EGF receptor

Effects of Mutation or Deletion

Table of contents

Egf-r and neurogenesis (part 1/2)

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).

Using the cell transplantation technique, a test was performed to see whether Egfr might be autonomously required for the specification and development of the NB lineages per se. The composition of complete lineages produced by single faint-little-ball (flb) (Egfr-) mutant progenitors was analyzed in a wild-type background. Early gastrula embryos (stage 7) of the flb IK35 mutant strain, which carries a null allele of Egfr, were used as donors. In one set of experiments, cells were taken from the ventral part of the neuroectoderm (vNE, 0-20% VD) of flb IK35 donors and transplanted isotopically into wild-type hosts at the same stage. 0-20% VD corresponds to the ventralmost region of the embryo. This region of the NE normally gives rise to medial and intermediate columns of NB lineages. In another set of experients, mutant cells were taken from the more dorsal 30-50% of the embryo and transplanted heterotopically into the ventral region of the wild-type embryos. In the first set of experiments, it was found that in the absence of Egfr function, the ability of vNE cells to assume the fate of intermediate NBs is reduced. In the second experiment, mutant cells from the more dorsal region of the neuroectoderm fail to establish an intermediate neuroblast fate when transplanted into the vNE, but instead establish a lateral neuroblast fate (refering to the lateral column of neuroblast lineages) (Udolph, 1998).

The results of isotopic cell transplantation experiments in conjunction with the antibody stainings show that Egfr activity is most crucial for the development of intermediate NBs. Intermediate NB lineages, like NB 4-2 and NB 7-3, are severely affected by the loss of Egfr function. The role of Egfr in specifying intermediate NB cell fates is also reflected by the expression pattern of the escargot (esg) gene (Yagi, 1997). esg, which encodes a zinc finger transcription factor, is expressed in two longitudinal stripes on each side of the wild-type embryo covering the medial and lateral regions of the NE. It is repressed specifically in the intermediate column of the NE. In Egfr mutants, however, esg becomes derepressed and can be found all over the dorsoventral axis of the NE as early as stage 6 (Yagi, 1997). It is interesting to note that, in tissue culture cells, esg interferes with the transcriptional activation function of the proneural Scute protein. Another possible target regulated by the Egfr pathway is the homeobox gene msh (muscle segment homeobox). Activation of the Egfr pathway is necessary to restrict Msh to the dorsal region of the NE, thereby prohibiting the expression within the more ventral regions of the NE. Ectopic expression of msh in the ventral NE severely affects the lineages of NBs derived from that region; thus, the Egfr-mediated repression of msh is crucial to allow normal ventral NE fate development (Udolph, 1998 and references).

Although loss of DER function has a severe effect on intermediate NBs, the NE still shows a D/V polarity since the lateral and most of the medial NB lineages appear to be normally determined. This Egfr-independent identity is clearly a cell autonomous property: cells from the vNE transplanted into dNE retain their ventral identity and Egfr mutant cells from the dNE still develop into a lateral fate when transplanted ventrally into wild-type hosts. Thus, this patterning is not only largely Egfr independent but is also already laid down at the time of transplantation (stage 7), i.e. before NB segregation. In accordance with this, there is a growing list of genes that are expressed in certain dorsoventral domains within the NE even before this stage. All of them are either directly or indirectly dependent on the gradient of nuclear Dorsal protein. For example, ventral nervous system defective (vnd/NK2), which encodes a homeobox protein, is expressed specifically in the ventral part of the NE before gastrula stage. Its early expression is Egfr independent and relies on the early dorsoventral patterning genes such as dorsal (dl), snail (sna), twist (twi) and decapentaplegic (dpp). Loss of vnd leads to loss of ventral proneural clusters and consequently to a loss of medial NBs. Thus, vnd is a good candidate for an Egfr-independent ventral patterning gene (Udolph, 1998 and references).

Despite the requirement of Egfr for intermediate and medial NB development, the determination of the adjacent mesectoderm to form the CNS midline is independent from Egfr signaling. Although cells taken from the dorsal NE of Egfr mutant donors are unable to take over the fate of medial NBs upon heterotopic transplantation into the vNE, they are induced to assume CNS midline cell fates upon heterotopic transplantation into the ventral midline. Thus, this capacity requires signaling mechanisms that are independent of Egfr. The nature of these mechanisms remains to be elucidated. There are indications that expression of single minded, the master gene of CNS midline development, is controlled in part by inductive influences mediated by the neurogenic gene Notch (Udolph, 1998 and references).

