Anaplastic lymphoma kinase: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - Anaplastic lymphoma kinase
Synonyms - dALK, DAlk53
Cytological map position - 53C--D
Function - receptor
Keywords - mesoderm, muscle-patterning pioneers
Symbol - Alk
FlyBase ID: FBgn0040505
Genetic map position -
Classification - receptor tyrosine kinase
Cellular location - surface transmembrane
|Recent literature||Wong, C. O., Palmieri, M., Li, J., Akhmedov, D., Chao, Y., Broadhead, G. T., Zhu, M. X., Berdeaux, R., Collins, C. A., Sardiello, M. and Venkatachalam, K. (2015). Diminished MTORC1-dependent JNK activation underlies the neurodevelopmental defects associated with lysosomal dysfunction. Cell Rep 12: 2009-2020. PubMed ID: 26387958
This study evaluated the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). Lysosomes promote the growth of neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. This paradox is attributed to anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. These findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment.
|Bai, L. and Sehgal, A. (2015). Anaplastic lymphoma kinase acts in the Drosophila mushroom body to negatively regulate sleep. PLoS Genet 11: e1005611. PubMed ID: 26536237
Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk), the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1) to regulate growth and associative learning in flies. This study shows that Alk mutants have increased sleep. Using a targeted RNAi screen the negative effects of Alk on sleep were located to the mushroom body, a structure important for both sleep and memory. Mutations in Nf1 were shown to produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes.
The secreted protein Jelly belly (Jeb) is required for an essential signalling event in Drosophila muscle development. In the absence of functional Jeb, visceral muscle precursors are normally specified but fail to migrate and differentiate. The structure and distribution of Jeb protein implies that Jeb functions as a signal to organize the development of visceral muscles. The Jeb receptor is the Drosophila homologue of anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase of the insulin receptor superfamily. Human ALK was originally identified as a proto-oncogene, but its normal function in mammals is not known. Drosophila Alk was identified using a degenerate PCR approach (Lorén, 2001). Like its mammalian counterpart, DAlk appears to be expressed in the developing CNS by in situ analysis. However, in addition to expression of DAlk in the Drosophila brain, careful analysis reveals an additional early role for DAlk in the developing visceral mesoderm where its expression is coincident with activated ERK (Lorén, 2001). In Drosophila, localized Jeb activates Alk and the downstream Ras/mitogen-activated protein kinase cascade to specify a select group of visceral muscle precursors as muscle-patterning pioneers. Jeb/Alk signalling induces the myoblast fusion gene dumbfounded (duf; also known as kirre) as well as optomotor-blind-related-gene-1 (Org-1), a Drosophila homologue of mammalian TBX1, in these cells (Lee, 2003).
Signalling molecules and their receptors orchestrate cell fate decisions essential to organogenesis. Studies of mesoderm development in Drosophila have highlighted the role of evolutionarily conserved signalling systems, and the transcription factors they regulate, in the elaboration of the mesoderm into its derivative tissues. The earliest cell fate assignments in the mesoderm are coordinated by inductive signals from the ectoderm. Decapentaplegic (Dpp), a Drosophila BMP signal, induces subjacent dorsal mesoderm to express Tinman (Tin), a homeodomain protein essential for heart, visceral and dorsal somatic mesoderm development. Dpp and Tin, together with Hedgehog, induce visceral mesoderm by activating the expression of two transcription factors, Bagpipe (Bap) and Biniou (Bin). A third signal, Wingless, antagonizes these visceral mesoderm-inducing activities. The combined actions of ectodermally derived Dpp, Hedgehog and Wingless generate segmental clusters of visceral mesoderm precursors in the dorsal mesoderm (Lee, 2003 and references therein).
The secreted protein Jeb is necessary for the subsequent rearrangement of these segmental clusters of visceral mesoderm precursors into bilateral longitudinal bands and for visceral muscle differentiation. Jeb is produced in ventral somatic mesoderm, locally secreted, and is specifically taken up by the visceral mesoderm cells. Its detailed developmental role, however, has not been defined. One critical function of Jeb signalling is to subdivide the pool of visceral mesoderm precursors into two distinct subtypes: muscle founders and fusion-competent cells. This subdivision is key to the muscle specification and fusion pathway, a hierarchical system for patterning muscles. As first shown for somatic muscle development in Drosophila, founder myoblasts are patterning pioneers. They establish specific muscles and recruit fusion-competent myoblasts to fuse with them into mature syncytial muscle fibers. Founder myoblasts and fusion-competent myoblasts are identified by the expression of functional components of the myoblast fusion pathway. Founder cells express Duf, a transmembrane protein necessary for recruitment of fusion-competent cells. Fusion-competent cells express Sticks and stones (Sns), a transmembrane protein also required for fusion (Lee, 2003 and references therein).
Positive regulation of duf and negative regulation of sns implies that Jeb signalling specifies visceral mesoderm founders. As assayed by the markers duf, org-1 and sns, no visceral muscle founders are specified in jeb mutant embryos. Instead all visceral mesoderm precursors become fusion-competent myoblasts. The consequence of absent visceral mesoderm founders, as shown by cell-lineage experiments, is fusion of visceral fusion-competent myoblasts with somatic muscle founders and loss of visceral musculature. Somatic muscle patterning, however, is unaffected (Lee, 2003).
Localized activation of the Ras/mitogen-activated protein kinase (MAPK) cascade in the visceral mesoderm has been noted previously. In the somatic muscle lineage this pathway is required for founder cell specification. It was therefore hypothesized that Jeb signals through the Ras/MAPK cascade in the visceral mesoderm. Activated MAPK is indeed detected in the visceral mesoderm precursors that take up Jeb. The observed overlapping signals for diphospho-MAPK and org-1, as well as the exclusive staining patterns for diphospho-MAPK and sns, confirm that the MAPK pathway is activated in presumptive visceral muscle founders. Moreover, Jeb signalling is necessary and sufficient to activate the Ras/MAPK cascade in visceral mesoderm precursors. Immunostaining of jeb mutant embryos demonstrates absent diphospho-MAPK in the ventral visceral mesoderm cells that normally accumulate Jeb and become founders. As with founder cell markers, ectopic Jeb produces ectopic diphospho-MAPK, but only in the visceral mesoderm (Lee, 2003).
The expanded expression of org-1 upon mesodermal expression of activated versions of Drosophila Ras and human Raf implicates the Ras pathway in MAPK activation and founder cell specification in the visceral mesoderm. If Jeb signals through the Ras/MAPK pathway, then activation of this pathway should rescue jeb mutations. This prediction is true. As judged by expression of Fasciclin III, a marker of visceral mesoderm differentiation, expression of activated Ras can substantially rescue jeb mutant embryos (Lee, 2003).
The observed effects of ectopic Jeb are limited to the visceral mesoderm. Together with the observation that uptake of Jeb into visceral mesoderm cells requires Shibire-mediated endocytosis, these data imply that Jeb acts through a tissue-specific receptor, which is coupled to the Ras/MAPK pathway. The receptor tyrosine kinase Drosophila Alk, a homologue of the human proto-oncogene anaplastic lymphoma kinase (ALK), is expressed in the early visceral mesoderm. It was therefore hypothesized that Drosophila Alk is the Jeb receptor. Alk messenger RNA is expressed in all cells of the trunk visceral mesoderm directly adjacent to the Jeb-expressing cells. In visceral mesoderm cells that both express Alk and take up Jeb1, diphospho-MAPK is detected (Lee, 2003).
Tested was the assumption that Alk activity, similar to Jeb, would be required for the specification of visceral mesoderm founder cells. Embryos homozygous for a deficiency uncovering the Alk locus lack org-1 expression in presumptive visceral mesoderm founders, a phenotype that can be rescued by expressing an Alk minigene in visceral mesoderm precursors. Mesodermal expression of a kinase-deficient, dominant interfering form of Alk produces an identical phenotype. RNA-mediated interference (RNAi) injection experiments further confirm that Alk is specifically required for visceral mesoderm founder specification. Gal staining of bap3-lacZ embryos injected with double-stranded (ds)Alk RNA demonstrates transformation of visceral into somatic muscle fates. Furthermore, injection of dsAlk RNA into duf-lacZ embryos results in strongly reduced or absent expression of this founder cell marker in the visceral mesoderm. These RNAi phenotypes resemble the phenotypes of jeb mutant embryos, although they are less severe (Lee, 2003).
The loss of duf expression and expansion of sns expression in the visceral mesoderm on expression of dominant-negative Alk is identical to a jeb null mutant phenotype as well. Conversely, the expansion of org-1 expression in the visceral mesoderm on expression of activated Alk (a fusion protein analogous to the human oncogenic version, NPM-ALK22) is indistinguishable from the effects of expression of ectopic Jeb, activated Ras and activated Raf. Finally, forced expression of activated Alk in homozygous jeb mutant backgrounds is able to rescue (and compared with wild type expand) org-1 expression in the visceral mesoderm and to restore midgut morphogenesis (Lee, 2003).
To confirm that Jeb signals through Alk, it was determined that Jeb binds Alk with high affinity, and that Jeb binding to Alk activates the Ras/MAP kinase cascade. In these experiments Jeb-alkaline phosphatase fusion proteins (Jeb-AP) was used. To establish qualitatively the binding of Jeb to Alk, the specific association of Jeb-AP with Alk-transfected mammalian tissue culture cells was visualized. Alk-transfected cells bind Jeb-AP. By contrast, Alk-transfected cells do not bind either an equivalent concentration of alkaline phosphatase alone or a Jeb-AP fusion protein that lacks the type-A LDL receptor repeat in Jeb. This truncated version of Jeb resembles a mutant protein encoded by a null allele of jeb. The truncated protein does not accumulate in visceral mesoderm cells. Binding of Jeb depends on Alk, as demonstrated with non-transfected cells that were incubated with full-length Jeb-AP (Lee, 2003).
A similar assay was used to demonstrate that the Jeb-Alk interaction is specific and has high affinity. Jeb binding to Alk-transfected cells is saturable at nanomolar concentrations. Scatchard analysis demonstrates a single class of high-affinity Jeb-binding site with a dissociation constant (Kd) of 2.2 nM. No binding was observed with either alkaline phosphatase alone or Jeb-AP that lacks the type-A LDL receptor repeat. Jeb-dependent activation of the Ras/MAP kinase cascade in this system was confirmed. The concentration dependence of Ras/MAP kinase activation by Jeb correlates well with binding data. Approximately half-maximal activation occurs in the range of 2-3 nM. As in vivo, removing the type-A LDL receptor repeat from Jeb abrogates Ras/MAP kinase activation (Lee, 2003).
This study has shown that Jeb activates the Ras/MAPK cascade both in vivo and in Alk-transfected tissue culture cells. Jeb binds Alk with high affinity. In vivo Jeb accumulates in visceral muscle founder cells and, in late-stage embryos, in axons of the central nervous system. These patterns of Jeb accumulation are absent from Alk-deficient embryos and in jeb mutants that produce an Alk-binding-deficient version of Jeb. Biochemical and genetic interference with Alk function produces phenotypes identical to jeb mutations. A critical function of Jeb signalling is to specify visceral muscle founder cells-patterning pioneers essential to midgut morphogenesis. Structurally Jeb belongs to a class of signalling molecules with type-A LDL receptor repeats as one of their functional domains. Others include Caenorhabditis elegans HEN-1 and MIG-13, and the mammalian proteins 8D6 and sco-spondin. Jeb is the first among these to have an identified signalling receptor and a defined biological pathway. It is anticipated that this discovery will lead to the identification of receptors and modes of action for other members of this class of signalling molecule (Lee, 2003).
The extracellular portions of mammalian and Drosophila Alk have common domain architectures. Their respective ligands are therefore also likely to share structural features. However, two closely related cytokines that are structurally unrelated to Jeb, pleiotrophin and midkine, have been identified by phage display as potential high-affinity ligands for human ALK. In Drosophila two clustered genes, miple1 and miple2, encode polypeptides related to midkine/pleiotrophin. Similar to the mammalian genes, Drosophila miple1 and miple2 are expressed widely during embryogenesis. So, unlike Jeb, Miple1 and Miple2 cannot control the spatially restricted activation of Alk in the visceral mesoderm, although they may have an auxiliary function in Alk activation. The potential functions of Jeb-related molecules in mammalian Alk activation and the possible contribution of midkine/pleiotrophin-related factors to Alk signalling in Drosophila can now be tested by genetic and molecular approaches. The characterization of the Jeb/Alk signalling pathway in Drosophila is also likely to enhance understanding of vertebrate Alk signalling in development and cancer. As most studies of mammalian Alk have focused on the role of oncogenic versions in cellular transformation, current understanding of Alk's normal function in mammals is rudimentary. In light of the known conservation of genetic pathways in the cardiac and splanchnic mesoderm, these insights into the regulation of org-1 expression in Drosophila are potentially relevant for the understanding of the regulation of human TBX1 and its roles in congenital cardiovascular and craniofacial disease. In addition, the specific expression of Drosophila and mouse Alk in the central nervous system suggests a conserved role of Alk signals in the development or function of neuronal tissues (Lee, 2003).
In Drosophila melanogaster, the receptor tyrosine kinase (RTK) Anaplastic lymphoma kinase (Alk) and its ligand Jelly belly (Jeb) are required to specify muscle founder cells in the visceral mesoderm. This study identified a critical role for the scaffolding protein Cnk (Connector enhancer of kinase suppressor of Ras) in this signaling pathway. Embryos that ectopically expressed the minimal Alk interaction region in the carboxyl terminus of Cnk or lacked maternal and zygotic cnk did not generate visceral founder cells or a functional gut musculature, phenotypes that resemble those of jeb and Alk mutants. Deletion of the entire Alk-interacting region in the cnk locus affected the Alk signaling pathway in the visceral mesoderm and not other RTK signaling pathways in other tissues. In addition, the Cnk-interacting protein Aveugle (Ave) was shown to be critical for Alk signaling in the developing visceral mesoderm. Alk signaling stimulates the MAPK/ERK pathway, but the scaffolding protein Ksr, which facilitates activation of this pathway, was not required to promote visceral founder cell specification. Thus, Cnk and Ave represent critical molecules downstream of Alk, and their loss genocopies the lack of visceral founder cell specification of Alk and jeb mutants, indicating their essential roles in Alk signaling (Wolfstetter, 2017).
