Effects of Mutation or Deletion (part 2/2)

Spitz and Mesoderm Development

The Drosophila ventral midline cells generate a discrete set of CNS lineages, required for proper patterning of the ventral ectoderm. The CNS midline cells also exert inductive effects on the mesoderm. Mesodermal progenitors immediately adjacent to the midline progenitor cells give rise to three types of structures: ventral somatic muscles, cells associated with the salivary glands (presumably fat cells), and a pair of unique cells that come to lie dorsomedially on top of the ventral nerve cord, the so-called dorsal median (DM) cells. DM cells are molecularly distinct mesodermal cells, as they express and require the homeobox gene buttonless in addition to a number of other markers (neuroglian, laminin, glutactin and collagen IV). Cell ablation as well as cell transplantation experiments indicate that formation of the DM cells is induced by midline progenitors in the early embryo. Each pair of DM cells derives from one mesodermal progenitor. In cyclin A mutants there is only one DM cell per segment, suggesting that in the wild type, the DM cells appear in the lineage after the second postblastodermal mitosis. Expression of a buttonless reporter construct reveals expression at early stage 11, in a cluster of 2-4 cells at the midline on top of the CNS. One of these cells expresses the marker significantly more strongly than the others and a short time later divides (Luer, 1997).

These results are corroborated by genetic analyses. Mutant single minded embryos lack the CNS midline as well as the DM cells. Transplanted ventral midline induce DM cells, which are recognized by their expression of a buttonless reporter. Embryos mutant for any of the spitz group genes, which primarily express defects in the midline glial cell lineages, show reduced formation of the DM cells. Since spitz group genes, includingpointed, star and rhomboid most prominently affect glial lineages, a test was made of the effect of mutation of orthodenticle on DM formation. otd determines midline neural cell fate. In all otd embryos inspected, DM cell numbrs correspond to wild type, suggesting that the glial lineage is responsible for DM induction. Directed overexpression of secreted Spitz by some or all CNS midline cells leads to the formation of additional DM cells. Supernumerary midline cells are triggered by additional mitoses and not by recruiting additional DM progenitors. DM cell development does not depend on the absolute concentration of the local inducer (likely to be Spitz), but appears to require a graded source of an inducing signal. Thus, the Drosophila CNS midline cells play a central inductive role in patterning the mesoderm as well as the underlying ectoderm (Luer, 1997).

Signalling by the Drosophila epidermal growth factor receptor is required for the specification and diversification of embryonic muscle progenitors

Muscle development initiates in the Drosophila embryo with the segregation of single progenitor cells, from each of which a complete set of myofibers arises. Each progenitor is assigned a unique fate, characterized by the expression of particular gene identities. The Drosophila Epidermal growth factor receptor (Egfr) provides an inductive signal for the specification of a large subset of muscle progenitors. In the absence of the receptor or its ligand, Spitz, specific progenitors fail to segregate. The resulting unspecified mesodermal cells undergo programmed cell death. In contrast, receptor hyperactivation generates supernumerary progenitors, as well as the duplication of at least one Spitz-dependent myofiber. The requirement for Egfr occurs early in muscle cell specification, as early as five to seven hours after fertilization. The development of individual muscles is differentially sensitive to variations in the level of signaling by the Epidermal growth factor receptor. Such graded myogenic effects can be influenced by alterations in the functions of Star and Rhomboid. In addition, muscle patterning is dependent on the generation of a spatially restricted, activating Spitz signal, a process that may rely on the localized mesodermal expression of Rhomboid. Thus, Epidermal growth factor receptor contributes both to muscle progenitor specification and to the diversification of muscle identities (Buff, 1998).

In a screen for lethal mutations that disrupt the normal embryonic muscle pattern, multiple alleles of two of the Drosophila spitz group genes were identified: Star and spitz. In a strong spi mutant approximately half of the normal myofibers are missing, while those that do develop have morphologies, positions and orientations that allow them to be assigned wild-type identities. For example, all of the lateral transverse muscles form normally in the absence of spi function, whereas gaps are present in the set of ventral longitudinal muscles. Only one of the normal three ventral oblique muscles is present in a spi mutant. Of note, muscle defects are not more severe in ventral regions where the spi group genes are known to be required for ectodermal patterning. Since spi encodes a ligand for the Drosophila Epidermal growth factor receptor, the Egfr loss-of-function phenotype was examined. A temperature-sensitive allele was used to examine the mutant Egfr muscle phenotype in an attempt to bypass the early pleiotropic requirements for Egfr signaling in embryonic development. Many of the spi-dependent muscles were also found to require Egfr function, but this analysis was limited by the finding that the temperature-sensitive period for Egfr involvement in myogenesis overlaps with that of other developmental roles for this receptor. To circumvent this problem, the myogenic function of Egfr was studied in isolation by targeting the expression of a dominant negative form (DNDER) to the mesoderm using the GAL4/UAS expression system. DNDER was constructed by deleting the intracellular domain of the protein, a strategy that has proved effective in other systems for inhibiting full-length RTKs (Buff, 1998).

The spi mutant muscle pattern is phenocopied by mesodermal expression of DNDER. The severity of this phenotype is dependent on the copy number of the DNDER transgene, and the specificity of the response to the truncated receptor is demonstrated by the ability of wild-type Egfr to reverse its effect. This strongly suggests that Spi signaling through Egfr is essential for normal myogenesis. The targeted ectopic expression of DNDER establishes that the receptor functions autonomously in mesodermal cells, as opposed to the known ectodermal abnormalities associated with loss of DER function having an indirect influence on mesoderm development (Buff, 1998).

To determine at what stage of muscle development spi and Egfr are required, the effect of loss-of-function of these genes was examined on the expression of several early myogenic markers. The mature myofibers seen in stage-16 embryos differentiate from muscle precursors that are formed by myoblast fusion starting at stage 12 and continuing through stage 15. Additional dorsal mesodermal cells segregate to become heart precursors during this time. The segmentation genes, even-skipped and Kruppel, are expressed in distinct but partially overlapping subsets of mesodermal precursors. The precursor of muscle DA1, which expresses both Eve and Kr, is missing in the absence of spi and DER functions. Additional Kr-positive muscle precursors, including LL1, VA2 and several other internal ventral precursors, are also spi/Egfr-dependent. However, the Eve-expressing pericardial cell precursors, as well as certain dorsal and lateral Kr-expressing muscle precursors (DO1, LT2 and LT4), form normally in these genetic backgrounds. The presence or absence of these precursors correlates completely with the mature myofiber pattern of spi and Egfr mutant embryos. These results demonstrate that spi and Egfr are required for the formation of some but not all muscle precursors. At an even earlier stage of mesoderm development, mononucleated progenitor cells segregate and divide to generate sibling founder cells, each sibling cell the founder for the formation of one muscle precursor. Progenitors initially express the proneural gene, lethal of scute (l'sc), as well as muscle identity genes such as S59, eve and Kr. The expression of identity genes persists while that of l'sc fades prior to progenitor division. This developmental sequence is illustrated for the two Eve progenitors, P2 and P15. P2 forms first and initially expresses both L'sc and Eve. By the time L'sc disappears from P2, P15 forms and co-expresses L'sc and Eve. Both progenitors then divide, each giving rise to two Eve-positive founder cells (F2 and F15). Eve is retained in only one founder cell of each pair. The F2 founder in which Eve persists divides again, giving rise to a pair of pericardial cells in each hemisegment, while the Eve-expressing F15 contributes to muscle DA1; the subsequent fates of the F2 and F15 founders that lose Eve expression remain unknown. With loss of either Spi or Egfr function, P15 does not develop, whereas P2 and its founders segregate normally. This is consistent with the prior finding that DA1 muscle precursors, but not the Eve pericardial cells, are dependent on spi and Egfr. Additional L'sc-expressing muscle progenitors also are missing from spi mutant embryos. Thus, Spi/Egfr signaling is involved in the earliest step of somatic myogenesis, the specification of muscle progenitors. It is shown that Spi/Egfr signaling specifies particular muscles at different developmental times and that unspecified mesodermal cells undergo programmed cell death in the absence of Spi/Egfr signaling. It is also shown that hyperactivation of Egfr generates supernumerary muscle founders and the duplication of a Egfr-dependent muscle (Buff, 1998).

