jelly belly : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - jelly belly
Cytological map position - 48E9
Function - ligand
Symbol - jeb
FlyBase ID: FBgn0086677
Genetic map position - 2-
Classification - secretory signal sequence and LDL receptor repeat motif
Cellular location - secreted
Inductive interactions subdivide the Drosophila mesoderm into visceral, somatic, and heart muscle precursors. The muscle precursors form organs by executing tissue-specific migrations and cell fusions. jelly belly (jeb) is required for visceral mesoderm development. jeb encodes a secreted protein that contains an LDL receptor repeat. In jeb mutants, visceral mesoderm precursors form, but they fail to migrate or differentiate normally; no visceral muscles develop. Jeb protein is produced in somatic muscle precursors and taken up by visceral muscle precursors. jeb reveals a signaling process in which somatic muscle precursors support the proper migration and differentiation of visceral muscle cells. Later in embryogenesis, jeb is transcribed in neurons and Jeb protein is found in axons (Weiss, 2001).
A screen was performed to identify genes that are transcriptionally regulated by the homeodomain protein Tinman (Tin). Tin, a member of the NK family of homeodomain proteins, is required for organogenesis of the embryonic heart and visceral mesoderm. It is one of a number of transcription factors whose functions in mesoderm development are conserved from insects to mammals. The screening method relies on genetic selection in yeast for a protein-DNA interaction. A library was screened that represents 15% of the Drosophila genomic DNA and six DNA fragments were obtained that satisfied genetic criteria in yeast for Tin binding sites. Most of the genomic DNA fragments were isolated multiple times. Sequence analysis has confirmed the presence of core recognition sites for NK class homeodomains in all of the fragments. To show that these fragments function as Tin-responsive enhancers in vivo, it was asked if they could drive expression of a reporter gene in patterns consistent with Tin regulation (Weiss, 2001).
The screen is surprisingly specific for genes regulated by Tinman (or closely related genes), as demonstrated both by the reporter-construct results and the genes that are located adjacent to the Tinman binding sites. Four fragments identified in the screen were inserted upstream of a lacZ reporter. Three of the four reporter constructs, tested as transgenes, are active in patterns consistent with Tin regulation. One fragment lies adjacent to jelly belly (jeb), a gene expressed in ventral, early mesoderm. The Tin binding site that led to the identification of jeb contains two Tin/NK2 class homeodomain recognition sites oriented as an imperfect inverted repeat. This genomic fragment was mapped to interval 48E9 of polytene chromosome 2R by in situ hybridization and based on the Drosophila genome sequence. The Tin binding sites lie adjacent to a P element insertion within a large intron of the jeb gene (Weiss, 2001).
Analysis of the jeb mutant phenotype reveals that jeb is required for visceral mesoderm development, but not for somatic muscle, fat body, or hemocyte development. To understand how Jeb might function biochemically, it was determined where, within the mesoderm, jeb is expressed in relation to early visceral mesoderm. jeb is clearly expressed in ventral and medial mesoderm immediately adjacent to the visceral mesoderm cells that depend on Jeb function. The cells that express jeb are somatic muscle precursors. jeb mRNA is initially produced in clusters of cells ventral to clusters of bagpipe-expressing cells. At stage 10, jeb-expressing cells surround the visceral mesoderm and fill in the gaps between the clusters of bap expression. By mid stage 11, jeb- and bap-expressing cells lie in juxtaposed layers (Weiss, 2001).
The signal sequence and LDL receptor repeat predicted in Jeb protein imply that Jeb is secreted from somatic mesoderm precursor cells and acts in the extracellular compartment. Specific Jeb antisera were used to monitor a possible Jeb signal from somatic to visceral mesoderm precursors. The antisera do not stain embryos homozygous for the P element excision allele. bap-expressing visceral mesoderm precursor cells that are dependent on jeb function, but do not transcribe jeb, clearly contain Jeb protein (Weiss, 2001).
Jeb protein is secreted from tissue culture cells. Extracts of Drosophila tissue culture cells producing Jeb were compared to protein found in their medium. The bulk of the Jeb protein was found outside the cells. The secreted protein migrates as a broad band. Thus, Jeb protein is clearly detectable in the culture medium, evidently in a posttranslationally modified form (Weiss, 2001).
