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

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

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

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

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

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

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

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).

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).

Regarding piRNA production, the results revealed that among the known piRNA machinery components, ago3, krimp, mael, and zuc genes are the major piRNA-related targets of Egg. Consistent with the previously suggested role of Egg for promoting piRNA production, all of the putative target genes were downregulated in egg mutant ovaries. In Drosophila, two piRNA processing pathways, primary processing and secondary processing, have been proposed; the primary processing pathway functions in both germline and somatic cells by processing precursor piRNAs into piRNAs whereas the secondary processing pathway functions only in germline cells in which piRNAs are amplified by the ping-pong cycle. Given the involvement of Egg in both germline and somatic piRNA production, tests were performed to see whether decreased expression of zuc is responsible for the ovarian phenotypes caused by loss of Egg. Phenotypic changes were investigated after introduction of wild-type zuc transgenes under the control of the actin5C-Gal4 driver into egg mutant background, but the ovaries of the genotypes (egg²¹³⁸/Df(2R)Dll-Mp; HA-tagged zuc (or EGFP-tagged zuc)/actin5C-Gal4) did not show any significant phenotypic changes compared with those of egg mutants. Ago3 is essentially involved in the secondary processing pathway along with Aub. The nuage, which surrounds the nuclei of nurse cells, has been proposed as a site for the ping-pong cycle. Various types of proteins, including Ago3, Krimp, and Mael, have been identified as nuage components. The decreased expression levels of the particular nuage components in egg mutant ovaries suggest that the reduction of germline piRNAs in egg mutants may result from not only a reduction of precursor piRNA transcription, as proposed previously, but also from a failure of the piRNA amplification pathway (Kang, 2018).

The data also revealed a previously unknown role of Egg as an lncRNA regulator. In Drosophila, the existence of lncRNAs has long been known, but only a few lncRNAs have been investigated. This study revealed that Egg is involved in regulating the expression of 100 potential lncRNA genes, and appears to play a repressive role in the expression of these lncRNAs. Among the upregulated lncRNAs in egg mutant ovaries, two well-known heat-inducible lncRNAs, αγ-element and hsr-ω are located in genomic regions where numerous TEs are found. Given the involvement of Egg in regulating piRNA production and thus TE mobilization, this raises an intriguing possibility of a link between the upregulation of αγ-element and hsr-ω in egg mutant ovaries and their genomic locations as TE hotspots. A strong upregulation of pncr003:2L and pncr004:X by loss of Egg is noteworthy, but the functional significance of these lncRNAs in the Drosophila ovary has not been determined. Although attempts were made to knock down pncr003:2L and pncr004:X using transgenic flies containing dsRNA for RNAi of pncr003:2L or pncr004:X under the control of the act5C-Gal4 driver, no phenotypic changes during oogenesis were detected. Interestingly, pncr003:2L was originally annotated as an lncRNA, but it was recently reported to encode two small functional peptides that are involved in regulating calcium transport in the Drosophila heart. Determination of the function of pncr003:2L in the Drosophila ovary is an interesting issue that needs to be addressed (Kang, 2018).

This study has demonstrated that dpp and dally were strongly upregulated in egg mutant ovaries and that dpp knockdown in ECs and early follicle cells in egg mutant background resulted in an increase in the size of the ovaries. Previously, by analysis of egg-RNAi knockdown in ECs, an EC-specific requirement for egg in controlling germ cell differentiation was suggested. Moreover, germ cell differentiation defects caused by egg knockdown in ECs was attributed to an increase in Dpp signaling because removal of one copy of dpp partially suppressed the tumorous phenotype caused by egg knockdown in ECs. The expression level of dally, an enhancer of Dpp signaling, may be maintained at a high level in the dpp knockdown in egg mutant background; therefore, the enhancement of Dpp signaling caused by loss of Egg may be maintained in the dpp knockdown in egg mutant germarium, thereby exhibiting similar differentiation defects as those observed in the egg mutant germarium. However, the defective cells in the dpp-knockdown in egg mutant background could be maintained for a longer time, which may be attributed to a reduction of the enhanced apoptosis in egg mutant ovaries because the increased expression levels of rpr and hid in egg mutant ovaries were significantly reduced in the dpp-knockdown in egg mutant background. Given that Egg is broadly expressed in germ cells and somatic cells in the ovary during oogenesis, dpp knockdown only in ECs and early follicle cells may not be sufficient to consistently counteract the overall apoptosis-promoting effect caused by dpp upregulation in egg mutant ovaries, which may explain the relatively low occurrence of the effect of the dpp knockdown in ECs and early follicle cells on the increase in ovary size (Kang, 2018).

A model is proposed in which loss of Egg may initially cause a relatively mild level of ectopic dpp and dally overexpression that may be sufficient to cause germ cell differentiation defects through repression of bam. Apoptosis may then be initiated when the level of ectopic dpp overexpression reaches a certain threshold level capable of inducing rpr and hid upregulation perhaps through a dpp positive-feedback loop. Alternatively, it cannot be ruled out that Egg represses dpp, dally, rpr and hid separately although the possibilities are not necessarily exclusive. Further studies are needed to determine the exact molecular mechanism by which Dpp signaling is increased in egg mutant ovaries. Given the role of BMPs in many mammalian stem cell systems and the existence of mammalian homologues of Egg and Dpp, the role of Egg in regulating Dpp signaling may provide important insights into their potential roles in mammalian stem cells (Kang, 2018).

One additional interesting non-autonomous phenomenon observed is that sometimes when a region of the wing disc has high pS6 levels, the rest of the disc loses its typically patchy pS6 pattern and becomes pS6 negative. This phenomenon is not understood, and future work will be necessary to understand it molecularly (Romero-Pozuelo, 2017).

Visual motion detection in sighted animals is essential to guide behavioral actions ensuring their survival. In Drosophila, motion direction is first detected by T4/T5 neurons. Their axons innervate one of the four lobula plate layers. How T4/T5 neurons with layer-specific representation of motion-direction preferences are specified during development is unknown. This study shows that diffusible Wingless (Wg) between adjacent neuroepithelia induces its own expression to form secondary signaling centers. These activate Decapentaplegic (Dpp) signaling in adjacent lateral tertiary neuroepithelial domains dedicated to producing layer 3/4-specific T4/T5 neurons. T4/T5 neurons derived from the core domain devoid of Dpp signaling adopt the default layer 1/2 fate. Dpp signaling induces the expression of the T-box transcription factor Optomotor-blind (Omb), serving as a relay to postmitotic neurons. Omb-mediated repression of Dachshund transforms layer 1/2- into layer 3/4-specific neurons. Hence, spatio-temporal relay mechanisms, bridging the distances between neuroepithelial domains and their postmitotic progeny, implement T4/T5 neuron-subtype identity (Apitz, 2018).

Visual signals received by the retina are generally not stationary because objects in the environment and/or the bodies of animals move. To detect motion, visual circuits perform complex spatio-temporal comparisons that convert luminance changes collected by photoreceptors into signals containing information about direction or speed. Despite the seemingly divergent anatomy of vertebrate and insect visual systems, they display remarkable parallels in the computations underlying motion vision and the neuronal elements performing them. In most sighted animals, this involves neurons that respond to motion signals in specific directions. Direction-selectivity emerges from differences in the connectivity of their dendrites. Motion-direction preferences by their axons are represented by layer-specific innervation. Thus, anatomical characteristics such as layer-specificity seem to be intricately linked with motion-directionality. However, how these are implemented during circuit development is poorly understood (Apitz, 2018).

