Gene name - thickveins
Synonyms - Dtfr
Cytological map position - 25D5-E1
Function - receptor kinase
Symbol - tkv
Genetic map position - 2-16
Classification - receptor tyrosine-kinase
Cellular location - surface
Thickveins is a receptor for the crucial Decapentaplegic morphogen. Decapentaplegic is responsibe for induction of dorsal-ventral polarity in the fly. Imagine the affects of its removal: the back of the fly fails to develop and instead develops into a neurogenic ectoderm resembling that usually found in the ventral portion of the trunk. The decapentaplegic pathway is also central to the establishment of segmentation and the morphogenesis of wing and eye.
The receptors for Decapentaplegic (Thickveins, Saxophone and Punt) mediate the transduction of DPP signals into the cell. Mutations in these genes have the same effect as mutation in dpp, since without DPP's receptors, its signals fail to be communicated into the cells that should receive them. Both TKV and SAX have similar structures, but they are only as closely related to each other as they are to their mammalian homologs. Both TKV and SAX require interaction with Punt to carry out their signaling process. For a discussion of the homology of TKV, SAX and Punt with mammalian TGF-beta receptors see the Punt site.
schnurri and mothers against dpp are the only identified genes located immediately downstream of the DPP receptor, a seeming paradox since this is a multi-potent, pervasive signaling pathway with ramifications for the expression of homeotic genes Ultrabithorax and labial, secreted proteins including Wingless and Decapentapletic, and other transcription factors like Pannier and Tinman.
SAX appears to be expressed more ubiquitously but required less ubiquitously than Thickveins. SAX also requires the function of both Thickveins and Punt (Wharton, 1995, and Ruberte, 1995). Thus, the primary receptors appear to be encoded by Punt and TKV.
Multiple BMPs are required for growth and patterning of the Drosophila wing. The Drosophila BMP gene, Tgfbeta-60A, exhibits a requirement in wing morphogenesis distinct from that shown previously for dpp. Tgfbeta-60A mutants exhibit a loss of pattern elements in the wing, particularly those derived from cells in the posterior compartment, consistent with the Tgfbeta-60A mRNA and protein expression pattern. Individuals homozygous for null alleles of the Tgfbeta-60A gene, exhibit embryonic defects in gut morphogenesis and result in early larval lethality. Tgfbeta-60A alleles have been shown to genetically interact with mutations in BMP type I receptor genes, tkv and sax. The Dpp signal is mediated by two different BMP type I receptors, Tkv and Sax, during wing morphogenesis as well as during other stages of development The possibility of a genetic interaction between alleles of Tgfbeta-60A and alleles of tkv or sax was investigated to address the relative importance of these receptors in mediating the signals resulting from the actions of Tgfbeta-60A and Dpp. Recombinants were constructed between gbb-60A 4 or gbb-60A 1 and several alleles of tkv and sax. The addition into a Tgfbeta-60A mutant background of a chromosomal deficiency that removes the tkv locus, results in a severe mutant wing phenotype with a dramatic loss of both the PCV and ACV and most of L4 and L5. In addition, distal gaps are present in L2 and L3. A less extreme phenotype is seen with tkv6, a hypomorphic allele that retains significant receptor function. The observed interaction between tkv and Tgfbeta-60A cannot be explained solely as a secondary consequence of lowering Dpp signaling readout by the mutation of a receptor that mediates Dpp signaling (Khalsa, 1998).
These data suggest that Tkv is able to mediate Tgfbeta-60A signaling and that it may do so in different ways at different times during development. The effect of reducing the Tgfbeta-60A copy number was investigated in flies compromised for functional Tkv receptor. Reducing Tgfbeta-60A in a tkv mutant background produces a further thickening of wing veins. This result suggests that Tgfbeta-60A may play a role in vein differentiation itself and/or in the tkv/dpp feedback loop important in defining the boundaries of the vein. Genetic combinations used to investigate the potential interaction between Tgfbeta-60A and sax alleles indicate a reduction in viability for Tgfbeta-60A mutant genotypes containing a single copy of a sax null allele. This reduction in viability suggests that lowering both Tgfbeta-60A and sax compromises development. The wing phenotype of the few viable adults recovered is similar to a very severe Tgfbeta-60A mutant wing phenotype, with a substantial loss of L5, complete loss of the PCV and ACV and loss of half of L4. Clearly the levels of Tgfbeta-60A signaling and Sax function are dependent on one another (Khalsa, 1998).