Receptor tyrosine kinase (RTK) signaling plays an essential role in development by transducing external signals into the nucleus and other cellular compartments, thereby altering gene expression and promoting intracellular responses. The hallmarks of RTK signaling are conserved among eukaryotic organisms and involve ligand-dependent activation of a transmembrane receptor protein tyrosine kinase and the recruitment of canonical intracellular signaling modules and cascades, such as the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Alk activation stimulates this pathway through the guanosine triphosphatase Ras and the serine-threonine kinases Raf [a MAPK kinase kinase (MAPKKK)], MEK (a MAPK kinase), and MAPK/ERK. Other factors that contribute to or modulate the activity of this pathway have been identified, such as the kinase suppressor of Ras (Ksr), which was identified by mutagenesis screens in Ras-sensitized genetic backgrounds in Drosophila melanogaster and Caenorhabditis elegans. Because of inconsistent findings regarding the catalytic activity of its kinase domain, the role of Ksr has remained controversial. Different models have proposed distinct roles for Ksr as an activator of Raf in parallel to or downstream of Ras or as a scaffolding protein for the assembly of Raf-MEK protein complexes. There is no evidence for a direct interaction between Ksr and Ras, but dimerization between Ksr and Raf can stimulate Raf activity in a manner that is independent of the kinase activity of Ksr, suggesting that Ksr may act as a scaffold in the context of RTK signaling (Wolfstetter, 2017).
A genetic modifier screen using ectopic expression of a dominant-negative, chimeric version of Ksr in the fly eye led to the identification of another critical factor for Ras-ERK signaling named Connector enhancer of kinase suppressor of Ras (Cnk). The cnk locus encodes a large protein of 1557 amino acids containing an N-terminal sterile α motif (SAM), followed by a conserved region in Cnk (CRIC), a PDZ domain, proline-rich motifs, and a pleckstrin homology (PH) domain. The protein structure suggests that Cnk acts as a multidomain protein scaffold. Like Ksr, Cnk functions downstream of various RTK signaling events including epidermal growth factor receptor (EGFR)-tribution of Cnk to embryonic Torso signaling, supporting the finding - and EGFR-dependent air sac development in the dorsal thorax. Ectopic expression of the Cnk N-terminal region enhances the effects of activated RasV12 independently of MAPK/ERK activation in the Drosophila eye. The C-terminal region contains a Raf inhibitory region (RIR) that binds to and represses Raf, which is released upon phosphorylation of Cnk by the Src family kinase Src42A. Thus, Cnk functions as a molecular scaffold to support Ksr-mediated Raf activation and to recruit and integrate additional signaling components such as Src42A (Wolfstetter, 2017).
During embryonic development in D. melanogaster, the visceral mesoderm (VM) gives rise to a lattice of midgut muscles that ensheaths the larval midgut. The VM consists of naïve myoblasts that become specified as either founder cells (FCs) or fusion competent myoblasts (FCMs). Subsequently, the FCs fuse one-to-one with FCMs and eventually form the binucleate visceral myotubes. Specification of VM cells requires the Drosophila ortholog of the receptor anaplastic lymphoma kinase (ALK), initially identified as part of a chimeric protein created by the 2;5 (p23:q35) translocation in human anaplastic large cell lymphoma cell lines. Drosophila Alk is expressed in the segmental clusters of the embryo that segregate from the dorsal trunk mesoderm to form the VM. Alk protein can be detected at the membrane of all VM cells, but only the distal arch within each cluster comes into direct contact with a secreted, small low-density lipoprotein domain ligand named Jelly belly (Jeb). Binding of Jeb to the extracellular part of Alk activates a downstream signaling cascade that results in ERK phosphorylation and triggers expression of an FC-specific subset of genes including Hand, optomotor-blind-related-gene-1 (org-1), and kin of irre (kirre; also referred to as dumbfounded or duf). Jeb-Alk signaling is crucial for visceral myoblasts to commit to the FC fate. In the absence of either ligand or receptor, neither ERK phosphorylation nor the expression of FC-specific marker genes in the VM occurs. Moreover, visceral cells fail to undergo myoblast fusion, and the VM subsequently disintegrates in jeb and Alk mutant embryos (Wolfstetter, 2017).
This study has identified the multidomain scaffolding protein Cnk as a potential Alk binding partner and essential component in the Alk signaling pathway. Cnk bound to the intracellular part of Alk by its C-terminal region. Loss of cnk function or expression of dominant-negative cnk constructs in Drosophila interfered with Alk signaling in multiple developmental contexts. Moreover, germline clone-derived embryos lacking maternal and zygotic Cnk failed to specify visceral FCs and did not develop a functional midgut. In agreement with its proposed function, epistasis experiments revealed that Cnk operates between Ras and Raf in the Alk signaling pathway. Further targeted deletion of a minimal Alk interaction region (AIR) in Cnk resulted in a specific decrease of Jeb-Alk-induced ERK phosphorylation within the visceral FC row. Deletion of the larger Alk interacting region blocked specification of visceral FCs in response to Alk activation. Although the SAM domain containing Cnk binding partner Aveugle (Ave) was essential for Alk signaling, it was found that Ksr is not essentially required to drive Alk signaling in the developing VM. Thus, Cnk and its binding partner Ave serve as critical components for Alk signaling in Drosophila (Wolfstetter, 2017).
This study uncovered an essential function for the protein scaffold Cnk in Alk signaling. The identification of multiple Cnk preys as AlkICD interactors in the Y2H analysis revealed a region in Cnk that likely mediates this interaction and allowed definition of a minimal AIR that was sufficient to bind Alk. The importance of Cnk in Alk signaling was supported by the loss of FCs in the VM of germline clone-derived cnk mutants [cnk (m-/z-)], which genocopied the embryonic Alk loss-of-function phenotype and the dominant-negative effect of ectopic CnkAIR expression on visceral FC specification. The tissue-specific decrease of ERK phosphorylation in the visceral FC row of cnkΔAIR mutants and the loss of visceral FCs upon deletion of the entire Alk interacting region identified by the Y2H approach further support a direct interaction between Cnk and Alk (Wolfstetter, 2017).
Various interactions between Cnk and membrane-associated factors (although not other RTKs) have been reported. In cultured mammalian cells, CNK1 promotes insulin signaling by binding to and localizing cytohesins at the plasma membrane, and binding of CNK1 to the transmembrane ligand EphrinB1 links fibronectin-mediated cell adhesion to EphrinB-associated JNK signaling. Moreover, mammalian CNK2 (also called MAGUIN), the mammalian CNK homolog most similar to Drosophila Cnk, binds to various members of the membrane-associated MAGUK family proteins and Densin-180. It will be interesting to determine whether the RTK binding capacities of Cnk are limited to Alk (Wolfstetter, 2017).
Cnk localizes close to the plasma membrane. Although the Alk-Cnk interaction appears to be important for ERK activation and visceral FC specification in the VM, Alk does not appear to be required for the subcellular localization of Cnk. Whether the Alk-Cnk interaction depends on Alk activity, potentially resulting in posttranslational modification of Cnk, will be interesting to pursue in further studies. Notably, Drosophila Cnk is tyrosine-phosphorylated upon coexpression with the activated form of the RTK Sevenless (SEVS11) in S2 cells, and activation of the platelet-derived growth factor receptor induces tyrosine phosphorylation of mammalian CNK1, leading to changes in CNK1 subcellular localization (Wolfstetter, 2017).
Cnk is generally abundant in the Drosophila embryo where its function is required in various RTK signaling pathways. However, the CnkAIR appears to be critical only for Alk signaling. Morphological analysis further reveals a minor contribution of Cnk to embryonic Torso signaling, supporting the finding that Torso signals are processed by three or more parallel branches. This finding also agrees with earlier analyses of embryonic Torso signaling in avem-/z- mutants, which form terminally derived structures. Notably, the differences in tracheal phenotypes caused by the cnk63F null allele and the strong, Raf-repressing cnksag alleles, which have C-terminal nonsense mutations and abrogate Alk signaling in the VM, indicate that distinct Cnk domains are involved in specific RTK-signaling pathways. This notion is also supported by the observation that the strong, Heartless-related phenotypes exhibited by cnkm-/z- null mutants were barely apparent in cnkΔY2H m-/z- embryos, which, on the other hand, displayed a complete loss of Alk-driven FC specification in the VM (Wolfstetter, 2017).
Selectivity for a requirement of CNK by different RTKs has also been observed in mammalian cells because CNK2 appears to be required for nerve growth factor, but not EGF-induced ERK activation in PC12 cells. Therefore, Cnk contributes to multiple signaling events, but its importance to different RTKs varies, perhaps reflecting differential wiring of downstream signaling in different developmental processes, an aspect that will be interesting to explore in future studies. Cnk has been described as a protein scaffold that facilitates Ras-Raf-MAPK signaling at the plasma membrane, allowing signal integration to enhance Raf and MAPK activation. The epistatic analysis presented in this study shows that Cnk is required downstream of activated Alk and RasV12 but upstream of activated Raf in the VM, which agrees with previous studies in Drosophila. Thus, activated Ras seems to require Cnk to transmit signaling to Raf in the VM (Wolfstetter, 2017).
Alk activation at the membrane of a prospective visceral FC by the ligand Jeb, which is secreted from the neighboring somatic mesoderm, induces the Raf/MAPK/ERK signaling cascade, eventually leading to the transcriptional activation of downstream targets including kirre, org-1, and Hand. Cnk and Ave are core components of the Alk signaling pathway that are required downstream of the receptor and upstream of Raf to mediate visceral FC specification. Although not essential for Jeb-Alk signaling, Ksr appears to be required for full activation of ERK (Wolfstetter, 2017).
Ave directly binds to Cnk through an interface formed by their SAM domains. This interaction is thought to be necessary to recruit Ksr to a complex that in turn promotes Raf activation in the presence of activated Ras. Although this study identified Ave as a critical component for Cnk function downstream of Alk, the single Ksr in Drosophila was not required for Alk-mediated FC specification. Cnk was originally identified in a Ksr-dependent genetic screen in Drosophila, and its function has been proposed to mediate the association between Ksr and Raf, suggesting that Ksr should also play an important role in Alk signaling. However, the role of Ksr is unclear, with early reports suggesting an inhibitory function rather than an activating potential in RTK signaling). Ksr requires the presence of additional factors such as 14-3-3 proteins or activated Ras , and loss of Ksr-1 suppresses RasE13-induced but not wild-type signaling during C. elegans vulva formation, suggesting altered affinities of Ksr for different variants of Ras. Because this analyses revealed a function for ksr in driving robust ERK phosphorylation, it is plausible that, although nonessential, Ksr might enhance Alk signaling by integrating signals from activated Ras to Cnk-associated Raf (Wolfstetter, 2017).
The importance of ERK activation in RTK-mediated signaling in the Drosophila embryo is difficult to address directly because the rolled locus (encoding the only MAPK/ERK1-2 ortholog in Drosophila) is located close to the centromere and therefore not accessible for standard germline clone analysis. The removal of the 42-amino acid AIR from Cnk by genomic editing of the cnk locus suggests that ERK phosphorylation, even at decreased amounts, is sufficient to promote Alk-induced specification of visceral FCs in vivo. Whereas the AIR is sufficient to mediate the Alk-Cnk interaction in vitro, the ability of the cnkΔAIR mutant to support Alk-driven FC specification in the developing embryo highlights additional requirements. The Y2H analysis supports a direct interaction between Alk and Cnk mediated by a more extensive binding interface within the Alk-Y2H binding region of Cnk. Accordingly, deletion of this region in the cnk locus (cnkΔY2H) blocks VM specification in vivo. However, the possibility cannot be excluded that the Y2H region of Cnk may form interactions with additional Alk binding proteins, which could mediate the Alk-Cnk interaction in an indirect manner (Wolfstetter, 2017).
In summary, this study has identified an interaction between Alk and Cnk mediated by an Alk binding region in Cnk. This region was specifically required for the activation of ERK and formation of FCs downstream of the activated Alk receptor in the Drosophila VM. Together with Ave, Cnk represents an important signaling module that is required for Alk-mediated signaling during embryogenesis. Cnk and Ave represent molecules identified downstream of Alk, whose loss genocopies the lack of visceral FC specification of Alk and jeb mutants. Further work should allow a better understanding of the importance of Cnk in Alk signaling and whether this is conserved in mammalian systems (Wolfstetter, 2017).
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).
Bidirectional trans-synaptic signals induce synaptogenesis and regulate subsequent synaptic maturation. Presynaptically secreted Mind the gap (Mtg) molds the synaptic cleft extracellular matrix, leading to a hypothesis that Mtg functions to generate the intercellular environment required for efficient signaling. In Drosophila secreted Jelly belly (Jeb) and its receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) are localized to developing synapses. Jeb localizes to punctate aggregates in central synaptic neuropil and neuromuscular junction (NMJ) presynaptic terminals. Secreted Jeb and Mtg accumulate and colocalize extracellularly in surrounding synaptic boutons. Alk concentrates in postsynaptic domains, consistent with an anterograde, trans-synaptic Jeb-Alk signaling pathway at developing synapses. Jeb synaptic expression is increased in Alk mutants, consistent with a requirement for Alk receptor function in Jeb uptake. In mtg null mutants, Alk NMJ synaptic levels are reduced and Jeb expression is dramatically increased. NMJ synapse morphology and molecular assembly appear largely normal in jeb and Alk mutants, but larvae exhibit greatly reduced movement, suggesting impaired functional synaptic development. jeb mutant movement is significantly rescued by neuronal Jeb expression. jeb and Alk mutants display normal NMJ postsynaptic responses, but a near loss of patterned, activity-dependent NMJ transmission driven by central excitatory output. It is concluded that Jeb-Alk expression and anterograde trans-synaptic signaling are modulated by Mtg and play a key role in establishing functional synaptic connectivity in the developing motor circuit (Rohrbough, 2010).