Star, which is known to interact with Egfr, modifies myogenic signaling by Egfr. Ectopic mesodermal expression of DNDER yields a sensitized background in which to quantitate genetic interactions with Star. One copy of UAS-DNDER caused a partial reduction in the development of DA1 and VA2. This effect is suppressed by co-expression of full-length Egfr or Star. Ectopic expression of Star or full-length Egfr in a wild-type genetic background had no effect on muscle development. The UAS-Star results also indicate that Star is required autonomously for Egfr function in the mesoderm. Star dominantly enhances the effect of DNDER on muscles DA1 and VA2, suggesting that Star is normally limiting for muscle development. Rhomboid is also required for muscle DA1 formation and is expressed in the mesoderm in proximity to the DA1 progenitor. As was found for spi, Star and Egfr, rho is also required for development of the Eve-expressing muscle DA1 precursor but not for formation of the adjacent pericardial cells. Because Rho is a positive regulator of Egfr and its expression is frequently localized to sites where Egfr signaling is active, the expression of rho in the vicinity of DA1 was examined during the course of its development. rho transcripts are found in segmentally repeated dorsal mesodermal cells in stage-11 embryos. These cells are located at the peaks of the mesodermal crests that lie between the tracheal pits, precisely where the Eve-expressing P2 and P15 progenitors and their founders arise. By double-labeling with Rho and Eve antibodies, it was found that Rho is co-expressed with Eve in P2. This is a particularly intriguing finding since the specification of P2 (the pericardial progenitor) precedes that of P15 (the muscle DA1 progenitor): these two cells segregate in very close proximity to each other, and only P15 is Egfr-dependent. Even under conditions where muscle DA1 forms in the absence of Eve pericardial cells, such as with partial inhibition of Heartless activity, Rho is expressed in a mesodermal cell that resembles a normal P2 but lacks Eve. Given the known effects of Rho in modifying Egfr activity in other developmental contexts, the temporal and spatial expression of Rho in the dorsal mesoderm is consistent with a functional role for Rho in the Egfr signaling responsible for P15 induction (Buff, 1998).

An interplay between two EGF-receptor ligands, Vein and Spitz, is required for the formation of a subset of muscle precursors in Drosophila

Activation of the Drosophila EGF-receptor (Egfr) is spatially and temporally controlled by the release of its various ligands. Egfr and its ligand Spitz mediate the formation of specific somatic muscle precursors. A second Egfr ligand, Vein, complements the activity of Spitz in the development of various somatic muscle precursors. In vn mutant embryos, the Egfr-dependent muscle precursors do not form in some of the segments. Double labeling with anti-Vein and anti-Kruppel antibodies reveal that the Kruppel-positive muscle precursors overlap. In vein mutant embryos at stage 12-13 of embryonic development DA1 muscle precursors are missing in one (or occasionally two) segments of mutant embryos. The Kruppel-positive precursors LL1 and VA2 are also missing in vein mutant embryos at similar frequencies as those observed for DA1. It is concluded that the loss of the various muscle precursor cells observed in vein mutant embryos is in line with the expression of Vein in these cells. This phenotype is significantly enhanced in embryos carrying only one copy of wild type spitz. This analysis suggests that Vein activation of Egfr differs qualitatively from that of Spitz in that it does not lead to the expression of the inhibitory protein Argos, possibly leading to a continuous activation of the Egfr signaling pathway. The results support the idea that the role of Vein in tissues where Spitz is the major ligand is to complement Spitz activity. A model of synergistic activation by the two ligands is not favored. This explains the extremely weak vein phenotype observed in comparison to a significant and measurable phenotype obtained in tissues where Vein functions as a single ligand (Yarnitzky, 1998).

The Drosophila homeobox genes zfh-1 and even-skipped are required for cardiac-specific differentiation of a numb-dependent mesodermal lineage decision

A series of inductive signals are necessary to subdivide the mesoderm in order to allow the formation of the progenitor cells of the heart. Mesoderm-endogenous transcription factors, such as those encoded by twist and tinman, seem to cooperate with these signals to confer correct context and competence for a cardiac cell fate. Additional factors are likely to be required for the appropriate specification of individual cell types within the forming heart. Similar to tinman, the zinc finger- and homeobox-containing gene zfh-1 is expressed in the early mesoderm and later in the forming heart, suggesting a possible role in heart development. zfh-1 is specifically required for formation of the even-skipped (eve)-expressing subset of pericardial cells (EPCs), without affecting the formation of their siblings, the founders of a dorsal body wall muscle (DA1). In addition to zfh-1, mesodermal eve itself appears to be needed for correct EPC differentiation, possibly as a direct target of zfh-1. Epistasis experiments show that zfh-1 specifies EPC development independent of numb, the lineage gene that controls DA1 founder versus EPC cell fate. The combinatorial control mechanisms that specify the EPC cell fate in a spatially precise pattern within the embryo are discussed (Su, 1999). zfh-1 and the components of the numb pathway are not the only factors required for specifying EPC or DA1 founder fates (or for eve expression characteristic of these fates). A transcription factor encoded by the lethal-of-scute gene is expressed in a cluster of mesodermal cells out of which the EPC and other muscle progenitors emerge aided by a laterally inhibitory mechanism. lethal-of-scute, however, as well as another transcription factor encoded by the Krüppel gene, which is expressed in the DA1 (and other muscle) founder cells, are only weakly required for the corresponding muscles to form. In contrast, the Drosophila EGF signal transduction pathway plays an essential role in DA1 specification. For example, in the absence of the secreted EGF-receptor ligand spitz, the number of EPCs is normal but nearly all the DA1 muscles fail to form. Since DA1 founders and EPCs are likely to derive from common precursors and the phenotype of spi mutants is the opposite of zfh-1, it was decided to determine whether or not zfh-1 and spitz function as part of a common genetic pathway. The phenotype of spitz;zfh-1 double mutants was examined. In these double mutants, neither EPC- nor DA1-specific eve expression is present, suggesting that the Egf-r pathway is required for DA1 differentiation independently of zfh-1. This raises the question of whether or not Egf-r pathway activation is required for providing the correct DA1 differentiation context in a way that is reminiscent of zfh-1 function, which provides a context for EPC differentiation. If yes, it would be expected that spitz, like zfh-1, functions independently of the numb pathway. Indeed, when numb is mesodermally overexpressed in spitz mutant embryos, a phenotype similar to that of spitz;zfh-1 double mutants is observed: neither EPC- and nor DA1-specific eve expression is observed. Taken together, these results suggest that correct cell type-specific differentiation depends on both asymmetric segregation of cell fate determinants during cell division as well as on the appropriate regional context. In this case, the context information (zfh-1 or Egf-r activity) does not need to be originating from a spatially localized source, but may act in concert with other mesodermal context determinants (e.g., tinman) (Su, 1999).

