thickveins

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion (part 2/2)

Thick veins in the wing

In the developing wing blade, somatic clones lacking the DPP receptors Punt or Thickveins, or lacking Schnurri, a transcription factor targeted by DPP signaling, fail to grow when induced early in larval development. tkv and shn are also required for vein cell differentiation. Drosophila wing veins are dorsoventrally asymmetrical, in that some protrude on the dorsal wing serface while others proturde on the ventral surface. tkv and shn mutant clones cause loss of veins when they are present on the dominant (protruding) side of the vein. Although dpp is expressed in only a subset of cells in the anterior compartment of the developing wing disc, all cells of the early prospective wing blade require tkv, punt and shn. This implies that DPP must diffuse away from its source to fulfill its function. Secreted DPP molecules would have to travel at least 4 cell diameters in order to signal to all future wing blade cells in the posterior compartment (Burke, 1996a).

The imaginal disk expression of the TGF-ß superfamily member 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 to understand how this localized DPP expression produces its global effects on appendage development. 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).

The gene homothorax is required for the nuclear import of Extradenticle, The functions of exd/hth and of the Hh/Wg/Dpp pathway are mutually antagonistic: exd blocks the response of Hh/Wg/Dpp target genes such as optomotor-blind and dachshund; high levels of Wg and Dpp eliminate exd function by repressing hth. This repression is mediated by the activity of Dll and dac. One prerequisite for appendage development is the inactivation of the exd/hth genes (Azpiazu, 2000 and references therein).

htx is originally expressed uniformly in the wing imaginal disc but, during development, its activity is restricted to the cells that form the thorax and the hinge, where the wing blade attaches to the thorax, and it is eliminated in the wing pouch, which forms the wing blade. Repression of hth in the wing pouch is a prerequisite for wing development; forcing hth expression prevents growth of the wing blade. Both the Dpp and the Wg pathways are involved in hth repression. Cells unable to process the Dpp signal (lacking thick veins or Mothers against Dpp activity) or the Wg signal (lacking dishevelled function) express hth in the wing pouch. vestigial has been identified as a Wg and Dpp response factor that is involved in hth control. In contrast to its repressing role in the wing pouch, wg upregulates hth expression in the hinge; teashirt is a positive regulator of hth in the hinge. tsh plays a role specifying hinge structures, possibly in co-operation with hth (Azpiazu, 2000).

In the second instar wing disc, the Hth product accumulates uniformly in the thoracic and appendage regions of the disc, but throughout the third larval period hth expression is downregulated and, by the late third instar, Hth only appears in the presumptive regions of the thorax and the wing hinge. The central part of the disc, which gives rise to the wing pouch, shows no hth expression. The repression of hth function is important for wing development, because if hth activity is forced in the wing pouch, the wing does not form. A similar observation has been made in the leg disc; hth or exd expression in the distal part results in a truncated appendage in which all the distal components are missing. In the leg, the subdivision between distal and proximal regions results from the antagonism between Hh signaling and exd/hth function. Hh response genes such as Dll and dac are instrumental in repressing hth (Azpiazu, 2000 and references therein).

The downregulation of hth in the wing pouch is a consequence of the activity of the Dpp and the Wg signaling pathways. In cells in which the response to the Dpp signal is prevented, as in tkv or Mad mutant cells, hth is expressed at high levels. Similarly, dsh minus cells, in which the transduction of Wg is blocked, show ectopic hth activity and consequently nuclear exd expression. These results also indicate that hth is latently active in the wing cells and has to be repressed by the continuous activity of the Dpp and Wg signals. The inability of cell clones to proliferate, cells in which the Dpp or the Wg pathways have been totally eliminated, may be due to high levels of hth expression. The Dpp and Wg pathways repress hth expression independently. This is illustrated by the experiments inducing dsh mutant clones: ectopic hth expression is only observed in clones located away from the AP border. This suggests that the high levels of Dpp expression near the AP border are sufficient to impede hth expression despite the removal of the control by Wg (Azpiazu, 2000).

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, Tkv^Q253D, 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 Tkv^Q253D 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 Tkv^Q253D 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 Tkv^Q253D-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, Tkv^Q253D, 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. Tkv^Q253D 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 Tkv^Q253D 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 Tkv^Q253D 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 Tkv^Q253D was expressed throughout the disc, indicating that the round bulging clonal phenotype is a consequence of abnormal heterotypic interactions between Tkv^Q253D-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 Tkv^Q253D-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 Tkv^Q253D-expressing cells proliferate 20% faster than controls. Tkv^Q253D-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 Tkv^Q253D-expressing cells. The GFP-negative cell population from the same discs was used as an internal control. Tkv^Q253D overexpression shifts the distribution of cells in the different phases of the cell cycle. A smaller proportion of the Tkv^Q253D-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 Tkv^Q253D 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 Tkv^Q253D, the expression patterns of String and Cyclin E protein were analyzed in discs containing Tkv^Q253D-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 Tkv^Q253D-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 Tkv^Q253D-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 Tkv^Q253D-expressing clones, and no non-autonomous effects were detected. This result implies that Tkv^Q253D stimulates cell proliferation cell-autonomously. No changes were detected in Cyclin E, String or phospho-Histone H3 expression levels in Tkv^Q253D-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. Tkv^Q253D-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, Tkv^Q253D-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 Tkv^Q253D-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 Tkv^Q253D to induce growth, areas of the disc epithelium encompassed by Tkv^Q253D-expressing clones were measured. Clones were induced early in larval development and analyzed at the end of the larval period. The average area of Tkv^Q253D-expressing clones was 2.5 times larger than that of control clones, indicating that Tkv^Q253D-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 Tkv^Q253D were 3.7 times larger than lateral control clones (Martín-Castellanos, 2002).

The cellular growth effects of Tkv^Q253D 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. Tkv^Q253D-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 Tkv^Q253D-expressing clones are much larger than controls, but consist of cells of roughly normal size, confirms that Tkv^Q253D 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)32^A1, 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 Tkv^Q253D, 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)32^A1/+ 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 Tkv^Q253D 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 Tkv^Q253D even in growth-impaired cells. Alternatively, if Tkv^Q253D were to promote cell cycle progression indirectly via stimulating cellular growth, the proliferative effect of Tkv^Q253D 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 Tkv^Q253D. Clones of cells that co-express Deltap60 and Tkv^Q253D 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 Tkv^Q253D, cells that co-express Deltap60 and Tkv^Q253D do not show the G1 delay characteristic of cells expressing Deltap60 alone. Thus, Tkv^Q253D 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 Tkv^Q253D may be Dp110 independent. However, the slight increase in size observed in cells co-expressing Deltap60 and Tkv^Q253D 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 Tkv^Q253D. spalt is probably not required, since Spalt protein is not induced by Tkv^Q253D 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. Tkv^Q253D can promote growth in the absence of Omb (Martín-Castellanos, 2002).

By contrast, Tkv^Q253D is not able to promote tissue growth in a null vg^83b27R 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 Tkv^Q253D 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 Tkv^Q253D 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 Tkv^Q253D 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 Tkv^Q253D causes an increase in overall rates of cell division. Thus, Tkv^Q253D 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 Tkv^Q253D-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 Tkv^Q253D-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 Tkv^Q253D-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 Tkv^Q253D-expressing clones. brinker mutant discs also exhibit a dramatic over-growth phenotype along the AP axis similar to discs that overexpress Tkv^Q253D 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 Tkv^Q253D 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 Tkv^Q253D 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 Tkv^Q253D to promote tissue growth, yet Vg protein is not up-regulated by ectopic Tkv^Q253D, and Tkv^Q253D 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).

In Drosophila wing discs, a morphogen gradient of Dpp has been proposed to be a determinant of the transcriptional response thresholds of the downstream genes sal and omb. Evidence is presented that the concentration of the type I receptor Tkv must be low to allow long-range Dpp diffusion. However, low Tkv receptor concentrations result in low signaling activity. To enhance signaling at low Dpp concentrations, a second ligand, Tgf-beta-60A, has been found to augment Dpp/Tkv activity. Tgf-beta-60A signals primarily through the type I receptor Sax, which synergistically enhances Tkv signaling and is required for proper Omb expression. Omb expression in wing discs is found to require synergistic signaling by multiple ligands and receptors to overcome the limitations imposed on Dpp morphogen function by receptor concentration levels (Haerry, 1998).

