EGF receptor

Effects of Mutation or Deletion

Table of contents

Egfr and gastrulation

In mutants of snail or twist, transcription of folded gastrulation is normal in the posterior midgut primordium but almost completely eliminated on the ventral side. Maternal-effect ventralizing mutations that expand the expression of twist and snail also expand the domain of fog transcription. In embryos from torpedoQY mutant mothers, Twist protein expression extends farther laterally, but the ventral furrow is usually split into two narrow ventrolateral invaginations by an unknown patterning mechanism. In this case, fog is transcribed in two separate ventrolateral stripes (Costa, 1994).

Egfr and ectoderm

During stages 8-9 of embryonic development [Images], the Drosophila EGF receptor is essential for determining the identity of cells within the ventral ectoderm. In the absence of Egfr activity at this phase, alterations in cell fate are observed: Ventral cells acquire more dorsal fates, as visualized by the expression profile of specific markers. The ventralizing effect of Egfr appears to function later than that of the dorsalizing dpp pathway; the spatial overlap between them is minimal (Raz, 1993).

The ventral epidermis is derived from longitudinal rows of ectodermal precursor cells that divide and expand to form the ventral embryonic surface. The spitz class genes are required for the proper formation of the larval ventral cuticle. Using a group of enhancer trap lines that stain subsets of epidermal cells, it has been shown that spitz class gene function is necessary for ventral epidermal development and gene expression. Analysis of single-minded mutant embryos implies that ventral epidermal cell fate is influenced by the CNS midline cells (Kim, 1993).

Signaling by the epidermal growth factor receptor (EGFR) plays a critical role in the segmental patterning of the ventral larval cuticle in Drosophila: by expressing either a dominant-negative EGFR molecule or Spitz (an activating ligand of EGFR), it is shown that EGFR signaling specifies the anterior denticles in each segment of the larval abdomen. rhomboid, spitz and argos are expressed in denticle rows 2 and 3, just posterior to denticle row 1 in the engrailed expression "posterior" domain of larval ectoderm. These denticles derive from a segmental zone of embryonic cells in which EGFR signaling activity is maximal. Expression of a dominant negative form of Egfr (DN-DER) leaves just two of the six rows of denticles, corresponding to rows 5 and 6. Expression of ectopic Egfr throughout the ventral epidermis results in larval cuticles showing considerably widened ventral denticle belts, with only narrow, naked stretches between them. Within these belts, the normal rows 1-4 are still recognisable by the morphology and orientation of their denticles. However, posterior to these, row 5 and 6 denticles are not apparent; these are replaced by a wide field of small denticles, apperently of the row 1-4 type. A similar phenotype of excessive denticles is seen after ubiquitous expression of an activated form of Ras (Stutz, 1997).

Both Egfr and spitz are expressed fairly uniformly throughout the embryonic epidermis. However, Spi appears to be incapable of activating Egfr unless it is processed into a secreted form; there is genetic evidence that the membrane-spanning products of the rhomboid and Star genes may be mediating this process. In young embryos (before the germ band is fully extended) rhomboid is expressed in distinct segmental stripes. These stripes remain visible until stage 16 by which time they fade away. They become circumferential, and one cell wide in the dorsal half of the embryo. In the ventral half, they are also one cell wide in the thoracic segments, a bit wider in the first abdominal segment, and at least two cells wide in all other segments. These stripes correspond roughly to the cells giving rise to denticle rows 2 and 3. argos is observed in, and adjacent to, cells expressing rhomboid, and indeed is expressed in circumferential stripes after completion of germ-band retraction. The segmental stripes of argos are fairly similar to the rhomboid stripes but a bit wider. Argos is an inhibitor of Egfr function, so Argos reduction is expected to result in overactivation of Egfr signaling. argos loss of function mutants result in an entire additional row of denticles anterior to row 1 (Szuts, 1997).

There is a competition between the denticle fate specified by EGFR signaling and the naked cuticle fate specified by Wingless signaling. The final pattern of the denticle belts is the product of this antagonism between the two signaling pathways. In the absence of Wg signaling, extra denticles form, instead of naked cuticle, whereas, ectopic activation of Wg causes naked cuticle to form, instead of denticles. Expressing DN-DER (that is removing Egfr) in wingless mutants (producing 'double-mutant' conditions) produces denticle belts with almost exclusively large denticles. Mostly based on their size, it is believed that the large denticles are of the row 5 type. This cuticle phenotype demonstrates that Egfr activity is responsible for the row 1-4 denticles that are seen in wingless mutants. There may be very little Egfr signaling in wingless loss of function mutants; the segmental stripes of rhomboid expression are weaker, and those of argos completely disappear. Thus, the segmental activation of Egfr signaling seems to be an indirect consequence of an early function of wingless. Egfr signaling activity can produce denticles in the complete absence of Wg signaling. Expressing Spitz ectopically produces denticles of the row 1-4 type. Egfr signaling does not require armadillo function to specify these small denticles. (Szuts, 1997).

High EGFR signalling activity depend on bithorax complex gene function. In mutants lacking abdominal-A and Ultrabithorax, rhomboid expression is very weak. In these mutants, there is very little expression of Argos. These homeotic genes account for the main difference in shape between abdominal and thoracic denticle belts (Szuts, 1997).

An allele of the yan locus was isolated as an enhancer of the Ellipse mutation of the Egfr gene. This yan allele is an embryonic lethal and also fails to complement the lethality of anterior open (aop) mutations (anterior open causes a severe head defect with an open anterior region of the cuticle). Phenotypic and complementation analysis reveal that aop is allelic to yan. Genetically, the lethal alleles act as null mutations for the yan gene. Analysis of the lethal alleles in the embryo and in mitotic clones shows that loss of yan function causes cells to overproliferate in the dorsal neuroectoderm of the embryo and in the developing eye disc. The role of yan is defined by the developmental context of the cells in which it functions. yan allows a cell the crucial choice between cell division and differentiation (Rogge, 1995).

A key step in development is the establishment of cell type diversity across a cellular field. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for this process. At each parasegment boundary, cells expressing the Wnt family member Wingless confront cells expressing the homeoprotein Engrailed. The Engrailed-expressing cells normally differentiate as one of two alternative cell types. In investigating the generation of this cell type diversity among the 2-cell-wide Engrailed stripe, it has been shown that Wingless, expressed just anterior to the Engrailed cells, is essential for the specification of anterior Engrailed cell fate. In a screen for additional mutations affecting Engrailed cell fate, anterior open (aop) (also known as yan) was identified, a gene encoding an inhibitory ETS-domain transcription factor that is negatively regulated by the Ras-MAP kinase signaling cascade. Anterior open must be inactivated for posterior Engrailed cells to adopt their correct fate. This is achieved by the EGF receptor (Egfr), which is required autonomously in the Engrailed cells to trigger the Ras1-MAP kinase pathway. Localized activation of Egfr is accomplished by restricted processing of the activating ligand, Spitz. Processing is confined to the cell row posterior to the Engrailed domain by the restricted expression of Rhomboid. These cells also express the inhibitory ligand Argos, which attenuates the activation of Egfr in cell rows more distant from the ligand source. Thus, distinct signals flank each border of the Engrailed domain, since Wingless is produced anteriorly and Spitz posteriorly. Since En cells have the capacity to respond to either Wingless or Spitz, these cells must choose their fate depending on the relative level of activation of the two pathways (O'Keefe, 1997).

The larval cuticle comprises a repeated array of precisely patterned denticle belts interspersed with smooth cuticle. In abdominal segments, each of these belts is made up of 6 rows of denticles, where each row is of a characteristic size and orientation reflecting fate decisions made by the underlying cells. Using a lacZ reporter gene expressed in the En cells, it has been demonstrated that the anterior En cells normally produce smooth cuticle, while the posterior En cells produce denticles and, thereby, form the first row of each belt. Thus, cells in the En domain adopt either a smooth or denticle fate depending on their position. To identify genes involved in specifying En cell fates, existing collections of mutants were screened for those in which anterior En cells inappropriately produce denticles. Ectopic denticles are observed immediately anterior to the denticle belts in aop mutants. The extra denticles are located at the lateral edges of denticle belts, and are more commonly observed in the posterior segments. To determine whether the En cell fates were altered in these mutants, the En cells were visualized with a lacZ reporter construct. Anterior En cells produce denticles instead of the normal smooth cuticle. Thus, aop function is required for some anterior En cells to adopt the smooth cell fate (O'Keefe, 1997).

