scraps play in
cellularization. Bnk is a small, highly basic protein that regulates the
dynamic restructuring of the actin cytoskeleton so as to control the timing of microfilament ring contraction during late cellularization. It is expressed during early cellularization and its level drops precipitously during the transition to the late phase (Schejter, 1993). During early cellularization, Bnk co-localizes
with myosin, but extends further apically in the furrow canal (Thomas, 2004).
The bnk phenotype is distinct to that of src64 or
scraps in that embryos homozygous for a bnk deficiency have
a hypercontractile phenotype. The microfilament rings are prematurely
constricted during early cellularization (Schejter,
1993). The rings squeeze the nuclei into dumbbell shapes during
early cellularization, trapping and dragging some of them along with the
advancing cellularization front during late cellularization. The microfilament rings of early cellularization and late cellularization bnk embryos have circularity indices of 0.92 and 0.91, respectively, values that do not differ from those of similarly staged wild-type embryos, even though initially they enclose a much smaller area of open cytoplasm (Thomas, 2004).
scraps; bnk double-mutant embryos show a mixture of the phenotypes of both scraps and bnk embryos. Like scraps embryos, scraps; bnk embryos fail to form actinmyosin rings, and instead show dense rod-like aggregates of myosin II lying between some of the non-rounded basal cellular openings. In spite of the absence of contractile rings, scraps; bnk embryos still display the premature hypercontraction phenotype characteristic of bnk embryos. The cytoskeleton
surrounding cells in scraps; bnk embryos is more contracted, in terms of both area and circularity, than the cytoskeleton surrounding cells in scraps embryos. Microfilaments are constricted around dumbbell-shaped nuclei that are trapped and dragged out of the periphery of the embryo by the cellularization front in scraps; bnk double-mutant embryos, as is characteristic of bnk embryos. The area enclosed by the microfilaments of scraps; bnk embryos is significantly less than that of scraps embryos, but is still larger than that of bnk embryos. During late
cellularization, the circularity index of the basal openings of scraps; bnk embryos is 0.90, similar to that of bnk embryos, but significantly different from the 0.73 value of scraps embryos. This difference suggests that the actin-myosin network can still contract in the absence of microfilament rings, but without the efficiency that is conferred by the organization of the cytoskeleton into
rings (Thomas, 2004).
The hypercontraction caused by the absence of Bnk protein, coupled with the loss of structural integrity of the cellularization network caused by the absence of anillin and microfilament rings, leads to the apparent tearing of parts of the cellularization network. Several regions of the cytoskeleton are either stretched thin or broken, leaving large gaps in the cellularization. This suggests
that the loss of anillin and microfilament rings results in a fragile
cytoskeletal structure that unravels in the absence of Bnk. These
double-mutant results suggest that Bnk and anillin both play structural roles in the cellularization front, but that neither are necessary for microfilament contraction itself (Thomas, 2004).
In restructuring the cytoskeleton during cellularization, Bnk controls the timing of microfilament ring contraction so that basal closure does not occur until after the cellularization front has passed the bases of the nuclei. bnk mutants have a prematurely hyperconstricted ring phenotype opposite that of the non-constricted ring phenotype of src64 mutants. src64 bnk double-mutant embryos look like src64 mutant embryos. The src64 bnk embryos have the large, non-constricted microfilament rings that appear to be under no tension. They have a circularity index of 0.85, similar to that of src64 embryos but different from that of bnk embryos. A few double-mutant
embryos showed some degree of microfilament ring contraction during late
cellularization; it is likely that these embryos are the result of some
residual activity of the reduction-of-function src64Delta17 allele. The analysis of src64
bnk double-mutant embryos demonstrates that the premature
hypercontraction of bnk requires src64 activity. The
interaction of bnk mutation with src64 and scraps
reveals the difference between the two genes: src64 is required for microfilament contraction and scraps (anillin) is not. This suggests that bnk regulates cytoskeletal contractility during cellularization by counteracting the src64-mediated contraction of the microfilament rings (Thomas, 2004).