The Drosophila visual system has emerged as a powerful model for elucidating the neural circuits and computations underlying motion detection. Photoreceptors (R-cells) in the retina extend axons into the optic lobe consisting of the lamina, medulla, lobula plate, and lobula. Neuronal projections in these ganglia are organized into retinotopically arranged columnar units. The medulla, lobula plate, and lobula are additionally subdivided into synaptic layers. They are innervated by more than a 100 neuronal subtypes that extract different visual features in parallel pathways. T4 and T5 lobula plate neurons are the first direction-selective circuit elements. Each optic lobe hemisphere contains ~5300 T4/T5 neurons. T4 dendrites arborize within medulla layer 10, and T5 dendrites in lobula layer Lo1. Their axons project to one of the four lobula plate layers, thereby defining four different neuron subtypes each. Axons segregate according to their motion-direction preferences. Thus, front-to-back, back-to-front, upward, and downward cardinal motion directions are represented in lobula plate layers. T4 neurons are part of the ON motion detection pathway reporting brightness increments, while T5 neurons are part of the OFF pathway reporting brightness decrements. Distinct neuron sets in the lamina and medulla relay ON and OFF information to T4 and T5 neurons. Direction-selectivity emerges within T4/T5 dendrites and involves the non-linear integration of input from these upstream neurons for enhancement in the preferred direction and suppression in the null-direction. Dendritic arbors of the four T4 neuron subtypes have characteristic orientations, that correlate with the direction preferences of lobula plate layers innervated by their axons. Thus, direction-selectivity involves the establishment of neuron subtypes, each with distinct spatial connectivities. This study addresses when and how T4 and T5 neuron subtypes with different layer identities are specified during development (Apitz, 2018).

Optic lobe neurons originate from two horseshoe-shaped neuroepithelia, called the outer and inner proliferation centers (OPC and IPC). These are derived from the embryonic optic lobe placode and expand by symmetric cell divisions during early larval development. At the late 2nd instar larval stage, neuroepithelial (NE) cells from the medial OPC edge begin to transform into medulla neural stem cells, called neuroblasts (Nbs). These undergo asymmetric divisions to self-renew and give rise to ganglion mother cells (GMCs), which divide to generate two neurons or glia. Apposing the OPC, two dorsal and ventral NE domains, called the glial precursor cell (GPC) areas, produce neuron subtypes associated with all ganglia. At the mid 3rd instar larval stage, the lateral OPC begins to generate lamina neurons (Apitz, 2018).

The IPC generates lobula and lobula plate neurons, including T4/T5 neurons from the early 3rd instar larval stage onward. Recent studies showed that NE cells in one domain, the proximal (p-)IPC, convert into progenitors in an epithelial-mesenchymal transition (EMT)-like process. Progenitors migrate to a second proliferative zone, the distal (d-)IPC, where they mature into Nbs. These transition through two competence windows to first produce C and T neurons, corresponding to C2 and C3 ascending neurons connecting the medulla and lamina, as well as T2/T2a and T3 neurons connecting the medulla and lobula, and then T4/T5 lobula plate neurons. Cross-regulatory interactions between Dichaete (D) and Tailless (Tll) control the switch in Nb competence defined by the sequential expression of the proneural bHLH transcription factors Asense (Ase) and Atonal (Ato). The latter is co-expressed with the retinal determination protein Dachshund (Dac). The molecular mechanisms that control layer-specific T4/T5 neuron subtype identities within this sequence of developmental events occurring at different locations have remained elusive (Apitz, 2018).

T4/T5 neuron diversity resulting in differential layer-specificity could be achieved by postmitotic combinatorial transcription factor codes upstream of distinct guidance molecules. Although not mutually exclusive, layer-specificity of T4/T5 neurons could also be determined by temporal differences in the expression of common postmitotic determinants, similar to the birth-order dependent R-cell growth cone segregation strategy described in the medulla. This study provides evidence for another mechanism, whereby layer-specific T4/T5 neuron subtype identity is determined early in the p-IPC neuroepithelium. Their specification depends on two relay mechanisms involving Wnt and Bone morphogenetic protein (Bmp) signaling and transcription factor interactions. These establish and translate the spatial patterning of NE cells into postmitotic neuronal subtype identities to bridge distances inherent to this particular neurogenesis mode (Apitz, 2018).

The spread of Wg is dispensable for patterning of many tissues. However, this study uncovered a distinct requirement for diffusible Wg in the nervous system, where it orchestrates the formation of T4/T5 neurons innervating lobula plate layers 3/4. Their generation depends on inductive mechanisms that are relayed in space and time. The spatial relay consists of a multistep-signaling cascade across several NE domains: Wg from the GPC areas induces wg expression in the s-IPC and Nb lineage adjacent to ventral and dorsal p-IPC subdomains; this secondary Wg source activates dpp expression. Dpp signaling mediates EMT of migratory progenitors from these subdomains. The p-IPC core produces Dac-positive layer 1/2 specific T4/T5 neurons. Dpp signaling in p-IPC NE subdomains triggers a temporal relay across intermediate cellular states by inducing omb. Omb in turn suppresses Dac, conferring layer 3/4 identity to postmitotic T4/T5 neurons (Apitz, 2018).

When Wg is membrane-tethered, the first step of this cascade is disrupted. This defect is not caused by decreased signaling activity of NRT-Wg protein in wg{KO;NRT-wg} flies. First, wild-type Wg signaling activity inside the GPC areas and the adjacent OPC was not affected. Second, in allele switching experiments, ectopic expression of a highly active UAS-NRT-wg transgene in the GPC areas was unable to rescue. By contrast, restoring wild-type wg function in the GPC areas was able to rescue, supporting the notion that Wg release and spread from the GPC areas are required to induce its own expression in the s-IPC and the Nb clone (Apitz, 2018).

Although Wg release is essential, the range of action is likely limited. Wg expression in the s-IPC commences in early 3rd instar larvae, when it is still in close proximity with the GPC. Half of the wg{KO;NRT-wg} flies showed residual dpp expression in one progenitor stream at the 3rd instar larval stage and a 25% reduction of T4/T5 neurons, correlating with three lobula plate layers in adults. The other half lacked dpp-lacZ expression and showed a 50% reduction of T4/T5 neurons correlating with two remaining layers. While this partial phenotypic penetrance is not fully understood, NRT-Wg likely partially substituted for Wg because of the initial close proximity of the GPC areas and the s-IPC and Nb clone. Occasional residual NRT-Wg expression in the s-IPC argues against an all-or-nothing inductive event and suggests a model, whereby cell-intrinsic signaling thresholds have to be reached. Theoretically, the dpp expression defect in the p-IPC of wg{KO;NRT-wg} flies could reflect the dependence on long-range Wg from the GPC areas. However, as this study has shown, IPC-specific wg knockdown leads to dpp loss in the p-IPC. Propagation of sequential Wnt signaling could explain long-range activities. Moreover, sequentially acting primary and secondary sources of Wg have been described in the developing Drosophila eye, suggesting that the regulatory mechanism observed in the optic lobe might be employed in several contexts. The different outcomes of early and late allele wg to NRT-wg allele switching indicate that Wg secretion is required for the induction but not long-term maintenance of wg expression in the s-IPC. The GPC areas become rapidly separated from the s-IPC and Nb clone by compact rows of newly generated neurons. As part of a relay system, diffusible Wg may therefore be required to bridge distances over a few cell diameters during the initial phase of neurogenesis. The s-IPC in wg{KO;NRT-wg} flies expressed Hth and generated two neuron clusters as in wild-type. Thus, the sole function of wg in the s-IPC is to relay the GPC-derived Wg signal to induce dpp expression in the p-IPC. Since Wg release is not required in the GPC areas to induce dpp in the adjacent OPC, this secondary wg function in the s-IPC is most likely juxtacrine (Apitz, 2018).