Since aop activity is required for anterior En cells to adopt the smooth cell fate, Aop activity was tested to see if it was sufficient to force posterior En cells to produce smooth cuticle instead of first row denticles. A constitutively active form of Aop was examined, where all eight MAP kinase consensus phosphorylation sites were mutated, and its expression was driven in the En cells using the UAS/GAL4 system. While En-GAL4 embryos carrying UAS-Aop WT exhibit normal denticle pattern, such embryos carrying UAS-Aop Act are missing the normal first denticle row of each belt. Thus, if posterior En cells express a form of Aop that can not be inhibited by MAP kinase, then these cells adopt the smooth fate. This suggests that, normally, Aop must be inactivated in the posterior En cells for them to adopt denticle fates. Given that the Ras1-MAP kinase cascade is responsible for inhibiting Aop function in other tissues, it became a good candidate for inactivating Aop in the posterior En cells. If this pathway is indeed involved, then inappropriate activation of the pathway should mimic the aop mutant phenotype and allow anterior En cells to incorrectly produce denticles. To test this, embryos expressing a constitutively active form of Ras (UAS-Ras1 val-12 ) in the En cells were examined. These embryos have an ectopic row of denticles anterior to the normal first row, corresponding to the location of the anterior En cells. Thus, the anterior En cells are mis-specified by ectopic Ras1-MAP kinase activity, similar to the effects of loss of aop function. This suggests that Ras1-MAP kinase activity may normally be responsible for inactivating Aop in the posterior En cells, allowing them to adopt the denticle fate (O'Keefe, 1997).

Since the Ras1-MAP kinase cascade is activated by receptor tyrosine kinases, a test was performed to see whether such a receptor could be involved in specifying En cell fate. For several reasons, the best candidate was the Drosophila EGF Receptor (Egfr). (1) In the eye, an allele of aop was isolated as an enhancer of mutations in Ellipse, a gain-of-function allele of Egfr. (2) Egfr is ubiquitously expressed epidermis throughout embryogenesis and is required early for ventral-to-lateral patterning, as is Aop. Finally, at later stages, Egfr is required for cells to adopt denticle fates (O'Keefe, 1997 and references).

To address whether Egfr function is required for posterior En cells to adopt their correct fate, a dominant negative form of Egfr was expressed specifically in En cells. These embryos lack the first denticle row, corresponding to the position of the posterior En cells. Therefore, Egfr is autonomously required for the posterior En cells to adopt a denticle fate. It was next determined whether there is a source of Egfr ligand positioned appropriately to signal to the En cells. The Spitz source is posteriorly adjacent to En cells. It seemed likely that Egfr would be activated by Spi, its ligand in many other contexts. Spi is ubiquitously expressed as an inactive membrane-bound molecule with homology to TGF-alpha. A processing event, which requires Rhomboid (Rho) activity, releases active ligand. Thus, the spatially regulated expression of Rho marks cells that are the source for active, secreted Spi. These cells can trigger activation of Egfr in adjacent cells. The expression of Rhomboid suggests that there is a novel source of active Spi ligand at the appropriate time and place to influence En cell fate (O'Keefe, 1997).

To test directly whether the Egfr pathway is activated in these transverse stripes, the spatial distribution of activated MAP kinase was examined, using an antibody that is specific to the di-phosphorylated (active) form of MAP kinase (dp-ERK). In late stage embryos (9.5 hours AEL), a stripe of activated MAP kinase is detected just posterior to the En cells. This stripe is dependent on Egfr, since it is selectively removed in flb mutant embryos. In wild type, active MAP kinase is detectable within the En cells themselves, although at low levels. Thus, it appears that Egfr activation indeed spreads into the En cells. It could not be determined whether there is a difference between the anterior and posterior En cells. Activation of the Egfr pathway was confirmed by testing for the induction of a Egfr target gene, argos, the expression of which is closely correlated with regions of maximal Egfr activation. For instance, during earlier ventral-to-lateral patterning, argos is expressed in the ventralmost 1- to 2-cell rows, the point of highest Egfr activation. However, at later stages Argos mRNA is expressed in a stripe of cells posterior to the En cells, coincident with the expression of Rho and the highest levels of activated MAP kinase. Taken together, these data demonstrate that a secreted Egfr ligand (probably secreted Spitz), produced by cells just posterior to En cells, activates Egfr. Furthermore, it appears that the activation of Egfr is graded; highest posterior to En cells and at lower levels within the En cells. This signaling corresponds to the time when fates of the En cells are being determined, which is consistent with a role for Egfr in determining the fates of En cells. Experiments were carried out that revealed that anterior En cells can, in fact respond to Spitz (O'Keefe, 1997).

Spitz and Wingless signaling have been shown to have competing affects on En cell fate. Anterior En cells assume a denticle fate when wg function is eliminated at 8 hours AEL. Wg is expressed just anterior to the En domain, in a region of smooth cuticle. Thus, while Wg input instructs cells to adopt the smooth fate, activation of Egfr instructs cells to adopt denticle fates. The opposite response of En cells to these two signals raises the question of what fate these cells would adopt in the absence of both signals. To determine this, Egfr signaling was blocked by expressing Aop Act in En cells while concomitantly removing wg function using a conditional allele. When wg ts embryos carrying both En-GAL4 and UAS-Aop Act are shifted to non-permissive temperature at 8 hours AEL, the En cells adopt smooth fates. This suggests that smooth cuticle is the default cell fate. Wg signaling in this context is required primarily for antagonizing the effect of DER signaling in anterior En cells (O'Keefe, 1997).

A model is presented for the cooperation between Wingless and Spitz in specifying cell fate in Engrailed expressing cells. The En-expressing cells are flanked anteriorly by a cell row producing Wg and posteriorly by a cell row expressing Rhomboid, which produces secreted Spitz. The En cell nearest the Spi source receives a higher concentration of Spi, and thus activates the Egfr pathway sufficiently to specify a denticle fate. Reciprocally, the En cell nearest the Wg source receives a higher concentration of Wg and adopts a smooth fate. Spi also activates the Egfr pathway in the Rho-expressing cell, which therefore produces and secretes Argos. Argos can inhibit Spi activation of the Egfr pathway at a distance. As a consequence, the Egfr pathway is not sufficiently activated in the anterior En cell to out compete Wg signaling in this cell, and it adopts a smooth fate. In fact, the specific targets of Egfr signaling responsible for conferring the denticle fate are unknown (O'Keefe, 1997).

Raf acts to elaborate dorsoventral pattern in the ectoderm of developing embryos

In the early Drosophila embryo the activity of the EGF-receptor (Egfr) is required to instruct cells to adopt a ventral neuroectodermal fate. Using a gain-of-function mutation it has been shown that D-raf acts to transmit this and other late-acting embryonic Egfr signals. A novel role for D-raf was also identified in lateral cell development using partial loss-of-function D-raf mutations. Thus, evidence is provided that zygotic D-raf acts to specify cell fates in two distinct pathways that generate dorsoventral pattern within the ectoderm. These functional requirements for D-raf activity occur subsequent to its maternal role in organizing the anterioposterior axis. The consequences of eliminating key D-raf regulatory domains and specific serine residues in the transmission of Egfr and lateral epidermal signals were also addressed in this study (Radke, 2001).

In the Drosophila embryo, Egfr activity is required to instruct a field of cells that lie on either side of the ventral furrow to adopt a ventral ectodermal fate. It is from this neuroectodermal cell population that the ventral nervous system and epidermis arise. At later times, Egfr functions in germband retraction and cuticle formation. Embryos that develop without Egfr activity fail to form ventral cuticular structures and show the 'faint little ball' phenotype. A constitutively active form of the D-raf protein, D-raf^tor4021, was used to bypass the requirements for Egfr function in embryos that lacked Egfr gene activity. For the generation of hyperactive D-raf^tor4021-proteins, the extracellular and transmembrane domains of the torso RTK gene were fused to the D-raf kinase domain. Chimera D-raf^tor4021proteins were shown to act independently of sevenless RTK gene function in developing photoreceptor cells: the chimeric proteins exhibited gain-of-function effects in the Tor signaling pathway (Radke, 2001).

Would this activated D-raf protein act independently of Egfr to rescue the embryonic lethality associated with homozygous mutations in the Egfr gene? In the case of noninjected control, 25% of the embryos derived from heterozygous Egfr parents (Egfr^-/+) failed to hatch, showed the faint little ball phenotype, and were homozygous for the Egfr mutation. D-raf^WT mRNA was used as a control for the injection procedure, and it was found that after injection 27% of the embryos from heterozygous Egfr parents failed to hatch. These embryos showed the Egfr mutant phenotype at 24 hr. When D-raf^tor4021 mRNA was injected into the central region of embryos collected from heterozygous Egfr parents, all aspects of defective Egfr signaling were rescued for some of the mutant Egfr embryos. Of the 258 embryos that received injection, 217 (84%) hatched out of their egg cases as larvae, while 41 (16%) remained within their eggshells. Thus, an increase in embryonic hatching and suppression of Egfr-induced lethality was observed after injection of D-raf^tor4021 mRNA. Partial rescue of the Egfr phenotype was found in unhatched embryos that had received D-raf^tor4021 mRNAs with ventral cuticular structures observed. It was concluded that constitutively active D-raf^tor4021 molecules can bypass the requirement for Egfr activity in the embryo and direct cells of the embryonic ectoderm to adopt a ventral fate. These results show that D-raf participates downstream of Egfr in developing embryos (Radke, 2001).

