thickveins: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - thickveins

Synonyms - Dtfr

Cytological map position - 25D5-E1

Function - receptor kinase

Keyword(s) - dpp pathway, dorsal-ventral polarity

Symbol - tkv

FlyBase ID:FBgn0003716

Genetic map position - 2-16

Classification - receptor tyrosine-kinase

Cellular location - surface

NCBI link: Entrez Gene

tkv orthologs: Biolitmine

Recent literature
Luo, L., Wang, H., Fan, C., Liu, S. and Cai, Y. (2015). Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche. J Cell Biol 209: 595-608. PubMed ID: 26008746
Stem cell self-renewal versus differentiation is regulated by the niche, which provides localized molecules that favor self-renewal. In the Drosophila melanogaster female germline stem cell (GSC) niche, Decapentaplegic (Dpp), a fly transforming growth factor β molecule and well-established long-range morphogen, acts over one cell diameter to maintain the GSCs. This study shows that Thickveins (Tkv; a type I receptor of Dpp) is highly expressed in stromal cells next to Dpp-producing cells and functions to remove excess Dpp outside the niche, thereby spatially restricting its activity. Interestingly, Tkv expression in these stromal cells was regulated by multiple Wnt ligands that were produced by the niche. These data demonstrate a self-restraining mechanism by which the Drosophila ovarian GSC niche acts to define its own boundary.

Inaba, M., Buszczak, M. and Yamashita, Y.M. (2015). Nanotubes mediate niche-stem-cell signalling in the Drosophila testis. Nature [Epub ahead of print]. PubMed ID: 26131929
Stem cell niches provide resident stem cells with signals that specify their identity. Niche signals act over a short range such that only stem cells but not their differentiating progeny receive the self-renewing signals. However, the cellular mechanisms that limit niche signalling to stem cells remain poorly understood. This study shows that the Drosophila male germline stem cells form previously unrecognized structures, microtubule-based nanotubes, which extend into the hub, a major niche component. Microtubule-based nanotubes are observed specifically within germline stem cell populations, and require intraflagellar transport proteins for their formation. The bone morphogenetic protein (BMP) receptor Tkv localizes to microtubule-based nanotubes. Perturbation of microtubule-based nanotubes compromises activation of Dpp signalling within germline stem cells, leading to germline stem cell loss. Moreover, Dpp ligand and Tkv receptor interaction is necessary and sufficient for microtubule-based nanotube formation. The study proposes that microtubule-based nanotubes provide a novel mechanism for selective receptor-ligand interaction, contributing to the short-range nature of niche-stem-cell signalling.

Li, W. et al. (2016). Angelman syndrome protein Ube3a regulates synaptic growth and endocytosis by inhibiting BMP signaling in Drosophila. PLoS Genet 12: e1006062. PubMed ID: 27232889
Altered expression of the E3 ubiquitin ligase UBE3A, which is involved in protein degradation through the proteasome-mediated pathway, is associated with neurodevelopmental and behavioral defects observed in Angelman syndrome (AS) and autism. However, little is known about the neuronal function of UBE3A and the pathogenesis of UBE3A-associated disorders. This study generated multiple mutations of ube3a, the Drosophila ortholog of UBE3A. A significantly increased number of total boutons and satellite boutons in conjunction with compromised endocytosis were found in the neuromuscular junctions (NMJs) of ube3a mutants compared to the wild type. Genetic and biochemical analysis showed upregulation of bone morphogenetic protein (BMP) signaling in the nervous system of ube3a mutants. An immunochemical study revealed a specific increase in the protein level of Thickveins (Tkv), a type I BMP receptor, but not other BMP receptors Wishful thinking (Wit) and Saxophone (Sax), in ube3a mutants. Ube3a is associated with and specifically ubiquitinated lysine 227 within the cytoplasmic tail of Tkv, and promotes its proteasomal degradation in Schneider 2 cells. Negative regulation of Tkv by Ube3a is conserved in mammalian cells. These results reveal a critical role for Ube3a in regulating NMJ synapse development by repressing BMP signaling. This study sheds new light onto the neuronal functions of UBE3A and provides novel perspectives for understanding the pathogenesis of UBE3A-associated disorders.