The phenotypic consequences of overexpressing constitutively active forms of Tkv and Sax receptors in the developing wing was investigated using the GAL4-UAS system. The A9-Gal4 line was used: this drives high-level expression of Gal4 in the entire wing disc before it is restricted to the dorsal pouch at late third instar stage. In wild-type discs, the Sal and Omb products are symmetrically expressed along the anterior/posterior (A/P) boundary in response to Dpp. Normally, the Sal domain is restricted to cells in the wing pouch that are in close proximity to the Dpp-expressing cells, while Omb responds to lower levels of Dpp and is expressed in cells further away from the A/P boundary. The anterior boundary of Sal has been shown to specify the location where the longitudinal vein 2 (L2) is formed, while the formation of L5 coincides approximately with the posterior boundary of the Omb domain, but a causal relationship has not yet been established. When Dpp is ubiquitously expressed in wing discs, they become overgrown and the expression of both Sal and Omb is expanded. Like Dpp, overexpression of constitutively active Tkv (TkvA) also leads to disc overgrowth and ectopic induction of Sal and Omb. All cells in wings derived from animals expressing either Dpp or activated Tkv appear to differentiate into vein tissue, as exemplified by production of vein-specific morphological markers such as dark pigment and longer bristles. In contrast to TkvA, expression of either one or two copies of SaxA or development at 30°C, which results in an approximately twofold increase of Gal4 activity, is not sufficient to expand either Sal or Omb and produces only weak adult phenotypes consisting primarily of ectopic and thickened veins with a small amount of wing blistering in the region of the posterior cross vein. This phenotype is similar to that seen in animals raised at 18°C, which express low levels of TkvA. Although these findings suggest that Sax function may be qualitatively similar to that of Tkv but simply weaker, higher levels of activated Sax (four copies) still cannot mimic the effects of activated Tkv, such as the expansion of Sal and Omb (Haerry, 1998).

When high levels of activated Sax activity are combined with low levels of activated Tkv, the result is more than additive. The combination of one copy of saxA and low levels of Tkv leads to overgrowth, with the expansion of Omb (but not Sal), and results in a strong wing phenotype. The interaction of Sax and Tkv is synergistic. Taken together, these data suggest that Sax and Tkv synergistically interact and control the expression of a common target gene, omb. Activation of omb expression requires a level of signaling that can be activated by either high levels of Tkv activity alone or by a synergistic interaction between low levels of Tkv and high levels of Sax activity. In contrast, Sal activation requires a higher level of signaling, which can only be achieved by high levels of Tkv activity (Haerry, 1998).

Since both Tkv and Sax, as well as the type II receptor Put, have been implicated in mediating Dpp signaling, whether the loss in signal activity of these receptors would cause similar patterning defects in the wing was investigated. If these three receptors all bind the same ligand and signal to the same sets of downstream genes, it would be expected that a reduction in the activity of any individual receptor should result in qualitatively similar phenotypes that differ in severity only. Increasing levels of dominant negative receptors were expressed in different regions of the developing wing disc. Similar to using an allelic series of hypomorphic mutations, it was expected that expression of increasing copy numbers of dominant negative receptors should result in progressively more severe phenotypes. Ubiquitous expression of 3-4 copies of either form of two dominant negative Tkv1 constructs results in small wings with partial loss of L4 and both cross veins. In addition, L2 and L3 are closer together and the triple-row margin bristles are shifted more distally/posteriorly, as expected if the level of Dpp signal is reduced by titration of Dpp into nonproductive complexes. At higher levels (6-8 copies) of dominant negative Tkv1, very small adult wings are produced that show fusion of L2 and L3 as well as L4 and L5. Similar phenotypes are produced by expressing dominant negative versions of the alternative Tkv isoform that have an N-terminal extended extracellular domain, and also by expression of dominant negative Put. Both the Sal and the Omb domains are strongly reduced: the adult wings show fusion of L2 with L3 and L4 with L5. The wing phenotypes obtained with increasing levels of dominant negative Tkv and Put resemble those of certain combinations of dpp loss-of-function alleles, which is consistent with the notion that Dpp is primarily signaling through the combination of the Tkv and Put receptors. In contrast to these observations, dominant negative Sax constructs produce different results. When increasing copy numbers (1-8 copies) of dominant negative Sax are expressed, the discs become smaller and the Omb domain is reduced to the size of the normal Sal domain. But unlike expressing dominant negative Tkv, the Sal domain is not affected. In the adult wing, L5 and the posterior cross vein are lost compared to losing L3 and L4 after expression of dominant negative Tkv or Put. In addition, L2 is shifted more proximally and the proximal triple-row bristles that expand more distally/posteriorly in dominant negative Tkv wings are replaced by more proximal costa bristles. While the distance between L3 and L4 is normal, the overall shape of the wing becomes more ‘strap-like’, suggesting loss of peripheral tissue rather than the central tissue that is deleted in animals expressing Tkv or Put dominant negative receptors. These results suggest that dominant negative Sax acts in a qualitatively different manner from dominant negative Tkv (Haerry, 1998).

These results indicate that while the reduction of Tkv and Put activity affects the whole disc (Sal, Omb and growth), the expression of dominant negative Sax only affects the peripheral region of the disc (Omb and peripheral growth). If the dominant negative receptors function primarily by titrating Dpp, then it is curious why the overexpression phenotypes of dominant negative Sax are different. One possibility is that these receptors do not simply signal in response to Dpp but also in response to the binding of other ligands as well. Of the other two BMP-type ligands that have been described in Drosophila, scw shows no detectable expression at this stage. However, Tgf-beta-60A is expressed broadly in wing discs, and mutant analyses indicate that Tgf-beta-60A is required for normal wing development. Given its role in wing patterning, the effects of heteroallelic Tgf-beta-60A mutations were examined on Sal and Omb expression. Similar to discs expressing dominant negative Sax, Sal expression in Tgf-beta-60A mutant discs is normal while the Omb domain is reduced, particularly in the dorsal compartment. These observations are consistent with the notion that a second BMP-type ligand, Tgf-beta-60A, is required in addition to Dpp for proper Omb expression. Furthermore, the similarity of the Tgf-beta-60A loss-of-function and the dominant negative Sax phenotypes is consistent with recently described genetic interactions between Tgf-beta-60A and sax mutations and suggests that Tgf-beta-60A could signal in part through Sax (Haerry, 1998).

Ubiquitous overexpression of moderate levels of Tgf-beta-60A does not result in excessive disc overgrowth and does not alter the distribution of Sal and Omb. The resulting wings are slightly larger and exhibit minor venation defects along L2 and L5. However, similar to Dpp or TkvA, higher levels of Tgf-beta-60A overexpression expands both Sal and Omb and results in blistered and pigmented adult wings. Since only activated Tkv but not Sax is able to expand Sal and Omb expression, these findings are consistent with the notion that expression of moderate levels of Tgf-beta-60A leads to signaling preferentially through Sax, producing relative mild phenotypes, while higher concentrations of Tgf-beta-60A may also result in signaling through Tkv, producing phenotypes similar to activated Tkv (Haerry, 1998).

An investigation was carried out to determine if Tgf-beta-60A contributes to wing development primarily in the form of homodimers or Tgf-beta-60A/Dpp heterodimers. Results: (1) the level of Tgf-beta-60A mRNA appears to be significantly less than that of DPP, based on RNA in situ hybridization, indicating that heterodimers are not likely to be very abundant assuming similar translational efficiencies. (2) Localized overexpression of Tgf-beta-60A in the dpp-expressing cells does not result in any mutant phenotypes. (3) Expression of Tgf-beta-60A in the posterior compartment results in overgrowth, an expansion of the Sal and Omb domains, and restriction all adult wing defects exclusively to the posterior compartment. Since Tgf-beta-60A expression in this experiment does not overlap with Dpp-secreting cells, no Dpp/Tgf-beta-60A heterodimers should form, since heterodimer formation requires expression of both proteins in the same cell. Therefore, Tgf-beta-60A functions most likely as a homodimer. This finding is consistent with recent genetic analysis showing that clones of Tgf-beta-60A mutant cells that do not include dpp-expressing cells nevertheless produce patterning defects. It has been shown that dominant negative Tkv is more potent than Sax for inhibiting Dpp signaling, while dominant negative Sax is a stronger suppressor than Tkv of Tgf-beta-60A signaling. High levels of Tkv receptor limit Dpp diffusion and restrict Omb expression (Haerry, 1998).

To determine whether modulation of Dpp signaling affects dally expression, the dally::lacZ expression was compared between wild-type and tkv heterozygous cells. Clones mutant for tkv were generated in a heterozygous background (tkv^a12/+) using the FLP-FRT system, which should, as a consequence, produce both mutant (tkv^a12/tkv^a12) and wild-type sister clones (+/+). However, tkv^– cells do not survive in the wing pouch since Tkv activity is indispensable for growth and, thus, only wild-type sister clones survive. In resultant mosaic discs with wild-type and tkv-heterozygous cells, dally expression is decreased cell autonomously in wild-type (+/+) clones at the AP border and peripheral to the border. To further confirm this result, the effects were examined of tkv-hypomorphic clones on dally expression. In such clones, where tkv activity is partially compromised, the levels of dally expression are elevated. In the notum region of the wing disc, tkv-null clones can be generated in which a substantial increase of dally expression is observed. Finally, the effect of increased Dpp signaling on dally expression was tested by using the FLP-OUT method to induce clones of cells that express tkv^Q253D, a constitutively active form of tkv, in the wing pouch. The level of dally::lacZ expression was found to be autonomously reduced in the tkv^Q253D-expressing clones. All of these results consistently indicate that dally expression in the wing disc is negatively regulated by Dpp signaling, as has been shown for tkv. Thus, dally and tkv are regulated by the same set of molecular pathways: Hh, En and Dpp signaling (Fujise, 2003).