Once it had been found that an activated form of the D-raf protein could suppress the effects of a loss-of-function Egfr allele, it was reasoned that embryos lacking maternal and zygotic D-raf activity would exhibit an Egfr-like phenotype. These embryos would also be expected to show defects associated with the loss of maternal D-raf function in Tor signaling. To determine whether the identities of cells in the ventral ectoderm were dependent on D-raf activity, marker gene expression patterns and cuticles produced by D-raf embryos were compared to those of wild-type and Egfr embryos. To generate these D-raf embryos, mosaic D-raf females were produced whose eggs lacked maternal D-raf proteins. Once fertilized, these eggs gave rise to two classes of embryos: the first class was composed of the paternally rescued D-raf torso embryos (D-raf^-/+) that had inherited a wild-type D-raf gene from their fathers: they were defective in Tor RTK signaling and were missing head and tail structures at 24 hr. These D-raf torso embryos lacked maternal but not zygotic D-raf activity. The second phenotypic class was composed of the D-raf null embryos (D-raf^-/Y) whose exoskeletons consisted of what appeared to be a small patch of dorsal cuticle. These embryos lacked maternal and zygotic D-raf activity throughout development. It was anticipated that this D-raf null embryonic class would exhibit the phenotypic characteristics consistent with defective Egfr signaling, a consequence of defective D-raf protein activity (Radke, 2001).

Initially, to determine whether the establishment of ventral cell identity by the maternal dorsal gene system occurred normally in D-raf embryos, the accumulation of rhomboid (rho) mRNAs between 4 and 6 hr (stages 9-12) of development was assayed. As visualized by in situ hybridization, a column of cells ~2-3 wide on either side of the ventral midline showed the accumulation of rho mRNAs. This temporal and spatial pattern of rho expression was observed in all embryos in the D-raf collections, with each embryo a member of either the D-raf torso (lacking maternal but not zygotic D-raf activity) or null class. An equivalent rho expression pattern was observed in wild-type and Egfr embryos. Thus, the initial step in the establishment of ventral cell identity, by dorsal and other maternal genes that act to define the dorsoventral embryonic axis, is not perturbed when these events take place in the absence of maternal or zygotic D-raf activity (Radke, 2001).

To determine whether EGR-receptor signaling occurs normally in D-raf embryos, expression of the orthodenticle (otd) gene was monitored. In wild-type control embryos, at 6 hr (stage 11) otd mRNAs accumulate in cells adjacent to the ventral midline and in the head. In embryos lacking Egfr activity, otd expression occurred only in those cells within the embryonic head. In D-raf embryo collections, two patterns of embryo staining were observed with approximately one-half of the embryos showing otd expression in cells along the ventral midline and in the head. For the remaining D-raf embryos, the accumulation of otd mRNAs was observed only in the head, similar to Egfr embryos (Radke, 2001).

To distinguish between torso and null embryos in D-raf collections, a ftz-ß-gal marker gene located on the paternal X chromosome was used. Males with the ftz-ß-gal gene were allowed to fertilize eggs from mosaic females that lacked D-raf activity. In this double-labeling experiment, embryos that showed a ftz pattern of ß-gal expression were assigned to the D-raf torso class. These embryos also displayed a wild-type pattern of otd expression. In those D-raf null embryos lacking ß-gal expression, otd mRNAs were detected only in cells of the head, similar to Egfr embryos (Radke, 2001).

Between 4 and 7 hr (stages 9-11) of development, wild-type and Egfr embryos accumulated decapentaplegic (dpp) mRNAs in cells that formed two lateral stripes, when embryos were viewed ventrally. A similar pattern of dpp mRNA accumulation is seen in D-raf mutant embryos at this developmental stage. However, the ventral distance between dpp stripes becomes smaller in Egfr embryos as they develop. The distance between lateral dpp stripes was recorded and compared in wild-type, Egfr, and D-raf embryos at 10 hr (stage 13) of development. For wild-type embryos the average stripe distance was 0.111 units. In the collection of Egfr embryos, ~75% showed an average dpp lateral stripe distance of 0.118 units, similar to wild type. This phenotypic class contained embryos that were heterozygous mutant (Egfr^-/+) or wild type with respect to the Egfr gene. In the remaining 25% of the embryos the average dpp stripe distance was reduced to 0.075 units as anticipated for homozygous mutant Egfr embryos (Radke, 2001).

Two phenotypic classes of D-raf embryos were also distinguished on the basis of a statistically relevant difference in dpp stripe distance. In approximately one-half of the embryos the average dpp lateral stripe distance was 0.120 units, with the remaining embryos showing an average separation of 0.064 units. It was speculated that this second phenotypic class contained the D-raf null embryos. To test this idea, the marker ftz-ß-gal X chromosome was again employed in a double-labeling experiment to distinguish between D-raf torso and null embryos. As anticipated, it was the male D-raf null embryonic class that showed the decrease in distance between lateral dpp stripes, indicative of a loss in ventral cell fates (Radke, 2001).

On the basis of this analysis of rho, otd, and dpp gene expression patterns in D-raf null embryos, it has been concluded that ventral ectoderm cells are specified incorrectly in the absence of D-raf activity. This loss results in the production of a mature D-raf null exoskeleton that is severely reduced in size and devoid of ventral structures, consistent with the Egfr embryonic phenotype. However, the distance between lateral dpp stripes in Egfr (0.075 units) and D-raf null (0.064 units) embryos was compared: it was smaller in D-raf null embryos. In addition, after cursory inspection, the size of the exoskeleton patch produced by D-raf null embryos appeared smaller than that from Egfr embryos. These differences could be biologically significant and the analysis was expanded to address this potentially interesting finding (Radke, 2001).

To better understand the role that D-raf plays in the ectoderm and to access its regulation in various developmental pathways partial loss-of-function alleles of D-raf generated in vitro were used. D-raf shares homology with family members in CR1 that contain (1) D-ras binding motifs; (2) CR2, a region rich in serine and threonine residues, and (3)the CR3 kinase domain. CR1 is thought to exhibit positive control in the regulation of the D-raf protein via its interaction with D-Ras, while CR2 appears to be involved in the negative regulation of the molecule. Whether conserved subdomains (CR1 and CR2) or putative phosphorylation sites (serine 388 or 743) are essential for the activity of D-raf in the embryo or involved in its positive or negative regulation was tested. These modifications of D-raf often result in decreased D-raf activity. Thus, by expressing partial loss-of-function D-raf alleles in D-raf null embryos the role D-raf plays in developing embryos could be deciphered (Radke, 2001).

Using a structure-function strategy, several modified forms of the D-raf protein were generated. The D-raf^WT and D-raf^K497M genes were constructed as positive and negative controls, respectively, with the D-raf^WT allele a full-length copy of a D-raf cDNA. D-raf^K497M lysine 497, which was shown to be critical for D-raf protein kinase activity and likely involved in ATP binding, was replaced with a methionine. The N-terminal and CR1 deletion mutation, D-raf^Delta315, was likely to show a partial loss-of-function in D-raf null embryos. For the D-raf^Delta445 mutation both positive (CR1) and negative (CR2) control elements were lost, and it was predicted that this form of D-raf would act in a manner similar to wild type or, on the basis of its structural similarity to oncogenic forms of Raf-1, and show a gain-of-function effect in the embryo. Of the five phosphorylation sites identified for the human Raf-1 kinase, two are conserved in the D-raf protein. Serine to alanine substitutions at these sites were generated and it has been shown that S388 (CR2) plays a negative role while S743 (CR3) is involved in the positive control of D-raf in the Tor pathway. It was predicted that the D-raf^S388A and D-raf^S743A proteins would show similar phenotypic consequences for developing cells in the embryo (Radke, 2001).

