Effects of Mutation or Deletion (part 1/2)

Mutations in the genes spitz, Star, single-minded, pointed , rhomboid (all zygotic), and sichel (maternal), collectively called the spitz group, cause similar pattern alterations in ventral ectodermal derivatives of the Drosophila embryo. The cuticle structures lacking in mutant embryos normally derive from longitudinal strips of the ventro-lateral blastoderm. Defects in these mutants are found in the median part of the central nervous system. Individual members of the spitz group can produce different effects. As shown by pole cell transplantations, spi and S are also required for normal development of the female germ line, while sim, and pnt appear to be exclusively zygotically expressed (Mayer, 1988).

Examination of mutant spitz embryos reveals that spi is involved in a number of unrelated developmental choices, for example, dorsal-ventral axis formation, glial migration, sensory organ determination, and muscle development. Mutants show a reduction in external sensory neurons and chordotonal neurons (Rutledge, 1992).

Egfr signaling is required in a narrow medial domain of the head ectoderm (here called 'head midline') that includes the anlagen of the medial brain (including the dorsomedial and ventral medial domain of the brain, termed DMD and VMD respectively), the visual system (optic lobe, larval eye) and the stomatogastric nervous system (SNS). These head midline cells differ profoundly from their lateral neighbors in the way they develop. Three differences are noteworthy: (1) Like their counterparts in the mesectoderm, the head midline cells do not give rise to typical neuroblasts by delamination, but stay integrated in the surface ectoderm for an extended period of time. (2) The proneural gene l’sc, which transiently (for approximately 30 minutes) comes on in all parts of the procephalic neurectoderm while neuroblasts delaminate, is expressed continuously in the head midline cells for several hours. (3) Head midline cells, similar to ventral midline cells of the trunk, require the Egfr pathway. In embryos carrying loss-of-function mutations in Egfr, spi, rho, S and pnt, most of the optic lobe, larval eye, SNS and dorsomedial brain are absent. This phenotype arises by a failure of many neurectodermal cells to segregate (i.e., invaginate) from the ectoderm; in addition, around the time when segregation should take place, there is an increased amount of apoptotic cell death, accompanied by reaper expression, which removes many head midline cells. In embryos where Egfr signaling is activated ectopically by inducing rho, or by argos (aos) or yan loss-of-function, head midline structures are variably enlarged. A typical phenotype resulting from the overactivity of Egfr signaling is a ‘cyclops’ like malformation of the visual system, in which the primordia of the visual system stay fused in the dorsal midline. The early expression of cell fate markers, such as sine oculis in Spitz-group mutants, is unaltered (Dumstrei, 1998).

The spitz class genes, pointed (pnt), rhomboid (rho), single-minded (sim), spitz (spi) and Star (S), as well as the Drosophila Epidermal growth factor receptor (Egfr) signaling genes, argos (aos), Egfr, orthodenticle (otd) and vein (vn), are required for the proper establishment of ventral neuroectodermal cell fate. The roles of the CNS midline cells, spitz class and Egfr signaling genes in cell fate determination of the ventral neuroectoderm were determined by analyzing the spatial and temporal expression patterns of each individual gene in spitz class and Egfr signaling mutants. This analysis shows that the expression of all the spitz class and Egfr signaling genes is affected by the sim gene, which indicates that sim acts upstream of all the spitz class and Egfr signaling genes. Overexpression of sim in midline cells fails to induce the ectodermal fate in the spi and Egfr mutants. In contrast, overexpression of spi and Draf causes ectopic expression of the neuroectodermal markers in the sim mutant. Ectopic expression of sim in the en-positive cells induces the expression of downstream genes such as otd, pnt, rho, and vn, which clearly demonstrates that the sim gene activates the Egfr signaling pathway and that CNS midline cells, specified by sim, provide sufficient positional information for the establishment of ventral neuroectodermal fate. These results reveal that the CNS midline cells are one of the key regulators for the proper patterning of the ventral neuroectoderm by controlling Egfr activity through the regulation of the expression of spitz class genes and Egfr signaling genes (Chang, 2001).

CrebA is thought to be epistatic to known dorsal/ventral patterning genes. Epistasis tests were done with the decapentaplegic gene and the spitz gene. In dpp/CrebA double mutants the entire cuticle is lateralized, while in dpp mutants the cuticle is ventralized. In spitz/CrebA double mutants narrower denticle bands are seen with some fusion of denticles between segments; however the denticles have the same morphology found in CrebA mutants alone. It is thought that near the end of both the Dpp- and Spi-signaling cascades, CrebA functions to translate the corresponding extracellular signals into changes in gene expression. The only determinant tested that shows altered expression patterns in CrebA mutants was Dsc73, a secreted protein expressed at late embryonic stages in the epidermal cells that produce denticles and hairs. There was a decrease in the levels of Dsc73 on the dorsal and ventral surfaces compared to levels of Dsc73 in lateral positions, which appear unchanged (Andrew, 1997).

The spitz-group mutants (spitz, rhomboid, and pointed) are embryonic lethal and have similar cuticle phenotypes; they are shorter than wild type and have deletions of ventral cuticle. vein mutant are shorter and the Keilin's organs and ventral black dots are closer together than in wild-type. Ventral cuticle is deleted between Keilin's organs. The deletions occur in a similar region in spitz-group mutants; spitz and rhomboid have a larger portion of ventral cuticle deleted than vein mutants, but pointed embryos have similar deletions. In vein mutants sensory hairs surrounding the pit structure of Keilin's organs are missing. Unlike the spi-group genes, vein is not critical for embryonic survival and head skeleton and sense organs are normal. Most vein mutants die either as embryos or as larvae, but a small number do pupariate. Individuals that survive to pupariate secrete a pupal case with pattern abnormalities (Schnepp, 1996).

vein and spitz show a strong genetic interaction suggesting a molecular interdependence. Reducing vein dose in a spi null genotype dramatically worsens the phenotype to produce a collapse embryo with an extruded head skeleton. However, the double mutants are not as severly affected as are Egf-R null mutants. Genetic interactions are also observed between vn, Egf-R and rolled. The gain of function alleles Egf-R-Ellipse and rolled-Sevenmaker rescue proliferation defects in strong and null vein mutants. These defects include a small wing disc and the size of the pupal case (Schnepp, 1996).

Signaling through the Drosophila EGF receptor (DER) is important for the growth and differentiation of the wing. These processes may be mediated by different DER ligands including Spitz and Vein . The roles of these ligands and other DER pathway components in wing disc development were investigated using in vivo culture to produce mutant discs from genotypes that are normally embryonic lethal. No role for spi in wing disc growth was found, whereas vn is essential. spi mutant wing discs are morphologically normal as judged by expression of the vein marker rhomboid and analysis of the differentiated wing tissue. spi embryos produce mature-size wing discs patterned with vein and intervein territories like wild-type discs and differentiated wing structures look normal. rho, Star and argos, all known to be involved in Spi/DER signaling are likewise not required for wing growth, whereas pointed, which acts at the end of the intracellular pathway, is required. The results suggest different ligands and molecular mechanisms control EGF-R signaling in wing growth and differentiation (Simcox, 1997).

Spalt modifies EGFR-mediated induction of chordotonal precursors in the embryonic PNS of Drosophila promoting the development of oenocytes

Genes of the spalt family encode nuclear zinc finger proteins. In Drosophila melanogaster, they are necessary for the establishment of head/trunk identity, correct tracheal migration and patterning of the wing imaginal disc. Spalt proteins display a predominant pattern of expression in the nervous system, not only in Drosophila but also in species of fish, mouse, frog and human, suggesting an evolutionarily conserved role for these proteins in nervous system development. Spalt works as a cell fate switch between two EGFR-induced cell types, the oenocytes and the precursors of the pentascolopodial organ in the embryonic peripheral nervous system. Removal of spalt increases the number of scolopodia, as a result of extra secondary recruitment of precursor cells at the expense of the oenocytes. In addition, the absence of spalt causes defects in the normal migration of the pentascolopodial organ. The dual function of spalt in the development of this organ, recruitment of precursors and migration, is reminiscent of its role in tracheal formation and of the role of a spalt homolog, sem-4, in the C. elegans nervous system (Rusten, 2001).