Compared to approximately 80 medulla neuron subtypes derived from the OPC, the specification of 13 distinct subtypes originating from the p-IPC appears simple. However, the distinct mechanisms employed are surprisingly complex. Previous work has shown that cross-regulatory interactions between D and tll regulate a Nb competence switch from generating early-born C2, C3, T2, T2a, and T3 neurons to eight distinct layer-specific T4/T5 subtypes. Ato and Dac are expressed in the second Nb competence window and depend on tll. Functional studies showed that dac mutant T4/T5 neurons adopted early-born T2/T3 neuron-like morphologies. Similarly, ato mutant T4/T5 neurons displayed neurite connectivity defects. Notably, simultaneous knockdown of dac and ato resulted in the absence of T4/T5 neurons, demonstrating that both are required together for the ability of d-IPC Nbs to produce new neuron subtypes in the second competence window (Apitz, 2018).

Dac is initially expressed in all T4/T5 neurons but only maintained in layer 1/2 innervating subtypes. This suggests that an essential step for the specification of layer 3/4 innervating neurons is the downregulation of Dac and the suppression of the T4/T5 default neuron fate, i.e., layer 1/2 identity. Although the mode of this inhibitory mechanism depends on the outcome of the Nb-specific switching mechanism in the d-IPC, it is already primed in p-IPC NE cells. Thus, layer-specificity and therefore motion-directionality are determined early in the NE precursors of T4/T5 neurons. Molecularly, it involves the Omb-mediated relay of Dpp-signaling-dependent NE cell patterning information across intermediate cell states to postmitotic T4/T5 neurons resulting in the repression of Dac. In contrast to the OPC, this study found no link between NE patterning in the p-IPC and Notch-dependent differential apoptosis of region-specific T4/T5 subtypes. Instead, Notch controls the choice between T4 and T5 identity, likely during the second competence window, indicating that the distinction between layer 1/2 and 3/4 fates precedes T4 and T5 neuron specification (Apitz, 2018).

The mechanisms controlling the maintenance of omb expression, and Omb-mediated downregulation of Dac are unclear. Hypotheses regarding the latter have to be reconciled with the fact that dac, together with ato, is required for the formation of all T4/T5 neurons and hence is expressed in all d-IPC Nbs during the second competence window. Omb and Dac are initially co-expressed in Nbs and young T4/T5 neurons, suggesting that Omb does not directly repress dac transcription. Yet, expression of the dacp7d23 enhancer trap Gal4 line showed that dac is only transcribed in layer 1/2 neurons in adults. A possible scenario is that Omb could break Dac autoregulation by triggering degradation of Dac. Since T-box genes can act as transcriptional activators and repressors and their effects are influenced by various co-factors, future studies will need to explore the molecular details underlying Omb-mediated repression of Dac. It will also be important to determine whether layer 3/4 specification is mediated solely by Dac downregulation, or whether omb has additional instructive roles (Apitz, 2018).

Consistent with the observation that C2 and C3 neurons have distinct developmental origins, this study found that Nbs derived from the Dpp-expression domain produce C2 and possibly T2a neurons during the first Nb competence window, while the core p-IPC generates C3, T2, and T3 neurons. dac mutant T4/T5 neurons adopt T2/T3-like morphologies suggesting that this is the default neuron fate in this neuron group. While Omb is maintained in C&T neurons derived from the Dpp-expression domain, Dac is not expressed, suggesting that Omb interacts with other molecular determinants in these neurons. While this study did not explore how layer 1 and 2 neurons or layer 3 and 4 neurons become distinct from each other because of the lack of specific markers, the data suggest a possible contribution of Ato/Dac and Notch signaling, as these are active within the d-IPC. Findings in a concurrent study of Pinto-Teixeira (2018) align with the current data concerning the role of Dpp and Notch signaling. Furthermore, a second study of Mora (2018) reported an additional role for Ato in controlling the transient amplification of d-IPC Nbs by symmetric cell division to ensure that the correct number of T4/T5 neurons is produced. It will be fascinating to identify the transcriptional targets of Notch, Ato/Dac, and Omb that mediate ganglion- and layer-specific targeting of T4/T5 dendrites and axons, respectively. Finally, future behavioral studies of layer 3/4-deficient flies will address to what extent direction selectivity is affected or compensatory mechanisms are in place (Apitz, 2018).

Signaling centers, also called organizers, pattern tissues in a non-autonomous fashion. The vertebrate roof plate and the cortical hem, for instance, both release Wnts and Bmps to pattern NE cells in the developing dorsal spinal cord and in the surrounding forebrain, respectively. In the Drosophila visual system, the GPC areas express wg and pattern the OPC by inducing dpp expression in adjacent dorsal and ventral OPC subdomains. Together with the current insights into the function of GPC-derived wg in IPC patterning and neurogenesis, this firmly establishes the GPC areas as local organizers of optic lobe development. At the onset of neurogenesis, wg is first expressed in the GPC areas followed by the s-IPC, explaining the well-established delay in neurogenesis between the IPC and OPC. Wg release from the GPC areas could coordinate the timely onset of neurogenesis in the OPC and IPC to safeguard the alignment of matching partner neurons across several retinotopically organized neuropils. The intercalation of new-born neurons between both neuroepithelia may have driven the need for a relay system using primary and secondary sources of Wg. Wg induces Dpp to subdivide the adjacent OPC and p-IPC NE into distinct regions as basis for generating neuronal diversity. The temporal relay mediated by Omb represents an efficient strategy to pass the memory of spatial NE patterning information by Dpp signaling on to postmitotic neurons generated at a distance. It is thus intricately tuned to the distinct neurogenesis mode of the p-IPC essential for spatially matching birth-order-dependent neurogenesis between the OPC and IPC. Interestingly, the progressive refinement of NE patterning by the induction of secondary signaling centers plays a central role in vertebrate brain development. Furthermore, similar signaling cascades have been recently identified in mammalian optic tissue cultures where sequential Wnt and Bmp signaling induces the expression of the Omb-related T-box transcription factor Tbx5 to specify dorsal retinal NE cells. Hence, such cascades could represent conserved regulatory modules that are employed repeatedly during invertebrate and vertebrate nervous system development (Apitz, 2018).

The Drosophila wing has served as a paradigm to mechanistically characterize the role of morphogens in patterning and growth. Wingless (Wg) and Decapentaplegic (Dpp) are expressed in two orthogonal signaling centers, and their gradients organize patterning by regulating the expression of well-defined target genes. By contrast, graded activity of these morphogens is not an absolute requirement for wing growth. Despite their permissive role in regulating growth, this study shows that Wg and Dpp are utilized in a non-interchangeable manner by the two existing orthogonal signaling centers to promote preferential growth along the two different axes of the developing wing. The data indicate that these morphogens promote anisotropic growth by making use of distinct and non-interchangeable molecular mechanisms. Whereas Dpp drives growth along the anterior-posterior axis by maintaining Brinker levels below a growth-repressing threshold, Wg exerts its action along the proximal-distal axis through a double repression mechanism involving the T cell factor (TCF) Pangolin (Barrio, 2020).