Based on genetic analysis and expression studies, it has been concluded that Tgfbeta-60A must signal primarily as a homodimer to provide patterning information in the wing imaginal disc. Tgfbeta-60A and dpp genetically interact and specific aspects of this interaction are synergistic while others are antagonistic. It is proposed that the positional information received by a cell at a particular location within the wing imaginal disc depends on the balance of Dpp to Tgfbeta-60A signaling. Furthermore, the critical ratio of Tgfbeta-60A to Dpp signaling appears to be mediated by both Tkv and Sax type I receptors (Khalsa, 1998).
The Hedgehog and Decapentaplegic pathways have several well-characterized functions in the developing Drosophila compound eye, including initiation and progression of the morphogenetic furrow. Other functions involve control of cell cycle and cell survival as well as cell type specification. This study used the mosaic clone analysis of null mutations of the smoothened and thickveins genes (which encode the receptors for these two signals) both alone and in combination, to study cell cycle and cell fate in the developing eye. It is concluded that both pathways have several, but differing roles in furrow induction and cell fate and survival, but that neither directly affects cell type specification (Vrailas, 2006a).
Interestingly, though Hedgehog signaling is required for Decapentaplegic expression, the two pathways are not completely redundant. The data demonstrate that for some aspects of eye development, the two pathways have separable and independent functions, such as Hedgehog signaling regulation of rough expression and S phase of the second mitotic wave. However, both pathways have redundant roles in the apical constriction of the actin cytoskeleton and proper expression of elements of the Egfr/Ras and Notch/Delta signaling pathways as well as in cell fate specification, though neither pathway is required for differentiation. Finally, the Decapentaplegic pathway is epistatic to the Hedgehog pathway for G1 arrest in the furrow and G1, G2 and M phases of the second mitotic wave. These various ways in which the Hedgehog and Decapentaplegic pathways work together (or not) demonstrate the complexity of pathway integration for proper eye development (Vrailas, 2006a).
A strong effect of loss of Hedgehog signaling was seen on the morphology of cells in the furrow, and it is suggested consequentially, in the distribution of the Egfr and Notch receptors. This disruption of the localization of elements of other signaling pathways, which is enhanced by the additional loss of thickveins, may explain some of the phenotypes observed. For example, cells at the edges of smoothened and double mutant clones near wild type tissue are still able to enter S phase. The Notch/Delta pathway has been shown to regulate the G1/S transition of the second mitotic wave with loss of pathway activity leading to a loss of S phase. Therefore, it may be that Notch/Delta signaling between cells in the wild type tissue and in the clone, allows for the S phases seen at the edges of the clones, while in the center of clones, where the Notch/Delta pathway is disrupted, S phase is lost. Cell fate specification can still occur at the edges of smoothened thickveins double mutant clones. It may be that the furrow does not really pass through the double mutant clones, but some signal from outside the clone can still induce photoreceptor cell fate, at least close to the clone margins. This is likely to be Spitz/Egfr signaling, which is present but disrupted in smoothened clones, since this signal can induce photoreceptor fate ectopically even anterior to the furrow and without the formation of R8/founder cells (Vrailas, 2006a).
This study reports the roles of Hedgehog and Decapentaplegic signaling in eye development, however, these pathways are also instrumental for patterning and proliferation in the developing wing. Studies in the wing have shown that as in the eye, decapentaplegic expression is downstream of hedgehog, suggesting that these pathways may also rely on each other for proper wing development. Though smoothened and thickveins have no role in ommatidial cell fate, Hedgehog signaling is required for specification of intervein and vein territories in the central region of the wing, and Decapentaplegic signaling has been shown to be required for vein cell fate in the developing pupal wing (Vrailas, 2006a).