Zhang, X., Rui, M., Gan, G., Huang, C., Yi, J., Lv, H. and Xie, W. (2017). Neuroligin 4 regulates synaptic growth via the Bone morphogenetic protein (BMP) signaling pathway at the Drosophila neuromuscular junction. J Biol Chem [Epub ahead of print]. PubMed ID: 28912273
The neuroligin (Nlg) family of neural cell adhesion molecules is thought to be required for synapse formation and development, and has been linked to the development of autism spectrum disorders in humans. In Drosophila melanogaster, mutations in the neuroligin 1-3 genes have been reported to induce synapse developmental defects at neuromuscular junctions (NMJs), but the role of neuroligin 4 (dnlg4) in synapse development has not been determined. This study reports that the Drosophila Neuroligin 4 (DNlg4) is different from DNlg1-3 in that it presynaptically regulates NMJ synapse development. Loss of dnlg4 results in reduced growth of NMJs with fewer synaptic boutons. The morphological defects caused by dnlg4 mutant are associated with a corresponding decrease in synaptic transmission efficacy. All of these defects could only be rescued when DNlg4 was expressed in the presynapse of NMJs. To understand the basis of DNlg4 function, we looked for genetic interactions, and found connections with the components of the bone morphogenetic protein (BMP) signaling pathway. Immunostaining and western blot analyses demonstrated that the regulation of NMJ growth by DNlg4 was due to the positive modulation of BMP signaling by DNlg4. Specifically, BMP type I receptor Tkv abundance was reduced in dnlg4 mutants, and immunoprecipitation assays showed that DNlg4 and Tkv physically interacted in vivo. This study demonstrates that DNlg4 presynaptically regulates neuromuscular synaptic growth via the BMP signaling pathway by modulating Tkv.
Xu, R., Li, J., Zhao, H., Kong, R., Wei, M., Shi, L., Bai, G. and Li, Z. (2018). Self-restrained regulation of stem cell niche activity by niche components in the Drosophila testis. Dev Biol. PubMed ID: 29679558
Most, if not all, stem cells reside in a defined microenvironment, called the niche. Short-ranged niche signal must be tightly controlled to be active only inside the niche to maintain the proper balance of stem cell self-renewal verse differentiation. However, how niche components restrict localized niche signal activation remains largely unknown. This study finds that Thickveins (Tkv, a type I receptor of the Dpp signaling pathway) in cyst stem cells (CySCs) of the testis niche prevents Dpp signaling activation outside of the niche. Tkv functions as Dpp trap/sink to spatially restrain Dpp signaling inside the niche. This self-restrained regulation of niche activity by Tkv in CySCs is independent of the canonical Dpp signaling pathway. These data demonstrate the critical roles of niche components (CySCs) in the self-restrained regulation of niche activity, which could be shed light on niche activity regulation in general.
Tseng, C. Y., Su, Y. H., Yang, S. M., Lin, K. Y., Lai, C. M., Rastegari, E., Amartuvshin, O., Cho, Y., Cai, Y. and Hsu, H. J. (2018). Smad-independent BMP signaling in somatic cells limits the size of the germline stem cell pool. Stem Cell Reports. PubMed ID: 30122445
In developing organisms, proper tuning of the number of stem cells within a niche is critical for the maintenance of adult tissues; however, the involved mechanisms remain largely unclear. This study demonstrated that Thickveins (Tkv), a type I bone morphogenetic protein (BMP) receptor, acts in the Drosophila developing ovarian soma through a Smad-independent pathway to shape the distribution of BMP signal within the niche, impacting germline stem cell (GSC) recruitment and maintenance. Somatic Tkv promotes Egfr signaling to silence transcription of Dally, which localizes BMP signals on the cell surface. In parallel, Tkv promotes Hh signaling, which promotes escort cell cellular protrusions and upregulates expression of the Drosophila BMP homolog, Dpp, forming a positive feedback loop that enhances Tkv signaling and strengthens the niche boundary. These results reveal a role for non-canonical BMP signaling in the soma during GSC establishment and generally illustrate how complex, cell-specific BMP signaling mediates niche-stem cell interactions.
Monedero Cobeta, I., Stadler, C. B., Li, J., Yu, P., Thor, S. and Benito-Sipos, J. (2018). Specification of Drosophila neuropeptidergic neurons by the splicing component brr2. PLoS Genet 14(8): e1007496. PubMed ID: 30133436
During embryonic development, a number of genetic cues act to generate neuronal diversity. While intrinsic transcriptional cascades are well-known to control neuronal sub-type cell fate, the target cells can also provide critical input to specific neuronal cell fates. Such signals, denoted retrograde signals, are known to provide critical survival cues for neurons, but have also been found to trigger terminal differentiation of neurons. One salient example of such target-derived instructive signals pertains to the specification of the Drosophila FMRFamide neuropeptide neurons, the Tv4 neurons of the ventral nerve cord. Tv4 neurons receive a BMP signal from their target cells, which acts as the final trigger to activate the FMRFa gene. A recent FMRFa-eGFP genetic screen identified several genes involved in Tv4 specification, two of which encode components of the U5 subunit of the spliceosome: brr2 (l(3)72Ab) and Prp8. This study focused on the role of RNA processing during target-derived signaling. brr2 and Prp8 were found to play crucial roles in controlling the expression of the FMRFa neuropeptide specifically in six neurons of the VNC (Tv4 neurons). Detailed analysis of brr2 revealed that this control is executed by two independent mechanisms, both of which are required for the activation of the BMP retrograde signaling pathway in Tv4 neurons: (1) Proper axonal pathfinding to the target tissue in order to receive the BMP ligand. (2) Proper RNA splicing of two genes in the BMP pathway: the thickveins (tkv) gene, encoding a BMP receptor subunit, and the Medea gene, encoding a co-Smad. These results reveal involvement of specific RNA processing in diversifying neuronal identity within the central nervous system.
