The mammalian receptor protein tyrosine kinase (RTK), Anaplastic Lymphoma Kinase (ALK), was first described as the product of the t(2;5) chromosomal translocation found in non-Hodgkin's lymphoma. While the mechanism of ALK activation in non-Hodgkin's lymphoma has been examined, to date, no in vivo role for this orphan insulin receptor family RTK has been described. This study describes here a novel Drosophila RTK, Alk, which maps to band 53 on the right arm of the second chromosome. Full-length Alk cDNA encodes a phosphoprotein of 200 kDa, which shares homology not only with mammalian ALK but also with the orphan RTK LTK. Analysis of both mammalian and Drosophila ALK reveals that members of the ALK family of RTKs contain a newly identified MAM domain within their extracellular domains. Like its mammalian counterpart, Alk appears to be expressed in the developing CNS by in situ analysis. However, in addition to expression of Alk in the Drosophila brain, careful analysis reveals an additional early role for Alk in the developing visceral mesoderm where its expression is coincident with activated ERK (Lorén, 2001).
These data provide evidence for the existence of a Alk RTK pathway in Drosophila and show that ERK participates in this pathway, and that it is activated by Alk in vivo. Expression patterns of Alk, together with activated ERK, suggest that Alk fulfils the criteria of the missing RTK pathway, leading to ERK activation in the developing visceral mesoderm (Lorén, 2001).
Mammalian Anaplastic Lymphoma Kinase (ALK) was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases (RTKs) which acquire their transforming capability when they are truncated and fused to nucleophosmin (NPM) in the t(2;5) chromosomal rearrangement associated with non-Hodgkin's lymphoma. To date, several chromosomal rearrangements leading to an activated ALK RTK have been described, including NPM-ALK which are constitutively dimerized through the fused domain. However, there are few insights into the normal structure and function of the ALK RTK. Full-length cDNA encoding the mammalian ALK RTK has been identified as a first step towards a functional assessment of the receptor. ALK is a member of the Insulin Receptor superfamily, most closely related to the orphan RTK leucocyte tyrosine kinase (LTK). In situ hybridization studies have revealed ALK expression in the developing nervous system and ALK is currently a novel orphan receptor tyrosine kinase that is suspected to play an important role in the normal development and function of the nervous system (Lorén, 2001).
Alk was identified using a degenerate PCR approach. Alk is a 200 kDa RTK that has strong homology with both ALK and LTK. Due to the conserved nature of many receptor signalling systems in Drosophila, ALK RTK mediated signalling may also be conserved from Drosophila to vertebrates. Drosophila has a smaller number of RTK genes than vertebrates, with ~21 RTKs now predicted to be encoded by the Drosophila melanogaster genome. In addition, since the sequencing of the Drosophila melanogaster genome has now been completed it can now be said that while an Insulin Receptor homologue is present, there appears to be no homologue for the ALK relative RTK, LTK in Drosophila melanogaster. Alk is expressed during early mesodermal development as well as within the developing nervous system. Interestingly, early expression of Alk in the mesoderm correlates with ERK activation in the developing embryo mesoderm in vivo. Furthermore, using the UAS-GAL4 expression system, together with clonal over-expression techniques, Alk is observed to indeed activate ERK in vivo (Lorén, 2001).
To identify novel PTKs in Drosophila melanogaster, a degenerate PCR-based approach was used. Highly conserved residues within subdomains VIb and IX of known PTKs were targeted for degenerate PCR primer design, leading to the identification of several novel putative Drosophila melanogaster PTKs. Multiple PCR products were obtained and sequenced, identifying novel as well as previously described PTKs. One of the novel PCR products, displayed the greatest similarity to members of the mammalian Insulin Receptor RTK superfamily (Lorén, 2001).
To characterize the Alk protein, pcDNA3:Alk was transiently expressed in 293 cells. Anti-Alk antibodies were used to detect Alk from cell lysates. Lysates were resolved on SDS-PAGE and analysed by immunoblotting for Alk. Alk antibodies specifically recognized a 200 kDa protein, which is present when the cells were transfected with pcDNA3:Alk. Lysates were also analysed by anti-phosphotyrosine immunoblotting; Alk was detected as a 200 kDa tyrosine phosphorylated protein, suggesting that Alk is indeed a PTK. Furthermore, anti-Alk antibodies recognize a doublet of endogenous Alk at approximately 200 kDa from whole embryo extracts. Currently, the nature of this doublet is unknown; it may reflect the phosphorylation status of Alk, although alternative splicing may also be responsible (Lorén, 2001).
Expression of Alk throughout embryonic development was examined by in situ analysis. Embryos from 0 to 24 h of age were analysed by in situ hybridization using a 3' digoxigenin Alk probe. The Alk transcript is found in the mesoderm and the dorsal ectoderm earlier in development (stages 10 and 11). From around 11 h of development to late embryogenesis, Alk mRNA is concentrated within the developing nervous system, and is observed in both the developing brain and ventral nerve cord (VNC) (Lorén, 2001).
To further define where Alk is expressed, monoclonal antibodies to the extracellular portion of the Alk protein were generated. Immunostaining with anti-Alk antibodies reveals that Alk is expressed in a striking pattern throughout development. All Alk expression patterns were abolished by competition with the original Alk immunogen. In particular, prominent Alk staining is observed in the visceral mesoderm at stage 11. It is first seen as segmental patches, before a fusion of the visceral arches from each segment, and is subsequently observed as a continuous waved line. The uniform pattern of Alk in the visceral mesoderm suggests that it may not be involved in the directed migration of these cells, but possibly in their differentiation (Lorén, 2001).