By analogy with the developing lch5, it was hypothesized that the oenocytes require Egfr signalling for proper development. Embryos mutants for Star and spitz were examined at different stages of development. Interestingly, in stage 11 embryos the sal pattern of expression remains unaltered in the cells surrounding the C1 precursor, as well as in the epidermis. However, later on, the development of the oenocytes is inhibited. These results indicate that sal regulation is independent of the Egfr pathway and that the oenocytes development depends on both sal and Egfr signaling activity. Furthermore, if the signaling arises from the precursor C1, the formation of oenocytes would be restrained in the absence of SOPs. Indeed, in ato mutant embryos oenocytes originate only in the segments where remnant SOPs develop (Rusten, 2001).

In conclusion, the results are consistent with a model where sal restricts the ability of C1-surrounding cells, receiving Egfr signaling, to adopt sensory organ precursor cell fate; these cells then develop as oenocytes rather than chordotonal organs (Rusten, 2001).

The Egfr pathway is implicated in the development of the chordotonal organs in Drosophila. The pathway is necessary for the second step of recruitment of SOPs from ectodermal precursors, and for the consequent increase of number of scolopodia in the lch5 and in the vchA/B organs. Thus, during development of the lch5 organ, where two secondary SOPs are recruited, removal of positive Egfr pathway components like rho, S, spi, pnt, sos, Drk, or Egfr itself, reduces the number of scolopodia in the lch5 from five to three. Conversely, mutations in negative regulators of Egfr signaling like argos, gap1 or spry result in an increase of secondary recruited SOPs in the thorax as well as in the abdominal segments (Rusten, 2001 and references therein).

Sal plays a role in the formation of the lch5 in parallel with the Egfr signaling pathway: the absence of sal generates supernumerary scolopodia, while the overexpression of Sal reduces the number of scolopodia from five to three. These results are consistent with the idea that under wild-type conditions, sal modifies the Egfr signaling output in the cells surrounding the primary precursor C1, which instead of becoming secondary SOPs adopt the oenocytes cell fate. Five lines of evidence support this idea. (1) Supernumerary support cells accompany the supernumerary neurons observed in sal mutants. Thus, the phenotype is not caused by cell fate transformation within the SOP lineage. (2) The C1-surrounding cells receive the Egfr signal (shown by the antibody staining for activated Rolled/MAPK) and, therefore, are capable of becoming secondary precursors. These cells are sal positive while the other potential secondary precursors, also showing activated Rolled and overlying the more ventrally located C2-C5, are not. Given that the number of cells receiving the Egfr signal is larger than the number of cells that become secondary SOPs (two for lch5 and one for vchA/B), the output of the Egfr pathway must be modified in the rest of the cells receiving the signal. (3) The analysis of allelic combinations between sal and Egfr pathway mutants reveals that the supernumerary neuronal phenotype observed in the absence of sal is Egfr dependent. (4) The oenocyte precursors depend on sal and Egfr signaling to develop, and (5) in the absence of primary precursors, oenocytes do not develop, as shown in ato mutants (Rusten, 2001).

The effects of sal loss- and gain-of-function are similar, but not identical, to the ones exhibited by corresponding changes in negative regulators of Egfr signaling. There are at least two important differences between the role of these regulators and sal. (1) aos, pnt and spry are expressed in all the cells receiving the Egfr signal from the primary SOPs, while sal is expressed only in a subset of them. Consistent with this, the loss of function of these regulators affects the secondary recruitment of SOPs to other chordotonal organs, like vchA/B and v'ch1, while sal seems to modify only lch5. (2) The increase of scolopodia numbers in lch5 is moderate in the spry and aos mutants, while in sal mutants, up to eight scolopodia are observed. In conclusion, sal is involved specifically in the formation of lch5 in a manner different from that of the Egfr pathway regulators that are involved in the development of all the chordotonal organs (Rusten, 2001).

The cells surrounding C1 migrate along the dorsoventral axis, closely associated with the pentascolopodial organ. These cells are easy to recognize by the elongated shape of their nuclei and the strong sal expression that they display. These cells occupy the location of oenocytes in late embryonic stages. It is then likely that sal plays a role in deciding the fate of the Egfr responding cells surrounding the C1 precursor. In the presence of sal these cells will become oenocytes while in the absence of sal (as is true for the presumptive secondary precursors overlying C2, C3, C4 and C5), the cells will become sensory organ precursors. Since the putative precursors of the oenocyte cells need Egfr signaling to accomplish some aspects of their development, sal is thought to act as a selector gene being necessary to direct them to their correct fate (Rusten, 2001).

spalt-dependent switching between two cell fates that are induced by the Drosophila EGF receptor

Signaling from the EGF receptor can trigger the differentiation of a wide variety of cell types in many animal species. The mechanisms that generate this diversity have been explored using the Drosophila peripheral nervous system. In this context, Spitz ligand can induce two alternative cell fates from the dorsolateral ectoderm: chordotonal sensory organs and non-neural oenocytes. The overall number of both cell types that are induced is controlled by the degree of Egfr signaling. In addition, the spalt gene is identified as a critical component of the oenocyte/chordotonal fate switch. Genetic and expression analyses indicate that the Sal zinc-finger protein promotes oenocyte formation and suppresses chordotonal organ induction by acting both downstream of and in parallel to the Egfr. To explain these findings, a prime-and-respond model is proposed. Here, sal functions prior to signaling as a necessary but not sufficient component of the oenocyte prepattern that also serves to raise the apparent threshold for induction by Spi. Subsequently, sal-dependent Sal upregulation is triggered as part of the oenocyte-specific Egfr response. Thus, a combination of Sal in the responding nucleus and increased Spi ligand production sets the binary cell-fate switch in favour of oenocytes. Together, these studies help to explain how one generic signaling pathway can trigger the differentiation of two distinct cell types (Elstob, 2001).

Oenocytes are induced from the ectoderm by an inductive signal that is generated in the developing PNS. The production of active Spi by the C1 precursor cell, under the control of ato and rho, triggers Egfr activation and thus oenocyte induction in adjacent ectoderm. Oenocyte induction by the PNS appears to be a short-range event with only the cells immediately surrounding C1 switching on oenocyte markers. In argos mutants, however, the range of the response is increased from one to two concentric rings of cells. Hence, as in photoreceptor recruitment, Spi ligand is not intrinsically limited to immediate neighbors but the response is nevertheless kept short-range by argos-mediated feedback inhibition of the receptor (Elstob, 2001).

In wild-type embryos and in all of the mutant backgrounds examined, the number of cells in the whorl at any one time is less than the final number of mature oenocytes. For example, a wild-type whorl contains 3-4 cells with sickle-shaped nuclei but the final oenocyte cluster contains on average 6 cells. The basis for this difference is not yet understood but it might be explained by cell division or by sequential delamination of oenocyte precursors (Elstob, 2001).

The specification of secondary COP and oenocyte fates requires the Egf pathway. In ato, rho, spi and EgfrDN backgrounds, where signaling is compromised, the induction of both cell types is blocked. Conversely, when the Egfr is hyperactivated, both cell types become more numerous. These results indicate that the number of recruited cells is controlled by the amount of Egf pathway signal. It is important to realize, however, that the level, duration and spatial extent of ligand production are all being altered in these experiments. More sophisticated methods would be needed to clearly distinguish which of these three signaling parameters is critical for controlling cell number (Elstob, 2001).