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 EGFR^DN were recovered that co-expressed EGFR^DN 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/Gal80^ts), 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).

Thick veins in the eye

The DPP requirement for cell fate specification and cell cycle synchronization in the developing Drosophila eye was examined by determining whether cells defective for thickveins, saxophone or schnurri show abnormalities in cell division or differentiation. Clones mutant for a null allele of tkv that are anterior or posterior to the morphogenetic furrow have amounts of cyclin B that are indistinguisable from those in surrounding cells. In contrast, tkv clones that span the MF maintain cyclin B expression in the anterior part of the furrow, even though the surrounding cells arrested in G1 have no detectable cyclin B. Maintenance of cyclin B is thought to indicate a failure of cell cycle progression, as cyclin B levels decline in M phase. In addition, mitotic figures are not observed in clones in the anterior half of the MF. The phenotype observed in the clones is similar to defects caused by mutations in division abnormally delayed (dally), which is required for G2-M progression ahead of the furrow. Mutations in dally and dpp display genetic interactions in development of the eye, antenna, and genitalia, which suggests that dally augments dpp function. The behavior of DPP-receptor mutant clones supports a role for DPP in controlling progression through G2-M as a means of synchronizing the divisions that accompany differentiation of the eye disc. Cell fate, however, is unaffected by receptor mutation as revealed by expression of atonal, a proneural gene required for retinal precursor cell 8 (R8) determination. Because atonal expression is maintained in tkv clones, hh must not act through dpp to induce its expression, and thus dpp mediates a subset of hh functions in the MF (Penton, 1997).

The progression of retinal morphogenesis in the Drosophila eye is controlled to a large extent by Hedgehog (HH), a signaling protein emanating from differentiating photoreceptor cells. Adjacent, more anterior cells in the morphogenetic furrow respond to HH by expressing dpp, suggesting that the relationship between HH and DPP might be similar to that in the limb imaginal discs where DPP mediates the organizing activity of HH. This study contradicts that suggestion. Analysis of somatic clones of cells lacking the DPP receptors Punt or Tkv reveals that DPP plays only a minor role in furrow progression and no critical role in subsequent ommatidial development. Within tkv and punt clones traversing the furrow at the time of dissection, neuronal differentiation, as shown by ELAV staining, is somewhat retarded, especially in the middle of large clones. The function of DPP in this context must be nonessential or redundant as the furrow is only slightly slowed, but not stopped. Normal ommatidial development occurs in the complete absence of DPP. In contrast, HH-independent dpp expression around the posterior and lateral margins of the first and second instar eye discs is important for the growth of the eye disc and for initiation of the morphogenetic furrow at these margins. Tkv and Punt are absolutely required for cell proliferation in the early developing eye imaginal disc. tkv clones are severly restricted in their ability to grow, implying a strong requirement for the DPP signal for cell proliferation in the early eye disc. There is a posterior requirement for punt function in eye development, which suggests a role for DPP signaling in the initiation of the furrow at the posterior margin Adult eyes containing predominantly punt mutant tissue are regularly observed, but such eyes always have some wild-type tissue at the posterior margin. Both punt and tkv clones cause local overproliferation and block neural differentiation. The tissue in these marginal clones must die, as loss of head cuticle and eye structures is observed in eyes containing mutant clones (Burke, 1996b).

Thick veins in the gonads

Stem cells are thought to occupy special local environments, or niches, established by neighboring cells that give them the capability for self-renewal. Each ovariole in the Drosophila ovary contains two germline stem cells surrounded by a group of differentiated somatic cells that express hedgehog and wingless. The BMP2/4 homolog decapentaplegic (dpp) is specifically required to maintain female germline stem cells and promote their division. Overexpression of dpp blocks germline stem cell differentiation. Overexpressing dpp for 3 days after eclosion produces tumorous germaria. Large germline cells filling germarial regions 1 and 2a contain spectrosomes but showed no evidence of cyst formation. In regions 2b and 3, 16-cell cysts are observed that probably derive from differentiated cystoblasts, or cysts that had formed before the first heat shock. This phenotype is very similar to that of bag of marbles (bam) and benign gonial cell neoplasm (bgcn) mutants. These results suggest that ectopic Dpp inhibits cystoblast differentiation but does not block cyst formation and maturation (Xie, 1998).

Mutations in dpp or its receptor (saxophone) accelerate stem cell loss and retard stem cell division. Mutant germline stem cell clones were constructed to show that the dpp signal is directly received by germline stem cells. punt, thickveins, mad, Medea and Dad are all shown to be required cell-autonomously for germline stem cell maintenance; punt, tkv, mad, and Med are shown to be required cell-autonomously to stimulate germline stem cell division. During aging, the number and activity of stem cells is thought to be reduced. The level of dpp signaling is shown to control the life span and division rate of germline stem cells. Reduced dpp signaling causes premature stem cell loss. Perhaps more surprising is the observation that putative increases in signaling, caused by removal of Dad activity from stem cells, causes them to be maintained longer. This finding suggests that dpp signaling not only is necessary, but may sometimes be rate limiting for stem cell maintenance. This is the first example where stem cell life span has been extended in an intact organism. Thus, dpp signaling helps define a niche that controls germline stem cell proliferation (Xie, 1998).

Stem cell niches are specific regulatory microenvironments formed by neighboring stromal cells. Owing to difficulties in identifying stem cells and their niches in many systems, mechanisms that control niche formation and stem cell recruitment remain elusive. In the Drosophila ovary, two or three germline stem cells (GSCs) have recently been shown to reside in a niche, in which terminal filaments (TFs) and cap cells are two major components. Signals from newly formed niches promote clonal expansion of GSCs during niche formation in the Drosophila ovary. After the formation of TFs and cap cells, anterior primordial germ cells (PGCs) adjacent to TFs/cap cells can develop into GSCs at the early pupal stage while the rest directly differentiate. The anterior PGCs are very mitotically active and exhibit two division patterns with respect to cap cells. One of these patterns generates two daughters that both contact cap cells and potentially become GSCs. Lineage tracing study confirms that one PGC can generate two or three GSCs to occupy a whole niche ('clonal expansion'). decapentaplegic is expressed in anterior somatic cells of the gonad, including TFs/cap cells. dpp overexpression promotes PGC proliferation and causes the accumulation of more PGCs in the gonad. A single PGC mutant for thick veins, encoding an essential Dpp receptor, loses the ability to clonally populate a niche. Therefore, Dpp is probably one of the mitotic signals that promote the clonal expansion of GSCs in a niche. This study also suggests that signals from newly formed niche cells are important for expanding stem cells and populating niches (Zhu, 2003).

This demonstrates how an adult GSC niche is populated with stem cells in the Drosophila ovary. Before niche formation, all PGCs proliferate as pre-stem cells and are undifferentiated. As niche formation starts, PGCs divide into two distinct subpopulations: anterior PGCs adjacent to cap cells start to acquire stem cell identity, and the remaining PGCs directly proceed to differentiation. GSCs in one niche can come from one PGC. Dpp is likely involved in stimulating clonal expansion of GSCs during niche formation. This study suggests that signals from newly formed niches are important for expanding GSCs and most likely for populating nascent adult GSC niches (Zhu, 2003).

How stem cell identity is established initially remains elusive even in the well-studied stem cell systems -- Drosophila ovary and testis. In the primitive female gonads before the pupal stage, PGCs appear to undergo symmetric division to generate germ cells with the identical pre-stem cell fate. Several studies suggest that GSCs are established at the early pupal stage. At the early pupal stage, there are 136 germ cells on average in each gonad. The adult ovary, which is composed of 12-16 ovarioles with two or three GSCs per ovariole (average of 2.5), contains about 30 to 40 GSCs. Therefore, at the most, 20%-30% of PGCs in the early pupal gonad are recruited to niches and turn into GSCs (Zhu, 2003).

How is a particular germ cell selected and recruited to niches, and how does it become a GSC? Positional information is known to be very important for cell-fate determination in various developmental processes. In this study, a developmental approach was taken to investigate when key niche components form, and how PGCs are subdivided into GSCs and differentiated germ cells. The expression of bam is associated with germ cell differentiation in the adult ovary. Using bam expression as an indicator for germ cell differentiation, it has been shown that no PGCs in late third instar larval gonads have differentiated. In early pupal gonads (about 0-4 hours after pupation), all the PGCs that are not in contact with TFs/cap cells are differentiated; therefore, the PGCs that contact newly formed cap cells remain undifferentiated and become GSCs. Possibly, newly formed TFs/cap cells directly prevent the most anterior PGCs from differentiation when an unknown developmental signal triggers PGC differentiation around the larval-pupal transition stage. This study demonstrates that the stem cell fate of PGCs is determined by their position, i.e. juxtaposition to TFs/cap cells (Zhu, 2003).

