schnurri


DEVELOPMENTAL BIOLOGY

Embryonic

SHN mRNA is expressed in a dynamic pattern in the embryo that includes most of the known target tissues of DPP, including the dorsal blastoderm, the mesodermal germlayer and parasegments 4 and 7 of the midgut. During the blastoderm stage, shn is restricted to the dorsal half of the embryo, similar to the pattern of DPP and TKV. Later, it is found in the presumptive mesoderm and the invaginating ventral furrow. In germ band retracted embryos [Images], shn is expressed in endoderm (Staehling-Hampton, 1995).

Larval and Adult

schnurri is transcribed in the eye antennal disc, the wing disc and the ovary (Arora, 1995).

Effects of Mutation or Deletion

Mutations in shn affect several developmental processes regulated by DPP, including induction of visceral mesoderm cell fate, dorsal/ventral patterning of the lateral ectoderm, and wing vein formation. Absence of shn function blocks the expanded expression of the homeodomain protein Bagpipe in the embryonic mesoderm caused by ectopic DPP expression, illustrating a requirement for shn function downstream of DPP action. SHN protein is the first identified downstream component of the signal transduction pathway used by DPP and its receptors (Staehling-Hampton, 1995).

The dorsal closure phenotype of schnurri mutant is accompanied by altered shape and cytoskeleton of dorsal epidermal cells (Arora, 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).

In the developing wing blade, somatic clones lacking the DPP receptors Punt or Thick veins, or lacking Schnurri, required for 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, 1996).

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).

This study investigated the role of the zinc finger transcription factor Schnurri (Shn) in mediating the nuclear response to Dpp during adult patterning. Using clonal analysis, it has been shown that wing imaginal disc cells mutant for shn fail to transcribe the genes spalt, optomotor blind, vestigial, and Dad, that are known to be induced by dpp signaling. shn clones also ectopically express brinker, a gene that is downregulated in response to dpp, thus implicating Shn in both activation and repression of Dpp target genes. Loss of shn activity affects anterior-posterior patterning and cell proliferation in the wing blade, in a manner that reflects the graded requirement for Dpp in these processes. Furthermore, shn is expressed in the pupal wing and plays a distinct role in mediating dpp-dependent vein differentiation at this stage. The absence of shn activity results in defects that are similar in nature and severity to those caused by elimination of Mad, suggesting that Shn has an essential role in dpp signal transduction in the developing wing. These data are consistent with a model in which Shn acts as a cofactor for Mad (Torres-Vazquez, 2000).

shn is broadly expressed in the wing imaginal disc, suggesting that it could be involved in the transcriptional regulation of Dpp target genes during larval development. In order to determine the contribution of shn to Dpp signaling, clones of homozygous mutant cells that lack shn activity were generated and their effect on adult wing patterning was examined. For all three alleles examined, clones were recovered at a significantly higher frequency when somatic recombination was induced during mid-late third larval instar, suggesting that shn clones induced earlier in development experience a growth disadvantage or do not survive to adulthood. The effect of loss of shn activity in the wing margin was examined using mutations in the yellow (y) gene to identify clones lacking shn function. The anterior wing margin differentiates three unique types of bristles that form excellent markers for anterior-posterior patterning. Each bristle type is found in a specific domain along the wing margin that corresponds to a distinct level of Dpp signaling. The anteriormost position is occupied by the distal costa bristles that are followed by the triple row bristles, and finally the double row bristles. The latter are closest to the A/P boundary and require the highest levels of Dpp signaling for specification. Although clones of shn induced at 90 h after egg lay can be recovered throughout the anterior wing margin, they are observed at a much higher frequency in the region containing the triple row bristles compared to the double row bristle domain. In addition, clones recovered in the triple row bristle domain tend to be larger than clones in the double row domain, as judged by the percentage of clones that differentiate at least three or more bristles within each region. Anterior shifts in cell fate were encountered in shn clones located in the double row bristle domain. Within these clones, margin cells differentiate the thicker triple row bristles instead of the finer bristles typical of the double row. The bristle transformations occur in a cell-autonomous manner and affect all bristles within the clone. Thus clones lacking shn function behave like cells further away from the source of Dpp. The phenotypes of these clones indicate a clear requirement for shn in anterior-posterior patterning of the wing. Mutant shn clones located in the region containing triple row bristles show no phenotypic consequences. In summary, the data demonstrate that both in cell proliferation and in A/P patterning, Shn activity is more stringently required in cells that are closer to the source of Dpp and thus exposed to higher levels of signaling (Torres-Vazquez, 2000).