A model is provided of the genetic network regulating the specification and differentiation of the EPC progenitors and their heart and muscle associated progeny (EPC and DA1). Initially, the spatially coincident activity of the transcription factor, Tinman, together with the mesoderm-specific response induced by the patterning signals, Wg and Dpp, are necessary to specify and position the most dorsal portion of the mesoderm, which includes the EPC progenitors and other cardiac precursors. The EPC progenitors then divide and produce two types of progeny cells under the control of the lineage gene numb. The daughter cell that inherits Numb protein will differentiate as the DA1 muscle founder, because the Notch and spdo encoded functions are inhibited, allowing Egf-r signaling (Spitz) to be effective (perhaps in conjunction with Eve). In the daughter cell without Numb, Notch signaling is operational and the transcription factors Zfh-1 together with and/or mediated by Eve can effectively contribute the correct differentiation of the EPC fate. Thus, three levels of information appear to cooperate in the specification of a particular cell fate: prepatterning or positional information, asymmetric lineages and tissue context information (Su, 1999).

Antagonistic roles of Rac and Rho in organizing the germ cell microenvironment

The capacity of stem cells to self renew and the ability of stem cell daughters to differentiate into highly specialized cells depend on external cues provided by their cellular microenvironments. However, how microenvironments are shaped is poorly understood. In testes of Drosophila, germ cells are enclosed by somatic support cells. This physical interrelationship depends on signaling from germ cells to the Epidermal growth factor receptor (Egfr) on somatic support cells. Germ cells signal via the Egf class ligand Spitz (Spi), and evidence is provided that the Egfr associates with and acts through the guanine nucleotide exchange factor Vav to regulate activities of Rac1. Reducing activity of the Egfr, Vav, or Rac1 from somatic support cells enhanced the germ cell enclosure defects of a conditional spi allele. Conversely, reducing activity of Rho1 from somatic support cells suppresses the germ cell enclosure defects of the conditional spi allele. It is proposed that a differential in Rac and Rho activities across somatic support cells guides their growth around the germ cells. These novel findings reveal how signals from one cell type regulate cell-shape changes in another to establish a critical partnership required for proper differentiation of a stem cell lineage (Sarkar, 2007).

In the male gonad of Drosophila, germ cells are surrounded by somatic cells that define their cellular microenvironmen. Germline stem cells (GSCs) are attached to a cluster of nondividing cells at the apical tip, called hub cells, and associated with cyst progenitor cells (CPCs) that act as stem cells for the somatic support cell lineage. Two CPCs extend their cytoplasm around one GSC, toward the hub, and toward each other such that each GSC appears to be completely enclosed in its cellular microenvironment. GSCc and CPCs generate differentiating daughters, called gonialblasts and cyst cells, respectively. The gonialblasts undergo transit amplification divisions to produce 16 spermatogonia, which become spermatocytes, grow in size, undergo the meiotic divisions, and differentiate into sperm. Two cyst cells grow cytoplasmic extensions around one gonialblast to form the germ cell cellular microenvironment that controls various aspects of germ cell differentiation (Sarkar, 2007).

Germ cells associated with somatic cells mutant for the Map-Kinase Raf fail to differentiate and accumulated as early-stage germ cells instead. A similar accumulation of early-stage germ cells was observed in Egfrts mutant testes shifted to nonpermissive temperature, and in testes from animals mutant for Stem cell tumor (Stet; Rhomboid2), a protease that cleaves Egfr ligands. However, stet mutant germ cells in addition fail to associate with somatic support cells, suggesting that the Egfr pathway is required for setting up the critical cellular microenvironment (Sarkar, 2007).

Loss of spi results in a failure of germ cells to differentiate, similar to the effects of loss of stet or the Egfr. Wild-type testes are long (~2 mm) tubular structures that contain germ cells in a spatio-temporal order along the apical-to-basal axis. Early germ cells (GSCs, gonialblasts, and spermatogonia) are small and have small, densely packed nuclei in DAPI-stained preparations. Spermatocytes are located basal to the spermatogonia, and differentiating spermatids fill the distal part of the testis (Sarkar, 2007).

Animals carrying a temperature-sensitive allele of spi, spi77-20, die when raised at 29°C. However, spi77-20 animals raised at a slightly permissive temperature (27°C) survive and have tiny testes. Most of these testes (40 of 50) contain only small cells, as seen at the tip of wild-type testes, and do not have spermatocytes or differentiating spermatids. Staining with molecular markers revealed that the testes contains increased numbers of GSCs, gonialblasts, and spermatogonia compared to wild-type. The remaining testes (10 of 50) have high numbers of early germ cells and a few spermatocytes, but no differentiating spermatids (Sarkar, 2007).

Testes from spi77-20 animals raised at an intermediate permissive temperature (25°C) are longer than testes from animals raised at 27°C, but significantly shorter (500 μm-1.5 mm) than wild-type testes. A substantial part of the testes is occupied by tumor-like aggregates of early-stage germ cells. However, spermatocytes and differentiating spermatids are also present (Sarkar, 2007).

spi activity is both sufficient and required within the germ cells. Expression of a cleaved version of Spi (sSpi) in germ cells but not in somatic support cells of spi77-20 testes restores the wild-type phenotype, and germ cell clones mutant for spi accumulate at early stages based on phase-contrast microscopy and DAPI-stained preparations) (Sarkar, 2007).

spi was also required for somatic support cells to associate with and enclose the germ cells. Germ cell clones mutant for the conditional spi77-20 allele from animals raised at 27°C either do not associate with somatic support cells or associated with only one somatic support cell (4 of 20 clones), based on staining with soma-specific antibodies, such as the transcription factor Traffic Jam (Tj). Tj labels the nuclei of somatic support cells that are normally associated with early-stage germ cells (Sarkar, 2007).

Germ cell enclosure can be investigated by labeling testes with molecular markers such as antibodies against the membrane-bound β-catenin Armadillo (Arm) that labels the cell membranes of somatic support cells as they surround the germ cells. In wild-type testes, each GSC, gonialblast, and cluster of developing germ cells is associated with and surrounded by two somatic support cells. In testes from spi77-20 animals raised at 27°C, Tj-positive cells did not form cytoplasmic extensions around the germ cells. Similar results were obtained with other markers, including a cytoplasmic UAS-Green Fluorescent Protein (UAS-GFP) expressed in somatic support cells under control of a soma-specific Gal4-driver. In control testes, GFP is detected in the cell bodies of the somatic cells surrounding the germ cells. In contrast, in spi77-20 testes from animals grown at 27°C, GFP is detected in balls, most likely small round cell bodies of somatic support cells. Occasionally, cytoplasmic extensions emerged from somatic support cells, but they remained short and did not enclose the germ cells (Sarkar, 2007).

The lack of cytoplasmic extensions from Tj-positive cells in spi77-20 mutant testes is similar to the phenotype observed in stet mutants. This suggests that the Egf class ligand Spi, expressed in germ cells and processed by Stet, stimulates the Egfr on somatic support cells, inducing them to send out cytoplasmic extensions to enclose the neighboring germ cells (Sarkar, 2007).

Association of germ cells with somatic support cells is sensitive to the level of Spi. Germ cell clones from spi77-20 animals raised at 25°C and germ cell clones from animals mutant for the spi2 allele often associated with more than two somatic support cells (Sarkar, 2007).

The growth of cytoplasmic extensions around the germ cells is also sensitive to the level of Spi. When spi77-20 animals are raised at 25°C, many Tj-positive cells form cytoplasmic extensions directed toward and/or around the germ cells. However, not every germ cell cluster appear to be associated with and/or surrounded by somatic support cells. Furthermore, many of the Tj-positive cells forme cytoplasmic extensions toward each other, suggesting that multiple somatic support cells may surround one tumor-like aggregate of germ cells. Similar abnormal associations of somatic support cells with germ cells are also seen in Egfrts mutants shifted to nonpermissive temperature. One possible explanation for the different phenotypes of loss compared to reduction of Egfr signaling is that different levels of Egfr stimulation may affect different cellular properties of somatic support cells, such as cell adhesiveness and/or growth (Sarkar, 2007).

