Effects of Mutation or Deletion

Table of contents

Rhomboid and the ventral midline

Mutations in rhomboid and single-minded appear to affect separate adjacent domains of cuticle. rhomboid mutants all show CNS defects localized near the ventral midline. Frequent breaks in major tracheal trunks and missing chordotonal neurons result. The late ectodermal expression correlates with deficiencies in cuticular structure. Muscle defects may reflect expression at segmental boundaries (Bier, 1990).

A good example of the function of EGF-R in regulating cell development is found by examining the role of EGF-R in midline glia maturation. The midline glial cells are required for correct formation of the axonal pattern in the embryonic ventral nerve cord. Initially, six midline cells form an equivalence group with the capacity to develop as glial cells. By the end of embryonic development three to four cells are singled out as midline glial cells. Midline glia development occurs in two steps, both of which depend on activation of the EGF-Receptor and subsequent Ras1/Raf-mediated signal transduction (See Drosophila Ras1). In the first step six midline cells in each segment, originating from the anterior-most three of a total of eight midline progenitor cells, are determined as the midline glia equivalence group. The process of generation of the midline glia equivalence group involves Notch function and segmentation genes. It might also depend on the function of single minded, the master regulatory gene of midline development. The single minded transcript accumulates in the midline glia and, depending on the context, can act either as a transcriptional activator or repressor. By the end of embryogenesis the final number of midline glial cells is about 3 to 4. Thus, the final number of cells has to be selected from the initially defined equivalence group in a second step (Scholz, 1997).

Egf-r mutants show a reduced number of midline glial cells and argos mutants, which possibly exhibit an increased activation of Egf-r in the midline, show an increased number of midline glial cells. Furthermore, expression of activated ras1 (or activated raf) in the midline results in the appearance of extra midline cells. This model suggests that activation of ras1 signaling in the entire midline glial equivalence group promotes survival of all cells in this cluster. Thus, in wild-type flies, about 2-3 cells in each group down-regulate Egf-r signaling and are destined for cell death. Both Rhomboid and Argos control activation of the EGF-R during midline glia development. It is thought that a graded activation of EGF-R is brought about by the activity of Rhomboid, which is thought to promote EGF receptor signaling, possibly by cell autonomous activation of the EGF-R ligand Spitz. Ectopic rhomboid leads to extra midline glial cells. EGF-R activates PointedP2 through phosphorylation; Pointed in turn activates the transcription of argos. Argos negatively regulates EGF-R signaling non-cell autonomously and competes with Spitz function. pointed mutants form extra glial cells. Yan antagonizes PointedP2A in midline glial cells, just as it does in the developing photoreceptor cells (Scholz, 1997).

Early midline glia survival and commissure formation requires the function of rhomboid and Star. In fused commissure mutants (rhomboid and Star) neuron number is reduced in the ventral unpaired median neuron lineage and the median neuroblast lineage before commissure formation (stage 12). Subsequent to these neuronal defects, the midline glia dies by apoptosis (stage 13). Commissure fusion and glial apoptosis may be triggered by the earlier perturbations in mesectodermal cell neuronal lineages (Sonnenfeld, 1994).

Cell death was examined within lineages in the midline of Drosophila embryos. Approximately 50% of cells within the anterior, middle and posterior midline glial (MGA, MGM and MGP) lineages die by apoptosis after separation of the commissural axon tracts. The initial number of MG within individual ventral nerve cord segments is increased by ectopic expression of the rhomboid gene, without changing axon number. Extra MGA and MGM are eliminated from the ventral nerve cord by apoptosis to restore wild-type numbers of midline glia. Ectopic rhomboid expression also shifts MGA and MGM cell death to an earlier stage of embryogenesis. One possible explanation is that axon-glia contact or communication promotes survival of the MG and that MG death may result from a competition for available axon contact (Sonnenfeld, 1995).

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 segmented portion of the Drosophila embryonic central nervous system develops from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after gastrulation in two sequential waves of neuroblast segregation and are arranged in three dorsoventral columns and four anteroposterior rows per hemisegment. Each neuroblast acquires a unique identity, based on gene expression and the unique and nearly invariant cell lineage that this expression produces. Little is known as to the control of neuroblast identity along the DV axis. The Drosophila Egfr receptor (Egfr) has been shown to promote the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Molecular markers identify particular neuroectodermal domains, composed of neuroblast clusters or single neuroblasts, and show that in Egfr mutant embryos (1) intermediate column neuroblasts do not form; (2) medial column neuroblasts often acquire identities inappropriate for their position, while (3) lateral neuroblasts develop normally. Active Egfr signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. The concomitant loss of rhomboid and vein yields CNS phenotypes indistinguishable from Egfr mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that Egfr plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS (Skeath, 1998).