The next important question is how these anterior PGCs populate niches. In this study, it has been show that the PGCs in contact with newly formed cap cells at the early pupal stage divide more frequently than the rest of the PGCs. The division patterns are very interesting: one division pattern generates two daughters that are both in contact with cap cells; the other pattern generates only one daughter that is in contact with cap cells. As in the adult ovary, two daughters that are in contact with cap cells can both become GSCs. This is verified by the observation that one marked PGC in the gonad at the late third-instar larval stage can generate two or three GSCs in a niche. The results also indicate that the stem cells in a niche can come from multiple PGCs. Whether GSCs in a niche come from one or multiple PGCs probably depends on whether one or multiple PGCs directly contact cap cells within the developing niche. If only one PGC contacts cap cells, it probably has an opportunity to generate two or three germ cells that contact cap cells and become GSCs. This study shows that newly formed niches do not simply recruit existing PGCs and turn them into GSCs, but also stimulate PGCs to proliferate and produce more GSCs (Zhu, 2003).

The clonal expansion of GSCs in a niche clearly requires the newly established stem cell to divide rapidly and generate a daughter that occupies the same niche, which further prevents other neighboring precursor cells from entering it. Consistent with this prediction, it was observed that the anterior row of germ cells at the early pupal stage is more mitotically active than the rest of the germ cells based on the BrdU incorporation assay. dpp is known to be important for maintaining GSCs and stimulating their division in the adult ovary. dpp is expressed in TFs/cap cells and other anterior somatic cells, and PGCs close to cap cells are capable of responding to dpp. Furthermore, overexpressing dpp promotes PGC proliferation. To demonstrate the necessity of dpp signaling in stimulating GSC clonal expansion, it has been shown that a PGC mutant for tkv, an essential dpp receptor, fails to clonally populate a niche. All these results demonstrate that dpp is probably a signal for stimulating GSC clonal expansion (Zhu, 2003).

As in the adult ovary, hh is also expressed in terminal filaments and cap cells in developing female gonads. Hh has been shown to play a minor role in modulating GSC division. Wingless (Wg) protein is expressed in terminal filaments and cap cells. Its expression in developing female gonads has not been examined. Because wg, dpp and hh often work together to regulate many developmental processes in Drosophila, it is possible that hh and wg could also cooperate with dpp to regulate PGC proliferation and modulate GSC clonal expansion in niches (Zhu, 2003).

PGCs in the gonad do not show any signs of differentiation until the larval-to-pupal transition. At the early pupal stage, only the PGCs in the anterior row remain undifferentiated, but the rest have already differentiated. It seems that a developmental signal(s) starts to appear and then induces the differentiation of PGCs during the transition from larva to pupa. Such a developmental signal could be mediated by a steroid-like hormone ecdysone. Interestingly, during most of the third instar larval stage, the ecdysteroid levels are very low but begin to rise and peak just before pupation. The ecdysteroid peak could be potentially responsible for the initial differentiation of germ cells in the gonad of the larva ready for pupation. It is also possible that the hormone is not a direct signal but controls the production of the signal(s). Somehow, the signals from the anterior somatic cells antagonize the differentiating signals and thus prevent the anterior row of the PGCs from differentiation. One of the signals that prevent PGCs from differentiation could be encoded by dpp. Dpp is known to prevent GSCs from differentiation in the adult ovary. In this study, 2.5% of the marked tkv mutant PGCs and none of the marked mad mutant PGCs before the third instar larval stage were recruited to niches or were maintained as GSCs before adulthood. The failure of tkv and mad mutant GSCs to be maintained in niches could be explained by the role of dpp in preventing PGCs from differentiation. It could also be explained by other possibilities, such as defects in the formation of adherens junctions between cap cells and GSCs. Whether dpp is a signal for maintaining the undifferentiated state of PGCs during early ovarian development remains undetermined. Therefore, the signals that maintain the undifferentiated state of PGCs from TFs/cap cells remain to be identified (Zhu, 2003).

BMP signaling is required for controlling somatic stem cell self-renewal in the Drosophila ovary: Glass bottom boat, signaling through thick veins, is essential for controlling somatic stem cell maintenance

BMP signaling is essential for promoting self-renewal of mouse embryonic stem cells and Drosophila germline stem cells and for repressing stem cell proliferation in the mouse intestine and skin. However, it remains unknown whether BMP signaling can promote self-renewal of adult somatic stem cells. In this study, BMP signaling is shown to be necessary and sufficient for promoting self-renewal and proliferation of somatic stem cells (SSCs) in the Drosophila ovary. BMP signaling, via the ligand Glass bottom boat, is required in SSCs to directly control their maintenance and division, but is dispensable for proliferation of their differentiated progeny. Furthermore, BMP signaling is required to control SSC self-renewal, but not survival. Moreover, constitutive BMP signaling prolongs the SSC lifespan. Therefore, this study clearly demonstrates that BMP signaling directly promotes SSC self-renewal and proliferation in the Drosophila ovary. This work further suggests that BMP signaling could promote self-renewal of adult stem cells in other systems (Kirilly, 2005)

FLP-mediated FRT recombination has revolutionized studies on diverse developmental processes in Drosophila. The mosaic clones marked by loss of armadillo (arm)-lacZ or ubiquitin (ubi)-GFP are routinely used to study Drosophila oogenesis. Two positive labeling methods, the tubulin-lacZ positive labeling system and the gal80-based mosaic analysis with a repressible cell marker (MARCM), have been developed to facilitate visualization of marked cells. The lacZ-positive labeling system is effective for identification of marked cells, but it is not ideal for manipulating gene function, while stable GAL80 protein may not allow rapid visualization of marked cells after one or two divisions due to its persistence. A new positively marked mosaic lineage (PMML) method has been developed to positively mark cells and allow for rapid expression of the UAS-GFP marker and any other UAS construct in the marked cells by using a combination of the GAL4-UAS and FLP-FRT systems. This PMML system uses the heat shock-inducible FLP to reconstitute a functional actin5C-gal4 gene from two complementary inactive alleles, actin5C FRT_52B and FRT_52B gal4. The actin5C-gal4 gene drives GFP expression to mark cells and can also activate or knock down gene function by using UAS constructs in the marked cells (Kirilly, 2005)

To test whether PMML is also suitable for marking SSCs and assisting in SSC identification in the Drosophila ovary, ovaries were immunostained with anti-GFP and anti-Fasciclin III (Fas3) antibodies 1 week after clone induction (ACI). Fas3 is expressed in SSCs at low levels and in differentiated follicle cell progenitor cells at higher levels. It takes about 4-5 days for transiently labeled GFP-positive follicle cells to completely exit the germarium. One week ACI, a typical GFP-positive SSC clone was easily observed with the GFP-marked follicle cells present in regions 2b and 3 of the germarium and in egg chambers. The marked SSC could be identified by its location (the GFP-positive somatic cell at the 2a/2b junction), low Fas3 expression, and the presence of GFP-marked follicle cells in the germarium and/or in the egg chambers. The GFP-marked inner germarial sheath (IGS) cells could also be readily identified by their location (the germarial regions 1 and 2a), the absence of marked differentiated follicle cells in the same ovarioles, and also the absence of Fas3 expression, since the IGS descendants do not pass beyond the 2a/2b junction. Therefore, this system can be applied effectively for labeling SSCs and their progeny and for further studying the function of any gene in the marked SSCs and their progeny by overexpression (Kirilly, 2005).

This study shows that SSCs in the adult ovary are capable of responding to BMP signaling. Genetic mosaic analyses demonstrate that known BMP downstream components are also required for SSC self-renewal, but not survival. Hyperactive BMP signaling enhances SSC self-renewal capacity. Glass bottom boat (Gbb) is essential for controlling SSC maintenance, at least in the GSC niche. Furthermore, BMP signaling appears to be specific to stem cells, since follicle cells mutant for BMP-specific downstream components proliferate and differentiate normally. In addition to participation in BMP signaling, Medea (Med) is likely involved in other TGF-β-like pathway(s) to control proliferation and size of differentiated follicle cells. The results from this study led to the proposal of a working model that Gbb perhaps as well as Dpp from neighboring somatic cells function as stem cell growth factors in vivo for promoting self-renewal of ovarian SSCs (Kirilly, 2005).

gbb and dpp are expressed in cap cells, inner germarial sheath (IGS) cells, and follicle cells. SSCs are located in the middle of the germarium and are likely exposed to both BMPs, since both Dpp and Gbb are diffusible molecules. gbb mutants exhibit severe SSC/follicle cell proliferation defects and SSC loss. Furthermore, SSCs mutant for BMP downstream components such as tkv, punt, and mad are lost faster and divide slower than wild-type ones. Although dpp mutants show much weaker mutant defects, it is still possible that it plays as important a role as does gbb, since only weak dpp mutations could be used for studying the regulation of adult SSCs due to its stringent requirements during early development. Therefore, these findings support the idea that Gbb, perhaps together with Dpp, controls SSC self-renewal and division. Studies on GSCs in the Drosophila ovary have shown that BMPs control GSC self-renewal by directly repressing transcription of differentiation-promoting genes such as bam. Possibly, BMP signaling also represses differentiation-promoting genes and thereby maintains SSC self-renewal. Meanwhile, BMP signaling could also positively regulate other genes that are important for maintaining the undifferentiated state of SSCs. This study also shows that BMP signaling also promotes SSC division. It has been shown that BMP signaling promotes GSC division. In order to better understand how BMP signaling controls SSC self-renewal and division, it is critical to identify the BMP target genes in SSCs, that are either repressed or activated by BMP signaling (Kirilly, 2005)