A number of observations point to the existence of early, midline-independent signals. (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).

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

In a screen to identify mutations that disrupt embryonic CNS development, one P element mutation, l(2)03033, was identified that causes a loss of essentially all Eve-positive RP2/RP2 sib neurons. This P element maps to cytological position 57F1-2 in the right arm of the second chromosome and is known to be inserted within the Egfr locus. To verify that lesions in Egfr result in a nearly complete loss of RP2 motoneurons, three additional Egfr mutants were obtained, including the Egfr null allele, flb 1K35Egfr allele (Skeath, 1998).

The first phase of CNS development, as gastrulation commences, involves the activation of the Ac-S proneural genes in a precise pattern of proneural clusters. To investigate whether Egfr regulates As-C expression in the neuroectoderm, the expression patterns of the achaete (ac) and lethal of scute (l’sc) genes were followed in Egfr mutant embryos. Loss of Egfr causes specific defects to the DV registration of ac and l’sc gene expression in the neuroectoderm; however, no defects to the AP registration for either ac or l’sc gene expression were found. In wild-type embryos during stages 8/9, ac is expressed in the medial and lateral, but not intermediate, clusters of rows 3 and 7; l’sc is expressed in the medial and lateral, but not intermediate, clusters of row 7 and in the medial, intermediate and lateral clusters of rows 1 and 5. A single neuroblast subsequently forms from each proneural cluster. In Egfr mutant embryos, ac gene expression expands into the intermediate column in rows 3 and 7 and l’sc expression expands into the intermediate column in row 7; l’sc is expressed normally in rows 1 and 5. The lateral limits of ac and l’sc gene expression in the neuroectoderm are unaltered in Egfr mutant embryos. The changes to the DV registration of ac and l’sc gene expression in Egfr mutant embryos suggest that neuroectodermal cells in the intermediate column change their fate. Both ac and l’sc are normally expressed in the medial and lateral columns in the affected rows, thus the phenotype is consistent with intermediate cells acquiring either a lateral or a medial fate. msh-1, which is expressed exclusively in the lateral column, expands into the intermediate column in Egfr mutant embryos. In this context, it appears that ac and l’sc expression expand from the lateral column into the intermediate column in the absence of Egfr (Skeath, 1998).

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

DER signals through the conserved RAS pathway. One of the final effectors of the Ras pathway is MAP kinase. MAP kinase is activated by the dual phosphorylation of threonine and tyrosine residues, carried out by MEK. The recent production of a monoclonal antibody that specifically recognizes the active, dual phosphorylated form of MAP kinase allows one to follow in situ the activation pattern of receptor tyrosine kinase pathways such as Egfr. To identify when and in which cells Egfr signaling is required to promote early CNS development, the presence of active MAP kinase was assayed in wild-type, Egfr, vein, spitz and rhomboid;vein mutant embryos. During early embryogenesis, active MAP kinase is present in two temporally and spatially distinct patterns within the neuroectoderm in wild-type embryos. Prior to and during gastrulation active MAP kinase is first found in two broad bilaterally paired longitudinal bands of cells that run down the length of the neuroectoderm. At stage 10, active MAP kinase is again expressed in the neuroectoderm in the most medial neuroectodermal cells that flank the midline. In Egfr and in rhomboid vein mutant embryos active MAP kinase is not present in either pattern. In embryos singly mutant for either vein or spitz, the first wave of active MAP kinase appears in its normal pattern although at reduced levels. However, the second wave of active MAP kinase is absent in spitz, but normal in vein, mutant embryos. Thus, early CNS defects correlate with the absence of the first but not the second wave of active MAP kinase as clear CNS defects occur in Egfr and rhomboid;vein but not in spitz mutant embryos. MAP kinase is found to be present in the medial and intermediate but excluded from the lateral neuroectodermal column. It is concluded that Egfr is required for the activation of proneural genes and functions to subdivide the neuroectoderm (Skeath, 1998).

