vein is expressed in blastoderm embryos in two ventrolateral stripes that are brought to the midline as gastrulation proceeds. These cells include precursors of the ventrolateral epidermis that is affected in vein and spitz/vein double mutants. Expression in midline cells persists but is progressively limited to single cells. In the germ-band retraction stage, cells in the CNS and epidermis express vein, and vein is expressed in the anlagen of the amnioserosa at late blastoderm and in the amnioserosa proper until the end of germ-band extention. Survival of amnioserosa cells is dependent of Egf-R. In late germ-band extended embryos, there is expression in some PNS precursors that include precursors of Keilin's organs and a subset of the cells of the chordotonal organs. Throughout development, vein is expressed in the head, in the clypeolabrum, the maxillary and labial lobes, and around the stomodeum. In late embryos vein expression decays in all ectodermal cells and appears in the segmental muscles and the gut wall (Schnepp, 1996). Although Vein mRNA is prominent in muscles, Vein protein expression is restricted to the sites of contact between muscles and the epidermal attachment cells. The protein localization at the junctional site betwen muscles and epidermal muscle attachment (EMA) cells, together with the abnormal muscle-dependent differentiation of the EMA cells in vein mutant embryo, support the possibility that Vein activity is required for tendon cell maturation (Yarnitzky, 1997).
In the Drosophila embryo, the correct association of muscle cells with their specific ectodermally derived tendon cells, also known as epidermal muscle attachment or EMA cells, is achieved through reciprocal interactions between these two distinct cell types. Vein, a neuregulin-like factor secreted by the approaching myotube, activates the EGF-receptor signaling pathway within the tendon cells to initiate tendon cell differentiation. kakapo is expressed in the tendons and is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. The expression of delilah, stripe, and beta1 tubulin is induced in the epidermal attachment cells as a result of the EGF-receptor pathway activation by the neuregulin-like growth factor, Vein (Yarnitzky, 1997). Vein is secreted by mesodermal cells underlying the EMA cells. Vein protein localization is restricted to the muscle-tendon junctional site in wild-type embryos. However, in kak mutant embryos, Vein protein is not localized and appears rather diffuse. This altered pattern of Vein may explain the multiple number of cells expressing delilah and stripe: since Vein is not strictly localized at a given muscle-tendon junction site, it apparently weakly activates the EGF-receptor pathway in neighboring cells as well. It is presumed that the only cells that can respond to the ectopic Vein protein are the competent population of EMA cells, defined by the early expression of stripe. These cells express stripe during early developmental stages in a muscle-independent manner and normally lose their stripe expression by stage 16 of embryonic development. When these competent EMA cells receive the muscle-derived Vein signal, the expression of stripe and delilah is reactivated. It appears that only this population of cells is capable of responding to Vein, since the pattern of the ectopic Stripe- or Delilah-expressing cells in the kak mutant embryos resembles that of the early population of Stripe-expressing cells. The reduced levels of beta1 tubulin mRNA in the mutant tendon cells may also result from the abnormal pattern of Vein localization, since lower levels of Vein may not be sufficient to induce maximal beta1 tubulin expression. It therefore appears that the primary defect in kak mutant embryos stems from the lack of Vein accumulation at the muscle-tendon junctional site (Strumpf, 1998).
How could this intracellular protein affect the localization of Vein at the extracellular matrix surrounding the EMA cell? At least two possibilities, which are not mutually exclusive, are considered. The first is the association of Kak with the unique cytoskeletal network of the EMA cell, which is critical for the cell's polarized organization. Tendon cell polarity may be essential for maintaining the characteristic junctional complexes formed between the basal surfaces of the EMA cell and the muscle cells. The space between these junctional complexes contains many extracellular matrix proteins, some of which may possess a Vein binding function. Impaired tendon cell polarity may lead to the loss of the putative Vein-binding component(s). Alternatively, Kak may be associated with a transmembrane protein(s) responsible for Vein localization either by direct binding or by association with additional extracellular matrix components that may directly bind Vein. Immunoprecipitation experiments with anti-Kak antibody indicated that Kakapo forms protein complexes containing the extracellular protein Tiggrin. These results favor the latter possibility that Kak is directly associated with protein complexes that may be important for Vein binding. The reduced amount of electron-dense material observed at the muscle-tendon junction site in the kak mutant embryos described in Prokop, et al. (1998) is in agreement with both mechanisms mentioned above (Strumpf, 1998).
