punt

DEVELOPMENTAL BIOLOGY

High levels of Punt are found in nurse cells and oocytes. The maternal mRNA persists into cellularization, where the level decreases and becomes asymmetric, with the highest levels found at the posterior end, underneath the pole cells [Images]. At the start of gastrulation punt expression is seen in the invaginating mesoderm. By stage 11, expression is seen throughout the gut. Expression is seen in imaginal disc cells of larvae (Childs, 1993).

Effects of Mutation or Deletion

Mutations in punt produce phenotypes similar to those exhibited by tkv, sax, and dpp mutants. (Letsou, 1995). Mutants completely lack dorsal branches in all the tracheal metamers, whereas the remaining tracheal system is established normally. Loss of punt activity causes a homeotic transformation of parasegement 7 to parasegment 6, exactly the same phenotype observed in tkv mutants (Ruberte, 1995).

Salivary gland formation in the Drosophila embryo is linked to the expression of the homeotic gene Sex combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when Scr is expressed everywhere, salivary glands form in new places. However, not every cell that expresses Scr is recruited to a salivary gland fate. Along the anterior-posterior axis, the posteriorly expressed proteins encoded by the teashirt (tsh) and Abdominal-B (Abd-B) genes block Scr activation of salivary gland genes. Along the dorsal-ventral axis, the secreted signaling molecule encoded by decapentaplegic prevents activation of salivary gland genes by Scr in dorsal regions of parasegment 2. Five downstream components in the Dpp signaling cascade required to block salivary gland gene activation have been identified: two known receptors (the type I receptor encoded by the thick veins gene and the type II receptor encoded by the punt gene); two of the four known Drosophila members of the Smad family of proteins which transduce signals from the receptors to the nucleus [Mothers against dpp (Mad) and Medea (Med)], and a large zinc-finger transcription factor encoded by the schnurri (shn) gene. The expression patterns of d-CrebA and Trachealess were examined in embryos missing zygotic function of schnurri. In embryos homozygous for shn, a dorsal expansion of salivary gland protein expression is observed. The presence of amnioserosa, an extreme dorsal cell type, suggests that embryos lacking zygotic shn function are not ventralized, as are embryos missing maternal and zygotic function of tkv, pt, Mad, or Med or missing zygotic function of dpp. These results reveal how anterior-posterior and dorsal-ventral patterning information is integrated at the level of organ-specific gene expression (Henderson, 1999).

Although the punt gene was originally identified based on its requirement for embryonic dorsal closure, multiple periods of punt activity have been documented throughout the Drosophila life cycle. Potentially related embryonic punt phenotypes, defects in dorsoventral patterning and dorsal closure are demonstrated that correspond to distinct maternal and zygotic requirements for punt. Using temperature-sensitive punt alleles, it has been demonstrated that the embryonic requirements for punt correspond to distinct time intervals; thus, the temporal requirement for punt during dorsal closure is separable from its earlier requirement for dorsoventral pattern formation. The demonstration of a dual requirement for Punt during embryogenesis indicates that the dorsal-open punt phenotype is a primary effect of the zygotic mutation and is consistent with the documented roles for Dpp and Tkv in dorsal closure (Simin, 1998).

Postembryonic requirements for punt activity are documented. Wing defects (ectopic wing veination) are detected in adult viable punt alleles. Temperature-shift manipulations were also employed to investigate postembryonic requirements for Punt. All punt homozygotes exhibit notal defects; these are almost exclusively medial notal clefts. Ninety-nine percent of punt homozygotes exhibit leg defects, including truncations, bifurcations, and abnormal twists. Distal pattern elements were deleted in at least one limb in 126 of 127 animals examined. All six legs are rarely affected to the same degree, and defects may be unilateral or bilateral with respect to each pair of legs. Although the posterior leg pair is more frequently affected than are the anterior pairs, duplicated sex combs are readily discernible on the forelegs of several mutants. Sex comb duplication is a hallmark of the ventralization of patterning in imaginal discs (Simin, 1998)

The comparison of punt homoallelic and heteroallelic phenotypes provides direct evidence for interallelic complementation. Taken together, these results suggest that the Punt protein functions as a dimer or higher order multimer throughout the Drosophila life cycle (Simin, 1998).

Dpp controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo. The requirement for Dpp is revealed by two manipulations: (1) the overexpression of Dpp using a heat-shock promoter and (2) use of mutations in the Dpp receptors thickveins and punt. The failure of tracheal cells to receive the DPP signal from adjacent dorsal and ventral cells results in the absence of dorsal and ventral migrations. Ectopic Dpp signaling can reprogram cells in the center of the placode to adopt a dorsoventral migration behavior. The effects observed in response to ectopic Dpp signaling are also observed upon the tracheal-specific expression of a constitutive active Dpp type I receptor (TKV[Q253D]). The alterations in migration behavior are similar for constitutively active receptor and for Dpp ectopic expression, indicating that the Dpp signal is received and transmitted in tracheal cells to control their migration behavior. Whereas, lack of Dpp signaling results in a failure of tracheal cells to migrate along the dorsoventral axis without significantly affecting anterior migrations, ubiquitous Dpp signaling suppresses anterior migrations without interfering with dorsoventral migration (Vincent, 1997).