spitz and vein are expressed in the early embryo and appear to function in independent pathways to activate Egfr during embyrogenesis. Thus, it is possible that either Spitz or Vein or both activate Egfr to promote neuroblast formation and specification. rhomboid and Star encode for transmembrane factors that appear to promote the production of secreted Spitz (s-Spitz) and to act in the same linear pathway as spitz. To investigate the extent to which spitz, rhomboid and Star, as well as vein participate in Egfr-mediated control of early CNS development, neuroblast formation and specification was assayed in embryos singly mutant for each gene. Early CNS development is essentially normal in embryos singly mutant for spitz, Star or vein. ac expression is restricted correctly to the medial and lateral columns and medial neuroblast specification appears normal. Embryos that lack rhomboid function exhibit more severe, yet still relatively mild, CNS defects. To determine the CNS phenotype of removing both spitz group activity and vein, a fly stock doubly mutant for rhomboid and vein was constructed. Removal of rhomboid and vein produces CNS defects virtually indistinguishable from those observed in Egfr mutant embryos: ac expression expands completely into the intermediate column in rows 3 and 7 and only two neuroblast columns form; the RP2 motoneuron almost never forms and roughly half of MP2s are mis-specified. These data suggest that the activity of the spitz group and vein are sufficient to account for all signals that activate Egfr during early CNS development (Skeath, 1998).

Neurogenesis in Drosophila melanogaster starts by an ordered appearance of neuroblasts arranged in three columns (medial, intermediate and lateral) in each side (right and left) of the neuroectoderm. In the intermediate column, the receptor tyrosine kinase Egfr represses expression of proneural genes achaete and scute, and is required for the formation of neuroblasts. Most of the early function of Egfr is likely to be mediated by the Ras-MAP kinase signaling pathway, which is activated in the intermediate column, since a loss of a component of this pathway leads to a phenotype identical to that of Egfr mutants. MAP-kinase activation is also observed in the medial column where escargot (esg) and proneural gene expression are unaffected by Egfr. The homeobox gene ventral nerve system defective (vnd) is required for the expression of esg and scute in the medial column. vnd acts through the negative regulatory region of the esg enhancer that mediates the Egfr signal, suggesting vnd's role is to counteract Egfr-dependent repression. Thus, the nested expression of vnd and the Egfr activator Rhomboid is crucial to subdivide the neuroectoderm into the three dorsoventral domains (Yagi, 1998).

To investigate the involvement of Egfr in neurogenesis, mutant phenotypes of Efgr and its activator rho were examined at various stages of neurogenesis. The dorsoventral subdivision of the neuroectoderm in stage-6 embryos is detectable by expression of esg, which is expressed in the lateral and medial columns but not in the intermediate column. A loss-of-function, temperature-sensitive mutation of Egfr and a null mutation of rho were used for analysis throughout this work. Egfr and rho mutations cause ectopic expression of esg in the intermediate column. Repression of esg in the intermediate column is likely to require a relatively high dose of Egfr signal. To examine the potential role of Egfr in neurogenesis, expression of the proneural genes ac and sc was carried out. These two proneural genes begin expression in the neuroectoderm of stage-7 embryos in a DV pattern of expression similar to that of esg in the previous stage. In Egfr and rho mutant embryos, ac and sc become ectopically expressed in the intermediate column. This phenotype is less penetrant and, occasionally, gaps of ac and sc expression are observed in the intermediate column. Since sc expression was similarly derepressed in Egfr mutant embryos, these phenotypes are likely to represent the near null phenotype of Egfr in the neuroectoderm. These data indicate that, in the intermediate column, the Egfr signal represses not only esg but also proneural genes, which are known to play key roles in neurogenesis. The effect of Egfr on neuroblast formation was monitored by the neuroblast marker Snail. Anti-Sna staining reveals three columns of SI neuroblasts in the control embryo: the intermediate column is distinguishable by the delayed onset of formation and number of Sna-positive cells. In Egfr and rho mutants, Sna-positive neuroblasts in the intermediate position are frequently missing, with a higher frequency of loss in Egfr embryos. In rho mutant embryos, the frequency of the loss of intermediate column neuroblasts is variable among embryos (Yagi, 1998).

