org Interactive Fly, Drosophila decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

Gene name - decapentaplegic

Synonyms -

Cytological map position - 22F1-2

Function - secreted morphogen

Keywords - dorsal-ventral patterning, imaginal disc development

Symbol - dpp

FlyBase ID:FBgn0000490

Genetic map position - 2-4.0

Classification - TGF-beta-like and BMP-like

Cellular location - secreted



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Arias, C., Fussero, G., Zacharonok, M. and Macias, A. (2015). Dpp-expressing and non-expressing cells: two different populations of growing cells in Drosophila. PLoS One 10: e0121457. PubMed ID: 25798905
Summary:
There are different models that explain growth during development. One model is based on insect and amphibian regeneration studies. This model proposes that growth is directed by pattern, and growth takes place by intercalation at a growth discontinuity; therefore, proliferation should surround the discontinuity. Currently, this model, apart from regenerative studies on mostly adult patterning, has not found supporting evidence in Drosophila that shows proliferation surrounding a discontinuity. Despite this lack of evidence, the importance of discontinuities has been shown in different experiments, even under wt conditions, more specifically in the formation of the leg joints because of the occurrence of cell death at their boundaries. This study shows the existence of a sharp discontinuity in Decapentaplegic (Dpp) in the genital discs at the third larvae stage (L3), which determines the upregulation in the Jun-NH2-Terminal-Kinase (JNK) pathway, reaper (rpr), head involution defective (hid) and active caspases from its boundaries. The proliferation and cell death surrounding the discontinuity suggest that growth can proceed by intercalation and competitive death takes place in this area. Finally, the Rpr, Grim and Hid (RGH) products are a few of the factors that define the growth discontinuity because they are negative regulators of growth, a new function that is unique from their known functions in apoptosis.

Chatterjee, R. N., Chatterjee, P., Kuthe, S., Acharyya-Ari, M. and Chatterjee, R. (2015). Intersex (ix) mutations of Drosophila melanogaster cause nonrandom cell death in genital disc and can induce tumours in genitals in response to decapentaplegic (dppdisk) mutations. J Genet 94: 207-220. PubMed ID: 26174668
Summary:
In Drosophila melanogaster, the mediator complex protein intersex (ix) is a terminally positioned gene in somatic sex determination hierarchy and functions with the female specific product of doublesex to implement female sexual differentiation. The null phenotype of ix is to transform diplo-X individuals into intersexes while leaving haplo-X animals unaffected. This study on the effect of different intersex mutations on genital disc development provides the following major results: (1) similar range of a characteristic array of morphological structures (from almost double sex terminalia to extreme reduction of terminal appendages) was displayed by the terminalia of ix flies; (2) an increased number of apoptotic cells were found to occur in a localized manner in mature third instar larval genital discs of ix individuals; (3) ix mutations can induce high frequency of neoplastic tumours in genitals in the presence of decapentaplegic mutations; and (iv) heteroallelic combinations of dpp mutations can also induce tumours in intersex genitals with variable expressivity. On the basis of these findings, it is suggested that: (1) loss of function of ix causes massive cell death in both male and female genital primordia of genital discs, resulting phenotype mimicking in male and female characteristics in genitals; and (2) at the discs, the apoptotic cells persist as 'undead' cells that can induce oncogenic transformation in the neighbouring disc cells when dpp signalling is blocked or reduced by dpp) mutations.

Ayyub, C., Banerjee, K. K. and Joti, P. (2015). Reduction of Cullin-2 in somatic cells disrupts differentiation of germline stem cells in the Drosophila ovary. Dev Biol. [Epub ahead of print] PubMed ID: 26206612
Summary:
Signaling from a niche consisting of somatic cells is essential for maintenance of germline stem cells (GSCs) in the ovary of Drosophila. Decapentaplegic (Dpp), a type of bone morphogenetic protein (BMP) signal, emanating from the niche, is the most important signal for this process. Cullin proteins constitute the core of a multiprotein E3-ligase important for their functions viz. degradation or modification of proteins necessary for different cellular processes. This study has found that a Cullin protein called Cullin-2 (Cul-2) expresses in both somatic and germline cells of the Drosophila ovary. Reduction of Cul-2 in somatic cells causes upregulation of Dpp signal and produces accumulation of extra GSC-like cells inside germarium, the anteriormost structure of the ovary. These results suggest that Cullin-2 protein present in the somatic cells is involved in a non cell-autonomous regulation of the extent of Dpp signaling and thus controls the differentiation of GSCs to cystoblasts (CBs).

Park, S. Y., Stultz, B. G. and Hursh, D. A. (2015). Dual role of Jun N-terminal kinase activity in bone morphogenetic protein-mediated Drosophila ventral head development. Genetics [Epub ahead of print]. PubMed ID: 26500262
Summary:
decapentaplegic (dpp) controls ventral head morphogenesis by expression in the eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. Palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the JNK pathway but in opposite ways. Slight reduction of both Jun N-terminal Kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal Kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal Kinase activity. It is concluded that formation of sensory vibrissae, rostral membrane and gena tissue requires the action of JNK in peripodial cells, while excessive Jun N-terminal Kinase signaling in these same cells inhibits the formation of maxillary palps.

Harmansa, S., Hamaratoglu, F., Affolter, M. and Caussinus, E. (2015). Dpp spreading is required for medial but not for lateral wing disc growth. Nature 527: 317-322. PubMed ID: 26550827
Summary:
Drosophila Decapentaplegic has served as a paradigm to study morphogen-dependent growth control. However, the role of a Dpp gradient in tissue growth remains highly controversial. Two fundamentally different models have been proposed: the 'temporal rule' model suggests that all cells of the wing imaginal disc divide upon a 50% increase in Dpp signalling, whereas the 'growth equalization model' suggests that Dpp is only essential for proliferation control of the central cells. To discriminate between these two models, morphotrap, a membrane-tethered anti-green fluorescent protein (GFP) nanobody was generated and used, that enables immobilization of enhanced (e)GFP::Dpp on the cell surface, thereby abolishing Dpp gradient formation. In the absence of Dpp spreading, wing disc patterning is lost; however, lateral cells still divide at normal rates. These data are consistent with the growth equalization model, but do not fit a global temporal rule model in the wing imaginal disc.