The Drosophila ventral nerve cord derives from a stereotypic population of about 30 neural stem cells (the neuroblasts) per hemineuromere. Previous experiments have provided indications for inductive signals at ventral sites of the neuroectoderm that confer neuroblast identities. Using cell lineage analysis, molecular markers and cell transplantation, it has been shown that Egf receptor (Egfr) signaling plays an instructive role in CNS patterning and exerts differential effects on dorsoventral subpopulations of neuroblasts. The Egfr is capable of cell autonomously specifiying medial and intermediate neuroblast cell fates (referring to neuroblasts arrayed in medial and intermediate columns in the ventral neuroblast proliferative zone). Egfr signalling appears to be most critical for proper development of intermediate neuroblasts and less important for medial neuroblasts. It is not required for the more lateral column of neuroblast lineages or for cells to adopt CNS midline cell fate. Thus, dorsoventral patterning of the CNS involves both Egfr-dependent and -independent regulatory pathways. Furthermore, there appear to be different phases of Egfr activation during neuroectodermal patterning with an early phase independent of midline-derived signals (Udolph, 1998).
A number of observations point to the existence of early, midline-independent signals in the establishment of the CNS. (1) The population of intermediate NBs that is preferentially affected in Egfr mutants (at least partially) derives from regions of the NE outside the range of Spitz diffusion. The fact that heterotopically transplanted cells can adjust to an intermediate NB fate due to Egfr function suggests, that Egfr can be activated in the intermediate NE region and has a direct role in this context. (2) In single minded mutants that lack midline-derived Spi, no loss of RP2 neurons that derive from the intermediate NB 4-2 can be detected. (3) Whereas local overexpression of Spi in the midline (from stage 7) leads to additional aCC/pCC neurons, no effect has been observed on other identified neurons, like the RP2 neurons. This is in marked contrast to the current finding that Egfr mutants show a nearly complete loss of RP2. Before gastrulation (stage 5/6) Egfr is indeed broadly activated within a region of the vNE that corresponds to the early Rhomboid expression domain and which probably includes the region from which the intermediate NBs originate. During gastrulation, this activity pattern is restricted more and more toward the midline. (4) vein is expressed in blastoderm embryos in two ventrolateral stripes that are brought to the midline as gastrulation proceeds and genetic data suggest it acts together with spi to achieve the required level of Egfr activation for normal development of ventrolateral cells. Thus, for the early neuroectodermal Egfr function, the midline is probably not the source of active ligands. The short-time expression of Rhomboid and Vein in more lateral positions and subsequent restriction to ventral sites could lead to a gradient of activating ligands that might be still present at stage 7, when cell transplantations were performed (Udolph, 1998).
Patterning of the Drosophila ventral epidermis is a tractable model for understanding the role of signalling pathways in development. Interplay between Wingless and EGFR signalling determines the segmentally repeated pattern of alternating denticle belts and smooth cuticle: spitz group genes, which encode factors that stimulate EGFR signalling, induce the denticle fate, while Wingless signalling antagonizes the effect of EGFR signalling, allowing cells to adopt the smooth-cuticle fate. Medial fusion of denticle belts is also a hallmark of spitz group genes, yet its underlying cause is unknown. This phenotype has been studied and a new function has been discovered for EGFR signalling in epidermal patterning. Smooth-cuticle cells, which are receiving Wingless signalling, are nevertheless dependent on EGFR signalling for survival. Reducing EGFR signalling results in apoptosis of smooth-cuticle cells between stages 12 and 14, bringing adjacent denticle regions together to result in denticle belt fusions by stage 15. Multiple factors stimulate EGFR signalling to promote smooth-cuticle cell survival: in addition to the spitz group genes, Rhomboid-3/roughoid, but not Rhomboid-2 or -4, and the neuregulin-like ligand Vein also function in survival signalling. Pointed mutants display the lowest frequency of fusions, suggesting that EGFR signalling may inhibit apoptosis primarily at the post-translational level. All ventral epidermal cells therefore require some level of EGFR signalling; high levels specify the denticle fate, while lower levels maintain smooth-cuticle cell survival. This strategy might guard against developmental errors, and may be conserved in mammalian epidermal patterning (Urban, 2004).