The P element that is integrated into the jeb locus interrupts the transcription unit in a large intron. Transcription of jeb upstream of the integration site should produce a protein of about 50 kDa. In mutant embryos, affinity-purified sera detect a truncated Jeb protein with an apparent molecular weight of 45 kDa. The predicted mutant protein would contain the secretory signal sequence but not the type A LDL receptor repeat. Antibody stains of jeb mutant embryos reveal two notable differences with respect to wild-type protein distribution: (1) the truncated, mutant protein accumulates to lower levels than wild-type protein; (2) visceral mesoderm precursors do not take up the truncated protein. The only detectable protein in mutant embryos is in or adjacent to the cells that make it. The type A LDL receptor repeat, missing from the mutant protein, thus appears to be necessary for Jeb function (Weiss, 2001).
The pattern of Jeb protein staining in the visceral mesoderm is qualitatively different from the staining observed in the Jeb-producing cells. It is exclusively punctate, in contrast to the diffuse staining observed in Jeb secreting cells. The punctate staining pattern suggests receptor-mediated endocytosis as a mechanism for Jeb accumulation in visceral mesoderm cells. To test this hypothesis, a temperature-sensitive allele of the gene shibire was used. shibire encodes a dynamin-related GTPase that is required for microtubule-mediated endocytosis. In shibire temperature-sensitive mutant embryos, raised at the nonpermissive temperature during the period of Jeb secretion and uptake, reduced or absent association of Jeb with the visceral mesoderm is found. This demonstrates that Shibire-mediated endocytosis is required for Jeb to accumulate in visceral mesoderm. It also suggests that a specific Jeb receptor may be required for uptake by the visceral mesoderm (Weiss, 2001).
Though Jeb protein is secreted from somatic muscle precursors and taken up by visceral muscle precursors, Jeb might act in somatic muscle precursors to produce a signal that is not Jeb. This possibility was ruled out by expressing Jeb in visceral muscle precursors in a jeb mutant background. Production of Jeb in the visceral mesoderm of mutants rescues early visceral mesoderm development. Robust Fas3 staining is restored in the visceral mesoderm of these rescued, mutant embryos. Despite the restoration of Fas3 production, subsequent visceral mesoderm migration is frequently abnormal. Longitudinal migration to form continuous bands is incomplete, resulting in gaps in the pattern of Fas3. Expression of Jeb in the visceral mesoderm is sufficient to rescue the differentiation and, to a lesser extent, migration, of visceral mesoderm precursors (Weiss, 2001).
The migration defect observed in the rescue experiment could mean that the normal location of the Jeb source conveys positional information to visceral muscle precursors. Consistent with this hypothesis, misexpression of jeb in the visceral mesoderm of jeb heterozygotes produces visceral mesoderm defects. Fas3 expression in these embryos is frequently discontinuous, in contrast to the linear expression in jeb heterozygotes in the absence of Jeb misexpression. These results show that jeb misexpression is sufficient to perturb the migration of visceral muscle precursors and support the model of Jeb functioning as a signal (Weiss, 2001).
The data are consistent with Jeb functioning primarily in visceral mesoderm migration, but it may also be required for visceral mesoderm differentiation. When jeb mutants were rescued by producing Jeb in discrete clusters of visceral mesoderm cells, local rescue of differentiation and subsequent gaps in the normally continuous, longitudinal bands of Fas3 expression were observed; this is presumably a defect in migration. This is consistent with ectopic jeb in discrete clusters of visceral mesoderm cells in a nonmutant embryo causing longitudinal gaps in the visceral mesoderm. The result is most readily explained if Jeb acts as a positive, positional cue for visceral mesoderm migration. An alternative would be that Jeb provides a permissive differentiation function necessary for migration (Weiss, 2001).
Jeb has a single LDL receptor repeat. LDL receptor repeats are found in several functional classes of proteins. One large class consists of a group of receptors and coreceptors (reviewed in Cooper, 1999; Tamai, 2000; Wehrli, 2000). All these proteins, many of which function cell autonomously in signaling systems, have transmembrane and intracellular domains. The absence of a transmembrane domain from Jeb, its non-cell-autonomous phenotype, and its translocation from synthesizing to responding cells argue against a similar receptor function for Jeb (Weiss, 2001).
Some secreted proteases and protease inhibitors contain LDL receptor repeats. The Drosophila protein Nudel, a secreted protease that carries out one step of a localized, signaling, protease cascade, contains an LDL receptor repeat that is highly related to the one in Jeb. Though Jeb has no apparent similarity to known proteases or protease inhibitors other than the type A LDL receptor repeat, it is possible that Jeb acts through a second, unknown signaling protein or protease (Weiss, 2001).