Akiyama, T. and Gibson, M. C. (2015). Decapentaplegic and growth control in the developing Drosophila wing. Nature 527: 375-378. PubMed ID: 26550824
Summary:
As a central model for morphogen action during animal development, the bone morphogenetic protein 2/4 (BMP2/4)-like ligand Decapentaplegic is proposed to form a long-range signalling gradient that directs both growth and pattern formation during Drosophila wing disc development. While the patterning role of Dpp secreted from a stripe of cells along the anterior-posterior compartmental boundary is well established, the mechanism by which a Dpp gradient directs uniform cell proliferation remains controversial and poorly understood. To determine the precise spatiotemporal requirements for Dpp during wing disc development, this study used CRISPR-Cas9-mediated genome editing to generate a flippase recognition target (FRT)-dependent conditional null allele. By genetically removing Dpp from its endogenous stripe domain, the requirement was confirmed of Dpp for the activation of a downstream phospho-Mothers against dpp gradient and the regulation of the patterning targets spalt, optomotor blind and brinker. Surprisingly, however, third-instar wing blade primordia devoid of compartmental dpp expression maintain relatively normal rates of cell proliferation and exhibit only mild defects in growth. These results indicate that during the latter half of larval development, the Dpp morphogen gradient emanating from the anterior-posterior compartment boundary is not directly required for wing disc growth.

Fried, P. and Iber, D. (2015). Read-out of dynamic morphogen gradients on growing domains. PLoS One 10: e0143226. PubMed ID: 26599604
Summary:
Quantitative data from the Drosophila wing imaginal disc reveals that the amplitude of the Decapentaplegic (Dpp) morphogen gradient increases continuously. It is an open question how cells can determine their relative position within a domain based on a continuously increasing gradient. This study shows that pre-steady state diffusion-based dispersal of morphogens results in a zone within the growing domain where the concentration remains constant over the patterning period. The position of the zone that is predicted based on quantitative data for the Dpp morphogen corresponds to where the Dpp-dependent gene expression boundaries of spalt (sal) and daughters against dpp (dad) emerge. The model also suggests that genes that are scaling and are expressed at lateral positions are either under the control of a different read-out mechanism or under the control of a different morphogen. The patterning mechanism explains the extraordinary robustness that is observed for variations in Dpp production, and offers an explanation for the dual role of Dpp in controlling patterning and growth. Pre-steady-state dynamics are pervasive in morphogen-controlled systems, thus making this a probable general mechanism for the scaled read-out of morphogen gradients in growing developmental systems.

Aggarwal, P., Gera, J., Mandal, L. and Mandal, S. (2016). The morphogen Decapentaplegic employs a two-tier mechanism to activate target retinal determining genes during ectopic eye formation in Drosophila. Sci Rep 6: 27270. PubMed ID: 27270790
Summary:
Understanding the role of morphogen in activating its target genes, otherwise epigenetically repressed, during change in cell fate specification is a very fascinating yet relatively unexplored domain. The in vivo loss-of-function genetic analyses in this study reveal that specifically during ectopic eye formation, the morphogen Decapentaplegic (Dpp), in conjunction with the canonical signaling responsible for transcriptional activation of retinal determining (RD) genes, triggers another signaling cascade. Involving dTak1 and JNK, this pathway down-regulates the expression of polycomb group of genes to do away with their repressive role on RD genes. Upon genetic inactivation of members of this newly identified pathway, the canonical Dpp signaling fails to trigger RD gene expression beyond a threshold, critical for ectopic photoreceptor differentiation. Moreover, the drop in ectopic RD gene expression and subsequent reduction in ectopic photoreceptor differentiation resulting from inactivation of dTak1 can be rescued by down-regulating the expression of polycomb group of genes. These results unravel an otherwise unknown role of morphogen in coordinating simultaneous transcriptional activation and de-repression of target genes implicating its importance in cellular plasticity. 

Neto, M., Aguilar-Hidalgo, D. and Casares, F. (2016). Increased avidity for Dpp/BMP2 maintains the proliferation of progenitors-like cells in the Drosophila eye. Dev Biol [Epub ahead of print]. PubMed ID: 27502436
Summary:
During organ development, the progenitor state is transient, and depends on specific combinations of transcription factors and extracellular signals. Not surprisingly, abnormal maintenance of progenitor transcription factors may lead to tissue overgrowth, and the concurrence of signals from the local environment is often critical to trigger this overgrowth. Therefore, identifying specific combinations of transcription factors/signals promoting -or opposing- proliferation in progenitors is essential to understand normal development and disease. This study used the Drosophila eye as a model where the transcription factors hth and tsh are transiently expressed in eye progenitors causing the expansion of the progenitor pool. However, if their co-expression is maintained experimentally, cell proliferation continues and differentiation is halted. It was shown that Hth+Tsh-induced tissue overgrowth requires the BMP2 Dpp and the abnormal hyperactivation of its pathway. Rather than using autocrine Dpp expression, Hth+Tsh cells increase their avidity for Dpp, produced locally, by upregulating extracellular matrix components. During normal development, Dpp represses hth and tsh ensuring that the progenitor state is transient. However, cells in which Hth+Tsh expression is forcibly maintained use Dpp to enhance their proliferation.

Li, X., Yang, F., Chen, H., Deng, B., Li, X. and Xi, R. (2016). Control of germline stem cell differentiation by polycomb and trithorax group genes in the niche microenvironment. Development [Epub ahead of print]. PubMed ID: 27510973
Summary:
Polycomb and Trithorax group (PcG and TrxG) genes function to regulate gene transcription by maintaining the repressive or active chromatin state, respectively. This antagonistic activity is important for body patterning during embryonic development, but whether this function module has a role in adult tissues is unclear. This study reports that in the Drosophila oogenesis, disruption of the Polycomb responsive complex 1 (PRC1) specifically in the supporting escort cells causes blockage of cystoblast differentiation and germline stem cell- like tumor formation. The tumor is caused by derepression of decapentaplegic (dpp)/i> which prevents cystoblast differentiation. Interestingly, activation of dpp in escort cells requires the function of TrxG gene brahma (brm), suggesting that loss of PRC1 in escort cells causes Brm-dependent dpp expression. This study suggests a requirement for balanced activity between PcG and TrxG in an adult stem cell niche, and disruption of this balance could lead to the loss of tissue homeostasis and tumorigenesis.
Redhai, S., et al. (2016). Regulation of dense-core granule replenishment by autocrine BMP signalling in Drosophila secondary cells. PLoS Genet 12: e1006366. PubMed ID: 27727275
Summary:
Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although how many secretagogues stimulate DCG release is understood, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. This study demonstrates that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, this study shows that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. This analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion.
Harmansa, S., Alborelli, I., Bieli, D., Caussinus, E. and Affolter, M. (2017). A nanobody-based toolset to investigate the role of protein localization and dispersal in Drosophila. Elife 6 [Epub ahead of print]. PubMed ID: 28395731
Summary:
The role of protein localization along the apical-basal axis of polarized cells is difficult to investigate in vivo, partially due to lack of suitable tools. This study presents the GrabFP system, a collection of four nanobody-based GFP-traps that localize to defined positions along the apical-basal axis. The localization preference of the GrabFP traps can impose a novel localization on GFP-tagged target proteins and results in their controlled mislocalization. These new tools were used to mislocalize transmembrane and cytoplasmic GFP fusion proteins in the Drosophila wing disc epithelium and to investigate the effect of protein mislocalization. Furthermore, the GrabFP system was used as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and showed that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.
BIOLOGICAL OVERVIEW

In a first phase of its developmentally crucial expression, decapentaplegic is responsible for dorsal/ventral polarity in the fly. In a second phase of its activity, as segments appear, dpp functions in the definition of boundaries between segmental compartments. As part of this process, dpp, along with wingless and hedgehog, defines the position of future limbs, including wings, legs and antenna. Dpp also has an independent role in the structuring of the mesoderm. Later, during the final process of appendage development, and acting downstream of engrailed and hedgehog, dpp defines boundaries between appendage compartments assuring correct anterior/posterior polarity. dpp has an analogous function in the development of the eye, where it is primarily responsible for the progression of the morphogenetic furrow, the induction site of the Drosophila retina.