Surprisingly, there is no parity between the numbers of excess oenocytes and lateral chordotonal organs that are produced by Egfr hyperactivation. Thus for a given increase in ligand, more oenocyte precursors than COPs are recruited. This implies the existence of an additional tier of control that restricts neural but not oenocyte induction. Such a selective inhibition process would ensure that the number of chordotonal organs is more tightly controlled than that of oenocytes, as is observed in wild-type embryos (Elstob, 2001).

The sal gene plays opposite roles in oenocyte and chordotonal induction. It is both necessary and sufficient for repressing secondary COP induction and it is necessary but not sufficient for promoting oenocyte formation. The lack of sufficiency for oenocyte induction is revealed when sal is misexpressed using the en-GAL4 driver. Oenocytes are not ectopically induced in ventral regions, even in the presence of excess Spitz. It is likely that other factors are required, together with Sal, to promote the oenocyte induction process (Elstob, 2001).

Using epistasis tests and gene expression analysis the regulatory relationship between sal and the Egf pathway in oenocyte and COP formation has been elucidated. These data allow the exclusion of the possibility that sal acts upstream of spi in the signaling cell. Importantly, the results indicate that sal functions in the responding ectoderm, either downstream of the Egfr or in a parallel pathway leading to oenocyte induction and secondary COP repression (Elstob, 2001).

In fact, it is probable that sal plays a dual role that is downstream and also in parallel to the Egfr. In rho and spi mutants, the normal upregulation of Sal in the vicinity of C1 is abolished. Conversely, Spitz misexpression produces ectopic Sal upregulation in dorsal locations. Both results indicate that sal lies downstream of the Egfr and that Sal protein levels are controlled by receptor activation. However, Sal is also expressed at moderate levels in presumptive oenocyte precursors prior to Egf pathway activation and this expression remains normal in rho and spi mutants. For these reasons, it is likely that at least part of the function of sal lies in a parallel pathway that, in conjunction with the Egf signal, promotes oenocyte induction and inhibits COP recruitment (Elstob, 2001).

A prime-and-respond model is presented to integrate the dual roles of sal downstream and also in parallel to the Egfr. In this model, sal functions in the parallel pathway as a competence switch. Thus, Sal prepatterns the dorsal ectoderm so that, on receipt of the Egf signal, oenocytes rather than COPs are induced. One consequence of the Sal oenocyte prepattern is to increase the apparent induction threshold in responding cells. This makes the prediction that the signaling cell inducing oenocytes needs to express more ligand than those that recruit secondary COPs, and this is indeed the case. C1 is known to express high levels of rho for longer than any of the other primary COPs. Thus, the Egf pathway does contribute to the cell-type specificity of the induction event in the sense that more Spi ligand is required to induce oenocytes than to recruit chordotonal organs (Elstob, 2001).

One of the early oenocyte-specific responses to the Sal prepattern is the subsequent upregulation of Sal itself. This, in turn, stimulates the expression of the sal target gene svp, one member of the repertoire of oenocyte early differentiation genes. A key feature of the prime-and-respond model is that moderate levels of sal expression serve to prime the responding cells to further upregulate Sal when they receive Spi ligand. In support of this priming mechanism, it has been demonstrated that upregulation in response to constitutive Spitz expression is restricted to those cells that have already expressed sal. Hence, Sal proteins provide a molecular link between the prepattern and the Egfr response (Elstob, 2001).

In the prime-and-respond model, it is implicit that the early and late phases of sal expression produce distinct effects on the responding cell. As the levels of Sal are different in the two phases, it may be that there are at least two different concentration-dependent effects for this transcription factor. In agreement with this, it has been shown that strong expression of the sal target gene, svp, correlates with the domain of sal upregulation and not with the lower-level prepattern. In another system, wing vein development, there is a very extreme example of a concentration difference, with low and high levels of Sal producing completely opposite transcriptional effects on the knirps target gene (Elstob, 2001 and references therein).

Regulation of cell number by MAPK-dependent control of apoptosis: A mechanism for trophic survival signaling

Trophic mechanisms in which neighboring cells mutually control their survival by secreting extracellular factors play an important role in determining cell number. However, how trophic signaling suppresses cell death is still poorly understood. The survival of a subset of midline glia cells in Drosophila depends upon direct suppression of the proapoptotic protein Hid via the Egf receptor/RAS/MAPK pathway. The TGF alpha-like ligand Spitz is activated in the neurons, and glial cells compete for limited amounts of secreted Spitz to survive. In midline glia that fail to activate the Egfr pathway, Hid induces apoptosis by blocking a caspase inhibitor, Diap1. Therefore, a direct pathway linking a specific extracellular survival factor with a caspase-based death program has been established (Bergmann, 2002).

The genetic requirement of mapk for MG survival and of hid for MG apoptosis prompted the assumption that MAPK promotes survival of the MG by inhibition of HID activity. According to this model, the MG would be unprotected from HID-induced apoptosis in mapk-deficient embryos, and die. Consistent with this idea, HID protein is detectable in the MG of late stage wild-type embryos. To test this further, embryos that were mutant for both mapk and hid were examined. In early stage mapk;hid double mutant embryos, the initial generation of the MG appears to be normal. However, in contrast to mapk mutants alone, the MG is rescued in mapk;hid double mutant embryos although the survival function of MAPK is missing in these embryos. Dissection revealed that the MG are located directly at the cuticle of the embryos. Because segmental fusions occur in these embryos, some of the MG cluster in groups of up to 20 cells. In individual segments, five to six MG are visible. This number is larger compared to wild-type (three MG per segment), and is remarkably similar to the number of surviving MG in hid mutant embryos alone, indicating that MAPK promotes MG survival largely through inhibition of HID (Bergmann, 2002).

The spi gene is required for MG survival and encodes a candidate trophic factor for MG survival. To prove that spi function is required to suppress hid activity, the fate of the MG in spi;hid double mutant embryos was determined. The MG survive in spi embryos if hid is removed as well, and it is concluded that the survival function of spi is mediated through suppression of hid-induced apoptosis (Bergmann, 2002).

The question arises as to which cells process mSPI and provide a source of sSPI for MG survival. Since spi is ubiquitously expressed, it is difficult to determine histochemically where sSPI, the active ligand, is generated. Therefore, a genetic approach was used; whether the loss of MG in spi mutant embryos can be rescued by expression of the membrane bound inactive precursor (UAS-mSPI) either in the MG (using the sim-GAL4 driver) or in neuronal axons (using the elav-GAL4 driver) was examined. It was reasoned that the MG would be rescued in spi mutant embryos only if mSPI is presented in the location where it is normally processed for MG survival in wild-type embryos. Presentation of mSPI by the MG itself does not result in rescue of the MG in spi mutant embryos, ruling out an autocrine mechanism. In contrast, expression of mSPI in neuronal axons appears to be sufficient for MG survival in spi embryos. This argues in favor of a paracrine mechanism. In control experiments, sSPI was examined using these two Gal4 drivers in wild-type embryos. With both GAL4 drivers an increase in the number of MG cells is detected, indicating that they are expressed at the right time and that the MG does not fail to secrete Spi once it has been processed (Bergmann, 2002).

A key regulator of Spi activation is rhomboid, a gene encoding a cell surface, seven-pass transmembrane protein that appears to function as a serine protease directly cleaving mSPI. rhomboid has been implicated in suppression of MG apoptosis. Ectopic expression of Rho in neurons (elav-Gal4/UAS-Rhomboid) promotes an excess of MG, suggesting that neurons have the capacity to process endogenous mSPI. Another essential protein for Spi processing is Star. Star mutants display an MG phenotype similar to spi. STAR regulates intracellular trafficking of mSPI. Expression of Star from the neurons but not from the MG rescues the Star phenotype in the MG. Thus, this analysis clearly demonstrates that the sSPI signal for MG survival is generated and secreted by neurons (Bergmann, 2002).

