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).
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).
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).
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).
Cooper, J. A. and Howell, B. W. (1999). Lipoprotein receptors: signaling functions in the brain? Cell 97: 671-674. 10380917
Gobron, S., et al. (1996). SCO-spondin: a new member of the thrombospondin family secreted by the subcommissural organ is a candidate in the modulation of neuronal aggregation. J. Cell Sci. 109: 1053-1061. 8743952
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
Li, L., et al. (2000). Identification of a human follicular dendritic cell molecule that stimulates germinal center B cell growth. J. Exp. Med. 191: 1077-1084. 10727470
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
Sym, M., Robinson, N. and Kenyon, C. (1999). MIG-13 positions migrating cells along the anteroposterior body axis of C. elegans Cell 98: 25-36. 10412978
Tamai, K., et al. (2000). LDL-receptor-related proteins in Wnt signal transduction. Nature 407: 530-535. 11029007
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 December 2004
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.