In the first process, the structuring of dorsal/ventral polarity, dpp is repressed in the ventral portion of the trunk by the dorsal gene product. Receptors for the secreted Dpp protein, Saxophone, Thick veins and Punt, are found in both the ectoderm and the underlying mesoderm. They mediate the transduction of the dpp signal to the interior of the cell through a phosphorylation cascade activating gene transcription (for review, see Raftery, 1999). Phosphorylation, the major mechanism of the cell for transduction of signals from protein to protein, involves the attachment of phosphate residues to appropriate target molecules.

The effects on the dorsal-most region of the fly are regulated by Saxophone in conjuction with Thick veins and Punt. Without Dpp signals through Saxophone, the amnioserosa, the most dorsal ectodermal tissue, does not develop properly and dorsal closure, the sealing of a dorsal "hole" in the developing embryo does not take place. The dorsal region is ventralized, and it develops characteristics of the ventral neuroectoderm. In a sense, the Saxophone-defective fly develops upside down, and the dorsal region starts to resemble ventral tissue.

The initial effects of Dpp on the heart and on visceral mesoderm (gut muscles) are mediated by Thick veins and Punt. An early and important event in the subdivision of the mesoderm is the restriction of tinman expression to dorsal mesodermal cells, the precursors of heart cells.

Whereas initial Dpp signals eminate from the ectoderm, later Dpp takes on a life of its own in the visceral mesoderm. It is activated by Ultrabithorax and repressed by abdominal-A. In the mesodermal midgut, the cluster of homeotic genes Sex combs reduced, Antennapedia, and abd-A are expressed in non-overlapping anterior/posterior domains. They are responsible for drawing string-like constrictions in three parts of the midgut and the outgrowth of pockets, or as they are termed, caeca. Dpp with its ability to define boundaries has a role in this process. Dpp secreted by the mesoderm also leads to the induction of labial in the endoderm. Thus Dpp also induces local differentiation in the endoderm (Manak, 1995). The actions of dpp on gene activation are not always positive. dpp actively suppresses the development of the proventriculus, confining it to the foregut.

The importance of Dpp expression cannot be overstated, both early in allocation of cells to appendages and later in compartment subdivision of these appendages. Secreted in an anterior to posterior stripe in the trunk, Dpp intersects wingless expressing cells under the control of hedgehog in segmentally repeated dorsal/ventral stripes. All three proteins are needed to allocate cells for the formation of imaginal discs, which will ultimately develop into appendages. This process involves induction of distal-less and aristaless, both of which are needed to specify the tips of appendages (Campbell, 1993 and Diaz-Benjumea, 1994).

The imaginal disk expression of Dpp in a narrow stripe of cells along the anterior-posterior compartment boundary is essential for proper growth and patterning of the Drosophila appendages. Dpp receptor function was examined in the formation of the wing to understand how this localized Dpp expression produces its global effects in appendage development. This work depicts in a very clear fashion how Dpp functions as a morphogen. Clones of saxophone (sax) or thick veins (tkv) mutant cells, defective in one of the two type I receptors for DPP, show shifts in cell fate along the anterior-posterior axis. In the adult wing, clones that are homozygous for a null allele of sax or a hypomorphic allele of tkv show shifts to more anterior fates when the clone is in the anterior compartment and to more posterior fates when the clone is in the posterior compartment. The effect of these clones on the expression pattern of the downstream gene spalt-major also correlates with these specific shifts in cell fate. The shift in cell fate is explained by assuming that the cells in mutant clones act as though they see a lower than normal Dpp concentration. Thus cell fate along the A/P axis is directly related to the perceived Dpp level. It is concluded that cell fate is directly related to the distance of cells from the source of Dpp at the A/P axis and that Dpp is responsible for patterning of the entire wing blade in direct response to the long-range Dpp signal. The similar effects of sax null and tkv hypomorphic clones indicate that the primary difference in the function of these two receptors during wing patterning is that Tkv transmits more of the Dpp signal than does Sax. These results are consistent with a model in which a gradient of Dpp reaches all cells in the developing wing blade to direct anterior-posterior pattern. While current evidence suggests that Tkv is absolutely required for Dpp signaling, there appears to be no such absolute requirement for Sax. Thus Dpp receptor complexes that lack a Tkv subunit cannot transmit a sufficient level of Dpp signal to trigger a biological response in the receiving cell. In contrast, receptor complexes lacking Sax subunits are still capable of significant signal reception and downstream signaling (Singer, 1997).

Dpp function in the wing imaginal disc

Cell proliferation and patterning must be coordinated for the development of properly proportioned organs. If the same molecules were to control both processes, such coordination would be ensured. This possibility has been investigated in the Drosophila wing using the Dpp signaling pathway. Previous studies have shown that Dpp forms a gradient along the AP axis that patterns the wing, that Dpp receptors are autonomously required for wing cell proliferation, and that ectopic expression of either Dpp or an activated Dpp receptor, TkvQ253D, causes overgrowth. These findings are extended with a detailed analysis of the effects of Dpp signaling on wing cell growth and proliferation. Increasing Dpp signaling by expressing TkvQ253D accelerates wing cell growth and cell cycle progression in a coordinate and cell-autonomous manner. Conversely, autonomously inhibiting Dpp signaling using a pathway specific inhibitor, Dad, or a mutation in tkv, slows wing cell growth and division, also in a coordinate fashion. Stimulation of cell cycle progression by TkvQ253D is blocked by the cell cycle inhibitor RBF, and requires normal activity of the growth effector, PI3K. Among the known Dpp targets, vestigial was the only one tested that was required for TkvQ253D-induced growth. The growth response to altering Dpp signaling varies regionally and temporally in the wing disc, indicating that other patterned factors modify the response (Martín-Castellanos, 2002).