Using P-element-mediated transformation, Drosophila lines were generated that contained an insertion of the D-raf^WT, D-raf^K497M, D-raf^Delta315, D-raf^Delta445, D-raf^S388A, or D-raf^S743A gene on either the second or third chromosome. Each of these modified D-raf genes were paternally introduced into D-raf embryos lacking maternal D-raf protein. The level and stability of D-raf proteins produced by expression of each paternally inherited D-raf^modified gene was tested. In this assay 100 embryos were collected for each sample and processed for Western analysis. Since the expression of each D-raf^modified gene was under the control of the hsp70 promoter, samples were processed from non-heat-shocked or heat-shocked embryos at 5 and 10 hr of development. These D-raf^modified proteins are variably stable and in D-raf null embryos show differences in the rescue of dorsoventral cuticular defects caused by the loss of D-raf maternal and zygotic function. The degree of phenotypic rescue observed in D-raf null embryos was as follows: D-raf^WT > D-raf^S388A > D-raf^Delta445 > D-raf^S743A > D-raf^Delta315 > D-raf^K497M (Radke, 2001).

The accumulation of D-raf protein was assayed in D-raf embryos that had inherited the D-raf^WT gene. For these embryos the accumulation of D-raf proteins after heat induction was approximately twofold greater than that found in wild-type embryos at 5 hr. At 10 hr, the level of the D-raf^WT protein was unchanged. The effect of D-raf^WT proteins on otd and dpp gene expression patterns was determined in D-raf embryos. As anticipated, induction of the D-raf^WT gene results in 100% of the D-raf null class showing wild-type ventral otd stripe expression and a normal pattern of dpp expression. Embryonic cuticles were examined at 24 hr to assess the ability of the D-raf^WT gene to promote signaling in the late-stage Egfr pathway responsible for epidermal differentiation and the final cuticular pattern. Of these D-raf null embryos that had inherited the D-raf^WT gene, 99% developed cuticles indistinguishable from their D-raf torso sisters. Thus, all ectodermal signaling pathways dependent on D-raf activity could be fully restored in null embryos by expression of the D-raf^WT gene (Radke, 2001).

In the phenotypic analysis, 84% of D-raf^S388A expressing D-raf null embryos showed rescue of Egfr-induced otd expression in ventral cells and the distance between dpp stripes appeared normal. By the completion of embryonic development, 97% of the D-raf null embryos showed the torso phenotype, while the remaining 3% showed a composite 'imperfect torso' phenotype. In addition to showing head and tail defects associated with the torso phenotype, embryos of the 'imperfect torso' class were twisted and had denticle bands of reduced width, indicative of partial loss of signaling in ventral cells that depend on the Egfr pathway for development. Since all of the D-raf null embryos showed some phenotypic rescue by D-raf^S388A, it was concluded that serine 388 is not essential for the function of D-raf in the ectoderm. Instead, it was thought likely that S388 plays a negative role in the regulation of D-raf similar to its function in Tor signaling (Radke, 2001).

For D-raf null embryos that inherited the D-raf^Delta445 gene, 52% showed rescue of the Egfr-induced otd expression pattern. This was approximately one-half the percentage rescued by the D-raf^WTgene, although the quantity of truncated ~38-kD D-raf protein in these embryos was equivalent to that observed for D-raf embryos expressing the D-raf^WT gene at 5 hr. For the human Raf-1 protein, removal of CR1 and CR2 resulted in unregulated kinase activity. Whether the D-raf^Delta445 protein acted ectopically to create a wide ventral otd stripe was tested, but all of the otd stripes were of wild-type width. When dpp mRNA patterns were analyzed in D-raf^Delta445 expressing null embryos the distance between lateral stripes in the third thoracic segment at 10 hr was similar to those that had inherited the D-raf^WT gene (Radke, 2001).

In the analysis of 24-hr cuticular patterns 52% of the D-raf^Delta445 embryos were rescued and showed the torso phenotype. For the remaining embryos, partial rescue was observed with signaling by the D-raf^Delta445 protein defective in the determination of the ventral ectoderm. Of these embryos, 18% showed the 'imperfect torso' phenotype and 30% showed the 'null with denticles' phenotype. These 'null with denticles' embryos were twisted, had faint cuticles with narrow denticle bands, and were phenotypically similar to Egfr embryos homozygous for intermediate defective alleles of Egfr. Overall, it was found that signal transmission by D-raf^Delta445 was less reliable when compared with D-raf^WT, although the D-raf^Delta445 protein had the potential to rescue all aspects of the embryonic D-raf null phenotype (Radke, 2001).

Analysis of D-raf embryos expressing the D-raf^S743A gene was somewhat complicated by the insertion of D-raf^S743A on the TM2 balancer chromosome. Thus, only one-half of the D-raf null embryos fertilized by D-raf^S743A transgenic males inherited the D-raf^S743A gene. The amount of D-raf^S743A protein that accumulated in D-raf embryos with the D-raf^S743A gene was determined; the D-raf^S743A protein was ~1.5-fold greater than that observed for those embryos that had inherited the D-raf^WT gene. Although greater levels of this modified D-raf protein accumulated in D-raf null embryos expressing the D-raf^S743A gene, otd stripe expression was not observed. Also, the distance between lateral dpp stripes in these D-raf^S743A embryos was diminished when compared with wild type, but not to the degree observed for embryos expressing the D-raf^Delta315 or D-raf^K497M genes. Thus, the specification of ventral cell fates at the midline requires the positive regulation of the D-raf protein at serine 743 (Radke, 2001).

Accordingly, 99% of the D-raf null embryos expressing the D-raf^S743A gene showed the 'imperfect torso' phenotype. To better assess the pattern deletions generated by the loss of epidermal cell fates in these D-raf^S743A embryos, epidermal sensory organs that develop in ventral and lateral domains of the embryo were scored. The separation between Keilin's organs and ventral black dots on the ventral surface was measured. Also, to determine whether patterning in lateral cells was normal for these embryos the distance between ventral and dorsal black dots was recorded. When compared with wild type, D-raf^S743A embryonic cuticles showed a decreased distance between Keilin's organs and ventral black dots. A decrease in the distance between ventral and dorsal black dot material was also observed. This latter finding proved very informative for it led to the hypothesis that a novel pathway, dependent upon the D-raf protein, was operating for signal transmission in cells undergoing lateral epidermal development. It appears that cell fate specification in the ventralmost ectoderm via the EGR receptor and proper development of a subpopulation of lateral cells requires an optimal level of D-raf activity that is not achieved by the D-raf^S743A protein (Radke, 2001).

Rescue of epidermal patterning defects was further diminished in D-raf null embryos that expressed the D-raf^Delta315 gene. Using Western analysis it was found that the D-raf^Delta315 protein migrated as an ~60-kD band detected at a level equivalent to that of the 90-kD D-raf^WT protein at 5 hr. Approximately 80% of this D-raf^Delta315 protein was present at 10 hr. When D-raf null embryos that inherited the D-raf^Delta315 gene were assayed for otd and dpp stripe expression, ventral otd expression was not observed and the distance between lateral dpp stripes was much reduced when compared with embryos expressing the D-raf^WT gene. Thus, a substantial decrease in the output of the Egfr-induced signal was detected. By the completion of development, 83 (81%) of the expected 102 D-raf null embryos with D-raf^Delta315 protein showed cuticles with the 'null with denticles' phenotype (Radke, 2001).

Epidermal sensory organs were scored in D-raf null embryos expressing the D-raf^Delta315 gene and their relative positions noted. Significantly, an absence of Keilin's organs was recorded and a corresponding expansion in the size of ventral black dot material was observed. The distance between these enlarged ventral dots was substantially reduced when compared with wild-type embryos. A reduction in the distance between ventral and dorsal black dot sensory organs was also observed. This finding again implicates D-raf in a pathway required for the development of lateral cells. Thus, by reducing the ability of the D-raf protein to act in signaling its role in the Egfr pathway has been verified and its function in a novel pathway involved in lateral cell development has also been uncovered (Radke, 2001).

As anticipated, D-raf-dependent pathways were not rescued when D-raf null embryos expressed the kinase defective D-raf^K497M gene (Radke, 2001). Thus, D-raf acts downstream of the Egfr for the specification of ventral ectodermal cell fates. D-raf also plays a second role in a novel pathway that is required for lateral cell development. In particular the D-raf^S743A and D-raf^Delta315 alleles generated in vitro proved useful in defining the function of D-raf in cells of the lateral epidermis. It is hypothesized that this novel pathway acts to specify cells of the lateral ectoderm subsequent to instructions received by nuclei from the dorsal maternal gene product. Thus, dorsoventral patterning in the embryo is likely dependent on the activity of three zygotic signaling pathways with Dpp that acts in dorsal cells, Egfr that directs cells in the ventral ectoderm, and a novel RTK pathway that specifies lateral cell fates (Radke, 2001).