Jeb and Alk are localized to pre- and postsynaptic junctions during embryonic synaptogenesis, predicting an inductive anterograde synaptic signaling role. Jeb-Alk RTK signaling at embryonic somatic-visceral mesoderm junctions similarly directs visceral muscle specification and differentiation. Jeb is the only identified Alk ligand, and Alk is the only identified Jeb receptor. It was recently shown that the C. elegans Alk ortholog SCD-2 is similarly neuronally expressed and activated by a Jeb-like secreted ligand, HEN-1, which contains an LDLa domain. Jeb-Alk anterograde signaling has recently been shown to regulate circuit formation in the Drosophila developing optic lobe (Rohrbough, 2010 and references therein).
Jeb-Alk NMJ and neuropil expression patterns indicate that anterograde signaling occurs at both peripheral and central synapses. Jeb localizes to NMJ presynaptic terminals and is secreted extracellularly, whereas Alk localizes to opposing postsynaptic membranes. The Jeb neuronal expression/trafficking profile suggests transport to the NMJ, rather than neuronal Jeb uptake from muscle, as previously suggested. Jeb and Alk display reciprocal expression levels at NMJ synapses, with lower Jeb levels at boutons expressing highest postsynaptic Alk levels. Jeb is also strongly increased at Alk mutant synapses, suggesting that internalization of secreted Jeb in postsynaptic cells requires Alk receptor function. This predicted synaptic signaling cascade therefore parallels the mechanism in mesoderm development (Rohrbough, 2010).
A working hypothesis predicts that the ECM environment modulates trans-synaptic ligand-receptor interactions. A key finding, therefore, is that the Jeb-Alk pathway is regulated by Mtg, a presynaptically secreted glycoprotein crucial for synaptic cleft ECM formation. In the absence of Mtg, postsynaptic Alk is strongly reduced and secreted Jeb is dramatically accumulated at NMJ synapses. Maintenance of Alk might be part of a larger role for Mtg in postsynaptic differentiation, as numerous postsynaptic components are lost/mislocalized in mtg mutants. Alternatively, Mtg might more directly regulate Alk, possibly by ECM tethering/anchoring of the Alk receptor. The Jeb upregulation should be partly attributable to the Mtg-dependent reduction in postsynaptic Alk. However, synaptic Jeb is upregulated to a much greater degree, despite a less severe downregulation of Alk, in mtg than in Alk null mutants. Jeb NMJ expression is also modulated independently of Alk by targeted neuronal or muscle Mtg overexpression, indicating that Mtg regulates Jeb independently of Alk. It is concluded that Mtg expression and function are highly likely to regulate developmental Jeb-Alk synaptic signaling. However, this interpretation must be verified in future studies by demonstrating a regulatory function for Mtg in previously established Jeb-Alk RTK molecular signaling pathways (Rohrbough, 2010).
Mtg and Jeb are co-expressed in developing NMJ presynaptic boutons, and are secreted to occupy largely overlapping domains within the synaptomatrix. The current findings suggest that Mtg normally acts at NMJ synapses to restrict localized Jeb accumulation within the synaptomatrix. It is suggested that the Mtg-dependent ECM might function as a barrier to maintain localized Jeb pools and/or as a scaffold that is required to appropriately present or proteolytically remove Jeb in the extracellular signaling space. It is presently unclear whether Mtg has a parallel regulatory role at developing central synapses, where Mtg is expressed in a more limited neuronal subset. Changes in central Jeb/Alk expression might be indirectly related to Mtg loss or overexpression in the CNS. Alternatively, changes in neuronal Mtg level might have greater effects on Jeb/Alk NMJ expression. Mammalian Alk candidate ligands, such as pleiotrophin, heparin affinity regulatory peptide (HARP), heparin-binding neurotrophic factor (HBNF), and midkine, are heparin-binding growth factors, further highlighting that Alk activation occurs via ligands that function within the complex and dynamic glycomatrix. It is proposed that Mtg-dependent modulation of extracellular space is critical for the signaling activity of multiple trans-synaptic signals (Rohrbough, 2010).
The Jeb-Alk pathway is not detectably required for embryonic axonal pathfinding, synapse morphogenesis or molecular assembly during synaptogenesis, including the proper localized expression of pre- and postsynaptic proteins. Likewise, Jeb-Alk function is not required for establishing functional NMJ synapses, including postsynaptic GluR domains. Jeb-Alk signaling is likely to have a role(s) during postembryonic NMJ development. The Alk receptor is required for expression and signaling of the TGFβ signaling component Dpp in developing endoderm, and Alk is similarly suggested to modulate a TGFβ pathway in C. elegans (Reiner, 2008). Therefore, Alk potentially regulates the TGFβ-dependent retrograde signaling pathway(s) involved in synaptic plasticity and function during larval NMJ development (Rohrbough, 2010).
The results indicate that Jeb and Alk have a role in the development of locomotion behavior. Jeb-Alk signaling regulates somatic as well as visceral muscle differentiation, with similar defects resulting from Alk removal or ectopic overexpression in muscle. Likewise, this study found that either muscle or neuronal Alk overexpression impairs locomotion and results in early larval lethality. However, jeb and Alk mutant muscle responds to direct stimulation and evoked NMJ transmission is normal, indicating that the primary locomotory impairment is not defective muscle or NMJ function. Moreover, jeb mutant locomotion is significantly rescued by neuronal, but not muscle, Jeb expression, consistent with a requirement for Jeb signaling from central neurons. Importantly, loss of Jeb-Alk signaling severely reduces endogenous NMJ neurotransmission by effectively reducing the occurrence of centrally generated, patterned synaptic output to the NMJ. The underlying excitatory synaptic drive onto motoneurons parallels the development of locomotion behavior. Central neuron recordings show functional excitatory synaptic input to jeb/Alk and mtg mutant motoneurons, which surprisingly show no significant loss of activity that might be suggested by the severe locomotion impairments. CNS dissection/recording conditions may effectively re-excite depressed motor activity, similar to the effect of direct stimulation in provoking mutant movement (Rohrbough, 2010).
The current results indicate that anterograde Jeb-Alk synaptic signaling is crucial for the maturation of locomotory behavior, and that Mtg regulatory activity intersects with the Jeb-Alk pathway during NMJ synaptic differentiation. It is proposed that Jeb-Alk signaling is essential for the functional differentiation of the central synaptic connections that drive motor circuit activity. Loss of Jeb-Alk signaling function impairs central excitatory synaptic transmission, resulting in a loss of endogenous central pattern generator activity driving motor output to the NMJ. Future studies will be directed towards dissecting the intersecting roles of Mtg and Jeb secreted signals in the functional differentiation of central motor circuits (Rohrbough, 2010).
Developing animals survive periods of starvation by protecting the growth of critical organs at the expense of other tissues. This study used Drosophila to explore the as yet unknown mechanisms regulating this privileged tissue growth. As in mammals, it was observed in Drosophila that the CNS is more highly spared than other tissues during nutrient restriction (NR). Anaplastic lymphoma kinase (Alk) efficiently protects neural progenitor (neuroblast) growth against reductions in amino acids and insulin-like peptides during NR via two mechanisms. First, Alk suppresses the growth requirement for amino acid sensing via Slimfast/Rheb/TOR complex 1. And second, Alk, rather than insulin-like receptor, primarily activates PI3-kinase. Alk maintains PI3-kinase signaling during NR as its ligand, Jelly belly (Jeb), is constitutively expressed from a glial cell niche surrounding neuroblasts. Together, these findings identify a brain-sparing mechanism that shares some regulatory features with the starvation-resistant growth programs of mammalian tumors (Cheng, 2011).
This study found that CNS progenitors are able to continue growing at their normal rate under fasting conditions severe enough to shut down all net body growth. Jeb/Alk signaling was identified as a central regulator of this brain sparing, promoting tissue-specific modifications in TOR/PI3K signaling that protect growth against reduced amino acid and Ilp concentrations. These findings highlight that a 'one size fits all' wiring diagram of the TOR/PI3K network should not be extrapolated between different cell types without experimental evidence. The two molecular mechanisms by which Jeb/Alk signaling confers brain sparing is discussed, and how they may be integrated into an overall model for starvation-resistant CNS growth (Cheng, 2011).
One mechanism by which Alk spares the CNS is by suppressing the growth requirement for amino acid sensing via Slif, Rheb, and TORC1 components in neuroblast lineages. An important finding of this study is that in the presence of Alk signaling Tor has no detectable growth input (evidence from Tor clones), but in its absence (evidence from UAS-AlkDN; Tor clones) Tor reverts to its typical role as a positive regulator of both growth and proliferation. The growth requirement for Slif/TORC1 is clearly much less in the CNS than in other tissues such as the wing disc but a low-level input cannot be ruled out due to possible perdurance inherent in any clonal analysis. Although Slif, Rheb, Tor, and Raptor mutant neuroblast clones attain normal volume, this reflects increased cell numbers offset by reduced average cell size. Atypical suppression of proliferation by TORC1 has also been observed in wing discs, where partial inhibition with rapamycin advances G2/M progression (Cheng, 2011).
Alk signaling in neuroblast lineages does not override the growth requirements for all TOR pathway components. The downstream effectors S6k and 4E-BP retain functions as positive and negative growth regulators, respectively. 4E-BP appears to be particularly critical in the CNS as mutant animals have normal mass, but mutant neuroblast clones are twice their normal volume. In many tissues, 4E-BP is phosphorylated by nutrient-dependent TORC1 activity. In CNS progenitors, however, 4E-BP phosphorylation is regulated in an NR-resistant manner by Alk, not by TORC1. Hence, although the pathway linking Alk to 4E-BP is not yet clear, it makes an important contribution toward protecting CNS growth during fasting (Cheng, 2011).
A second mechanism by which Alk spares CNS growth is by maintaining PI3K signaling during NR. Alk suppresses or overrides the genetic requirement for InR in PI3K signaling, which may or may not involve the direct binding of intracellular domains as reported for human ALK and IGF-IR (Shi, 2009). Either way, in the CNS, glial Jeb expression stimulates Alk-dependent PI3K signaling and thus neural growth at similar levels during feeding and NR. In contrast, in tissues such as the salivary gland, where PI3K signaling is primarily dependent upon InR, falling insulin-like peptides concentrations during NR strongly reduce growth (Cheng, 2011).
The finding that Alk signals via PI3K during CNS growth differs from the Ras/MAPK transduction pathway described in Drosophila visceral muscle. However, a link between ALK and PI3K/Akt/Foxo signaling during growth is well documented in humans, both in glioblastomas and in non-Hodgkin lymphoma. Similarities with mammals are less obvious with regard to Alk ligands, as there is no clear Jeb ortholog and human ALK can be activated, directly or indirectly, by the secreted factors Pleiotrophin and Midkine (Cheng, 2011).
A comparison of these results with those of previous studies indicates that CNS super sparing only becomes fully active at late larval stages. Earlier in larval life, dietary amino acids are essential for neuroblasts to re-enter the cell cycle after a period of quiescence. This nutrient-dependent reactivation involves a relay whereby Slif-dependent amino acid sensing in the fat body stimulates Ilp production from a glial cell niche (Sousa-Nunes, 2011). In turn, glial-derived Ilps activate InR and PI3K/TOR signaling in neuroblasts thus stimulating cell cycle re-entry. Hence, the relative importance of Ilps versus Jeb from the glial cell niche may change in line with the developmental transition of neuroblast growth from high to low nutrient sensitivity (Cheng, 2011).
The results of this study suggest a working model for super sparing in the late-larval CNS. Central to the model is that Jeb/Alk signaling suppresses Slif/ RagA/Rheb/TORC1, InR, and 4E-BP functions and maintains S6k and PI3K activation, thus freeing CNS growth from the high dependence upon amino acid sensing and Ilps that exists in other organs. The CNS also contrasts with other spared diploid tissues such as the wing disc, in which PI3K-dependent growth requires a positive Tor input but is kept in check by negative feedback from TORC1 and S6K. Alk is both necessary (in the CNS) and sufficient (in the salivary gland) to promote organ growth during fasting. However, both Alk manipulations produce organ-sparing percentages intermediate between the 2% salivary gland and the 96% neuroblast values, arguing that other processes may also contribute. For example, some Drosophila tissues synthesize local sources of Ilps that could be more NR resistant than the systemic supply from the IPCs. In mammals, this type of mechanism may contribute to brain sparing as it has been observed that IGF-I messenger RNA (mRNA) levels in the postnatal CNS are highly buffered against NR. It will also be worthwhile exploring whether mammalian neural growth and brain sparing involve Alk and/or atypical TOR signaling. In this regard, it is intriguing that several studies show that activating mutations within the kinase domain of human ALK are associated with childhood neuroblastomas. In addition, fetal growth of the mouse brain was recently reported to be resistant to loss of function of TORC1. Finally, a comparison between the current findings and those of a cancer study, highlights that insulin/IGF independence and constitutive PI3K activity are features of NR-resistant growth in contexts as diverse as insect CNS development and human tumorigenesis (Cheng, 2011).
Neural circuit formation relies on interactions between axons and cells within the target field. While it is well established that target-derived signals act on axons to regulate circuit assembly, the extent to which axon-derived signals control circuit formation is not known. In the Drosophila visual system, anterograde signals numerically match R1-R6 photoreceptors with their targets by controlling target proliferation and neuronal differentiation. This study demonstrates that additional axon-derived signals selectively couple target survival with layer specificity. Jelly belly (Jeb) produced by R1-R6 axons was shown to interact with its receptor, Anaplastic lymphoma kinase (Alk), on budding dendrites to control survival of L3 neurons, one of three postsynaptic targets. L3 axons then produce Netrin, which regulates the layer-specific targeting of another neuron within the same circuit. It is proposed that a cascade of axon-derived signals, regulating diverse cellular processes, provides a strategy for coordinating circuit assembly across different regions of the nervous system (Pecot, 2014).