Chen, Z. (2019). The formation of the Thickveins (Tkv) gradient in Drosophila wing discs: A theoretical study. J Theor Biol. PubMed ID: 30998935
The development of wing imaginal disc is commonly adopted for the studies of patterning and growth which are two fundamental problems in developmental biology. Decapentaplegic (Dpp) signaling regulates several aspects of wing development, such as the anterior (A)-posterior (P) patterning, cellular growth rate, and cell adhesion. The distribution and activity of Dpp signaling are controlled in part by the expression level of its major type I receptor, Thickveins (Tkv). This paper focuses on theoretically investigating mechanisms by which the highly asymmetric pattern of Tkv is established in Drosophila wing disc. To that end, a mathematical model of Hh signaling and Dpp signaling is proposed and validated by comparisons with experimental observations. This model provides a comprehensive view of the formation of Tkv gradients in wing discs. It was found that engrailed (En), Hedgehog (Hh) signaling, and Dpp signaling cooperate to establish the asymmetric gradients of Tkv and pMad in the wing disc. Moreover, the model suggests a Brinker-mediated mechanism of Dpp-dependent repression of Tkv. Based on this mechanism, a couple of predicted experimental observations have been provided for further lab confirmation.
Chen, Z. and Zou, Y. (2019). Anterior-Posterior patterning of Drosophila wing disc I: A baseline mathematical model. Math Biosci. PubMed ID: 31085191
Wing imaginal disc of Drosophila is one of the commonly used model systems for the studies of patterning, growth, and scaling. Development of the wing disc involves many interacting components as well as a variety of compound processes whose underlying mechanisms are still under investigation. For instance, it remains unclear about how to form compound expersmentally-measured patterns of Decapentaplegic (Dpp) type-I receptor Thickveins (Tkv), as well as phosphorylated Mothers Against Dpp (pMad) which is the indicator of Dpp signaling activities. This work proposes a baseline mathematical model that integrates established experimental facts to investigate the formation of pMad and Tkv gradients. This model is validated by the accurate reproduction of complex asymmetric profiles of Tkv and pMad in both anterior and posterior compartments of wing disc. Moreover, using this model as a numerical platform, specific roles played by engrailed (En), Hedgehog (Hh) and Dpp were examined in the establishment of Tkv and pMad profiles. It turns out that all En, Hh, Dpp play an essential role in the formation of pMad and Tkv patterns. In particular, the proposed model supports the crucial part of the downregulation of Tkv by Dpp. Besides, dual negative regulations of Tkv by both Hh and Dpp simultaneously prevent the Dpp signaling from interfering and expand the effective range of Dpp gradients. Finally, parameter sensitivity was carried out to ensure that these results and conclusions are robust against specific choices of parameter values.
Tracy Cai, X., Li, H., Safyan, A., Gawlik, J., Pyrowolakis, G. and Jasper, H. (2019). AWD regulates timed activation of BMP signaling in intestinal stem cells to maintain tissue homeostasis. Nat Commun 10(1): 2988. PubMed ID: 31278345
Precise control of stem cell (SC) proliferation ensures tissue homeostasis. In the Drosophila intestine, injury-induced regeneration involves initial activation of intestinal SC (ISC) proliferation and subsequent return to quiescence. These two phases of the regenerative response are controlled by differential availability of the BMP type I receptor Thickveins (Tkv), yet how its expression is dynamically regulated remains unclear. This study shows that during homeostasis, the E3 ubiquitin ligase Highwire and the ubiquitin-proteasome system maintain low Tkv protein expression. After ISC activation, Tkv is stabilized by proteasome inhibition and undergoes endocytosis due to the induction of the nucleoside diphosphate kinase Abnormal Wing Disc (AWD). Tkv internalization is required for the activation of the Smad protein Mad, and for the return to quiescence after a regenerative episode. These data provide insight into the mechanisms ensuring tissue homeostasis by dynamic control of somatic stem cell activity.
Ladyzhets, S., Antel, M., Simao, T., Gasek, N., Cowan, A. E. and Inaba, M. (2020). Self-limiting stem-cell niche signaling through degradation of a stem-cell receptor. PLoS Biol 18(12): e3001003. PubMed ID: 33315855
Stem-cell niche signaling is short-range in nature, such that only stem cells but not their differentiating progeny receive self-renewing signals. At the apical tip of the Drosophila testis, 8 to 10 germline stem cells (GSCs) surround the hub, a cluster of somatic cells that organize the stem-cell niche. Previous work has shown that GSCs form microtubule-based nanotubes (MT-nanotubes) that project into the hub cells, serving as the platform for niche signal reception; this spatial arrangement ensures the reception of the niche signal specifically by stem cells but not by differentiating cells. The receptor Thickveins (Tkv) is expressed by GSCs and localizes to the surface of MT-nanotubes, where it receives the hub-derived ligand Decapentaplegic (Dpp). The fate of Tkv receptor after engaging in signaling on the MT-nanotubes has been unclear. This study demonstrates that the Tkv receptor is internalized into hub cells from the MT-nanotube surface and subsequently degraded in the hub cell lysosomes. Perturbation of MT-nanotube formation and Tkv internalization from MT-nanotubes into hub cells both resulted in an overabundance of Tkv protein in GSCs and hyperactivation of a downstream signal, suggesting that the MT-nanotubes also serve a second purpose to dampen the niche signaling. Together, these results demonstrate that MT-nanotubes play dual roles to ensure the short-range nature of niche signaling by (1) providing an exclusive interface for the niche ligand-receptor interaction; and (2) limiting the amount of stem cell receptors available for niche signal reception.