The expression of Alk was further investigated through an analysis of the putative enhancer promoter region. The Alk transcription unit is composed of eight coding exons and two 5' alternatively spliced noncoding exons spanning approximately 15 kb. Since both exon1A and exon1B mapped within a 6.5-kb EcoRI fragment 5' of the ORF of Alk, this fragment was used to generate a transgenic fly in which the putative enhancer region of Alk was placed upstream of Gal4 in the P-element vector RSBSK (pRSBSK:AlkEI6.5-GAL4). The resulting transgenic flies were then used to drive UAS-GFP reporters. As judged by reporter gene expression, the 6.5 kb EcoRI genomic fragment drives reporter gene expression in the visceral mesoderm (VM) at stages 11/12 in a pattern similar to that seen for the Alk protein. At later stages (from stage 13 onwards) of embryonic development, Alk was found to be expressed within the developing brain and ventral nerve cord. The expression of Alk within the CNS persists through larval instar stages where Alk is highly expressed within the brain and ventral nerve cord (Lorén, 2001).
A 1997 study conducted by Gabay (1996) produced a detailed 'atlas of MAPK activation' in vivo. This study used antibodies that were specific for activated phospho-ERK as a tool for dissecting ERK activation throughout Drosophila embryonic development. It was noted that most aspects of the phospho-ERK pattern observed could be accounted for by known Drosophila RTK pathways. However, several of the patterns revealed were novel with respect to the receptor they are triggered by. It was speculated that these patterns may be induced by unknown RTKs that may activate ERK. In particular, prominent phospho-ERK staining was observed in the visceral mesoderm at stage 11. It was first seen as segmental patches, before fusion of the visceral arches from each segment, and subsequently observed as a continuous waved line. Furthermore, this phospho-ERK pattern in the visceral mesoderm was not dependent upon the Heartless RPTK. Since Alk expression was seen in the visceral mesoderm, whether Alk expression coincided with the phospho-ERK pattern in the visceral mesoderm was examined in vivo (Lorén, 2001).
In order to confirm that Alk and phospho-ERK were expressed in the visceral mesoderm during development, wild-type embryos were collected and stained for Alk and phospho-ERK. In both cases, expression was observed in the visceral mesoderm at stages 11/12 in a similar pattern. Subsequently, embryos were collected and double-stained for activated phospho-ERK and Alk. Co-localization of both activated phoshpo-ERK and Alk could clearly be observed in the visceral mesoderm (Lorén, 2001).
So far it has not been possible to obtain Alk mutants and so it was not possible to examine whether Alk is responsible for ERK activation in the developing visceral mesoderm in vivo. However, it was ask if Alk was capable of driving ERK activation in vivo by utilizing the GAL4-UAS system. Alk cDNA was cloned into P element expression vectors under the control of yeast GAL4 upstream activating sequences (UAS) and P element-mediated germ-line transformation was used to generate UAS:Alk transgenic fly lines. When Alk was expressed ectopically under the control of the Actin5C promoter driving GAL4 (Actin5C-GAL4) the result was 100% embryonic lethality. In order to examine whether the Alk RTK is capable of driving ERK activation in vivo, pGMR-GAL4, which drives expression in all photoreceptor cells, was employed to express Alk in the developing eye disc. A very clear effect of Alk expression on ERK activation was observed: normally prominent ERK activation is seen within the morphogenetic furrow, with lower levels in the differentiated third instar eye disc. In contrast, high levels of ERK activation in vivo were observed when Alk was expressed. Further conformation of Alk driven ERK activation in vivo was achieved using a combination of the FLP-out system and the GAL4-UAS system. In this system, a fragment of DNA bracketed by FRT sites and containing transcription stop signals is inserted between the Actin5C promoter and GAL4. Heat shock induction of Flippase activity induces recombination in which the transcription stop segment is flipped out, thereby allowing the Actin5C promoter to drive the GAL4 expression. This system allows the creation of clones of cells expressing Alk, which are marked by GFP expression. The expression of Alk, as judged by immunostaining, and GFP were coincident, demonstrating that the system works for Alk as well as establishing the specificity of the anti-Alk antibodies. While endogenous Alk protein is expressed in the third instar brain during normal development, levels of Alk within over-expressing clones are clearly observed over endogenous levels. Alk over-expressing clones also display increased levels of phosphotyrosine, consistent with the over-expressed Alk being active and either directly or indirectly leading to protein phosphorylation in these clones. Furthermore, larger clones were observed to disrupt the normal tissue structure, leading to abnormal disc development. Animals carrying Alk over-expression clones did not survive to adulthood. Further analysis of Alk clones in discs isolated from third instar larva indicates that Alk leads to ERK activation in situ. Thus, Alk has the capacity to drive activation of ERK in vivo, and is therefore a prime candidate for the 'missing' RTK driving ERK activation within the developing visceral mesoderm in vivo (Lorén, 2001).
The Drosophila melanogaster gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian Alk, which encodes a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). In humans, the t(2;5) translocation, which involves the ALK locus, produces an active form of ALK, which is the causative agent in non-Hodgkin's lymphoma. The physiological function of the Alk RTK, however, is unknown. Loss-of-function mutants in the Drosophila Alk gene are described that cause a complete failure of the development of the gut. It is proposed that the main function of Drosophila Alk during early embryogenesis is in visceral mesoderm development (Lorén, 2003).