The requirement for shn in cell proliferation is apparent in the underrepresentation of mutant cells with respect to wild-type cells in the wing blade as well. shn clones marked with prickle are smaller than the corresponding twin spots marked with shavenoid. In addition, in several instances shavenoid clones in the wing are not accompanied by a corresponding prickle clone. In agreement with the idea that cells lacking shn experience a growth disadvantage, larger clones of shn mutant cells were recovered when they were generated in the presence of a Minute mutation on the shn chromosome. In these animals, a loss of wing blade tissue and margin structures is obvserved, phenotypes that are likely to result from cell death or a failure of cell proliferation. In a number of cases small patches of shn mutant cells were recovered immediately adjacent to the missing portion of the wing, suggesting that the deleted region comprised cells lacking shn function that did not survive to adulthood (Torres-Vazquez, 2000).

A third class of phenotypic defects has been observed in shn mutant clones that abut or bisect a vein. Mutant cells within such clones do not differentiate vein tissue. shn clones on the dominant (protruding) surface of the vein are associated with interruptions in the vein tissue. Clones on the nonprotruding surface do not appear to disrupt the vein, although it is possible that the loss of vein tissue is obscured by the stronger pigmentation on the dominant surface. The loss of venation is almost exclusively cell autonomous. However, in a few rare instances clones on one surface have been observed that apparently impact vein differentiation on both sides of the wing. This is intriguing since there is no evidence to suggest that dpp is involved in communication between the dorsal and the ventral wing surfaces. A possible explanation could be that loss of vein fates impacts the expression of other signals that are involved in interactions between the two wing surfaces. The failure of shn clones to adopt vein cell fate is observed throughout the wing and thus is not specific to any particular vein position or compartment. In addition, an interesting non-cell-autonomous effect has been observed associated with small shn clones that impinge on veins. In these situations vein differentiation is disrupted within the mutant clone, but wild-type cells surrounding the clone differentiate ectopic veins. Such clones can result in branching and disruption of endogenous veins, as well as bifurcations that encapsulate the mutant tissue. In all such instances the ectopic vein is comprised of wild-type cells entirely. In rare cases ectopic veins formed by wild-type cells have been observed that do not appear to be in the vicinity of a mutant clone. Presumably in these cases, the shn clone associated with the ectopic vein did not survive to adult stages. Taken together, these results demonstrate that shn activity is required for Dpp responsiveness in multiple aspects of wing development: A/P patterning of the imaginal disc, cell survival, and vein differentiation in pupal wings (Torres-Vazquez, 2000).

Finally, wings in which shn clones are induced frequently differentiate small circular arrays of cells, which are pigmented like vein cells but do not possess a compact linear form. The genotype of these cells could not be determined since they lack trichome hairs and thus are not labeled by any of the clonal markers used. The clusters appear more frequently in the anterior compartment and are encountered both adjacent to endogenous veins and in the intervein region (Torres-Vazquez, 2000).

Stromal cells are thought to generate specific regulatory microenviroments or 'niches' that control stem cell behavior. Characterizing stem cell niches in vivo remains an important goal that has been difficult to achieve. The individual ovarioles of the Drosophila ovary each contain about two germ line stem cells that maintain oocyte production. Anterior ovariolar somatic cells comprising three cell types act as a germ line stem cell niche. Germ line stem cells lost by normal or induced differentiation are efficiently replaced, and the ability to repopulate the niche increases the functional lifetime of ovarioles in vivo. These studies implicate one of the somatic cell types, the cap cells, as a key niche component (Xie, 2000).