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).

Hedgehog and Decapentaplegic pathway function in the developing Drosophila compound eye: smoothened, thickveins and the genetic control of cell cycle and cell fate

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).

smoothened and thickveins regulate Moleskin/Importin 7-mediated MAP kinase signaling in the developing Drosophila eye

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).

Nanotubes mediate niche-stem-cell signalling in the Drosophila testis

Stem cell niches provide resident stem cells with signals that specify their identity. Niche signals act over a short range such that only stem cells but not their differentiating progeny receive the self-renewing signals. However, the cellular mechanisms that limit niche signalling to stem cells remain poorly understood. This study shows that the Drosophila male germline stem cells form previously unrecognized structures, microtubule-based nanotubes, which extend into the hub, a major niche component. Microtubule-based nanotubes are observed specifically within germline stem cell populations, and require intraflagellar transport proteins for their formation. The bone morphogenetic protein (BMP) receptor Tkv localizes to microtubule-based nanotubes. Perturbation of microtubule-based nanotubes compromises activation of Dpp signalling within germline stem cells, leading to germline stem cell loss. Moreover, Dpp ligand and Tkv receptor interaction is necessary and sufficient for microtubule-based nanotube formation. The study proposes that microtubule-based nanotubes provide a novel mechanism for selective receptor-ligand interaction, contributing to the short-range nature of niche-stem-cell signalling (Inaba, 2015).