This study demonstrates that Jeb/Alk signaling regulates the survival of L3 neurons, one of several postsynaptic targets of R1-R6 neurons. Jeb is expressed in R1-R6 growth cones and acts at short range, prior to synapse formation, through the Alk receptor tyrosine kinase localized on budding L3 dendrites within the lamina neuropil. Jeb/Alk signaling is highly selective, as the survival of other R1-R6 postsynaptic targets (i.e., L1 and L2) is not affected when signaling is disrupted. This study also showed that, at a later stage of development, L3 growth cones produce Netrin within the medulla, which is required for the targeting of R8 growth cones to the M3 layer. It is speculated that a cascade of growth-cone-derived signals acting across different brain regions provides a general strategy for the assembly of neural circuits (Pecot, 2014).
In many regions of the developing nervous system, neurons are produced in excess, and significant cell death occurs after axons innervate their targets. In vertebrates, it is well established that target-derived neurotrophins, such as nerve growth factor, regulate neuronal numbers. These factors are produced by target neurons in limiting amounts and locally promote survival in a retrograde manner through receptors localized on axon terminals, providing a mechanism for matching the number of axons to targets. In recent years, diverse classes of molecules have been shown to control neuronal survival during development. Anterograde sources of trophic factors may also regulate survival, as denervation has been shown to induce excessive target neuron cell death. Indeed, several signals, including BDNF, are transported, in some contexts, in an anterograde manner within axons. In addition, the overexpression of BDNF in afferents can rescue cell death within the target field, and the disruption of BDNF through function blocking antibodies has been reported to decrease the number of target neurons within the rat superior colliculus. As BDNF may be produced by both axons and cells within the superior colliculus, it remains unclear whether endogenous axon-derived BDNF, and thus anterograde signaling, is required to regulate neuron survival (Pecot, 2014).
Although a role for target-derived retrograde trophic factors in vertebrate neural development was established many decades ago, trophic factors have only recently been shown to regulate neuronal development in Drosophila. Three Drosophila proteins, Neurotrophin 1, Neurotrophin 2, and Spatzle, are distantly related to vertebrate neurotrophins, and it has been shown that, like their vertebrate counterparts, they function as target-derived retrograde survival signals. Unlike their vertebrate homologs, however, which act through receptor tyrosine kinases, fly neurotrophins promote cell survival through Toll-like receptors (Pecot, 2014).
Although Jeb bears no significant homology to fly or vertebrate neurotrophins, Jeb acts through a receptor tyrosine kinase, Alk, which is distantly related to vertebrate neurotrophin receptors or Trks. Alk was originally identified as part of a fusion protein associated with large cell anaplastic lymphoma. Its role in mammals remains poorly understood. Drosophila Alk was initially found to regulate visceral mesoderm development through interaction with Jeb, and subsequently, Jeb/Alk signaling has been shown to regulate diverse cellular processes. Recent studies in vertebrates and Drosophila demonstrated that disrupting Alk function causes a decrease in the number of neurons. While in the vertebrate studies Alk's mechanism of action was not established, in Drosophila, Alk was shown to antagonize pathways that restrict neurogenesis under conditions of nutrient deprivation. Whether Jeb and Alk regulate neuronal survival in contexts outside of L3 development is not known, although Alk is widely expressed in the developing visual system, and Jeb is expressed by several populations of neurons, in addition to photoreceptors (Pecot, 2014).
The cellular specificity of the Jeb/Alk requirement is particularly surprising. Indeed, at all R1-R6 synapses containing L3 postsynaptic elements, L1 and L2 neurons each contribute a single postsynaptic element juxtaposing the same presynaptic site on R cell axons. In the absence of Jeb/Alk signaling, however, only L3 neurons die. The mechanisms that underlie this selectivity are not known. Alk is broadly expressed in the lamina, suggesting specificity may be controlled at the level of downstream signaling or that other trophic signals act redundantly with Jeb to control L1 and L2 survival. Collectively, the findings reported in this study demonstrate that anterograde Jeb/Alk signaling acts selectively to control L3 survival, providing direct evidence that anterograde signaling regulates target neuron survival in vivo (Pecot, 2014).
Several lines of evidence indicate that signaling between Jeb, expressed by R1-R6 growth cones, and Alk, localized to budding L3 dendrites, controls L3 survival between 20-40 hr APF. First, Alk mutant L3 neurons, or wild-type L3 neurons innervated by jeb mutant R1-R6 axons, die between 20-40 hr APF. Second, R cell populations containing only R1-R6 neurons are sufficient for L3 survival. Third, Alk and Jeb are expressed in a complementary fashion at the appropriate time on budding L3 dendrites and R1-R6 growth cones, respectively. And finally, L3 degeneration begins within budding L3 dendrites juxtaposed to R1-R6 growth cones. The temporal requirement for Alk/Jeb signaling corresponds to a critical and fascinating phase of lamina circuit assembly (Pecot, 2014).
R1-R6 growth cones form connections with lamina neurons in three discrete steps. First, R1-R6 growth cones from the same ommatidium associate with a single cartridge of differentiating lamina neurons. Second, through a highly stereotyped reassortment process occurring between 24-38 hr APF, these six growth cones diverge from one another and project locally to six different developing cartridges. As a consequence of this rearrangement, the R1-R6 cells that 'see' the same point in space form connections with L1, L2, and L3 neurons within the same cartridge. And third, R1-R6 then commence synapse formation at 45 hr APF, and this process continues until eclosion (~96 hr). Thus, L3 death in jeb and Alk mutants occurs prior to synapse formation, during the process of R1-R6 growth cone rearrangement. The suppression of L3 death by expression of the caspase inhibitor p35 argues that during normal development Jeb/Alk signaling acts to inhibit caspase activity. Which caspases contribute to L3 death, and whether caspases antagonize other cellular processes necessary for wiring, is not known. Regardless of how Jeb/Alk signaling functions at the molecular level, it acts to ensure that visual input from R1-R6 neurons is transmitted to the L3 pathway (Pecot, 2014).
These findings and the work of others suggest a logic underlying neural circuit assembly within the Drosophila visual system. The retina, lamina, and medulla are distinct yet interconnected regions comprising columnar modules (i.e., ommatidia, cartridges, and columns, respectively) that are matched topographically between each region. Within each module, intrinsic mechanisms and intercellular interactions control cell fate determination. For instance, R8 neurons provide a discrete locally acting signal to induce R7 development in the developing retina, while in the medulla, Notch/Delta interactions between daughter cells generated from the same ganglion mother cell promote acquisition of distinct cell fates. Superimposed upon these interactions are axon-derived signals that coordinate development between matched modules from different regions. Together, these mechanisms organize the assembly of columnar units in multiple regions (i.e., super columns), each processing visual information captured from a discrete region of the visual field. Indeed, the modular assembly of these super columns spanning different regions of the visual system reflects the function of these circuits in the parallel processing of visual information (Pecot, 2014).
R cell growth cones produce signals that regulate diverse cellular processes in the developing lamina. Hedgehog drives lamina neuronal precursors through their final division; cell adhesion proteins promote the association of columns of lamina neurons with R cell axon fascicles; EGF induces lamina neuron differentiation; a yet-to-be-identified signal regulates the development of lamina glia; and Jeb selectively regulates L3 survival. Thus, axon-derived signals act at multiple levels and in a cell-type-specific manner to regulate target development (Pecot, 2014).
Axon-derived signals also coordinate circuit assembly across topographically matched modules. Within medulla columns, L3 growth cones produce Netrin in the M3 layer, which controls the targeting of R8 growth cones to M3. Importantly, Netrin production by L3 occurs after Jeb, released from R1-R6 cells in topographically corresponding lamina cartridges, promotes L3 survival. Thus, Netrin indirectly relies upon prior Jeb signaling. As the L3 and R8 axon terminals within each medulla column transmit information captured from the same point in space to the same layer (M3) and share several postsynaptic targets, the developmental mechanisms giving rise to this circuit may reflect functional relationships between these neurons. Thus, signals produced by axons coordinate assembly of circuits between different brain regions (Pecot, 2014).
It is envisioned that intercellular signaling cascades, analogous to what are described in this study, organize other circuit modules in the fly visual system [e.g., ON (L1) and OFF (L2) circuits] comprising different cell types. As many regions of the vertebrate nervous system, including the neocortex, spinal cord, and retina, are also arranged in a hierarchically repetitive fashion, this raises the intriguing possibility that similar strategies may coordinate the development of these structures (Pecot, 2014).
This study evaluated the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). Lysosomes promote the growth of neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. This paradox is attributed to Anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. These findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment (Wong, 2015).
Mucolipidosis type IV (MLIV) and Batten disease are untreatable
lysosomal storage diseases (LSDs) that cause childhood neurodegeneration. MLIV
arises from loss-of-function mutations in the gene encoding
TRPML1, an endolysosomal cation channel belonging to the
TRP superfamily. The absence of TRPML1 leads to defective lysosomal storage and autophagy, mitochondrial damage, and macromolecular aggregation, which together initiate the protracted neurodegeneration observed in MLIV). Batten disease arises from the absence of a lysosomal protein, CLN3), and results in psychomotor retardation. Both diseases cause early alterations in neuronal function. For instance, brain imaging studies revealed that MLIV and Batten patients display diminished
axonal development in the cortex and corpus callosum, the causes of which remain
unknown (Wong, 2015).
To better understand the etiology of MLIV in a genetically tractable model, flies were generated lacking the TRPML1 ortholog. The trpml-deficient (trpml1) flies have led to insight into the mechanisms of neurodegeneration and lysosomal
storage (Wong, 2015).
This study reports that trpml1 larvae exhibit diminished synaptic
growth at the NMJ, a well-studied model synapse. Lysosomal function supports
Rag GTPases and MTORC1 activation, and this is essential for
JNK phosphorylation and synapse development (Wong, 2015).
Drosophila larvae and mice lacking CLN3 also exhibit diminished Rag/
MTORC1 and JNK activation, suggesting that alterations in
neuronal signaling are similar in different LSDs and are evolution-
arily conserved. Interestingly, the NMJ defects in the two fly LSD
models were suppressed by the administration of a high-protein
diet and a drug that is currently in clinical trials to treat certain
forms of cancer. These findings inform a pharmacotherapeutic
strategy that may suppress the neurodevelopmental defects
observed in LSD patients (Wong, 2015).
This study shows that lysosomal dysfunction in
Drosophila MNs results in diminished bouton numbers at the larval NMJ. Evidence is presented that lysosomal dysfunction results in decreased activation of the amino-acid-responsive cascade involving Rag/MTORC1, which are critical for normal NMJ development (Wong, 2015).
Despite the requirement for MTORC1 in NMJ synapse development, previous studies and the current findings show that bouton numbers are independent of S6K and 4E-BP1. Rather,
MTORC1 promotes NMJ growth via a MAP kinase cascade
culminating in JNK activation. Therefore, decreasing
lysosomal function or Rag/MTORC1 activation in hiwND8
suppressed the associated synaptic overgrowth. However, the
'small-bouton' phenotype of
hiwND8 was independent of
MTORC1. Thus, MTORC1 is required for JNK-dependent regulation of bouton numbers, whereas bouton morphology is independent of MTORC1. Furthermore, although both
rheb expression and hiw loss result in Wnd-dependent elevation in bouton
numbers, the supernumerary boutons in each case show distinct
morphological features. Additional studies are needed for deciphering the complex interplay between MTORC1-JNK in regulating the NMJ morphology (Wong, 2015).
Biochemical analyses revealed that both JNK phosphorylation
and its transcriptional output correlated with the activity of
MTORC1, which are consistent with prior
observations that cln3 overexpression promotes JNK activation and that
tsc1/tsc2 deletion in flies
result in increased JNK-dependent transcription. These findings point to the remarkable versatility of
MTORC1 in controlling both protein trans lation and gene transcription (Wong, 2015).
Using an in vitro kinase assay, this study demonstrates that Wnd is a target of MTORC1.
Because axonal injury activates both MTORC1 and DLK/JNK, these findings imply a functional connection between these two pathways. Interestingly, the data also suggest that MTORC1 contains additional kinases besides MTOR that can phosphorylate Wnd. One possibility is that ULK1/Atg1,
which associates with MTORC1, could be the kinase that phosphorylates Wnd. Consistent
with this notion, overexpression of Atg1 in the Drosophila
neurons has been shown to promote JNK signaling and NMJ synapse
overgrowth via Wnd) (Wong, 2015).
This study also found that developmental JNK activation in axonal
tracts of the CC and pJNK levels in cortical neurons were
compromised in a mouse model of Batten disease. Thus, the
signaling deficits identified in
Drosophila are also conserved in mammals. The activity of DLK (the mouse homolog of Wnd)
and JNK signaling are critical for axonal development in the
mouse CNS. Therefore, decreased neuronal
JNK activation during development might underlie the thinning
of the axonal tracts observed in many LSDs (Wong, 2015).
Although the findings of this study demonstrate a role for an amino-acid-
responsive cascade in the synaptic defects associated with
lysosomal dysfunction, simply elevating the dietary protein content was not sufficient to rescue these defects. These findings
were reminiscent of an elegant study that showed that the
growth of Drosophila neuroblasts is uncoupled from dietary
amino acids owing to the function of ALK, which suppresses
the uptake of amino acids into the neuroblasts (Cheng, 2011). Indeed, simultaneous administration of an ALK inhibitor and a high-protein diet partially rescued the synaptic growth defects associated with the lysosomal dysfunction, and improved the rescue of pupal lethality associated with
these studies do not causally link the defects in synapse development with pupal lethality, they do raise the intriguing possibility
that multiple phenotypes associated with LSDs could be targeted using ALK inhibitors along with a protein-rich diet (Wong, 2015).