This study also shows that tkv is a major type I BMP receptor for controlling SSC self-renewal in the Drosophila ovary. The SSCs mutant for sax⁴, a null allele of sax, behave close to normal wild-type ones, while the SSCs mutant for a strong tkv allele, tkv⁸, are lost rapidly, indicating that Tkv is a major functional receptor to control SSC self-renewal. Given the evidence that gbb signaling is essential for maintaining SSCs, this study strongly supports the idea that Gbb signals mainly through Tkv to control SSC self-renewal in the Drosophila ovary. A recent study on Drosophila spermatogenesis also suggests that Gbb signaling primarily functions through Tkv, but not Sax. In the Drosophila testis, gbb and tkv are both essential for maintaining GSCs, but sax is not. Although one study on dominant-negative tkv and sax receptors suggests that dpp and gbb signal preferentially through tkv and sax, respectively, another more recent study has shown that both dpp and gbb use tkv, but not sax, control the process of vein promotion during pupal development and disc proliferation and vein specification during larval development. Taken together, the results from this study and the previous studies indicate that Gbb can use Tkv as a major receptor for its signal transduction in Drosophila (Kirilly, 2005).

Although Gbb/BMP signaling plays a critical role in controlling SSC self-renewal and division, it appears that it is dispensable for SSC survival, follicle cell proliferation, and cell size control. For example, expression of the baculovirus antiapoptotic gene p35 could not rescue the mutant punt SSC loss; the follicle cell clones mutant for strong tkv and mad alleles, tkv⁸ and mad¹², proliferate normally, and the sizes of the mutant follicle cells are quite normal. In contrast, p35 expression can rescue the Med²⁶ SSC loss to the levels of the mutant punt, tkv, and mad mutant SSC loss. The partial rescue indicates that Med is required for SSC survival in a BMP-independent pathway. The Med mutant follicle cell clones proliferate slower than wild-type, and the size of follicle cells is also smaller than that of wild-type, suggesting that Med is required for follicle cell proliferation and growth. Since BMP signaling is not involved in the control of SSC survival, follicle cell proliferation, and growth, these findings further suggest that Med must participate in other TGF-β-like pathways controlling these processes. In mammalian systems, SMAD4 has been shown to be a common SMAD for all TGF-β-like signaling pathways, including TGF-β, Activin, and BMP. A likely candidate TGF-β-like signaling pathway includes Activin and TGF-β. Activin and TGF-β molecules exist in Drosophila. Activin-like signaling has been shown to be involved in regulating growth control and neuronal remodeling. However, the role of TGF-β signaling in Drosophila remains a mystery. It could not be completely ruled out, however, that Med is involved in other signaling pathways unrelated to TGF-β-like pathways to control SSC survival, follicle cell proliferation, and growth. In the future, it is very important to figure out which pathway Med takes part in for controlling SSC survival, follicle cell proliferation, and growth control (Kirilly, 2005).

In a variety of systems, stem cells have been proposed to be regulated by signals from niches. SSCs are anchored to the posterior group of IGS cells through DE-cadherin-mediated cell adhesion. Elimination of the anchorage leads to rapid SSC loss, suggesting that the posterior IGS cells function as a SSC niche. This study shows that gbb is expressed in the somatic cells, including IGS cells and follicle cells, and plays an important role in maintaining SSCs. Hh and Wg are expressed in the cap cells and play essential roles in controlling SSC self-renewal, suggesting that the SSC niche is composed of IGS cells and cap cells. In Drosophila imaginal development, these three pathways often regulate one another to control patterning, cell proliferation, and differentiation. In the Drosophila ovary, disruption of Hh, Wg, and BMP signaling cascades causes rapid SSC loss, while hyperactive signaling results in abnormal proliferation and differentiation of SSC progeny. Interestingly, their downstream transcriptional factors are also required for controlling SSC maintenance, suggesting that integration of these pathways likely takes place at or after transcription of their target genes. This study has shown that hyperactive BMP signaling can substitute for Wg signaling, but not Hh signaling, in controlling SSC self-renewal. However, it still remains unclear how hyperactive BMP signaling bypasses Wg signaling in SSCs. An important task in the future is to define their target genes in SSCs and to further figure out how these three signal transduction pathways interact with each other to control expression of these target genes (Kirilly, 2005).

In mammals, Shh, Wnt, and BMP pathways have been shown to regulate stem cell behavior directly or indirectly. BMP signaling directly represses activities of stem cells in the intestine and the hair follicle and promotes self-renewal of ES cells and spermatogonial stem cells. BMP signaling can also indirectly regulate haematopoeitic stem cells (HSCs) by controlling niche size. Wnt signaling has been shown to control self-renewal of HSCs, ES cells, intestinal stem cells, and possibly hair follicle stem cells. Shh signaling is required for proliferation of stem cells/progenitor cells in the lung airway. Studies from Drosophila and mice have shown that different stem cell types may utilize a combination of different growth factors to control their self-renewal, proliferation, and differentiation. Interestingly, Wnt and BMP signaling pathways promote ES self-renewal in mice and ovarian SSC self-renewal in Drosophila. Future studies of how different signaling pathways are integrated in Drosophila ovarian SSCs may also shed light on how these same pathways control stem cell self-renewal in mammals (Kirilly, 2005).

Hemocyte-secreted type IV collagen enhances BMP signaling to guide renal tubule morphogenesis in Drosophila

Details of the mechanisms that determine the shape and positioning of organs in the body cavity remain largely obscure. This study shows that stereotypic positioning of outgrowing Drosophila renal tubules depends on signaling in a subset of tubule cells and results from enhanced sensitivity to guidance signals by targeted matrix deposition. VEGF/PDGF ligands from the tubules attract hemocytes, which secrete components of the basement membrane to ensheath them. Collagen IV sensitizes tubule cells to localized BMP guidance cues. Signaling results in pathway activation in a subset of tubule cells that lead outgrowth through the body cavity. Failure of hemocyte migration, loss of collagen IV, or abrogation of BMP signaling results in tubule misrouting and defective organ shape and positioning. Such regulated interplay between cell-cell and cell-matrix interactions is likely to have wide relevance in organogenesis and congenital disease (Bunt, 2010).

As the renal tubules extend through the body cavity, two processes occur; they elongate through cell rearrangements and they make precise, guided movements with respect to other tissues. A major source of the motive force required for tubule extension is the convergent-extension movements of the tubule cells themselves. As the tubules are continuous with the hindgut and thus have a fixed point proximally, these movements result in a distal-directed extensive force. This study shows that in addition the normal morphogenesis of the anterior tubules depends on tissue guidance involving the coordinated activity of the PDGF/VEGF and BMP signaling pathways. Abrogation of either pathway has no effect on convergent-extension movements in the tubules but leads to failure of their normal pathfinding through the body cavity (Bunt, 2010).

PVF ligands (Pvf1-3) expressed by the tubules attract migrating hemocytes to form short-term associations with them, during which hemocytes secrete components of the BM. The presence of collagen IV in the matrix ensheathing anterior tubule cells primes their response to local sources of the BMP pathway ligand, Dpp. Thus, interference with hemocyte secretion of collagen IV, whether by preventing hemocyte migration, by preventing their attraction to the tubules, or by abrogating hemocyte expression and/or processing of collagen IV, results in failure of BMP pathway activation in tubule cells and consequent misrouting of the anterior tubules. The tissue interactions that govern the guided outgrowth of the anterior tubules are summarized in Tissue Interactions Underlie Anterior Tubule Morphogenesis (Bunt, 2010).

As the tubules elongate, a distinct but dynamic subset of cells in the kink region responds sequentially to Dpp guidance cues from dorsal epidermal cells, the midgut, and, more anteriorly, gastric cecal visceral mesoderm and leads forward extension. Activation of the pathway targets, pMad and Dad, in these leading cells ensures that as the tubules project through the body cavity they take a stereotypical route. Loss of Dpp expression in the midgut or repression of BMP signaling in the tubules leads to stalling of their forward movement. Misexpression of Dpp is sufficient to cause tubule misrouting, in which the kink regions project toward the ectopic source. In accordance with these findings defective tubule morphogenesis has been described in embryos lacking the BMP receptors Thick veins (type 1) or Punt (type II), as well as in embryos mutant for schnurri, which encodes a pathway transcriptional regulator shown to be active during embryogenesis (Bunt, 2010 and references therein).