To further examine the effect of the loss of Egfr signaling on the late events of neurogenesis, the progeny was traced for one of the intermediate neuroblasts, NB4-2. NB4-2 gives rise to the RP2 motor neurons, which can be identified by the expression of Even-skipped (Eve) and its unique position. Loss of RP2 neurons in stage 13 is observed (over half the cases examined) with the frequency of loss slightly higher in Egfr than in rho mutants, reflecting the earlier defect in neuroblast formation in stage 9. It is known that the Ras-MAPK signaling cascade is the major target of Egfr in many tissues. To understand whether Ras-MAPK signaling also mediates the Egfr signal in the neuroectoderm and to determine the relative contribution of each component of the pathway, the expression of esg and sc was examined in embryos lacking one of the Ras-MAPK signaling components. The phenotype of mutants lacking either Sos, Ras1, Draf or Dsor1 was examined. As in wild-type embryos, embryos mutant for any of the four genes examined express esg in three separate domains: procephalic neurogenic region, amnioserosa and neuroectoderm. In all cases, the anterior limit of the procephalic expression and the posterior limit of neuroectodermal expression are expanded to the terminus, consistent with the fact that Ras-MAPK is required for the terminal fate specification controlled by Torso receptor tyrosine kinase. All mutants exhibit specific defects within the neuroectoderm where esg expression is derepressed in the intermediate column. Essentially the same phenotype is also observed with sc expression, suggesting the loss of Ras-MAPK signaling has the same consequence as the loss of Egfr. All four Ras pathway mutants show, qualitatively, the same phenotype in the neuroectoderm. The neuroectoderm phenotype in Ras1 mutants is not rescued by a paternal copy of the wild-type gene, suggesting that a relatively high dose of the Ras signal is required for repression of esg and sc in the neuroectoderm (Yagi, 1998).

Rhomboid (rho) is initially expressed in the medial half of neuroectoderm, but repression of esg, ac and sc transcription by Egfr and Ras-MAPK occurs only in the intermediate column, posing a question as to whether or not the site of MAPK activation and the site of transcriptional repression exactly correspond. The spatial and temporal pattern of MAPK activation has been described by the use of an antibody that specifically reacts with the phosphorylated and activated form of MAPK (diphospho-MAPK=dpMAPK), which shows that dpMAPK is distributed in a broad domain in the neuroectoderm in stage 5-7 embryos. dpMAPK is distributed in an 8- to 10- cell-wide area in the neuroectoderm in stage-5 embryos and becomes restricted to the ventral region at the end of gastrulation. This rapidly evolving pattern of dpMAPK expression made it difficult to determine the exact correlation between distribution of dpMAPK and the DV subdomains in the neuroectoderm. A protocol was used to double label embryos with dpMAPK and antisense RNA probes to study the spatiotemporal relationship between expression of dpMAPK, its activator Rhomboid (Rho) and its downstream target, esg. Initial expression of dpMAPK overlaps with that of Rho in stage-5 embryos; dpMAPK expression remains in this broad domain when Rho expression became restricted to the medial column at gastrulation in stage 6, and finally narrows down to a 2- to 3-cell-wide stripe abutting the stripe of Rho at stage 7. Comparison with the mesodermal marker sna shows that the ventral border of dpMAPK expression abuts the neuroectoderm-mesoderm border. Examination of histochemically stained material reveals a sharp ventral border of dpMAPK expression, which gradually declines in the dorsal direction, resembling the pattern of Rho expression. In Egfr mutant embryos, dpMAPK staining is not detectable. These results demonstrate that MAPK activation in the neuroectoderm is dependent on Egfr and follows the spatial expression pattern of Rho, but persists for some time after termination of Rho transcription. The latter observation may reflect perdurance of Rho or its target protein, Spitz (Spi). Alternatively, a ligand other than Spi, such as Vein, might be activating Egfr. The dorsal limit of dpMAPK expression was determined relative to the three separate columns of neuroectoderm revealed by esg expression. In stage 5, the dorsal limit of dpMAPK reaches halfway within the intermediate column and subsequently retracts to the medial column in stage 6 and 7. These data indicate MAPK is activated at least in the ventral half of the intermediate column of the neuroectoderm when it is required to repress transcription of esg. It is concluded that transcription of esg is repressed by a marginal level of MAPK activation (Yagi, 1998).