The Sox-domain-containing gene Dichaete/fish-hook plays a crucial role in patterning the neuroectoderm along the DV axis. Dichaete is expressed in the medial and intermediate columns of the neuroectoderm, and mutant analysis indicates that Dichaete regulates cell fate and neuroblast formation in these domains. Molecular epistasis tests, double mutant analysis and dosage-sensitive interactions demonstrate that during these processes, Dichaete functions in parallel with ventral nerve cord defective and intermediate neuroblasts defective, and downstream of EGF receptor signaling to mediate its effect on development. These results identify Dichaete as an important regulator of dorsoventral pattern in the neuroectoderm, and indicate that Dichaete acts in concert with ventral nerve cord defective and intermediate neuroblasts defective to regulate pattern and cell fate in the neuroectoderm (Zhao, 2002).

vnd, ind and Egfr are key factors that regulate pattern and cell fate along the DV axis of the neuroectoderm. To ask if Egfr pathway activity depends on vnd or ind, MAPK activity was assayed in homozygous vnd or ind single mutant embryos. In both backgrounds, the initial activation of Egfr signaling in the medial and intermediate columns is normal. Thus, in the early neuroectoderm, Egfr acts either upstream or in parallel to vnd and ind. To investigate whether Egfr acts upstream of vnd or ind, vnd and ind expression was assayed in embryos homozygous mutant for the Egfr null allele flb^IK35 (referred to as Egfr mutant embryos). ind expression is absent in Egfr mutant embryos, indicating that Egfr activates ind expression in the intermediate column. By contrast, vnd expression in Egfr mutant embryos appears normal through the onset of stage 8. However, during stage 8, vnd expression begins to dissipate in medial column cells, and by early stage 10 these cells no longer express vnd. Conversely, medial column NBs that form in Egfr mutant embryos express vnd normally and retain vnd expression throughout embryogenesis. Thus, Egfr functions to maintain vnd expression in the neuroectoderm but is dispensable for vnd expression in NBs. These data indicate that Egfr resides atop the genetic hierarchy known to subdivide the neuroectoderm along the DV axis (Zhao, 2002).

These results suggest that Egfr patterns the neuroectoderm, at least in part, through its regulation of vnd and ind. To determine if additional genes act downstream of Egfr in this process, the phenotypes of embryos singly mutant for Egfr and ind were compared. It was reasoned that if Egfr patterns the intermediate column solely through regulation of ind, then Egfr and ind mutant embryos should exhibit identical intermediate column phenotypes. To compare the early CNS phenotypes of Egfr and ind, a precise analysis of msh expression and the NB pattern was carried out. In both cases, Egfr exhibits a more severe phenotype than ind. msh expression expands more medially in Egfr mutant embryos than in ind mutant embryos. In addition, lateral NBs are most often separated from medial NBs by a gap in ind mutant embryos, while lateral NBs develop immediately adjacent to medial NBs in Egfr mutant embryos. These data indicate a greater disruption to the intermediate column in Egfr mutant embryos than in ind mutant embryos. These phenotypic differences are consistent with the presence of additional genes acting downstream of Egfr and in parallel to ind to control cell fate in the intermediate column. However, Egfr maintains vnd expression in the neuroectoderm; thus, these data do not exclude the possibility that the differences in phenotype between Egfr and ind arise due to the late regulation of vnd expression by Egfr (Zhao, 2002).

To test whether the phenotypic differences between ind and Egfr mutant embryos are an indirect result of the regulation of vnd expression by Egfr, it was asked whether these differences are equalized in double mutants where vnd function is also removed. In vnd;ind mutant embryos, msh is expressed throughout the neuroectoderm, although its expression is higher in the lateral column relative to the medial column. By contrast, msh is expressed at uniformly strong levels throughout the neuroectoderm in vnd;Egfr mutant embryos. Thus, removal of vnd and Egfr causes a stronger derepression of msh in the neuroectoderm than loss of vnd and ind. These results suggest that additional gene(s) act downstream of Egfr and in parallel to vnd and ind to regulate DV pattern in the neuroectoderm. They also suggest that in the absence of vnd and Egfr function, the entire neuroectoderm acquires a lateral column fate (Zhao, 2002).

ind normally represses ac expression in the intermediate column, because in ind mutant embryos, ac expression is completely derepressed within rows 3 and 7 of the intermediate column. The Dichaete and ind phenotypes demonstrate that both genes are necessary for intermediate column fates. To determine if Dichaete and ind function in a linear pathway to regulate intermediate cell fates, ind expression was followed in Dichaete mutant embryos and Dichaete expression in ind mutant embryos. ind expression is normal in Dichaete mutant embryos and Dichaete expression is normal in ind mutant embryos. Thus, ind and Dichaete are regulated independently of each other (Zhao, 2002).