The Drosophila ovary is a tissue where stem cells can be studied at the cellular and molecular level in vivo. Near the beginning of each developing egg string (or ovariole) within the ovary reside about two germ line stem cells (GSCs) whose progeny differentiate into eggs within 8 days as they move at predictable rates along the ovariole. These stem cells are surrounded by three differentiated somatic cell types -- terminal filament, cap, and inner sheath cells -- that help make up an anatomically simple tubular structure known as the germarium. GSCs are easily identified by size, location, and the shape of the fusome, an intracellular structure rich in membrane skeleton proteins. Stem cells usually contain a round fusome, but display a distinctive elongated fusome after division when they remain transiently connected with their daughter cell. Under appropriate conditions, GSCs divide about once per day and are randomly lost by differentiation, with a half-life of 4 to 5 weeks. It has been proposed that the somatic cells at the tip of the ovariole are organized into a niche that maintains and controls GSCs (Xie, 2000).

Ovariolar anatomy is consistent with the existence of a niche at the anterior tip. After stem cell division, the daughter that lies closer to the terminal filament and cap cells remains a stem cell, whereas the daughter that more closely adjoins the inner sheath cells differentiates into a cystoblast. Anatomical asymmetry may ensure that equivalent stem cell daughters receive different fate-determining signals. GSCs require a signal mediated by Dpp, a homolog of human bone morphogenetic proteins 2 and 4, in order to remain as stem cells and to divide at a normal rate. Two other proteins needed to maintain GSCs, Piwi and Fs(1)Yb (Yb), act outside the germ line. However, a requirement for intercellular signals does not by itself indicate the presence of a niche. A true niche should function independently of resident stem cells and be able to reprogram newly introduced cells to become stem cells. Consequently, it was investigated whether the microenvironment at the ovariolar tip can specify cells to become GSCs (Xie, 2000).

Ovarioles normally lose GSCs by differentiation, but the low rate of GSC loss and the possibility that rapid replacement quickly restores the original GSC configuration complicate observing such events. To study germaria with recently lost stem cells, individual GSCs were genetically marked and destabilized. FRT-mediated recombination was used to generate mutant clones of schnurri (shn), a gene that is likely to reduce GSC lifetime by disrupting dpp signaling, under conditions where the mutant cells also lose an armadillo-lacZ marker. Because cystoblasts require 4 to 5 days to exit the germarium, the only remaining lacZ- cells 1 week after transiently activating the hs-FLP transgene by means of a heat shock will be clones consisting of shn mutant stem cells and their progeny of 4 to 5 days. With this marking system, marked shn GSCs that differentiate during the last 4.5 days can be recognized because lacZ- germ cells will remain in the germarium; moreover, the developmental age of the least mature such cell will indicate the elapsed time since GSC loss (Xie, 2000).

The results demonstrate that shn mutant stem cells are lost at an increased rate and are rapidly replaced by wild-type cells. Seventy-nine germaria were found that retained lacZ- germ cells, revealing that a shn stem cell had been recently lost. In every case they contained two wild-type stem cells, indicating that the lost lacZ- stem cell had been replaced by a wild-type stem cell. Even when the stem cell was lost so recently that it remained a cystoblast, two lacZ+ stem cells were present at the tip. These stem cells are connected by an elongated fusome, indicating that they are recently divided sister cells in early interphase; the fusome is oriented in an unusual manner, perpendicular to the anterior/posterior (A/P) axis. These observations suggest a specific model for GSC replacement. After one GSC is lost, its neighboring stem cell divides perpendicular to the A/Paxis, causing a daughter cell to occupy the environment recently vacated by the departed GSC. For this mechanism to work, the environment at the site of the lost GSC must be capable of programming the incoming cell to become a GSC rather than a cystoblast. Observations indicate that it is capable of doing so, and hence that GSCs reside in a true stem cell niche (Xie, 2000).