To identify novel players in germ cell enclosure, the sensitized background of the spi77-20 allele was used to search for genetic modifiers. It was found that impaired activity of the small monomeric GTPase (small GTPase) Rac1 enhances the spi77-20 testes phenotype. Activity of Rac1 was impaired by two strategies-either by removing one copy of the rac1 gene or by expressing a dominant-negative version of Rac1 (dnRac1) in somatic support cells of testes from spi77-20 animals raised at 25°C. In either case, the enhanced testes are shorter than testes from spi77-20 animals raised at 25°C. In 12 of 20 enhanced testes, Tj-positive cells do not enclose the germ cells, and early-stage germ cells accumulate (Sarkar, 2007).

Reducing activity of Vav, a guanine nucleotide exchange factor for Rac-type small GTPases, from somatic support cells by antisense expression also enhances the spi77-20 testes phenotype from animals raised at 25°C. 11 of 20 enhanced testes were tiny and contained mostly early-stage germ cells that were not surrounded by somatic support cells. The enhanced phenotypes caused by impairing Rac or Vav raises the possibility that Rac1 and Vav act downstream of the Egfr in somatic support cells and that Vav plays a role in regulating somatic support cell-shape changes associated with germ cell enclosure (Sarkar, 2007).

In mammalian cells, autophosphorylation of specific Vav-binding motifs within the cytoplamic tail of the Egfr allows for binding and phosphorylation of mammalian Vav2. Phosphorylation of Drosophila Vav has been shown to depend on Egfr stimulation in both mammalian and Drosophila cultured cells, and Drosophila Vav bound to mammalian Egfr (Sarkar, 2007 and references therein).

Consistent with a role for Drosophila Vav in Egfr signaling in testes, Vav protein immunoprecipitates from testis extracts with an antibody against the Egfr. Vav does not immunoprecipitate from testis extracts that had been pretreated with phosphatase, suggesting that the interaction between Vav and the Egfr is phosphorylation dependent. The immunoprecipitated Vav band comigrates with a band detected by antibodies against phospho-tyrosine, suggesting that Vav is phosphorylated when in a complex with the Egfr (Sarkar, 2007).

In the classical view of the Drosophila Egfr pathway, only one docking protein-Downstream receptor kinase (Drk)-binds to the stimulated Egfr and activates a MAP-Kinase cascade for transcription of target genes. However, genetic and biochemical data suggest that the Egfr pathway is branched at the level of docking proteins and that the adaptor protein Vav binds to the Egfr to activate the small GTPase Rac1. These data suggest that Rac regulates cell-shape changes associated with germ cell enclosure, and studies on Raf suggested that it regulates the transcription of target genes. However, the possibility of crosstalk between the two branches cannot be excluded: Vav may contribute to transcriptional regulation and Map-Kinases may contribute to germ cell enclosure. A possible crosstalk is consistent with findings that in cultured Drosophila cells (Hornstein, 2003), Vav can contribute to Erk phosphorylation (Sarkar, 2007).

Surprisingly, impairing activity of the Rho-type small GTPase Rho1 has the opposite effect to impairing Rac1. Testes from spi77-20 animals raised at 27°C that express dominant-negative Rho1 (dnRho1) in somatic support cells are long and appear almost wild-type. In contrast to somatic support cells in spi77-20 testes from animals raised at 27°C without dnRho1 expression, the somatic support cells expressing dnRho1 enclose the germ cells. The same dominant suppression is observed in spi77-20, rho1/+ testes, indicating that expression of dnRho1 reflects loss of Rho1 activity (Sarkar, 2007).

These data raise the possibility that Rac and Rho have antagonistic effects on germ cell enclosure. Rac appears to be required for somatic support cells to grow cytoplasmic extensions around the germ cells, and Rho appears to suppress this growth. Antagonistic roles for Rac and Rho have also been reported in cultured mammalian cells, where Rac and Rho regulate cell-shape changes and growth via different effects on the actin cytoskeleton. Prominent readouts for small GTPase activities on the actin cytoskeleton are the appearances of ruffles and lamellipodia in the cell membranes (Sarkar, 2007).

To address a potential role of Rac and Rho in shape changes of somatic support cells, dominant-negative Rac or Rho were expressed in somatic support cells of otherwise wild-type testes, and transmission electron microscopy (TEM) was used to investigate changes in the membranes of somatic support cells surrounding single germ cells and spermatogonia at the apical tip of the testes. Germ cells and somatic support cells can be identified based on their different shapes and density of staining in TEM. In wild-type, the somatic support cells surrounding single germ cells and spermatogonia exhibit wavy plasma membranes, possibly analogous to membrane ruffles accompanying cellular growth and rearrangements of the actin cytoskeleton in cultured cells (Sarkar, 2007).

Somatic support cells expressing dnRac1 have much smoother plasma membranes than do wild-type somatic support cells. Conversely, somatic support cells expressing dnRho1 have lamellipodia-like extensions in their membranes. Lamellipodia-like extensions were not detected in somatic support cell membranes in serial sections of wild-type testes or in testes expressing dnRac1. In mammalian cells, formation of lamellipodia depends on Rac-type small GTPases. The presence of lamellipodia-like extensions in somatic support cells expressing dnRho1 suggests that Rac may become hyperactive in the absence of Rho. Based on these TEM data, it is hypothesized that, just as their mammalian counterparts do in cultured cells, Drosophila small GTPases may act on the cytoskeleton of somatic support cells to mediate cell-shape changes and growth of cellular extensions and that the effects of Rac and Rho are antagonistic (Sarkar, 2007).

This model predicts that expression of a constitutively active Egfr ligand in somatic support cells might compromise the differential in smGTPase activities. Indeed, forced expression of cleaved ligand in somatic support cells, but not in germ cells, closely mimics the effect of dnRho expression: the somatic support cells formed lamellipodia-like structures in their membranes (Sarkar, 2007).

This research on the Drosophila gonad provides a striking example how one cell type in tissue communicates with another cell type to induce and direct the formation of a proper cellular microenvironment: a signal from one cell induces subcellular changes throughout the body of another cells. This mechanism underlying the formation of a cellular microenvironment may be conserved across species (Sarkar, 2007).

Qian, Y., Dominado, N., Zoller, R., Ng, C., Kudyba, K., Siddall, N. A., Hime, G. G. and Schulz, C. (2014). Ecdysone signaling opposes epidermal growth factor signaling in regulating cyst differentiation in the male gonad of Drosophila melanogaster. Dev Biol 394(2):217-27. PubMed ID: 25169192

Ecdysone signaling opposes epidermal growth factor signaling in regulating cyst differentiation in the male gonad of Drosophila melanogaster

The development of stem cell daughters into the differentiated state normally requires a cascade of proliferation and differentiation steps that are typically regulated by external signals. The germline cells of most animals, in specific, are associated with somatic support cells and depend on them for normal development. In the male gonad of Drosophila melanogaster, germline cells are completely enclosed by cytoplasmic extensions of somatic cyst cells, and these cysts form a functional unit. Signaling from the germline to the cyst cells via the Epidermal Growth Factor Receptor (EGFR) is required for germline enclosure and has been proposed to provide a temporal signature promoting early steps of differentiation. A temperature-sensitive allele of the EGFR ligand Spitz (Spi) provides a powerful tool for probing the function of the EGRF pathway in this context and for identifying other pathways regulating cyst differentiation via genetic interaction studies. Using this tool, this study showed that signaling via the Ecdysone Receptor (EcR), a known regulator of developmental timing during larval and pupal development, opposes EGF signaling in testes. In spi mutant animals, reducing either Ecdysone synthesis or the expression of Ecdysone signal transducers or targets in the cyst cells resulted in a rescue of cyst formation and cyst differentiation. Despite of this striking effect in the spi mutant background and the expression of EcR signaling components within the cyst cells, activity of the EcR pathway appears to be dispensable in a wildtype background. It is proposed that EcR signaling modulates the effects of EGFR signaling by promoting an undifferentiated state in early stage cyst cells (Qian, 2014).