Given the results of the present work showing that vnd counteracts the negative regulatory effect of Egfr, a model is proposed for the DV structuring of the neuroectoderm. A gradient of nuclear localized Dorsal protein induces expression of dorsoventrally regulated genes such as dpp, sna, and twi, which determine the extent of the neuroectoderm, and the nested expression domains of rhomboid and vnd. rho determines the domain of MAPK activation, which covers the medial and intermediate columns. vnd is expressed in the medial column where it counteracts the Egfr signal to allow expression of esg. Thus the three columns in the stage 5-6 neuroectoderm are distinguished by unique combinations of activated MAPK and vnd expression. In the lateral column, neither of them are activated or expressed, and esg transcription is activated by default. In the intermediate column, MAPK is activated and represses esg transcription. In the medial column, vnd counteracts activated MAPK to allow the default pathway to activate esg transcription. It is possible that proneural genes are also regulated by the same mechanism. Loss of the Egfr signal leaves two domains, one with and the other without expression of vnd, the pattern likely to be reflected in the appearance of only two neuroblast columns in the later stage. Thus it is proposed that the primary role of Egfr signal in this stage is to define the intermediate domain to the neuroectoderm which is otherwise separated into two domains. It is possible that Egfr signal and vnd have later roles in promoting neuroblast formation in the intermediate and medial columns, respectively (Yagi, 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).

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose interacts with Egfr and N signaling pathways (Shamloula, 2002).

Star, rhomboid, and spitz belong to the 'spitz' group of genes and encode an essential function necessary for ventral midline development. In addition to the recessive lethal embryonic phenotype, S mutations are haplo-insufficient and show a dominant, rough eye phenotype. During development, S is required in a wide variety of tissues and S mutations show genetic interactions with genes from multiple signaling pathways. S encodes a putative membrane protein that, in combination with Rhomboid (rho), participates in the processing of the EGFR ligand, Spitz. In the modifier screen an S deficiency was identified as a strong enhancer of the rugose rough phenotype. A number of S alleles have been tested for their interactions with multiple alleles of rugose. Mutations at the S locus act as strong enhancers of the rugose eye phenotype and conversely heat-shock promoter-driven overexpression of the wild-type Star protein acts as a dominant suppressor of the rugose mutant phenotype. Consistent with these results, rhomboid (rho) mutations act as dominant enhancers of the rough eye phenotype of rugose mutants. rho encodes a novel intramembrane serine protease and is involved in the proteolytic processing of the EGFR ligand Spitz. A single copy of the rho mutation acts as an enhancer of the rg eye phenotype and a single copy of the hs-rho acts as a weak suppressor (Shamloula, 2002).

Rhomboid and recruitment of wing and leg precursors

Wing and leg precursors of Drosophila are recruited from a common pool of ectodermal cells expressing the homeobox gene Dll. Induction by Dpp promotes this cell fate decision toward the wing and proximal leg. The receptor tyrosine kinase Egfr antagonizes the wing-promoting function of Dpp and allows recruitment of leg precursor cells from uncommitted ectodermal cells. By monitoring the spatial distribution of cells responding to Dpp and Egfr, it has been shown that nuclear transduction of the two signals peaks at different positions along the dorsoventral axis when the fates of wing and leg discs are specified and that the balance of the two signals assessed within the nucleus determines the number of cells recruited to the wing. Differential activation of the two signals and the cross talk between them critically affect this cell fate choice (Kubota, 2000).

In a screen for genes expressed in the embryonic limb primordia, rhomboid was found to be transiently expressed in the central part of Dll-expressing limb primordia in stage 11 embryos. rho transcription is the rate-limiting step of the activation of an EGFR ligand Spitz. As expected from the role of rho as a stimulator of Egfr, a transient expression of an activated, phosphorylated form of MAPK (dpMAPK) is detected in the nucleus of limb primordial cells surrounding the rho-expressing cells. The dpMAPK expression starts after the initiation of Dll transcription and diminishes before the separation of the wing and leg disc primordium. The dpMAPK expression is undetectable in null mutants of rho or Egfr. The peak of dpMAPK expression is located ventrally to the cells expressing dpp. The results suggest that rho-mediated stimulation of Egfr and MAPK occurs at the time of cell fate specification of wing and leg discs (Kubota, 2000).

To study the role of Egfr at the stage of wing and leg cell fate determination, specific marker gene expression was examined in Egfr signaling mutants. DLL mRNA is expressed in the entire limb primordium at stage 11 and becomes restricted to distal leg cells at stage 15. Esg protein expression was used to detect both wing and proximal leg cells. In rho mutants, the size of limb primordia at stage 11 is the same as the control, but the later development of leg discs is abnormal. The number of leg disc cells expressing Dll and/or Esg at stage 15 is reduced, and these cells no longer show the circular arrangement typical of leg disc precursors. Amorphic mutation of Egfr cause a ventral expansion of limb primordia as a result of a loss of the early function of Egfr, but the expression of leg markers is severely reduced at stage 15. A similar phenotype is observed in mutants lacking both maternal and paternal copies of Dsor1, which encodes a MAP kinase kinase. In all cases described above, Esg-expressing cells at the dorsal part of leg discs are most frequently lost, suggesting that the development of dorsoproximal leg cells is most sensitive to the loss of Egfr activity. In contrast, wing and leg disc development is normal in vein mutants, suggesting the putative ligand of Egfr encoded by this gene is dispensable. These results suggest that MAPK activation induced by Rho and Egfr is essential for normal leg development (Kubota, 2000).