The Drosophila testis represents an excellent model system to study niche-stem-cell interactions because of its well-defined anatomy: eight to ten germline stem cells (GSCs) are attached to a cluster of somatic hub cells, which serve as a major component of the stem cell niche. The hub secretes at least two ligands: the cytokine-like ligand Unpaired (Upd), and a BMP ligand Decapentaplegic (Dpp), both of which regulate GSC maintenance. GSCs typically divide asymmetrically, so that one daughter of the stem cell division remains attached to the hub and retains stem cell identity, while the other daughter, called a gonialblast, is displaced away from the hub and initiates differentiatio. Given the close proximity of GSCs and gonialblasts, the ligands (Upd and Dpp) must act over a short range so that signalling is only active in stem cells, but not in differentiating germ cells. The basis for this sharp boundary of pathway activation remains poorly understood (Inaba, 2015).

Using green fluorescent protein (GFP)-α1-tubulin84B expressed in germ cells (nos-gal4>UAS-GFP-αtub), this study found that GSCs form protrusions, referred to as microtubule-based (MT)-nanotubes hereafter, that extend into the hub. MT-nanotubes are sensitive to fixation similar to other thin protrusions reported so far, such as tunnelling nanotubes, explaining why they have escaped detection in previous studies. MT-nanotubes appear to be specific to GSCs: 6.67 MT-nanotubes were observed per testis in the GSC population (or 0.82 per cell). The average thickness and length of MT-nanotubes are 0.43 ± 0.29 µm (at the base of MT-nanotube) and 3.32 ± 1.6 µm, respectively. These GSC MT-nanotubes are uniformly oriented towards the hub area. By contrast, differentiating germ cells showed only 0.44 MT-nanotubes per testis (or <0.002 per cell), without any particular orientation when present. MT-nanotubes were sensitive to colcemid, the microtubule-depolymerizing drug, but not to the actin polymerization inhibitor cytochalasin B, suggesting that MT-nanotubes are microtubule-based structures. MT-nanotubes were not observed in mitotic GSCs, and GSCs form new MT-nanotubes as they exit from mitosis. By contrast, MT-nanotubes in interphase GSCs were stably maintained for up to 1 h of time-lapse live imaging. Although cell-cycle-dependent formation of MT-nanotube resembles that of primary cilia, MT-nanotubes are distinct structures, in that they lack acetylated microtubules and are sensitive to fixation. Furthermore, a considerable fraction of GSCs form multiple MT-nanotubes per cell (54% of GSCs with MT-nanotubes), and MT-nanotubes are not always associated with the centrosome/basal body, as is the case for the primary cilia (Inaba, 2015).

To examine the geometric relationship between MT-nanotubes and hub cells further, MT-nanotubes were imaged in combination with various cell membrane markers, followed by three-dimensional rendering. Although the MT-nanotubes are best visualized in unfixed testes that express GFP-αTub in germ cells, adding a low concentration (1 μM) of taxol to the fixative preserves MT-nanotubes, allowing immunofluorescence staining. First, Armadillo (Arm, β-catenin) staining, which marks adherens junctions formed at hub cell/hub cell as well as hub cell/GSC boundaries, revealed that adherens junctions do not form on the surface of MT-nanotubes. Using FM4-64 styryl dye, it was found that the MT-nanotubes are ensheathed by membrane lipids. Furthermore, myristoylation/palmitoylation site GFP (myrGFP), a membrane marker, expressed in either the germline or hub cells illuminated MT-nanotubes, suggesting that the surface membrane of a MT-nanotube is juxtaposed to hub-cell plasma membrane (Inaba, 2015).