As in the eye, Hedgehog and Decapentaplegic signaling have been implicated in cell cycle regulation in the developing wing. Studies in the wing found that overexpression of the Hedgehog signal induces proliferation through upregulation of Cyclin D and Cyclin E, as well as specifically promotes S phase in the wing margin. FACS analysis of wing discs revealed that thickveins loss of function clones (tkv7) have a reduced number of cells in S phase and an increase in the number of cells in G1 phase. Additionally, inhibition of the Hedgehog signal results in decreased growth and cell proliferation rates, and loss of Decapentaplegic pathway signaling results in small clones, suggesting that these pathways are important in cell survival and/or proliferation in the wing (Vrailas, 2006a).
It appears that both tissues use Hedgehog signaling to promote S phase and possibly cell survival, since inhibiting Hedgehog signaling results in cell death in the eye and decreased growth in the wing. Additionally, the two tissues may use Hedgehog signaling to regulate the G1 phase, though this regulation may have subtle differences. In addition, Decapentaplegic signaling also appears to be necessary for proliferation in the developing eye and wing, though these tissues may use this signal to regulate the cell cycle differently. This is not surprising, since the developing third instar eye and wing discs may have fundamental differences in cell cycle regulation; the eye has a coordinated second mitotic wave and the wing does not. For example, the eye may utilize some factors that are not present in the wing disc to prevent the build up of too much Cyclin E. Therefore, Cyclin E levels are decreased in the eye but not in the wing. Additionally, thickveins appears to be responsible for G1 arrest in the furrow, while in the wing, G1 arrest in the zone of nonproliferating cells is mediated by Wingless signaling. However, it may be that the eye and wing regulate the cell cycle using Hedgehog and Decapentaplegic signaling in much the same way, but the techniques used to examine this phenomenon in the different tissues do not allow for a direct comparison of results. For example, it may be that FACS analysis is a more sensitive technique than immunohistochemistry, and thus subtle changes in the cell cycle that were observed in the wing were not observed in the eye. Alternatively, the FACS analysis was performed on wing discs that contained thickveins clones in a Minute background in order to achieve a larger sample of thickveins mutant cells. However, dying cells, such as those homozygous for Minute mutations, have been shown to have non-autonomous effects on the biology of the surrounding cells in the wing. Indeed, one study has reported that Minute mutations can non-autonomously affect pattering of photoreceptors in the developing eye. It may be that the Minute background partially masked the thickveins cell cycle phenotypes and the eye and wing may not be as different as it initially appears (Vrailas, 2006a).
The data also shows that the Hedgehog and Decapentaplegic pathways are only partially redundant in the eye, which has also been shown in the wing. Hedgehog signaling alone is required for specification of veins 3 and 4 and the sensory organ precursors (SOPs) near the anterior/posterior boundary of the developing wing, whereas Decapentaplegic signaling mediated by Hedgehog promotes some SOP formation in the notum and some other regions of the wing (Vrailas, 2006a).
In some instances, the data contrasts with previous reports from others. In one case, in which different alleles of smoothened (smo3 versus smoD16) were examined, phenotypic variation may be a result of allele specific effects. However, in another case, the same allele was used by two groups, and it may be that some other aspect of the genetic background of the stocks differed that influenced the results observed. The effects of removing a receptor (Smoothened) may also differ in some cases from those of removing a downstream element (Ci). It was also observed that clones the remove thickveins or smoothened and thickveins together often appear to be re-specified as other structures, resembling appendage discs. This may be due to other functions of the Decapentaplegic pathway on the disc margins and in defining the limits of the eye field. The interpretations of others may have been confounded by such re-specification in some cases. Indeed, in the developing wing, cells lacking Decapentaplegic pathway function actually leave the epithelium. Some care was taken to analyze only those small clones near the center of the eye field that do not have these characteristics. Indeed, the fact that photoreceptor specific markers were observed in some cells that lack both smoothened and thickveins demonstrates that even the double mutant clones do not always re-specify (Vrailas, 2006a and references therein).
In summary, it is concluded that the Hedgehog pathway has important roles in inducing furrow initiation and progression. The Hedgehog and Decapentaplegic pathways have redundant roles in actin constriction in the morphogenetic furrow, expression of Egfr, Notch and Delta, and differentiation with neither pathway essential for cell type specification. Likewise, no role was found for either Hedgehog or Decapentaplegic signaling in ommatidial rotation or chirality. It is also suggested that the Hedgehog pathway alone is required for rough expression and the G1/S transition in the second mitotic wave and provides a protective function against apoptosis. In contrast, the Decapentaplegic pathway appears critical for furrow initiation at the disc margins (but not progression in the center). In addition, the Decapentaplegic pathway is epistatic to Hedgehog signaling for maintenance of G1 arrest in the furrow and regulation of G1 phase and the G2/M transition in the second mitotic wave (Vrailas, 2006a).