Strikingly only cells in the kink show pathway activation. The current evidence suggests that leading kink cells respond directionally to local gradients of Dpp and that they receive the highest level of ligand, which would account for the restricted domain of activation. However, as the kink region extends beyond the Dpp source, more posterior cells experience high levels of signal but show no pathway activation, indicating that other factors must differentiate between the leading and trailing cells. Segregation into leading and following populations is a common feature of collective cell migration and tubule branching and extension during organogenesis. Leading cells in outgrowing Drosophila trachea, migrating border cells and mammalian ureteric bud formation show distinct patterns of gene expression, respond differentially to external signals, and may repress pathway activation in their neighbors. Thus, tubule kink cells could themselves restrict the domain of pathway response (Bunt, 2010)

As well as their roles in determining cell fate, survival, and growth in Drosophila, TGF-β superfamily signals regulate tissue morphogenesis and have been shown to influence the invasive behavior of metastatic tumors. This study shows, through loss- and gain-of-function analysis, that Dpp also acts as a chemoattractant during organogenesis to determine the path of renal tubule extension though the body cavity. TGF-β superfamily signaling can induce epithelial-to-mesenchymal (EMT) transition through the expression of Snail- and ZEB-family members, which act to repress cell adhesion and polarity, leading to increased motility and, in the case of cancers, to single-cell metastatic activity. Such changes in kink cells could explain their role in pathfinding. However, recent evidence suggests that collective cell migration of epithelial tissues can occur without full EMT and kink cells remain polarized, ensheathed in ECM during tubule elongation (Bunt, 2010).

Ninov (2010) has shown that pathway activation through pMAD leads to increased actin dynamics and E-cadherin turnover in outgrowing histoblasts, resulting in reduced cell adhesion and enhanced cell motility through filopodial/lamellipodial extensions. The current results reveal similar lamellipodial extensions in kink cells, in line with Vasilyev (2009), who demonstrated directional basal lamellipodia in cells of the extending pronephric tubules of zebrafish. It is possible that the production of lamellipodia and tubule navigation also depends on Mad-independent effects on cytoskeletal regulators such as cdc42 (Bunt, 2010).

The current analysis reveals that deposition of ECM is a prerequisite for BMP signaling in tubule guidance. TGF-β/BMP signaling can be modified both by soluble ECM components such as HSPGs and also by architectural, fibrillar elements. The current evidence indicates that for normal tubule outgrowth collagen IV is the crucial component of the BM; it is deposited before tubule elongation (cf. perlecan deposited after elongation), is uniquely contributed by the hemocytes (the tubules express laminins as well as the hemocytes), and the effects of collagen IV loss of function mimic the failure of hemocyte migration to the tubules (whether in collagen IV mutants or in embryos lacking the function of lysyl hydroxylase or dSparc, factors that are required for normal collagen IV processing and deposition) (Bunt, 2010).

Collagen IV sharpens the dorsoventral gradient of BMP signaling in early Drosophila embryos through enhanced ligand-mediated activation (Wang, 2008), which depends on a conserved BMP-binding domain in the C-terminal region of collagen IV. Wang (2008) propose a two-step process in which the binding of Dpp/Screw ligand hetereodimers to collagen IV facilitates the formation of a complex between Dpp/Scw dimers, Sog, and Tsg. Tolloid cleavage of the complex releases ligand dimers, which become active on rebinding to collagen IV dorsally where Sog is absent. This study now shows that basement membrane collagen IV also acts during organogenesis to facilitate BMP signaling in a specialized region of tubule cells. Whereas the mechanism of activation could be as outlined by Wang (2008), early requirements for Dpp signaling in tubule development (Hatton-Ellis, 2007) complicate further analysis (Bunt, 2010).

Although the forward extension of the anterior tubules is important for their morphogenesis, it is likely that other factors regulate their navigation through the body cavity. The kink region dips ventrally and the distal tips extend dorsally late in embryogenesis so that specialized cells at the distal tip contact dorsal structures. Further, morphogenesis of the posterior tubules is unaffected by the repression of BMP signaling; they migrate posteriorly, crossing the hindgut and adopt their normal position in the body cavity, with their tip cells contacting hindgut visceral nerves. It is probable that the coordination of multiple inputs controls the morphogenetic movements of all four tubules (Bunt, 2010).

This study has highlighted the importance of multiple tissue interactions in the outgrowth of Drosophila renal tubules, between the tubules and hemocytes, and, as a consequence of this interaction, with guidepost tissues such as the midgut visceral mesoderm. Similar interactions occur during the specification and recruitment of renal tubule cells, in the branching of the ureteric bud and in the formation of the glomerulus. In vertebrate nephrogenesis kidney medullary and cortical tubules extend, taking up stereotypical positions with respect to blood vessels, with which they later interact to maintain tissue homeostasis. TGF-β superfamily signaling plays multiple roles early in vertebrate kidney development so that analysis of signaling during renal tubule morphogenesis requires conditional alleles or specialized reagents. Such studies reveal requirements for TGF-β superfamily signaling in the morphogenesis of the pronephric tubules and duct in Xenopus, and for the maintenance and morphogenesis of mammalian nephrogenic mesenchyme. VEGF is expressed in early renal mesenchyme and ureteric bud and later in glomeruli, where it is essential for glomerular capillary growth. It will be exciting to discover whether a combination of VEGF/PDGF ligands in renal tissues and spatially restricted TGF-beta superfamily guidance cues underpins the coordinated morphogenesis of these spatially linked renal/blood systems, as has now been shown ro occur in Drosophila (Bunt, 2010).

Drosophila spichthyin inhibits BMP signaling and regulates synaptic growth and axonal microtubules

To understand the functions of NIPA1, mutated in the neurodegenerative disease hereditary spastic paraplegia, and of ichthyin, mutated in autosomal recessive congenital ichthyosis, their Drosophila melanogaster ortholog was studied. Spichthyin (Spict) is found on early endosomes. Loss of Spict leads to upregulation of bone morphogenetic protein (BMP) signaling and expansion of the neuromuscular junction. BMP signaling is also necessary for a normal microtubule cytoskeleton and axonal transport; analysis of loss- and gain-of-function phenotypes indicate that Spict may antagonize this function of BMP signaling. Spict interacts with BMP receptors and promotes their internalization from the plasma membrane, implying that it inhibits BMP signaling by regulating BMP receptor traffic. This is the first demonstration of a role for a hereditary spastic paraplegia protein or ichthyin family member in a specific signaling pathway, and implies disease mechanisms for hereditary spastic paraplegia that involve dependence of the microtubule cytoskeleton on BMP signaling (Wang, 2007).

Axonal abnormalities, including impairment of transport, are a hallmark of many neurological and neurodegenerative diseases. These include the hereditary spastic paraplegias (HSPs), a heterogeneous set of diseases characterized by degeneration of corticospinal tract axons and spasticity of the lower extremities. Different forms of the disease are termed either pure or complicated, depending on whether other mainly neurological symptoms are present. The mechanisms of degeneration in HSPs are unknown, but over twenty causative loci (SPG loci) have been mapped and thirteen cloned. Some SPG products are implicated in microtubule function or transport, including the microtubule motor protein kinesin, and the microtubule-severing protein spastin. Since microtubules are the route for fast axonal transport, the most distal portions of axons are likely to be most sensitive to impairments of microtubule function. A second class of SPG products are mitochondrial proteins, but it is not known how mutations in these cause axonal degeneration. A third class of SPG products are apparently associated with endosomes, judged by immunolocalization or the presence of domains such as MIT or FYVE. HSP is also caused by some mutations in the amyotrophic lateral sclerosis gene ALS2, which encodes alsin, a guanine-nucleotide-exchange-factor for the early endosomal GTPase Rab5. However, the mechanism by which impairment of endosomal membrane traffic might cause axonal degeneration is unknown. (Wang, 2007).

One membrane protein encoded by an SPG gene is SPG6, mutations in which cause a dominant pure form of HSP, and which is widely expressed, although enriched in brain tissue. SPG6 is a member of a protein family (Pfam: DUF803) predicted to have between seven and nine transmembrane (TM) domains. Three different amino-acid substitutions are known, one of which is found in ethnically disparate families and another caused by different nucleotide substitutions in the same codon, suggesting a dominant gain-of-function disease mechanism that can be mediated by only a few mutations in the protein. This protein family includes another human disease protein, ichthyin, mutations in which cause autosomal recessive congenital ichthyosis (ARCI), a skin disorder whose cellular basis is not understood. Ichthyin is widely expressed, although with high expression in keratinocytes, and little or no expression in brain, and at least six recessive alleles are known that cause substitutions of mainly conserved amino acids in different parts of the protein. In summary, little is known of the cellular roles of the SPG6 and ichthyin family (Wang, 2007).

To understand the normal role of the SPG6 and ichthyin protein family, and how changes in their function might lead to cellular defects, their Drosophila homolog, spichthyin (Spict) was have studied. Spict shows preferential localization on early endosomes. It regulates growth of the neuromuscular junction (NMJ) presynaptically, by inhibition of BMP (Bone Morphogenic Protein)/TGF-β (Transforming Growth Factor-β) signaling. BMP signaling regulates synaptic growth, function and stabilization at the NMJ. This study shows a novel role for BMP signaling in maintenance of microtubules and axonal transport, and that this function is also inhibited by Spict. These data suggest that Spict inhibits BMP signaling by regulating BMP receptor traffic. These findings provide a cellular role for the Spict family of proteins, and suggest potential mechanisms for the pathology of HSPs and ARCI that include dependence of microtubules on BMP signaling (Wang, 2007).