In contrast to the severe defects in leg discs, none of the mutations in Egfr signaling interfer with wing disc formation. In these mutants, wing primordia consistently express Esg and another wing disc marker Vestigial, and invaginate to form discs. However, an increase in the number of wing disc cells has been noted in Egfr signaling mutants. This effect was analyzed in rho mutants; unlike Egfr mutants, in rho mutants the number of limb primordial cells at stage 11 is the same as the control. The number of Esg-expressing wing disc cells in rho mutants is increased compared to the control, while the number of the proximal leg disc cells is severely reduced. It is concluded that Egfr signaling is required to limit wing disc cell differentiation in limb primordial cells that are not yet fully committed. It is inferred that a subset of prospective leg cells that do not receive a sufficient amount of Egfr signaling fail to differentiate as proximal leg and instead adopt a wing fate (Kubota, 2000).

The increase in the number of wing disc cells in rho mutants resembles the overexpression phenotype of Dpp and raises a possibility that Egfr might prevent wing disc development by negatively regulating Dpp signaling. Such a cross talk could occur at several levels including the following: (1) regulation of dpp transcription, (2) signal transduction from Dpp receptors to the nucleus, and (3) transcriptional regulation of downstream target genes. The analyses excluded the first two possibilities for two reasons. (1) The expression pattern of DPP mRNA is unaffected by the mutation of rho. A previous report showing an expansion of dpp expression in Egfr mutants probably reflects the global patterning role of Egfr in the earlier stage. (2) pSSVS (activated Mad) expression around limb primordia does not change in rho mutants. Conversely, the expression pattern of dpMAPK is not changed by a null mutation of tkv. These results suggest that the differential distribution of cells responding to Dpp and Egfr is set up independently of each other's activity (Kubota, 2000).

The antagonism between Dpp and Egfr during wing disc development raises a question: what is the default state of the wing and leg primordia in the absence of the two signals? Double mutant phenotypes of Dpp and Egfr signaling were examined. tkv mutants lack wing discs and their leg discs are malformed. This phenotype reflects a disc cell autonomous requirement for Dpp signaling, because the phenotype is reproduced by the disc-specific inhibition of Dpp signaling by dad, which inhibits Mad. The phenotype of either tkv;rho or tkv;Egfr double mutants is a simple addition of each mutation, in which wing discs are lost completely and leg discs are severely reduced. Since Dll-expressing limb primordial cells are present in tkv;Egfr double mutants in stage 11, it has been concluded that these cells fail to differentiate as wing discs and their ability to differentiate as leg discs is also compromised. A few Esg-positive cells remain at the position of the leg, and it is speculated that this reflects the presence of a second leg-inducing signal. These results suggest that Dpp is absolutely required for wing disc development irrespective of the activity of Egfr (Kubota, 2000).

The finding that Egfr is activated in the limb primordium and prevents wing disc formation suggests that Egfr is a key factor in the diversification of the wing and leg fate. It is proposed that the differential activation of Dpp and Egfr, and the dorsal cell migration brings a subset of limb primordial cells out of the range of Egfr signaling, and thereby allows Dpp to induce wing development. It follows that dorsally migrating cells acquire the wing cell identity only after the separation from leg-promoting signals. Consistent with this idea, expression of wing-specific markers Vg and Sna, start only after the separation of the two primordia. Mechanisms that promote the dorsal cell migration remain to be identified. Given that the basic genetic components for the induction of the wing and leg have been identified in the model organism Drosophila, it can now be asked how the genetic mechanism of wing and leg specification has evolved by comparing the expression and function of these genes in limb primordial cells of primitive insects (Kubota, 2000).