Double labeling Dichaete mutant embryos for ac and ind, and double labeling ind mutant embryos for ac and Dichaete reveals an interdependent relationship between Dichaete and ind. In Dichaete mutant embryos, a significant number of row 3 and 7 intermediate column cells and NBs co-express ac and ind -- an occurrence never observed in wild-type embryos. Thus, the ability of ind to repress ac in the intermediate column requires Dichaete activity. Reciprocally, in ind mutant embryos, all row 3 and 7 intermediate column cells co-express ac and Dichaete. Thus, the ability of Dichaete to repress ac in the intermediate column requires ind activity (Zhao, 2002).

These loss of function analyses have identified Dichaete as a regulator of DV pattern and cell fate in the neuroectoderm. To place Dichaete within the known genetic regulatory hierarchy that governs DV pattern in the neuroectoderm, systematic molecular epistasis tests were performed for Dichaete, ind, vnd and Egfr. Initially, vnd and ind expression, as well as Egfr activity was assayed in Dichaete mutant embryos. Dichaete mutant embryos exhibit no obvious defects to the expression of vnd or ind, or the activity of Egfr. Thus, Egfr, vnd and ind function upstream or in parallel to Dichaete (Zhao, 2002).

To investigate whether Egfr, vnd or ind regulate Dichaete, Dichaete expression was assayed in embryos mutant for each gene. No alterations were observed to the initial pattern of Dichaete expression in vnd or ind mutants, or in embryos doubly mutant for vnd and ind. Dichaete expression remains normal in ind mutant embryos throughout embryogenesis. However, by stage 11 in vnd and vnd; ind mutant embryos, Dichaete expression narrows inappropriately to an irregularly patterned stripe two-to-four cells wide immediately adjacent to the ventral midline. These results show that Dichaete is regulated independently of ind and is activated independently of vnd, but that vnd helps maintain Dichaete expression in the neuroectoderm (Zhao, 2002).

In contrast to vnd and ind, the initial pattern of Dichaete in Egfr mutant embryos is greatly reduced in the intermediate column and moderately reduced in the medial column during early neurogenesis. By stage 11, Dichaete expression narrows inappropriately to a thin and irregular stripe zero-to-three cells wide immediately adjacent to the ventral midline; Dichaete expression in the ventral midline is normal. These data identify Egfr as a key positive regulator of Dichaete in the neuroectoderm, and indicate that at least one other gene acts with Egfr to activate Dichaete expression in the medial column (Zhao, 2002).

To investigate whether vnd acts with Egfr to promote Dichaete expression in the medial column, Dichaete expression was followed in vnd; Egfr mutant embryos. The initial pattern of Dichaete in these embryos is the same as that observed in Egfr mutant embryos. However, by stage 11, Dichaete expression is completely absent from the neuroectoderm, although Dichaete expression is normal in the ventral midline. These results indicate that vnd and Egfr collaborate to maintain Dichaete expression in the neuroectoderm (Zhao, 2002).

To determine if Egfr activity is sufficient to activate Dichaete expression, the GAL4/UAS system system was used to activate Egfr signaling throughout the early Drosophila embryo. Ubiquitous Egfr signaling activates Dichaete expression throughout the neuroectoderm but not in the dorsal ectoderm. Thus, Egfr is necessary and sufficient to activate Dichaete in the neuroectoderm. However, in the dorsal ectoderm, either factors exist that inhibit the ability of Egfr to activate Dichaete or this domain lacks co-factors required for Egfr to activate Dichaete. Molecular epistasis tests place Egfr upstream of Dichaete and indicate that vnd, ind and Dichaete function largely in parallel to regulate pattern and cell fate in the neuroectoderm (Zhao, 2002).

The parallel genetic activities of Dichaete, vnd and ind, the co-expression of Dichaete with vnd and ind, and the similarity of the early Dichaete CNS phenotype to those of vnd and ind led to a test of whether Dichaete interacts genetically with vnd and ind. To ascertain whether Dichaete interacts with vnd, the double mutant vnd;Dichaete was made and the formation of medial column SIII NBs 4-1 and 6-1 was assayed. In Dichaete mutant embryos, NBs 4-1 and 6-1 formed in 69.1% and in 92.1% of hemisegments, respectively. In vnd mutant embryos it was found that NBs 4-1 and 6-1 formed in 39.3% and 35.5 of hemisegments, respectively. In vnd; Dichaete mutant embryos NBs 4-1 and 6-1 formed in 10.8% and 9.1% of hemisegments, respectively. The increased defects in NB formation in vnd; Dichaete mutant embryos relative to either single mutant confirms that Dichaete and vnd do not act in a linear pathway to regulate NB formation -- rather, they demonstrate that Dichaete and vnd function in parallel to control NB formation in the medial column (Zhao, 2002).