The Alk locus in Drosophila was mutated by first creating a designer deletion over the Alk53 locus, which was subsequently used in an ethylmethane sulphonate (EMS) screen with the aim of identifying Alk mutant animals. Eleven independent Alk mutants were identified using this approach, and each showed similar phenotypes, as described below. All the mutations identified were located in amino acids that are conserved between Drosophila, mouse and human Alk. The Alk11 mutant line carried several amino-acid mutations (Lorén, 2003).
The mutations could be divided into three groups: truncations (Alk1 and Alk8), point mutations within the extracellular domain (Alk2 to Alk7) and point mutations within the protein tyrosine-kinase (PTK) domain (Alk9 and Alk10 ). Alk1 is a mutation of Gln 306 at the beginning of the first MAM (named after mephrins, A-5 protein and receptor protein tyrosine phosphatase mu) domain, which creates a stop codon and results in a truncated protein. This protein is estimated to have a molecular weight of 33 kDa and, consistent with this, analyses of heterozygous Alk1 mutant animals showed the presence of a truncated protein. The Alk1 protein lacks any recognizable domain: this allele is considered to be an Alk RTK functional null. The second mutation that causes a truncation, Alk8, arises from a splice-donor-site mutation and is predicted to generate a truncation just after the transmembrane domain. Since no Alk mutant phenotypes are seen in Alk8 heterozygous animals, it seems that the mutant protein expressed does not act in the predicted dominant-negative manner, at least when expressed at endogenous levels. Interesting conclusions about the functional importance of the various Alk extracellular regions can be made from the Alk point mutations that lie within the extracellular domain (Lorén, 2003).
Alk is the only RTK that contains a MAM domain in its extracellular region (Lorén, 2001). MAM domains are comprised of 160 amino acids, and are present in transmembrane proteins such as the meprins and receptor protein-tyrosine phosphatases, in which they seem to function in cell-cell interactions. In Drosophila Alk, the second MAM domain seems to be important, since Alk2 was identified as a mutation of Asp 681 in this domain. More surprisingly, the Drosophila Alk mutant screen underscores the importance of the glycine-rich region, a region that contains stretches of up to six glycines in a row, which the Alk RTK shares with its relative, Ltk. In Alk4, Alk5, Alk6 and Alk7 mutants, a single glycine within the glycine-rich domain is mutated to an acidic amino acid. All of the glycines that are mutated in the Drosophila Alk mutants are conserved not only between the Drosophila and human Alks, but also in the Ltk RTK, thus suggesting an important role for this domain and highlighting the intolerance of an acidic residue in the stretches of glycine in this domain (Lorén, 2003).
The third class of Alk mutants have point mutations in the intracellular domain. It is interesting to note that no mutations were found in the six potential phosphotyrosine motifs that lie outside the PTK domain, and although this may simply be due to chance, it may also indicate some plasticity in the signalling pathways downstream of the Drosophila Alk receptor. Both Alk9 and Alk10 have mutations that lie in the conserved PTK catalytic domain of the receptor, thus indicating that in the case of Drosophila Alk, the PTK activity of the receptor is indeed essential for its in vivo action. This is an important observation, since PTK activity is not essential for at least one RTK in Drosophila. Alk9 is a mutation in the conserved sub-domain III of the kinase domain, in which the invariant glutamate (Glu 1244 in Drosophila Alk) in the C-helix, which is responsible for stabilizing the catalytic lysine and the alpha- and ß-phosphates of Mg-ATP, is mutated to lysine. In a fourth class of Alk mutants, Alk10 has a mutation of the aspartic acid (Asp 1347 in Drosophila Alk) of the highly conserved triplet Asp-Phe-Gly (DFG), in subdomain VII, to asparagine. This aspartic acid is an invariant residue in protein kinases and is essential for activity, functioning to orientate the gamma-phosphate of Mg-ATP for transfer to the substrate. Thus, from the ten Alk mutant alleles identified, the importance of the different domains in the Drosophila Alk RTK can be inferred: functionally, the second MAM domain, the glycine-rich domain and the PTK domain are of crucial importance for Drosophila Alk function (Lorén, 2003).
Fifty per cent of animals homozygous for Alk mutations died as embryos, and the rest died as first-instar larvae. In no case did an Alk mutant animal survive past the first-instar larval stage. Despite expression of Alk in the brain in mice and flies (Iwahara, 1997; Lorén, 2001; Morris, 1997), the central nervous system of mutant larvae seems to be generally normal, as visualized by staining with monoclonal antibody 22C10. Both human and Drosophila Alk have been reported to be expressed in the gut (Lorén, 2001; Morris, 1994), with Drosophila Alk being highly expressed in the developing visceral mesoderm during embryogenesis (Lorén, 2001; Lorén, 2003).
Visceral muscles are important components of many internal organ systems, particularly the gastrointestinal and urogenital tracts, respiratory tract and vascular system. In Drosophila, the visceral musculature is less diverse and primarily consists of the musculature of the digestive tract. Following the early subdivision of the mesoderm in the embryo, cells are specified to contribute to distinct tissues, which then perform coordinated migrations to form organs. The Drosophila visceral mesoderm is composed of two sets of muscles, an inner layer of circular muscles derived from cells along the trunk of the embryo, and an outer layer of longitudinal muscles derived from the posterior end of the embryo. The presumptive visceral mesoderm can first be identified as 12 metameric clusters. The cells of these clusters migrate longitudinally to form two parallel bands along most of the length of the embryo, then ventrally and dorsally to form a closed tube, which is lined by endoderm. Longitudinal visceral-muscle precursors migrate over the circular muscle cells (Lorén, 2003).