A mammalian protein, the 8D6 antigen, structurally resembles Jeb in that it is secreted and contains two LDL receptor repeats. 8D6 is synthesized in follicular dendritic cells of the immune system and stimulates germinal center B cell proliferation. 8D6 may function as a signal from follicular dendritic cells to B cells in immune responses (Li, 2000; Weiss, 2001).
One other well-characterized LDL receptor repeat-containing protein may be functionally related to Jeb -- the product of the C. elegans gene Mig-13, which, like Jeb, contains a single LDL receptor repeat (Sym, 1999). Structurally, Mig-13 differs from Jeb in that it contains both a CUB and a transmembrane domain not found in Jeb. Mig-13 function, however, resembles Jeb in two notable ways: (1) Mig-13 is required non-cell-autonomously, like Jeb; (2) Mig-13 is a positive migratory factor necessary for anterior migration of developing neurons in C. elegans, a function similar to Jeb's. Mig-13 is produced locally along the anterior-posterior body axis under the control of specific Hox genes, and appears to guide migrations in a concentration-dependent manner (Weiss, 2001).
Whether Jeb signaling is conserved in evolution is not simple to determine. Outside the LDL receptor repeat, a motif shared by a number of extracellular proteins, no unambiguous vertebrate Jeb homologs have been identified in the public databases. Either the LDL receptor repeat is the essential functional, and therefore conserved, portion of Jeb, or Jeb signaling is not widespread in the animal kingdom. The former hypothesis if favored because every known signaling system in Drosophila has also been found in vertebrates. The sequence of Sco-Spondin, a secreted protein, is significantly similar to Jeb (Gobron, 1996). Jeb signaling may therefore be an evolutionarily conserved process, a possibility that is now being investigated using the mouse Sco-spondin gene (Weiss, 2001).
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).
jelly belly (jeb) was identified as a putative Tinman target gene. jeb expression in tin mutant embryos is scarcely different from wild-type, though it may be somewhat reduced. Tin activation of jeb transcription is likely to be redundant with other regulators of mesoderm development. To test the sufficiency of Tin for activating jeb, embryos in which tin was ectopically expressed were assessed for ectopic jeb expression. Misexpression of tin in the ectoderm with an engrailed GAL4 driver does not alter jeb expression. Misexpression of tin throughout the mesoderm is sufficient to activate jeb expression at a late time (stage 12) when it is not expressed in wild-type embryos, and in cells where jeb is not normally expressed. A cofactor in the mesoderm may be required for Tin-mediated activation of jeb transcription. The expression domains of tin and jeb imply that Tin's role in the regulation of jeb is restricted to the earliest stages of jeb expression, since at late stage 10, Tin is only in dorsal mesoderm and Jeb is in ventral mesoderm (Weiss, 2001).
The ability of Tin to activate jeb transcription ectopically in the mesoderm implies that Tin plays an early and redundant function in the regulation of jeb. Other regulators that may play roles in the regulation of jeb include the bHLH protein Twist and the Pax domain protein Pox Meso (Weiss, 2001).
Fragile X syndrome (FXS), the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1) gene product (FMRP), an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1) null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs): GPI-anchored glypican Dally-like protein (Dlp) and transmembrane Syndecan (Sdc). Earlier work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg) ligand abundance and downstream Frizzled-2 (Fz2) receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb), and downstream ERK phosphorylation (dpERK) are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb) and downstream signaling via phosphorylation of the transcription factor MAD (pMAD) seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1) Wg and Jeb trans-synaptic signaling, and (2) synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state (Friedman, 2013).
FXS is widely considered a disease state arising from synaptic dysfunction, with pre- and postsynaptic defects well characterized in the Drosophila disease model. There has been much work documenting FXS phenotypes in humans as well as in animal models, but there has been less progress on mechanistic underpinnings. This study focuses on the extracellular synaptomatrix in FXS owing to identification of pharmacological and genetic interactions between FMRP and secreted MMPs, a mechanism that is conserved in mammals. Other studies have also highlighted the importance of the synaptomatrix in synaptogenesis, particularly the roles of membrane-anchored HSPGs as co-receptors regulating trans-synaptic signaling. Importantly, it has been shown that FMRP binds HSPG mRNAs, thereby presumably repressing translation. Based on these multiple lines of evidence, this study hypothesized that the FMRP-MMP-HSPG intersection provides a coordinate mechanism for the pre- and postsynaptic defects characterizing the FXS disease state, with trans-synaptic signaling orchestrating synapse maturation across the synaptic cleft (Friedman, 2013).