In vivo analysis of argos structure-function. Sequence requirements for inhibition of the Drosophila epidermal growth factor receptor

The Drosophila Argos protein is the only known extracellular inhibitor of the epidermal growth factor receptor (EGFR). It is structurally related to the activating ligands, since it is a secreted protein with a single epidermal growth factor (EGF) domain. An investigation was carried out to determine which regions of the Argos protein are essential for inhibition. A series of deletions were made and tested in vivo; analyzing chimeric proteins between Argos and the activating ligand, Spitz (a transforming growth factor-alpha-like factor), would disclose what makes one protein inhibitory and the other activating (Howes, 1998).

In one set of chimeras, the entire EGF domains of Spitz and Argos were swapped. The SAS (Spitz N-terminal; Argos EGF domain; Spitz C-terminal) and ASA pair (in which only the EGF domains themselves were swapped) were designed to test the possibility that the EGF domain is solely an EGFR binding domain, with no role in activation or inhibition. If this were the case, the EGF domains would be interchangeable and SAS might behave like Spitz, and ASA like Argos. In the SA and AS pair, the complete C termini of Spitz and Argos were swapped, including the EGF domains. If the Argos EGF domain and the adjacent C terminus were sufficient to confer the Argos protein's inhibitory function, the SA chimera (Spitz N terminus with the Argos EGF domain and C terminus) might be expected to behave like Argos. Similarly, since the N terminus of Argos appears not to contain sufficient information to confer inhibitory function, the AS chimera (Argos N terminus with the Spitz EGF domain and C terminus of the secreted form) might retain Spitz activity. However, neither SAS nor ASA has any activity; they neither suppress nor enhance the argos mutant phenotype. This suggests that the EGF domain is more than simply an EGFR binding domain; instead, it participates in specifying whether the receptor is activated or inhibited. SA also has no Argos activity (nor, as expected, is it Spitz-like). This implies that sequences N-terminal to the Argos EGF domain are necessary for its function. The AS chimera activates the EGF receptor, suggesting its function is similar to Spitz, not Argos. When overexpressed in the wing, it produces the characteristic extra vein phenotype of EGFR activation. In this same assay, overexpression of Argos causes the opposite phenotype, namely loss of wing veins. These results indicate that the large N-terminal region of Argos is not sufficient to convert Spitz into an inhibitor. AS, however, rescues spitz eye mutants only partially, suggesting that Spitz's potency is reduced by the addition of 355 amino acids of the Argos N terminus (Howes, 1998).

The spacing between the six essential cysteines in EGF domains varies only slightly. In particular, between C3 and C4 (the B-loop), there are usually 10 amino acids in EGF-like ligands; the range is 8-13 in all known EGF repeats. In Argos, there are 20 residues between C3 and C4, and this extension might account for Argos's unusual inhibitory function. Indeed, although the function of this B-loop remains uncertain, many studies have implicated it in EGF binding and activation of the receptor. The C-loop of EGF domains, between cysteines 5 and 6, is also critical for the function of EGF-like ligands. In Argos, the C-loop is also atypical. It is only five residues long, compared with eight in Spitz and all the mammalian activating ligands, and it has two extra positive charges. To examine the functional importance of the Argos B- and C-loops, four chimeras were constructed between Argos and Spitz in which the B- and C-loops were exchanged, and tested for whether they activate or inhibit the EGFR. The extended B-loop is necessary for Argos function, whereas the C-loop can be replaced with the equivalent Spitz region without substantially affecting this inhibitory function. Comparison of the argos genes from Drosophila and the housefly, Musca domestica, supports this structure-function analysis. These studies are a prerequisite for understanding how Argos inhibits the Drosophila EGFR and provide a basis for designing mammalian EGFR inhibitors (Howes, 1998).

Proneural and abdominal Hox inputs synergize to promote sensory organ formation in the Drosophila abdomen

The atonal (ato) proneural gene specifies a stereotypic number of sensory organ precursors (SOP) within each body segment of the Drosophila ectoderm. Surprisingly, the broad expression of Ato within the ectoderm results in only a modest increase in SOP formation, suggesting many cells are incompetent to become SOPs. This study shows that the SOP promoting activity of Ato can be greatly enhanced by three factors: the Senseless (Sens) zinc finger protein, the Abdominal-A (Abd-A) Hox factor, and the epidermal growth factor (EGF) pathway. First, it was shown that expression of either Ato alone or with Sens induces twice as many SOPs in the abdomen as in the thorax, and does so at the expense of an abdomen-specific cell fate: the larval oenocytes. Second, Ato was shown to stimulate abdominal SOP formation by synergizing with Abd-A to promote EGF ligand (Spitz) secretion and secondary SOP recruitment. However, it was also found that Ato and Sens selectively enhance abdominal SOP development in a Spitz-independent manner, suggesting additional genetic interactions between this proneural pathway and Abd-A. Altogether, these experiments reveal that genetic interactions between EGF-signaling, Abd-A, and Sens enhance the SOP-promoting activity of Ato to stimulate region-specific neurogenesis in the Drosophila abdomen (Gutzwiller, 2010).

How proneural pathways that specify sensory precursor cells throughout the body are integrated with region-specific patterning genes to yield the correct type and number of sensory organs is not well understood. This study shows that three factors enhance the ability of Ato to promote ch organ SOP cell fate in the Drosophila abdomen; the EGF pathway mediated by the Spi ligand, the Abd-A Hox factor, and the Sens zinc finger transcription factor (Gutzwiller, 2010).

EGF signaling is used reiteratively throughout development to specify the formation of distinct cell types along the body plan. In the embryonic Drosophila abdomen, EGF signaling initiated by the activation of rhomboid (rho) in a set of ch organ SOP cells induces the formation of both a cluster of abdomen-specific oenocytes as well as a set of 2° ch organ SOP cells. But how does the EGF-receiving cell know whether to become a larval oenocyte that is specialized to process lipids or a ch organ SOP cell that forms part of the peripheral nervous system? Previous studies have shown that oenocyte specification requires at least two inputs: (1) the reception of relatively high levels of EGF signaling and (2) the expression of the Spalt transcription factors. Hence, oenocytes develop in close proximity to the abdominal C1 SOP cells that lie within a Spalt expression domain and express high levels of rho. In contrast, 2° SOP cells require less EGF signaling and form if the receiving cells lack Spalt. Consistent with this model, genetic studies have shown that oenocytes fail to develop and one to two additional ch organ SOP cells are specified in Spalt mutant embryos, whereas ectopic Spalt expression in the ventral ectoderm inhibits the recruitment of 2° SOP cells. Thus, Spalt promotes oenocyte development and antagonizes 2° ch organ specification in the Drosophila embryo (Gutzwiller, 2010).