Dpp signaling determines localized gene expression patterns in the developing tracheal placode, and is also required for the dorsal expression of the recently identified Branchless (Bnl) guidance molecule, the ligand of the Breathless (Btl) receptor. spalt (sal) is strongly expressed in dorsal trunk cells in stage 14 embryos and is necessary for the directed anterior migration of these cells. sal is expressed in the dorsal trunk in punt and tkv mutant embryos, indicating that Dpp does not regulate sal expression. However, embryos in which the Dpp signaling pathway has been activated in all tracheal cells at the placode stage fail to accumulate Sal. This lack of Sal expression correlates with the absence of the dorsal trunk upon ectopic Dpp signaling. In contrast to sal the gene knirps is activated in the developing tracheal system in all the branches (dorsal branch, ganglionic branch, and lateral trunk) that are thought to be under the control of Dpp. kni expression is lost in tkv mutants; (kni expression only persists in the visceral branches of tkv mutants). kni expression is turned on in all tracheal cells after constitutive Dpp signaling. The requirement for Dpp for the correct expression of the ligand branchless (bnl) was revealed using tkv and punt mutants. The dorsal-most patches of bnl expression which prefigure the formation of the dorsal branches, are severely reduced in punt mutant embryos and absent in tkv mutants. Thus, Dpp plays a dual role during tracheal cell migration. It is required to control the dorsal expression of the Bnl ligand. In addition, the Dpp signal recruits groups of dorsal and ventral tracheal cells and programs them to migrate in dorsal and ventral directions (Vincent, 1997).

In the developing tracheal system of Drosophila, six major branches arise by guided cell migration from a sac-like structure. The chemoattractant Branchless/FGF (Bnl) appears to guide cell migration and is essential for the formation of all tracheal branches, while Decapentaplegic signaling is strictly required for the formation of a subset of branches, the dorsal and ventral branches. Using in vivo confocal video microscopy, it has been found that the two signaling systems affect different cellular functions required for branching morphogenesis. Bnl/FGF signaling affects the formation of dynamic filopodia, possibly controlling cytoskeletal activity and motility as such, and Dpp controls cellular functions allowing branch morphogenesis and outgrowth (Ribeiro, 2002).

The formation of tracheal branches via directed cell migration requires input from other signaling systems in addition to Bnl/FGF. Activation of the Dpp signal transduction cascade is essential in dorsal and ventral tracheal cells prior to migration for the subsequent formation of dorsal and ventral (ganglionic and lateral trunk anterior and posterior) branches. In the absence of the Dpp receptors Thick veins (Tkv) or Punt (Put), dorsal branches completely fail to develop and ventral branches are strongly affected. Dpp induces the expression of the genes kni and knrl in the ventral and dorsal cells of the placode; in the absence of these two nuclear proteins, dorsal branches are absent and ventral branches are strongly abnormal (Ribeiro, 2002).

Knowing that Bnl/FGF acts as a chemoattractant for tracheal cells, and having shown above that Bnl/FGF signaling induces filopodial activity, one must wonder why cells need input from the Dpp signaling cascade for a directed movement to the Bnl/FGF source. Is the Dpp response a prerequisite for the subsequent induction of filopodia by Bnl/FGF? Or do dorsal branch cells respond to Bnl/FGF with the formation of filopodia even in the absence of Dpp signaling input, yet fail to migrate properly? In order to find out how these different signaling systems interact in vivo, the cytoskeletal activity of tracheal cells was examined in the absence of Dpp signaling, with particular emphasis on dorsal branches. However, both tkv and put mutants lack dorsal expression of bnl; therefore, they not only lack the Dpp signaling input but also the Bnl/FGF signaling input. In line with the absence of dorsal bnl expression, cellular extensions were not observed in dorsal tracheal cells in put mutants when analyzed in vivo using the GFP-actin fusion protein (Ribeiro, 2002).

To circumvent the problem of the absence of dorsal bnl expression in mutants defective in Dpp signaling, use was made of the inhibitory SMAD protein encoded by the Drosophila Daughters against dpp (Dad) gene. Specific inhibition of Dpp signaling in tracheal cells via trachea-specific ectopic expression of Dad led to the absence of dorsal branches, despite the presence of bnl expression on the dorsal side of the embryo. Consistent with the absence of dorsal branches upon ectopic expression of Dad, kni expression was not detectable in dorsal tracheal cells. Loss of dorsal branches was also readily visible in the later larval stages; no dorsal branches were observed in third instar larvae upon the expression of Dad in the tracheal system during embryogenesis. In embryos and in larvae expressing Dad, stump-like dorsal outgrowths were occasionally observed at positions where dorsal branches form in wild-type animals. It is argued that these stumps are misrouted dorsal trunk outgrowths; such outgrowths are never observed in tkv or put mutants, presumably due to the lack of bnl expression dorsal to the invaginating placode. It is concluded from these experiments that ectopic expression of Dad mimics the tkv and put mutant phenotypes with regard to the lack of dorsal branch formation, and that dorsal branches fail to form through guided cell migration in this particular Dpp loss-of-function situation despite the presence of dorsal bnl expression (Ribeiro, 2002).