The lateral epidermis consists of two narrow stripes of tissue on the left and right sides of the embryo extending from the anterior head to the posterior tail region. For the meta- and meso-thoracic regions this lateral tissue gives rise to epidermal cuticular structures that form between dorsal and ventral black dot sensilla. Along the circumference of each abdominal segment these two regions of lateral cuticle can be subdivided into dorsolateral and ventrolateral domains. Normally in late-stage embryos the dorsolateral region is characterized by numerous discontinuous rows of long slender hairs that have a pattern similar to that found for region b of the dorsal epidermis. These dorsolateral hairs are most similar in size and morphology to a subset of dorsal hairs, the 4° hairs. The ventrolateral domain is characterized by a segmental organization of naked cuticle alternating with two to three sparse rows of denticles similar to those found in the ventral belts although not as strongly pigmented (Radke, 2001).

Several findings have indicated that a novel pathway acts in the determination of lateral ectodermal cell fates and are consistent with a role for D-raf in this pathway. Embryos that developed in the absence of dpp and dorsal activity are lateralized. Mutations in the Drosophila dCREB-A gene are also important for defining lateral embryonic regions. In the absence of dCREB-A gene function, embryos show development of only lateral epidermal structures. Two consequences of lateral cell induction have also been identified: activation of the MAP kinase protein and expression of the msh gene encoding a homeodomain protein product. Using D-raf proteins with partial function it has been found that D-raf also participates in the development of the lateral epidermis most likely to specify cellular fates in the lateral ectoderm (Radke, 2001 and references therein).

Is there a receptor tyrosine kinase responsible for triggering the activation of the D-raf protein and MAP kinase in cells of the lateral ectoderm? In mammalian systems, mitogenic signaling by insulin in fetal rat, brown adipocyte, and primary cultures involves the activation of Ras and Raf-1 proteins. Insulin also triggers an increase in Raf-1 activity in several cell lines that expressed large numbers of insulin receptors (Radke, 2001 and references therein).

The Raf-MEK-MAP kinase cascade acts in a variety of cells to transmit RTK-generated signals during Drosophila development. The protein kinase activity of D-raf is required to elicit distinct ventral cell fates specified by the EGR receptor in early embryos. Using partial loss-of-function mutations in D-raf, cell fates normally specified by high levels of Egfr activity were lost while those that required lower receptor activity appeared normal or were expanded (Radke, 2001).

How is a graded pattern of cell types within a developmental field generated by a receptor tyrosine kinase? It has been hypothesized that the main function of the Raf-MEK-MAPK phosphorylation cascade is to amplify RTK-initiated signals. In this case, the quantity of activated Raf, MEK, and MAPK molecules is directly proportional to the number of receptor molecules activated, in the absence of feedback mechanisms. This information is then translated into position-dependent gene expression patterns that lead to morphological changes and cellular development. In this model, the quantity of activated RTK receptors defines the determined state of the cell. However, a number of studies in Drosophila reveal the existence of parallel signaling pathways emanating from a receptor during embryonic development. To extend the amplification hypothesis, the Raf-MEK-MAP kinase cascade may also act to integrate signals received from these parallel pathways and ultimately define precise transcriptional outcomes using a multistep mechanism. In mammalian cells, Raf-1 is regulated by a variety of inputs including the enzymatic function of PKC, Src, and Jnk kinases that upregulate activity. Autophosphorylation also plays a role in regulating Raf-1, as well as binding to Ras, 14-3-3, KSR, hsp90, and p50 proteins. In addition, PKA, Atk (PKB), and phosphatases have been implicated in the downregulation of Raf-1 function (Radke, 2001).

This study has addressed the consequences of eliminating key D-raf regulatory domains or specific serine residues that might act to integrate distinct signaling pathways in the Egfr pathway for ventral cell determination. In general, signal transmission was less reliable for D-raf proteins that lacked the negative regulatory site S388 (D-raf^S388A) or the regulatory sequences CR1 and CR2 associated with the N-terminal one-half of the molecule (D-raf^Delta445). However, both proteins showed the potential to transmit the highest level of ventral signal. This phenomenon was perhaps indicative of an important role played by the D-raf protein in the assembly of multiprotein complexes with components derived from parallel pathways. The full-length wild-type D-raf molecule, which contains several conserved motifs, may serve to bring parallel-signaling components together. Thus, the structural integrity of the D-raf protein may be important for the efficiency of complex assembly or its stability. In this model only complete and stable-signaling complexes achieve the highest level of signal output. It is speculated that in the case of D-raf^S388A and more often for D-raf^Delta445 proteins, complete signaling complexes were not built, leading to the phosphorylation of fewer D-MEK molecules, decreased signal output, and fewer cell fate choices specified within the Egfr developmental field (Radke, 2001).

In contrast, the Egfr signal was severely compromised when transmitted by either D-raf^S743A or D-raf^Delta315 proteins. The range of cell types specified by these mutant D-raf molecules was dramatically reduced from the wild type. In both cases, the establishment of cell fates that require the highest level of Egfr activity was consistently lost. Serine 743 may be important for the formation of D-raf dimers or oligomers as has been suggested for Raf-1. This type of complex may be essential for the generation of the highest level of ventral signal. In embryos that developed with D-raf^Delta315 proteins, cell fates were generated that required substantially lower levels of Egfr activity. It is speculated that the wild-type D-raf protein undergoes release from negative regulation imparted by the CR2 domain via its N-terminal and CR1 sequences. In the case of the D-raf^Delta315 protein, maintenance of the negative regulatory function of CR2 severely limited the ability of D-raf molecules to activate D-MEK. These results point to a multistep process in the generation of active D-raf molecules with multiple upstream factors acting in parallel. The highest level of D-raf signal was generated when all inputs were received. In the absence of one or several interactions the signaling potential of the D-raf protein was reduced, but not abolished (Radke, 2001).

Egfr and trachea

The formation of the tracheal network in Drosophila is driven by stereotyped migration of cells from the tracheal pits. No cell divisions take place during tracheal migration and the number of cells in each branch is fixed. This work examines the basis for the determination of tracheal branch fates, prior to the onset of migration. The EGF receptor pathway is activated by localized processing of the ligand Spitz in the tracheal placodes and is responsible for the capacity to form the dorsal trunk and visceral branch. Prominent double phosphorylation of Erk (Rolled) is detected in the tracheal placodes at stage 10-11. This pattern is Efgr-dependent and is abolished in rhomboid mutants. The double phosphorylated Erk domain is broader than the region of rhomboid expression. Since Rhomboid is known to regulate Spitz processing, this pattern probably reflects the diffusion of the secreted form of Spitz originating within the rhomboid-expressing cells, in the central part of the placode. In mutants for Egfr, tracheal pits appear normal, although certain tracheal branches fail to develop: specifically, the dorsal trunk and visceral branch are missing or incomplete. spalt mutants show specific defects in the migration of dorsal trunk cells, pointing to an important role for spalt in subdivision of tracheal fates. The Dpp pathway is induced in the tracheal pit by local presentation of Dpp from the adjacent dorsal and ventral ectodermal cells. This pathway patterns the dorsal and lateral branches. Elimination of both Dpp and Egfr pathways blocks migration of all tracheal branches. Antagonistic interactions between the two pathways are demonstrated. The opposing activities of two pathways may refine the final determination of tracheal branch fates. Egfr-dependent activation of Erk (Rolled) in the tracheal placode precedes the activation of the same pathway by Breathless. Only after Egfr induction is diminished, does a new double phosphorylated Erk pattern appear, induced by Breathless. It is proposed that two opposing gradients of Dpp and Spitz are operating within the placode. the cells in the center of the tracheal placode encounter high concentrations of secreted Spitz, and low or negligable levels of Dpp. Conversely, the cells located at the dorsal and ventral domains of the placode encounter high concentrations of Dpp and low levels of secreted Spitz. Therefore, induction by the Egfr and Dpp pathways creates three subsets of cells: dorsal, central and ventral (Wappner, 1997).

EGF-receptor ligands act as chemoattractants for migrating epithelial cells during organogenesis and wound healing. Evidence suggests that Rhomboid 3/EGF signalling, which originates from the midline of the Drosophila ventral nerve cord, repels tracheal ganglionic branches and prevents them from crossing the midline. rho3 acts independently from the main midline repellent Slit, and originates from a different sub-population of midline cells: the VUM neurons. Expression of dominant-negative Egfr or Ras induces midline crosses, whereas activation of the Egfr or Ras in the leading cell of the ganglionic branch can induce premature turns away from the midline. This suggests that the level of Egfr intracellular signalling, rather than the asymmetric activation of the receptor on the cell surface, is an important determinant in ganglionic branch repulsion. It is proposed that Egfr activation provides a necessary switch for the interpretation of a yet unknown repellent function of the midline (Gallio, 2004).