Although LSDs result in lysosomal dysfunction throughout the
body, neurons are exceptionally sensitive to these alterations. The cause for this sensitivity remains incompletely understood. Given the findings of this study that mature
neurons do not efficiently take up amino acids from the extracellular medium, lysosomal degradation of proteins serves as a major source of free amino acids in these cells. Therefore, disruption
of lysosomal degradation leads to severe shortage of free amino
acids in neurons, regardless of the quantity of dietary proteins,
thus explaining the exquisite sensitivity of neurons to lysosomal
dysfunction (Wong, 2015).
Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. The role of Miple proteins was investigated in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). This study shows that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally it was shown that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, the data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo (Hugosson, 2014: PubMed).
This study evaluated the mechanisms underlying the neurodevelopmental deficits in Drosophila and mouse models of lysosomal storage diseases (LSDs). Lysosomes promote the growth of neuromuscular junctions (NMJs) via Rag GTPases and mechanistic target of rapamycin complex 1 (MTORC1). However, rather than employing S6K/4E-BP1, MTORC1 stimulates NMJ growth via JNK, a determinant of axonal growth in Drosophila and mammals. This role of lysosomal function in regulating JNK phosphorylation is conserved in mammals. Despite requiring the amino-acid-responsive kinase MTORC1, NMJ development is insensitive to dietary protein. This paradox is attributed to Anaplastic lymphoma kinase (ALK), which restricts neuronal amino acid uptake, and the administration of an ALK inhibitor couples NMJ development to dietary protein. These findings provide an explanation for the neurodevelopmental deficits in LSDs and suggest an actionable target for treatment (Wong, 2015).
Mucolipidosis type IV (MLIV) and Batten disease are untreatable lysosomal storage diseases (LSDs) that cause childhood neurodegeneration. MLIV arises from loss-of-function mutations in the gene encoding TRPML1, an endolysosomal cation channel belonging to the TRP superfamily. The absence of TRPML1 leads to defective lysosomal storage and autophagy, mitochondrial damage, and macromolecular aggregation, which together initiate the protracted neurodegeneration observed in MLIV). Batten disease arises from the absence of a lysosomal protein, CLN3), and results in psychomotor retardation. Both diseases cause early alterations in neuronal function. For instance, brain imaging studies revealed that MLIV and Batten patients display diminished axonal development in the cortex and corpus callosum, the causes of which remain unknown (Wong, 2015).
To better understand the etiology of MLIV in a genetically tractable model, flies were generated lacking the TRPML1 ortholog. The trpml-deficient (trpml1) flies have led to insight into the mechanisms of neurodegeneration and lysosomal storage (Wong, 2015).
This study reports that trpml1 larvae exhibit diminished synaptic growth at the NMJ, a well-studied model synapse. Lysosomal function supports Rag GTPases and MTORC1 activation, and this is essential for JNK phosphorylation and synapse development (Wong, 2015).
Drosophila larvae and mice lacking CLN3 also exhibit diminished Rag/ MTORC1 and JNK activation, suggesting that alterations in neuronal signaling are similar in different LSDs and are evolution- arily conserved. Interestingly, the NMJ defects in the two fly LSD models were suppressed by the administration of a high-protein diet and a drug that is currently in clinical trials to treat certain forms of cancer. These findings inform a pharmacotherapeutic strategy that may suppress the neurodevelopmental defects observed in LSD patients (Wong, 2015).
This study shows that lysosomal dysfunction in Drosophila MNs results in diminished bouton numbers at the larval NMJ. Evidence is presented that lysosomal dysfunction results in decreased activation of the amino-acid-responsive cascade involving Rag/MTORC1, which are critical for normal NMJ development (Wong, 2015).
Despite the requirement for MTORC1 in NMJ synapse development, previous studies and the current findings show that bouton numbers are independent of S6K and 4E-BP1. Rather, MTORC1 promotes NMJ growth via a MAP kinase cascade culminating in JNK activation. Therefore, decreasing lysosomal function or Rag/MTORC1 activation in hiwND8 suppressed the associated synaptic overgrowth. However, the 'small-bouton' phenotype of hiwND8 was independent of MTORC1. Thus, MTORC1 is required for JNK-dependent regulation of bouton numbers, whereas bouton morphology is independent of MTORC1. Furthermore, although both rheb expression and hiw loss result in Wnd-dependent elevation in bouton numbers, the supernumerary boutons in each case show distinct morphological features. Additional studies are needed for deciphering the complex interplay between MTORC1-JNK in regulating the NMJ morphology (Wong, 2015).
Biochemical analyses revealed that both JNK phosphorylation and its transcriptional output correlated with the activity of MTORC1, which are consistent with prior observations that cln3 overexpression promotes JNK activation and that tsc1/tsc2 deletion in flies result in increased JNK-dependent transcription. These findings point to the remarkable versatility of MTORC1 in controlling both protein trans lation and gene transcription (Wong, 2015).
Using an in vitro kinase assay, this study demonstrates that Wnd is a target of MTORC1. Because axonal injury activates both MTORC1 and DLK/JNK, these findings imply a functional connection between these two pathways. Interestingly, the data also suggest that MTORC1 contains additional kinases besides MTOR that can phosphorylate Wnd. One possibility is that ULK1/Atg1, which associates with MTORC1, could be the kinase that phosphorylates Wnd. Consistent with this notion, overexpression of Atg1 in the Drosophila neurons has been shown to promote JNK signaling and NMJ synapse overgrowth via Wnd) (Wong, 2015).
This study also found that developmental JNK activation in axonal tracts of the CC and pJNK levels in cortical neurons were compromised in a mouse model of Batten disease. Thus, the signaling deficits identified in Drosophila are also conserved in mammals. The activity of DLK (the mouse homolog of Wnd) and JNK signaling are critical for axonal development in the mouse CNS. Therefore, decreased neuronal JNK activation during development might underlie the thinning of the axonal tracts observed in many LSDs (Wong, 2015).
Although the findings of this study demonstrate a role for an amino-acid- responsive cascade in the synaptic defects associated with lysosomal dysfunction, simply elevating the dietary protein content was not sufficient to rescue these defects. These findings were reminiscent of an elegant study that showed that the growth of Drosophila neuroblasts is uncoupled from dietary amino acids owing to the function of ALK, which suppresses the uptake of amino acids into the neuroblasts (Cheng, 2011). Indeed, simultaneous administration of an ALK inhibitor and a high-protein diet partially rescued the synaptic growth defects associated with the lysosomal dysfunction, and improved the rescue of pupal lethality associated with trpml1. Although these studies do not causally link the defects in synapse development with pupal lethality, they do raise the intriguing possibility that multiple phenotypes associated with LSDs could be targeted using ALK inhibitors along with a protein-rich diet (Wong, 2015).
Although LSDs result in lysosomal dysfunction throughout the body, neurons are exceptionally sensitive to these alterations. The cause for this sensitivity remains incompletely understood. Given the findings of this study that mature neurons do not efficiently take up amino acids from the extracellular medium, lysosomal degradation of proteins serves as a major source of free amino acids in these cells. Therefore, disruption of lysosomal degradation leads to severe shortage of free amino acids in neurons, regardless of the quantity of dietary proteins, thus explaining the exquisite sensitivity of neurons to lysosomal dysfunction (Wong, 2015).
Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. The role of Miple proteins was investigated in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). This study shows that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally it was shown that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, the data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo (Hugosson, 2014: PubMed).
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).
Anaplastic lymphoma kinase (Alk) has been proposed to regulate neuronal development based on its expression pattern in vertebrates and invertebrates; however, its function in vivo is unknown. This study demonstrated that Alk and its ligand Jelly belly (Jeb) play a central role as an anterograde signaling pathway mediating neuronal circuit assembly in the Drosophila visual system. Alk is expressed and required in target neurons in the optic lobe, whereas Jeb is primarily generated by photoreceptor axons and functions in the eye to control target selection of R1-R6 axons in the lamina and R8 axons in the medulla. Impaired Jeb/Alk function affects layer-specific expression of three cell-adhesion molecules, Dumbfounded/Kirre, Roughest/IrreC, and Flamingo, in the medulla. Moreover, loss of flamingo in target neurons causes some R8-axon targeting errors observed in Jeb and Alk mosaic animals. Together, these findings suggest that Jeb/Alk signaling helps R-cell axons to shape their environment for target recognition (Bazigou, 2007).
These genetic studies in Drosophila provide functional evidence in vivo that Alk plays a crucial role in the developing central nervous system. This study shows that Alk and its cognate ligand Jeb form an anterograde signaling pathway in the fly visual system, which is required for target selection by R cell axons within the lamina and medulla. It is proposed that R cell axons release Jeb to activate Alk signaling in target neurons and, through direct or indirect regulation of downstream guidance molecules, contribute to creating the appropriate environment for target recognition (Bazigou, 2007).
In the visual system, R cell axons provide two known anterograde signals to the optic lobe to promote neuronal proliferation and differentiation of target neurons during the third instar larval stage. R cell-derived Hh induces mitotic divisions of lamina precursor cells (LPCs), as well as expression of the early neuronal marker Dachshund in both LPCs and postmitotic lamina neurons. Dachshund in turn is required to control the expression of the EGF receptor in lamina neurons, thus making them competent for the second anterograde R cell-derived signal Spitz that induces the next step of lamina neuron differentiation. A third so far unidentified signal controls glial cell development and migration in the optic lobe. The current findings show that R cell axons provide an unexpected fourth anterograde signal -- Jeb -- that is required to mediate target selection of R cell axons during pupal development. Unlike the Hh and Spitz signals, Jeb represents an anterograde signal delivered by R cell axons not only to the lamina but also to the medulla (Bazigou, 2007).
That Jeb and Alk form an anterograde signaling pathway in the visual system that is supported by three lines of evidence: first, Jeb and Alk are expressed in a largely complementary pattern from the third instar larval to midpupal stages. The ligand Jeb is produced in R cells, whereas the receptor Alk is specifically expressed by target neurons. Since the Jeb protein has been shown to be secreted in vitro, it is highly likely released from R cell growth cones. Second, jeb is genetically required in R cells, whereas Alk functions in target neurons. Third, in the converse experiment, removal of jeb function in the target or Alk in the eye does not produce any conspicuous targeting phenotypes (Bazigou, 2007).
It is proposed that Jeb/Alk signaling plays a role in regulating late events of target-neuron maturation to control R1-R6 axons in the lamina and R8 axons in the medulla. Consistent with this model, the data indicate that loss of Jeb/Alk signaling affects the expression of three guidance molecules, Duf/Kirre, Rst/IrreC, and Fmi, in the R8 recipient layer of the medulla, while Caps, LAR, PTP69D, and CadN appear normal at this level of resolution. Interestingly, animals lacking Jeb/Alk signaling display similar R8 projection defects as fmi and caps eye mosaics. It was further shown that loss of fmi in target neurons causes R8-targeting defects, which qualitatively resemble those observed in Jeb/Alk mosaics. As Jeb/Alk signaling acts upstream of multiple cell-adhesion molecules, loss of one factor likely results in milder targeting defects. In support of this notion, it was observed that phenotypes in jeb or Alk mosaics were more frequent in comparison to fmi knockdown or fmi ELF mosaics. Moreover, loss of fmi in the target appeared to cause one prevalent targeting defect, i.e., the fasciculation of R8 axons with processes in adjacent medulla columns. Notably, loss of sec15 in R cells, which encodes an exocyst component regulating the localization of cell-adhesion molecules to axon terminals, also causes distinct targeting errors. This is consistent with the model that regulating the precise expression of guidance molecules by Jeb/Alk signaling is indeed important for axon targeting in the visual system (Bazigou, 2007).
R cell-targeting defects occurred in both null and kinase domain mutant alleles of Alk, showing that tyrosine kinase activity is essential. Furthermore, studies of vertebrate Alk in vitro, as well as Drosophila Alk in vivo, demonstrate that this RTK drives an ERK/MAPK-mediated signaling pathway, suggesting that Alk may also act through this pathway in the visual system. There are three possible mechanisms as to how Jeb/Alk signaling could regulate downstream guidance molecules: (1) Jeb and Alk may directly regulate the expression of guidance molecules, (2) they could indirectly regulate the expression pattern of guidance molecules via the activation of transcriptional programs determining target neuron identities, or (3) they could separately control both the expression of guidance molecules and transcription factors. Such mechanisms would be analogous to what has been observed in the developing visceral mesoderm, where Jeb/Alk signaling induces the expression of both Duf/Kirre and Org-1, a transcription factor and mammalian Tbx1 homolog, to drive muscle fusion. At present, it cannot be excluded that Alk additionally modulates the activity of downstream targets (Bazigou, 2007).
Anterograde Jeb/Alk signaling would make it possible to coordinate the timing of R cell growth-cone extension with local expression of guidance factors in the target. These in turn could directly regulate afferent axon targeting. Alternatively, guidance factors may be required to shape dendritic and axonal arbors of target neurons and to mediate R cell-targeting decisions. Fmi could indeed take part in both processes, as it can control dendrite development, as well as axon guidance by afferent/afferent and afferent/target interactions. Similar to CadN or LAR eye mosaics, some R1-R6 axons lacking jeb function failed to extend from their original bundle. Extension and cartridge assembly phenotypes were also detected in jeb eye or Alk target mosaics, which qualitatively resembled those described for fmi eye mosaics. Future studies will require the identification and validation of (other) downstream guidance molecules, as well as the isolation of transcriptional regulators controlling target neuron subtype specificity in both the lamina and medulla to provide further insights into the mechanisms underlying Jeb/Alk function (Bazigou, 2007).
It was observed that ectopic expression of Jeb in the visual system strongly reduces the number of activated Caspase 3-positive cells in the medulla at 24 hr APF, when many postmitotic medulla neurons normally undergo apoptosis in wild-type. Thus, Jeb/Alk signaling may also mediate cell survival in parallel to neuronal maturation. The mechanism could be similar to the pleiotropic function of EGF-receptor signaling, which, depending on low or high level of activation regulates cell-cycle withdrawal, mitosis, cell survival, and differentiation in the developing eye imaginal disc of Drosophila (Bazigou, 2007).