To investigate the possible cell shape changes or cytoskeletal rearrangements in dorsal tracheal cells in the absence of Dpp signaling in vivo, confocal imaging was performed of living embryos expressing both a Dad and a GFP-tagged actin transgene in the developing trachea. Confirming the observations made in fixed embryos and in third instar larvae, the phenotype observed in late embryonic stages (stages 15 and 16) in vivo is the complete absence of dorsal branches. However, analysis of a time-lapse study of three-dimensional reconstructions, in which tracheal GFP-actin dynamics were recorded in an interval of 5 min for 135 min, revealed a strikingly different picture. Unlike put mutants, embryos in which Dpp signaling is inhibited specifically in tracheal cells by ectopic expression of Dad clearly show dorsal outgrowths and filopodial activities in positions where dorsal branches normally form. These outgrowths look bud-like and showed dynamic filopodial extensions, but never refine to single-cell diameter, tubular dorsal branches. Although tracheal cells migrated dorsally, they never migrated over a large distance, and in most cases all the cells forming these buds eventually reintegrated into the main dorsal trunk, leading to a general absence of dorsal branches (Ribeiro, 2002).

These results demonstrate that in the absence of Dpp signaling, tracheal cells close to the dorsal bnl-expressing ectodermal cells are able to form actin-containing filopodial extensions and initiate dorsal migration. However, the lack of Dpp signaling, which results in the lack of expression of the kni/knrl target genes, leads to failure to form a dorsal branch, and the short, bud-like dorsal outgrowths eventually reintegrate into the main dorsal trunk. Consistent with this interpretation, cells forming the initial dorsal outgrowth in Dad-expressing embryos in rare cases generated a dorsal trunk-sized lumen. These dorsally directed stumps of dorsal trunk were also visible in third instar larvae. Such dorsal trunk-like buds are also seen in mutants that lack Dpp-induced kni/knrl in the tracheal system, indicating that dorsal migration also takes place in these mutants. These buds are never observed in put mutants, presumably due to the lack of dorsal expression of the chemoattractant Bnl/FGF (Ribeiro, 2002).

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

Multiple BMPs are required for growth and patterning of the Drosophila wing. The Drosophila BMP gene, Tgfbeta-60A, exhibits a requirement in wing morphogenesis distinct from that shown previously for dpp. Tgfbeta-60A mutants exhibit a loss of pattern elements in the wing, particularly those derived from cells in the posterior compartment, consistent with the Tgfbeta-60A mRNA and protein expression pattern. Individuals homozygous for null alleles of the Tgfbeta-60A gene, exhibit embryonic defects in gut morphogenesis and result in early larval lethality. Tgfbeta-60A alleles have been shown to genetically interact with mutations in BMP type I receptor genes, tkv and sax. The Dpp signal is mediated by two different BMP type I receptors, Tkv and Sax, during wing morphogenesis as well as during other stages of development The possibility of a genetic interaction between alleles of Tgfbeta-60A and alleles of tkv or sax was investigated to address the relative importance of these receptors in mediating the signals resulting from the actions of Tgfbeta-60A and Dpp. Recombinants were constructed between gbb-60A 4 or gbb-60A 1 and several alleles of tkv and sax. The addition into a Tgfbeta-60A mutant background of a chromosomal deficiency that removes the tkv locus, results in a severe mutant wing phenotype with a dramatic loss of both the PCV and ACV and most of L4 and L5. In addition, distal gaps are present in L2 and L3. A less extreme phenotype is seen with tkv6, a hypomorphic allele that retains significant receptor function. The observed interaction between tkv and Tgfbeta-60A cannot be explained solely as a secondary consequence of lowering Dpp signaling readout by the mutation of a receptor that mediates Dpp signaling. These data suggest that Tkv is able to mediate Tgfbeta-60A signaling and that it may do so in different ways at different times during development. The effect of reducing the Tgfbeta-60A copy number was investigated in flies compromised for functional Tkv receptor. Reducing Tgfbeta-60A in a tkv mutant background produces a further thickening of wing veins. This result suggests that Tgfbeta-60A may play a role in vein differentiation itself and/or in the tkv/dpp feedback loop important in defining the boundaries of the vein. Genetic combinations used to investigate the potential interaction between Tgfbeta-60A and sax alleles indicate a reduction in viability for Tgfbeta-60A mutant genotypes containing a single copy of a sax null allele. This reduction in viability suggests that lowering both Tgfbeta-60A and sax compromises development. The wing phenotype of the few viable adults recovered is similar to a very severe Tgfbeta-60A mutant wing phenotype, with a substantial loss of L5, complete loss of the PCV and ACV and loss of half of L4. Clearly the levels of Tgfbeta-60A signaling and Sax function are dependent on one another (Khalsa, 1998).