These analyses suggest that src64 and tec29 are required
for tension in the cellularization front during early cellularization, and for the constriction of the basal microfilament rings during late cellularization. Src64 and Tec29, which are present at higher levels in the microfilament rings, might activate actin-myosin contraction or be essential for the ability of the actin-myosin network to contract. Despite a general similarity of form,
the cellularization microfilament ring and the oocyte-nurse cell complex ring canal differ substantially in structure and dynamics. In the ovary,
src64, and presumably tec29, control ring canal expansion by regulating actin polymerization and cross-linking. It is unlikely that myosin can play a role in this process since myosin-driven sliding of actin filaments would lead to contraction rather than expansion. Although myosin is localized to the ring canal, and null mutations in regulatory myosin light chain cause defects in the ring canals, these defects are not severe and do not prevent ring canal assembly or expansion. Thus, despite a similar involvement of src64 and tec29, it is unlikely that microfilament ring constriction and ring canal expansion are mechanistically similar (Thomas, 2004).
Anillin, which localizes to the cellularization front and shows higher
concentration in the contractile microfilament rings, is required for proper cellularization. Anillin bundles actin filaments and may stabilize these filaments during actin-myosin contraction. On
the basis of these observations, it is concluded that in the absence of anillin, stable contractile microfilament rings do not form; instead the contractile protein myosin is irregularly distributed in aggregates throughout the cellularization front. Strikingly, loss of anillin in bnk embryos does not suppress the severe early contraction defect seen in bnk embryos. In the absence of the structure provided by these rings, the contraction of the microfilaments is uneven, leading to increasing defects in the shape of the basal openings as cellularization progresses. This suggests that anillin is not required for the ability of the microfilaments of the cellularization network to contract, only for their organization into stable rings (Thomas, 2004).
The phenotypes presented in this study support a model in which
src64 and bnk oppose each other to control contraction of
the early cellularization network. Double-mutant analysis reveals that
src64 is epistatic to bnk. Bnk acts only to
restrain and partially redirect Src64-mediated ring constriction. The fact that cellularization proceeds in src64 and tec29 mutants suggests that a force other than microfilament ring contraction is sufficient to drive cellularization front invagination. This force may be a result of the insertion of membrane, or may be due to the action of plus-end directed microtubular motors, or some combination of both (Thomas, 2004).
Most models for cellularization invoke a role for myosin contraction during the ring constriction and basal closure that occurs during late stages of the process; a role during early stages is more controversial. The early phenotype of src64 mutants, if the above interpretation is correct, suggests a role for microfilament ring contraction in the early stages as well, acting both to coordinate the invagination of the furrow canals by maintaining tension along the cellularization front and to direct their invagination inward. This force is a product of the interaction of src64-dependent, myosin-mediated contraction of the microfilament rings and resistance to this contraction exerted by Bnk protein, which acts as a linker between the rings. These forces
oppose each other at all points along the contractile microfilament ring
network, generating a dynamic tension over the entire network, keeping it taut and driving the minimization of its surface area. The addition of these force vectors acting on a cross-section of one ring on a curved surface produces a resultant vector directed both toward the interior of the embryo (the center of the circular cross-section) and toward the center of the microfilament ring. The first component of the resultant force vector is the src64-mediated force that provides direction to the invagination that follows the increase in surface area produced by membrane insertion during early cellularization. The other component of the resultant force vector is in the plane of the microfilament ring, coordinating constriction about the entire circumference of the embryo and driving a small degree of constriction consistent with the decrease in cellularization front surface area during invagination (Thomas, 2004).
As the cellularization front passes the bases of the nuclei and
cellularization shifts into its late phase of rapid progression,
Bnk expression is shut off and the protein is rapidly degraded and removed from the cellularization network. In the absence of Bnk protein, there is no force resisting microfilament ring contraction, so it no longer contributes to driving cellularization front invagination. The src64-mediated force is now directed along the radii of the rings, leading to their constriction. This constriction pulls the membrane toward the center of the base of the cell, expanding the furrow canals and leading to basal closure. The src64-independent force (membrane addition or microtubular motors)
may be the only force now driving the inward invagination of the
cellularization front (Thomas, 2004).