The ability of the ovariole tip to act as a stem cell niche is likely to be biologically important. Females produce eggs for months, despite the 4- to 5-week half-life of an individual stem cell. To investigate whether stem cell replacement occurs normally, the number of stem cells and somatic niche cells was measured in aging females. During the first 5 weeks of adult life the average number of GSCs per germarium declines from about 2.5 to 2.0, significantly less than the 50% reduction expected in the absence of replacement. Replacement stem cells must function efficiently because the rate of stem cell loss does not increase with age. Some of the ovarioles that did lose a stem cell started with three GSCs, because the number of such ovarioles declined over the same period (Xie, 2000).

One of the three somatic cell types, cap cells, interacts with stem cells in a manner that suggests they play a role in niche function. Over the 36-day period, the number of cap cells and GSCs remained closely correlated at about 2.5 cap cells per GSC. Moreover, GSCs were observed to always make special contacts with cap cells that characteristically align with the A/P axis of the ovariole. The GSC's fusome remains adjacent to the GSC/cap cell interface during most of the cell cycle. In contrast, the behavior of inner sheath cells and terminal filament cells does not correlate closely with GSCs. As germaria age, terminal filament cells decrease in number from an average of 9.2 (3 days) to 5.0 (36 days) and change from a linear to a ball-like arrangement. Likewise, the relative number of inner germarium sheath (IGS) cells and GSCs vary. However, the number of IGS cells is closely correlated with the number of differentiating germ cells. A functional connection between IGS cells and germ cell cysts has been previously suggested, because ovariole tips that develop without germ cells lack IGS cells (Xie, 2000).

To investigate the role of IGS cells in adult germaria females carrying a hs-bam transgene, whose stem cells can be induced to differentiate, were studied. Over the course of several days after heat shock, GSCs are lost and all germ line cysts completed development and left the germarium. Such germaria also lose all IGS cells, further indicating that developing germ cells control IGS cell number. In contrast, terminal filament and cap cells do not change in the absence of germ cells. Somatic cell divisions continue in their vicinity as in germaria that form in the absence of germ cells. Despite their presence near the GSC niche, these dividing somatic cells do not become GSCs (Xie, 2000).

Because the number of cap cells correlates closely with the number of GSCs, whether they might function by preferentially sending a dpp signal was investigated. Suitable antibodies to Dpp are unavailable, so whole-mount in situ hybridization was used to determine which cells at the ovariole tip express dpp mRNA. These experiments detected low levels of dpp mRNA in both cap cells and inner sheath cells, as well as higher levels in prefollicle cells farther posterior in the germarium. No dpp mRNA was seen in terminal filament cells or in any germ line cells, including GSCs. These results show that cap cells are one of several cell types located near the GSCs that express dpp. Moreover, it does not appear to be the absence of contact with a dpp-expressing cell that causes the posterior stem cell daughter to differentiate as a cystoblast (Xie, 2000).

These studies suggest a working model for a GSC niche. It is proposed that cap cells are critical to the formation, maintenance, and regulation of the GSC niche. Cap cells and terminal filament cells form a characteristic structure with sufficient internal surface area to contact two or three GSCs. A special cell-cell junction is likely to form between GSCs and cap cells to explain their intimate juxtaposition throughout adult life. Such a junction likely holds a GSC at the anterior and prevents it from moving away from the ovariolar tip where it might receive differentiation cues. An intercellular signal, possibly dpp, would be needed to maintain this junction and control the rate of GSC division, but need not be spatially graded. Additional signals appear to be involved in niche function as well. Terminal filament cells and/or cap cells express hedgehog, wingless, and armadillo, although roles for these signaling molecules in regulating GSCs remain unclear. Yb and piwi function outside the germ line in maintaining GSCs. The combined action of these genes ensures that precisely one of the GSC daughters loses cap cell contacts and differentiates (Xie, 2000).