Using immunohistochemistry, the expression of Alk protein in the developing visceral mesoderm in detail was examined. Alk expression is first detected at germband extension (stage 10) as two Alk-positive cell groups per segment in the metameric clusters that correspond to presumptive visceral mesoderm. During germband retraction (stage 11), the clusters on each side of the embryo fuse into a continuous band. At the end of germband retraction (stage 13), Alk is expressed in a broad band of visceral mesoderm on each side of the embryo. During stage 14, these two bilateral bands begin their expansion. By late embryogenesis, the cells of the visceral mesoderm have spread out dorsally and ventrally to encircle the entire gut. Alk mutant animals that survived to first-instar stages were analysed using a gut-coloration assay. Whereas heterozygous sibling animals are robust, with healthy appetites, Alk mutant animals do not seem to ingest food. On fine dissection, these animals were found to lack discernable intestinal structures. Further analysis of the developing gut in Alk mutant animals showed that the Alk-positive visceral mesoderm is severely disrupted and that no functional midgut is formed (Lorén, 2003).
The function of Alk in visceral mesoderm development was further analysed using the immunoglobulin domain adhesion molecule Fasciclin III (FasIII), which is a marker for differentiated visceral mesoderm in the Drosophila embryo. In wild-type embryos, Alk and FasIII expression patterns overlap perfectly in the visceral mesoderm as the midgut forms. Further analysis of Alk mutant embryos shows that there is a complete loss of Alk-positive and FasIII-positive cells, whereas FasIII staining in the epidermis was normal. Similar results were obtained when antibodies to Drosophila Myocyte enhancer factor 2 (Mef2), which is produced in all muscle lineages of the Drosophila embryo, were used. Furthermore, anti-myosin-heavy-chain (MHC) staining, which showed the thin layer of gut mesoderm in wild-type embryos, was absent from Alk mutant animals (Lorén, 2003).
To test whether Alk is sufficient for FasIII activation, UAS-Alk (an Alk transgene under the control of the yeast Gal4 upstream activating sequence) was expressed using the mesodermal twist-Gal4 driver. Indeed, Alk induces ectopic expression of FasIII, supporting the idea that Alk controls FasIII expression. This is an exciting possibility, since the forkhead-domain transcription factor, Drosophila FoxF/Biniou, has been reported to drive expression of visceral mesoderm markers, including FasIII, and Drosophila FoxF/Biniou could potentially be activated by an Alk RTK signalling pathway, since Alk is a member of the Insulin receptor RTK superfamily (Lorén, 2001). Since induction of FasIII expression was seen upon Alk expression, Alk mutant embryos were re-examined. Using confocal microscopy, it was possible to locate scattered Alk-positive cells in Alk1 mutant animals. This is possible because the anti-Alk antibodies are raised against the extreme amino-terminal end of Alk and therefore the Alk1 truncated protein could be detected. On closer inspection, these cells are also seen to be weakly FasIII-positive. Thus, although FasIII expression seems to be significantly reduced in visceral mesoderm cells in Alk mutants, it is not absent. Nevertheless, full FasIII expression may still require Alk signalling through a FoxF/Biniou-mediated pathway, especially since it has been reported that there may be a positive-feedback pathway that reinforces FasIII expression (Lorén, 2003).
It is concluded that the Drosophila Alk RTK is indeed crucial for ERK activation and for downstream ERK-mediated events in the visceral mesoderm. The endogenous Alk RTK has a function in controlling gut development in Drosophila. The targets of Alk-mediated signalling remain to be discovered. The exciting possibility exists that Alk is the receptor for the newly discovered Jelly belly protein, which has been shown to be required for mesoderm migration and differentiation. Exploring these Alk pathways and targets is an important task for the future (Lorén, 2003).
The visceral muscles of the Drosophila midgut consist of syncytia and arise by fusion of founder myoblasts with fusion-competent myoblasts (fcms), as described for the somatic muscles. A single-step fusion results in the formation of binucleate circular midgut muscles, whereas a multiple-step fusion process produces the longitudinal muscles. A prerequisite for muscle fusion is the establishment of myoblast diversity in the mesoderm prior to the fusion process itself. Evidence is provided for a role of Notch signalling during establishment of the different cell types in the visceral mesoderm, demonstrating that the basic mechanism underlying the segregation of somatic muscle founder cells is also conserved during visceral founder cell determination. Searching for genes involved in the determination and differentiation of the different visceral cell types, two independent mutations were identified causing loss of visceral midgut muscles. In both of these mutants, visceral muscle founder cells are missing and the visceral mesoderm consists of fusion-competent myoblasts only. Thus, no fusion occurs resulting in a complete disruption of visceral myogenesis. Subsequent characterization of the mutations revealed that they are novel alleles of jelly belly (jeb) and the Drosophila Alk homolog named milliways (miliAlk or just plain Alk). The process of founder cell determination in the visceral mesoderm depends on Jeb signalling via the Milliways/Alk receptor. Moreover, it has been demonstrated that in the somatic mesoderm determination of the opposite cell type, the fusion-competent myoblasts, also depends on Jeb and Alk, revealing different roles for Jeb signalling in specifying myoblast diversity. This novel mechanism uncovers a crosstalk between somatic and visceral mesoderm leading not only to the determination of different cell types but also maintains the separation of mesodermal tissues, the somatic and splanchnic mesoderm (Stute, 2004).