In conclusion, these data define the differing roles that src64,
tec29 and anillin play in the cytoskeletal dynamics of
Drosophila cellularization, and reveal more precisely the role that the cytoskeleton plays in the formation of the cellular blastoderm. These data establish that microfilament ring organization and contraction are crucial to basal closure of the blastoderm cells during cellularization. However, these data also suggest that membrane invagination can proceed, though abnormally and less efficiently, in the absence of microfilament organization or
contraction. It will be interesting to determine what the comparative roles and contributions of membrane insertion and microtubular motors are to the progression of the cellularization front (Thomas, 2004).
Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. The Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk–/– overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. The data provide in vivo evidence that Src activity requires JNK and STAT function (Read, 2004).
Partial reduction of Src64B, Src42A, or Btk29A activity suppresses the dCsk–/– phenotype, providing functional data to support the view that the imaginal disc overgrowth, defective larval and pupal development, and lethality of dCsk–/– mutants results from inappropriate activation of the Src-Btk signal transduction pathways. Mutations in Btk29A more strongly suppress dCsk phenotypes than either Src42A or Src64B mutations, perhaps reflecting that (1) Src paralogs act redundantly to each other in Drosophila as in mammals and (2) Btk29A has been shown to act downstream of Src family kinases (SFK) in flies and in mammals. In vivo evidence is provided that loss of Csk function hyperactivates Btk to drive cell cycle entry in development, demonstrating that Tec-Btk family kinases are critical to SFK-mediated proliferation. The data raise the possibility that partial reduction of Tec-Btk kinase activity could reduce proliferation in other cellular contexts in which overgrowth is driven by hyperactivated SFKs, such as in colon tumors (Read, 2004).
Tissue culture models show that constitutively activated SFK signal transduction modulates the function of numerous downstream effector molecules and pathways. Using a loss-of-function approach to identify effectors that mediate the dCsk overgrowth phenotypes, some of these pathways were not implicated in dCsk function. For example, SFKs up-regulate the SOS-Ras-ERK pathway in multiple tissue culture studies and Drosophila overexpression models. However, although dRas1 signaling is active throughout retinal development, reduced dEGFR, Sos, and Jra (c-jun) gene dosage failed to affect the dCsk phenotype. dCsk mutations also failed to modify a hypermorphic allele of dEGFR. Levels of doubly phosphorylated and activated ERK appeared unaltered in dCsk–/– tissue. Moreover, the dCsk phenotype failed to phenocopy defects caused by Ras pathway hyperactivation. For example, constitutively active dRas1 causes increased cell size and patterning defects in the developing imaginal discs, defects that were not observed in dCsk mutant eye tissues. These data argue that not every signal transduction pathway implicated in SFK tissue culture models necessarily functions as predicted within a developing epithelial tissue (Read, 2004).
These studies emphasize the importance of two signaling pathways in dCsk and SFK function. Since certain defects in dCsk–/– animals, such as a split notum, resembled those of hep (JNKK) mutants, it is suspected that JNK pathway activity is involved in dCsk function. Phenotypic and FACS analysis established that reduced JNK (bsk) function suppresses the phenotypes and cell cycle defects caused by loss of dCsk. These results confirm studies indicating that JNK functions downstream of the Src-Btk pathway in Drosophila and mammalian tissue culture cells. Components of the JNK pathway are required for Src-dependent cellular transformation, but the exact role of JNK in these cells is unknown. Importantly, the data show that the JNK pathway mediates proliferative responses to Src signaling in vivo. Further work will be needed to precisely understand its role in proliferation (Read, 2004).