Two orthogonal signaling centers, corresponding to the AP and DV compartment boundaries and expressing the Dpp and Wg morphogens, regulate growth and patterning of the developing wing along the AP and PD axes, respectively. Whereas graded activity of these morphogens defines the spatial location of longitudinal veins and sensory organs that decorate the adult wing along these two axes, their graded activity is not an absolute requirement for its growth-promoting role. Despite the non-instrumental role of Wg and Dpp gradients in regulating tissue size, this study presents evidence that these two morphogens control the size of the adult wing along two orthogonal axes by mediating the growth-promoting activities of compartment boundaries in a non-interchangeable manner through the use of morphogen-specific molecular mechanisms. While Dpp regulates growth along the AP axis by maintaining the levels of the transcriptional repressor Brinker below a growth-repressing threshold, Wg regulates growth along the PD axis by counteracting the activity of TCF as a transcriptional repressor. At the time TCF was molecularly identified in flies, it was shown that clones of cells mutant for TCF are poorly recovered in the primordium of the wing pouch and proposed to be a consequence of TCF promoting proliferative growth. However, later studies identified cell competition as the mechanism to eliminate cells with steep differences in Wg signaling in the wing primordium. The Warts-Hippo signaling pathway governs organ size in animals, and the upstream regulators include the atypical cadherins Fat and Dachsous. Surprisingly, inactivation of the Warts-Hippo signaling pathway was unable to rescue the tissue size defects caused by morphogen depletion. These data indicate that for wing blade cells to grow along the PD and AP axes, cells need first to lose TCF and Brinker, and it is proposed that Hippo signaling can then modulate the amount of growth of those cells in which these two repressors are not active or expressed. The experimental data are consistent with a model whereby a minimal amount of signaling from the two morphogens, sufficient to maintain the activity levels of the two transcriptional repressors below a growth-repressing threshold, regulate the physical size of the adult wing primordium along the AP and PD axes. The mechanistic similarities of how Dpp and Wg morphogens, their gradients, and their range of activity regulate the patterning and growth of the fly wing are remarkable and might shed light on the role of morphogens in regulating proliferative growth and patterning in vertebrates (Barrio, 2020).

Experimental conditions in developing wings in which proliferation rates are either increased or reduced have shown that a perfectly normal-sized wing can be obtained with fewer or more cells. Similarly, experimental randomization of the orientation of cell divisions in the growing wing primordium can give rise to well-shaped adult wings. These results suggest that the ability of compartment boundaries, and their dedicated morphogens, to drive anisotropic growth and regulate the width and length of the adult wing blade does not rely only on the control of cell division or oriented cell divisions. Several experimental data indicate that it is the range of the morphogen and not the total amount of it that regulates the physical size, and not the number of cells, of each axis. How do Wg and Dpp regulate growth preferentially along a certain axis and not the other? Restricted expression of these two morphogens along the two existing orthogonal boundaries does not appear to be essential as their ability to drive anisotropic growth is still observed when they are ubiquitously overexpressed in all wing cells. The experimental data indicate that the capacity of Wg and Dpp to drive anisotropic growth relies on the existence of morphogen-specific and non-interchangeable molecular mechanisms mediating their growth-promoting activities and the requirement of the presence of the two of them to drive growth. In this regard, each morphogen promotes growth only along a particular axis, as the distance to the source of the other morphogen has to be maintained to get sufficient levels of the two of them to promote wing growth. The data also indicate that the Wg gradient contributes to orient growth along the PD axis. However, this contribution does not appear to play an essential role since well-shaped elongated wings can be obtained upon uniform expression of Wg (Barrio, 2020).

While the growth-promoting role of Dpp emanating from the AP compartment boundary has been experimentally validated and recently clarified, previous experimental characterization of the growth-promoting role of Wg emanating from the DV compartment boundary reached opposing conclusions. This study presents experimental evidence that Wg mediates the organizing activity of the DV boundary in terms of growth, as uniform expression of this morphogen rescues the extreme growth defects caused by the absence of a DV signaling center. Moreover, the data indicate that Wg is the main growth-promoting Wnt in the developing wing, the DV boundary is the main source of Wg driving proliferative growth of the primordium of the wing appendage, and boundary Wg regulates tissue growth and proliferation rates equally in distal and proximal regions of the developing wing appendage, throughout development and independently of its potential role as survival factor. This latter observation questions the proposal that Wg drives wing growth, at least in part, by promoting cell survival. This proposal was based on the ability of apoptotic inhibitors to rescue the poor recovery and growth of clones of cells unable to transduce the Wg signal, but cell competition was subsequently shown to be the mechanism used to eliminate cells with steep differences in Wg signaling. The experimental observation that even late depletion of Wg expression has an effect on wing size questions the proposal that continuous exposure to Wg is not an absolute requirement for wing cells to grow. Recently, a membrane-tethered form of the Wg protein was shown to be able to substitute for the endogenous Wg protein in producing normally patterned wings of nearly the right size. Either the activity of cellular extensions at a distance, higher stability of the membrane-tethered form of Wg, or emerging compensatory mechanisms should be able to facilitate or extend in time the exposure of all wing cells to the morphogen in the absence of secretion, thus fulfilling its continuous growth-promoting role (Barrio, 2020).

Morphogen gradients specify cell fates during development, with a classic example being the bone morphogenetic protein (BMP) gradient's conserved role in embryonic dorsal-ventral axis patterning. This study elucidates how the gradient of Dpp is interpreted in the Drosophila embryo by combining live imaging with computational modeling to infer transcriptional burst parameters at single-cell resolution. By comparing burst kinetics in cells receiving different levels of BMP signaling, this study shows that BMP signaling controls burst frequency by regulating the promoter activation rate. Evidence is provided that the promoter activation rate is influenced by both enhancer and promoter sequences, whereas Pol II loading rate is primarily modulated by the enhancer. Consistent with BMP-dependent regulation of burst frequency, the numbers of BMP target gene transcripts per cell are graded across their expression domains. It is suggested that graded mRNA output is a general feature of morphogen gradient interpretation and discuss how this can impact on cell-fate decisions (Hoppe, 2020).

A gradient of bone morphogenetic protein (BMP) signaling patterns ectodermal cell fates along the dorsal-ventral axis of vertebrate and invertebrate embryos. In Drosophila, visualization of Decapentaplegic (Dpp), the major BMP signaling molecule, reveals a shallow graded distribution in early embryos that subsequently refines to a peak of Dpp at the dorsal midline. BMP-receptor activation leads to phosphorylation of the Mothers against dpp (Mad) transcription factor, which associates with Medea (Med) to activate or repress target gene transcription. A stripe of phosphorylated Mad (pMad) and Med centered at the dorsal midline has been visualized in the early Drosophila embryo, similar to that observed for Dpp, although lower nuclear pMad levels are also detectable in a few adjacent dorsal-lateral cells. The BMP/pMad gradient activates different thresholds of gene activity, including the peak target gene hindsight (hnt) and the intermediate target u-shaped (ush) (Hoppe, 2020).