Rhomboid and Wingless Degradation

Embryos have evolved various strategies to confine the action of secreted signals. Using an HRP-Wingless fusion protein to track the fate of endocytosed Wingless, it has been shown that degradation by targeting to lysosomes is one such strategy. Wingless protein is specifically degraded at the posterior of each stripe of wingless transcription, even under conditions of overexpression. If lysosomal degradation is compromised genetically or chemically, excess Wingless accumulates and ectopic signaling ensues. In the wild-type, Wingless degradation is slower at the anterior than at the posterior. This follows in part from the segmental activation of signaling by the Epidermal growth factor receptor, which accelerates Wingless degradation at the posterior, thus leading to asymmetrical Wingless signaling along the anterior-posterior axis (Dubois, 2001).

The spatial and temporal regulation of Wingless degradation implies the existence of one or several regulatory genes that are activated at the posterior and not at the anterior of each wingless stripe. The activity of hedgehog, as well as that of its downstream effector cubitus interruptus, are needed to prevent the spread of Wingless toward the posterior. In hedgehog (or cubitus interruptus), mutant embryos that overexpress wingless under the control of engrailed-gal4, many Wingless-containing vesicles are detected at the posterior and this correlates with increased Wingless signaling there. In light of the present results, it is presumed that a target of hedgehog is needed to accelerate Wingless degradation at the posterior. One candidate target gene is rhomboid, because it is only expressed at the posterior of each stripe of engrailed (and hedgehog) expression. Moreover, segmental expression of rhomboid commences around stage 11, roughly the stage when the second phase of Wingless degradation begins. By analogy with the experiments with hedgehog and cubitus interruptus, rhomboid mutants that overexpress Wingless under the control of engrailed-gal4 were examined and increased Wingless staining was found both within and at the posterior of each engrailed stripe. Thus, in the absence of rhomboid, Wingless degradation is impaired. This result implicates Egfr signaling, since rhomboid encodes a limiting factor needed for the activation of the Egfr ligand Spitz. A null mutation in Egfr leads to extensive morphological defects, thus making staging and analysis difficult. Nevertheless, in embryos that could be analyzed (of the genotype engrailed-gal4 UAS-Wingless EGFR-), excess Wingless is detected at the posterior of the expression domain. The role of Egfr could be mediated by a target gene of the MAP kinase pathway. Alternatively, or in addition, a nontranscriptional response to Egfr signaling could lead to Wingless degradation. The role of Pointed, a transcription factor that mediates many activities of the Egfr in Drosophila, was examined. In embryos of the genotype engrailed-gal4 UAS-Wingless pointed -, excess Wingless accumulates posterior to the source. Therefore, it appears that a transcriptional target of Egfr signaling is involved in modulating Wingless degradation (Dubois, 2001).

rhomboid mutants were used to assess the role of EGFR signaling when wild-type levels of Wingless are produced. This cannot be done by simply looking at rhomboid mutants because the first phase of Wingless clearance (following transcriptional repression of frizzled and frizzled2) already brings Wingless below detection level. Receptor expression was therefore artificially maintained in rhomboid mutants (engrailed-gal4 UAS-Frizzled2 rhomboid -). The general morphology of such embryos is again somewhat aberrant. Nevertheless, it could be clearly seen that Wingless-containing vesicles linger within the engrailed domain, even as late as stage 12, thus confirming the role of rhomboid in targeting Wingless to lysosomes. Although this is hard to prove formally, Wingless accumulation in rhomboid mutants appears to be in intracellular vesicles. In particular, the subcellular distribution of Wingless is clearly different from that seen in clathrin mutants or in embryos expressing Deltafrizzled2-GPI; two situations when Wingless accumulates at the cell surface. In conclusion, it is suggested that Rhomboid (and Egfr) regulate the transfer of Wingless from endosomes to degradative structures. However, it is unlikely to be the sole regulator since not all engrailed-expressing cells accumulate Wingless in the rhomboid mutant. Additional regulators might include a redundant homolog of rhomboid or a gene controlling a parallel degradation pathway (Dubois, 2001).

Rhomboid and the peripheral nervous system

The argos null mutation causes an increase in chordotonal (Ch) organs in both the thoracic and the abdominal segments, whereas overexpression of the argos gene results in a decrease in these organs. Argos transcripts are expressed transiently in the cells surrounding the Ch organ precursor, and rhomboid, which is involved in the regulation of the number of Ch organs, acts epistatically to argos in this event. These findings suggest that argos plays a role in Ch organ precursor formation and regulates the final number of Ch organs (Okabe, 1996).

The function of Atonal is best illustrated by its role in chordotonal organ development. A scolopidium, the basic unit of chordotonal organs, consists of four cells: a neuron with a single dendrite, the scolopale cell, cap cell and ligament cell. The scolopale cell (a glial cell) forms a sheath around the dendrite, while the cap cell and ligament cell mediate the attachment of the chordotonal organ to the body cell. Expression of atonal is restricted to a subset of atonal-requiring chordotonal precursors, called founder precursors (zur Lage, 1997).