Based on genetic analysis and expression studies, it has been concluded that Tgfbeta-60A must signal primarily as a homodimer to provide patterning information in the wing imaginal disc. Tgfbeta-60A and dpp genetically interact and specific aspects of this interaction are synergistic while others are antagonistic. It is proposed that the positional information received by a cell at a particular location within the wing imaginal disc depends on the balance of Dpp to Tgfbeta-60A signaling. Furthermore, the critical ratio of Tgfbeta-60A to Dpp signaling appears to be mediated by both Tkv and Sax type I receptors (Khalsa, 1998).

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

punt and schnurri function in somatic cells of the testis; they regulate a signal from somatic to germline cells that restricts proliferation of committed progenitors in the germline. In the testis, germ line stem cells and their primary and secondary spermatogonial cell progeny are surrounded by two somatic cyst cells. These two cyst cells, produced by asymmetric division of cyst progenitor cells, completely envelop the primary spermatogonial cell, forming a cyst. Cyst cells do not proliferate, but continue to envelop the germ cells throughout spermatogenesis. The primary spermatogonial cell is the mitotic founder of a cyst of secondary spermatogonial cells. There are four mitotic divisions, each with incomplete cytokinesis, producing 16 interconnected spermatogonial cells. The spermatogonial cells then undergo premeiotic S-phase, becoming spermatocytes and entering a prolonged G2 period and increasing in volume 25-fold. Subsequently, the spermatocytes undergo meiosis and spermatid differentiation. Progression through this sequence is accompanied by progressive displacement of the cyst through the tubular testis (Matunis, 1997).

By screening for mutants in which daughter germline cells fail to stop dividing, it was found that schnurri and punt are required to limit transient amplification of germ cells. Producing similar phenotypes to bag of marbles (bam) and benign gonial cell neoplasm (bgcn), mosaic analysis demonstrates that punt and schnurri act within the somatic cyst cells that surround germ cells, rather than in germ cells. shn mutant cyst cells are morphologically normal and appropriately express cyst cell markers. Thus, cyst cells themselves are not deleteriously affected by loss of shn function, suggesting that the principal role of shn function in cyst cells is to regulate the soma-to-germline signal. bam and bgcn are candidates for germ cell intrinsic components of this pathway and are likely be the targets of the punt and sch signal. Punt and Schnurii are components of a Dpp signal transduction pathway in other differentiation events. But in the testis system, a Dpp signal initiating from stem cells is probably not involved in triggering the punt/sch pathway, since a reporter gene that mimics Dpp expression shows no expression within the testis, and loss of Dpp function does not lead to overproliferation. Although the TGF-beta signal from germline cells is not known, there are two other known TGF-beta family members in Drosophila: screw and 60A. These are currently being examined to elucidate their role in this system. Thus, a signal relay operating in somatic cells regulates cell proliferating in the germline stem cell lineage (Matunis, 1997).

During Drosophila embryogenesis the Malpighian tubules evaginate from the hindgut anlage and in a series of morphogenetic events form two pairs of long narrow tubes, each pair emptying into the hindgut through a single ureter. Some of the genes that are involved in specifying the cell type of the tubules have been described. Mutations of previously described genes were surveyed and ten were identified that are required for morphogenesis of the Malpighian tubules. Of those ten, four block tubule development at early stages; four block later stages of development, and two, rib and raw, alter the shape of the tubules without arresting specific morphogenetic events. Three of the genes, sna, twi, and trh, are known to encode transcription factors and are therefore likely to be part of the network of genes that dictate the Malpighian tubule pattern of gene expression (Jack, 1999).

Mutations of punt interfere with rearrangement without apparently reducing the number of cells in the tubules. The tubules of punt embryos are more than two cells wide in various locations along their length. punt encodes the type II receptor for Dpp. The punt type II receptor and the type I receptor encoded by tkv typically act together to transduce the Dpp signal, and a transcription factor encoded by shn is a downstream effector of Dpp signaling. Mutations of tkv or shn normally have phenotypes nearly identical to put. However, thickened Malpighian tubules have not been observed in either tkv or shn mutants. Although the difference in phenotype could be due to residual activity in the tkv and shn 1 alleles examined, these results suggest that other proteins may be involved in transmitting the signal through Punt to cause tubule elongation (Jack, 1999).