Spitz and Malpighian tubules

The Drosophila Malpighian tubules (MTs), form a simple excretory epithelium comparable in function to kidneys in vertebrates. MTs function as the insect kidney both in the larva and the adult. They consist of two pairs of blind ending tubes that are composed of a single cell-layered epithelium made up of a tightly controlled number of cells. The tubules float in the hemolymph from where they take up nitrogenous waste that is excreted as uric acid. During embryogenesis, MTs evert as four protuberances from the hindgut primordium, the proctodeum. The everting tubules grow by cell proliferation, which takes place in a few cells along the tubules and extensively in a distal proliferation domain located in the tip region of the tubules. Cell ablation experiments and studies on the pattern of cell division have shown that a single large cell at the distal end of each tubule, termed the tip cell, is decisive for controlling the proliferation of its neighboring cells. The tip cell that differentiates into a cell with neuronal characteristics during later stages of development arises by division of a tip mother cell that is selected in the tubule primordium by lateral inhibition involving the Notch signaling pathway and the transcription factor Krüppel (Kr). It has been suggested that the tip cell sends a mitogenic signal to adjacent cells in the distal proliferation zone. It has remained elusive, however, what the signal is or what its target molecules in the signal-receiving cells could be and how cell proliferation during MT morphogenesis is regulated. Seven-up is shown to be a key component that becomes induced in response to mitogenic EGF receptor signaling activity emanating from the tip cell. Seven-up (Svp) in turn is capable of regulating the transcription of cell cycle regulators (Kerber, 1998).

To identify the nature of the mitogenic tip cell signal a screen was carried out for genes specifically active in the tip cells. The genes rhomboid (rho) and Star (S), which encode transmembrane proteins involved in epidermal growth factor receptor (EGFR) signaling, are expressed in the tip cells and both are required for MT growth. Rho and S process a membrane-bound form of the activating ligand of the receptor, the TGFalpha-like Spi protein, to generate the secreted form of Spi (sSpi). sSpi is then proposed to diffuse to neighboring cells, bind to the receptor, and activate target genes via the Ras/Raf signaling cassette; these include the primary target gene pointedP1 (pntP1), encoding an ETS domain transcription factor, and the secondary target gene argos (aos), encoding a negatively acting ligand of the receptor. These downstream components of the pathway are also active during tubule development. pntP1 and aos are expressed during stage 10 in six to eight cells on one side of the MTs overlapping the rho and S expression domains and later, weakly in several cells in the tip region. In amorphic aos mutants a slightly larger number of tubule cells are observed, whereas amorphic pnt mutants show a decrease of tubule cells. These results indicate that for controlling cell proliferation and cell determination, the same key components of the EGFR cascade are required (Kerber, 1998).

These findings suggest that the EGFR pathway provides the mitogenic tip cell signal that activates svp expression and regulates cell division. To test this hypothesis, svp expression was analyzed in EGFR mutants and ectopic expression studies were performed with various members of the pathway using the UAS-Gal4 system. svp is absent in mutants for the Egfr. It is still expressed, however, in amorphic pnt mutants, suggesting that Svp is a transcriptional regulator that is likely to be activated in parallel to the primary transcription factor PntP1 in the signaling cascade. If sSpi activity is provided ectopically in all of the tubule cells, the svp expression domain becomes dramatically expanded and an increase of the tubule cell number is observed. Similar, although slightly weaker effects on svp transcription and the number of tubule cells could be observed upon ubiquitous expression of other components of the EGFR pathway, like Rho, activated Ras, or Raf.Conversely, when a dominant-negative Ras allele is ectopically expressed in all of the tubule cells, svp transcription became strongly reduced. Ectopic expression of svp in an Egfr mutant background restores the tubule cell number to a considerable extent. These results provide strong evidence that svp is a downstream target gene of EGFR signalling in the tubules (Kerber, 1998).

Spitz and the stomatogastric nervous system

The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. RTK signaling mediated by the Epidermal growth factor receptor plays a key role in two consecutive steps during early SNS development. Like Wg, Egfr signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the Egfr ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage (González-Gaitán, 2000).

In order to investigate the role of RTK signaling in SNS development, lack-of-function mutants of the Egfr ligand Spitz were examined. In spitz mutants, the formation of the four SNS ganglia is strongly impaired. The SNS anlage, however, forms normally. In addition, the expression domain of wg and proneural AS-C genes is indistinguishable from a wild-type SNS anlage. At the stage when the three ac-expressing cells were singled-out within the wild-type SNS anlage, only one ac positive cell is found in spitz mutants. The same phenotype has been observed in wg mutants or mutants lacking an integral component of the wg pathway. Since no altered wg pattern was found in the spitz mutant SNS anlage, Spitz-dependent RTK signaling may act in parallel or in combination with wg to adjust the proper range of Notch-dependent lateral inhibition. In contrast to wg mutants, however, no invagination fold is observed. This observation indicates that the singled-out ac-expressing cell of spitz mutants has lost the ability to function as a tip cell and possibly fails to induce morphogenetic movements within the SNS anlage (González-Gaitán, 2000).

spitz, like other genes encoding components of the Egfr signaling pathway such as Egfr, Ras, Raf and the cascade of MAP kinases, is ubiquitously expressed. Local activation of Egfr signaling requires the transmembrane protein Star, which is necessary for the secretion of Spitz. Star is expressed in restricted patterns corresponding to the Spitz secreting cells. In the SNS anlage, it was noted that Star becomes restricted to the three tip cells and is maintained in these cells when invagination takes place. As in spitz mutants, the Star mutant SNS anlage is established normally; only one ac-expressing cell is selected and no invagination occurs. Consistently, Star mutants fail to develop the proper set of SNS ganglia and the associated nerves. These observations suggest that tip cells are a Star-dependent source of Spitz activity that triggers Egfr-dependent RTK signaling in the neighboring cells within the SNS anlage. This conclusion is supported by the finding that phosphorylated MAPK, a cellular marker for RTK signaling activity, is indeed activated in cells of the invagination folds, whereas phosphorylated MAPK does not appear in the Star mutant or in the spitz mutant SNS anlage (González-Gaitán, 2000).

To examine whether activated Spitz is sufficient to induce cell movements within the SNS anlage, use was made of the GAL4/UAS system to misexpress secreted Spitz in an ectopic pattern. This was achieved through the expression of activated Spitz from a UAS promotor driven transgenethat was activated by Gal4 under the control of the actin promotor. Under the conditions applied, scattered UAS-dependent transgene expression is observed throughout the early embryo, including the SNS anlage. When activated Spitz is expressed in such a pattern, a variable number of supernumerary infoldings within the SNS anlagen are observed, indicating that activated Spitz is sufficient to initiate cell movements. This result, in conjunction with the observation that the invaginated cells express phosphorylated MAPK, provides evidence that tip cell-derived activated Spitz triggers RTK signaling to initiate the invagination process. This proposal was tested by blocking Egfr signaling in the anterior most region of the SNS anlage that gives rise to the first invagination fold. For this, a GAL4 driver (SNS1-Gal4) was used that causes UAS-dependent gene expression in the corresponding region of the SNS anlage. SNS1-Gal4-mediated expression of a dominant-negative Egfr mutant form from a UAS-controlled transgene causes a specific suppression of the anterior most invagination fold without affecting the others (González-Gaitán, 2000).