A BLASTP search using human SPG6 identified one Drosophila homolog, CG12292. A search using CG12292 identified four predicted human proteins that were 40-50% identical to it: SPG6 (NIPA1), NIPA2, ichthyin and NPAL1. Two more distantly related human proteins, NPAL2 and NPAL3 are more closely related to plant and fungal homologs than to CG12292, and probably represent a subfamily lost from the Drosophila lineage. Since Drosophila CG12292 appears orthologous to both SPG6 and ichthyin, it was designated spichthyin (spict).

To generate spict mutant flies, transposase-mediated imprecise excision was used of a P element, EP(2)2202, inserted in the spict 5' untranslated region. One imprecise excision, spict^mut, had lost the entire coding region, and was therefore a null allele of spict. Several precise excision events were recovered; one of these was used as a wild-type control in most subsequent experiments, and is referred to as spict⁺. Homozygous spict^mut flies were viable and fertile, and took about a day longer than spict⁺ flies to reach adulthood (Wang, 2007).

To determine where Spict might act, its expression pattern and subcellular localization was examined. spict mRNA was found ubiquitously during embryogenesis, with elevated expression in some tissues, including CNS and muscles. EGFP-Spict and Spict-EGFP fusions both showed punctate distributions in Drosophila S2 cells, that overlapped substantially with the early endosome compartment detected using anti-Rab5, but showed no striking overlap with the late endosomal/multivesicular body marker Hook, the recycling endosomal marker Rab11, or the late endosomal/lysosomal markers Spinster and LysoTracker. A Spict-mRFP fusion protein also showed a punctate cytoplasmic distribution in wild-type and spict^mut third instar larvae, which also overlapped substantially with Rab5, but not with late endosomal/lysosomal markers, in muscles and NMJs. Trypsin digestion of N-terminally and C-terminally tagged Spict, redistibuted to the plasma membrane by blockage of endocytosis, suggested that the N-terminus of Spict is in the endosome lumen, and its C-terminus in the cytosol. This result is consistent with previous suggestions that Spict family members might either have nine transmembrane domains, or be divergent members of the 7-TM superfamily. Attempts to raise an antibody that recognized endogenous Spict in immunomicroscopy were unsuccessful. However, since Spict-EGFP and EGFP-Spict fusions had apparently identical localizations in S2 cells, the Spict-mRFP fusion could rescue a spict^mut phenotype and cause the same overexpression phenotypes as wild type Spict, these fusions are likely to have the same localization as endogenous Spict (Wang, 2007).

Since tagged Spict proteins localized with Rab5, tests were performed to see whether Rab5 staining is normal when Spict is lacking. Rab5 staining was less intense in spict^mut NMJ boutons compared to wild-type; these phenotypes were rescued by ubiquitous expression of UAS-spict. Rab5 staining was also reduced in muscles but not obviously affected in neuronal cell bodies and axons of spict^mut larvae, or in S2 cells treated by spict RNAi. Therefore, Spict is essential for a normal Rab5 compartment at the NMJ, but not in all situations (Wang, 2007).

One of the signaling pathways with the largest effects on synaptic size at the Drosophila NMJ is the BMP pathway, which stimulates synaptic growth. The expanded NMJ phenotype of spict^mut is similar to that of spinster (also known as benchwarmer), which also shows defects in endosomal-lysosomal trafficking and requires an active BMP/TGF-β signaling pathway for NMJ expansion. It is also similar to the increase in bouton number of highwire NMJs. Highwire encodes a putative E3 ubiquitin ligase that appears to affect multiple signaling pathways including JNK and BMP. To determine whether the synaptic overgrowth of spict^mut larvae requires BMP signaling, key BMP signaling components were genetically removed from spict^mut larvae. Mutations affecting the type I receptor subunits Tkv (Thickvein) and Sax (Saxophone), the type II receptor subunit Wit (Wishful Thinking), the type II receptor ligand Gbb (Glass Bottom Boat), or the co-Smad Med (Medea) all suppressed the NMJ overgrowth of spict^mut larvae. In all cases, the synaptic undergrowth in larvae that were doubly homozygous for spict^mut and BMP pathway mutations was indistinguishable from that of homozygous BMP pathway mutations alone. In addition, all heterozygous BMP pathway mutations tested partly suppressed the NMJ expansion of spict^mut larvae, but had no effect on NMJ bouton number in a wild type background. Therefore, BMP signaling is essential for the excessive NMJ growth of spict^mut larvae (Wang, 2007).

The contrasting phenotypes of spict^mut and loss of BMP signaling, and the genetic interactions between spict and BMP signaling mutants, suggest that Spict antagonizes BMP signaling in the control of NMJ growth. Nevertheless, alternative models are possible: for example, highwire mutations interact with BMP signaling mutations, but Highwire affects synaptic size primarily through a MAPK signaling pathway. However, evidence strongly supports a direct effect of Spict on BMP signaling. During BMP signaling in neurons, the R-Smad protein Mad is phosphorylated by active BMP receptors, and phosphorylated Mad (PMad) is then translocated to the nucleus and acts as a transcription factor. At the NMJ, PMad overlaps mainly with the presynaptic marker cysteine string protein (CSP), but also with the largely postsynaptic marker Discs-large (Dlg). PMad is also found in cell body nuclei in the larval CNS. PMad levels were significantly higher in spict^mut than in spict⁺ larvae, both at the NMJ and in CNS cell bodies, and this phenotype was fully rescued by neuronal expression of UAS-spict. Therefore, BMP signaling is upregulated at spict^mut neurons, in contrast to highwire neurons. Next the possibility of upregulation of BMP receptors at spict^mut NMJs was tested. HA-tagged Tkv was found mainly in a punctate distribution in the periphery of synaptic boutons, at or close to the plasma membrane, and at higher levels in spict^mut than in spict⁺ boutons. Wit was barely detectable in spict⁺ boutons, but was present at higher levels in spict^mut boutons, also in a punctate pattern mainly at or close to the plasma membrane. The effect of spict^mut on Tkv-HA and Wit levels was rescued by neuronal expression of UAS-spict. No effect was found of spict^mut on levels of other neuronal membrane proteins (Fasciclin II, Syntaxin), or on the neuronal surface antigen recognized by anti-Horseradish Peroxidase (HRP) at the NMJ. Therefore, Spict action specifically lowers the levels of BMP receptors at the presynaptic NMJ (Wang, 2007).

The opposing effects of Spict and BMP signaling on NMJ and neuronal microtubules suggest that Spict is a novel antagonist of BMP signaling. BMP signaling acts both presynaptically and postsynaptically at the NMJ; rescue experiments show that Spict acts presynaptically to regulate NMJ expansion. The data suggest a direct effect of Spict on the presynaptic BMP signaling machinery. First, elevated levels of PMad and BMP receptors are seen at spict^mut NMJs. Second, Spict can be co-immunoprecipitated with Wit. Third, Spict shows partial colocalization with the BMP receptors Tkv-HA or Wit at NMJ boutons. Fourth, Spict promotes relocalization of Wit from the surface of S2 cells to the Rab5 early endosomal compartment. Therefore, these data suggest strongly that Spict antagonizes BMP signaling by regulating its receptor traffic. This is in contrast to Highwire - while synaptic overgrowth in highwire mutants can be suppressed by BMP signaling mutants, the highwire phenotype is more completely suppressed by loss of the Wallenda MAP kinase kinase kinase, and there is no apparent upregulation of PMad in highwire mutants (Wang, 2007).

The posterior crossveinless phenotype in some spict^mut adult wings is also typical of reduced BMP signaling in pupal wing discs. At first sight a crossveinless phenotype is inconsistent with Spict being an antagonist of BMP signaling. However, lowered BMP signaling in the posterior crossvein primordium could be due not only to direct downregulation of signaling, but also to upregulation of receptors that reduces diffusion of BMP ligands. No changes were detected in the level of BMP signaling about the time when the posterior crossvein primordium develops, but this could be due to either the partial penetrance of the phenotype, or the robustness of the regulatory and feedback mechanisms that translate smooth gradients of BMP ligands into more sharply defined developmental features (Wang, 2007).

How might an endosomal protein regulate BMP signaling? Membrane trafficking from the plasma membrane to lysosomes regulates many signaling pathways including BMP/TGF-β. For example, mutations that impair endosome to lysosome traffic cause an increase in BMP signaling, in at least some cases accompanied by increased levels of Tkv. However, the predominant localization of Spict on early endosomes, and its ability to internalize Wit to this compartment suggest that Spict functions at some step of plasma membrane to endosome traffic. (1) Rab5 compartments fail to accumulate at spict^mut NMJs, rather than enlarge as in Hrs mutants. (2) Spict overexpression in S2 cells redistributes Wit mainly to early endosomes, rather than to late endosomes or lysosomes. (3) There is no obvious degradation of Wit in Spict-overexpressing cells that internalize Wit, suggesting that Spict does not directly target Wit for degradation, at least in S2 cells. While levels of BMP receptors are elevated locally in NMJ boutons that lack Spict, this could be either to altered trafficking or degradation, and BMP signaling in S2 cells can be affected by Spict, without detectable changes in levels of BMP receptors. Therefore, Spict might inhibit BMP signaling by internalizing vacant receptors and thus preventing them from responding to ligand; since clathrin RNAi treatment redistributes Spict to the plasma membrane, Spict probably appears at least transiently at the plasma membrane. However, more complex models are possible. For example, Spict might sequester BMP receptors in a compartment from which they cannot signal; Notch receptors apparently have to reach a specific endosomal compartment before they can signal (Wang, 2007).