EGF receptor signaling is required in neural recruitment during formation of Drosophila chordotonal sense organ clusters. A total of five neural precursors express atonal in abdominal segments, and this number is too few to explain the formation of the eight scolopidia in each abdominal segment. The remaining precursors require Egf-R signaling for their selection. Signaling by the founder precursors is initiated by atonal activating (directly or indirectly) rhomboid expression in the founder cells. It should be noted that in some developmental processes, rhomboid appears to function in the signal-receiving cells, such as in the patterning of ovarian follicle cells. It is not believed that this is the case in chordotonal-precursor formation, because rho is expressed in precursors that do not require rhomboid function (C1-C5 are formed even in rhomboid mutants). Signaling by these founder precursors, presumably through the EGF receptor ligand Spitz, then provokes a response in the surrounding ectodermal cells, as shown by the activation of expression of the Egf-R target genes pointed and argos. The signal and response then leads to recruitment of some of the ectodermal cells to the chordotonal precursor cell fate. Egf-R hyperactivation by misexpression of rhomboid results in excessive chordotonal precursor recruitment. Argos functions in a feedback mechanism to prevent the excess recruitment of additional ectodermal cells. The increase in the number of scolopidia caused by Egf-R hyperactivation is confined to an enlargement of existing cluster sizes: no new chordotonal clusters are formed. A two step mechanism is postulated for the formation of clusters of chordotonal precursors. In the first step, precursors C1-C5 are selected as founder precursors by the conventional route of proneural gene expression and lateral inhibition. In a distinct second phase, these precursors then signal to adjacent ectodermal cells via the Egf-r pathway, inducing some of them to become chordotonal precursors (secondary or recruited precursors). This two-step process is strongly reminiscent of the way atonal acts in neurogenesis in compound eyes. Here, atonal expression is initially refined by lateral inhibition, until atonal is expressed in only the founding R8 precursor, which then recruits R1-R7 in a mechanism that does not require the activation of atonal in these cells (zur Lage, 1997).

Tests were made of growth cone choices of Drosophila motoneurons in response to muscle fiber mismatch. In rhomboid mutants muscle fiber 7 does not develop. Despite the loss of one of its targets, RP3 faithfully innervated the remaining muscle fiber 6 in over 80% of the observed cases. Furthermore, neuron RP1 accurately innervated muscle fiber 13, although it traversed one less fiber to reach it. Laser ablation of muscle fiber 7 confirmed the target choices shown by the motoneurons. These results indicate that each motoneuron growth cone has a primary target preference, which is retained even when the numbers of the muscle fibers, and therefore their relative positions, are altered (Chiba, 1993).

Rhomboid and mesodermal development

The homeobox genes ladybird in Drosophila and their vertebrate counterparts Lbx1 genes display restricted expression patterns in a subset of muscle precursors, and both of them are implicated in diversification of muscle cell fates. In order to gain new insights into mechanisms controlling conserved aspects of cell fate specification, a gain-of-function (GOF) screen was performed for modifiers of the mesodermal expression of ladybird genes using a collection of EP element carrying Drosophila lines. Among the identified genes, several have been previously implicated in cell fate specification processes, thus validating the strategy of the screen. Observed GOF phenotypes have led to the identification of an important number of candidate genes, whose myogenic and/or cardiogenic functions remain to be investigated. Among them, the EP insertions close to rhomboid, yan and rac2 suggest new roles for these genes in diversification of muscle and/or heart cell lineages. The analysis of loss and GOF of rhomboid and yan reveals their new roles in specification of ladybird-expressing precursors of adult muscles (LaPs) and ladybird/tinman-positive pericardial cells. Observed phenotypes strongly suggest that rhomboid and yan act at the level of progenitor and founder cells and contribute to the diversification of mesodermal fates. Analysis of rac2 phenotypes clearly demonstrate that the altered mesodermal level of Rac2 can influence specification of a number of cardiac and muscular cell types, including those expressing ladybird. The finding that in rac2 mutants ladybird and even skipped-positive muscle founders are overproduced, indicates a new early function for this gene during segregation of muscle progenitors and/or specification of founder cells. Intriguingly, rhomboid, yan and rac2 act as conserved components of Receptor Tyrosine Kinase (RTK) signalling pathways, suggesting that RTK signalling constitutes a part of a conserved regulatory network governing diversification of muscle and heart cell types (Bidet, 2003).