New insights into transcriptional activation have been obtained by studying this process in single cells using quantitative and live imaging approaches, including single-molecule fluorescence in situ hybridization (smFISH) and the MS2/MS2 coat protein (MCP) system (Pichon, 2018). The latter allowed the first direct visualization of pulses or bursts of transcriptional activity. Enhancers have been shown to regulate the frequency of transcriptional bursts, with strong enhancers generating more bursts than weaker enhancers. In addition, the detection of simultaneous bursts of transcription of two linked reporters by a single enhancer argues against the classic enhancer-promoter looping model (Fukaya, 2016; Hoppe, 2020).

Recently, Notch target genes in Drosophila and C. elegans have been shown to undergo transcriptional bursting, with Notch controlling burst size through effects on duration (Falo-Sanjuan, 2019; Lee, 2019). However, it is unclear whether this is a general mechanism by which signals control transcriptional bursting. Therefore, to provide insight into BMP gradient interpretation at single-cell resolution, live imaging and quantitative analysis was used to determine the kinetics of endogenous BMP target gene transcription in the Drosophila embryo. These data reveal that BMP signaling modulates the promoter activation rate and therefore burst frequency. The different burst frequencies of BMP target genes, depending on cellular position, result in a gradient of mRNA numbers per cell. Overall these data reveal how a signaling gradient is decoded with different transcriptional kinetics to impart positional information on cells (Hoppe, 2020).

This study has analyzed the transcriptional burst kinetics of the endogenous hnt and ush genes at single-cell resolution. Cells were shown to interpret different BMP signaling levels by modulating burst frequency via k_on . For ush, k_on is unchanged when the hnt promoter is introduced, suggesting that features of the enhancer, most likely transcription factor-binding sites, dictate k_on. Transcription factor binding is coupled to initiation of a burst (Donovan, 2019), and burst frequency depends on the time it takes a transcription factor to find its binding site (Larson, 2011), providing an explanation for why high BMP/pMad levels increase k^on. Consistent with this, other studies have found transcription factor concentration and enhancer strength to regulate k_on. For ush, this study provides evidence that k_on is at a maximum in cells receiving peak signaling. A burst frequency ceiling has been described for tumor necrosis factor (TNF) targets, although the k_on ceiling for ush appears to be gene specific, as it is below that observed for hnt and other Drosophila genes studied (Lammers, 2020; Zoller, 2018). Perhaps k_on for ush is limited by slow recruitment of pMad or fewer productive activation events between its enhancer and promoter. Current ideas for enhancer-promoter communication include a dynamic kissing model that invokes transient interactions or models based on the proximity of regulatory elements in space, potentially in phase separated condensates. In these models, the Mediator coactivator may act as a dynamic molecular bridge between the enhancer and promoter, as activator-recruited Mediator at the enhancer can also make contact with the promoter (Hoppe, 2020).

When the hnt and ush promoters are swapped, mean Pol II loading rate is unchanged in both cases suggesting that it is predominantly dictated by the enhancer, although increased variability is detected in loading rate for ush>hnt. This suggests that transcription factors bound to the enhancer control Pol II loading rate, which is consistent with a previous study that found loading rate to be influenced by the strength of the transcription factor's activation domain. Loading rate is unchanged by altered BMP levels, with a constant loading rate also described for bursting of gap genes (Zoller, 2018), suggesting it is not a major regulated step of transcription in the Drosophila embryo. Despite the similar bursting behavior between ush and hnt>ush, introducing the ush promoter into hnt reduces k_on. This suggests that the hnt promoter has some feature that is important in the hnt genomic context, for example a TATA box or highly paused Pol II, as the ush promoter lacks both. TATA increases burst frequency in the presence of interferon signaling; however, other studies have linked TATA promoters to burst size, and the hnt promoter does not increase frequency when introduced into ush. Another possibility is that the ability of paused Pol II at the hnt promoter to prevent encroachment of nucleosomes increases k_on. In support of this, introduction of nucleosome disfavoring sequences around a promoter and linked transcription factor-binding site was found to increase burst frequency. It has also been shown that forcing interactions between a β-globin enhancer and its promoter increases burst frequency (Bartman, 2019). As enhancer-promoter compatibility has been proposed, perhaps the hnt enhancer is more compatible with its cognate promoter. In terms of compatibility, Mediator and the p300 acetyltransferase, both of which are recruited by Smads, most strongly activate TATA-containing promoters (Haberle, 2019). While further work is required to understand how the core promoter influences bursting, these results suggest a role for both the enhancer and promoter in influencing k_on and burst frequency, thereby allowing greater flexibility in controlling the transcriptional response (Hoppe, 2020).

hnt transcriptional bursts are shorter and of higher frequency and amplitude than the ush bursts. The resulting hnt promoter occupancy is around half that for ush, providing a molecular explanation for the observed threshold responses of these genes to the BMP gradient. Unlike for ush, low BMP signaling levels are insufficient to maintain the hnt promoter in an active state, resulting in a narrow expression pattern. Burst duration, although not responsive to Dpp levels, is around 4 times longer for ush than for hnt. Transcription factor dwell time, which is limited by binding site affinity and nucleosomes, controls burst duration. As the Smad proteins have low affinity for DNA and weak specificity, they cooperate with other DNA-binding proteins. The ush and hnt enhancers have yet to be characterized, but the pioneer factor Zelda and homeodomain protein Zerknüllt may be pMad cofactors for intermediate (ush) and peak (hnt) Dpp targets, respectively. It is possible that at the hnt enhancer the Zerknüllt-pMad complex has a shorter residency time, or the pMad-binding sites are weaker affinity, resulting in shorter duration bursts (Hoppe, 2020).

The lack of a contribution of burst duration (1/k_off ) to decoding BMP signaling is in stark contrast to the findings that Notch alters the duration, but not frequency, of transcription bursts in Drosophila and C. elegans (Falo-Sanjuan, 2019; Lee, 2019). Increasing gene expression through high k_on rates can decrease noise, whereas lengthening burst duration is associated with more noise (Wong, 2018). Regulation of burst frequency may also allow genes to respond more sensitively to activator concentration (Li, 2018). Therefore, perhaps regulation of BMP target genes via k_on has the advantage of allowing more sensitive regulation with less noise. It remains to be determined whether other signals will be interpreted through changes in k_on and burst frequency or duration (Hoppe, 2020).

The different burst kinetics of BMP target gene transcription across the expression domain explain why cells at the edge are frequently captured with a single active allele in the single molecule florescent in situ hybridization (smFISH) data. sog and brk exhibit transcriptional bursting (Esposito, 2016), and the current data suggest that sog and brk bursting is regulated across their expression domains. Allele by allele repression has been observed in the Drosophila embryo, potentially because repressors are better able to act in the refractory period following a burst (Esposito, 2016). Such allele by allele repression can also explain why nuclei with one active allele are observed at the ventral borders of the sog and brk expression domains, where dorsal activator levels are high. Single-allele transcription has also been reported for zygotic hunchback (hb) transcription, which is activated by the Bicoid gradient, particularly at the borders of the expression domain. It is suggested that infrequent transcriptional bursting, with a concomitant reduction in mRNA number, is a general feature of gradient interpretation for cells receiving low signal (Hoppe, 2020).

The ush mRNA distribution reflects the spatial BMP gradient as the central 6 rows that receive peak BMP signaling have the highest mRNA number/cell, with subsequent declining mRNA numbers mirroring a reduction in Dpp levels. Additionally, modeling suggests that the concentration of BMP-receptor complexes at the dorsal midline doubles between 20 min and 30 min into nc14. This corresponds to the onset times of ush and hnt, respectively, suggesting that hnt transcription requires more activated receptors. Furthermore, BMP-receptor levels peak at ~45 min into nc14, which broadly coincides with the observed maximum fluorescence output detected for ush and hnt (Hoppe, 2020).