The Drosophila Mitogen Activated Protein Kinase (MAPK) Rolled is a key regulator of developmental signaling, relaying information from the cytoplasm into the nucleus. Cytoplasmic MEK phosphorylates MAPK (pMAPK), which then dimerizes and translocates to the nucleus where it regulates transcription factors. In cell culture, MAPK nuclear translocation directly follows phosphorylation, but in developing tissues pMAPK can be held in the cytoplasm for extended periods (hours). This study shows that Moleskin antigen (Drosophila Importin 7/Msk), a MAPK transport factor, is sequestered apically at a time when lateral inhibition is required for patterning in the developing eye. It is suggested that this apical restriction of Msk limits MAPK nuclear translocation and blocks Ras pathway nuclear signaling. Ectopic expression of Msk overcomes this block and disrupts patterning. Additionally, the MAPK cytoplasmic hold is genetically dependent on the presence of Decapentaplegic (Dpp) and Hedgehog receptors (Vrailas, 2006b).
Early in eye development, all cells anterior to the furrow (phase 0) are primed for Ras-induced neural differentiation; ectopic activation of the pathway causes all cells to differentiate as photoreceptors, even without atonal. Normally these cells are thought to receive only low levels of Egfr-mediated Ras signaling, supporting proliferation but not differentiation. Later, in the furrow (phase 1), Delta-induced, Notch-mediated lateral inhibition progressively restricts Atonal expression to single founder cells. Suspension of Ras signaling is required for this inhibition in order to avoid premature neuronal differentiation, and it has been proposed that this inhibition is mediated by MAPK cytoplasmic hold. However, this block to the Ras pathway must be released in phase 2 (posterior to the furrow) to allow for developmental induction by the R8 cell. To better understand how MAPK cytoplasmic hold is maintained in phase 1, the role was examined of the pMAPK nuclear transport factor Drosophila Importin 7/Msk, in eye development (Vrailas, 2006b).
It is suggested that in wild-type eye discs, the level of pMAPK antigen is a very misleading reporter of Egfr/Ras pathway activity, because cytoplasmic hold in phase 1 allows even a relatively low level of pathway activity to build up high levels of pMAPK antigen. A system has been developed to reveal MAPK nuclear translocation without the use of an antibody (MG-driven reporter gene expression that reveals MAPK nuclear translocation). [Note: MG (Mapk-Gal4vp16) contains the entire sequence of Rolled, followed by the yeast GAL4 DNA binding domain (which is not known to contain a nuclear localization signal) with an acidic activation domain from herpes simplex virus protein 16]. However, it has been since found that under all conditions tested, MG-driven reporter expression does not reveal nuclear MAPK in phase 0, where Ras pathway activation is required. MG-driven reporter expression is reliably see in phase 2, where there is thought to be high (or sustained) levels of Ras pathway activity. In phase 1, the level of pathway signaling may be insufficient for expression, and thus MG-driven reporter expression may reveal only high (or sustained) levels of nuclear MAPK. Alternatively, this could be caused by a technical limitation: the hsp70 promoter drives the expression of only low levels of MG protein. Therefore, two less direct assays were used, that together, are interpreted as revealing the loss of MAPK cytoplasmic hold in the furrow: (1) loss of Atonal expression (as previously demonstrated by fusing an SV40 NLS to MAPK and by the ectopic expression of Rasv12); and (2) loss of pMAPK antigen, which may be due to exposure to a nuclear phosphatase/protease (Vrailas, 2006b).
