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).
The cDNA sequences and developmental RNA blots of jeb demonstrate two size classes of transcripts derived from the jeb locus during early to mid embryogenesis. Later in embryogenesis, a third, larger, transcript is detected. The two early embryonic transcripts contain the same open reading frame. They differ only in 5' and 3' untranslated regions. The predicted protein product of the jeb locus contains a secretory signal sequence and a single LDL receptor repeat motif. In the region of the LDL receptor repeat, Jeb is most similar to two bovine proteins, Sco-spondin and enterokinase (Weiss, 2001).
Currently jeb corresponds to two predicted genes of the Berkeley Drosophila genome project, CG13180 and CG13182. The LDL receptor repeat is found in CG13183.
date revised: 15 December 2001
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