The results demonstrate that RTK signaling participates in the selection of tip-cell-dependent invagination centers in the SNS anlage and is subsequently required to initiate morphogenetic movements resulting in invagination folds. This study does not focus on how RTK signaling ties into the wg-modulated Notch signaling process previously shown to be necessary for the selection of the three SNS invagination centers. The data indicate, however, that RTK signaling acts either in parallel or in combination with wg signaling to adjust the proper range of Notch-dependent lateral inhibition. Although in both wg and Egfr signaling mutants, only one ac-expressing cell is singled-out, the selected cells differ with respect to whether they function as tip cells or not. In wg mutants, the single cell causes an invagination, whereas in Egfr signaling mutants, the selected cell fails to provide this feature of SNS invagination centers. The results, therefore, consistently argue that tip cell-derived Spitz triggers local RTK signaling and thereby initiates the formation of invagination folds each headed by the Spitz-secreting tip cell. Thus, Egfr-dependent RTK signaling in Drosophila does not only participate in cell fate decisions and cell proliferation, but also triggers morphogenetic movements within an epithelium, as has been recently demonstrated for fibroblast growth factor (FGF) signaling. It will be interesting to see whether the role of the EGF pathway in cell migration differs at the cellular level from cell migration events triggered by activated FGF receptors (González-Gaitán, 2000).

Non-autonomous crosstalk between the Jak/Stat and Egfr pathways mediates Apc1-driven intestinal stem cell hyperplasia in the Drosophila adult midgut

Inactivating mutations within adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, are responsible for most sporadic and hereditary forms of colorectal cancer (CRC). This study used the adult Drosophila midgut as a model system to investigate the molecular events that mediate intestinal hyperplasia following loss of Apc in the intestine. The results indicate that the conserved Wnt target Myc and its binding partner Max are required for the initiation and maintenance of intestinal stem cell (ISC) hyperproliferation following Apc1 loss. Importantly, it was found that loss of Apc1 leads to the production of the interleukin-like ligands Upd2/3 and the EGF-like Spitz in a Myc-dependent manner. Loss of Apc1 or high Wg in ISCs results in non-cell-autonomous upregulation of upd3 in enterocytes and subsequent activation of Jak/Stat signaling in ISCs. Crucially, knocking down Jak/Stat or Spitz/Egfr signaling suppresses Apc1-dependent ISC hyperproliferation. In summary, these results uncover a novel non-cell-autonomous interplay between Wnt/Myc, Egfr and Jak/Stat signaling in the regulation of intestinal hyperproliferation. Furthermore, evidence is presented suggesting potential conservation in mouse models and human CRC. Therefore, the Drosophila adult midgut proves to be a powerful genetic system to identify novel mediators of APC phenotypes in the intestine (Cordero, 2012).

Using the Drosophila adult midgut as a model system this study has uncovered a key set of molecular events that mediate Apc-dependent intestinal hyperproliferation. The results suggest that paracrine crosstalk between Egfr and Jak/Stat signaling is essential for Apc1-dependent ISC hyperproliferation in the Drosophila midgut (Cordero, 2012).

Previous studies have demonstrated that Myc depletion prevents Apc-driven intestinal hyperplasia in the mammalian intestine. This study provides evidence that such a dependency on Myc is conserved between mammals and Drosophila. It was further demonstrated that endogenous Myc or Max depletion causes regression of an established Apc1 phenotype in the intestine. Taken together, these data highlight the importance of developing Myc-targeted therapies to inhibit Apc1-deficient cells. Since not all roles of Myc are Max dependent, present efforts are focused on developing inhibitors that interfere with Myc binding to Max and would therefore be less toxic. These data provide the first in vivo evidence in support of the Myc/Max interface as a valid therapeutic target for CRC (Cordero, 2012).

Recent work showed that loss of the tuberous sclerosis complex (TSC) in the Drosophila midgut leads to an increase in cell size and inhibition of ISC proliferation. Reduction of endogenous Myc in TSC-deficient midguts restored normal ISC growth and division. These results might appear contradictory to the current work, where Myc is a positive regulator of ISC proliferation. However, in both scenarios, modulation of Myc levels restores the normal proliferative rate of ISCs (Cordero, 2012).

Previous work in mouse showed that Myc upregulation is essential for Wnt-driven ISC hyperproliferation in the intestine. However, Myc overexpression alone only recapitulates some of the phenotypes of hyperactivated Wnt signaling. This study shows that overexpression of Myc is capable of mimicking some aspects of high Wnt signaling in the Drosophila midgut, such as the activation of Jak/Stat, but is not sufficient to drive ISC hyperproliferation. Multiple lines of evidence have shown that forced overexpression of Myc in Drosophila and vertebrate models results in apoptosis partly through activation of p53. Therefore, driving ectopic myc alone is unlikely to parallel Apc deletion in the intestine, where the activation of multiple pathways downstream of Wnt signaling is likely to contribute cooperatively to hyperproliferation (Cordero, 2012).

Understanding the contribution of Jak/Stat signaling to the Apc phenotype in the mammalian intestine has been complicated by genetic redundancy between Stat transcription factors. Constitutive deletion of Stat3 within the intestinal epithelium slowed tumor formation in the ApcMin/+ mouse, but the tumors that arose were more aggressive and ectopically expressed Stat1. Using the Drosophila midgut, direct in vivo evidence is provided that activation of Jak/Stat signaling downstream of Apc1/Myc mediates Apc1-dependent hyperproliferation (Cordero, 2012).

The data on the Drosophila midgut and in mouse and human tissue samples suggest that blocking Jak/Stat activation could represent an efficacious therapeutic strategy to treat CRC. Currently, there are a number of Jak2 inhibitors under development and it would be of great interest to examine whether any of these could modify the phenotypes associated with Apc loss (Cordero, 2012).

Previous studies have demonstrated that enterocytes (ECs) are the main source of Upds/interleukins in the midgut epithelium. The results show that activation of Wnt/Myc signaling in ISCs leads to non-autonomous upregulation of upd3 within ECs. Furthermore, Spitz/Egfr signaling appears to mediate the paracrine crosstalk between Wnt/Myc and Jak/Stat in the midgut. Overexpression of a dominant-negative Egfr in ECs blocks upd3 upregulation and ISC hyperproliferation in response to high Wnt signaling. A previous EC-specific role for Egfr has been demonstrated during midgut remodeling upon bacterial damage. Nevertheless, the downstream signaling that mediates such a role of Egfr remains unclear given that the activation of downstream MAPK/ERK occurs exclusively within ISCs. Therefore, the current evidence would suggest that Egfr activity in ECs does not involve cell-autonomous ERK activation. Consistent with these observations, p-ERK (Rolled -- FlyBase) localization was not detected outside ISCs in response to either Apc loss or overexpression of wg in the Drosophila midgut. Reports on the Apc murine intestine have also failed to detect robust ERK activation. Since MAPK/ERK is only one of the pathways activated downstream of Egfr, it is possible that ERK-independent mechanisms are involved. It is important to explore this further because ERK-independent roles of Egfr signaling have not yet been reported in Drosophila. Thus, what mediates Upd3 upregulation in ECs in response to Egfr signaling activation and whether Spitz-dependent upregulation of Upd3 involves a direct role of Egfr in ECs remain unclear. A potential alternative explanation is that intermediate factors induced in response to Spitz/Egfr activation in ISCs might drive Upd3 expression (Cordero, 2012).

In summary, this study has elucidated a novel molecular signaling network leading to Wnt-dependent intestinal hyperproliferation. Given the preponderance of APC mutations in CRC, the integration of Egfr and Jak/Stat activation might be a conserved initiating event in the disease (Cordero, 2012).