Evidence that ato has the opposite effect as Spalt: it promotes ch organ SOP cells at the expense of oenocyte specification. Witt (2010) showed that ato loss-of-function results in decreased expression of activity of the rho enhancer, RhoBAD (Witt, 2010), in C1 SOP cells and induces fewer oenocytes. These data are consistent with EGF signaling being compromised in ato mutant embryos and oenocyte specification being dependent upon the reception of high levels of Spi. This study shows that Ato gain-of-function stimulates RhoBAD expression yet results in the inhibition of oenocyte formation. Importantly, the loss of oenocytes is not due to decreased EGF signaling as similar whorls of phospho-ERK-positive cells and even extra phospho-ERK staining are observed in Ato-expressing segments compared with non-expressing segments. In addition, no difference was detected in cell death between Ato-expressing and non-Ato-expressing segments (using an anti-cleaved Caspase3 marker), indicating the oenocyte loss is not due to apoptosis. Instead, Ato promotes the formation of additional ch organ SOP cells in abdominal segments that normally form oenocytes. Moreover, while the broad activation of EGF signaling (PrdG4;UAS-Rho) induces many extra oenocytes and a few scolopodia, the co-expression of Ato and Rho induces many scolopodia and few oenocytes. These data suggest that if the Spi-receiving cell expresses high Ato relative to Salm then ch organ development occurs whereas if the Spi-receiving cell expresses high Salm relative to Ato then oenocytes are formed. Thus, Ato plays a role in both the Spi-secreting (induction of rho expression) and Spi-receiving cell to dictate the choice of cell fate (Gutzwiller, 2010).

The broad expression of Ato within the ectoderm revealed differences in sensory organ competency between the thorax and abdomen. In particular, it was found that Ato induced approximately twice as many ch organ SOP cells in the abdomen as in the thorax. Moreover, the co-expression of Ato with the Abd-A Hox factor induced significantly more ch organ cell formation than expression of either factor alone (none by Abd-A, four by Ato, and eight by Ato/Abd-A). These data suggest that Ato and Abd-A synergize to enhance ch organ SOP formation in the abdomen, an prompted an examination whethere these SOP cells are predominantly 1° or 2° cells. This problem was first addressed by first showing that the co-expression of Ato and Abd-A stimulates Rho enhancer activity (RhoAAA) within additional cells and results in enhanced phospho-ERK staining. Second, it was shown that Ato and Abd-A require the EGF pathway to enhance ch organ development as co-expression of both factors in a spi mutant embryo failed to promote more ch organs than expression of Ato alone. These data indicate that the co-expression of Ato and Abd-A enhances the ability of 1° ch organ SOP cells to activate rho, stimulates Spi secretion and, since the receiving cell expresses Ato, 2° SOPs form instead of oenocytes. The net result is that Ato and Abd-A synergize to activate the EGF pathway to promote region-specific neurogenesis within the Drosophila abdomen (Gutzwiller, 2010).

The Sens transcription factor is essential for the formation of much of the peripheral nervous system in Drosophila and previous studies revealed that Sens can stimulate the sensory bristle-forming activity of the Scute and Achaete proneural factors in the wing disc. Similarly, it was found that Sens stimulates the ability of Ato to generate internal stretch receptors in the embryo and that Ato and Sens promote more sensory organ development in the abdomen than in the thorax. In addition, while the overall number of ch organs formed by Ato and Sens co-expression is decreased in spi mutant embryos, significantly more ch organ SOP cells in the abdomen than in the thorax are observed in this EGF-compromised genetic background. Thus, Ato and Sens can stimulate abdominal ch organ SOP cell development in the presence or absence of Spi-mediated cell signaling (Gutzwiller, 2010).

So, what is the relationship between Ato, Sens, and Abd-A in regulating both EGF signaling and region-specific sensory organ formation? It was previously found that Ato, Sens, and Abd-A control EGF signaling through the regulation of a cis-regulatory element within the rhomboid (rho) locus (RhoBAD) (Li-Kroeger, 2008; Witt, 2010). RhoBAD acts in abdominal C1 SOP cells to induce oenocyte formation, and Ato and Abd-A both stimulate RhoBAD expression, at least in part, by limiting the ability of Sens to repress RhoBAD activity. Moreover, they do so using different mechanisms. An Abd-A Hox complex containing Extradenticle and Homothorax directly competes with the Sens repressor for overlapping binding sites in RhoBAD (Li-Kroeger, 2008). In contrast, Ato does not directly bind RhoBAD but does directly interact with Sens to limit its ability to bind and repress Rho enhancer activity (Witt, 2010). Consequently, SOPs that co-express Ato and Abd-A are likely to limit the ability of Sens to repress Rho and thereby increase the number of ch organ SOP cells that secrete Spi. Consistent with this prediction, the co-expression of Ato and Sens preferentially stimulates Rho enhancer activity within abdominal segments compared to thoracic segments. Each SOP cell that expresses rho would further enhance sensory organ development through the recruitment of 2° SOP cells via Spi-mediated signaling. Hence, the genetic removal of spi results in a significant decrease in the number of ch organ SOP cells that develop in response to Ato and Sens. Thus, the ato-sens genetic pathway, which is used throughout the body to promote SOP formation, interacts with an abdominal Hox factor to stimulate EGF signaling and promote additional cell fate specification in the abdomen (Gutzwiller, 2010).

While the above model fits well with most of the data, two unexpected findings were observed when comparing the ability of Ato-Sens co-expression to induce ch organ development in the presence and absence of spi function: First, it was predicted that Ato-Sens co-expression in the thoracic regions, which lack Abd-A, should predominantly induce the formation of 1° ch organ SOP cells that do not require EGF signaling for their development. However, it was found that significantly fewer ch organs form in the thorax of spi mutants, indicating that EGF signaling can enhance 2° sensory organ formation within thoracic segments that co-express Ato and Sens. Interestingly, previous studies have shown that both rho and the Rho enhancers are weakly active within thoracic C1 SOP cells, but their levels do not reach a high enough threshold to induce oenocyte formation. However, it is possible that the co-expression of Ato and Sens sufficiently sensitizes the receiving cells to respond to low levels of EGF signaling and become ch organ SOP cells. The second unanticipated finding is that Ato and Sens co-expression still induced significantly more ch organ development within the abdomen (5-6 extra SOP cells) relative to the thorax (1-2 extra SOP cells) in the absence of Spi-mediated signaling. This finding suggests that Ato and Sens can genetically interact with the Abd-A Hox factor to promote sensory organ development in an Spi-independent manner. Currently, it is not understood how Abd-A enhances the proneural activity of the Ato-Sens factors in the absence of Spi signaling. One possibility is that Abd-A and Ato use similar mechanisms to limit Sens-mediated repression of additional target genes besides rho to stimulate ch organ development. Alternatively, Abd-A could independently regulate other factors such as those involved in the Notch-Delta pathway to enhance the competency of the ectoderm to respond to the Ato-Sens pathway. Intriguingly, a Hox factor (lin-39) in C. elegans has been shown to directly regulate Notch signaling during vulval development, and the vertebrate Hoxb1 factor regulates neural stem cell progenitor proliferation and maintenance by modulating Notch signaling. Since differential Notch-Delta signaling is a key pathway in deciding neural versus non-neural cell fates, the ability of Hox factors to modify this pathway could result in segmental differences in neurogenesis (Gutzwiller, 2010).

Spitz and Eye Development

Recessive spitz loss-of-function mutations affect compound eye development. In mosaic clones, spitz function is required in the differentiation of the R8 photoreceptor cells, the first of eight photoreceptor cells to differentiate in each ommatidium. spitz loss-of-function mutations are dominant suppressors of EgfrE gain-of-function mutations of the epidermal growth factor-receptor gene. These data suggest that the spitz product is a precluster promoting factor. spitz transcription increases abruptly in the morphogenetic furrow, the obverse of Egfr expression (Tio, 1994).