The morphogenesis of the embryonic tracheal network depends on the charted migration of ~2000 epithelial cells deriving from 20 epidermal invaginations. These cells undergo three successive rounds of branching to generate a tubular network that extends along stereotyped paths towards specific target tissues. The last branching event produces thin, unicellular terminal branches that associate with distinct organs. The ventral nerve cord (VNC) is invaded by 20 ganglionic branches (GBs), which sprout from the lateral trunk of the trachea. GB migration towards and inside the CNS is highly stereotyped. Each GB initially tracks along the inter-segmental nerve and toward the CNS. GB1, the leading cell of the ganglionic branch, enters the nerve cord and changes substrate to track along the segmental nerve, proceeding ventrally on top of the longitudinal fascicles and towards the CNS midline. Finally, after reaching the midline, GB1 takes a sharp turn and migrates dorsally through the dorsoventral channel and then turns posteriorly on the dorsal side of the VNC. At the end of embryogenesis, GB1 will have trailed a remarkable 50 µm inside the CNS. Genetic analysis has uncovered a number of factors that are necessary for this fixed migratory path: the FGF homolog Branchless is required to guide the GBs towards the CNS and to induce them to enter it, in part by inducing the expression of the nuclear protein Adrift. Once inside the CNS, Slit, the main repulsive cue for axons at the midline, becomes a key guiding cue for the migrating GBs. Slit controls several, distinct aspects of ganglionic branch pathfinding into the CNS: it is first required to attract GBs toward the CNS, an effect mediated by Slit's receptor Robo2, and then to prevent GBs from crossing the midline once they reach it: this is also mediated by Robo (Gallio, 2004 and references therein).

A collection of 2640 P-element insertions was screened for mutants affecting the pathfinding of the ganglionic branch (GB) into the CNS. One of the recovered mutants, named inga (from ingen återvändo, meaning 'no turning back' in Swedish) was characterised by a specific midline-cross phenotype: at stage 16.3-4, upon approaching the CNS midline, a significant number of inga GBs failed to turn posteriorly and dorsally at the midline and crossed to the other side, or remained lingering on it. No other defect was detected in the tracheal system of inga embryos. Sequence analysis of the genomic region surrounding the transposon in inga mutants showed that the P-element was inserted in roughoid/rho3, and all available ru/rho3 alleles as well as inga/Df(3L)Ar14-8 embryos (a chromosomal deficiency removing the 61-62 region). These showed the same tracheal phenotype as inga. Therefore, it was concluded that inga is an allele of rho3 and subsequent analysis focused on a previously characterised null allele (Gallio, 2004),

The essential components of the Egfr signalling pathway were associated with ventral nerve cord development soon after their discovery. rhomboid, spitz and pointed mutants were originally identified for their effect on the ventral ectodermal region. Egfr signalling also plays a central role in the development of the VNC midline, where it is first required for cell differentiation and positioning of midline glia and later for their survival during the late stages of embryogenesis (Gallio, 2004)

The expression of rho3 in VUMs and its function in GB1 guidance away from the midline identifies a new role for Egfr signalling in the VNC. Unlike rho1, rho3 mutants have a normal VNC pattern in which longitudinal connectives and glial populations appear normal, suggesting that rho3 is specifically required for GB1 guidance. Expression of dominant-negative forms of the EGF receptor or Ras in GB1 phenocopies the rho3 guidance phenotype. In addition, overactivation of Egfr signalling in the trachea is sufficient to redirect GB1 and induce early turn phenotypes. Finally, rho3 is required in parallel to slit, the main repulsive cue deriving from midline glia. Taken together, these results suggest that rho3 mutant GB1s are misrouted because of reduced levels of Egfr/Ras signalling in GB1 cells, rather than to indirect, subtle defects of midline patterning or signalling capacity in rho3 mutants. This led to the proposal of a simple model in which Rho3 activates one or more Egfr ligands secreted by the midline cells. Reception of this signal by migrating GBs is mediated by Egfr and Ras, and promotes turning away from the midline (Gallio, 2004)

Three Drosophila Egfr ligands are activated by Rhomboid proteases: Gurken (which is only present in oocytes), Spitz and Keren, the latter expressed in embryos below the detection level of in situ hybridisation or antibody staining. Thus, the ligand activated by Rho3 to guide GB1 migration is very likely Spitz; it is expressed and is functional at the midline, but a contribution by Keren cannot be firmly excluded (Gallio, 2004)

The mammalian EGF receptors regulate migration in a variety of contexts, but in all known examples they appear to promote responses to chemoattractants. They do so by directly affecting cytoskeletal organisation, mainly through the PI3K, PKC or PLC pathways. The proper activation of the fly Egfr is also necessary for the migration of border cells toward the source of Gurken in the dorsal part of the oocyte. During this migration Egfr activation is coordinated with the activation of the fly PDGF/VEGF receptor homologue and requires the conserved adaptor protein Mbc (Dock 180/CED-5). Mbc provides a link to activated Rac and actin re-arrangements, which leads to the stereotyped attraction of the border cells towards the oocyte. It is, however, unclear whether Egfr provides the necessary spatial information for border cells during their pathfinding, or if it is required for the interpretation of positional cues provided by Pdgf/Vegfr or other receptors (Gallio, 2004 and references therein)

Egfr signalling is mediated by a number of downstream effectors in different cell types. In order to determine which one is used in GB1 pathfinding, a panel of mutants and dominant-negative constructs of known downstream effectors were analysed for their effect on GB migration. myoblast city (mbc) is a conserved adaptor necessary for the chemo-attractant function of Gurken during border cell migration in the ovary. mbc alleles had no defects in GB pathfinding. Since mbc has negligible maternal contribution and is not readily detected in tracheal tissues, it is concluded that it is unlikely to have a role in Egfr-mediated GB repulsion from the midline. Two additional effectors were tested that have been implicated in Egfr-elicited migratory responses in other systems: PLCgamma and PI3K. The fly PLCgamma is encoded by the small wing (sl) locus. small wing embryos had extra terminal sprouts emanating from the primary tracheal branches but show no specific defects in GB migration inside the VNC. Deltap60 is a deletion variant of the adaptor p60, which has dominant-negative effects on PI3K activity in vivo and in vitro. SRF-Gal4 driven expression of Deltap60 resulted in a stalling phenotype of 19% of the GBs but not midline crosses. This may reflect a requirement of PI3-K in the early extension of the GBs toward the midline, which is also impaired by the expression of the dominant-negative form of Egfr in GB1 (Gallio, 2004).

The activation of Ras is a necessary step in many of the cellular responses induced by Egfr signalling in Drosophila. It leads to the activation of Raf, and culminates with activation of the Ets-transcription factor Pointed and the nuclear export of Yan, another Ets protein that antagonises Pnt in the activation of target genes. SRF-Gal4-directed expression of a dominant-negative form of Ras results in stalled branches inside or outside the VNC. Importantly, a significant number of GBs was grossly misrouted (8%) or crossed the midline (4%) suggesting that Ras is required in the GB1 cells for their turn away from the midline. The large proportion of arrests in cell migration observed in these experiments might reflect a broader requirement for these common effectors in tracheal cell migration and sprouting (Gallio, 2004).

To analyse whether Egfr mediated repulsion of GB1 from the midline requires Raf or downstream pathway components, a dominant-negative form of Raf and an activated form of Yan were expressed under the control of SRF-Gal4. These constructs caused many of the branches to stall or misroute but in neither case could any branches that crossed the VNC midline be found. As an example, expression of the activated Yan construct stalled the migration of 45% of the GBs, and misrouted an additional 7%, but not a single midline cross was observed (Gallio, 2004).

In summary, activation of Ras appears to be required for repulsion of GB1 from the midline, whereas the remaining components of the pathway are required for tracheal cell extension inside the VNC but not for the decision to cross the midline barrier (Gallio, 2004).