To address the cell autonomous effects of the Dpp signaling pathway, the Flp/Gal4 method was used to activate or suppress Dpp signaling in clones of cells marked with GFP. First, a mutant version of the Dpp type I receptor Thick veins, TkvQ253D, containing a point mutation in the glycine/serine rich domain (GS) was expressed. This mutation mimics the receptor phosphorylation that occurs upon ligand binding, and therefore renders the receptor constitutively active and ligand independent. TkvQ253D expression strongly activates the Dpp signaling pathway, inducing high levels of the phospho-Mad transducer and expression of two Dpp targets, omb and spalt. Initially, clones of cells that expressed TkvQ253D were induced in early second instar larvae (at 48 hours AED) and the cells were allowed to proliferate until the end of larval development (120 hours AED). Wing cell clones expressing TkvQ253D showed smooth borders compared with control clones, which showed jagged borders, and were also larger than control clones. This phenotype is stronger in lateral areas of the disc, far from the endogenous Dpp source. Approximately half of the lateral clones were completely round and bulged out of the disc epithelium, which generated extra folds around the clones. This phenotype was not seen when TkvQ253D was expressed throughout the disc, indicating that the round bulging clonal phenotype is a consequence of abnormal heterotypic interactions between TkvQ253D-expressing cells and wild-type cells (Martín-Castellanos, 2002).

Induction of clones by heat shock allowed the age of the clones to be controlled, and also allowed the inference of cell proliferation rates from the number of cells per clone. Since cell death was observed by Acridine Orange staining in TkvQ253D-expressing clones, the apoptotic inhibitor p35 was expressed to block cell death. This was necessary to obtain accurate proliferation rate measurements, which are confounded by cell death. Clones were induced and allowed to proliferate for a short time in the period of larval development when imaginal wing cells proliferate exponentially. Because of the regional phenotype described above, the number of cells per clone was counted in lateral and medial areas, as well as in the entire presumptive wing region. Cells over-expressing the activated Dpp receptor proliferate faster than control cells. This phenotype is stronger in lateral areas, where TkvQ253D-expressing cells proliferate 20% faster than controls. TkvQ253D-expressing cells proliferate 10% faster than controls in the medial region. This regional phenotype reflects the graded activity of endogenous Dpp signaling; lateral areas normally low in Dpp are more sensitive to signaling activation (Martín-Castellanos, 2002).

To further analyze the cellular phenotype, flow cytometry (FACS) was performed using co-expressed GFP to identify TkvQ253D-expressing cells. The GFP-negative cell population from the same discs was used as an internal control. TkvQ253D overexpression shifts the distribution of cells in the different phases of the cell cycle. A smaller proportion of the TkvQ253D-expressing cells are in the G1 phase and greater proportion in G2. These data, together with the shorter doubling time of these cells, suggests that TkvQ253D preferentially promotes G1/S progression. This cell cycle phenotype is more severe if the activated receptor is expressed for a longer period of time (Martín-Castellanos, 2002).

To address more carefully the autonomy of the effects of TkvQ253D, the expression patterns of String and Cyclin E protein were analyzed in discs containing TkvQ253D-expressing clones. String and Cyclin E limit progression of the imaginal disc cell cycle through G2/M and G1/S transitions, respectively. S-phase progression in TkvQ253D-expressing clones was also assessed using BrdU incorporation, and mitosis by phospho-Histone H3 detection. The BrdU incorporation assay yielded a result consistent with increased proliferation within TkvQ253D-expressing clones in lateral regions of the discs: these clones show a uniform increase in BrdU uptake. Increased BrdU incorporation is limited to within the TkvQ253D-expressing clones, and no non-autonomous effects were detected. This result implies that TkvQ253D stimulates cell proliferation cell-autonomously. No changes were detected in Cyclin E, String or phospho-Histone H3 expression levels in TkvQ253D-expressing clones or surrounding cells (Martín-Castellanos, 2002).

Although raising the levels of Dpp signaling increases rates of cell proliferation, it does not appear to bypass the developmentally programmed proliferation arrest that occurs at the end of larval development. TkvQ253D-expressing clones induced late in larval development (96 hours AED) contain the same number of cells as control clones. The same result was obtained when p35 was co-expressed. In addition, TkvQ253D-expressing clones induced early (48 hours AED) and analyzed in pupae (168 hours AED) do not contain mitotic cells. This suggests that a dominant, developmentally programmed signal prevents TkvQ253D-expressing cells from continuing to divide beyond the normal proliferation stage (Martín-Castellanos, 2002).

Induction of cell proliferation does not necessarily indicate increased growth. To more directly assess the ability of TkvQ253D to induce growth, areas of the disc epithelium encompassed by TkvQ253D-expressing clones were measured. Clones were induced early in larval development and analyzed at the end of the larval period. The average area of TkvQ253D-expressing clones was 2.5 times larger than that of control clones, indicating that TkvQ253D-expressing cells grow faster than wild-type cells. This phenotype depends on the position of the clone in the anterior-posterior axis. Clones in the lateral areas, far from the source of endogenous Dpp, showed the strongest phenotype. Fifty percent of these lateral clones were larger than the largest control clone. On average, lateral clones expressing TkvQ253D were 3.7 times larger than lateral control clones (Martín-Castellanos, 2002).

The cellular growth effects of TkvQ253D were further assessed using FACS analysis to measure cell size. The ratio of the mean forward light scatter (FSC) of GFP+ cells versus GFP- cells was measured as a cell size indicator. GFP expression did not cause a significant change in cell size. TkvQ253D-expressing cells analyzed by FACS generally showed a size that was not significantly different from wild-type cells. In some experiments, however, these cells were slightly larger than controls. The fact that TkvQ253D-expressing clones are much larger than controls, but consist of cells of roughly normal size, confirms that TkvQ253D accelerates cell cycle progression. Taking the in situ and FACS analyses together, it is concluded that activation of Dpp signaling coordinately increases both rates of cell proliferation and cell growth (Martín-Castellanos, 2002).

To complement these experiments, the effects of autonomously inhibiting Dpp signaling were analyzed by overexpressing the pathway-specific inhibitor Dad, or by generating cell clones mutant for tkv. Dad is an inhibitory Smad protein that, when overexpressed, blocks omb expression and the adult wing phenotypes induced by ectopic Dpp signaling. It is normally activated by Dpp signaling and expressed in a broad domain centered on the AP axis. When Dad was overexpressed using the Flp/Gal4 method, clones were not recovered in the dorsomedial area of wing blade. However, Dad-expressing clones were recovered in medial areas when the apoptotic inhibitor p35 was co-expressed. These clones contained fewer cells than controls, indicating that Dad overexpression impairs proliferation of cells at medial positions. The cell doubling time of Dad overexpressing medial cells was more than 3 hours (22%) longer than the control doubling time. Slow-growing cells are eliminated by a mechanism known as cell competition when normal growing cells surround them. Because Dad overexpressing cells proliferate slowly, this may explain why they are not recovered unless the apoptotic inhibitor p35 is co-expressed (Martín-Castellanos, 2002).