Genes were examined that regulate primary cilia and cytonemes for their possible involvement in MT-nanotube formation. RNA interference (RNAi)-mediated knockdown of oseg2 (IFT172), osm6 (IFT52) and che-13 (IFT57), components of the intraflagellar transport (IFT)-B complex that are required for primary cilium anterograde transport and assembly, significantly reduced the length and the frequency of MT-nanotubes. Knockdown of Dlic, a dynein intermediate chain required for retrograde transport in primary cilia<, also reduced the MT-nanotube length and frequency. Knockdown of klp10A, a Drosophila homologue of mammalian kif24 (a MT-depolymerizing kinesin of the kinesin-13 family, which suppresses precocious cilia formation), resulted in abnormally thick/bulged MT-nanotubes. No significant changes were observed in MT-nanotube morphology upon knockdown of IFT-A retrograde transport genes, such as oseg1 and oseg3 (Inaba, 2015).

Endogenous Klp10A localized to MT-nanotubes both in wild-type testes and in GFP-αTub-expressing testes. GFP-Oseg2 (IFT-B), GFP-Oseg1, GFP-Oseg3 (IFT-A) and Dlic also localized to the MT-nanotubes when expressed in germ cells. The localization of IFT-A components to MT-nanotubes, without detectable morphological abnormality upon mutation/knockdown, is reminiscent of the observation that most of the genes for IFT-A are not required for primary cilia assembly. Expression of a dominant negative form of Dia (DiaDN) or a temperature-sensitive form of Shi (Shits) in germ cells (nos-gal4>UAS-diaDN or UAS-shits), which perturb cytoneme formation, did not influence the morphology or frequency of MT-nanotubes in GSCs. Taken together, these results show that primary cilia proteins localize to MT-nanotubes and regulate their formation (Inaba, 2015).

In search of the possible involvement of MT-nanotubes in hub-GSC signalling, it was found that the Dpp receptor, Thickveins (Tkv), expressed in germ cells (nos-gal4>tkv-GFP) was observed within the hub region, in contrast to GFP alone, which remained within the germ cells. A GFP protein trap of Tkv (in which GFP tags Tkv at the endogenous locus) also showed the same localization pattern as Tkv-GFP expressed by nos-gal4. By inducing GSC clones that co-express Tkv-mCherry and GFP-αTub, it was found that Tkv-mCherry localizes along the MT-nanotubes as puncta. Furthermore, using live observation, Tkv-mCherry puncta were observed to move along the MT-nanotubes marked with GFP-αTub, suggesting that Tkv is transported towards the hub along the MT-nanotubes. It should be noted that, in the course of this study, it was noticed that mCherry itself localized to the hub when expressed in germ cells, similar to Tkv-GFP and Tkv-mCherry. Importantly, the receptor for Upd, Domeless (Dome), predominantly stayed in the cell body of GSCs, demonstrating the specificity/selectivity of MT-nanotubes in trafficking specific components of the niche signalling pathways. A reporter of ligand-bound Tkv, TIPF localized to the hub region together with Tkv-mCherry, in addition to its reported localization at the hub-GSC interface. Furthermore, Dpp-GFP expressed by hub cells co-localized with Tkv-mCherry expressed in germline. These results suggest that ligand (Dpp)-receptor (Tkv) engagement and activation occurs at the interface of the MT-nanotube surface and the hub cell plasma membrane. Knockdown of IFT-B components (oseg2RNAi, che-13RNAi or osm6RNAi), which reduces MT-nanotube formation, resulted in reduction of the number of Tkv-GFP puncta in the hub area, concomitant with increased membrane localization of Tkv-GFP. A similar trend was observed upon treatment of the testes with colcemid, suggesting that MT-nanotubes are required for trafficking of Tkv into the hub area. By contrast, knockdown of Klp10A, which causes thickening of MT-nanotubes, led to an increase in the number of Tkv-GFP puncta in the hub area. Taken together, these data suggest that Tkv is trafficked into the hub via MT-nanotubes, where it interacts with Dpp secreted from the hub (Inaba, 2015).