The denticle belt fusion phenotype is one of the distinguishing features of the spitz group genes (Mayer, 1988), yet its developmental basis has remained mysterious, since no function has been known for EGFR signalling in the smooth cuticle, which is the affected tissue. Analysis of this phenotype has revealed its cause and uncovered a previously unrecognized function for EGFR signalling in Drosophila epidermal development. Spitz is the primary EGFR ligand in epidermal patterning, and is activated by proteolysis in three rows of rhomboid-1-expressing cells in the future denticle region. High EGFR signalling is required for cells to adopt the denticle fate, and other signalling pathways are used to elaborate the different denticle morphologies. The Wingless signal emanates from one posterior row of each parasegment and spreads anteriorly, suppressing the denticle fate and thus allowing cells to secrete a smooth cuticle. These future smooth-cuticle cells also require signalling through the EGFR for viability, and its absence results in apoptosis of future smooth-cuticle cells and thus denticle belt fusions. This survival signalling is mediated by low-level stimulation of the EGFR by cooperation between the ligands Vein and Spitz, which is activated by Rhomboid-1, Rhomboid-3 and Star (Urban, 2004).
The ventral epidermis is patterned in multiple stages during development, with cell fate specification occurring late, through antagonism between EGFR and Wingless signalling around stages 12-14. Direct phenotypic analysis indicates that EGFR signalling is required for smooth-cuticle cell survival during these fate specification stages and not earlier or later: epidermal cell apoptosis is greatly elevated in mutant embryos at stages 12-14, and the fusion phenotype first becomes apparent around stage 15 as curvature of Engrailed stripes (Urban, 2004).
This direct phenotypic analysis is also supported by several independent genetic observations. EGFR signalling is not required for survival in future smooth-cuticle cells early, when the ventrolateral fates are being specified (stage 10/11) since removing Rhomboid-1 expression at only this stage using the single-minded mutation never results in denticle belt fusions (Mayer, 1988). Defects at this early stage also cause ventral narrowing in spitz group genes (Mayer, 1988), and since rhomboid-3 does not enhance this phenotype, this suggests that rhomboid-3 cooperates with rhomboid-1 only later in development. Vein acts independently of spitz group genes to suppress denticle belt fusions, and this cannot occur at stage 10/11 since at this early stage Vein expression is dependent on EGFR signalling through a positive feedback loop. Finally, the fusion phenotype itself suggests that it forms late since denticle cells are being pulled into smooth cuticle regions and, as such, their denticle fate must have already been determined and cannot be altered by receiving signals from these smooth domains (Urban, 2004).
Thus, two thresholds with different outcomes exist for EGFR signalling in patterning the ventral epidermis. The level of EGFR signalling that a cell receives is presumably dependent on its distance from the Spitz-processing cells; activated MAPK staining indicates that these rows of cells receive high levels of EGFR signalling. High levels of EGFR signalling are required to induce the denticle fate, while lower levels that reach smooth-cuticle cells are sufficient to suppress apoptosis. All ventral epidermal cells therefore require EGFR signalling, but the exact level, together with antagonism of shavenbaby transcription by Wingless signalling, determines the biological outcome. Importantly, these functions may be separate, since Wingless signalling is known to antagonize shavenbaby transcription to repress the denticle fate, but may not repress EGFR signalling itself in smooth-cuticle cells: activated MAPK staining suggests that some smooth-cuticle cells in the midline may also receive higher levels of EGFR signalling (Urban, 2004).
These results indicate that cells require EGFR signalling for their survival only when they are starting to differentiate. A similar pattern was also observed in the developing eye imaginal disc where removing the EGFR resulted in cell death only once the morphogenetic furrow had passed. These observations raise the intriguing possibility that establishing a requirement for survival signals may be inherent in the differentiation program itself, perhaps for protecting against developmental errors. However, the observation that the requirement for survival signalling is restricted to the central region of the ventral epidermis implies that either this requirement is not ubiquitous, or that another signal is also involved (Urban, 2004).
Drosophila proprioceptors (chordotonal organs) are structured as a linear array of four lineage-related cells: a neuron, a glial cell, and two accessory cells, called cap and ligament, between which the neuron is stretched. To function properly as stretch receptors, chordotonal organs must be stably anchored at both edges. The cap cells are anchored to the cuticle through specialized lineage-related attachment cells. However, the mechanism by which the ligament cells at the other edge of the organ attach is not known. The identification of specialized attachment cells is reported that anchor the ligament cells of pentascolopidial chordotonal organs (lch5) to the cuticle. The ligament attachment cells are recruited by the approaching ligament cells upon reaching their attachment site, through an EGFR-dependent mechanism. Molecular characterization of lch5 attachment cells demonstrates that they share significant properties with Drosophila tendon cells and with mammalian proprioceptive organs (Inbal, 2004).