Defects in NB formation in vnd; Dichaete mutant embryos are more severe than would be expected if these genes functioned independently. For example, if two genes act independently to promote NB formation, then the frequency of NB formation in the double mutant would be the product of the individual probabilities that the indicated NB will form in each single mutant. Thus, if vnd and Dichaete function independently, it would be expected that NB 4-1 would form 27.2% of the time (0.393 x 0.691=0.272) and NB 6-1 to form 32.7% of the time (0.355 x 0.921=0.327) in vnd; Dichaete mutant embryos. However, NBs 4-1 and 6-1 form ~10% of the time in vnd; Dichaete mutant embryos -- roughly threefold more severe than predicted for independently acting genes. These results reveal a genetic interaction between Dichaete and vnd. Furthermore, these results are interpreted to suggest that the activities of vnd and Dichaete are more convergent than parallel with respect to NB formation (Zhao, 2002).

Next, genetic interactions between Dichaete and ind were tested. The partial derepression of ac expression and the incomplete loss of an Eve-positive RP2 neuron are the most sensitive assays for Dichaete function in the intermediate column. However, strong alleles of ind cause a complete derepression of ac expression, and a complete loss of RP2 neurons in this domain. Thus, an analysis of Dichaete ind double mutant embryos using these markers would be uninformative. To circumvent this problem, a test was performed to see whether ind dominantly enhances the Dichaete intermediate column ac and RP2 phenotypes. Embryos heterozygous for ind exhibit wild-type ac expression and RP2 formation. However, Dichaete ind/Dichaete + mutant embryos exhibit enhanced derepression of ac expression and an approximately threefold enhancement of the RP2 loss phenotype relative to Dichaete mutant embryos. The dominant enhancement of the Dichaete phenotype by ind reveals a genetic interaction between Dichaete and ind (Zhao, 2002).

Initial interest in Dichaete arose from the observation that vnd; Egfr mutant embryos exhibit a more severe neuroectodermal phenotype than vnd; ind mutant embryos. This suggests that at least one other gene acts downstream of Egfr, and in parallel to vnd and ind to pattern the early neuroectoderm: this led to the analysis of Dichaete. To determine if the continued function of Dichaete in vnd; ind mutant embryos can explain the phenotypic differences between vnd; ind and vnd; Egfr mutant embryos, msh expression was followed in vnd;Dichaete;ind triple mutant embryos. In this background, a complete and uniform derepression of msh expression was observed throughout the neuroectoderm. The msh phenotype of vnd; Dichaete; ind embryos is essentially identical to that of vnd; Egfr embryos, and more severe than that of vnd; ind embryos. Thus, with respect to msh expression the difference between the vnd; ind and vnd; Egfr mutant phenotypes appears to result from the persistent function of Dichaete in vnd; ind mutant embryos (Zhao, 2002).

The results in this paper indicate that Dichaete is a key regulator of DV pattern in the neuroectoderm. Dichaete is expressed in the medial and intermediate columns and regulates cell fate and NB formation in these domains. Within the neuroectoderm, Dichaete acts downstream of Egfr and in parallel to vnd and ind. Together with biochemical research on Sox-domain-containing genes in vertebrates this work supports a model in which Dichaete protein physically associates with Vnd and Ind to regulate target gene expression and NB formation in distinct neuroectodermal columns (Zhao, 2002).

Interest in Dichaete arose owing to the observation that removal of vnd and Egfr function caused a stronger derepression of msh expression in the neuroectoderm than removal of vnd and ind function. These results contrast slightly with previous research that did not identify a phenotypic difference between vnd; ind and vnd; Egfr mutant embryos. This work analyzed msh expression in the neuroectoderm at a later stage (late stage 9) than the current work. At late stage 9, identical alterations to msh expression were also observed in vnd; ind mutant embryos relative to vnd; Egfr mutant embryos. However, the msh expression pattern is dynamic -- rapidly changing from uniform expression in the lateral column during stage 8 to a segmentally modulated pattern of cell clusters located within the lateral half of the neuroectoderm by stage 10. The differences in these observations are attributed to the different stages used to assay the effects of vnd, ind and Egfr on neuroectodermal development in the two studies (Zhao, 2002 and references therein).