In testing this hypothesis, a dramatic upregulation of GPI-anchored glypican Dlp and transmembrane Sdc HSPGs was discovered at dfmr1 null NMJ synapses. Indeed, these are among the largest synaptic molecular changes reported in the Drosophila FXS disease model. Importantly, HSPGs have been shown to play key roles in synaptic development. For example, the mammalian HSPG Agrin has long been known to regulate acetylcholine receptors, interconnected with a glycan network modulating trans-synaptic signaling. In Drosophila, Dlp, Sdc and Perlecan HSPGs mediate axon guidance, synapse formation and trans-synaptic signaling. Previous work on dlp mutants reports elevated neurotransmission, paradoxically similar to the Dlp overexpression phenotype shown in this study. However, the previous study does not show Dlp overexpression electrophysiological data, although it does show increased active zone areas consistent with strengthened neurotransmission. The same study reports that Dlp overexpression decreases bouton number on muscle 6/7, which differs from finding in this study of increased bouton number on muscle 4. Because HSPG co-receptors regulate trans-synaptic signaling, dfmr1 mutants were tested for changes in three established pathways at the Drosophila NMJ. Strong alterations in both Wg and Jeb pathways were found, with anterograde signaling being downregulated in both cases. In contrast, no change was found in the retrograde BMP Gbb pathway, suggesting that FMRP plays specific roles in modulating anterograde trans-synaptic signaling during synaptogenesis (Friedman, 2013).
The defect in Jeb signaling seems to be simple to understand, with decreased synaptomatrix ligand abundance coupled to decreased dpERK nuclear localization. However, there is no known link to HSPG co-receptor regulation. It has been shown earlier that Jeb signaling is regulated by another synaptomatrix glycan mechanism, providing a clear precedent for this level of regulation. In contrast, the Wnt pathway exhibits an inverse relationship between Wg ligand abundance (elevated) and Fz2-C nuclear signaling (reduced). This apparent contradiction is explained by the dual activity of the Dlp co-receptor, which stabilizes extracellular Wg to retain it at the membrane, but also competes with the Fz2 receptor. This ‘exchange-factor mechanism’ is competitively dependent on the ratio of Dlp co-receptor to Fz2 receptor, with a higher ratio causing more Wg to be competed away from Fz2. Indeed, it has been demonstrated that the same elevated Wg surface retention couples to decreased downstream Fz2-C signaling in an independent HSPG regulative mechanism at the Drosophila NMJ. This study suggests that in the dfmr1 null synapse, highly elevated Dlp traps Wg, thereby preventing it from binding Fz2 to initiate signaling (Friedman, 2013).
Dysregulation of the Wg nuclear import pathway (FNI) provides a plausible mechanism to explain synapse development defects underlying the FXS disease state, with established roles in activity-dependent modulation of synaptic morphogenesis and neurotransmission. FXS has long been associated with defects in activity-dependent architectural modulation, including postsynaptic spine formation, synapse pruning and functional plasticity. Although it is surely not the only player, aberrant Wg signaling could play a part in these deficiencies. Importantly, it has been shown that the FNI pathway is involved in shuttling large RNA granules out of the postsynaptic nucleus, providing a potential intersection with the FMRP RNA transport mechanism. However, the Wg FNI pathway is not the only Wnt signaling at the Drosophila NMJ, with other outputs including the canonical, divergent canonical and planar cell polarity pathways, which could be dysregulated in dfmr1 nulls. For example, a divergent canonical retrograde pathway proceeds through GSK3β (Shaggy) to alter microtubule assembly, and the FXS disease state is linked to dysregulated GSK3β and microtubule stability misregulation via Drosophila Futsch/mammalian MAP1B. Moreover, it has been shown that the secreted HSPG Perlecan (Drosophila Trol) regulates bidirectional Wnt signaling to affect Drosophila NMJ structure and/or function, via anterograde FNI and retrograde divergent canonical pathways. It is also important to note that previous studies show that a reduction in the FNI pathway, due to decreased Fz2-C trafficking to the nucleus, leads to decreased NMJ bouton number. Future work is needed to fully understand connections between FMRP, HSPGs, the multiple Wnt signaling pathways and the established defects in the synaptic microtubule cytoskeleton in the FXS disease state (Friedman, 2013).