These experiments show that a small group of stromal cells located at the tip of the Drosophila ovariole acts as a stem cell niche. Stem cells in many different tissues and organisms may be regulated in a similar manner. In the Drosophila testis, five to seven stem cells are anchored on terminally differentiated somatic hub cells, suggesting that both the ovary and testis could use similar strategies to regulate their stem cells. In Caenorhabditis elegans, distal tip cells have been directly implicated in the maintenance of the GSC population. In the Arabidopsis shoot meristem, an organizing center located nearby is required to maintain meristem stem cells. The reported plasticity of some mammalian stem cells may result from the existence of niches that can reprogram stem cell identity. The studies presented here provide a basis for detailed comparisons between the structure and regulatory properties of niches supporting different stem cells and will assist efforts to elucidate the molecular signals that control stem cell division and differentiation (Xie, 2000).

Transforming growth factor ß signaling mediated by Decapentaplegic and Screw is known to be involved in defining the border of the ventral neurogenic region in the fruitfly. A second phase of Decapentaplegic signaling occurs in a broad dorsal ectodermal region. The dorsolateral peripheral nervous system forms within the region where this second phase of signaling occurs. Decapentaplegic activity is required for development of many of the dorsal and lateral peripheral nervous system neurons. Double mutant analysis of the Decapentaplegic signaling mediator Schnurri and the inhibitor Brinker indicates that formation of these neurons requires Decapentaplegic signaling, and their absence in the mutant is mediated by a counteracting repression by Brinker. Interestingly, the ventral peripheral neurons that form outside the Decapentaplegic signaling domain depend on Brinker to develop. The role of Decapentaplegic signaling on dorsal and lateral peripheral neurons is strikingly similar to the known role of Transforming growth factor ß signaling in specifying dorsal cell fates of the lateral (later dorsal) nervous system in chordates (Halocythia, zebrafish, Xenopus, chicken and mouse). It points to an evolutionarily conserved mechanism specifying dorsal cell fates in the nervous system of both protostomes and deuterostomes (Rusten, 2002).

The second phase of Dpp signaling, covering most if not all the dorsal ectoderm, starts at stage 9 and lasts until stage 10/11 [3.40 to 5.20 hours after egg laying (AEL)]. Initially proneural clusters (PNCs) and later sensory organ precursors (SOPs), singled out within each PNCs, can be visualized by the expression of the proneural genes achaete (ac, 4.20-7.20 hours AEL), atonal (ato, 5-6.30 hours AEL) and amos (amo, 5.20-6 hours AEL). Thus, the second wave of Dpp signaling precedes and overlaps with the development of the PNCs and SOPs. The domain of Dpp signaling was examined using an enhancer trap lacZ line inserted in the gene daughters against dpp (dad), a target of Dpp. Double immunofluorescence staining shows that dorsally located Ac and Ato positive PNCs and SOPs originate inside the dad-lacZ positive region, suggesting that they have received, or still receive, Dpp signaling. However, a subset of PNCs and SOPs are ventral to the dad-lacZ domain. As the PNS neuronal precursors differentiate close to the position where they originate, it can be concluded that a part of the dorsal PNS forms within an active Dpp signaling region (Rusten, 2002).