The process of lateral inhibition involving Notch and its ligand Delta plays a role in determining the founder myoblasts and fusion-competent myoblasts (fcms) of the somatic musculature. Since many of the processes involved in the development of the somatic musculature also seem to affect the development of the visceral muscles, whether the mechanism of determination of founder cells and fcms is also conserved was examined. In Notch mutant embryos more founder cells appear to be present in the visceral mesoderm. The visceral fcms seem to be reduced compared with the wild-type expression of sticks and stones (sns) as a marker for these cells. This reduction is not as severe as in the somatic mesoderm but still quite obvious. In Delta mutants, the number of founder cells also seems to be increased in comparison with the wild type and the fcms are reduced in mutant embryos (Stute, 2004).
These observations cannot exclude the possibility that the observed phenotypes are induced by secondary effects from defects in other tissues, among others the lack of fcms in the somatic mesoderm. Therefore overexpression studies were undertaken using the UAS-GAL4 system. The GAL4 and UAS lines employed in this study also carry rP298-lacZ, which serves to mark the founder cells. As a driver line bap-GAL4 was used to drive expression in the entire trunk visceral mesoderm. Expression of UAS-Notch+Delta, which contains the entire coding regions of both genes or UAS-Notchintra, which represents a constitutively active form of Notch, in the visceral mesoderm, both result in a distinct phenotype. In midgut preparations of these embryos the founder cells of the circular visceral mesoderm are strongly reduced and later on, no functional visceral mesoderm can be observed. By contrast, the founder cells of the longitudinal visceral muscles, which have a different origin at the posterior tip of the embryo, are still present. Interestingly,bap-GAL4-driven expression of the Notch ligand Delta does not result in fewer founder cells in the visceral mesoderm (Stute, 2004).
To exclude the possibility that the described defects are due to non-endogenous effects induced by the overexpression of the examined genes in the wrong tissue, wild-type Notch expression was analyzed and found to be expressed in the visceral mesoderm. Notch is localized at cell membranes in the entire visceral mesoderm during stage 11, with expression becoming weaker in the fcms of the visceral mesoderm, that continue to express bap-lacZ after the determination process is finished. This reduction of Notch expression in the fcms after the establishment of the founder cells is similar to its expression in the somatic mesoderm, where Notch expression is also highest in the progenitor cell after the determination process is completed. Surprisingly, the analysis of Delta expression exhibits that this Notch ligand is not expressed in the visceral mesoderm during founder cell formation. Delta expression was found in adjacent, probably somatic cells and might be needed there to participate in the visceral determination process, as indicated by the increased number of founder cells and reduced number of fcms in Delta mutants. Even though Dl is expressed in the cells surrounding the visceral mesoderm, ectopic expression of UAS-Dl in these cells with a twi-GAL4 driver line does not result in an obvious phenotype, which might be due to the fact that the amount of Delta in this tissue is not the limiting factor that restricts Notch signalling. Another explanation for a missing Delta expression in the visceral mesoderm might be that a different factor acts as a ligand for Notch in the visceral mesoderm and that the observed phenotype in Delta mutants is due to secondary effects (Stute, 2004).
Since the ectopic expression causes such a severe phenotype, the lethality of these embryos was tested. Most of the progeny of the cross between the bap-GAL4 driver line and UAS-N+Dl or UASNintra develop and hatch but die as first larvae (78% or 70%), presumably owing to the fact that they cannot ingest any food. Ectopic expression of UAS-Dl alone also increased lethality compared with the UAS and GAL4 lines alone, but still ~65% of the larvae survive (Stute, 2004).
To confirm these results, a dominant-negative form of Notch (UAS-dnN) was overexpressed specifically in the visceral mesoderm with a bap-GAL4 driver. The embryos exhibit an obvious duplication of most visceral founder cells but still some fcms remain (Stute, 2004).
From these results, it is concluded that Notch plays a role in the determination of the founder cells and fcms in the visceral mesoderm. Delta, which is expressed in the cells surrounding the visceral mesoderm, might serve as the ligand in this process but it is also possible that another factor takes over this role. Hence, not only is the fusion mechanism between the founder cells and the fcms in the somatic and visceral mesoderm conserved, but so is the initial mechanism of determination of these two cell types (Stute, 2004).
To find out more about the mechanisms involved in the formation of the visceral muscles, a collection of EMS mutagenised flies was screened in order to search for genes involved in the determination of the two visceral cell types as well as in other aspects of visceral mesoderm differentiation. Mutant embryos were stained and analysed with Fasciclin 3 (Fas3), which marks the complete visceral mesoderm and allowed the two cell types to be distinguished. Founder cells show a strong Fas3 expression and are characterized by a more columnar shape, while the more globular fcms show a clearly weaker Fas3 expression. Using this approach, several mutants were identified with various defects in the development of the visceral musculature (Stute, 2004).
A subgroup of mutants consisted of two independent mutations, wellville (weli) and milliways (mili), with the same, distinct phenotype. In these two mutants, the continuous band of the visceral mesoderm in stage 11 is formed, but when stained with Fas3, the more columnar shaped founder cells with the stronger Fas3 expression are absent. Thus, it appears that the founder cells of the circular visceral muscles are not determined in either of these mutants. Using the enhancer trap line rP298-lacZ, which shows a ß-galactosidase pattern reflecting the expression of Duf/Kirre, it was indeed shown that in both mutants this founder cell marker is not expressed in the visceral mesoderm. In contrast to these observations, the determination of founder cells in the somatic mesoderm is not affected, and the somatic muscle pattern shows only mild fusion defects, which are especially obvious in the dorsal and ventral muscles. At later stages no visceral mesoderm is present in either mutant (Stute, 2004).