The MAPK nuclear transport factor Drosophila Importin 7/Msk is apically sequestered in phase 1, the time when pMAPK nuclear access is blocked. Furthermore, ectopic Msk is sufficient to break the cytoplasmic hold in the furrow, as seen by loss of pMAPK antigen and suppression of the early stages of Atonal expression. However, this transient expression of Msk is unable to promote the precocious neural differentiation or the increase in rough expression, as has been seen with hs:rasv12 or nuclear-directed MAPK. Because ectopic rasv12 produces an increase in pMAPK, and the phosphorylation state of nuclear-directed MAPK is not required for nuclear translocation, it may be that the available pool of pMAPK that can be imported into the nucleus by Msk is enough to affect Atonal expression, but not to affect Elav or Rough expression. Genetic evidence shows that the MAPK cytoplasmic hold depends on the Hedgehog receptor Smo and is enhanced by the loss of the Dpp receptor Tkv. smo loss-of-function clones reduce Atonal and pMAPK expression, whereas tkv clones have much weaker effects. However, the loss of smo and tkv together completely abolishes both pMAPK and Atonal expression in the furrow. This is consistent with a previous report of the loss of Atonal expression in smo tkv clones. Additionally, MAPK cytoplasmic hold in smo tkv clones is rescued by the additional loss of msk. Thus, msk genetically antagonizes pMAPK levels in the morphogenetic furrow: msk gain-of-function reduces pMAPK and msk loss-of-function (in smo tkv clones) increases it (Vrailas, 2006b).
Hedgehog signaling has also been reported as a positive regulator of Atonal on the anterior side of the furrow and as a negative regulator (perhaps through Rough or Bar) on the posterior side. However, the inductive effect of Hedgehog on Atonal appears to be independent of the Hedgehog pathway transcription factor Ci, which is consistent with an indirect effect through the MAPK cytoplasmic hold. smo tkv msk triple mutant clones were used to show that msk is genetically epistatic to smo and tkv in the furrow, and suggest that Msk sequestration in the furrow is required for MAPK cytoplasmic hold, and that smo and tkv are genetically upstream of this sequestration of Msk. Indeed, loss of smo and tkv results in a disruption of the actin cytoskeleton in the furrow, as well as of expression of Egfr and other signaling molecules. The loss of apical constriction may therefore disrupt Msk apical sequestration in such a way as to allow precocious Msk-mediated pMAPK nuclear import (Vrailas, 2006b).
What is more surprising is that differentiation and ommatidial assembly, which are known to require Ras signaling and MAPK nuclear translocation, occur normally in the absence of Msk in phase 2. It may be that cytoplasmic MAPK targets are important for ommatidial assembly or that pMAPK can translocate into the nucleus by some Ran-independent mechanism. However, the possibility is favored that, in phase 2, other (possibly redundant) transport factors are expressed (Vrailas, 2006b).
Like the Ras pathway, msk plays a role in ommatidial rotation but not chirality. It may be that in the absence of Msk, enough pMAPK can translocate into the nucleus for ommatidial assembly, but not enough for proper rotation. Additionally, in phase 0, Msk is found to be required for proliferation, which also requires Ras signaling. Therefore, Msk is required for some pMAPK nuclear translocation in phase 0 and phase 2, but is not necessary in phase 1, in order to allow for the initial specification of the Atonal-positive R8 (Vrailas, 2006b).
To conclude, the apical sequestration of Drosophila Importin 7/Msk in the morphogenetic furrow has been identified and it is suggested that this may be required for the MAPK cytoplasmic hold in the developing eye. Cytoplasmic hold is required to allow initial patterning through lateral inhibition and the focusing of the proneural factor Atonal. It is further suggested that this is mediated by the combined action of Hedgehog and Dpp (Vrailas, 2006b).
The structural properties and expression patterns of TKV can be compared with the DPP receptor encoded by Saxophone. While the sax gene is expressed ubiquitously, tkv is expressed in a highly localized and dynamic pattern during development. Some, but not all, of the tkv expression pattern parallels that of dpp. Ubiquitous expression of a tkv transgene rescues both tkv and sax loss-of-function mutations. Thus, there is at least partial functional overlap of the SAX and TKV receptors in vivo (Brummel, 1994).
The serine-tyrosine kinase domains of the Drosophila and vertebrate receptors are 78% homologous, the two fly genes being no more closely related to one another than they are to their vertebrate homologs. The extracellular domains show resemblence only in the spacing of the cysteine residues (Nellen, 1994 and Xie, 1994).
date revised: 25 MAR 97
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.