There are substantial differences in the ways by which Egfr controls migration in GB1 and in border cells. This analysis indicates that Egfr signalling is not a chemotactic cue for tracheal pathfinding -- rather, it reveals a surprising role in mediating repulsion from the signalling source. In addition, mbc mutants do not show any midline crossing phenotypes that would resemble the phenotypes of rho3 or the ones generated by inactivation of the receptor. Furthermore, the increase of signalling levels in GB1, either by the expression of Rho1, activated receptor or activated Ras, results in a significant phenotype opposite that of the rho3 mutants: such treatments induced GBs to turn early before reaching the midline. This suggests that at the appropriate distance from the midline, Egfr activation becomes a switch to initiate the turn of GB1 away from it. Hence, an experimental increase of signalling levels can shift the crucial switch further away from the midline, while decreased signalling causes midline crosses. In essence, migrating GBs use Egfr activation to efficiently compute their relative distance from the midline, fine-tuning their response to the repulsive and attractive cues originating from it (Gallio, 2004)

Migration in general, and axonal pathfinding at the midline in particular, is known to rely on a number of guidance signals, at times redundant ones. The major midline repulsive signal for GB1 is Slit, yet a genetic test shows that rho3 acts in parallel to Slit. It is hypothesized that Egfr works in an analogous manner by activating a second, yet undiscovered, signalling system for GB repulsion. Such a guidance cue may be specific for GB1 migration, since axonal fascicles are not affected in rho3 mutants. Alternatively, the activation of Egfr in GB1 provides an epithelial specific regulation of a common repulsive signal used by both axons and GB1 (Gallio, 2004)

What could this repulsive signal be? Likely candidates fall in the short list of conserved signals repelling axons and non-neural cells in different systems: Netrins, Semaphorins and Ephrins. Netrins are involved in the repulsion of motor axons in both vertebrates and invertebrates and both Drosophila Netrins are expressed at the CNS midline, where they mediate attraction of commissural axons. Semaphorins and Ephrins are also capable of repelling axons and non-neural cells in different contexts, and they therefore represent possible guiding cues for GBs. Intriguingly, each family uses receptor tyrosine kinases as receptors (in the case of Ephrin) or co-receptors (in the case of Semaphorins). Most of these signals are bi-functional, they can elicit both attractive and repulsive responses on the receiving cells depending on context. Egfr activation in GB1 may lead to the post-translational modifications that activate a repellent receptor or inactivate an attractant one and may represent a general 'switch' mechanism for changing the orientation of cell migration depending on the strength of RTK signalling (Gallio, 2004)

Branching morphogenesis is a widespread mechanism used to increase the surface area of epithelial organs. Many signaling systems steer development of branched organs, but it is still unclear which cellular processes are regulated by the different pathways. The development of the air sacs of the dorsal thorax of Drosophila was used to study cellular events and their regulation via cell-cell signaling. Two receptor tyrosine kinases play important but distinct roles in air sac outgrowth. Fgf signaling directs cell migration at the tip of the structure, while Egf signaling is instrumental for cell division and cell survival in the growing epithelial structure. Interestingly, Fgf signaling requires Ras, the Mapk pathway, and Pointed to direct migration, suggesting that both cytoskeletal and nuclear events are downstream of receptor activation. Ras and the Mapk pathway are also needed for Egf-regulated cell division/survival, but Pointed is dispensable (Cabernard, 2005).

The air sac of the dorsal thorax grows from a bud that arises during the third larval instar from a wing disc-associated tracheal branch. To illustrate the development of the air sac, a GFP trap line was used that rather ubiquitously expressed membrane bound GFP; tracheal cells were counterstained with an mRFP1-moesin construct under the direct control of the trachea-specific breathless (btl) enhancer. From the early to late third instar stage, a bud-like structure grows out of the transverse connective and spreads on the wing imaginal disc epithelium; this outgrowth corresponds to the primordium of the air sac of the dorsal thorax (Cabernard, 2005).

It has been proposed that the air sac of the dorsal thorax forms de novo from a small group of wing imaginal disc cells, and that the resulting sac subsequently generates a tracheal lumen by an unknown process (Sato, 2002). Since, in the early Drosophila embryo, the lumen arises from an epithelial invagination via cell migration, it was asked whether the cells in the growing air sac are epithelial in nature with a clear apical/basal polarity. For this purpose, a Dα-Catenin-GFP (Dα-Cat-GFP) fusion construct was expressed in the developing air sac and the distribution of GFP from early to late third instar larvae was analyzed. Dα-Cat-GFP labels the adherens junctions (AJs) of epithelial cells. Clearly, the growth of the air sac was accompanied from the early stages onward by an out-bulging of an AJ network, suggesting that most or all of the cells in the growing bud were epithelial in nature, and that a luminal space was generated at the apical side of the epithelial tracheal cells during outgrowth. To confirm this interpretation, use was made of the recent identification of a protein, Piopio (Pio), which is apically secreted into the tracheal lumen in the embryo (Jazwinska, 2003). Indeed, the prospective luminal space in the outgrowing air sac is filled with Pio protein, demonstrating that the air sacs consist of a sac-like epithelial sheet, generating a luminal space as they grow (Cabernard, 2005).

To test whether all cells maintained an apical-basal polarity during air sac budding, single tracheal cells were labelled by using a recently developed assay system that allows for the visualization (and manipulation) of individual tracheal cells in vivo (Ribeiro, 2004). When this scenario was used in the presence of a UAS-Dα-Catenin-GFP chromosome, it was found that, in virtually all cases, such individually labeled air sac cells contacted the lumen and formed AJs with neighboring cells, even when these cells were located at the tip of the outgrowing air sac. The same conclusion was reached when the expression of GFP-moesin was analyzed in single air sac cells; cells at the tip made clear contact with the lumen. Therefore, it is concluded that the air sac is sculpted from an epithelial cell layer, which expands and at the same time generates an apical luminal space filled with secreted proteins (Cabernard, 2005).

It was of interest to better understand how Ras can be used in the same tissue at the same time for different cellular processes. egfr mutant cells can contribute to the tip of the growing air sac, although the clones are relatively small. In the stalk of the air sac, cells lacking Egfr often appeared fragmented, a sign of cell death. Indeed, when egfr mutant cells were sustained with anti-Drice, a marker for apoptotic cells, a strong accumulation of this protein was found. When p35, a viral antiapoptotic protein, was expressed in egfr mutant cell clones, these clones grew to larger sizes and were able to populate the air sac tip at a significantly higher frequency than in the absence of p35. These experiments establish that Egfr is dispensable for migration, and that migration is exclusively triggered by one of the two RTKs, Btl/Fgfr. The experiments also demonstrate that, during the growth phase, Btl/Fgfr signaling is dispensable for cell division; clones can grow to large sizes, although they fail to populate the tip. This same result was obtained with two other components, which are exclusively used by the Fgfr signaling pathway in the air sac (and not the Egfr pathway), namely, Dof and Pointed. Thus, migration and cell division are controlled by two different RTKs, but both RTKs signal via the activation of Ras and the Map kinase pathway to regulate these different cellular outcomes (Cabernard, 2005).

How does Ras control cell migration in the tip and cell division in the remaining air sac? To start to address these questions, whether high levels of constitutive active Ras were compatible with directional cell migration was tested and RasV12 was expressed in wild-type tissue in small cell clones. Interestingly, such clones expanded considerably and grew to large sizes in the center of the air sac or in the stalk, resulting in bulgy outgrowths; however, clones expressing RasV12 never contributed to the tip of the air sac. This finding suggests that unrestricted levels of Ras in a cell perturb its capacity to read out the migratory cues (presumably the Bnl/Fgf ligand); wild-type cells were apparently much better in taking up the leading position. In line with this interpretation, expression of an activated version of Btl (Torso-Btl/Fgfr) also resulted in bulky outgrowths. In addition, cells expressing the chimeric Btl receptor never populated the tip. Quite in contrast, activated Egfr (Egfr fused to a lambda dimerization site) was not able to perturb air sac guidance, but it also triggered higher division rates in clones, generating bulgy outgrowths (Cabernard, 2005).

To test whether single cells expressing activated receptor constructs changed their behavior with regard to cytoskeletal dynamics, the expression of either the activated version of Fgfr or Egfr was induced in early third instar stages and the behavior of such cells was analyzed with live imaging of cultured discs. Cells in the stalk of the air sac expressing activated Fgfr showed extremely dynamic cytoskeletal activity and formed large lamelipodia extending away from the air sac, similar to cells at the tip. Quite in contrast, cells expressing activated Egfr did not show increased lamelipodia formation, and their basal side remained relatively inactive (Cabernard, 2005).

Since the expression of constitutive active versions of the two different RTKs during air sac growth had different effects, whether the endogenous receptors activated the Ras/Mapk pathway to different levels in wild-type air sacs was investigated. In order to monitor the strength of Mapk signaling, an antibody recognizing the double-phosphorylated form of Erk, dpErk were used. Indeed, high levels of dpErk was detected in the nucleus of tip cells; lower dpErk levels were found in the cells in the center of the air sac, and dpErk was mostly cytoplasmic (Cabernard, 2005).