By studying Spict, this study has identified a role for BMP signaling in maintenance of axonal microtubules. Notably, local loss of presynaptic microtubules has also been seen in loss of BMP signaling at the NMJ, and apical microtubule arrays are eliminated in tkv mutant clones in wing imaginal discs. Since BMP signaling promotes synaptic growth and synaptic strength at the NMJ, it would be logical for it also to stimulate the additional transport of materials and organelles that a larger more active synapse requires (Wang, 2007).

If human SPG6 alleles are dominant gain-of-function, then the HSP that they cause would resemble the situation of Spict overexpression in Drosophila, and axonal degeneration in HSP could then be caused by inhibition of BMP signaling, loss of axonal microtubules, and impaired axonal transport. Given the effect of BMP signaling on axonal microtubules, other HSP gene products apart from SPG6 may affect BMP signaling and thus maintenance of axonal microtubules. (Wang, 2007).

In contrast to SPG6, ARCI appears to be caused by loss of ichthyin function (Lefevre, 204). Identification of a role for the ichthyin ortholog Spict in inhibiting BMP signaling suggests upregulation of BMP signaling as a possible disease mechanism in ARCI. Indeed, the BMP-like ligand TGF-β1 has complex roles in maintenance of skin, and its overexpression can cause psoriasis, a condition that bears some resemblance to ichthyosis. Inhibitors of BMP signaling may therefore be candidates for therapeutic purposes in ARCI or similar conditions. (Wang, 2007).

In conclusion, this study has established a cellular role for the SPG6 and ichthyin family of proteins, thus identifying a novel group of players in BMP signaling, and providing a framework for future understanding of diseases caused by mutations that affect these proteins (Wang, 2007).

DPP-mediated TGFβ signaling regulates juvenile hormone biosynthesis by activating the expression of juvenile hormone acid methyltransferase

Juvenile hormone (JH) biosynthesis in the corpus allatum (CA) is regulated by neuropeptides and neurotransmitters produced in the brain. However, little is known about how these neural signals induce changes in JH biosynthesis. This study reports a novel function of TGFβ signaling in transferring brain signals into transcriptional changes of JH acid methyltransferase (jhamt), a key regulatory enzyme of JH biosynthesis. A Drosophila genetic screen identified that Tkv and Mad are required for JH-mediated suppression of broad (br) expression in young larvae. Further investigation demonstrated that TGFβ signaling stimulates JH biosynthesis by upregulating jhamt expression. Moreover, dpp hypomorphic mutants also induces precocious br expression. The pupal lethality of these dpp mutants is partially rescued by an exogenous JH agonist. Finally, dpp is specifically expressed in the CA cells of ring glands, and its expression profile in the CA correlates with that of jhamt and matched JH levels in the hemolymph. Reduced dpp expression was detected in larvae mutant for Nmdar1, a CA-expressed glutamate receptor. Taken together, it is concluded that the neurotransmitter glutamate promotes dpp expression in the CA, which stimulates JH biosynthesis through Tkv and Mad by upregulating jhamt transcription at the early larval stages to prevent premature metamorphosis (Huang, 2011).

The functions of the TGFβ superfamily and other morphogens in regulating insect metamorphosis are rarely reported. In two independent genetic screens, it was discovered that Drosophila TGFβ signaling controls two different aspects of insect metamorphosis. In a previous study, it was found that Baboon (Babo) and dSmad2-mediated TGFβ signaling regulates larval neuron remodeling, which is part of the insect central nervous system metamorphosis induced by 20E during the pupal stage. Further investigation revealed that Babo/dSmad2-mediated TGFβ signaling controls larval neuron remodeling through regulating the expression of EcR-B1, a specific isoform of the 20E receptor (Huang, 2011).

This paper reports several findings. First, br is precociously expressed in 2nd instar tkv and Mad mutant larvae. Second, the precocious br expression phenotype in tkv and Mad mutant larvae can be suppressed by exogenous JH agonist (JHA). Third, Tkv and Mad repressed br expression in a non-cell-autonomous manner. Fourth, the presence of Mad in the CA is sufficient to repress br expression in the fat body (FB). Fifth, jhamt mRNA levels and JHAMT activity were significantly reduced in the Mad-deficient larvae. These results demonstrate that Tkv- and Mad-mediated signaling is required in the CA to activate jhamt expression and thus JH biosynthesis, which in turn controls insect metamorphosis (Huang, 2011).

The Drosophila genome encodes two TGFβ type II receptors, Punt (Put) and Wishful thinking (Wit). The genetic screen failed to identify a role for either of these receptors in the regulation of JH biosynthesis. Put and Wit are most probably functionally redundant in this biological event, as in the case of TGFβ-mediated mushroom body neuron remodeling (Huang, 2011).

Dpp is a key morphogen that controls dorsal/ventral polarity, segmental compartment determination and imaginal disc patterning. Dpp function usually depends on its gradient distribution. In an attempt to identify the ligand for Tkv/Mad-mediated TGFβ signaling in the CA, a novel, gradient-independent role for Dpp was discovered that controls JH biosynthesis. Dpp is the ligand of Tkv, which regulates jhamt transcription. Loss of Dpp, even RNAi reduction of Dpp in the CA specifically, causes precocious br expression at the early larval stages, which phenocopies tkv and Mad mutants. Phenotypes of dpp, including precocious br expression and lethality, are at least partially rescued by JHA treatment or ectopic jhamt expression in the CA. Notably, dpp-lacZ is strictly expressed in the CA cells, but not in the other two types of endocrine cells in the ring gland: the prothoracic gland and corpus cardiacum cells. The developmental expression profile of dpp in the CA is always consistent with that of jhamt. Finally, dpp expression in the CA may be directly controlled by neurotransmitter signals in the brain, which is supported by reduced dpp and jhamt transcription levels in the Nmdar1 mutant wandering larvae (Huang, 2011).

Several lines of evidence suggest that Met is a crucial regulator at or near the top of a JH signaling hierarchy, possibly acting as a JH receptor. However, null Met mutants of Drosophila are completely viable, which is unexpected if Met is a JH receptor. A recent investigation indicated that another Drosophila bHLH-PAS protein, Germ cell-expressed (Gce), which has more than 50% homology to Met, may function redundantly to Met in transducing JH signaling (Baumann, 2010). Because Met is on the X chromosome in the fly genome, it was not covered by the genetic screen. The br protein in the FBs of a Met null allele, Met²⁷, was tested at the 2nd instar larval stage, and precocious br expression was observed. Importantly, this precocious br expression phenotype could not be suppressed by exogenous JHA. This result not only supports the previous reports regarding the function of Met in transducing JH signaling but also suggests that the precocious br expression is a more sensitive indicator for the reduced JH activity in Drosophila compared with precocious metamorphosis, lethality and other phenotypes (Huang, 2011).

Kr-h1 was reported to act downstream of Met in mediating JH action. Studies in both Drosophila and Tribolium reveal that, at the pupal stages, exogenous JHA induces Kr-h1 expression, which in turn upregulates br expression. The genetic screen successfully identified that Kr-h1 is cell-autonomously required for the suppression of br expression at young larval stages. Precocious br expression occurred in the FBs of Kr-h1 mutants and was not suppressed by JHA treatment. Therefore, these studies further suggest that Kr-h1 functions as a JH signaling component in mediating insect metamorphosis. However, the finding shows that, at the larval stages of Drosophila, the JH-induced Kr-h1 suppresses, rather than stimulates, br expression. This result is consistent with the facts that Kr-h1 functions to prevent Tribolium metamorphosis and Br is a crucial factor in promoting pupa formation (Huang, 2011).

In summary, this study has found a novel function of Dpp, Tkv and Mad-mediated TGFβ signaling in controlling insect metamorphosis. As summarized in a model, the brain sends neurotransmitters, such as glutamate, to the CA through neuronal axons. Glutamate interacts with its receptor (NMDAR) on the surface of CA cells to induce dpp expression. Dpp protein produced and secreted by CA cells forms a complex with TGFβ type I receptor (Tkv) and type II receptor on the membrane of CA cells, followed by phosphorylation and activation of Tkv. Activated Tkv in turn phosphorylates Mad, which is imported into the nucleus together with co-Smad and stimulates jhamt expression. JHAMT in CA cells transforms JH acid into JH, which is released into hemolymph. The presence of JH in young larvae prevents premature metamorphosis through Met/Gce and Kr-h1 by suppressing the expression of br, a crucial gene in initiating insect metamorphosis (Huang, 2011).

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