Although Jeb shares some sequence similarity with proteins such as the secreted bovine glycoprotein Sco-Spondin , no Jeb homolog has been isolated so far in vertebrates. However, the growth factors Pleiotrophin and Midkine have been reported to act as ligands for Alk in vertebrates, and both have been linked to neuronal development and neurodegenerative diseases. Therefore, Alk may work with different ligands in the vertebrate nervous system. The C. elegans homolog of Alk is localized presynaptically at the neuromuscular junction and has been proposed to mediate synapse stabilization. Also, the vertebrate homologs of Alk are strongly expressed in the developing and adult nervous systems. This includes motor-neuron columns in the spinal cord and, intriguingly, also the superior colliculus, a higher-order processing center for visual information in the brain. That Alk may play a role in neuronal development in vertebrates is further supported by in vitro studies indicating that activated Alk can promote neuronal differentiation and neurite outgrowth in specific cell line. These observations suggest that the function of Alk in regulating specific aspects of neuronal development may be conserved (Bazigou, 2007).
Anaplastic Lymphoma Kinase (Alk) is a Receptor Tyrosine Kinase (RTK) activated in several cancers, but with largely unknown physiological functions. This study reports two unexpected roles for the Drosophila ortholog dAlk, in body size determination and associative learning. Remarkably, reducing neuronal dAlk activity increased body size and enhanced associative learning, suggesting that its activation is inhibitory in both processes. Consistently, dAlk activation reduced body size and caused learning deficits resembling phenotypes of null mutations in dNf1, the Ras GTPase Activating Protein-encoding conserved ortholog of the Neurofibromatosis type 1 (NF1) disease gene. dAlk and dNf1 co-localize extensively and interact functionally in the nervous system. Importantly, genetic or pharmacological inhibition of dAlk rescued the reduced body size, adult learning deficits, and Extracellular-Regulated-Kinase (ERK) overactivation dNf1 mutant phenotypes. These results identify dAlk as an upstream activator of dNf1-regulated Ras signaling responsible for several dNf1 defects, and they implicate human Alk as a potential therapeutic target in NF1 (Gouzi, 2011).
The prominent expression of Alk in the mammalian and Drosophila CNS and presence of the dAlk ligand Jeb in the embryonic fly CNS, provided the first indication that Alk and Jeb likely participate in the development of the nervous system. Subsequent in vitro studies demonstrated that Alk promoted neuronal differentiation of PC12 or neuroblastoma cell lines, and work in C. elegans implicated its Alk ortholog, scd-2, in the inhibition of presynaptic neuronal differentiation in vivo. Several functions have also been attributed to Drosophila dAlk, including roles in the specification of the intestinal musculature, in retinal axon targeting, and in signaling at the larval neuromuscular junction. The current results establish two novel in vivo functions for the Drosophila dAlk/Jeb receptor/ligand pair, in the regulation of organismal growth and associative learning (Gouzi, 2011).
The results presented in this study lead to a hypothesis that dAlk and dNf1 have opposite roles in controlling neuronal ERK activity during larval development, and therefore determine overall organismal size in a non-cell autonomous manner. In support of this hypothesis, dAlk and dNf1 co-localize extensively in larval neurons, both proteins control ERK activity, and both modulate growth by regulating cell size. In agreement with this conclusion, transgenic neuronal expression of the constitutively active ERK, RlSEM, is sufficient to reduce Drosophila size (Gouzi, 2011).
dAlk is the second active RTK implicated in Drosophila growth control. Previous work demonstrated that the fly homolog of the insulin/IGF1 receptor dInr, regulates both body and organ size. In peripheral tissues, dInr is activated by a family of Insulin-like proteins (dILPs), leading to the activation of the IRS (Chico), PI(3)K (Dp110), PTEN (dPTEN), and Akt/PKB, (dAkt1/dPKB), signaling pathway. Ablating the Insulin Producing Cells (IPCs), or silencing the function of dInr pathway components in the larval CNS resulted in severe growth defects. Notwithstanding the similar growth phenotypes, several lines of evidence argue that dAlk and dInr control growth in fundamentally different ways. Most importantly, organismal growth is impaired when dInr activity or signaling is reduced, whereas a similar phenotype is observed upon dAlk activation. Secondly, dAlk affects organism growth by specifically regulating cell size in a non cell-autonomous manner. In contrast, dInr signaling affects both cell size and number cell-autonomously and non-autonomously. Finally, expression of Jeb or dAlk transgenes in neuroendocrine IPCs using the dILP2-Gal4 driver did not modify pupal size. Thus, although both dAlk and dInr RTKs are involved in body size determination, their mechanisms and sites of action are distinct. This interpretation is consistent with results with the C. elegans Alk homolog Scd-2 shown to function in parallel with or converge with TGF-β signaling, but act independently of the Insulin cascade in dauer determination (Reiner, 2008). However, given that dInr and dAlk are members of the same subfamily of RTKs, a potential explanation for the lack of rescue of dNf1 mutant homozygous larvae with systemic administration of 100 µM of the selective inhibitor TAE684, may be off-target inhibition of dInr at the higher drug concentration (Gouzi, 2011).
Interestingly, S6K (dS6K) resides on a downstream branch of the dInr/PI(3)K signaling pathway and regulates cell size without impinging on cell number. Although the dS6K loss-of-function phenotype resembles the dAlk gain-of-function and dNf1 loss-of-function phenotypes, its mode of action is cell-autonomous. However, it is still tempting to speculate that dAlk and dNf1 ultimately affect neuroendocrine signals that affect dS6K activity in peripheral tissues (Gouzi, 2011).
Increasing signaling through the cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway has been reported to suppress the size defect of dNf1 mutants. This among other findings, have led some to propose that dNf1 regulates Ras activity and cAMP levels independently. In contrast, an investigation of the cAMP/PKA sensitive dNf1 mutant growth defect argued that aberrantly upregulated Ras/ERK signaling in Ras2-expressing larval neurons was its proximal cause. The current results further support the latter explanation implicating a Ras/ERK signaling defect downstream of dAlk as the cause of size defects in dNf1 mutants. Then, how could elevated cAMP/PKA signaling rescue decreased body size? Because neuroendocrine signals can activate the cAMP pathway, it is possible that defective dAlk/Ras/ERK signaling in dNf1 mutants may lead to a neuroendocrine deficiency, which is restored by increasing cAMP/PKA signals (Gouzi, 2011).
In Drosophila, the dAlk/Jeb receptor-ligand pair has been shown to act in an antrerograde signaling pathway essential for assembly of the neuronal circuitry of the fly visual system. However, loss of either Alk or Jeb did not appear to impair assembly of functional synapses with normal postsynaptic responses at the larval neuromuscular junction, indicating that they do not participate in CNS development. In agreement, pan-neuronal, spatially restricted attenuation or unregulated activation of Alk throughout development did not appear to yield gross structural defects in the adult brain or alter naïve behavioral responses to the stimuli used for conditioning. Hence, it is unlikely that the learning phenotypes described in this study are the result of developmental alterations in the CNS. In fact, dAlk seems to be acutely required for normal learning as limiting modulation of its activity to the adult CNS results in phenotypes on its own and also modifies the learning deficits of dNf1 mutants. Moreover, the function of dAlk and dNf1 in associative learning is independent of body size as the learning reverted to normal by dAlk abrogation in the small-sized dNf1 mutant homozygotes. Interestingly, the C. elegans Jeb homolog Hen-1 is required non-cell autonomously in the mature nervous system for sensory integration and associative learning (Ishihara, 2002). Collectively then, these studies in C. elegans, mice and the current data strongly support an evolutionary conserved role for Alk signaling in adult associative learning and memory (Gouzi, 2011).
Elevated dAlk/Jeb signaling outside the MBs impaired olfactory learning, while its abrogation increased learning efficiency. These are results are consistent with the enhanced performance in a hippocampus-dependent task described for Alk knockout mice (Bilsland, 2008). It is proposed then, that Alk signaling normally functions to limit the strength of the CS/US associations, in effect providing a putative threshold required to be overcome for specific and efficient association of the stimuli. A GABAergic neuron outside the MBs, the Anterior Paired Lateral (APL), was recently reported to similarly suppress olfactory learning and its silencing yielded enhanced performance. Interestingly, a decrease in presynaptic GABA release or abrogation of the GABAA receptor, RDL in the post-synaptic mushroom body neurons resulted in enhanced learning. Whether Ras2-Gal4 is expressed in the APL neuron and dAlk also functions in this neuron to suppress learning are questions currently under investigation (Gouzi, 2011).
Interestingly, a recent study suggested that the learning defects of Nf1+/− mice are attributed to increased ERK-mediated phosphorylation of synapsin I in hippocampal inhibitory neurons and concomitant increase in GABA release. In accord, a GABAA receptor antagonist enhanced learning in Nf1+/− mice and controls, and reversed LTP defects in the mutants. Similarly, elimination of the dAlk-mediating inhibition in Drosophila Ras2-expressing neurons enhanced learning, potentially via attenuation of ERK phosphorylation. In support of this notion, this study shows that constitutive activation of ERK in adult Ras2-expressing neurons precipitates learning deficits. Collectively, these results together with the reported learning deficits of Drosophila synapsin mutants, suggest that a mechanism similar to that proposed for vertebrates may also regulate Nf1-dependent learning in flies (Gouzi, 2011).
In mice, a decrease in Nf1 levels in heterozygous mutants increased Ras/ERK signaling and precipitated Long-Term Potentiation (LTP) and spatial learning deficits. These deficits were reversed upon genetic or pharmacological inhibition of Ras signaling. The current results demonstrate that dAlk inhibition reversed the impaired learning of dNf1 mutants and since this is the first 'kinase-active' RTK shown to be involved in this process in flies, it provides independent support for Ras/ERK hyperactivation as causal of these learning defects. Then, how can the reported phenotypic reversal of Nf1 learning deficits by expressing the PKA catalytic subunit throughout the fly be explained? It is hypothesized that the MBs are functionally downstream of the dAlk/dNf1 neurons and elevated PKA activity within the former could result in normal learning. Future work will focus on addressing the merits of these hypotheses regarding the mechanisms underlying the size and learning defects of dNf1 mutants (Gouzi, 2011).
A recent report Buchanan (2010) suggested that dNf1 mRNA is found in the mushroom bodies and in agreement, immunohistochemical results of the current study demonstrate that dNf1 is present within the mushroom body calyces. Protein synthesis-dependent memory defects in Nf1 mutants were rescued upon MB-limited expression of the same full-length transgene as was used in this study. Since memory deficits were not examined in the current work, this complements the current data and suggests a function for dNf1 within the MBs. In contrast, the current data indicate that dNf1 expression in the MBs is not sufficient for learning/3 min memory. Three common MB drivers including the most specific MB247 and the most broadly expressed OK107-Gal4, did not rescue learning in Nf1 mutants by expressing dNf1. It is suggested therefore that rescue described by Buchanan and Davis was mediated largely by c739-Gal4 transgene expression in neurons extrinsic to MBs where Elav, Ras2 and Alk(38)-Gal4 are expressed, perhaps in combination with expression within MB-intrinsic neurons. The neuronal circuits where dNf1 and dAlk are required for normal learning are the subject of ongoing investigations (Gouzi, 2011).
This study identifies dAlk as the first RTK to functionally interact with Nf1 in Drosophila, raising the important question whether a similar functional relationship exists in mammals. Suggestive evidence argues that this may indeed be the case. Thus, Alk and NF1 extensively colocalize in the mammalian CNS during the same developmental periods. Additionally, excess Alk expression or activation has been reported in astrocytomas, gliomas, neuroblastomas and pheochromocytomas, in which loss of NF1 expression has also been found. Based on identification of Alk as a bona-fide RTK that initiates a Ras/ERK cascade regulated by Nf1, this suggests that Alk inhibition may rescue not only the phenotypes reported in this study, but also other symptoms that have been previously associated with Nf1 loss and ERK over-activation. It was recently reported that knockdown of NF1 expression renders a neuroblastoma cell line resistant to retinoic acid-induced differentiation, and that NF1 deficient neuroblastoma tumors have a poor outcome. The current results suggest that Alk inhibition may provide an intervention strategy in such cases. Finally, the findings reported in this study, combined with the lack of overt abnormalities in Alk knock-out mice, provide a rationale for further explorations of Alk as a potential therapeutic target in NF1 (Gouzi, 2011).
In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ), and a conserved intercellular signaling function was been postulated. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans-synaptic signaling. This study shows that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb-Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild-type postsynaptic Alk expression in Alk partial loss-of-function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb-Alk signaling triggers the Ras-MAP kinase cascade in both pre- and postsynaptic compartments. These novel roles for Jeb-Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis (Rohrbough, 2012).
The results support an anterograde signaling model in which presynaptically secreted Jeb activates postsynaptic Alk. The data to support this hypothesis derives from multiple tests. First, immunolabeling shows Jeb is concentrated within presynaptic boutons, while Alk is present in the surrounding postsynaptic subsynaptic reticulum (SSR) (Rohrbough, 2011). Second, targeted postsynaptic Alk expression in Alk LOF mutants is sufficient to rescue synaptic transmission defects, a strong demonstration that Alk is required in the postsynaptic muscle to regulate neurotransmission. Third, post-synaptic inhibtion of Alk by tissue specific RNAi results in 2- fold increased accumulation of perisynaptic Jeb. Fourth, the MARCM clonal approach demonstrates Jeb may be required within presynaptic motor neurons to regulate postsynaptic molecular assembly. Fifth, elevated presynaptic Jeb expression activates postsynaptic Ras/MAPK/ERK activation, while inhibition of postsynaptic Alk reduces Ras/MAPK/ERK activitation (Rohrbough, 2012).