In Drosophila, imaginal wing discs, Wg and Dpp, play important roles in the development of sensory organs. These secreted growth factors govern the positions of sensory bristles by regulating the expression of achaete-scute (ac-sc), genes affecting neuronal precursor cell identity. Earlier studies have shown that Dally, an integral membrane, heparan sulfate-modified proteoglycan, affects both Wg and Dpp signaling in a tissue-specific manner. dally is required for the development of specific chemosensory and mechanosensory organs in the wing and notum. dally enhancer trap is expressed at the anteroposterior and dorsoventral boundaries of the wing pouch, under the control of hh and wg, respectively. dally affects the specification of proneural clusters for dally-sensitive bristles and shows genetic interactions with either wg or dpp signaling components for distinct sensory bristles. These findings suggest that dally can differentially regulate Wg- or Dpp-directed patterning during sensory organ assembly. For pSA, a bristle on the lateral notum, dally shows genetic interactions with iroquois complex (IRO-C), a gene complex affecting ac-sc expression. Consistent with this interaction, dally mutants show markedly reduced expression of an iro::lacZ reporter. These findings establish dally as an important regulator of sensory organ formation via Wg- and Dpp-mediated specification of proneural clusters (Fujise, 2001).

Wg and Dpp have been shown to affect prepatterning of sensory organs by governing the expression of proneural genes, such as ac-sc. dally has been shown to affect the signaling levels of either Wg or Dpp. Therefore, an examination was made to determine whether dally affects sensory organ formation via either Wg or Dpp signaling pathways. Genetic experiments provided evidence that, in the prospective notum region of the wing disc, dally selectively influences Wg signaling to form the pPA bristle and Dpp signaling to form the pSA and DC bristles. dally genetically interacts with punt in the production of a bristle phenotype. It is particularly intriguing that, during development of DC macrochaetae, dally genetically interacts with only Dpp signaling, while the formation of these bristles requires both Wg and Dpp activities. It has been indicated that the A/P coordinates of the DC cluster are limited by Dpp signaling. In dally homozygous wing discs, the DC cluster is apparently shorter in the A/P coordinates compared with wild-type discs, suggesting that dally regulates Dpp signaling activity to limit the A/P length of the DC cluster. What are the mechanisms that can account for the selective interactions of dally and specific growth factor signaling? One obvious interpretation of genetic experiments on DC macrochaetae is that differences in dose effects between dpp and wg are responsible for the apparent specificity. It is also possible that the ligand-specificity of Dally is controlled at the cellular level through modification of heparan sulfate structures (Fujise, 2001).

Dally, Dpp, and IRO-C genetically interact with each other during the formation of the pSA macrochaete. Although interactions between Dpp signaling and IRO-C have been suggested, evidence is provided that Dpp signaling components interact with the genes of IRO-C. Ectopic Dpp signaling using a constitutively active type I receptor, tkv, leads to an ectopic induction of the pSA macrochaete, supporting the idea that Dpp signaling is required for prepatterning for this bristle. Significant reductions in the expression of iro enhancer trap is observed in dally mutant wing discs. Expression of the iro at the lateral notum region is critical for the proneural cluster formation and bristle development in this region. Taken together, these findings suggest that dally mediates Dpp signaling to control expression of the genes of IRO-C during the formation of the pSA bristle (Fujise, 2001).

Metamorphosis of the Drosophila brain involves pruning of many larval-specific dendrites and axons followed by outgrowth of adult-specific processes. From a genetic mosaic screen, two independent mutations were recovered that block neuronal remodeling in the mushroom bodies (MBs). These phenotypically indistinguishable mutations affect Baboon function, a Drosophila TGF-ß/activin type I receptor, and Smad on X (Smox, or dSmad2), its downstream transcriptional effector. Punt and Wit, two type II receptors, act redundantly in this process. In addition, knocking out Activin-beta (dActivin) around the mid-third instar stage interferes with remodeling. Binding of the insect steroid hormone ecdysone to distinct Ecdysone receptor isoforms induces different metamorphic responses in various larval tissues. Interestingly, expression of the Ecdysone receptor B1 isoform (EcR-B1) is reduced in activin pathway mutants, and restoring EcR-B1 expression significantly rescues remodeling defects. It is concluded that the Drosophila Activin signaling pathway mediates neuronal remodeling in part by regulating EcR-B1 expression (Zheng, 2003).

It was of interest to identify possible ligands that participate in the remodeling process. Seven TGF-β type ligands are present in the Drosophila genome. Three of these, dpp, scw, and gbb, are clearly of the BMP family. The remaining, maverick (mav), myoglianin (myo), dActivin (dAct), and activin-like-protein (alp), have not been assigned either genetically or biochemically to a particular family or signaling pathway. Phylogenetic considerations place dAct clearly within the Activin subfamily, while Myo is most similar to BMP-11 and GDF-8, and Mav and Alp are equidistant from both the BMP and TGF-β/Activin subgroups. Therefore, possible involvement of dAct in the Babo signaling was examined (Zheng, 2003).