Both mutations, weli and mili, are located on the second chromosome. Complementation tests were subsequently performed with mutants on the second chromosome, which are known to affect visceral mesoderm development. Surprisingly, this analysis revealed that weli is a new jelly belly (jebweli) allele. jeb encodes a secreted protein that is produced in the somatic mesoderm but is needed for proper visceral mesoderm formation and has been proposed to be essential for the migration and differentiation of the visceral mesoderm (Weiss, 2001). The phenotype of the specific loss of founder cells of the circular visceral muscles has not been described (Stute, 2004).
mili displays the same distinct phenotype as jeb and it was reasoned that it is likely that both genes are involved in the same pathway. Since Jeb is a secreted protein the most promising candidate for mili was Drosophila Alk, a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs), that is expressed in the nervous system and the visceral mesoderm (Lorén, 2001). Alk is considered (Lorén, 2003) to be a possible receptor for jeb signalling (Stute, 2004).
In order to further analyze whether mili is indeed an allele of Alk, a newly created deficiency in the region (Df(2R)AlkDelta21), in which Drosophila Alk has been removed (Lorén, 2003), was tested. Indeed, mili is allelic to Df(2R)AlkDelta21, and furthermore, embryos transheterozygous for Df(2R)AlkDelta21 and mili show the same phenotype as mili mutant embryos on Fas3 analysis. mili was then directlt tested against the newly generated Alk1 allele (Lorén, 2003) and indeed, this confirmed that mili is a new Alk allele, now termed miliAlk. The analysis of miliAlk mutants with the help of Alk antibodies (Lorén, 2001) reveals that the mutant Alk protein is found in the cytoplasm instead of its normal localization at the cell membrane. Therefore it was concluded that the mutation is a phenotypic null allele. Furthermore, the specific loss of founder cells in the visceral mesoderm could be rescued by ectopic expression of UAS-Alk in the miliAlk mutant background using bap-GAL4 as driver. Thus, the two newly identified mutants, both of which display the same, very distinct, phenotype of loss of founder cells in the visceral mesoderm, turn out to be novel jelly belly and Alk alleles (Stute, 2004).
The cells of the visceral mesoderm in jebweli and miliAlk mutants do not express the founder cell marker rP298-lacZ and exhibit exclusively a globular shape upon Fas3 staining: this is characteristic for fcms. This raised the question of whether the cells indeed are determined to become fcms or remain undifferentiated. To clarify this question, in situ hybridization was performed with sns as probe. sns is expressed in all fcms, both in the somatic and in the visceral mesoderm. In the wild type during stage 11, two bands of sns-expressing cells can be observed in the mesoderm; the cells are connected in a ladder like pattern and represent the fcms of the somatic and visceral mesoderm. In jebweli and miliAlk mutants, only one band is present whereas the other band is missing. As indicated by the location of the connecting cells ventral to the present band, the dorsal band consisting of the fcms of the visceral mesoderm is still present. Thus, the remaining cells in the visceral mesoderm differentiate as fcms and express genes that are characteristic for this differentiated cell type (Stute, 2004).
The findings that in jebweli and miliAlk mutants (namely, the lack of founder cells in the visceral mesoderm, and the observation that fcms of the somatic mesoderm do not express fcm specific genes like sns) were interesting since only mild defects in the somatic muscles are observed. To explain this phenotype, a closer look was taken of Alk expression in wild-type embryos. In addition to the expression of Alk in the cells of the visceral mesoderm, additional patches can be found in the neuroectoderm and the somatic mesoderm during stages 10 and 11. It is concluded that these patches of Alk expression in the somatic mesoderm are essential for the development of the somatic fcms because in Alk mutant embryos, which are unable to activate the RTK pathway, these cells do not express fcms-specific genes. Furthermore, jeb signalling is also required for this process, because the same phenotype can be observed in jeb mutants. Therefore the RTK signalling pathway involving Jeb and Alk is not only needed for founder cell specification of the visceral mesoderm but also for the differentiation of the fcms in the somatic mesoderm (Stute, 2004).
Having found that sns is no longer expressed in the fcms of the somatic mesoderm, an examination was made of the transcription factor lame duck/myoblast incompetent/gleeful (lmd/minc/glee), which is expressed in the somatic and visceral fcms and is responsible for their determination. The expression pattern of Lmd in the wild type is similar to that of sns and the protein is present in two bands at stage 11-12. In both jebweli and miliAlk mutants, the Lmd expression pattern is present not only in the fcms of the visceral mesoderm but also in the somatic ones, even though it seems as if it is slightly weaker in the ventral part in the mutants than in the wild type. These data suggest that in jebweli and miliAlk mutants the initial determination of the fcms in the somatic mesoderm takes place, but the subsequent differentiation is blocked. Therefore, the Alk-RTK signalling pathway in the somatic mesoderm seems to be essential for the differentiation of the fcms but not for the initial determination (Stute, 2004).
Because most of the fcms of the somatic mesoderm do not express sns in jebweli and miliAlk mutants, a closer look was taken at defects in this tissue. ß-galactosidase antibody staining in mutants carrying the founder cell marker rP298-lacZ shows a regular pattern of somatic founder cells compared with the wild type in the somatic mesoderm. Only in some of the mutant embryos were local distortions detected because of the defects in the visceral mesoderm. ß3 tubulin antibody staining shows some mild fusion defects in the dorsal and ventral muscles in jebweli and miliAlk mutants, indicated by unfused myoblasts in this region and long thin projections of the muscles (Stute, 2004).
The development of the visceral mesodermal cells cannot be followed with Fas3 staining because it disappears in the mutants after stage 11. Therefore, the fate of the fcms was visualized using the visceral mesoderm marker bap-lacZ, which normally is expressed exclusively in the visceral mesoderm throughout embryonic development. jebweli and miliAlk mutants carrying this marker show ß-galactosidase expression in the somatic mesoderm from late stage 12 onwards (Stute, 2004).