Tendon cells are induced by the approaching myotubes, which secrete the EGFR ligand Vein and activate the EGFR pathway in the tendon precursor cells that they contact. This activation results in the expression of tendon cell-typical markers and terminal differentiation of tendon cells. Since the approaching ligament cells seem to induce the LA cells that share many properties with tendon cells, whether the EGFR pathway plays a role in the process of the ectodermal LA (ligament attachment) cell induction was tested. Activation of the EGFR pathway within the developing LA cells was tested by costaining wild-type embryos for the activated form of MAP kinase (dp-ERK) and for alpha85E-tub. In stage 16 embryos, low levels of dp-ERK could be detected in the LA cells but not in any of the other lch5 cells. Higher levels of dp-ERK were detected in the LA cells of stage 17 embryos. These observations demonstrate that the MAP-kinase pathway is activated in the LA cells at the time of their formation. To establish whether this pathway is necessary for the induction of these cells and whether it is mediated through EGFR activation, the EGFR pathway was blocked specifically by expressing a dominant-negative form of the receptor (DN-DER). The DN-DER was expressed throughout the ectoderm using the 69B-Gal4 driver or, in all of the Stripe-expressing cells, including the LA cells, using a sr-Gal4 driver. In both cases, the expression of DN-DER abolished the formation of LA cells, indicating that activation of the EGFR pathway is necessary for LA cell development. To establish whether activation of the pathway plays a permissive or an instructive role in the formation of LA cells, whether higher levels of EGFR activation can lead to the formation of supernumerary LA cells was tested. To elevate the level of EGFR activation locally, the EGFR ligand Vein or a secreted form of the ligand Spitz (sSpi) was expressed in the ligament cells under the regulation of repo-Gal4. This excessive activation results in the formation of increased numbers of LA cells, indicating that the EGFR pathway plays an instructive role in the induction of lch5 LA cells. Expression of sSpi throughout the ectoderm led to the induction of multiple ectopic Sr-expressing cells; however, these cells did not express the alpha85E-tub protein, suggesting that EGFR pathway activity is required but not sufficient to determine the identity of an LA cell (Inbal, 2004).
The data suggest that Vein is expressed in the lch5 ligament cells in late stages of embryogenesis and that Vein is the major ligand responsible for EGFR activation in the prospective LA cells. The ability of Vein to induce supernumerary LA cells when overexpressed in the ligament cells is consistent with this conclusion. The role of Vein in the induction of LA cells extends the molecular similarity between the development of lch5 organs and mammalian proprioceptors. It has been shown that Neuregulin1, a Vein homolog, is secreted from proprioceptive afferent nerve endings and is required for the expression of Egr3 and differentiation of muscle spindles in the mouse (Inbal, 2004).
Intercellular signaling through the EGF receptor (EGFR) patterns the Drosophila egg. The TGF alpha-like ligand Gurken signals from the oocyte to the receptor in the overlying somatic follicle cells. In the dorsal follicle cells, this initial paracrine signaling event triggers an autocrine amplification by two other EGFR ligands: Spitz and Vein. Spitz becomes an effective ligand only in the presence of the multitransmembrane domain protein Rhomboid. Consequent high-level EGFR activation leads to localized expression of the diffusible inhibitor Argos, which alters the profile of signaling. This sequential activation, amplification, and local inhibition of the EGFR forms an autoregulatory cascade that leads to the splitting in two of an initial single peak of signaling, thereby patterning the egg (Wasserman, 1998).
In other tissues Rhomboid appears to activate Spitz/Egfr signaling, leading to the suspicion that Rhomboid might mediate autocrine Spitz signaling in the follicle cells. Consistent with this idea, the phenotype caused by loss of Spitz from the follicle cells is similar to that caused by loss of Rhomboid. Expression of antisense rhomboid causes loss of dorsal tissue and fusion of the appendages in eggs from heat-shocked females expressing HS-as-rho. Unmarked follicle cell clones of a rhomboid null mutation also give fused appendage phenotypes; as with spitz clones, these range from mild to severe fusions. Like Spitz and the Egfr, Rhomboid is not needed in the oocyte, implying that it, too, is only required in the follicle cells (Wasserman, 1998).