It is suggested that altered transcriptional burst kinetics and graded mRNA numbers in response to morphogen gradients can impact on cell-fate decisions. It is proposed that cells on the edge of an expression domain synthesize sufficient mRNAs to adopt a particular cell fate, whereas cells in the center have a surplus of transcripts. This model can explain the lack of robustness when shadow enhancers are deleted. Perturbation of the system, such as removal of a shadow enhancer, would lead to a further reduction in mRNA number per cell so that those on the edge would only just exceed the threshold level. Another challenge, such as high temperature or reduced activator level, would further decrease the transcriptional output such that there are insufficient mRNAs to specify the correct cell fate. It will be interesting in the future to test how the different numbers of mRNAs per cell from key BMP target genes impact on the robustness of dorsal ectoderm cell-fate decisions (Hoppe, 2020).

Hedgehog (Hh) and bone morphogenetic proteins (BMPs) pattern the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. This study shows that a previously unknown Hh-dependent mechanism fine-tunes the activity of BMPs. Through genome-wide expression profiling of the Drosophila wing imaginal discs, this study identified nord as a novel target gene of the Hh signaling pathway. Nord is related to the vertebrate Neuron Derived Neurotrophic Factor (NDNF) involved in Congenital Hypogonadotropic Hypogonadism and several types of cancer. Loss- and gain-of-function analyses implicate Nord in the regulation of wing growth and proper crossvein patterning. At the molecular level, biochemical evidence ia presented that Nord is a secreted BMP-binding protein and localizes to the extracellular matrix. Nord binds to Decapentaplegic (Dpp) or the heterodimer Dpp-Glass bottom boat (Gbb) to modulate their release and activity. Furthermore, this study demonstrates that Nord is a dosage-depend BMP modulator, where low levels of Nord promote and high levels inhibit BMP signaling. Taken together, it is proposed that Hh-induced Nord expression fine tunes both the range and strength of BMP signaling in the developing Drosophila wing (Yang, 2022).

In Drosophila, the short-range morphogen Hh and the long-range morphogen BMP function together to organize wing patterning. It has been previously shown that the Hh signal shapes the activity gradient of BMP by both inducing the expression of Dpp and simultaneously downregulating the Dpp receptor Tkv, resulting in lower responsiveness to Dpp in cells at the A/P compartment border. This study showed that the activity of BMP is further fine-tuned by another previously unknown Hh-dependent mechanism. Using a genome-wide expression profiling of the Drosophila wing imaginal discs, this study identfied nord as a novel target gene of the Hh signaling pathway. Nord and its homolog NDNF belong to a family of secreted proteins that can exist in two distinct pools: diffusible Nord/NDNF proteins that can reach a longer distance and membrane/matrix-associated Nord/NDNF proteins spreading within a short distance from the source cells. During larval and early pupal wing development, Nord is expressed together or in close proximity with the BMP ligand Dpp along the A/P compartment boundary. Elimination of nord caused a reduction of overall wing size and resulted in ectopic posterior crossvein (PCV) formation. Both of these phenotypes are attributable to alterations of BMP signaling activity as monitored by the level of Mad phosphorylation, yet in opposite directions: loss of nord led to decreased pMad in larval wing discs, whereas ectopic pMad surrounded the primordial PCV in nord mutant pupal wings. Moreover, expressing exogenous Nord at different levels and during different developmental stages and contexts showed that Nord is a dosage-dependent modulator of BMP signaling both in wing growth and crossvein patterning. At the molecular level, it was further demonstrated that Nord is a BMP-binding protein that directly enhances or inhibits BMP signaling in cultured S2 cells (Yang, 2022).

Combining the genetic and biochemical evidence, it is proposes that Nord mediates BMP signaling activity through binding of the BMP ligands Dpp and Dpp-Gbb. Depending on the levels of Nord proteins and the source/types of BMP ligands, Nord-mediated binding of Dpp and Dpp-Gbb may promote or repress BMP signaling activity. Additionally, the existence of two spatially distinct pools of diffusible and membrane/matrix-associated Nord proteins may introduce further complications in Nord-mediated BMP signaling regulation. In the wild-type wing discs, expressed in a subset of Dpp-secreting cells along the A/P boundary, Nord binds and enhances the local BMP signaling activity by augmenting ligand concentration near the Nord/Dpp-secreting cells. Meanwhile, Nord also impedes the mobilization of Dpp, especially the long-range BMP signaling mediator Dpp-Gbb heterodimer. Loss of nord simultaneously led to reduced local BMP and increased long-range BMP activities, and therefore gave rise to the seemingly opposite phenotypes of reduced wing size and ectopic PCV. In contrast, low levels of ectopic Nord in the P compartment autonomously increased BMP signaling activity, whereas high levels of Nord, either in the P compartment or throughout the wing pouch, inhibited BMP signaling activity likely through interfering with the normal BMP reception. Taken together, it is proposed that Hh-induced Nord expression provides an exquisite regulation of the strength and range of BMP signaling in the developing Drosophila wing (Yang, 2022).

The activity of TGF-β type factors, including the BMP subfamily, is modulated by a large variety of binding proteins that can either enhance or inhibit their signaling in a context-dependent manner. These modulator proteins vary broadly in structure, location, and mechanism of action. Well-known extracellular and freely diffusible proteins include Noggin, Tsg, Follistatin, the CR (cysteine-rich) domain containing proteins such as Chordin/Sog, and the Can family named after two founding members, Dan and Cerberus. With the exception of Tsg and Tsg/Sog or Tsg/Chordin complexes that in some cases can promote BMP signaling, all of these factors behave as antagonists, where BMP binding prevents association of the ligand with the receptor complex (Yang, 2022).

The other broad category of BMP-binding proteins includes membrane-bound or matrix-associated proteins and, in contrast to the highly diffusible class of BMP-binding factors, these proteins often act as either agonists or antagonists depending on context. These proteins are also structurally diverse, but to date, none contain FN3 or DUF2369 domains that are characteristic of Nord and NDNF, its vertebrate counterpart. From a mechanistic point of view, perhaps the two most instructive Drosophila members of this class of modulators are the heparan sulfate proteoglycan (HSPG) Dally and the CR-containing protein Cv-2. HSPGs are well characterized as modulators of growth factor signaling. In the case of FGFs, HSPGs act as true co-receptors in which they form a tripartite complex with ligand and FGFR, the signaling receptor. However, they can also mediate signaling in other ways. Analysis of dally loss-of-function clones in imaginal discs demonstrates that it has both cell-autonomous and non-autonomous effects with respect to BMP signaling. In general, low levels tend to promote signaling while high doses attenuate signaling. Many models have been put forth to explain these opposing effects and often come down to balancing ligand sequestration and diffusion properties. For instance, in the absence of HSPGs, Dpp may more freely diffuse away from the disc epithelial cell surface. In this case, HSPG acts to enhance signaling by keeping Dpp tethered to the cell surface where it can engage its signaling receptors. On the other hand, a high level of HSPG may compete with signaling receptors for BMP binding and thereby reduce signal (Yang, 2022).