Spitz and Oogenesis

During Drosophila oogenesis two distinct stem cell populations produce either germline cysts or the somatic cells that surround each cyst and separate each formed follicle. From analyzing daughterless (da) loss-of-function, overexpression and genetic interaction phenotypes, several specific requirements have been identified for da+ in somatic cells during follicle formation. (1) da is a critical regulator of somatic cell proliferation. (2) da is required for the complete differentiation of polar and stalk cells, and elevated da levels can even drive the convergence and extension that is characteristic of interfollicular stalks. (3) da is a genetic regulator of an early checkpoint for germline cyst progression: loss of da function inhibits normally occurring apoptosis of germline cysts at the region 2a/2b boundary of the germarium, while da overexpression leads to postmitotic cyst degradation. Collectively, these da functions govern the abundance and diversity of somatic cells as they coordinate with germline cysts to form functional follicles (Smith, 2002).

The initial requirement for da during follicle formation is somatic cell proliferation. The straightforward observation that one extra copy of da+ results in excess somatic cell production, while da loss of function leads to an insufficient number of somatic cells for germline cyst envelopment, demonstrates a role for da in proliferation. The genetic interactions between da and every component of the EGFR pathway, which is also known to regulate somatic proliferation through mid-oogenesis, suggest that da and the EGFR pathway cooperate to control cell division. Gurken has been identified as the germline-localized ligand for this proliferative function; however, grk null ovaries have a relatively low frequency of follicle formation defects. The genetic interaction observed between da and spi implicates Spi as a second ligand for EGFR in proliferation, although this must be a somatic signal, since spi expression is restricted to somatic cells. Finally, although there is no evidence for da acting during specification of the somatic stem cells, it may control their proliferation once founded. Alternatively, da control of proliferation may be limited to the progeny of the stem cells. Either possibility is consistent with suppression of the ectopic hh phenotype in da heterozygotes and the complete epistasis of dalyh (Smith, 2002).

The jing and ras1 pathways are functionally related during CNS midline and tracheal development as revealed by genetic interations with spitz and Star

The Drosophila jing gene encodes a zinc finger protein required for the differentiation and survival of embryonic CNS midline and tracheal cells. There is a functional relationship between jing and the Egfr pathway in the developing CNS midline and trachea. jing function is required for Egfr pathway gene expression and MAPK activity in both the CNS midline and trachea. jing over-expression effects phenocopy those of the Egfr pathway and require Egfr pathway function. Activation of the Egfr pathway in loss-of-function jing mutants partially rescues midline cell loss. Egfr pathway genes and jing show dominant genetic interactions in the trachea and CNS midline. Together, these results show that jing regulates signal transduction in developing midline and tracheal cells (Sonnenfeld, 2004).

The effect of a reduction in EGFR signaling on the jing gain-of-function phenotype was examined in the midline glia. sim-Gal4 and sli-Gal4 drivers were used to over-express jing specifically in the CNS midline in heterozygous and homozygous spi and S mutant backgrounds. The number of sli-lacZ-expressing midline glia in each nerve cord segment was quantified during stage 13 and compared to that in wild-type embryos over-expressing jing. Expression of two copies of the UAS-jing transgene in the midline glia of wild-type or heterozygous spi and S embryos resulted in an average of 12 midline glia instead of the normal 8 during stage 13. In contrast, UAS-jing transgene expression was unable to induce 12 midline glia in homozygous spi and S mutant backgrounds. In these embryos, there was an average of 1.5 midline glia in each nerve cord segment after jing over-expression; this is similar to the number of midline glia present in homozygous spi and S mutant embryos during stage 13 (Sonnenfeld, 2004).

The jing ectopic expression phenotype was dominantly suppressed by a 50% reduction in the levels of spi(spi1) and Df(2L)TW50 or Egfr deficiency [Df(2R)Egfr5]. After spi reduction, ommatidia were more organized and more abundant, although the position of the photoreceptors was not like that in controls. This interaction was not influenced by activation of the glass promoter in the heterozygous spi background (P[GMR-Gal4]/spi1). These results suggest that there is a dosage-sensitive interaction between the Egfr pathway and jing function in the eye, where increased jing activity can be suppressed by a reduction in downstream components such as spi and Egfr. Given that sim and trh are not expressed in third instar larval eye discs, these experiments suggest that jing can have an effect on the Egfr pathway in the absence of sim or trh and support the model that jing works as an independent regulator in bHLH-PAS pathways (Sonnenfeld, 2004).

Gene dosage experiments were used to determine the effects of simultaneously altering the levels of jing and genes of the Egfr pathway. Mutations in spi and its regulator Star, have been characterized for their midline and tracheal phenotypes. To determine whether jing and Egfr function is inter-dependent, the development of the CNS midline and trachea was analyzed in double heterozygotes of jing and S or spi. The basis for this experiment is that if the Egfr and jing pathways are inter-dependent then simultaneous reduction of only one copy of each gene should alter CNS midline and tracheal function. Multiple jing alleles balanced with wg-lacZ Cyo were crossed to SIIN23/wg-lacZ Cyo or spi1/wg-lacZ Cyo flies and their progeny were double stained with anti-Sim or anti-Trh and anti-β-Gal (Sonnenfeld, 2004).

The number of CNS midline cells was reduced from wild-type in embryos homozygous and double heterozygous for jing, spi or S and stained with anti-Sim. Since some of the Sim-positive nuclei appeared to be fragmenting, their fate was determined by TUNEL labeling to identify apoptotic cells. In wild-type embryos, cell death is uncommon in the CNS midline during stage 12 with an average of 6(±2) Sim-positive apoptotic nuclei per embryo. In contrast, in homozygous jing stage 12 mutant embryos, there was an average of 35(±3) Sim-positive apoptotic nuclei per embryo, therefore, displaying a significant increase over that in wild-type embryos. In embryos double heterozygous for mutations in jing and S or spi there was an average of 25(±2) and 30(±3) SIM-positive apoptotic nuclei per embryo during stage 12, respectively. This is consistent with the time period for the requirement of Egfr function in CNS midline glia. Embryos heterozygous for either jing, spi or S mutations did not alter the normal events of midline cell apoptosis. In summary, these results suggest that proper dosage of both jing and spi group gene function is required for midline cell survival (Sonnenfeld, 2004).

Soma-germline interactions coordinate homeostasis and growth in the Drosophila gonad

The ability of organs such as the liver or the lymphoid system to maintain their original size or regain it after injury is well documented. However, little is known about how these organs sense that equilibrium is breached, and how they cease changing when homeostasis is reached. Similarly, it remains unclear how, during normal development, different cell types within an organ coordinate their growth. This study shows that during gonad development in Drosophila the proliferation of primordial germ cells (PGCs) and survival of the somatic intermingled cells (ICs) that contact them are coordinated by means of a feedback mechanism composed of a positive signal and a negative signal. PGCs express the EGF receptor (EGFR) ligand Spitz, which is required for IC survival. In turn, ICs inhibit PGC proliferation. Thus, homeostasis and coordination of growth between soma and germ line in the larval ovary is achieved by using a sensor of PGC numbers (EGFR-mediated survival of ICs) coupled to a correction mechanism inhibiting PGC proliferation. This feedback loop ensures that sufficient numbers of PGCs exist to fill all the stem-cell niches that form at the end of larval development. It is proposed that similar feedback mechanisms might be generally used for coordinated growth, regeneration and homeostasis (Gilboa, 2006).

Each ovary in the adult fruitfly is composed of 16-18 units called ovarioles. At the anterior of each ovariole, two or three germline stem cells (GSCs) interact with somatic cells that affect their establishment, maintenance and differentiation. These somatic niche cells develop across the larval gonad at the third larval instar and are separated into ovarioles during early pupal development. The GSCs are derived from primordial germ cells (PGCs) that form in the early embryo. During the three larval instars, the entire gonad grows. The number of PGCs increases eightfold, from about 12 PGCs in each embryonic gonad to about 100 by the middle of third instar (ML3). PGCs double their numbers every 24 h during first (L1) and second (L2) instar, and division rates are slightly slower during the next 24 h. By ML3, sufficient PGCs exist to fill all the somatic GSC niches that form at that time (Gilboa, 2006).