A lack of sns expression in fcms in the somatic mesoderm has been shown to result in strong defects in the somatic musculature where the founder cells become blocked at the point of myoblast fusion. Because such a strong phenotype was not detected in miliAlk and jebweli mutants, and because bap-lacZ-expressing cells are present in the somatic mesoderm, it is concluded that the sns-expressing cells from the visceral mesoderm become incorporated into the somatic mesoderm and replace at least a fraction of the somatic fcms (Stute, 2004).
Since jeb is a secreted protein, it was of interest to see whether the localization of Alk controls the specification of the more ventral cells of the visceral mesoderm to become founder cells whereas the others develop into fcms. Staining with anti-Alk antibodies (Lorén, 2003) shows that in the wild type the protein is localized at the cell membranes in the visceral mesoderm. Surprisingly, Alk can be found in the founder cells of the circular visceral muscles and in the fusion-competent myoblasts, which are not obviously affected in miliAlk mutants (Stute, 2004).
In jebweli mutants, the localization of Alk is not affected. As in the wild type, it localizes at the cell membranes and is also present in all cells of the visceral mesoderm, that persist in these mutants. In miliAlk mutant embryos, however, the Alk protein is still detectable in all cells of the visceral mesoderm, but it is not correctly localized at the cell membrane and is instead found in the cytoplasm. Because of this mislocalization and the fact that the embryos transheterozygous for Df(2R)AlkDelta21 and miliAlk display the same phenotype in the visceral mesoderm as the miliAlk mutant embryos alone, it is concluded that the mutation is a phenotypic null allele even though the N-terminal part of the protein, against which the antibody was raised, is still present. Since the Alk receptor is not properly localized, the founder cells cannot receive the Jeb signal and thus the signal transduction pathway leading to the activation of duf/kirre in the visceral founders is disturbed (Stute, 2004).
Since Alk is localized at the membranes of all visceral cells and not only in the founder cells, it was reasoned that the localization of the Jeb protein must be responsible for the activation of the RTK pathway only in visceral founder cells. Therefore a co-localization of both proteins only at the prospective founder cells is postulated. The double immunolabelling with Jeb and Alk (Loren, 2003) antibodies demonstrates that Jeb protein co-localizes with Alk protein at the membranes of only the visceral founder cells. Moreover, this specific interaction cannot be found in miliAlk mutants where, owing to the mislocalization of the receptor protein, no Jeb uptake takes place. Therefore these mutants display an inactive RTK pathway (Stute, 2004).
Previous work has shown that Jeb is secreted from the ventromedial cells of the somatic mesoderm; these cells are close to the visceral mesoderm. Because all cells of the visceral mesoderm express the Alk RTK, it is theoretically possible that all are able to respond to jeb signalling. The fact that only the most ventral cells of the visceral mesoderm display an active RTK pathway as a result of this interaction and later become the founders of the visceral mesoderm could be explained by the fact that these cells are closest to the cells that secrete the Jeb signal, which is suggested to be the limiting factor. Therefore, a test was performed to see whether increased levels of Jeb could change the fate of the more dorsally located visceral fcms, which also express the receptor Alk, to become founder cells. The UAS-GAL4 system was used to expressed UAS-jeb in the entire mesoderm with a twi-GAL4 driver. As expected, nearly all cells of the visceral mesoderm are now converted to founder cells. Even though fcms are missing, the founders are able to form visceral muscles that later on encircle the midgut. This ability to form muscles is one of the characteristics of founder cells. From sns and mbc mutants, it is known that even though no fusions take place, mini-muscles are formed in the somatic mesoderm that display the right orientation and attachment sites. This has also been shown for the founder cells of the visceral mesoderm. In sns mutants, apart from the first gut constriction, the visceral mesoderm develops normally. On closer inspection just the founder cells differentiate, whereas the fcms remain undifferentiated. Thus, apart from the increased number of founder cells no defects are visible. The same phenotype can be observed if UAS-jeb is ectopically expressed only in the visceral mesoderm (Stute, 2004).
sns in situ hybridization confirms that through the overexpression of UAS-jeb in either the entire or just the visceral mesoderm, the fate of the fcms is changed so that they no longer express fcm-specific gene products such as SNS. This seems to be the reason why the band of fcms of the visceral mesoderm is missing in these embryos. The somatic mesoderm shows no defects as indicated by an anti-ß3 tubulin staining. Overexpression of UAS-jeb in a Alk mutant background shows that the UAS-jeb overexpression phenotype is suppressed in the Alk mutants. Therefore jeb is dependent upon Alk as a receptor to activate the downstream signalling pathway (Stute, 2004).
In the wild type, the limited amount of the Jeb signal appears to restrict founder cell determination to the most ventral cells of the visceral mesoderm. However, these findings prove that in principle all cells of the visceral mesoderm are able to respond to jeb signalling. Furthermore, no difference was found when the signal was produced from the somatic or the visceral mesoderm (Stute, 2004).