The situation with respect to signal modulation becomes even more complex for factors such as Nord that bind both HSPGs and BMPs. An instructive example to consider is Cv-2, a secreted factor that, like Nord, binds both to HSPGs and BMPs and is also induced by BMP signaling. Like Dally, Cv-2 also has dose-dependent effects on signaling in wing imaginal discs, where low levels enhance while high levels inhibit BMP signaling. By virtue of being bound to HSPGs, it may simply function as an additional tethering molecule that keeps BMPs localized near the cell surface. However, Cv-2 has the unique property that it is also able to bind Tkv, a Drosophila BMPR type I receptor. This has led to speculation that it could act as an exchange factor that aids in handing off a BMP ligand from the HSPG pool to the type I receptor. Mathematical modeling showed that this mechanism can produce a biphasic signal depending on affinities of the various BMP-binding proteins involved and their concentrations (Yang, 2022).

In the case of Nord, its mechanism of action is likely compatible with a variety of these and/or alternative models. While this study has shown that Nord is a BMP-binding protein and Akiyama (2021) have shown that it also binds HSPGs, it is not clear whether the BMP and HSPG-binding sites overlap or are distinct and where they are positioned relative to the FN3 and DUF2369 domains. This is an important issue to consider with respect to the two CRISPR mutants that were generated that truncate Nord within the DUF2369 domain. Interestingly, the nord^3D allele appears to retain some function since it does not generate ectopic crossveins as do the nord^MI06414 or nord^22A alleles, yet nord^3D still produces small wings in transheterozygous combination with a deficiency or nord^22A, consistent with having lost the BMP growth-promoting ability. The discrepancy in crossvein patterning between the different nord alleles may be explained by a difference in residual function of the various truncated Nord protein products. Because the nord^MI06414 allele yields a much shorter predicted Nord peptide compared to the two CRISPR alleles, it is likely to behave as a protein null with a stronger phenotype. The two nord CRISPR alleles, although similar in the sequence deleted from the C-terminus, differ in how many non-nord encoded amino acids occur between the frameshift and the stop codon. The nord^22A allele has additional 14 amino acids relative to nord^3D. Perhaps this extension of the truncated fragment destabilizes or interferes with residual function found in the nord^3D allele. Additional biochemical studies defining the BMP and HSPG-binding sites, the stability of truncated Nord fragments, and whether Nord can also associate with either the type I or II receptors will aid in formulating a more precise mechanistic model (Yang, 2022).

Nord shows some sequence similarity to the NDNF family of proteins. Based on a very recent study, like many other neurotrophic factors, NDNF arose in the ancestor of bilaterians or even later. In agreement, by analyzing the genome and EST sequences from various organisms, this study found that nearly all bilaterian animals have either single or multiple orthologous genes for Nord/Ndnf. Of note, no Ndnf homologs were identiied in the flatworm Planarian, but these factors are highly conserved across vertebrates. All vertebrate family members contain a signal peptide, two FN3-like repeats, and a domain of unknown function (DUF2369) that is now referred to as the NDNF domain. The NDNF domain partially overlaps with the first FN3 but shows some additional conservation that extends between the two FN3 domains. The FN3 module is quite diverse in sequence but is thought to exhibit a common fold that is used as an interaction surface or spacer. The function of the NDNF domain is not clear, but it may also provide a protein interaction surface (Yang, 2022).

Although the vertebrate NDNFs are highly conserved throughout the entire protein length, the Caenorhabditis elegans and Drosophila relatives are quite divergent in primary sequence and show little conservation beyond a few key residues that define the second FN3 and NDNF domains. Notably, the Drosophila protein is missing the first FN3 domain, and therefore it is not clear the extent to which Nord and the vertebrate NDNFs may exhibit functional conservation. Ironically, the original human NDNF clone was identified on the basis of domain structure conservation with Drosophila Nord, which was identified via enhancer trapping to be a gene expressed in mushroom bodies and whose loss leads to defects in olfactory learning and memory (Dubnau et al., 2003). Unfortunately, that particular LacZ enhancer trap line that disrupted the nord locus is no longer available. The use of these new alleles should prove helpful for either confirming or eliminating the involvement of Nord as a modulator of learning and memory and/or other neuronal functions in larva and adult Drosophila (Yang, 2022).

In the mouse, NDNF is highly expressed in many neurons of the brain and spinal cord. Studies using cultured mouse hippocampal neurons revealed that it promotes neuron migration and neurite outgrowth, hence its name. In later studies, NDNF was also found to be upregulated in mouse endothelial cells in response to hindlimb ischemia, where it promotes endothelial cell and cardiomyocyte survival through integrin-mediated activation of AKT/endothelial NOS signaling. Additionally, recent studies have shown that NDNF expression is significantly downregulated in human lung adenocarcinoma (LUAD) and renal cell carcinoma (RCC), indicating that NDNF may also provide a beneficial function as a tumor suppressor (Yang, 2022).

Taken together, these studies have suggested some possible functions for vertebrate NDNF. However, they have primarily relied on in vitro cell culture models, and only recently have in vivo loss-of-function studies been reported. Remarkably, NDNF mutants were discovered in the genomes of several probands with congenital hypogonadotropic hypogonadism (CHH), a rare genetic disorder that is characterized by absence of puberty, infertility, and anosmia (loss of smell). This phenotype is very similar to that produced by loss of the anos¹, which also encodes an FN3 superfamily member and is responsible for Kallmann syndrome, a condition that similarly presents with CHH and anosmia due to lack of proper GnRH and olfactory neuron migration. Although in vitro studies indicated that NDNF modulates FGFR1 signaling after FGF8 stimulation, the in vivo molecular mechanism responsible for the neuronal migration defects is not clear. The results of the current study on the function of Drosophila Nord raise the issue of whether any of the ascribed vertebrate NDNF functions could involve alterations in BMP signaling. In the case of angiogenesis and EMT, BMPs, as well as other TGF-β family members, participate at many levels. At present, however, no involvement of BMP or TGF-β signaling has been implicated in migration of the GnRH neurons, although BMP signaling does define neurogenic permissive areas in which the olfactory placode forms. A clear objective for the future is to determine if the vertebrate NDNF factors bind BMPs and/or HSPG proteins such as Dally-like glypicans to modulate BMP signaling activity. On the Drosophila side, additional non-BMP-modulating roles for Nord should also be examined (Yang, 2022).

Akiyama, T., Seidel, C. W. and Gibson, M. C. (2022). The feedback regulator Nord controls Dpp/BMP signaling via extracellular interaction with Dally in the Drosophila wing. Dev Biol 488: 91-103. PubMed ID: 35609633

The Drosophila BMP 2/4 homologue Decapentaplegic (Dpp) acts as a morphogen to regulate diverse developmental processes, including wing morphogenesis. Transcriptional feedback regulation of this pathway ensures tightly controlled signaling outputs to generate the precise pattern of the adult wing. Nevertheless, few direct Dpp target genes have been explored and understanding of feedback regulation remains incomplete. This study employed transcriptional profiling following dpp conditional knockout to identify nord, a novel Dpp/BMP feedback regulator. nord mutants generated by CRISPR/Cas9 mutagenesis produce a smaller wing and display low penetrance venation defects. At the molecular level, nord encodes a secreted heparin-binding protein, and this study shows that its overexpression is sufficient to antagonize Dpp/BMP signaling. Mechanistically, it was demonstrated that Nord physically interacts with the Dpp/BMP co-receptor Dally and promotes its degradation. In sum, it is proposed that Nord fine-tunes Dpp/BMP signaling by regulating Dally availability on the cell surface, with implications for both developmental and disease models (Akiyama, 2022).