Adding to the complications of FXS trans-synaptic signaling regulation, it was shown that two trans-synaptic signaling pathways are suppressed in parallel: the Wg and Jeb pathways. Possibly even more promising for clinical relevance, it has been established that the Jeb signaling functions as a repressor of neurotransmission strength at the Drosophila NMJ, with jeb and alk mutants presenting increased evoked synaptic transmission. Consistently, loss of FMRP leads to increased EJC amplitudes, which could be due, at least partially, to misregulated Jeb-Alk signaling. Importantly, it has been shown that dfmr1 null neurotransmission defects are due to a combination of pre- and postsynaptic changes, and that there is a non-cell-autonomous requirement for FMRP in the regulation of functional changes in the synaptic vesicle (SV) cycle underlying neurotransmission strength. Additionally, jeb and alk mutants exhibit synaptic structural changes consistent with this FMRP interaction, including a larger NMJ area and synaptic bouton maturation defects, which are markedly similar to the structural overelaboration phenotypes of the FXS disease state. These data together suggest that altered Jeb-Alk trans-synaptic signaling plays a role in the synaptic dysfunction characterizing the dfmr1 null. The study proposes that Wg and Jeb signaling defects likely interact, in synergistic and/or antagonistic ways, to influence the combined pre- and postsynaptic alterations characterizing the FXS disease state (Friedman, 2013).
Although trans-synaptic signaling pathways, and in particular both Wnt and Jeb-Alk pathways, have been proposed to be involved in the manifestation of a number of neurological disorders, this study provides the first evidence that aberrant trans-synaptic signaling is causally involved in an FXS disease model. The study proposes a mechanism in which FMRP acts to regulate trans-synaptic ligands by depressing expression of membrane-anchored HSPG co-receptors. HSPG overexpression alone is sufficient to cause both synaptic structure and function defects characterizing the FXS disease state. Increasing HSPG abundance in the postsynaptic cell is enough to increase the number of presynaptic branches and synaptic boutons, as well as elevate neurotransmission. Correlation with these well-established dfmr1 null synaptic phenotypes suggests that HSPG elevation could be a causal mechanism. Conclusively, reversing HSPG overexpression in the dfmr1 null is sufficient to correct Wnt and Jeb signaling, and to restore normal synaptic structure and function. Because there is no dosage compensation, HSPG heterozygosity offsets the elevation caused by loss of dfmr1. Correcting both Dlp and Sdc HSPGs in the dfmr1 background restores Wg and Jeb signaling to control levels. Correcting Dlp levels by itself restores synaptic architecture, but both Dlp and Sdc have to be corrected to restore normal neurotransmission in dfmr1 null synapses. Taken together, these results from the Drosophila FXS disease model provide exciting new insights into the mechanisms of synaptic phenotypes caused by the loss of FMRP, and promising avenues for new therapeutic treatment strategies (Friedman, 2013).
The pattern of jeb transcription was determined by whole-mount in situ hybridization to embryos. jeb mRNA is first detectable at stage 8 in repeated, segmental clusters of ventral mesoderm cells. These cells are precursors of somatic muscle. Subsequently, jeb is transcribed in two roughly parallel, continuous bands in the ventral mesoderm. At stage 12, jeb mRNA is no longer detectable in the mesoderm (Weiss, 2001).
Developmental signals often play multiple roles. jeb appears to function as a novel signal and, like other signals, is likely to be employed in multiple contexts. At stage 16, jeb mRNA is detected in a subset of embryonic neurons that are distributed throughout the ventral nerve cord. Jeb protein appears in a small set of longitudinal axons of the CNS as well as some lateral axons that exit to the PNS. Jeb signaling in the CNS and PNS may be used for communication among a restricted group of neurons (Weiss, 2001).
In the P element-induced jeb mutant, the protein distribution is strikingly different from wild-type. In the jeb mutants, the protein distribution resembles the pattern of mRNA expression. By extrapolation from the mesoderm results, the altered protein distribution in jeb mutants implies that the axonal staining observed in wild-type embryos represents transport of the protein in neurons that have taken up the protein, as opposed to Jeb secreting cells. This signal transport resembles that observed for Hh in the developing eye (Weiss, 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).
Two alleles of jeb have been isolated: the P element transposon insertion that interrupts the jeb transcription unit and a P element excision derivative from the insertion line that results in loss of detectable jeb mRNA and Jeb protein. Both mutations cause a recessive lethal phenotype. The phenotypes of the two alleles are indistinguishable from each other and from a heterozygote with the excision allele in trans to the original P element allele. The mutant phenotype can be rescued by driving expression of a jeb cDNA transgene in mutant embryos. These results demonstrate that the phenotype is attributable solely to loss of Jeb function (Weiss, 2001).