First, in situ hybridization revealed that dAct is widely expressed in larval brains. Next, when conditioned media from cells expressing dAct was added to S2 cells transfected with Smox, it was found that this ligand is able to stimulate phosphorylation of Smox, while the prototypical BMP ligand Dpp is not. Finally, attempts were made to knock out dAct activity using two independent approaches and dAct, like Babo, was found to be essential for both optic lobe development and EcR-B1 expression in larval brains. Since dAct mutations are currently unavailable, attempts were made to produce a partial loss-of-function condition by overexpression of a dominant-negative form of the protein or RNAi. All TGF-β type ligands that have been examined dimerize and are processed prior to secretion. Previous studies have shown that overexpression of a cleavage-defective form of a particular ligand can interfere with processing and secretion of endogenous ligand. Therefore, a cleavage defective form of dAct (CMdAct) was expressed using either a general GAL4 driver (tubP-GAL4) or an MB-specific driver (GAL4-OK107). CNS development was observed to be retarded only when the CMdAct is ubiquitously expressed and not when it is expressed in MBs. This suggests that dAct does not function within MBs in an autocrine-like fashion. Poor development of the optic lobes is apparent in the tubP-GAL4>CMdAct larval brains, similar to that observed in babo and punt mutant larvae. More importantly, EcR-B1 expression is largely absent in γ neurons of animals that ubiquitously express CMdAct, similar to what is observed in babo mutants. Hs-GAL4-mediated transient expression of CMdAct around the mid-third instar stage also blocks both optic lobe development and EcR-B1 expression (53%). Consistent results are obtained after induction of RNAi using a hairpin-loop dAct construct (UAS-HLdAct). For instance, no EcR-B1 expression was detected in 65% of the late third instar larval brains that were heat shocked to express UAS-HLdAct transiently around the mid-third instar stage. Again, absence of EcR-B1 expression is tightly associated with poor optic lobe development. Similar treatments yield no detectable phenotypes when UAS-CMdAct/UAS-HLdAct is absent or replaced with other UAS-transgenes, such as UAS-mCD8-GFP and UAS-antisense dActivin. In addition, punt mutants, despite having small brains, continue to show EcR-B1 expression. Taken together, these results suggest that dAct, like Babo and dSmox, is indispensable for EcR-B1 expression in the CNS of wandering larvae (Zheng, 2003).

In Drosophila, recent data suggest that a BMP signaling pathway controls synaptic growth and function at the neuromuscular junction (NMJ). Whether this pathway also contributes to activity-dependent remodeling at the NMJ remains to be determined. It is interesting to note, however, that in this pathway Wit acts as a BMP receptor, and it can not be substituted for by Punt. In contrast, the activin pathway described here appears to be able to utilize either Punt or Wit for signaling. This may reflect selectivity in the binding of some ligands to one receptor, but not the other. Additional studies will be required to resolve this issue. Since many components of several different TGF-β signaling pathways show pronounced expression in different parts of the developing and postnatal rodent brain, the demonstration that TGF-β/Activin signaling cell-autonomously controls plasticity of MB neurons may provide novel insights into how neuroplasticity is dynamically regulated in higher organisms (Zheng, 2003).

Traditional screens aiming at identifying genes regulating development have relied on mutagenesis. A new gene has been identified involved in bristle development, identified through the use of natural variation and selection. Drosophila melanogaster bears a pattern of 11 macrochaetes per heminotum. From a population initially sampled in Marrakech, a strain was selected for an increased number of thoracic macrochaetes. Using recombination and single nucleotide polymorphisms, the factor responsible was mapped to a single locus on the third chromosome, poils au dos (French for 'hairy back'), that encodes a zinc-finger-ZAD protein. The original, as well as new, presumed null alleles of poils au dos are associated with ectopic achaete-scute expression that results in the additional bristles. This suggests a possible role for Poils au dos as a repressor of achaete and scute. Ectopic expression appears to be independent of the activity of known cis-regulatory enhancer sequences at the achaete–scute complex that mediate activation at specific sites on the notum. The target sequences for Poils au dos activity were mapped to a 14 kb region around scute. In addition, pad has been shown to interact synergistically with the repressor hairy and with Dpp signaling in posterior and anterior regions of the notum, respectively (Gibert, 2005).

Mutations in very few genes have been shown to induce ectopic bristles in the anterior region of the notum. Some ectopic bristles can be induced in this region by reduction in Dpp signaling late in development. A genetic interaction between pad and Dpp signaling was tested using mutations in the receptors punt (put) and thickveins (tkv). A strong genetic interaction was observed between pad¹ and put^P1. Trans-heterozygous put^P1/pad¹ flies have ectopic DC bristles whereas each of the single heterozygotes displays a wild-type pattern. Flies homozygous for the hypomorphic mutation tkv¹ occasionally have ectopic bristles anterior to the aDC at 18°C. The phenotype is strongly enhanced in the anterior region of the notum of double mutant tkv¹; pad¹ flies grown at 25°C. In particular, many more ectopic bristles are visible around the prescutal suture than in pad¹ alone (Gibert, 2005).