The presented rho, yan and rac2 gain and loss-of-function phenotypes, clearly demonstrate that these genes play critical roles in the specification of lb-expressing mesodermal lineages. When over-expressed, the regulator of EGF-ligand maturation rho is able to induce specification of an increased number of lb-positive lateral adult muscle precursors (LaPs). Consistent with this observation, the GOF of a negative effector of RTKs signalling yan leads to the loss of LaPs. Interestingly, the large number of LaPs in rho GOF embryos suggests that during segregation of the LaPs progenitor, the Notch-mediated lateral inhibition is affected. Antagonistic activities of the EGFR and the Notch signalling pathways have been reported, thus indicating that the excess of EGFR signalling can overrule the lateral inhibition during specification of muscular progenitors. The highly restricted mesodermal expression of rho suggests, however, that in wild type embryos the rho-triggered EGF signals can interfere with lateral inhibition only in a subset of promuscular clusters. This indicates that other RTKs contribute to the negative interactions with Notch. Taking into consideration all the available information, it is speculated that the ectopically expressed rho induces the EGFR pathway that antagonizes Notch dependent lateral inhibition, specifically during segregation of the LaP progenitor. This results in promoting the LaP fate. Since in rho and yan mutants the segmental border muscle (SBM) is duplicated, it is proposed that during specification of SBM founder the repressive action of yan is relieved by a Rho/EGFR-independent RTK pathway (Bidet, 2003).

These data also demonstrate new roles for rho, yan and rac2 in the specification of cardiac lineages. Interestingly, mutations of rho and rac2 affect specification of pericardial cells with no major effects on cardioblast identity. yan loss and GOF leads to even more pronounced phenotypes suggesting that, in addition to EGFR, other RTKs are involved in diversification of cardiac fates. rho and Ras/MAPK pathway have been shown to influence specification of eve-expressing pericardial cells. In addition, this study shows that rho represses and yan promotes specification of lb-positive pericardial cells. Surprisingly, in rho mutants, the supernumerary lb-positive pericardial cells co-express eve, a situation never observed in wild type embryos because of mutual repressive activities of eve and lb. This suggests that cross-repression requires the co-ordinated action of identity gene products and effectors of RTK signalling pathway. The overproduction of tin/eve-positive pericardial cells observed in rho GOF and in rac2 loss of function mutants suggests that the diversification of this particular cell type involves a rac2-dependent trafficking of EGF receptor. A future challenge will be to unravel whether Drosophila rac2 indeed co-operates with cell fate specification machinery by controlling the intracellular processing of EGFR and others RTKs (Bidet, 2003).

Rhomboid belongs to a large family of intermembrane serine proteases regulating the EGF-like ligand maturation in different species from prokaryotes to Human. One of the mouse rho homologs, ventrhoid, exhibits a very dynamic expression in central nervous system and forming somites, suggesting it may regulate early cell fate specification genes in a manner similar to that in which rho regulates lb in Drosophila. Several yan-like genes have also been identified in vertebrates. Two human yan homologs, named tel1 and tel2 share similar mesodermal embryonic expression pattern restricted to hematopoietic lineages. In addition, in adult mouse, tel1 is expressed in the heart and in skeletal muscles. As in Drosophila, yan functions with its closely related partner pointed. It is important to note that the vertebrate pnt genes ets-1 and ets-2 are involved in early embryonic heart and muscle development. The numerous vertebrate homologs of the third candidate gene of this study, rac2, control a variety of cellular processes including actin polymerization, integrin complex formation, cell adhesion, membrane trafficking, cell cycle progression, and cell proliferation. The majority Rho-GTPases are ubiquitously expressed, including the developing muscular and cardiac tissues, but their myogenic functions have not yet been investigated. The vertebrate Rac2 gene is specifically required for hematopoiesis. Its mutation in mice leads to the defective neutrophil cellular functions reminiscent of human phagocyte immunodeficiency. The only described link between Rho-GTPases and muscle concerns the binding and activation of a Serine/Threonine protein kinase homologous to myotonic dystrophy kinase by a small GTP binding protein Rho. It is speculated, however, that given the involvement of RhoB in EGFR trafficking, the vertebrate Rho GTPase can contribute to RTK-controlled myogenic pathways (Bidet, 2003).

Altogether, these data suggest that the RTK signalling involving rho, yan and rac2 might play an important and at least partially conserved role in diversification of cardiac and muscular lineages (Bidet, 2003).

Table of contents

rhomboid: Biological Overview | Regulation | Protein Interactions | Developmental Biology | References

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