To better understand the basis of this proliferative defect, tkv- clones were generated by mitotic recombination. A recessive lethal allele, tkv7, was used that carries a point mutation in a conserved glutamate residue in the kinase domain and results in loss of expression of Dpp targets. In the medial wing pouch, tkv7 clones survive for 36 hours but are lost within 48 hours of induction (in the 72-120 hours AED interval). In lateral areas, tkv7 mutant clone survival is greater, however mutant clones are still small compared with wild-type twin spots, and show round morphology. This lateral-medial survival phenotype reflects the lower requirement for Dpp signaling in lateral areas of the wing imaginal disc (Martín-Castellanos, 2002).

Flow cytometry was used to analyze tkv7 cells. To counteract cell competition and enrich the population of mutant cells, a cell lethal Minute mutation, M(2)32A1, was used that carries a lesion in ribosomal protein S13, and slows growth when heterozygous. Since M-/- cells are not viable, only M+/+ cells were recovered after mitotic recombination. These M+/+ cells were tkv7 homozygous. In the Minute background, tkv7 cells survive at least 4 days and colonize more tissue than in a wild-type background. However, they are still growth impaired relative to wild-type cells growing in the same Minute+/- background, and they still appear mainly in lateral areas. Approximately 30% of the tkv7 discs showed an aberrant morphology, probably caused by abnormal adhesive interactions between mutant and wild-type cells. tkv7 cells show a cell cycle profile consistent with a proliferation defect; the S phase fraction is extremely reduced and the G1 fraction is increased. This phenotype is opposite that of cells overexpressing TkvQ253D, which has a shortened G1. FACS analysis also showed that tkv7 cells are not detectably different in size from control cells. Previous studies indicate that when cell cycle progression is specifically delayed, cell size increases since cells continue to grow at the normal rates. Since tkv7 cells proliferate very slowly while maintaining a normal cell size, evidently they are impaired for growth as well as cell cycle progression (Martín-Castellanos, 2002).

Interestingly, M(2)32A1/+ cells are larger than wild-type cells. This suggests that these cells divide more slowly than they grow, and thus that the growth defect caused by the Minute mutation affects cell cycle progression preferentially. In fact, in both budding and fission yeast cell cycle control genes are sensitive to translational conditions. Studies using another Minute mutation that encodes a ribosomal protein, M(3)95A, detected no size alteration in M/+ cells, and thus this effect may be gene specific (Martín-Castellanos, 2002).

Using a third approach to avoid the effects of cell competition, Dad was induced ubiquitously throughout the wing disc using the A9-Gal4 driver. This causes a reduction of disc size. This size reduction is especially pronounced along the AP axis and thus is opposite that of the phenotype resulting from TkvQ253D expression using the same driver, which enlarges the wing disc preferentially along the AP axis. These results show that inhibition of Dpp signaling reduces growth and impairs proliferation, whereas activation of Dpp signaling increases growth and accelerates proliferation (Martín-Castellanos, 2002).

If growth and cell cycle progression are independently regulated by Tkv, one would expect to detect the proliferative effect of TkvQ253D even in growth-impaired cells. Alternatively, if TkvQ253D were to promote cell cycle progression indirectly via stimulating cellular growth, the proliferative effect of TkvQ253D should be inhibited when cell growth is impaired (Martín-Castellanos, 2002).

To suppress cell growth a truncated version of p60, Deltap60, was expressed. This is an adaptor molecule for the class I Phosphoinositide 3-Kinase (PI3K/Dp110 in Drosophila. Dp110 signaling is a potent growth inducer. Adaptor molecules, such as p60, bind to the Dp110 kinase and recruit it to the Insulin Receptor, allowing full activation of the enzyme. Deltap60 binds the Insulin Receptor but cannot bind Dp110, and thus inhibits Dp110 signaling in a dominant-negative manner. When expressed in wing cells, Deltap60 reduces cell size and strongly delays G1 progression. Flp/Gal4 clones expressing Deltap60 contain very few cells compared with controls. Overexpressed Deltap60 also dominantly blocks the growth and proliferation effects of TkvQ253D. Clones of cells that co-express Deltap60 and TkvQ253D contain as few cells as those expressing Deltap60 alone, and these cells are just slightly larger than those expressing Deltap60 alone. Thus, loss of growth resulting from loss of PI3K activity cannot be rescued by hyperactivating Dpp signaling, and cell proliferation induced by Dpp probably requires Dp110 activity. These results are consistent with the model in which Dpp-driven cell growth indirectly promotes cell cycle progression (Martín-Castellanos, 2002).

Although clonal growth is blocked by co-expressing Deltap60 and TkvQ253D, cells that co-express Deltap60 and TkvQ253D do not show the G1 delay characteristic of cells expressing Deltap60 alone. Thus, TkvQ253D appears to be able to promote G1/S progression even in the presence of Deltap60. This suggests that some aspects of cell cycle progression induced by TkvQ253D may be Dp110 independent. However, the slight increase in size observed in cells co-expressing Deltap60 and TkvQ253D makes it difficult to rule out the possibility that this effect on G1/S progression also occurs indirectly, as a consequence of increased growth (Martín-Castellanos, 2002).

In the wing imaginal disc, omb, spalt and vestigial (vg) have been reported to respond to Dpp signaling. It was of interest to know which if any of these genes was involved in controlling tissue growth effected by TkvQ253D. spalt is probably not required, since Spalt protein is not induced by TkvQ253D expression in the lateral areas of the wing disc, where the strongest overgrowth effects are observed. In the case of omb and vg, null alleles were used as a genetic background in which the expression of the activated Dpp receptor was induced. TkvQ253D can promote growth in the absence of Omb (Martín-Castellanos, 2002).

By contrast, TkvQ253D is not able to promote tissue growth in a null vg83b27R background. This result points to Vg as a possible effector of growth induced by Dpp signaling. Consistently, ectopic Vg expression induces wing-like outgrowths in imaginal discs. However, it was surprising to find that clones expressing TkvQ253D do not show increased levels of Vg protein, regardless of their position in the disc. Some lateral clones express Vg, but these most probably originate in the Vg expression domain. In fact, clones in lateral positions where Vg is expressed over-grow better than in other regions. These results suggest that activation of Dpp signaling is not sufficient to induce Vg expression, but that TkvQ253D and Vg might synergize to effect tissue growth (Martín-Castellanos, 2002).