Punt and spermatogenesis

The continuous and steady supply of transient cell types such as skin, blood and gut depends crucially on the controlled proliferation of stem cells and their transit amplifying progeny. Although it is thought that signaling to and from support cells might play a key role in these processes, few signals that might mediate this interaction have been identified. During spermatogenesis in Drosophila, the asymmetric division of each germ line stem cell results in its self-renewal and the production of a committed progenitor that undergoes four mitotic divisions before differentiating while remaining in intimate contact with somatic support cells. TGF-ß signaling pathway components punt and schnurri have been shown to be required in the somatic support cells to restrict germ cell proliferation. This study showns, by contrast, that the maintenance and proliferation of germ line stem cells and their progeny depends upon their ability to transduce the activity of a somatically expressed TGF-ß ligand, the BMP5/8 ortholog Glass Bottom Boat. TGF-ß signaling represses the expression of the Bam protein, which is both necessary and sufficient for germ cell differentiation, thereby maintaining germ line stem cells and spermatogonia in their proliferative state (Shivdasani, 2003).

In order to test the requirement for TGF-β signaling in the germ line, the behavior of marked germ line clones lacking the activity of various TGF-β signaling pathway components was investigated. Germ line stem cells mutant for tkv or put (a type II TGF-β receptor) and spermatocytes lacking the activity of tkv, put, or mad (a transcription factor required for the regulation of TGF-β target genes) were generated but did not persist to the same extent as wild-type clones, as evidenced by assessing the ratio of the number of testes containing at least one germ line clone to the number of testes containing wild-type control clones. Sporadically (approximately 4% of cases), cysts containing eight, rather than 16, spermatocytes were observed, implying that the fourth spermatogonial division had not been complete. Such a scenario might have arisen due to the transient persistence of Tkv, Mad, or Put protein after the respective wild-type allele was lost. Together, these clonal analysis data suggest that TGF-β signaling is required for both germ line stem cell maintenance and spermatogonial proliferation. No requirement was found for schnurri (shn), the product of which is frequently required in Dpp signaling, in the germ line for these processes (Shivdasani, 2003).

Male gametes are produced throughout reproductive life by a classic stem cell mechanism. However, little is known about the molecular mechanisms for lineage production that maintain male germ-line stem cell (GSC) populations, regulate mitotic amplification divisions, and ensure germ cell differentiation. The Drosophila system has been used to identify genes that cause defects in the male GSC lineage when forcibly expressed. A gain-of-function screen was conducted using a collection of 2050 EP lines and 55 EP lines were found that causes defects at early stages of spermatogenesis upon forced expression either in germ cells or in surrounding somatic support cells. Most strikingly, analysis of forced expression indicated that repression of bag-of-marbles (bam) expression in male GSC is important for male GSC survival, while activity of the TGFß signal transduction pathway may play a permissive role in maintenance of GSCs in Drosophila testes. In addition, forced activation of the TGFß signal transduction pathway in germ cells inhibits the transition from the spermatogonial mitotic amplification program to spermatocyte differentiation (Schulz, 2004).

The TGFß signal transduction pathway clearly plays a role in regulating the transition from the spermatogonial-amplifying mitotic division program to spermatocyte differentiation. However, exactly how TGFß signaling acts to govern this transition remains a puzzle. Mosaic analysis demonstrated that cysts of wild-type spermatogonia undergo extra rounds of mitotic divisions and fail to become spermatocytes when associated with a somatic cyst cell mutant for either punt, the TGFß type 2 receptor, or schnurri, a transcription factor downstream of TGFß signaling during embyrogenesis. These data suggest that receipt of a TGFß class signal by somatic cyst cells induces the somatic cells to send a signal of unknown nature to the germ cells that they enclose, either inducing or permitting the spermatogonia to initiate differentiation as spermatocytes (Schulz, 2004).

Forced expression of the TGFß class signaling molecule dpp specifically in germ cells has effects similar to loss of function of the signal transduction pathway in somatic cyst cells: failure of spermatogonia to stop mitotic divisions and become spermatocytes. This result is surprising, since one would expect that forced expression of a ligand might cause a phenotype opposite that of a receptor's loss of function. One explanation might be that precise levels of the dpp ligand may be critical, for example, for proper temporal or spatial control of activation of the pathway in somatic cyst cells. Another possibility is that dpp may not be the normal ligand, but that high levels of dpp secreted from germ cells may bind to TGFß receptors on cyst cells and block their ability to respond to the normal ligand. Consistent with this hypothesis, the TGFß type II receptor punt and both TGFß type I receptors sax and tkv have been demonstrated to bind dpp in transfected Cos cells. The TGFß homolog Maverick, rather than dpp, may be the ligand normally expressed in spermatogonia for activation of the TGFß signal transduction pathway in surrounding cyst cells, since Maverick mRNA but not dpp mRNA was detected in early germ cells in wild-type testes by in situ hybridization (Schulz, 2004).