Genetic studies also highlight the importance of the Jak/Stat signal transduction pathway. dCsk proves a negative regulator of Jak/Stat signaling; for example, dCsk mutant tissues show up-regulation of Stat92E protein, a hallmark of Jak/Stat activation in Drosophila. Stat92E, the sole Drosophila STAT ortholog, is most similar to mammalian STAT3. In mammalian cells, Src directly phosphorylates and activates STAT3 and STAT3 function and activation are required for Src transforming activity. Conversely, overexpression of Csk blocks STAT3 activation in v-Src transformed fibroblasts. Activating mutations in STAT3 can also promote oncogenesis in mice. However, the physiological significance of these interactions within developing epithelia remains unclear (Read, 2004).
dCsk; Stat92E double mutant clones reveal that blockade of STAT function in dCsk mutants severely reduces Src-dependent overgrowth and promoted apoptosis of mutant tissue. dCsk–/–; Stat92E–/– EGUF adult eyes (the EGUF method produces genetically mosaic flies in which only the eye is exclusively composed of cells homozygous for the mutation) are nearly identical to phenotypes caused by overexpression of Dacapo, the fly ortholog of the cdk inhibitor p21, and PTEN, a negative regulator of cell proliferation and growth. Importantly, removing Stat92E function in dCsk mutant tissue led to a synthetic small eye phenotype and did not simply rescue the dCsk–/– proliferative phenotype. This outcome distinguishes Stat92E from mutations in Src64B, Btk29A, or bsk, which rescue dCsk-mediated defects toward a normal phenotype. The loss of tissue in dCsk–/–; Stat92E–/– clones indicates that Src-Btk signaling provokes apoptosis in the absence of Stat92E function. Consistent with this interpretation, reduced Btk29A function rescued the dCsk–/–; Stat92E–/– EGUF phenotype to a more normal phenotype, demonstrating that the reduced growth and increased apoptosis observed in the dCsk–/–; Stat92E–/– tissues is indeed Src-Btk pathway dependent (Read, 2004).
The data suggest the existence of a Src-dependent proapoptotic pathway that is normally suppressed by STAT. One possible component of this pathway is JNK, given that JNK signaling is an important activator of apoptosis in both flies and mammals. Perhaps Src-dependent hyperactivation of Bsk (JNK) in dCsk–/–; Stat92E–/– tissue contributes to cell death in the absence of proliferative and/or survival signals provided by Stat92E. However, a number of other candidate pathways may also mediate this response. The further characterization and identification of these pathways may have important implications for interceding in Src-mediated oncogenesis (Read, 2004).
Together, these observations indicate that, in tissue that contains hyperactive Src or reduced Csk, blocking STAT function is sufficient to trigger apoptosis and decrease proliferation in the absence of any further mutations or interventions. Reduced STAT3 function can promote apoptosis within breast and prostate cancer cells that show elevated SFK activity, but the molecular pathways driving apoptosis in these cells are unknown. These cells may require survival signals provided by STAT3 to counteract apoptosis due to chromosomal abnormalities or other defects. Alternatively, these cells may die because of proapoptotic signals provided by hyperactive SFKs in the absence of STAT3 function. The data argue that the latter may be true, which suggests the intriguing possibility that therapeutic blockade of STAT function in tumors with activated Src may actively provoke Src-dependent apoptosis and growth arrest in tumor tissues (Read, 2004).
Formins are involved in a wide range of cellular processes that require the
remodeling of the actin cytoskeleton. This study analyzes a novel
Drosophila formin, belonging to the recently described DAAM
subfamily. In contrast to previous assumptions, it is shown that DAAM
plays no essential role in planar cell polarity signaling, but it has striking
requirements in organizing apical actin cables that define the taenidial fold
pattern of the tracheal cuticle. These observations provide evidence the first
time that the function of the taenidial organization is to prevent the
collapse of the tracheal tubes. The results indicate that although
DAAM is regulated by RhoA, it functions upstream or parallel to the non-receptor tyrosine kinases Src42A and Tec29 to organize the actin cytoskeleton and to determine the cuticle pattern of the Drosophila respiratory system (Matusek, 2006).