Dominant Ellipse mutant alleles of the Drosophila EGF receptor homolog (Egfr) dramatically suppress ommatidium development in the eye and induce ectopic vein development in the wing. This phenotype suggests a possible role for Egfr in specifying the founder R8 photoreceptor cells for each ommatidium. Ellipse mutations have been used to probe the role of Egfr in eye development: Elp mutations result from a single amino acid substitution in the kinase domain, which activates tyrosine kinase activity and MAP kinase activation in tissue culture cells. Transformant studies confirm that the mutation is hypermorphic in vivo, but the Egfr function is elevated less than by ectopic expression of the ligand Spitz. Ectopic Spi promotes photoreceptor differentiation, even in the absence of R8 cells. Pathways downstream of Egfr activation were assessed to explore the basis of these distinct outcomes. Elp mutations cause overexpression of the Notch target gene E(spl) mdelta and require the function of Notch to suppress ommatidium formation. E(spl) mdelta is known to be expressed in cell clusters in the morphogenetic furrow of wild type eyes. The Elp phenotype also depends on the secreted protein Argos and therefore, in Elp;aos double mutants, the phenotype is reverted. Complete loss of Egfr function in clones of null mutant cells leads to delay in R8 specification and subsequently to the loss of mutant cells. The Egfr null phenotype is distinct from that of either spitz or vein mutants, suggesting that a combination of these or other ligands is required for aspects of Egfr function. No delay in ato refinement is found for spi mutant eyes, and vn mutants have vestigial eye discs that usually fail to differentiate any type of eye cell. In normal development, Egfr protein is expressed in most retinal cells, but at distinct levels. Antibody specific for diphospho-ERK as well as expression of the Egfr target gene argos was used to assess the pattern of Egfr activity; highest activity was found in the intermediate groups of cells in the morphogenetic furrow. However, studies of mutant genotypes suggest that this activity may not be required for normal ommatidium development. Since distinct phenotypic effects for four different levels of Egfr activity associated with wild-type, null mutant, Elp mutant, or fully activated DER function are seen, it is proposed that multiple thresholds separate several aspects of Egfr function. These include activation of N signaling to repress R8 specification; turning on argos expression, and recruiting photoreceptors R1-R7. It is possible that during normal eye development these thresholds are attained by different cells, contributing to the pattern of retinal differentiation. It is suggested that Egfr activation by Spi is the only signal that the R8 cell needs to provide for most photoreceptor cells and that ubiquitous targeted Spi expression converts every retinal cell to an R1-R7 photoreceptor, at the expense of R8 and other cell types. Argos is required for loss of ommatidia in Elp. Nonautonomy of Elp mutations suggests that aos acts nonautonomously to inhibit ommatidium formation (Lesokhin, 1999).

Receptor tyrosine kinases such as the EGF receptor transduce extracellular signals into multiple cellular responses. In the developing Drosophila eye, Egfr activity triggers cell differentiation. This study focuses on three additional cell autonomous aspects of Egfr function and their coordination with differentiation, namely, withdrawal from the cell cycle, mitosis, and cell survival. Whereas differentiation requires intense signaling, dependent on multiple reinforcing ligands, lesser Egfr activity maintains cell cycle arrest, promotes mitosis, and protects against cell death. Each response requires the same Ras, Raf, MAPK, and Pnt signal transduction pathway. Mitotic and survival responses also involve Pnt-independent branches, perhaps explaining how survival and mitosis can occur independently. These results suggest that, rather than triggering all or none responses, Egfr coordinates partially independent processes as the eye differentiates (Yang, 2003).

Focus was placed on three aspects of Egfr function that occur at two developmental stages. These are the withdrawal from the cell cycle that accompanies the first fate specifications and the mitosis and survival of cells that pass unspecified through a 'second mitotic wave' (SMW) before later recruitment to retinal cell fates. The onset of advancing differentiation is defined by a morphogenetic furrow, which sweeps anteriorly from the posterior part of the eye imaginal disc. Just anterior to the morphogenetic furrow, cells arrest in G1 of the cell cycle. Within the morphogenetic furrow some of the arrested cells are specified as individual R8 photoreceptor cells, the founders of each ommatidium. Each R8 cell then produces ligands that activate Egfr in four neighboring cells. These neighbors are recruited to become the R2, R3, R4, and R5 photoreceptor cells of each ommatidium. While these five cells maintain their G1 arrest and differentiate in response to Egfr activity, the surrounding cells in which Egfr is inactive reenter the cell cycle and begin S phase DNA synthesis. Entry into this SMW occurs around retinal column 1, also the stage at which 'preclusters' of R8, R2, R3, R4, and R5 cells first become morphologically recognizable. The SMW produces unspecified cells that will be recruited to take the remaining 14 retinal cell fates. Egfr is required for survival and G2/M progression of SMW cells as well as later cell fate specification (Yang, 2003).

R2-5 differentiation requires the Egfr ligand Spitz (Spi) and the Ras/Raf/MAPK pathway. Spitz is not required to maintain R2-5 in G1, even though experiments with a temperature-sensitive allele show that Egfr is required simultaneously to maintain G1 arrest and to initiate differentiation. Any function of the neuregulin-like Egfr ligand Vein (Vn) in cell cycle withdrawal was ruled out from the study of spi; vn double mutants. The role of another zygotic ligand, Keren (Krn), was evaluated through mutations in rhomboid1 (rho) and rhomboid3 (a.k.a. roughened [ru]), two proteases required to cleave transmembrane precursors of the Keren and Spitz proteins. Cell cycle progression was assessed in clones of homozygous ru; rho1 double mutant cells with Cyclin B as a marker. Cyclin B protein accumulates in cells that have passed the G1/S checkpoint but not yet divided. All cells except R8s reentered the cell cycle in ru; rho1 double mutant clones. It is inferred that Krn must be necessary and sufficient to maintain R2-5 cells in G1 in the absence of spi (Yang, 2003).

To investigate the difference between Egfr activation by Spi and by Krn, whether the Ras/Raf/MAPK/Pnt pathway is required to maintain G1 arrest was examined. Mosaic analysis of null alleles was used. For Ras and Raf the result was the same as that for Egfr. All these genes are required to prevent cells from reentering the cell cycle. It was not possible to generate clones of cells lacking MAPK using the FLP/FRT system for mosaic analysis because of the centromeric location of the MAPK gene rolled proximal to all FRT transgene insertion sites. The transcription factor Pnt is the next component downstream in photoreceptor differentiation. Only R8 cells remain in G1 in cells deleted for the entire pnt gene. Thus, keeping R2-5 in G1 requires Egfr signaling through the same Ras/Raf/Pnt pathway as taken during differentiation (Yang, 2003).

Since spi activates the Ras/Raf/MAPK/Pnt cascade, it was asked whether Spitz could also maintain cells in G1. A secreted Spi protein (sSpi) that does not require Rho function was overexpressed to test this. The GMRGal4 driver was used to target expression posterior to column 1 of the eye disc. The GMR>sSpi eye discs lacked any secondary mitotic wave (SMW). Neither mitotic figures nor cells reentering the cell cycle were seen. Thus, not only does maintaining R2-5 cells in G1 require the same effectors as differentiation, but the differentiation ligand Spi can also maintain G1 arrest (Yang, 2003). sim dichaete drifter

Estes, P., Fulkerson, E. and Zhang, Y. (2008). Identification of motifs that are conserved in 12 Drosophila species and regulate midline glia vs. neuron expression. Genetics 178(2): 787-99. PubMed Citation: 18245363

Identification of motifs that are conserved in 12 Drosophila species and regulate midline glia vs. neuron expression

Functional complexity of the central nervous system (CNS) is reflected by the large number and diversity of genes expressed in its many different cell types. Understanding the control of gene expression within cells of the CNS will help reveal how various neurons and glia develop and function. Midline cells of Drosophila differentiate into glial cells and several types of neurons and also serve as a signaling center for surrounding tissues. This study examined regulation of the midline gene, wrapper, required for both neuron-glia interactions and viability of midline glia. A region upstream of wrapper required for midline expression was identified that is highly conserved (87%) between 12 Drosophila species. Site-directed mutagenesis identifies four motifs necessary for midline glial expression: (1) a Single-minded/Tango binding site, (2) a motif resembling a Pointed binding site, (3) a motif resembling a Sox binding site, and (4) a novel motif. An additional highly conserved 27 bp are required to restrict expression to midline glia and exclude it from midline neurons. These results suggest short, highly conserved genomic sequences flanking Drosophila midline genes are indicative of functional regulatory regions and that small changes within these sequences can alter the expression pattern of a gene (Estes, 2008).