Alternatively, TGFß signaling may be required in germ cells. Indeed, forced expression of the activated tkv receptor in early germ cells also caused spermatogonia to continue mitotic proliferation rather than differentiate as spermatocytes. The apparently cell autonomous effect of forced expression of the activated tkv receptor in germ cells suggests a direct role for the TGFß signaling pathway in germ cells. However, results that marked clones of germ cells mutant for the TGFß receptor sax differentiate as spermatocytes, along with similar findings that marked clones of germ cells mutant for punt, schnurri, or Mothers against dpp differentiate as spermatocytes with the normal number of 16 spermatocytes per cyst, indicate that the TGFß signaling pathway may not normally be required in germ cells for proper execution of the spermatogonia-to-spermatocyte transition. These observations raise the possibility that forced expression of dpp or the activated tkv receptor in early germ cells blocks the transition from the spermatogonial mitotic division program to spermatocyte differentiation by artificial and abnormal interference with the germ cell autonomous mechanisms that regulate this critical cell fate transition. The only Drosophila genes previously known to be required cell autonomously in the germ line for spermatogonia to exit the spermatogonial division program and become spermatocytes are bam and its partner, bgcn. The phenotype of males haplo-insufficient for bam suggests that the level of bam expressed in male germ cells is important for the correct transition from spermatogonia to spermatocytes. One model proposed for the female germ line is that dpp secreted from somatic cap cells at the tip of the germarium blocks expression of bam in GSCs, allowing stem cell maintenance. Strikingly, the Pro-bam-GFP reporter was expressed at reduced levels in spermatogonia from males in which UAS-tkv* or UAS-dpp were forcibly expressed in early male germ cells under control of the nos-gal4 germ-line-specific transgene driver, suggesting that activated Tkv or Dpp may suppress bam expression in males as well. It is tempting to speculate that, in the male, forced expression of dpp in spermatogonia may alter levels of bam expression so that bam protein does not reach a critical threshold required for the transition to spermatocyte differentiation. However, consistent with the production of many cysts of differentiating interconnected spermatogonia in UAS-tkv*; nos-gal4 and UAS-dpp; nos-gal4 males, some expression of the Pro-bam-GFP reporter was detected. The expression of the Pro-bam-GFP reporter even in the presence of the activated tkv receptor suggests that there may be mechanisms at work in spermatogonia that can override silencing of bam expression by the TGFß signaling pathway. Because the Pro-bam-GFP transcriptional reporter was expressed in spermatogonia even in cells expressing the activated tkv receptor, these mechanisms are likely either to interfere with the TGFß signal transduction pathway downstream of receptor activation or to act independently of and/or override the TGFß signaling effect. Forced expression of activated tkv in spermatogonia may also somehow affect expression or stability of Bam protein, since no accumulation of BamC protein was detected in spermatogonial cysts in testes from UAS-tkv*; nos-gal4 animals (Schulz, 2004).

Forced expression of the TGFß class signaling molecule dpp or the activated tkv receptor in early male germ cells leads to a mild increase in the number of male GSCs and gonialblasts around the apical hub and to reduced expression levels of the Pro-bam-GFP transcriptional reporter in spermatogonia. If TGFß signaling normally acts on the silencer element in the bam gene to repress expression of bam in male GSCs, as has been shown for female GSCs, then forced activation of TGFß signaling in male early germ cells might delay the transition from stem cell to spermatogonial differentiation by delaying the accumulation of bam protein. However, the effect of activation of TGFß class signaling on male GSCs was much more subtle than the effects noted in female germ cells. The difference between the sexes in this regard may reflect the fundamental difference in the role of bam in male vs. female early germ cells. Loss of function of TGFß class signaling in male GSCs has a subtle, but opposite, effect. Germ-line clones homozygous mutant for the TGFß class receptor sax appear at lower frequency and tend to produce fewer differentiating cysts compared to control clones. Of course, data from clonal analysis must always be interpreted with caution because of the possibility of effects from secondary recessive mutations on the chromosome arm. However, given the observations on the effects of forced expression of bam, it is tempting to speculate that loss of function of sax from germ cells allows bam to be expressed too early in male GSCs and gonialblasts, slowing or arresting differentiation of spermatogonial cysts and eventually leading to early germ cell loss. It is noted that some sax mutant germ-line clones did persist over time, again suggesting that male GSCs appear less sensitive than female early germ cells to either loss of function of TGFß signaling or overactivation of the receptor (Schulz, 2004).

Although parallels between the male and female GSC systems are beginning to emerge, bam and the TGFß signaling pathway appear to play fundamentally different roles in male vs. female early germ cells. In both cases, male GSCs appear to be less sensitive than female GSCs to perturbations. It is proposed that this difference relates, at least in part, to the difference in the primary role of bam in the two sexes. In the female germ line, expression of bam appears to be the key event that produces a cystoblast and drives it to embark on cystocyte differentiation. Thus the mechanisms that suppress bam expression in GSCs and allow it in cystoblasts are likely to be key instructive determinants in the decision between stem cell self-renewal and the onset of differentiation. In contrast, in the male germ line, wild-type function of bam is primarily required at a later step in the differentiation pathway for cessation of the amplifying mitotic spermatogonial divisions and the transition to spermatocyte differentiation. In this case, the mechanisms that block bam protein expression in the GSCs may play a permissive rather than instructive role in allowing stem cell maintenance (Schulz, 2004).