Transcriptomic analyses using dpp^FO revealed a novel Dpp/BMP target gene nord. Analysis of mRNA FISH and a Gal4-based transcriptional reporter revealed strong nord expression in the stripe of cells along with the A/P compartment and little or weak expression in the D/V boundary in the wing disc. A similar Nord expression pattern was observed using the Nord-GFP protein trap. Surprisingly, unlike other well-studied Dpp/BMP targets, nord expression was restricted in the anterior compartment. Dpp/BMP signaling activity was essential but not sufficient to induce nord expression, indicating that other factors are required for its activation. Indeed, a recent study reports that Hh signaling regulates nord expression in the developing wing (Yang, 2022). Thus, nord expression is positively regulated by both Dpp/BMP and Hh inputs, although the molecular basis for nord suppression at the D/V boundary remains elusive. Interestingly, these signaling pathways also control dally expression. dally expression is activated by Hh signaling in the central stripe region of the wing disc, while Dpp/BMP signaling downregulates dally expression outside the stripe domain. Thus, nord and dally are co-expressed in part of the central stripe region through transcriptional regulation. This expression pattern may allow Nord to efficiently modulate functions of HSPG Dally to fine-tune the Dpp/BMP signaling pathway in the extracellular space (Akiyama, 2022).

BMP positive feedback loop mechanisms provide an optimal extracellular environment that allows BMP ligands to precisely distribute through the wing vein lumen and direct posterior crossvein (PCV) cell fate. Accordingly, partial attenuation of the pathway often results in an ectopic PCV formation. This study found that while nord^Δ1728 homozygotes rarely exhibited ectopic vein formation in the PCV region under normal conditions, PCV phenotypes were enhanced by genetic and environmental stress. It is inferred that BMP feedback mechanisms are sensitive to stress, thus enhancing the nord PCV phenotype. Intriguingly, Nord protein was detected in hemocytes distributed within the wing veins even though it was only expressed in a row of distal anterior pupal wing cells. These observations indicate that either wing cells or other sources deposit Nord protein on hemocytes. Similarly, although crossveinless d (cv-d), which encodes a vitellogenin-like lipoprotein, is expressed in the fat body and other non-imaginal disc tissues, Cv-d proteins circulate to the pupal wing via hemocytes, bind BMPs and HSPGs, and regulate PCV development. It is thus inferred that Nord may travel far from its site of expression via hemocytes and play a critical role in establishing a BMP positive feedback loop to ensure proper PCV formation (Akiyama, 2022).

Lastly, while performing the genetic interaction experiments, we noticed that nord^Δ1728 ; dally^gem double mutant animals exhibited a male larval gonad developmental defect. Further, it was found that the nord transcriptional reporter, nord-Gal4, was strongly expressed in male-specific somatic gonadal precursor cells and the terminal epithelium of testis derived from the precursor cells. These results suggest that Nord is not only essential for wing development but also plays an essential role with the HSPG Dally during testis development. It is of great interest to examine how nord and dally are involved in testis development in the future (Akiyama, 2022).

Extracellular regulation of HSPGs, such as shedding of membrane-localized HSPGs from the cell surface and digestion and modification of HS chains, is tightly regulated during development and homeostasis, and its misregulation has been recognized as a hallmark of cancer. Matrix Metallopeptidase 9 cleaves a transmembrane HSPG syndecan-1 from the cell membrane, facilitating tumor growth, metastasis, and angiogenesis. In addition, the Drosophila endo-6-O-sulfatase Sulf1, which selectively removes 6-O-sulfate groups from the HS chains in the extracellular space, controls molecular interactions between HSPGs and signaling molecules including BMP and Wg, and regulates diverse biological processes such as wing development and intestinal stem cell activity. In the developing Drosophila wing, transcription of the secreted Dpp/BMP feedback regulator Pent is restricted to the lateral regions of the wing disc by Dpp/BMP-dependent repression in the central domain. Pent-mediated endocytosis and degradation of glypicans shape the Dpp and Wg morphogen gradients. This study identified the novel secreted factor Nord through transcriptional profiling of Dpp/BMP target genes. Nord inhibits Dpp/BMP signaling and interacts with the HSPG Dally. Subsequent analyses revealed that Nord promotes Dally internalization for degradation. It was further demonstrated that Nord neutralizes the ability of Dally to promote Dpp/BMP signaling. Based on these results, a model is proposed in which 1) Dpp/BMP signaling induces nord expression at the A/P compartment boundary, 2) Nord is secreted into the extracellular space and binds to the co-receptor Dally, and 3) Nord enhances Dally degradation via endocytosis, thus downregulating the Dpp/BMP signaling pathway. Although the precise molecular mechanisms underlying the phenotypic enhancements observed in nord^Δ1728; dally^gem double mutant animals need to be addressed in the future, the current model explains how nord mutations suppress the L5 vein defect observed in dally mutants. In this scenario, the lack of Nord function would help to stabilize Dally protein in a dally^gem hypomorphic mutant background, and this slight increase in Dally activity is in turn sufficient to rescue the L5 vein phenotype. Further, it is speculated that the same molecular mechanism can account for the downregulation of Wg signaling observed in wing discs overexpressing nord. In this case, nord overexpression destabilizes Dally on the cell surface, leading to reduced Wg signaling activity. Lastly, a recent study demonstrated that Nord also directly interacts with BMPs and acts as a biphasic Dpp/BMP regulator that promotes the pathway at low levels but inhibits it at high concentrations. In the future, deciphering how Nord controls BMP ligand and co-receptor availability in the extracellular space with other secreted factors will provide a more concrete picture of the molecular mechanisms regulating the Dpp/BMP morphogen gradient formation. It will be particularly exciting to investigate how Nord and Pent cooperate to modulate glypican activity to fine-tune Dpp/BMP signaling since they possess a similar protein function with distinct expression patterns in the developing wing disc (Akiyama, 2022).

nord encodes a Drosophila NDNF protein. Human NDNF acts as a tumor suppressor. NDNF expression is decreased in lung adenocarcinoma, and downregulation of NDNF promotes tumor growth in a mouse xenograft model . NDNF is also known as a causative gene for congenital hypogonadotropic hypogonadism (CHH), which is characterized by infertility and delayed/absence of puberty (Messina, 2020). CHH is rooted in the abnormal development of gonadotropin-releasing hormone (GnRH) neurons. Many CHH-linked genes are involved in the regulation of the Fibroblast growth factor (FGF) signaling pathway. Indeed, NDNF overexpression inhibits FGF signaling in cell culture, and ndnf mutant mice exhibit a GnRH neuronal migration defect. Further, NDNF improves cardiac function after ischemia and myocardial infarction by reducing cardiomyocyte apoptosis and promoting angiogenesis through activation of the AKT signaling pathway. Nevertheless, the molecular mechanisms by which NDNF modulates multiple signaling pathways remain unclear. Intriguingly, it has been shown that HSPGs play essential roles in these biological processes. For instance, mutations in heparan sulfate 6-O-sulfotransferase-1 and anosimin-1 are found in CHH patients. Anosmin-1 is an HSPG interacting protein and regulates GnRH neuronal migration by promoting FGF signaling. Taken together, it is speculated that vertebrate NDNF may modulate HSPG activity to fine-tune multiple downstream signaling pathways. In this regard, the present study not only reveals a new mode of extracellular morphogen regulation, but also opens up a new avenue for investigating the molecular basis for NDNF-associated disorders in humans (Akiyama, 2022).