To facilitate the identification of sequences responsible for wrapper expression in the midline glia of Drosophila, the genomic region flanking the wrapper transcription unit was examined to determine the degree of conservation between the 12 available Drosophila species. The regions most likely to contain regulatory control elements (motifs) of wrapper are tractable; the genomic regions flanking the transcription unit and the first intron are relatively small. The results of this analysis highlighted a region between -492 and -326 upstream of the transcription start site of wrapper that is highly conserved in all Drosophila 12 species examined, particularly a 70-bp region. To test if these sequences are responsible for the wrapper expression pattern in embryos, this genomic region was amplified within a 884-bp fragment, and then fused it to the green fluorescent protein (GFP) reporter gene within the pHstinger vector, which contains a minimal Hsp70 promoter. This DNA construct (wrapper W:GFP) was injected into D. melanogaster embryos using P element-mediated transformation to generate stable fly lines. Embryos containing this construct express GFP in midline glia beginning at stage 12 of embryogenesis and throughout larval stages. It was confirmed that GFP was expressed in midline glia by staining embryos simultaneously with either (1) wrapper and GFP or (2) sim and GFP. Because wrapper protein is found at the surface of midline glial cells, but the GFP produced by pHstinger localizes to the nucleus, wrapper protein encircles the GFP in these cells. The wrapper W:GFP reporter construct also drives expression in a few additional cells within the lateral CNS and muscles, a pattern that differs from the endogenous wrapper expression pattern. This suggests that the W fragment, although sufficient to drive high levels of expression in midline glia, lacks certain sequences that exclude expression in lateral CNS cells. To confirm the midline expression pattern generated by the reporters, all subsequent experiments were performed by staining embryos with both sim and GFP at stage 16 of embryogenesis. These experiments revealed that GFP generated by the wrapper W:GFP reporter gene was indeed expressed in the midline glia, but not in the cells that develop into midline neurons (Estes, 2008).

Next, to determine the minimal sequences required to provide expression in midline glia, this 884-bp region was divided into several subregions, fused to GFP within the pHstinger vector and tested for the ability to drive midline expression in transgenic embryos. Region E, extending from sequences -756 to -286, is sufficient to drive high levels of GFP expression in midline glia. Moreover, a smaller 166-bp (-492 to -327) G fragment, and an even smaller 119-bp (-492 to -374) internal K fragment, that both include the highly conserved region, are also sufficient to drive GFP expression in midline glia, but the level of expression is reduced compared to that of the E fragment and the intact 884-bp W fragment. None of the other reporter constructs drove GFP expression in the midline. The K fragment is also expressed in a subset of midline neurons, including progeny of the median neuroblast, suggesting that the larger W, E, and G fragments contain a silencer, which is absent from the K fragment and normally represses expression in these midline neurons (Estes, 2008).

Next, to determine if the observed conservation at the sequence level between Drosophila species reflects conservation in function, the corresponding E region from D. virilis was tested to see if it could drive GFP reporter expression in the midline glia of D. melanogaster. The E region is also located upstream of wrapper in D. virilis and is 476 bp in length, while it is 462 bp in melanogaster. The entire E region is 58.4% identical in the two species, and the 70-bp highly conserved section differs by only six nucleotides. The midline expression pattern provided by the D. virilis wrapper E:GFP construct in D. melanogaster flies is indistinguishable from that of the corresponding D. melanogaster E region. These results suggest that the location and function of the regulatory sequences of wrapper have been conserved between D. melanogaster and D. virilis (Estes, 2008).

To determine if previously identified midline transcription factors affect wrapper through these regulatory sequences, the wrapper W:GFP reporter gene was tested in a number of mutant backgrounds. First, the effect of sim mutations on the reporter gene was tested by placing the 884-bp wrapper W:GFP transgene into a simH9 mutant background, a mutation that eliminates Sim protein expression. In this background, GFP expression was abolished in most cells, suggesting that sim expression is required for wrapper transcriptional activation in the midline. A few remaining cells did express GFP and these are likely lateral CNS cells also observed in wild-type embryos containing the wrapper W:GFP reporter (Estes, 2008).

Next, the reporter gene was tested in a spitz (spi) mutant background. Spi is a signaling molecule that plays multiple roles during Drosophila development. Wrapper protein is normally found on the surface of midline glia where it mediates direct contact with the lateral CNS axons that cross the midline and promotes survival of midline glia. In wrapper mutant embryos, this intimate interaction cannot occur and additional midline glia die. The amount of spi signaling provided by lateral CNS axons determines how many midline glia survive in each segment. The spi mutation severely disrupted CNS development so that the sim positive cells remained on the ventral surface of the embryo. Only a few of the sim positive cells also express GFP driven by wrapper regulatory sequences, suggesting these are the remaining midline glia. The cells expressing sim, but not GFP, are likely midline neurons, while cells expressing GFP and not sim are lateral glia, because they also express reversed polarity (repo), a marker of lateral CNS glia. These results indicate spi mutations reduce the number of midline glia in the embryo and also reduce expression of the wrapper W:GFP reporter gene (Estes, 2008).

In addition to sim and tgo, the transcription factors Dichaete (D), a Sox HMG protein, and Dfr, a POU domain protein, regulate genes expressed in midline glia. The D protein directly interacts with the PAS domain of Sim and the POU domain of Dfr and all three genes activate expression of slit in midline glial . The wrapper W:GFP construct was tested in both a D and dfr mutant background. In both cases, the number and behavior of midline cells was altered and they did not migrate to the dorsal region of the ventral nerve cord, as they normally do. While development of midline cells was disrupted in these mutant backgrounds and fewer midline glia were present, robust GFP expression was still observed from the reporter construct in the midline cells that remained, suggesting that (1) D and Dfr do not directly activate wrapper via these regulatory sequences, (2) additional, redundant factors exist that can substitute for them, or (3) they can substitute for one another (Estes, 2008).

In summary, midline cell development was disrupted in sim, spi, D, and dfr mutant backgrounds. The simH9 mutation eliminated midline glia and neurons, while a mutation in spi eliminated most midline glia. As predicted, both sim and spi mutations severely reduced the number of cells expressing GFP driven by the wrapper W:GFP reporter gene. In the D and dfr mutants, the number of midline glia was reduced and the remaining midline glia expressed high levels of GFP (Estes, 2008).

Ectopic sim expression converts neuroectodermal cells into midline cells and activates downstream, midline genes. To test the effect of ectopic sim on wrapper expression, sim was overexpressed using the UAS/GAL4 system and it was found that wrapper was expressed in neuroectodermal cells outside of the midline, but not in all cells that overexpress sim. In the UAS-sim/da-GAL4 embryos, wrapper is activated in cells that correspond to the lateral edges of the CNS and the cells in the anterior of each segment, with gaps in the expression pattern. Next, whether overexpression of the secreted form of spi could expand wrapper to cells outside the midline was tested. Ectopic expression of secreted spi with the da-GAL4 driver also expanded wrapper expression. To determine if it is possible to expand the expression domain of wrapper further, sim was overexpressed together with spi. This caused additional expansion of the wrapper domain into broad stripes within ectodermal cells. In addition, overexpression of either sim or spi causes severe disruption in embryonic development (Estes, 2008).