In structurally normal NMJs, strong effects on neurotransmission were found as a consequence of perturbations in Jeb-Alk signaling. The clearest, most consistent results derive from techniques that activate or inhibit Jeb-Alk signaling postsynaptically. Postsynaptic hyperactivation of Alk weakens NMJ synaptic transmission. This functional phenotype parallels the negative regulation of synaptic growth by postsynaptic Alk activation. Consistent with the inhibitory effect of Alk activation on neurotransmission, enhanced neurotransmission was observed as a consequence of muscle specific reductions in Alk levels by transgenic RNAi. Additional confirmation for Alk-dependent inhibition of neurotransmission is provided by analysis of a hypomorphic temperature sensitive allele of Alk. Partial loss of Alk function results in strongly increased NMJ neurotransmission. The implication is that Alk activity limits or negatively regulates synaptic strength. It was also shown that muscle-specific Alk expression in the strongest alkts/alkf01491 partial loss of function genotype rescues reduced neurotransmission to near wild-type levels, a conclusive demonstration that postsynaptic Alk function negatively regulates the strength of NMJ neurotransmission. This function is novel: Jeb-Alk transynaptic signaling is the only known negative regulator of synaptic transmission (Rohrbough, 2012).
Presynaptic manipulation of Jeb yields less strong though still consistent results. Transmission is uneffected by increased pan-neuronal Jeb expression, though this activates Ras/MAPK/ERK both centrally and presynaptically at the NMJ and, to a lesser degree, within the postsynaptic muscle. Motor neuron electrical activity activates neuronal Ras/MAPK/ERK signaling, and this presynaptic Ras/MAPK/ERK activation is positively linked to both structural and functional NMJ synaptic remodeling. Motor neuron specific over expression of Jeb does produce a modest but statistically significant reduction in neuromuscular transmission. Ectopic expression of Jeb in muscle results in substantial inhibiton of neuromuscular transmission. One hypothesis that may account for the diffence between panneuronal and motor neuron or muscle specific manipulation of Jeb-Alk signalling is that the effects of manipulating pan-neuronal Jeb represent a composite of central and peripheral effects on the motor neuron. In first instar larvae it was found that both jeb and alk mutants display impaired central output to motor neurons most consistent with a central synaptic defect (Rohrbough, 2011). The integrated physiologic function subserved by Jeb-Alk signaling in the NMJ, which has yet to be determined, will provide the essential context for interpretting these results (Rohrbough, 2012).
The novel inhibitory role of Jeb-Alk signaling in NMJ transmission implies that it is part of a transynaptic regulatory network that integrates neuronal activity and responses with other homeostatic mechanisms. This study provides indirect evidence that Jeb secretion is regulated. The physiologic regulation of Jeb secretion is a critical missing component of understanding how Jeb-Alk signaling fits into the regulation of synaptic plasticity. Jeb-Alk signaling regulates postembryonic NMJ synaptic growth and patterning Jeb-Alk signaling is not required for embryonic NMJ synaptogenesis or differentiation, although jeb and alk null mutants display impaired locomotion and reduced NMJ transmission (Rohrbough and Broadie 2011). At later developmental stages, removing Jeb in motor neurons strongly disrupts late larval NMJ synaptic terminal architecture and bouton morphology. Postsynaptic Dlg scaffolding and GluR clustering are strongly perturbed in association with jeb mutant terminals. The mosaic analysis supports a cell-autonomous, anterograde signaling function for Jeb. One mechanistic hypothesis is that Jeb-Alk nerve-to-muscle signaling regulates NMJ morphogenesis by recruiting or regulating cell adhesion molecules (CAMs). In the developing adult visual system, anterograde Jeb-Alk signaling induces the expression of postsynaptic adhesion molecules Dumbfounded/Kirre, Roughest/IrreC and Flamingo to shape the optic neuropil target environment. At the larval NMJ, adhesion molecules such as fasciclins and integrins regulate activity-dependent synaptic growth and structural remodeling. The current results imply that Jeb-Alk signaling either directly regulates Dlg localization or indirectly drives Dlg-dependent postsynaptic differentiation. Dlg has demonstrated roles in NMJ morphogenesis and GluR expression and field regulation, and directly binds and regulates fasciclin II and βPS integrin. Future work will test the hypothesis that Jeb-Alk signaling organizes or regulates adhesion receptors and postsynaptic scaffolding to control bouton differentiation and shape functional synaptic architecture (Rohrbough, 2012).
In other systems, Jeb-Alk signaling has been studied primarily at the level of behavior. In C. elegans, the Jeb homolog Hen-1 was identified in a forward genetic behavioral screen for impaired ability to integrate conflicting sensory input (Ishihara, 2002). The Hen-1 phenotype is non-developmental and can be rescued only by adult Hen-1 expression. There is no uniquely identified mammalian Jeb/Hen-1 homolog, but ALK is expressed in the mammalian nervous system during development and at maturity. Alk is expressed in the mouse hippocampus and Alk loss of function enhances behavioral performance in tests dependent on hippocampal function. Similarly, Drosophila learning has shown a dependence on the Ras/MAPK/ERK cascade, which is activated by Jeb-Alk signaling and is probably inhibited by Drosophila neurofibromin (dNf1). Genetic or pharmacologic inhibtion of Jeb-Alk signaling enhances associative learning while increased Jeb-Alk signaling or loss of dNf1 impairs learning. Inhibition of Alk rescues dNf1 mutant learning deficits. These studies suggest that the Jeb-Alk trans-synaptic pathway acts in concert with other, negative regulators of Ras/MAPK/ERK signaling to balance developmental and learning-related synaptic structural and functional changes. Strikingly, a whole-genome association study recently identified human ALK as one of a small number of genes associated with sporadic amyotrophic lateral sclerosis (ALS), a devistating neurodegerative disease of central motor units. If Alk has a conserved inhibitory role in synaptic physiological regulation, hypofunctional human Alk variants may result in augmented motor unit activity and contribute to excitotoxicity and progressive motor unit degeneration in ALS. Pharmacologic activation of Alk has already been hypothesized to have therapeutic benefit in treating ALS. Further insight from future studies should be gained into the mechanism by which the Jeb-Alk signaling pathway regulates synaptic adaptivity in both normal and pathological states (Rohrbough, 2012).
Neurofibromatosis type 1 (NF1), a genetic disease that affects 1 in 3,000, is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein (GAP) for Ras. Among Drosophila Nf1 (dNf1) null mutant phenotypes, learning/memory deficits and reduced overall growth resemble human NF1 symptoms. These and other dNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3'-5' cyclic adenosine monophosphate (cAMP) dependent Protein Kinase A (PKA) pathway, or phenocopied by inhibiting this pathway. However, whether dNf1 affects cAMP/PKA signaling directly or indirectly remains controversial. To shed light on this issue 486 1st and 2nd chromosome deficiencies that uncover >80% of annotated genes were screened for dominant modifiers of the dNf1 pupal size defect, identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi) knockdown. Validating the screen, identified suppressors include the previously implicated dAlk tyrosine kinase, its activating ligand jelly belly (jeb), two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in the dNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor. Providing mechanistic clues, it was shown that dAlk, jeb and CCKLR-17D1 are among mutants that also suppress a recently identified dNf1 neuromuscular junction (NMJ) overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone (AKH)-producing cells at the base of the neuroendocrine ring gland restore the dNf1 growth deficiency. Finally, supporting the contention that ALK might be a therapeutic target in NF1, this study reports that human ALK is expressed in cells that give rise to NF1 tumors and that NF1 regulated ALK/RAS/ERK signaling appears conserved in man (Walker, 2013).
Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk), the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1) to regulate growth and associative learning in flies. This study shows that Alk mutants have increased sleep. Using a targeted RNAi screen the negative effects of Alk on sleep was localized to the mushroom body, a structure important for both sleep and memory. Mutations in Nf1 produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes (Bai, 2015).
Though a few studies implicate Alk orthologs in regulating behaviors such as decision-making, cognition, associative learning and addiction, most functional studies demonstrate various developmental roles for Alk. This study acutely induce a long-sleep phenotype by taking advantage of a temperature-sensitive allele, Alkts, revealing that Alk regulates sleep directly rather than through developmental processes. Mutations in Nf1, a gene encoding a GAP that regulates the Ras/ERK pathway activated by ALK, also cause a sexually dimorphic short-sleep phenotype. Thus this study establishes a novel in vivo function for both Alk and Nf1 and shows they interact with each other to regulate sleep (Bai, 2015).
Many downstream signaling pathways have been proposed for ALK, among them Ras/ERK, JAK/STAT, PI3K and PLCγ signaling. ERK activation through another tyrosine receptor kinase Epidermal growth factor receptor (EGFR) has been linked to increased sleep, while this study shows that Alk, a positive regulator of ERK, inhibits sleep. It is noted that ERK is a common signaling pathway targeted by many factors, and may have circuit- specific effects, with different effects on sleep in different brain regions. Indeed, neural populations that mediate effects of ERK on sleep have not been identified. The dose of ALK required for ERK activation might also differ in different circuits. Region-specific effects of Alk are supported by a GAL4 screen, in which down-regulation of Alk in some brain regions even decreased sleep. The overall effect, however, is to increase sleep, evident from the pan-neuronal knockdown. It was found that the mushroom body, a site previously implicated in sleep regulation and learning, requires Alk to inhibit sleep. Interestingly, the expression patterns of Alk and Nf1 overlap extensively in the mushroom body, suggesting that they may interact here to regulate both sleep and learning. However, it was previously shown that Alk activation in the mushroom body has no effect on learning. The mushroom body expression in that study was defined with MB247 and c772, both of which also had no effects on sleep when driving Alk RNAi. The spatial requirement for Nf1 in the context of learning has been disputed in previous studies with results both for and against a function in the mushroom body. The discrepancies between these studies could result from: 1) varied expression of different drivers within lobes of the mushroom body, with some not even specific to the mushroom body; 2) variability in the effectiveness and specificity of MB-Gal80 in combination with different GAL4s. It was confirmed that the MB-Gal80 manipulation eliminated all mushroom body expression and preserved most if not all other cells with 30Y, 386Y and c309. Future work will further define the cell populations in which Alk and Nf1 interact to affect sleep (Bai, 2015).
A substantial sleep decrease was observed in Nf1 male flies compared to control flies. However, sleep phenotypes in Nf1 female flies are inconsistent. It is unlikely that unknown mutations on the X chromosome cause the short-sleeping phenotype because 7 generation outcrosses into the control iso31 background started with swapping X chromosomes in Nf1P1 and Nf1P2 male flies with those of iso31 flies. In support of a function in sleep regulation, restoring Nf1 expression in neurons of Nf1 mutants reverses the short sleep phenotype to long sleep in both males and females. This does not result from ectopic expression of the transgene as expressing the same UAS-Nf1 transgene in wild-type flies has no effect. It was hypothesized that Nf1 promotes sleep in some brain regions and inhibits it in others, and sub-threshold levels of Nf1, driven by the transgene in the mutant background, tilt the balance towards more sleep. As reported in this study, Alk also has differential effects on sleep in different brain regions, as does protein kinase A, thus such effects are not unprecedented. Severe sleep fragmentation was also observed in Nf1 mutants, which suggests that they have trouble maintaining sleep (Bai, 2015).
The sex-specific phenotypes of Nf1 mutants may reflect sexually dimorphic regulation of sleep. A recently published genome-wide association study of sleep in Drosophila reported that an overwhelming majority of single nucleotide polymorphisms (SNPs) exhibit some degree of sexual dimorphism: the effects of ~80% SNPs on sleep are not equal in the two sexes. Interestingly, sex was found to be a major determinant of neuronal dysfunction in human NF1 patients and Nf1 knock-out mice, resulting in differential vision loss and learning deficits. The sex-dimorphic sleep phenotype in Nf1 flies provides another model to study sex-dimorphic circuits involving Nf1. Interestingly, a prevalence of sleep disturbances have recently been reported in NF1 patient, suggesting that NF1 possibly play a conserved function in sleep regulation (Bai, 2015).
An attractive hypothesis for a function of sleep is that plastic processes during wake lead to a net increase in synaptic strength and sleep is necessary for synaptic renormalization. There is structural evidence in Drosophila to support this synaptic homeostasis hypothesis (SHY): synapse size and number increase during wake and after sleep deprivation, and decrease after sleep. However, little is known about the molecular mechanisms by which waking experience induces changes in plasticity and sleep. FMRP, the protein encoded by the Drosophila homolog of human fragile X mental retardation gene FMR1, mediates some of the effects of sleep/wake on synapses. Loss of Fmr1 is associated with synaptic overgrowth and strengthened neurotransmission and long sleep. Overexpressing Fmr1 results in dendritic and axonal underbranching and short sleep. More importantly, overexpression of Fmr1 in specific circuits eliminates the wake-induced increases in synapse number and branching in these circuits. Thus, up-regulation of FMR accomplishes a function normally associated with sleep (Bai, 2015).
It is hypothesized that Alk and Nf1 similarly play roles in synaptic homeostasis. They are attractive candidates for bridging sleep and plastic processes, because: 1) Alk is expressed extensively in the developing and adult CNS synapses. In particular, both Alk and Nf1 are strongly expressed in the mushroom body, a major site of plasticity in the fly brain. 2) Functionally, postsynaptic hyperactivation of Alk negatively regulates NMJ size and elaboration. In contrast, Nf1 is required presynaptically at the NMJ to suppress synapse branching. 3) Alk and Nf1 affect learning in adults and they functionally interact with each other in this process. It is tempting to speculate that in Alk mutants, sleep is increased to prune the excess synaptic growth predicted to occur in these mutants. Such a role for sleep is consistent with the SHY hypothesis. The SHY model would predict that Alk flies have higher sleep need, which is expected to enhance rebound after sleep deprivation. While the data show equivalent quantity of rebound in Alk mutants, this study found that they fall asleep faster than control flies the morning after sleep deprivation, suggesting that they have higher sleep drive. Increased sleep need following deprivation could also be reflected in greater cognitive decline, but this has not yet been tested for Alk mutants. It is noted that Nf1 mutants have reduced sleep although their NMJ phenotypes also consist of overbranched synapses. It is postulated that their sleep need is not met and thus results in learning deficits. Clearly, more work is needed to test these hypotheses concerning the roles of Alk and Nf1 in sleep, learning, and memory circuits (Bai, 2015).