Thus cell growth and cell cycle progression are coordinately regulated. These findings extend earlier studies that indicated a role for Dpp signaling in tissue growth. The 'balanced' effects on cell growth and cell proliferation caused by TkvQ253D differ markedly from results obtained when other growth stimulatory factors are manipulated in the developing wing. Ras, Myc and PI3K stimulate wing cell growth. Growth mediated by ectopic expression of these factors leads to a truncated G1 phase, which in the case of Ras and Myc has been attributed to post-transcriptional upregulation of the G1/S regulator Cyclin E. However, hyperactivation of Ras, Myc or PI3K signaling does not increase overall rates of wing cell proliferation, apparently because of a failure to stimulate G2/M progression. Consequently, these factors drive 'unbalanced' growth characterized by substantial increases in cell size. By contrast, ectopic TkvQ253D causes an increase in overall rates of cell division. Thus, TkvQ253D must induce G2/M as well as G1/S progression. Although no changes in Cyclin E or String levels have been detected by immunofluorescence, it is possible that small differences not detectable by antibody staining are responsible for G1/S and G2/M promotion (Martín-Castellanos, 2002).

Although early studies of wing development suggested that gradients of signaling might be the driving force that promotes cell growth in the wing, recent work has suggesting that Dpp signaling need not be employed in a gradient to stimulate growth. Dpp signaling in TkvQ253D-expressing clones is intense and homogenous, as assayed by anti-phospho-Mad staining, even in lateral areas. This suggests that gradients of Dpp signaling within these clones have been obliterated. Nevertheless, a variety of assays indicate that cell proliferation is promoted uniformly and autonomously throughout the clones, rather than at their edges, where sharp differentials of signaling intensity occur. Gradient models also predict non-autonomous effects on growth in regions bordering TkvQ253D-expressing clones. Although cell growth rates were not directly analyzed in these regions, inspection of markers for cell cycle progression did not detect major non-autonomous effects on cell proliferation. Thus, all these observations suggest that absolute intracellular levels of Dpp signaling, rather than gradients, are important for growth (Martín-Castellanos, 2002).

Survival of tkv-cells is better in regions of the wing that experience low level Dpp signaling. However, even in lateral regions far from the Dpp source, tkv- cells have a growth and proliferation defect. This suggests that all cells in the wing disc, including lateral cells, receive and require at least low levels of a Tkv ligand for normal growth. This led to the suggestion that some of the Dpp targets that mediate its growth effects might not have regionalized, nested expression patterns like two well-characterized Dpp targets, spalt and omb (which appear not to be mediators of TkvQ253D-induced growth). Instead, it seems plausible that some of the Dpp targets that mediate cell growth and proliferation are more uniformly expressed in regions where Dpp is required (Martín-Castellanos, 2002).

How might Dpp, expressed in a gradient, drive expression of growth regulatory targets more uniformly? It has been proposed that induction of target genes in cells receiving low levels of Dpp must overcome the activity of the transcriptional repressor, Brinker. brinker mutant clones in lateral areas of the wing disc exhibit a round morphology and over-growth phenotypes that are similar to TkvQ253D-expressing clones. brinker mutant discs also exhibit a dramatic over-growth phenotype along the AP axis similar to discs that overexpress TkvQ253D ubiquitously. Thus, it seems plausible that all wing cells require a threshold level of Dpp activity to grow, and that in lateral regions this threshold is equal to the amount of signaling activity needed to overcome repression of Dpp growth targets by Brinker. When Brinker is lost or TkvQ253D is expressed in lateral regions, this threshold level of signaling may be greatly surpassed, causing increased expression of growth regulators and acceleration of cell growth rates beyond normal levels (Martín-Castellanos, 2002).

The growth response of a cell to altered Dpp signaling varies according to its location in the disc. Ectopic TkvQ253D causes the strongest over-growth phenotypes in lateral regions, far from the source of endogenous Dpp, whereas inhibition of Dpp signaling has the strongest phenotypes in medial areas of the disc, where Dpp levels are normally high. Similar region-specific responses have been observed in experiments in which Notch or Wingless signaling is activated ectopically using cell autonomous effectors, or ligands. What is the significance of these region-specific responses? Without knowing the pertinent growth regulatory targets of these signaling systems, it is only possible to speculate. Perhaps the differential responses reflect cooperation between several regionally expressed signals that affect tissue growth, both positively and negatively, in a combinatorial fashion. Observations relating to vg seem consistent with this possibility. vg is required by TkvQ253D to promote tissue growth, yet Vg protein is not up-regulated by ectopic TkvQ253D, and TkvQ253D is capable of promoting overgrowth in wing regions where Vg is not detectable. The complex growth responses of cells to Dpp signaling illustrate how much is unknown about mechanisms of growth control. New, more global, approaches to studies of growth modulation will be required before its regulation by patterning signals can be understood. Important tasks for future studies include identifying the Dpp targets that stimulate cellular metabolism to effect growth, and determining how these targets integrate input from other patterning signals such as Wingless, Notch, Hedgehog and the Egfr ligands (Martín-Castellanos, 2002).

Specificity of Drosophila cytonemes for distinct signaling pathways

Cytonemes are types of filopodia in the Drosophila wing imaginal disc that are proposed to serve as conduits in which morphogen signaling proteins move between producing and target cells. The specificity was investigated of cytonemes that are made by target cells. Cells in wing discs made cytonemes that responded specifically to Decapentaplegic (Dpp) and cells in eye discs made cytonemes that responded specifically to Spitz (the Drosophila epidermal growth factor protein). Tracheal cells had at least two types: one made in response to Branchless (a Drosophila fibroblast growth factor protein, Bnl), to which they segregate the Bnl receptor, and another to which they segregate the Dpp receptor. It is concluded that cells can make several types of cytonemes, each of which responds specifically to a signaling pathway by means of the selective presence of a particular signaling protein receptor that has been localized to that cytoneme (Roy, 2011).

Cells in developing tissues are influenced by multiple signals that they process and integrate to control cell fate, proliferation, and patterning. An example is in the Drosophila wing imaginal disc, where cells depend on several signaling systems that are intrinsic to the disc. Dpp, Wingless (Wg), Hedgehog (Hh), and epidermal growth factor (EGF) are produced and released by different sets of disc cells, and receipt of these signaling proteins programs their neighbors to develop and grow. The mechanisms by which morphogen signaling proteins influence target cells must ensure both specificity and accuracy, and one possibility is that these proteins transfer at points of direct contact. Imaginal discs are flattened sacs that have a monolayer of columnar cells on one side and squamous peripodial cells on the other. Many cells in wing discs make filopodial extensions that lie along the surfaces of the monolayers, oriented toward morphogen-producing cells. These extensions have been termed cytonemes to denote their appearance as cytoplasmic threads and to distinguish them as specialized structures that polarize toward morphogen-producing regions (Roy, 2011).