From all of the above-mentioned data, it is concluded that air sac development makes use of two distinct RTKs to control directed organ extension via cell migration (Fgfr) and organ growth via cell division (Egfr). This study carefully analyzed air sac outgrowth from early to late stages, by using a number of different markers labeling either membranes or AJs of individual air sac cells, or the apical luminal compartment. It was found that the thoracic air sac is modeled out of the existing tracheal epithelium, and that a luminal space is generated by the migration of a few cells away from the cuticle of the existing tracheal branch; the luminal space is then expanded by increasing the cell number in the sac-like epithelial structure via cell division. During this process, all cells remain within the epithelium and only round up when they divide. Even those cells that send out filopodia and lamelipodia and migrate in the direction of Bnl/Fgf remain embedded within the epithelium, contact the lumen, and form AJs with their neighbors. Thus, the directed outgrowth of the thoracic air sac during larval development is very similar to the budding of tracheal branches in the early embryo, in that epithelial cells form extensions from the basal side, ultimately resulting in cell movement toward the Fgf ligand. In contrast, during tubule formation of MDCK cells in culture, cells initially depolarize and migrate to form chain-like structures before they repolarize and form the luminal cavity; tubulogenesis is thus accompanied with partial epithelial-to-mesenchymal as well as mesenchymal-to-epithelial transitions. The tube-forming process has been subdivided into different stages such as cyst, extension, chain, cord, and tubule. In the case of the MDCK model system, growth factors have been proposed to trigger branching by inducing a dedifferentiation that allows the monolayer to be remodeled via cell extension and chain formation. Similar to the MDCK system, it was found in Drosophila that growth factor signaling induces the formation of cellular extensions, the first sign of outgrowth. Also, in both systems, cell division is an integral part of the process, but it occurs randomly throughout the structure and not locally at the point of outgrowth. However, two different RTKs are used in the air sac to control extension (migration) and cell division, and chain and chord stages are not observed. It thus appears that both similarities and differences exist between these different cellular systems (Cabernard, 2005).

It has already been reported that cells divide during air sac formation. The cell division rates have been semiquantified and it was found that the elongating structure does not grow preferentially at the tip. The genetic analysis demonstrates that the Egfr is essential for cells to divide and survive efficiently in the air sac. Egfr signals via Ras and the Mapk pathway, but it does not require the Pnt transcription factor to regulate cell division. It is not yet known which ligand activates Egfr, and whether expression of this ligand is induced at early stages of development by Fgf signaling. As shown before (Sato, 2002), the complete lack of Fgfr signaling results in the absence of air sacs; Fgf signaling might thus be used at the onset of the budding process to initiate or trigger cell division, but it is clearly dispensable in later stages. Since cells in the tracheal branch, which gives rise to the air sac primordium, also divide in the absence of Fgf signaling, it is possible that the role of Fgf signaling consists in generating an outgrowth via directed cell movement, triggering cell division indirectly (Cabernard, 2005).

Interestingly, a recent study addressing the role of GDNF/Ret signaling in kidney branching morphogenesis in vivo has shown that ret mutant cells (which are unable to respond to GDNF) can contribute to the primary outgrowth of the ureteric bud, but are excluded from the ampulla that forms at its tip. Apparently, a Ret-dependent proliferation of tip cells under the influence of GDNF controls branch outgrowth. This study found that in Drosophila, in the developing air sac, cells lacking Fgfr are also excluded from the tip. However, evidence is provided that Fgf signaling is translated into directed migration in the leading structure and not into a local increase in cell proliferation. The isolation and cultivation of wing imaginal discs allows for using 4D imaging to document cell behavior during air sac growth. It was found that numerous tip cells extend long filopodia and lamelipodia, similar to the findings reported earlier (Sato, 2002). Tip cells not only produce extensions, but they indeed change their respective position with time, and move forward over the substrate in the direction of the filopodia/lamelipodia. Thus, tip cells are clearly motile and migrate in the direction of Bnl/Fgf. Cell clones incapable of responding to different families of ligands were produced and marked and were examined with regard to their capacity to populate the air sac tip. Among the receptors analyzed, only Btl/Fgfr was strictly required for cells to populate the leading tip of the air sac. Considering the observation that cells in the tip actively migrate, that Btl/Fgfr signaling is required for tracheal cell migration in the embryo, that tracheal cells migrate to ectopic sources of Bnl/Fgf in the embryo and the larva (Sato, 2002), and that cells form numerous filopodia and lamelipodia upon constitutive activation of the Fgf signaling pathway, it is concluded that Fgf steers cell migration in the tip of the air sac and leads to its directional outgrowth on the surface of the wing imaginal disc. The demonstration that the MARCM system can be used to analyze gene function with regard to cell migration in the developing air sac prompted an investigation of the role of Ras and the Mapk pathway in Fgf-directed cell movement (Cabernard, 2005).

Using the MARCM system, it was found that Ras activation is essential for cells to migrate at the tip of the air sac. The requirement for Cnk and Ksr strongly suggests that one important branch downstream of Ras in the control of cell migration is the Mapk pathway. This interpretation is supported by the somewhat surprising finding that the transcription factor Pnt is also strictly required for cell migration. In the Drosophila embryo, genes regulated by Fgf signaling at the transcriptional level and essential for migration have not been identified so far; although both pnt itself and blistered/DSrf are targets of Fgf signaling with important functions in tracheal morphogenesis, they are not required for migration. One possible target of Fgf signaling in the dorsal air sac cells might be the btl/fgfr gene itself. Attempts were made to rescue the pointed defects in air sac development by supplementing a btl transgene under the control of UAS sequences. It was found that even when Btl/Fgfr is provided by the transgenes, pnt mutant clones do not reach the tip. A second gene that might have been a transcriptional target of Pointed is dof; however, it was found that Dof protein is still present in pnt mutant clones (Cabernard, 2005).

The results demonstrate that the outgrowth of the dorsal air sac along the underlying wing imaginal disc is controlled by Btl/Fgfr and Egfr. Fgf signaling is required for directional outgrowth via cell migration, and Egf signaling is required for organ size increase sustaining cell division/cell survival. Both signals use the Ras/Mapk pathway to elicit their cellular responses. To what extent these two pathways regulate different downstream targets is not known at present. However, this study shows that Pointed is only required downstream of Fgf signaling in the control of directed cell migration, and not downstream of Egf signaling in the control of cell division/survival. Since the activation of the Map kinase pathway is much stronger in the cells at the tip as compared to the cells in the central portion or in the stalk of the air sac (according to the levels of dpErk), it is thought that the local availability of the Bnl/Fgf ligand results in a local signaling peak. Egf signaling in more central and proximal cells does not result in a strong activation of the Map kinase pathway, yet this activation is apparently sufficient to control cell division and survival. The independent regulation of cell migration and cell division by two different RTKs might be even more important in later stages of dorsal air sac development, when the growing tip is yet farther away from the main body of the air sac. It will be interesting to find whether other growing branched tissues use similar mechanisms to uncouple directional expansion and size increase (Cabernard, 2005).

Egfr and Malpighian tubules

Defects in the locus Egfr affect the development of the Malpighian tubules. In mutants they form much shorter structures than in wild-type embryos, and contain a reduced number of cells. The severity of this phenotype in seven alleles correlates with other embryonic defects caused by Egfr mutations. Interestingly, the severity of defects in the two pairs of tubules differs: a greater reduction in cell numbers occurs in the posterior pair as compared to the anterior pair. Temperature shift experiments indicate a role for this receptor in the regulation of tubule cell division. It is possible an additional role for Egfr in the allocation of cells to the tubule primordia will be found (Baumann, 1993).

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

To identify the nature of the mitogenic tip cell signal a screen was carried out for genes specifically active in the tip cells. The genes rhomboid (rho) and Star (S), which encode transmembrane proteins involved in epidermal growth factor receptor (EGFR) signaling, are expressed in the tip cells and both are required for MT growth. When the tubules start to evert, rho and S are expressed in the tip mother cell; subsequently rho is strongly expressed in the tip cell and S in the tip cell and its former sister cell. An analysis of the MTs in the corresponding amorphic mutants reveals a strong decrease of cells in rho mutants and a weaker decrease in S mutants. In a rho;S double mutant, the tubules are barely detectable, indicating that rho and S activities are essential (albeit redundant) components controlling MT growth. The tubule phenotype of rho;S double mutants is very similar to that of EGFR mutants, which also show a drastic decrease in the tubule cell number. As in svp mutants, the allocation and the differentiation of the tip cells are normal in the receptor mutants, indicating that receptor activity is not required for tip cell determination and differentiation. The reduction of the tubule cell number in EGFR mutants is due to a failure of proper cell divisions. No BrdU incorporation occurs in EGFR mutants in the outbudding tubules at the time when cells divide in wild-type embryos. However, BrdU incorporation occurs again much later during the endomitotic cycles, indicating that in EGFR muants, a specific defect in DNA replication exists in cells that would normally divide (Kerber, 1998).

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

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

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