Drosophila DAAM is required to organize an array of
parallel running actin cables beneath the apical surface of the tracheal cells
that define the taenidial fold pattern of the cuticle. The actin
ring pattern corresponds exactly to that of the taenidial fold pattern, and it is
proposed that the actin rings organized by DAAM define the position of
taenidial fold formation. The genetic interaction and epistasis data are
consistent with a model that DAAM activity is regulated by
RhoA. In addition, DAAM works together with the
non-receptor tyrosine kinases Src42A and Tec29 to regulate
the actin cytoskeleton of the Drosophila tracheal system (Matusek, 2006).
The basic structure of the insect tracheal system is a highly conserved tubular network in every species. The most important function of this network
is to allow oxygen flow to target cells. Thus, tracheal tubes need to be both
rigid enough, to ensure continuous air transport, and flexible enough along
the axis of the tubes, to prevent the break down of the tube system when body
parts or segments move relative to each other. These requirements are mainly
ensured by the tracheal cuticle, which covers the luminal surface of the tubes
and displays cuticle ridges (making the overall structure similar to the
corrugated hose of a vacuum cleaner). Analysis of DAAM mutants
provides the first direct evidence that this hypothesis is correct. The data
demonstrate that in the absence of DAAM the taenidial fold pattern is
severely disrupted, often leading to the collapse of the tubes and to
discontinuities in the tubular network. In addition, the analysis revealed
that the remarkably ordered cuticle pattern, displayed in the wild-type
trachea tubes, depends on DAAM-mediated apical actin organization. Apical
actin is organized into parallel-running actin cables, much the same way
teanidial folds run in the cuticle. Significantly, the formation of these actin bundles precedes the onset of cuticle secretion, and the number and phasing of the actin rings correspond exactly to that of the taenidial folds in the cuticle. Thus these studies revealed a novel aspect of apical actin organization in the tracheal cells that has not been appreciated before (Matusek, 2006).
The DAAM gene encodes a novel member of the formin family of
proteins, involved in actin nucleation and polymerization. Consistent with
this, DAAM is colocalized with apical actin in the tracheal cells, and the
activated form of DAAM is able to induce actin assembly when expressed in
tracheal cells and in other cell types (unpublished). In DAAM mutant tracheal cells, apical actin is still
detected, albeit at a somewhat lower level than in wild type, but the bundles
formed in the mutant are much shorter and thinner than in wild type, and often
appear to be crosslinked to each other. Most strikingly, global actin
organization is almost completely lost, although some local order can still be
detected. Remarkably, the cuticle pattern in mutant tracheal cells still
follows the underlying aberrant actin pattern. Overall, in DAAM
mutants, both the tracheal cuticle and the apical actin pattern resemble a
mosaic of locally ordered patches that failed to be coordinated and aligned
with each other and the axis of the tracheal tubes. It is thus proposed that the
apical actin bundles play a key role in patterning the tracheal cuticle by
defining the place of taenidial fold formation. Regarding the function of
DAAM, the results suggest that the major role of this formin in the tracheal
cells is to organize the actin filaments into parallel running actin rings or
spirals instead of simply executing the well characterized formin function
related to actin assembly. However, whether this is a direct effect on actin
organization, and thus represents a novel formin function, needs to be further
elucidated. An alternative model could be that DAAM is primarily required for
actin polymerization but tightly coupled to an actin 'organizing' protein. In
such scenario, the polymerization activity should be a redundant requirement,
whereas the link to the organizing protein would be a DAAM-specific function,
thereby explaining the presence of unorganized actin bundles in DAAM
mutant tracheal cells (Matusek, 2006).
In the case of the main tracheal airways, which are multicellular along
their periphery, it is striking that in wild type the run of the actin bundles
is perfectly coordinated across cell boundaries. In addition, the run is
always perpendicular to that of the tube axis. How does DAAM ensure
the coordination of these two aspects of actin organization? Because the DAAM
protein and the apical actin cables are both found at the level of the
adherens junctions, it is possible that DAAM regulates the coordination of the
actin cables at the cell boundaries through a direct interaction with
junctional protein complexes. However, other explanations are also possible,
and further experiments will be required to elucidate the molecular mechanism
of this regulatory function. The fact that actin cables normally run
perpendicular to the tube axis seems to suggest that tracheal cells are able
to sense a global orientation cue and align their actin bundles accordingly.