When embryos contain very few PGCs (either when few PGCs are transplanted into embryos lacking germ cells, or in certain genetic backgrounds), they nonetheless develop into fully fertile females. To test whether PGC proliferation might be regulated during larval growth, PGC numbers were counted in germcell-less (gcl) and oskar (osk) mutant embryos, in which fewer PGCs form during embryogenesis. In gcl mutants, in which only two PGCs on average were incorporated into the embryonic gonad, an average of about 60 PGCs was reached by ML3. This division rate (on average five divisions in three days) is higher than in the wild type (three divisions in three days). In osk mutants, in which 3 PGCs were incorporated into the gonad, the division rate was faster than in the wild type until the end of L2 (EL2), but as PGCs approached wild-type numbers, their proliferation rate decreased markedly. Thus, PGC proliferation is regulated, and can increase or decrease to achieve wild-type numbers (Gilboa, 2006).

To determine how PGC division rate is controlled, the Gal4/UAS system was used to express dominant-active or dominant-negative signalling pathway components in the larval gonad. For somatic expression the soma-specific C587-Gal4 was used. For PGC-specific expression nos-Gal4-VP16 was used. A large increase was observed in PGC numbers when the dominant-negative form of EGFR (UAS-EgfrDN) was expressed in somatic gonadal cells by C587-Gal4. Similarly, somatic overexpression of Ras85D.N17, a dominant-negative form of Ras85D, which acts downstream of EGFR, yielded an expansion of PGCs. This indicates that EGFR might function in somatic gonadal cells to regulate PGC divisions (Gilboa, 2006).

To analyse how EGFR signalling affects PGC numbers, the gonadal expression of different EGFR signalling components was determined. Consistently with the somatic effect of UAS-EgfrDN, EGFR and the phosphorylated form of mitogen-activated protein kinase (pMAPK) were detected by antibody staining in somatic cells adjacent to PGCs. Enhancer traps in both vein and argos, which are known transcriptional targets of EGFR signalling, were also expressed in these cells. Enhancer trap expression of the EGFR ligand Spitz was found in PGCs. Gurken, an additional EGFR ligand, which is expressed in oocytes, could not be detected in PGCs. Ligand presentation requires Rhomboid family members (Rhomboid or Stet) and Star in the ligand-producing cells. Both star and stet, but not rhomboid, were detected by enhancer trap expression in PGCs. These results indicate that PGCs might produce Spitz and activate EGFR signalling in neighbouring somatic cells (Gilboa, 2006).

Very little is known about the origin or function of the somatic cells adjacent to PGCs, which have been termed intermingled cells (IC). These cells express the MA33 enhancer trap at L3. ICs also express the protein Traffic Jam (TJ), although before L3 TJ expression is not limited to ICs. The results demonstrate that the EGFR signalling pathway is activated in ICs. To determine its role in ICs, a temperature-sensitive allelic combination of EGFR (Egfr(ts)) was used to overcome the earlier, embryonic, requirement for EGFR. PGCs overproliferated in Egfr(ts) larvae grown at the restrictive temperature, whereas the gonads of their wild-type siblings remained normal. Although ICs were readily observed in heterozygous gonads, fewer of them were present in Egfr(ts) gonads. A similar reduction in IC numbers was observed in UAS-EgfrDN gonads. Antibodies against cleaved caspase 3, an apoptotic marker, revealed a significant increase in dying cells in UAS-EgfrDN gonads at EL2 in comparison with those expressing C587-Gal4 alone,. Other aspects of somatic differentiation and morphogenesis seemed normal, because components of the niche such as terminal filament and cap cells differentiated normally in Egfr(ts) and UAS-EgfrDN flies. Thus, EGFR signalling is required for IC survival. A similar role for EGFR signalling in cell survival has been described in the Drosophila nervous system, in which neuronal cells secrete Spitz and protect midline glial cells from death. To determine whether IC death resulted directly from the abrogation of EGFR signalling, or indirectly from PGC overproliferation, UAS-CycD, UAS-Cdk4 were mis-expressed in PGCs. Under these conditions, PGCs overproliferated extensively, without loss of ICs. This indicates that PGC overproliferation in UAS-EgfrDN and in Egfr(ts) might have been the effect rather than the cause of IC death (Gilboa, 2006).

To ask more directly whether PGC production of Spitz is required for IC survival, Spitz production was reduced by RNA interference (UAS-spiRNAi) in either somatic gonadal cells or PGCs. Expression of UAS-spiRNAi in PGCs resulted in a significant increase in PGC numbers, whereas somatic expression had no effect. As could be expected, reducing Spitz production in PGCs resulted in reduced IC numbers. Then EGFR signalling was examined in gonads lacking PGCs altogether. At L1 and L2, pMAPK was absent, pMAPK was detected in a subpopulation of migrating cells but not where ICs are located. In gcl mutants containing PGCs, the strongest pMAPK staining was observed in somatic cells contacting PGCs. In gcl gonads lacking PGCs, ICs could be detected by TJ expression, but in greatly reduced numbers. This is not due to a general reduction in somatic cell numbers, because similar numbers of terminal filaments form in gcl and in wild-type gonads. The reduction in IC number resembles the disappearance of inner-sheath cells from adult germaria lacking germ cells. MA33 could not be detected in gcl gonads. The difference could be due to weaker staining of MA33 than that of TJ, or because MA33-positive cells represent a subpopulation of ICs that disappears in gonads lacking PGCs. Taken together, these results indicate that PGCs produce Spitz and activate EGFR signalling in ICs, which is necessary for their survival. In return, ICs inhibit PGC proliferation. It is suggested that this feedback mechanism allows the gonad to monitor and correct PGC numbers during larval growth. When very few PGCs form, Spitz production is low, leading to reduced IC numbers. This, in turn, leads to increased PGC proliferation. Compensation of PGC numbers by the end of larval development ensures that sufficient PGCs are present to occupy the adult niches (Gilboa, 2006).

To test whether EGFR signalling in ICs has an additional role to that of promoting survival, EGFR signalling was increased by mis-expressing constitutively active forms of EGFR signalling components (UAS-EgfrCA, UAS-Ras85D.G12V or UAS-phl.gof) in the soma, or mis-expressing the secreted form of Spitz in PGCs (UAS-sSpi). PGC numbers were significantly reduced in these cases. Interestingly, IC numbers remained unchanged. The restriction of TJ expression to ICs was also unchanged. Because increasing EGFR signalling resulted in decreased PGC numbers, without an apparent effect on gonad morphogenesis or IC numbers, it is suggest that EGFR signalling in ICs might be directly required for the inhibition of PGC proliferation (Gilboa, 2006).

Soma-germline interactions through EGFR signalling are a recurring motif in Drosophila. In females they serve to pattern the eggshell and localize the oocyte nucleus. In males they serve to restrict GSC proliferation and promote differentiation. The rhomboid homologue stet is required in both males and females for GSC differentiation and for proper connections between somatic cells and germ cells. The signals originating in the somatic cells and perceived by germ cells remain unknown. In this study it has been show that EGFR has a central role in a feedback loop coordinating IC survival and PGC proliferation. The properties of this loop make it ideal for regulating homeostasis and for coordinating the growth of different cell populations in any organ. In the liver, for example, several cell types proliferate after injury. It has been suggested that hepatocytes provide the mitogenic stimuli for other liver cells, such as Kupffer cells, hepatic stellar cells and biliary ductular cells. The production of transforming growth factor β by hepatic stellar cells may, in turn, limit hepatic growth. Similar feedback loops may apply in other cases during normal development or after injury (Gilboa, 2006).

back to Spitz Effects of Mutation part 1/2

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

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