Anti-Alk stainings on embryos carrying the visceral mesoderm marker bap-lacZ show that Alk is expressed in all cells of the visceral mesoderm, some neuroectodermal cells and transiently in stage 10 to 11 in cell clusters in the somatic mesoderm. The consequences of ectopic expression of UAS-Alk in the entire mesoderm were examined. Surprisingly, the overexpression of UAS-Alk by a twi-GAL4 driver produces a similar phenotype to that in miliAlk or jebweli mutant embryos. In early stage 11 only fcms are visible in Fas3 stainings and later on there is no evidence of the presence of visceral mesoderm any more. In the somatic mesoderm, defects can be seen by an anti-ß3 tubulin antibody staining. Several muscles are small and display a spindle-like shape with long and thin projections, indicating that only few myoblasts fuse to form the muscles. In comparison with jebweli and miliAlk mutants, in the Alk overexpressing embryos the muscle defects are stronger. Another surprising finding was that in this overexpression situation the sns-expressing cells of the somatic mesoderm are again missing (Stute, 2004).
It remains an unanswered question why the overexpression of Alk in the entire mesoderm results in a similar phenotype as that in jebweli and miliAlk mutants. One possible explanation for the visceral phenotype is that because of the absence of sns-expressing cells in the somatic mesoderm, jeb is not secreted anymore, which results in the absence of an active RTK pathway in the visceral founder cells. Therefore, anti-Jeb antibody stainings were carried out with these embryos. In stage 10 wild-type embryos, jeb is expressed in two bands in the somatic mesoderm and disappears in stage 12 from all mesodermal derivatives. In embryos overexpressing Alk in the entire mesoderm, only one small group of jeb-expressing cells was observed per hemisegment. The reduced amount of the ligand Jeb thus phenocopies a jeb mutant situation where the visceral founders are not determined (Stute, 2004).
A distinct difference between the founder cells and the fcms in the somatic mesoderm is the expression of lmd/minc/glee in the fcms. It was assumed that in the wild type, only the fcms, which are characterised by this expression, are able to respond to the Jeb/Alk signalling pathway, which promotes the further differentiation of the somatic fcms. These in turn continue to secrete Jeb, which is required for the induction of the signalling pathway in the visceral mesoderm (Stute, 2004).
It is assumed that in the somatic mesoderm it is mainly the fcms that express Alk, and it is suggested that the overexpression of Alk in the entire somatic mesoderm enables all cells of the mesoderm to take up the Jeb signal. Therefore, the signal necessary for the further differentiation of the fcms in the somatic mesoderm is downregulated through the increased Jeb uptake of the cells now ectopically expressing Alk. Another possibility to explain the visceral phenotype obtained by overexpressing UAS-Alk in the whole mesoderm is that the overexpression of Alk itself leads to a strong downregulation of Jeb. As a consequence, the visceral founder cells are not specified, again owing to the lack of Jeb signal (Stute, 2004).
A further indication for the relevance of these changes in the somatic mesoderm for the visceral phenotype arises from the overexpression of Alk just in the visceral mesoderm with a bap-GAL4 driverline. This does not result in the phenotype described above. In this case, the founder cells in the visceral mesoderm are present and seem to be even increased in number. It is assumed that due to the Alk overexpression,` additional cells of the visceral mesoderm are now able to take up some of the limited amount of Jeb signal from the somatic mesoderm and thus become founder cells. In this case, Jeb expression in the somatic mesoderm is not affected (Stute, 2004).
Gabay, L., Seger, R., Shilo, B.Z. (1997) MAP kinase in situ activation atlas during Drosophila embryogenesis. Development 124: 3535-3541. 9342046
Iwahara, T., Fujimoto, J., Wen, D., et al. (1997) Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene 14: 439-449. 9053841
Lee, H. H., Norris, A., Weiss, J. B. and Frasch, M. (2003), Jelly belly protein activates the receptor tyrosine kinase Alk to specify visceral muscle pioneers. Nature. 425(6957): 507-12. 14523446
Liao, E. H., Hung, W., Abrams, B. and Zhen, M. (2004). An SCF-like ubiquitin ligase complex that controls presynaptic differentiation. Nature. 430(6997): 345-50. 15208641
Lorén, C. E., Scully, A., Grabbe, C., Edeen, P. T., Thomas, J., McKeown, M., Hunter, T. and Palmer, R. H. (2001). Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives ERK activation in vivo. Genes Cells 6: 531-544. 11442633
Lorén, C. E., Englund, C., Grabbe, C., Hallberg, B., Hunter, T. and Palmer, R. H. (2003). A crucial role for the Anaplastic lymphoma kinase receptor tyrosine kinase in gut development in Drosophila melanogaster. EMBO Rep. 4: 781-786. 12855999
Morris, S.W., Kirstein, M.N., Valentine, M.B., et al. (1994) Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 263: 1281-1284. 8122112
Morris, S.W., Naeve, C., Mathew, P., et al. (1997) ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK). Oncogene 14: 2175-2188. 9174053
Motegi, A., Fujimoto, J., Kotani, M., Sakuraba, H. and Yamamoto, T. (2004). ALK receptor tyrosine kinase promotes cell growth and neurite outgrowth. J Cell Sci. 117(Pt 15): 3319-29. 15226403
Reiff, T., et al. (2011). Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition. Development 138(21): 4699-708. PubMed Citation: 21989914
Stute, C., Schimmelpfeng, K., Renkawitz-Pohl, R., Palmer, R. H. and Holz, A. (2004). Myoblast determination in the somatic and visceral mesoderm depends on Notch signalling as well as on milliways (miliAlk) as receptor for Jeb signalling. Development 131(4): 743-54. 14757637
Weiss, J. B., Suyama, K. L., Lee, H.-H. and Scott, M. P. (2001). Jelly belly: A Drosophila LDL receptor repeat-containing signal required for mesoderm migration and differentiation. Cell 107: 387-398. 11701128
date revised: 15 December 2011
Home page: The Interactive Fly © 2003 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.