A thin layer of mesoderm overlying the yolk in the gut of wild-type embryos as revealed by anti-myosin heavy chain antibody staining. In jeb mutants, no differentiated visceral mesoderm is detectable. Other muscular components of the mesoderm, the somatic muscles and dorsal vessel or heart, and other mesoderm tissues, fat body and hemocytes, develop normally in jeb mutants (Weiss, 2001).
Only differentiated muscle contains myosin heavy chain, so the jeb mutation could affect differentiation or a prior step in visceral mesoderm development. To look at earlier stages, jeb mutant embryos were stained with an antibody against D-Mef2. D-Mef2 is produced early in all muscle lineages of the Drosophila embryo. Early D-mef2 expression is normal in the mesoderm of jeb mutants. Later, D-Mef2 staining in visceral mesoderm is absent, though the somatic mesoderm makes D-Mef2 normally (Weiss, 2001).
Endoderm development in jeb mutants is not primarily or severely affected. Antibodies against Hindsight protein, a marker of midgut endoderm, were used to follow endoderm development in jeb mutants. Despite the absence of visceral mesoderm, the endoderm is specified normally and migrates to form two longitudinal bands. Subsequent dorsal and ventral endoderm migration is abnormal in jeb mutants, presumably because dorsal and ventral migration depends on the visceral mesoderm (Weiss, 2001).
Specification of visceral mesoderm requires Decapentaplegic (Dpp) and Hedgehog (Hh), produced by the overlying ectoderm, to induce Bagpipe (Bap), a homeodomain protein related to Tin, in the precursors. Bap protein accumulates normally in jeb mutants. In wild-type embryos during stage 11, bap-expressing cells, initially specified as segmentally repeated, discrete clusters, commence midgut morphogenesis by migrating longitudinally to form two parallel continuous bands. In jeb mutants, bap-expressing cells fail to migrate normally to form these two continuous bands. Instead, the cells persist as discrete clusters through the end of stage 11. Shortly after the longitudinal migration of the bap-expressing cells, Bap protein decays, and Fas3, a mid-stage marker of visceral mesoderm, is produced. Fas3, a structural protein, is at this stage made only in the visceral part of the mesoderm, and is a useful marker of early differentiation. In jeb mutants, Fas3 is weakly and transiently produced. At the germ band retraction stage, when Fas3 production is robust in wild-type embryos, it is absent in jeb mutants (Weiss, 2001).
jeb is transcribed in somatic mesoderm cells, yet Bap staining shows that visceral mesoderm precursors form but fail to migrate normally in the absence of jeb function. There is no evidence of visceral mesoderm in jeb mutants after stage 11, so what becomes of the bap-expressing cells? They could undergo programmed cell death. Transcription patterns of three genes that serve as markers of apoptosis (grim, hid, and reaper) are the same in jeb mutants as in wild-type embryos. TUNEL staining confirmed the result; no evidence of increased programmed cell death was found (Weiss, 2001).
Since the cells in question do not express known markers of visceral mesoderm, it is difficult to follow their fates in jeb mutants. D-Mef2 stains of jeb mutant embryos show increased numbers of nuclei in positions consistent with an increase in somatic muscle precursors. Anti-myosin staining of jeb mutants shows that no major disruption of somatic muscle patterning occurs in jeb mutants. In jeb mutants, the visceral mesoderm precursor cells may default to a somatic mesoderm fate and become incorporated into the normal somatic muscle pattern, as in bap mutants (Weiss, 2001).
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).
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).
Search PubMed for articles about Drosophila jelly belly
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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
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Friedman, S.H., Dani, N., Rushton, E. and Broadie, K. (2013). Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila. Dis Model Mech 6: 1400-1413. PubMed ID: 24046358
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Ishihara, T., Y. Iino, et al. (2002). HEN-1, a secretory protein with an LDL receptor motif, regulates sensory integration and learning in Caenorhabditis elegans. Cell 109(5): 639- 49. PubMed Citation: 12062106
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
Lorén, C. E., Scully, A., Grabbe, C., Edeen, P. T., Thomas, J., McKeown, M., Hunter, T. and Palmer, R. H. (2001). Identification and charakterization 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
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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
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
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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
Wehrli, M., et al. (2000). arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 407: 527-530. 11029006
Weiss, J. B., Suyama, K. L., Lee, H.-H. and Scott, M. P. (2001). Jelly belly: A Drosophila LDL receptor repeat-containing signal required for mesoderm migration and differentiation. Cell 107: 387-398. 11701128
date revised: 10 June 2015
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