In wing discs dissected from third instar larvae, cytonemes can be seen as filaments extending from randomly generated somatic clones engineered to express a fluorescent protein such as soluble, cytoplasmic green fluorescent protein (GFP) or a membrane-bound form such as mCD8:GFP (the extracellular and transmembrane domains of the mouse lymphocyte protein CD8 fused to GFP). To image disc cytonemes, unfixed discs were placed peripodial side down on a coverslip, covered with a 1-mm-square glass, and mounted over a depression slide with the disc hanging from the coverslip. Because fluorescence levels in cytonemes were low relative to background, recorded images were processed to increase intensity and were subjected to de-convolution. Expression of CD8:GFP in wing disc clones revealed cytonemes emanating from both the apical and basal surfaces of columnar cells, as well as from peripodial cells (whose apical and basal surfaces could not be distinguished). Most cytonemes were perpendicular to the anterior/posterior (A/P) axis of the disc and oriented toward the cells that produce Dpp at the A/P compartment border; others were oriented toward the cells that produce Wingless at the dorsal/ventral (D/V) compartment border. Disc-associated myoblasts also had filopodia (Roy, 2011).

In the eye disc, cells in the columnar layer organize into ommatidial clusters as a wave of differentiation [the morphogenetic furrow (MF)] passes from posterior to anterior. A second axis, centered at the equator, is orthogonal to the MF and defines a line of mirror-image symmetry where dorsal and ventral ommatidia are juxtaposed. The columnar cells divide during the third instar period but stop or divide only once after the MF passes. CD8:GFP expression was induced in somatic clones and the columnar cells were examined. Whereas clones of six to eight cells were present on both sides of the MF, only cells anterior to the MF had visible cytonemes. Cytonemes emanating from these clones oriented either toward the axis defined by the MF or toward the axis defined by the equator. Single clones with cytonemes oriented both toward the MF and toward the equator were not observed, and there was no apparent correlation between clone position and cytoneme orientation or cytoneme length. Cells in the peripodial layer of the eye disc also had cytonemes (Roy, 2011).

The EGF pathway is a key signaling system for eye development, and cells in the MF express the EGF protein Spitz (Spi). Because one of the two types of anterior cell cytonemes extended toward the MF and to explore the distribution of membrane-bound receptor proteins, clones were induced that expressed an epidermal growth factor receptor:GFP (EGFR:GFP) fusion protein. Anterior cells expressing EGFR:GFP had cytonemes that oriented toward the MF, and most of these cytonemes had fluorescent puncta; no cytonemes that were marked by EGFR:GFP oriented toward the equator. Other than their 'furrow-only' orientation, the cytonemes marked by EGFR:GFP were similar to those marked by CD8:GFP. In contrast, co-expression of CD8:GFP with (nonfluorescent) EGFR marked both furrow-directed and equator-directed cytonemes. Thus, expression of EGFR:GFP does not eliminate the equator-directed cytonemes, suggesting that the specific localization of EGFR:GFP to furrow-directed cytonemes is not a consequence of ectopic (over)expression of this fusion protein (Roy, 2011).

Evidence that the furrow-directed cytonemes depend on Spi/EGF signaling was obtained by expressing a dominant negative form of EGFR. Although EGFR is required for cell proliferation in the disc, small clones expressing EGFRDN were recovered that co-expressed EGFRDN and CD8:GFP; in these clones, only cytonemes that appeared to be randomly oriented were present, indicating that the long, furrow-directed cytonemes may require EGFR signal transduction in the cytoneme-producing cells (Roy, 2011).

Wing disc-associated tracheal cells also make cytonemes. The transverse connective (TC) is a tracheal tube that nestles against the basal surface of the wing disc columnar epithelium and that sprouts a new branch [the air sac primordium (ASP)] during the third instar period in response to Branchless (Bnl) expressed by the wing disc. Tracheal tubes are composed of a monolayer of polarized cells whose apical surfaces line a lumen. Expression of CD8:GFP throughout the trachea (btl-Gal4 UAS-CD8:GFP) made it possible to detect GFP fluorescence in several types of cytonemes emanating from the basal surfaces of the TC and ASP. Cytonemes at the tip of the ASP (length range, 12 to 50 μm; average length of 23 μm) contained the Breathless (Btl); the Drosophila fibrobast growth factor receptor (FGFR) and appeared to contact disc cells that express Bnl. Short cytonemes (length range, 2 to 15 μm; average length of 8.5 μm) extended from the TC cells in the vicinity of the ASP (Roy, 2011).

Tests were carried out to se whether Dpp, Spi, Bnl, and Hh affected wing disc, eye disc, and tracheal cytonemes differentially. Ubiquitous expression of Spi, Bnl, or Hh (induced by heat shock) did not alter the A/P-oriented apical cytonemes in the wing disc, and, in the eye disc, the long cytonemes of the columnar layer were unaltered after ubiquitous expression of Dpp, Bnl, or Hh. In contrast, long oriented cytonemes were absent in wing discs after ubiquitous expression of Dpp, and only short cytonemes that appeared to be randomly oriented were observed. Similarly, 0.5 to 3 hours after cSpi, a constitutively active form of EGF, was expressed ectopically by heat shock induction, clones expressing CD8:GFP in the eye disc had many short cytonemes that lacked apparent directional bias; in contrast to controls, no long cytonemes oriented toward the MF were observed. Cytonemes with normal orientation and length (including MF-directed cytonemes) were present in eye discs that were examined later, 8 hours after a pulse of cSpi expression. To monitor EGFR-containing cytonemes for sensitivity and responsiveness to Spi, cSpi was expressed by heat shock induction, and cells in clones expressing EGFR:GFP were examined. After a pulse of cSpi expression, the extensions oriented outward without apparent directional bias, and the EGFR:GFP puncta were present in all cytonemes (Roy, 2011).

To examine responses of the ASP tip cytonemes, Hh, Spi, Dpp, and Bnl were overexpressed by heat shock and GFP-marked cytonemes at the ASP tip were examined. No differences in number of cytonemes were detected until about 3 hours after heat shock. Four to 5 hours after heat shock, expression of Bnl increased the number of tip cytonemes by ~2.6 times, and although most of the cytonemes were <30 μm, the cytonemes >30 μm also increased (~3.2 times). Most of the long cytonemes in these preparations were oriented in directions other than toward the cells that normally express Bnl. The number of long cytonemes >30 μm did not change after overexpression of Hh, Spi, and Dpp (0.6 to 0.8 times); the number of short cytonemes increased after Dpp overexpression (~1.7 times) but not after overexpression of Hh or Spi (Roy, 2011).