Next, the ability of sim and spi, either alone or together, to expand expression of the wrapper reporter genes was tested. Expression from both the full-length reporter construct, wrapper W:GFP, and the smaller wrapper G:GFP construct expanded in the UAS-sim/da-GAL4 embryos to a greater extent than the endogenous wrapper gene. The expression pattern provided by the reporter constructs differs from the endogenous wrapper expression pattern, suggesting that either (1) some of the sequences that normally repress wrapper in tissues outside the midline glia may be missing in these wrapper W and G constructs, or (2) ectodermal cells overexpressing sim may undergo cell death and the GFP marker may be more stable in these dying cells compared to wrapper. Overexpression of spi alone also expanded reporter gene expression driven by both the wrapper W:GFP and wrapper G:GFP constructs. The GFP expression domain was expanded to a greater extent in embryos overexpressing sim together with spi compared to those overexpressing either gene alone. Taken together, the results indicate that (1) limiting the wrapper regulatory sequences and (2) increasing the cells that express sim and spi converts the highly specific expression pattern of wrapper from a single strip of CNS cells to a more general pattern throughout the ectoderm of the embryo. In addition, these results suggest that both the sim transcription factor and spi signaling molecule can activate transcription through these sequences derived from the regulatory region of wrapper (Estes, 2008).

To both (1) identify functionally important motifs needed for wrapper expression and (2) determine if all the invariant nucleotides within the conserved 70-bp region of wrapper are essential for the observed midline glial expression pattern, effects of select mutations within the wrapper G region were tested. Previous studies have demonstrated the importance of sim/tgo, D, dfr, and spi for the expression of midline glial genes and, therefore, possible binding sites for these factors were sought. To examine both predicted binding sites, as well as other conserved sequences that may contain binding sites for novel factors, the region was divided into eight motifs that were tested for their effect on midline glia expression (Estes, 2008).

Each of these conserved motifs was tested by changing 2-3 nucleotides in the context of the D. melanogaster G fragment. The altered G fragments were then inserted independently into the pHstinger vector and injected into fly embryos to test their ability to drive midline expression (Estes, 2008).

Despite the high degree of conservation within this region, only four of the eight mutations that were tested (G1, G2, G5, and G7) caused a noticeable reduction in reporter expression. Two of the mutation sets destroyed midline expression of the G reporter construct. The putative Sim/Tgo binding site (G2: CACGT) was needed for midline expression, because changing this sequence to GAAGT eliminated midline glial expression. In addition, another sequence, ATTTTATC (G5), located upstream of the G2, was required for expression of the reporter gene in wild-type embryos and changing this sequence to ATTGGATC eliminated midline glial expression. Two additional sites within the G fragment of wrapper are needed for midline expression: CGGAGAG (G7) and CACAAT (G1). If either of these motifs is altered, midline glial expression is greatly reduced, but not completely eliminated (Estes, 2008). In contrast, the other four sets of mutations had no detectable negative effect on midline glial expression of the reporter gene, even though these sequences are conserved in all 12 Drosophila species. Mutation sets G4, and G8 did cause a low level of reporter gene activation in some midline neurons, suggesting that repressor proteins present in midline neurons may interact with these regions of the wrapper regulatory region. Finally, mutation G3 had no detectable positive or negative effect on expression of the reporter gene, despite being conserved in all 12 Drosophila species. In summary, the various mutations had three different effects on expression driven by the wrapper regulatory sequences: (1) some reduced midline glial expression, (2) some caused the inappropriate activation of the wrapper reporter in midline neurons, and (3) one was conserved, but apparently had no effect on wrapper regulation, in the context of the experiments presented here (Estes, 2008).

Therefore, these experiments suggest that Sim/Tgo heterodimers may directly regulate wrapper gene expression. (1) Activity of the wrapper W:GFP reporter gene is severely reduced in a sim mutant background, suggesting sim is necessary for expression of this transgene and that sim regulates wrapper by activating transcription through these sequences. (2) Midline activity of the wrapper reporter gene is abolished by eliminating the single CME (CACGT) present within this region. (3) wrapper reporter gene expression is expanded in sim overexpression embryos. Future biochemical studies will determine if Sim/Tgo heterodimers directly interact with the wrapper regulatory motif identified in this study (Estes, 2008).

The studies described in this study demonstrate that the wrapper reporter genes are sensitive to levels of spi signaling. Mutations in spi reduce wrapper reporter gene expression and overexpression of the secreted form of Spi, together with Sim expands, not only the expression domain of the endogenous wrapper gene, but the wrapper reporter genes as well. Spi binds the Epidermal Growth Factor Receptor in midline glia, leading to MAPK activation and subsequent activation of the ETS transcription factor, pnt. Therefore, it may be Pnt that directly activates wrapper transcription through the regulatory sequences studied in this study. One of the identified motifs needed for transcriptional activity of wrapper is: CGGAGAG, which loosely conforms to the consensus binding site for ETS transcription factors (C/A)GGA(A/T)(A/G)(C/T). However, further experiments are needed to determine if Pnt directly interacts with these regulatory sequences, as well as the precise mechanism whereby spi signaling regulates wrapper. Taken together with previous studies, these results suggest that the spi signaling pathway may play at least two roles in promoting survival of midline glia: (1) activating wrapper, needed for neuron-glial interactions and (2) phosphorylating, thereby inactivating Head involution defective, which would otherwise cause programmed cell death in midline glia (Estes, 2008).

Many genes expressed in the CNS of metazoan organisms are regulated through synergistic interactions between Sox HMG-containing proteins and POU domain proteins. Recently, many vertebrate genes expressed in the developing CNS have been shown to contain highly conserved noncoding DNA regions enriched for binding sites for three classes of transcription factors: Sox, POU, and homeodomain proteins. Experiments indicated that Sox and POU proteins work together to activate, while homeodomain proteins repress and limit expression of CNS genes. Interestingly, several motifs identified in this study as important for regulation in midline glia of Drosophila resemble binding sites for Sox (G1: CACAAT), POU (G4: ATGCAAAT, G6: ATGCAACA, and G8: ATGCGTGG), and homeodomain proteins (G5: ATTTTATC) (Estes, 2008).

That the wrapper K:GFP, but not the wrapper G:GFP construct is expressed in certain midline neurons, identifies a midline neural silencer in the 43-bp region present in the G fragment, but absent in the K fragment. Within this region, 27 bp are highly conserved in all 12 Drosophila species and two of the three mutations in the G fragment that cause slight activation of reporter gene expression in midline neurons are found within the 43-bp region. All three sites that lead to activation in midline neurons, G4, G6, and G8, conform to a POU domain binding site, suggesting a POU domain protein expressed in midline neurons may bind to one or more of these sites to keep the wrapper gene silent (Estes, 2008).

One POU domain protein, Dfr, binds to the sequence ATGCAAAT in other gene regulatory regions to activate transcription, including those of two genes expressed in midline glia: dfr itself and slit. This sequence is found at site G8 in the wrapper regulatory region, but when changed to ATGCTAGC, caused a low level of activation in midline neurons, rather than reducing expression in midline glia. Although the number of midline glia is reduced in a dfr mutant background, those that remain express a high level of reporter gene expression driven by wrapper sequences and the results suggest dfr is not absolutely required for wrapper reporter gene expression in midline glia (Estes, 2008).

Mutations in the POU domain motifs within the wrapper regulatory sequences suggest a notable difference between the CNS genes studied previously in vertebrates and the midline glial gene studied here. The POU domain binding sites appear to limit expression in midline neurons (rather than activate expression as in vertebrate CNS genes), and it is the Sox and homeodomain binding sites that are needed for activation. This may reflect a key difference in regulatory control of glial vs. neural genes and it is plausible that other midline glial genes excluded from midline neurons will contain silencer elements similar to the one identified in this study, but further experiments are needed to confirm this (Estes, 2008).

Spitz and Mesoderm Development

Continued: Spitz Effects of Mutation part 2/2

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

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