How might Dichaete exhibit region specific effects on putative target genes? Work from vertebrate systems suggests that individual Sox-domain-containing proteins exhibit a widespread ability to partner with different transcription factors. Thus, Dichaete protein could exhibit column-specific functions via its association with different transcription factors in different domains. The formation of distinct protein complexes containing Fish could alter the output of Fish activity in at least two ways. Different protein complexes that contain Fish could exhibit different effects on transcription: repression versus activation. Alternatively, different Fish-containing protein complexes could exhibit distinct DNA-binding properties and therefore bind distinct recognition sites. These two possibilities are not mutually exclusive, and different Fish-containing protein complexes may both bind different recognition sites and exert different transcriptional effects on target genes (Zhao, 2002).

Examples of both forms of regulation are known. In the early Drosophila embryo, the transcription factor Dorsal activates one set of target genes ventrally and represses a distinct set dorsally. On its own, Dorsal functions as a transcriptional activator. However, in the dorsal region of the embryo, the interaction of Dorsal with a co-factor that binds to adjacent sites on target promoters converts Dorsal to a repressor. Although less well-defined mechanistically, the vertebrate Sox2 protein appears capable of activating or repressing target gene expression depending on cell-type and the target promoter (Botquin, 1998). In addition, work on vertebrate Sox domain proteins indicates that the composition of Sox-protein containing complexes modulates the DNA-binding specificity of these complexes. For example, in lens cells, Sox2 interacts with the DNA-binding factor deltaEF3 and binds to a bipartite recognition site on the delta-crystallin enhancer (Kamachi, 1998; Kamachi, 1999). In embryonic stem cells, Sox2 interacts with Oct3/4 and binds to a different recognition site in the Fgf4 minimal enhancer (Ambrosetti, 1997). In both enhancers, Sox2 binds to the same individual sequence. However, the specificity for the entire recognition site in one enhancer over the other arises as a consequence of the interaction of Sox2 with different transcription factors in different cell types and the distinct DNA-binding preferences of the entire complex (Zhao, 2002).

Based on these data, Dichaete is expected to associate with different transcription factors in the medial and intermediate columns to carry out its column-specific effects on target genes. The results in this paper identify Vnd and Ind as excellent candidates to be column-specific factors that associate with Dichaete and enable Dichaete to regulate transcription in a region specific manner: (1) Dichaete is co-expressed with Vnd in the medial column and Ind in the intermediate column; (2) the neuroectodermal Dichaete mutant phenotype is similar to those of vnd and ind; (3) Dichaete functions in parallel to vnd and ind in the neuroectoderm; (4) Dichaete exhibits dose-sensitive interactions with ind and genetic interactions with vnd, consistent with these proteins interacting physically. Based on these data, it is speculated that physical interactions between Dichaete and Vnd in the medial column and Dichaete and Ind in the intermediate column mediate the ability of distinct Dichaete protein complexes to bind to and either activate or repress distinct target genes. Validation of this model awaits the determination of whether Dichaete associates with Vnd or Ind, and how these proteins regulate target gene activity. However, recent results provide precedence for the model since genetic interactions between Dichaete, single-minded and drifter during midline development in the Drosophila CNS have led to experiments that show Dichaete physically associates with the Single-minded and Drifter proteins (Zhao, 2002).

These results place Dichaete within the known genetic regulatory hierarchy that controls pattern and cell fate along the DV extent of the neuroectoderm. In the future, it is expected that many additional genes will be joined into this pathway. For example, the Sox-domain-containing gene sox-neuro is expressed throughout the entire neuroectoderm and it may exhibit region-specific effects in the neuroectoderm in a manner similar to that proposed for Dichaete. In addition, the Ras-pathway antagonist yan is expressed in the lateral half of the neuroectoderm during early neurogenesis and may help regulate pattern and cell fate in this domain. A complete understanding of the genetic and molecular mechanisms that pattern the neuroectoderm requires the identification of all such genes and the elucidation of how these genes interact to regulate cell fate along the DV axis of the neuroectoderm (Zhao, 2002).

Egfr and neurogenesis part 2/2

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