The 2;5 chromosomal translocation occurs in most anaplastic large-cell non-Hodgkin's lymphomas arising from activated T lymphocytes. This rearrangement fuses the NPM nucleolar phosphoprotein gene on chromosome 5q35 to a protein tyrosine kinase gene, ALK, on chromosome 2p23. In the predicted hybrid protein, the amino terminus of nucleophosmin (NPM) is linked to the catalytic domain of anaplastic lymphoma kinase (ALK). Expressed in the small intestine, testis, and brain but not in normal lymphoid cells, ALK shows greatest sequence similarity to the insulin receptor subfamily of kinases. Unscheduled expression of the truncated ALK may contribute to malignant transformation in these lymphomas (Morris, 1994).
The 2;5 chromosomal translocation is frequently associated with anaplastic large cell lymphomas (ALCLs). The translocation creates a fusion gene consisting of the alk (anaplastic lymphoma kinase) gene and the nucelophosmin (npm) gene: the 3' half of alk derived from chromosome 2 is fused to the 5' portion of npm from chromosome 5. A recent study shows that the product of the npm-alk fusion gene is oncogenic. To help understand how the npm-alk oncogene transform cells, it is important to investigate the normal biological function of the alk gene product, ALK. cDNAs for both the human and mouse ALK proteins have been cloned. The deduced amino acid sequences reveal that ALK is a novel receptor protein-tyrosine kinase having a putative transmembrane domain and an extracellular domain. These sequences are absent in the product of the transforming npm-alk gene. ALK shows the greatest sequence similarity to LTK (leukocyte tyrosine kinase) whose biological function is presently unknown. RNA blot hybridization analysis of various tissues reveals that the alk mRNA is dominantly detected in the brain and spinal cord. Immunoblotting with anti-ALK antibody shows that ALK is highly expressed in the neonatal brain. Furthermore, RNA in situ hybridization analysis shows that the alk mRNA is dominantly expressed in neurons in specific regions of the nervous system such as the thalamus, mid-brain, olfactory bulb, and ganglia of embryonic and neonatal mice. These data suggest that ALK plays an important role(s) in the development of the brain and exerts its effects on specific neurons in the nervous system (Iwahara, 1997).
Anaplastic Lymphoma Kinase (ALK) was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused to nucleophosmin (NPM) in the t(2;5) chromosomal rearrangement associated with non-Hodgkin's lymphoma, but further insights into its normal structure and function are lacking. A full-length normal human ALK cDNA and its product have been characterized, and the pattern of expression of its murine homologue has been determined in embryonic and adult tissues as a first step toward the functional assessment of the receptor. Analysis of the 6226 bp ALK cDNA identified an open reading frame encoding a 1620-amino acid (aa) protein of predicted mass approximately 177 kDa that is most closely related to leukocyte tyrosine kinase (LTK), the two exhibiting 57% aa identity and 71% similarity over their region of overlap. Biochemical analysis demonstrates that the approximately 177 kDa ALK polypeptide core undergoes co-translational N-linked glycosylation, emerging in its mature form as a 200 kDa single chain receptor. Surface labeling studies indicate that the 200 kDa glycoprotein is exposed at the cell membrane, consistent with the prediction that ALK serves as the receptor for an unidentified ligand(s). In situ hybridization studies reveal Alk expression beginning on embryonic day 11 and persisting into the neonatal and adult periods of development. Alk transcripts are confined to the nervous system and included several thalamic and hypothalamic nuclei; the trigeminal, facial, and acoustic cranial ganglia; the anterior horns of the spinal cord in the region of the developing motor neurons; the sympathetic chain, and the ganglion cells of the gut. Thus, ALK is a novel orphan receptor tyrosine kinase that appears to play an important role in the normal development and function of the nervous system (Morris, 1997).
Anaplastic lymphoma kinase (ALK) is a receptor-type protein tyrosine kinase that is expressed preferentially in neurons of the central and peripheral nervous systems at late embryonic stages. To elucidate the role of ALK in neurons, an agonist monoclonal antibody (mAb) was developed against the extracellular domain of ALK. Here, mAb16-39 is shown to elicit tyrosine phosphorylation of endogenously expressed ALK in human neuroblastoma (SK-N-SH) cells. Stimulation of these cells with mAb16-39 markedly induces the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), Shc, and c-Cbl and also their interaction with ALK and activation of ERK1/2. Furthermore, continuous incubation with mAb16-39 induces the cell growth and neurite outgrowth of SK-N-SH cells. These responses are completely blocked by MEK inhibitor PD98059 but not by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, indicating an essential role of the mitogen-activated protein kinase (MAP kinase) signaling cascade in ALK-mediated growth and differentiation of neurons (Motegi, 2004).
Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase plays important roles in the pathogenesis of several malignancies. Although it promotes the growth of stimulated hematopoietic cells, a direct role of IGF-IR in malignant lymphoma has not been identified. Anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK(+) ALCL) is a unique type of T-cell lymphoma. Approximately 85% of ALK(+) ALCL cases harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. This study explored a possible role of IGF-IR in ALK(+) ALCL. The results demonstrate that IGF-IR and IGF-I are widely expressed in ALK(+) ALCL cell lines and primary tumors. Importantly, novel reciprocal functional interactions were identified between IGF-IR and NPM-ALK. Antagonism of IGF-IR decreased the viability, induced apoptosis and cell-cycle arrest, and decreased proliferation and colony formation of ALK(+) ALCL cell lines. These effects could be explained by alterations of cell survival regulatory proteins downstream of IGF-IR signaling. These findings improve current understanding of the biology of IGF-IR and NPM-ALK and have significant therapeutic implications as they identify IGF-IR signaling as a potential therapeutic target in ALK(+) ALCL and possibly other types of malignant lymphoma (Shi, 2009).
During synapse formation, specialized subcellular structures develop at synaptic junctions in a tightly regulated fashion. Cross-signalling initiated by ephrins, Wnts and transforming growth factor-beta family members between presynaptic and postsynaptic termini are proposed to govern synapse formation. It is not well understood how multiple signals are integrated and regulated by developing synaptic termini to control synaptic differentiation. FSN-1 is novel F-box protein that is required in presynaptic neurons for the restriction and/or maturation of synapses in Caenorhabditis elegans. Many F-box proteins are target recognition subunits of SCF (Skp, Cullin, F-box) ubiquitin-ligase complexes. fsn-1 functions in the same pathway as rpm-1, a gene encoding a large protein with RING finger domains. FSN-1 physically associates with RPM-1 and the C. elegans homologues of SKP1 and Cullin to form a new type of SCF complex at presynaptic periactive zones. Evidence is provided that T10H9.2, which encodes the C. elegans receptor tyrosine kinase ALK (anaplastic lymphoma kinase), may be a target or a downstream effector through which FSN-1 stabilizes synapse formation. This neuron-specific, SCF-like complex therefore provides a localized signal to attenuate presynaptic differentiation (Liao, 2004).
Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and arises from cells of the developing sympathoadrenergic lineage. Activating mutations in the gene encoding the ALK tyrosine kinase receptor predispose for NB. This study focused on the normal function of Alk signaling in the control of sympathetic neuron proliferation, as well as on the effects of mutant ALK. Forced expression of wild-type ALK and NB-related constitutively active ALK mutants in cultures of proliferating immature sympathetic neurons results in a strong proliferation increase, whereas Alk knockdown and pharmacological inhibition of Alk activity decrease proliferation. Alk activation upregulates NMyc and trkB and maintains Alk expression by an autoregulatory mechanism involving Hand2. The Alk-ligand Midkine (Mk) is expressed in immature sympathetic neurons and in vivo inhibition of Alk signaling by virus-mediated shRNA knockdown of Alk and Mk leads to strongly reduced sympathetic neuron proliferation. Taken together, these results demonstrate that the extent and timing of sympathetic neurogenesis is controlled by Mk/Alk signaling. The predisposition for NB caused by activating ALK mutations may thus be explained by aberrations of normal neurogenesis, i.e. elevated and sustained Alk signaling and increased NMyc expression (Reiff, 2011).
The mammalian kidney and male reproductive system are both derived from the intermediate mesoderm. The spatial and temporal expression of bone morphogenetic protein (BMP) 2 and BMP4 and their cognate receptor, activin like kinase 3 (ALK3), suggests a functional role for BMP-ALK3 signaling during formation of intermediate mesoderm-derivative organs. This study defined cell autonomous functions for Alk3 in the kidney and male gonad in mice with CRE-mediated Alk3 inactivation targeted to intermediate mesoderm progenitors (Alk3IMP null). Alk3-deficient mice exhibit simple renal hypoplasia characterized by decreases in both kidney size and nephron number but normal tissue architecture. These defects are preceded by a decreased contribution of Alk3-deleted cells to the metanephric blastema and reduced expression of Osr1 and SIX2, which mark nephron progenitor cells. Mutant mice are also characterized by defects in intermediate mesoderm-derived genital tissues with fewer mesonephric tubules and testicular Leydig cells, epithelial vacuolization in the postnatal corpus epididymis, and decreased serum testosterone levels and reduced fertility. Analysis of ALK3-dependent signaling effectors revealed lineage-specific reduction of phospho-p38 MAPK in metanephric mesenchyme and phospho-SMAD1/5/8 in the testis. Together, these results demonstrate a requirement for Alk3 in distinct progenitor cell populations derived from the intermediate mesoderm (Di Giovanni, 2011).
Search PubMed for articles about Drosophila Anaplastic lymphoma kinase
Bai, L. and Sehgal, A. (2015). Anaplastic lymphoma kinase acts in the Drosophila mushroom body to negatively regulate sleep. PLoS Genet 11: e1005611. PubMed ID: 26536237
Bazigou, E., Apitz, H., Johansson, J., Loren, C. E., Hirst, E. M., Chen, P. L., Palmer, R. H. and Salecker, I. (2007). Anterograde Jelly belly and Alk receptor tyrosine kinase signaling mediates retinal axon targeting in Drosophila. Cell 128: 961-975. Pubmed: 17350579
Bilsland, J. G., et al. (2008). Behavioral and neurochemical alterations in mice deficient in anaplastic lymphoma kinase suggest therapeutic potential for psychiatric indications. Neuropsychopharmacology 33: 685-700. PubMed Citation: 17487225
Buchanan, M. E. and Davis, R. L. (2010). A distinct set of Drosophila brain neurons required for neurofibromatosis type 1-dependent learning and memory. J. Neurosci. 28: 10135-10143. PubMed Citation: 20668197
Cheng, L. Y., Bailey, A. P., Leevers, S. J., Ragan, T. J., Driscoll, P. C. and Gould, A. P. (2011). Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila. Cell 146: 435-447. PubMed ID: 21816278
Di Giovanni, V., Alday, A., Chi, L., Mishina, Y. and Rosenblum, N. D. (2011). Alk3 controls nephron number and androgen production via lineage-specific effects in intermediate mesoderm. Development 138(13): 2717-27. PubMed Citation: 21613322
Gabay, L., Seger, R., Shilo, B.Z. (1997) MAP kinase in situ activation atlas during Drosophila embryogenesis. Development 124: 3535-3541. 9342046
Gouzi, J. Y., et al. (2011). The receptor tyrosine kinase Alk controls neurofibromin functions in Drosophila growth and learning. PLoS Genet. 7(9): e1002281. PubMed Citation: 21949657
Hugosson, F., Sjogren, C., Birve, A., Hedlund, L., Eriksson, T. and Palmer, R. H. (2014). The Drosophila Midkine/Pleiotrophin homologues Miple1 and Miple2 affect adult lifespan but are dispensable for Alk signaling during embryonic gut formation. PLoS One 9: e112250. PubMed ID: 25380037
Ishihara, T., et al. (2002). HEN-1, a secretory protein with an LDL receptor motif, regulates sensory integration and learning in Caenorhabditis elegans. Cell 109: 639-649. PubMed Citation: 12062106
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
Pecot, M. Y., Chen, Y., Akin, O., Chen, Z., Tsui, C. Y. and Zipursky, S. L. (2014). Sequential axon-derived signals couple target survival and layer specificity in the Drosophila visual system. Neuron 82: 320-333. PubMed ID: 24742459
Reiff, T., et al. (2011). Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition. Development 138(21): 4699-708. PubMed Citation: 21989914
Reiner, D. J., Ailion, M., Thomas, J. H. and Meyer, B. J. (2008.) C. elegans anaplastic lymphoma kinase ortholog SCD-2 controls dauer formation by modulating TGF-beta signaling. Curr Biol. 18: 1101-1109. PubMed Citation: 18674914
Rohrbough, J. and K. Broadie (2011). Anterograde Jelly belly ligand to Alk receptor signaling at developing synapses is regulated by Mind the gap. Development 137(20): 3523-33. PubMed Citation: 20876658
Rohrbough, J., Kent, K. S., Broadie, K. and Weiss, J. B. (2012). Jelly belly trans-synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture. Dev Neurobiol. Pubmed: 22949158
Shi, P., Lai, R., Lin, Q., Iqbal, A. S., Young, L. C., Kwak, L. W., Ford, R. J. and Amin, H. M. (2009). IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells. Blood 114: 360-370. PubMed ID: 19423729
Sousa-Nunes, R., Yee, L. L. and Gould, A. P. (2011). Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila. Nature 471: 508-512. PubMed ID: 21346761
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
Walker, J. A., Gouzi, J. Y., Long, J. B., Huang, S., Maher, R. C., Xia, H., Khalil, K., Ray, A., Van Vactor, D., Bernards, R. and Bernards, A. (2013). Genetic and functional studies implicate synaptic overgrowth and ring gland cAMP/PKA signaling defects in the Drosophila melanogaster neurofibromatosis-1 growth deficiency. PLoS Genet 9: e1003958. PubMed ID: 24278035
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Wolfstetter, G., Pfeifer, K., van Dijk, J. R., Hugosson, F., Lu, X. and Palmer, R. H. (2017). The scaffolding protein Cnk binds to the receptor tyrosine kinase Alk to promote visceral founder cell specification in Drosophila. Sci Signal 10(502). PubMed ID: 29066538
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date revised: 15 December 2015
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