Thus, the responses of apical wing disc cytonemes to overexpressed Dpp, of eye disc cytonemes to ubiquitous Spi, and of ASP tip cytonemes to exogenous Bnl (Drosophila FGF) are similar. These results suggest that the cytonemes detected in the wing discs and eye discs may have orientations and lengths that are dependent specifically on the respective sources of Dpp and Spi, whereas the ASP may extend cytonemes in response to more than one signaling protein. These results are, however, complicated by the heat shock mode of induction because both the cells that expressed GFP (and extended marked cytonemes) as well as the surrounding cells expressed the signaling proteins. To overcome this problem, a method was developed to induce two types of somatic clones in the same tissue, one that expressed GFP and another that expressed Dpp (Roy, 2011).

The GAL4 system was used to label cytonemes with CD8:GFP. Clones of GAL4-expressing cells were generated with heat shock-induced flippase (FLP recombinase). The second type of clone expressed a Dpp:Cherry fusion and was generated with a variant Cre-progesterone receptor recombinase that could be activated with a regime of heat shock and RU486. By adjusting the timing and strength of induction, wing discs were produced with small, independent, and relatively infrequent clones. In discs with clones that expressed ectopic Dpp as well as clones that expressed CD8:GFP, apical cytonemes tagged with GFP were detected that oriented toward nearby Dpp:Cherry-expressing cells and not toward either the A/P or D/V signaling centers. Such 'abnormally directed' cytonemes were never observed in control discs. The abnormally oriented cytonemes suggest that apical cytonemes in the wing blade respond directly to sources of Dpp and that their orientation reflects extant sources of signaling protein (Roy, 2011).

To characterize the relationship between tracheal ASP tip cytonemes and FGF signaling from the wing disc, the distribution of Btl (FGFR) was examined in ASP cells and in ASP cytonemes. In preparations from larvae with tracheal expression of both CD8:GFP and Btl:Cherry (btl-GAL4 UAS-CD8:GFP;UAS-Btl:Cherry), cytonemes were marked by CD8:GFP, some of which had fluorescent Btl:Cherry puncta. Each ASP had only a few long (>30 μm) cytonemes, most of which contained Btl:Cherry puncta. Few of the more numerous short cytonemes (<30 μm) contained Btl:Cherry puncta. To characterize Btl:Cherry after overexpression of Bnl, focus was placed on preparations obtained 1 to 2 hours post-induction (genotype btl-GAL4 UAS-CD8:GFP/HS-Bnl;UAS-Btl:Cherry/Gal80ts), because during this time interval the ASP morphology was close to normal but cytonemes had changed. ASPs were ignored after longer postinduction intervals because of major malformations to ASP morphology after 3 to 4 hours. Long cytonemes with Btl:Cherry puncta were present 1 hour after a pulse of Bnl expression; but 2 hours after the pulse, most ASPs had no long cytonemes, and the number of short puncta-containing cytonemes increased at the tip and along the shaft of the ASPs. After control heat shock or heat shock-induced expression of Dpp, the distribution of Btl:Cherry puncta in the ASP tip cytonemes was similar to normal controls: Long cytonemes had Btl:Cherry puncta, but most short cytonemes did not (Roy, 2011).

Because the number of small cytonemes at the ASP tip may have increased after ectopic Dpp expression, whether the thickveins (tkv) gene, which encodes a subunit of the Dpp receptor, is expressed in the ASP was investigated. Expression of the tkv reporter, tkv-lacZ (P{lacW}tkv16713), was detected in the ASP. When Tkv:GFP and Btl:Cherry were expressed together, Tkv:GFP and Btl:Cherry segregated to separate tip cytonemes at the ASP tip. Whereas Tkv-containing cytonemes were short (<30 μm), most of the Btl-containing cytonemes were longer (three of four of the Btl:Cherry-containing cytonemes were longer than 30 μm), and they lay in focal planes closer to the disc. These properties were consistent in all preparations examined in which both green Tkv and red Btl cytonemes were intact. Imaging these marked ASPs revealed that overexpressed Tkv:GFP and Btl:Cherry were present not only in the plasma membranes (as expected) but also in separate puncta in the cell bodies. This shows that Tkv and Btl receptors also segregated to separate locations in the ASP cell bodies (Roy, 2011).

These findings suggest that the ASP has long cytonemes that are specific to Bnl and specifically harbor Btl-containing puncta and that the ASP also has cytonemes that are specific to Dpp and specifically harbor Tkv. Similarly in the eye disc, the presence of EGFR:GFP in furrow-oriented cytonemes and not in equator-oriented cytonemes suggests that cytonemes in the eye disc also selectively localize receptors. And as was previously shown, apical cytonemes in the wing disc selectively localize Tkv. The apparent ligand specificities and contrasting makeup of these cytonemes suggest a diversity of functionally distinct subtypes: Cells appear to make cytonemes that respond specifically to the Dpp, EGF, or Bnl signaling proteins. The basal filopodia implicated in Delta-Notch signaling in the wing disc may represent yet another type (Roy, 2011).

The mechanism that endows cytonemes with specificity for a particular signaling protein cannot be based solely on tissue-specific expression of a receptor. Spi, Dpp, and Hh are active in eye discs, but only changes in Spi signaling affected the furrow-directed cytonemes. And in the wing disc, both the Hh and EGF signal transduction pathways are active in cells at the A/P compartment border, but the apical cytonemes only responded to overexpressed Dpp. The findings that tracheal cells in the ASP respond to both Dpp and Bnl and that the Tkv and Btl receptors are present in different cytonemes that the ASP cells extend suggest that specificity may be a consequence of the constitution of the cytoneme, not on which receptors the cells make. The mechanism that localizes receptors to different cytonemes is not known, but because the marked receptors that were expressed also segregated to different intracellular puncta, the processes that concentrate these receptors in separate locations may not be exclusive to cytonemes. There is a precedent for segregation of proteins to different cellular extensions, neurons segregate proteins to dendrites or axons, so extending projections with specific and distinct attributes may be a general property of cells (Roy, 2011).


GENE STRUCTURE

cDNA clone length - There are 3 major transcripts ( 3.3, 3.8 and 4.3 kb). Transcript A is present early and reaches a peak at between 4 and 12 hours. Transcript B is detected from 8 to 12 hours and remains high throughout development. Transcript C accumulates early and remains at high levels. Minor transcripts are detected as well. Major transcripts differ in the first exon, which makes up most of the 3'UTR. Thus each of these different transcripts have different proximal promoters and each shares a second and third exon. The common second and third exons code for the single protein species of Dpp (St. Johnston, 1990).

Bases in 5' UTR - variable

Exons - three

Bases in 3' UTR - 1041 and variable


PROTEIN STRUCTURE

Amino Acids - 588

Structural Domains

The carboxy-terminal 100 amino acids have 25-40% homology to human and porcine TGFbeta, Inhibin A and Inhibin B. The homologous region is preceded by three arginine dimers that each function as proleolytic cleavage sites (Padget, 1987).


decapentaplegic : Evolutionary Homologs | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References
date revised: 26 Dec 96 

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