The embryonic abdominal (A) PNS of Drosophila consists of three bilateral clusters of neurons (ventral, lateral and dorsal) per segment, which can be most especially appreciated in the serially homologous segments A1-A7. In order to investigate whether the second phase of Dpp signaling is necessary for patterning the PNS, mutant alleles for a gene involved in the Dpp signaling pathway, schnurri (shn), were examined. This gene encodes a zinc-finger transcription factor that is necessary for the transcription of some Dpp target genes and binds directly to the main Dpp mediator Mothers against Dpp (Mad). Unlike the zygotic mutants of dpp, scw, tolloid (tld) or mad, shn mutants have no effect on the initial dpp/scw governed dorsoventral patterning of the blastoderm. They express normally the early Dpp target genes, such as pannier (pnr, stage 7), dpp itself in the dorsal ectoderm (stage 9) and Krüppel (Kr) (which is a marker for the amnioserosa), showing that the dorsal ectoderm is correctly specified. By contrast, several Dpp target genes that are expressed following the second phase of Dpp signaling are affected in shn zygotic mutants: at stage 11, the expression of genes responsive to Dpp signaling, such as dad, pnr, spalt or dpp itself is reduced or lost. Thus, any failures in PNS formation, which are observed in shn mutant embryos, must originate from the second rather than the first phase of Dpp signaling and are likely to be mediated by Shn. PNS malformations were sought in strong shn zygotic mutant embryos using the ubiquitous PNS neuronal marker 22C10. Homozygous shn1 and shnk00401 fail to undergo dorsal closure and show severe defects of PNS development. A strong reduction in number of neurons is observed, especially in the dorsal and lateral PNS clusters, although it is difficult to determine exactly which neurons are affected because of the dorsal closure failure. Therefore, another allele, shnk04412, which does undergo dorsal closure, was also examined. In these embryos, position and identity of PNS neurons could be more clearly assigned. In homozygosity, as well as in transheterozygosity over shn1, this mutant shows a reduction in the number of dorsal and lateral neurons, similar to the other mutants analyzed. These results are consistent with a role for Shn-mediated Dpp signaling in the formation of the dorsal and lateral PNS (Rusten, 2002).

In all these mutant backgrounds the dorsal and lateral PNS clusters show a severe reduction in the number of neurons. No major differences are found depending on the neuronal type: the percentage of external sensory organ neurons lost is similar to the loss of neurons in the chordotonal organs. The penetrance of this effect, as measured in the differentiated PNS clusters, varies among abdominal segments. The average reduction in neuronal number ranges from 25% (HS-ssog) to 41% (shnk00401) in the dorsal cluster and 8% (HS-ssog) to 52% (shnk00401) in the lateral cluster. By contrast, the ventral cluster is less affected because it shows 2% (HS-ssog) to 18% reduction (shnk00401). The lateral pentascolopodial organ shows migration defects in these embryos, but the other sensory organs are located in their expected relative positions (Rusten, 2002).

The reduced number of neurons observed in the dorsal and lateral PNS when Dpp signaling is impeded could result from lack of proneural gene expression, which is known to be necessary for PNC and SOP formation. The expression of ato and ac was analyzed to examine the specification of progenitor cell subclasses in mutant backgrounds defective for Dpp signaling. The development of the serially homologous abdominal segments A1 to A7 is similar and very synchronous. Thus, in the wild type, whenever a specific number of PNCs and SOPs appear in one abdominal segment, a similar pattern is observed in the other abdominal segments as well. This is not true for shnk04412 mutants and for embryos expressing ubiquitous ssog, where the numbers of Ac and Ato positive SOPs and PNCs vary among the abdominal segments. This is consistent with the variably penetrant phenotypes observed in differentiated PNS among abdominal segments. In embryos expressing Kr-Gal4;UAS-brk, loss of Ato- and Ac-positive PNCs and SOPs was observed specifically in the abdominal segments A1-A3 where brk was misexpressed, when compared with abdominal segments A4-A7 that served as an internal reference. The reduced numbers of Ato- and Ac-positive neuronal progenitors appear to result from failure of PNC formation rather than an increase in cell death ratio: apoptosis does not appear to increase in segments expressing brk compared with the other abdominal segments. Taken together, these results suggest that reduction in the number of neurons is produced by failure in proneural gene expression (Rusten, 2002).

A misexpression screen reveals effects of bag-of-marbles and TGF ß class signaling on the Drosophila male germ-line stem cell lineage

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).


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schnurri: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 10 August 2010

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