Knockdown of klp10A (klp10ARNAi) led to elevated phosphorylated Mad (pMad) levels, a readout of Dpp pathway activation, in GSCs. By contrast, RNAi-mediated knockdown of oseg2, osm6 and che-13 (IFT-B components), which causes shortening of MT-nanotubes, reduced the levels of pMad in GSCs. Dad-LacZ, another readout of Dpp signalling activation, exhibited clear upregulation upon knockdown of klp10A. GSC clones of che-13RNAi, osm6RNAi or oseg2452 were lost rapidly compared with control clones, consistent with the idea that MT-nanotubes help to promote Dpp signal transduction. Knockdown of oseg2, che-13 and osm6 did not visibly affect cytoplasmic microtubules, suggesting that GSC maintenance defects upon knockdown of these genes are probably mediated by their role in MT-nanotube formation. Global RNAi knockdown of these genes in all GSCs using nos-gal4 did not cause a significant decrease in GSC numbers , indicating that compromised Dpp signalling due to MT-nanotube reduction leads to a competitive disadvantage in regards to GSC maintenance only when surrounded by wild-type GSCs (Inaba, 2015).

When klp10ARNAi GSC clones were induced, pMad levels specifically increased in those GSC clones, indicating that Klp10A acts cell-autonomously in GSCs to influence Dpp signal transduction. Importantly, klp10ARNAi spermatogonia did not show a significant elevation in pMad level compared with control spermatogonia, demonstrating that the role of Klp10A in regulation of Dpp pathway is specific to GSCs. pMad levels did not change in spermatogonia upon manipulation of MT-nanotube formation. GSC clones of klp10ARNAi or klp10A null mutant (klp10A24) did not dominate in the niche, despite upregulation of pMad, possibly because of its known role in mitosis. Importantly, these conditions did not significantly change STAT92E levels, which reflect Upd-JAK-STAT signalling in GSCs, revealing the selective requirement of MT-nanotubes in Dpp signalling. Together, these results demonstrate that MT-nanotubes specifically promote Dpp signalling and their role in enhancing the Dpp pathway is GSC specific (Inaba, 2015).

Since cytonemes are induced/stabilized by the signalling molecules themselves, the possible involvement of Dpp in MT-nanotube formation was explored First, it was found that a temperature-sensitive dpp mutant (dpphr56/dpphr4) exhibited a dramatic decrease in the frequency of MT-nanotubes (0.067 MT-nanotubes per GSC) and the remaining MT-nanotubes were significantly thinner. Knockdown of tkv (tkvRNAi) in GSCs also resulted in reduced length and frequency of MT-nanotubes. Conversely, overexpression of Tkv (tkvOE) in germ cells led to significantly longer MT-nanotubes. Interestingly, expression of a dominant negative Tkv (tkvDN), which has intact ligand-binding domain but lacks its intracellular GS domain and kinase domain, resulted in thickening of MT-nanotubes, rather than reducing the thickness/length. This indicates that ligand-receptor interaction, but not downstream signalling events, is sufficient to induce MT-nanotube formation. Strikingly, upon ectopic expression of Dpp in somatic cyst cells (tj-lexA>dpp), spermatogonia/spermatocytes were observed to have numerous MT-nanotubes, suggesting that Dpp is necessary and sufficient to induce or stabilize MT-nanotubes in the neighbouring germ cells. In turn, MT-nanotubes may promote selective ligand-receptor interaction between hub and GSCs, leading to spatially confined self-renewal (Inaba, 2015).

This study shows that previously unrecognized structures, MT-nanotubes, extend into the hub to mediate Dpp signalling. It is proposed that MT-nanotubes form a specialized cell surface area, where productive ligand-receptor interaction occurs. In this manner, only GSCs can access the source of highest ligand concentration in the niche via MT-nanotubes, whereas gonialblasts do not experience the threshold of signal transduction necessary for self-renewal, contributing to the short-range nature of niche signalling. In summary, the results reported here illuminate a novel mechanism by which the niche specifies stem cell identity in a highly selective manner (Inaba, 2015).


The tkv transcript is alternatively spliced. The splice variants differ in their 5' exons. Both variants have identical 3' regions derived from two exons that code for protein (Penton, 1994).


Amino Acids - 563

Structural Domains

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).

thickveins: Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised: 25 MAR 97 

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