The nature and source of this cue is unknown, as is the mechanism by which
DAAM is involved in the read-out of this signal. Nevertheless, it is
interesting that in DAAM and btl-Gal4/UAS-C-DAAM mutant
trachea, the main pattern of the cuticle phenotype is often changing from one
segment to the other, suggesting that the effect of the 'global' orientation
cue is limited to metameric units (Matusek, 2006).
Sequence comparisons of FH2 proteins suggest a close phylogenetic
relationship between the DRF, FRL and DAAM subfamilies. Members of these three subfamilies have a high level of conservation in the FH2 domain, and importantly, also in the region of the GBD and DAD domains,
suggesting that the FRL and DAAM family formins are also regulated by
autoinhibition and RhoGTPases, like the DRFs. Further evidence is presented in
support of this view. First, DAAM and RhoA display a strong
genetic interaction. Second, C-DAAM (an N-terminally truncated form of DAAM)
behaves like an activated form much the same way DRF family formins behave.
Third, epistasis experiments with C-DAAM and RhoA suggest that RhoA
acts upstream of DAAM. Thus, the data support the model in which DAAM, at
least in the Drosophila tracheal system, is regulated by
autoinhibition that can be relieved by RhoGTPases (Matusek, 2006).
This conclusion, however, contradicts the observation that human DAAM1 is
required for Wnt/Fz/Dvl dependent RhoA activation in cultured cells and that
xDaam1 appears to mediate Wnt-11 dependent RhoA activation in Xenopus
embryos. These results suggested that DAAM functions upstream of RhoA in non-canonical Wnt/Fz-PCP signaling. An explanation for these distinct conclusions might be related to the fact that DAAM, in contrast to xDaam1, does not appear to be required for Fz/Dsh-PCP signaling. Hence, it is possible that the
Drosophila ortholog is regulated in the same way as the DRF formins,
while the vertebrate family members can be regulated in a different way, once
bound by Dsh/Dvl and recruited into PCP signaling complexes (Matusek, 2006 and references therein).
Genetic interactions with the hypomorphic DAAMEx1
allele identified two non-receptor tyrosine kinases, Src42A and
Tec29, as strong interacting partners. Although both of these kinases
play multiple roles during embryogenesis, single mutants
for both affect the tracheal cuticle pattern in a similar way to
DAAM. These results suggest that DAAM and the Src family
kinases work together to regulate the actin cytoskeleton and cuticle pattern
in tracheal cells. Although it is not known whether DAAM physically binds
Src42A and/or Tec29, it has been established that the FH1 region of DRFs and
other formins can bind SH3 domains, including those of the Src family kinases. In
agreement with these data that DAAM acts upstream of Src42A and Tec29
in tracheal cells, cytoskeleton remodeling and SRF activation mediated by
mouse Dia1 and mouse Dia2 requires Src activity.
Moreover, a recent report suggests that RhoD and human DIA2C regulate endosome
dynamics through Src activation, proposing that Src activity is stimulated via
human DIA2C dependent recruitment to early endosomes. Similarly, the Limb deformity protein (a formin) interacts with Src on the perinuclear membranes of primary mouse fibroblasts. Based on these examples, it is speculated that in Drosophila tracheal cells the RhoA/DAAM/Src module may not only be required to organize apical actin bundles, but additionally it might represent a link to secretory vesicles and to the regulation of exocytosis. Future studies will be required to test this hypothesis, and to unravel the mechanisms whereby DAAM family formins and Src family kinases contribute to cytoskeletal remodeling in the Drosophila tracheal system and in other tissues (Matusek, 2006).
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date revised: 23 July 2014
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