In the absence of Egfr signaling, rhomboid expression is lost and, conversely, it is ectopically expressed in fs(1)K10 egg chambers. These expression profiles of spitz and rhomboid are consistent with Gurken signaling from the oocyte activating the expression of rhomboid in the follicle cells. This may in turn allow Spitz to become an autocrine ligand in the follicle cells and thus establish an autocrine amplification of the initial paracrine signal. The expression of the neuregulin-like Egfr ligand vein was also examined. It is also expressed in two stripes of follicle cells at stage 10b. Interestingly, vein expression is dependent on Egfr signaling: it is ectopically expressed in fs(1)K10 eggs and absent from gurken null eggs, establishing another potentially important feedback mechanism. This suggests that the autocrine amplification of Egfr signaling also involves Vein, although in this case the feedback occurs by direct transcriptional activation of the ligand (Wasserman, 1998). vein expression has also been found to be dependent on Egfr signaling during embryogenesis (T. Volk, personal communication to Wasserman, 1998).
The expression of the secreted Egfr inhibitor, Argos, is dependent on Egfr signaling in many tissues. Consistent with this, argos is expressed in the dorsal-anterior follicle cells at the time when Egfr signaling occurs. At stage 11 the RNA is detectable in a single, T-shaped group of cells centered on the dorsal midline, and by stage 13, argos, like rhomboid and vein, is found in two groups of cells: one on either side of the midline. As elsewhere, argos expression is dependent on Egfr activation: in gurken mutant egg chambers it is lost, and it is ectopically expressed in fs(1)K10 egg chambers. Is argos expression dependent on Spitz amplification of Egfr signaling? An examination was performed to see if Spitz contributes to a signaling threshold required to induce argos expression. argos expression is normal in eggs from mothers with reduced Ras1, but when Spitz is halved, dorsal-anterior argos expression is abolished in most egg chambers. Therefore, there is indeed a threshold of Egfr signaling required to switch on argos, and both Gurken and Spitz participate in reaching this threshold (Wasserman, 1998).
The initial expression of argos at the dorsal midline led to the speculation that it might cause a reduction of Egfr signaling near the midline, thereby splitting the single signaling peak in two. The resulting twin peaks of Egfr activation would then specify the location of the dorsal appendages. A prediction of this model is that loss of Argos should remove inhibition of the Egfr at the midline and produce a single peak of signaling, leading to the formation of a fused appendage phenotype. The eggs from females with hypomorphic argos mutations were examined. A significant proportion of these eggs have a partially or, in the most severe cases, fully fused phenotype. The same fused appendage phenotype is observed in follicle cell clones of an argos null mutation. These data imply that there is a requirement for Argos in eggshell patterning and that, as with Spitz, Rhomboid, and the Egfr, this requirement is confined to the follicle cells (Wasserman, 1998).
It is proposed that Argos modifies the initial Egfr activation profile in the follicle cells, producing twin peaks of activity displaced from the midline. These specify the position of the dorsal appendages. Direct evidence for a transition from one to two peaks of signaling was obtained with an antibody that recognises only the activated, diphosphorylated form of MAP kinase, a key member of the signal transduction pathway downstream of the receptor. At stages 9-10, there is a single domain of activated MAP kinase in the follicle cells, centered on the dorsal midline. By stage 11, two domains, are observed: one on each side of the dorsal midline. From their position, these cells correspond to the cells that will form the dorsal appendages. In Egfr hypomorphs, which have a fused appendage phenotype, the single peak of activated MAP kinase does not split in two. These results clearly demonstrate that Egfr signaling does indeed evolve from a single peak into twin peaks of activation. This is supported by examining the expression pattern of known Egfr target genes in the follicle cells. By stage 11 these targets (pointed, rhomboid, argos, vein, and Broad) are expressed in two dorsal anterior domains, one on each side of the midline. This is taken as additional evidence for twin peaks of Egfr activation. Earlier, pointed, rhomboid, and argos are all also detectable in a single peak at the dorsal midline (Wasserman, 1998).
Egfr signaling specifies the dorsoventral axis and patterns the eggshell. It is suggested that these two functions are controlled by temporally separate phases of Egfr activation. When amplification and splitting of Egfr signaling do not occur, eggs have only a single, fused appendage. Surprisingly, larvae emerge from these eggs at the frequency predicted by Mendelian principles, and those that emerge have no apparent dorsoventral defects. When follicle cell clones of a spitz null are induced, the hatching rate of eggs with fused appendages os 82% of the predicted number. Similarly, all of the predicted number of eggs with a single fused appendage hatch from mutant females. The same is true of eggs with fused appendages caused by follicle cell clones of argos null mutations. Therefore, disruption of the amplifying and splitting process does not perturb dorsoventral axis specification, implying that the initial Gurken signal to the Egfr is sufficient to specify the axis. The subsequent cascade of amplification and splitting then patterns the eggshell (Wasserman, 1998).
Continued: see vein Developmental Biology part 2/2
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