Transcriptional interpretation of the EGF receptor signaling gradient

Epidermal growth factor receptor (EGFR) controls a wide range of developmental events, from body axes specification in insects to cardiac development in humans. During Drosophila oogenesis, a gradient of EGFR activation patterns the follicular epithelium. Multiple transcriptional targets of EGFR in this tissue have been identified, but their regulatory elements are essentially unknown. This study reports the regulatory elements of broad (br) and pipe (pip), two important targets of EGFR signaling in Drosophila oogenesis. br is expressed in a complex pattern that prefigures the formation of respiratory eggshell appendages. This pattern is generated by dynamic activities of two regulatory elements, which display different responses to Pointed, Capicua, and Mirror, transcription factors involved in the EGFR-mediated gene expression. One of these elements is active in a pattern similar to pip, a gene repressed by EGFR and essential for establishing the dorsoventral polarity of the embryo. This similarity of expression depends on a common sequence motif that binds Mirror in vitro and is essential for transcriptional repression in vivo (Fuchs, 2012).

Current models of pattern formation in Drosophila oogenesis involve multiple components, signaling pathways, and network motifs. Critical tests of these models require direct analysis of the cis-regulatory sequences of genes comprising the network. As a first step in this direction, this study identified the regulatory elements of br, a gene that plays a key role in eggshell patterning and morphogenesis. The dynamic pattern of br was found to be generated by superposition of the activities of two distinct regulatory regions, which drive br expression in nonoverlapping regions of space and display differential sensitivity to three transcription factors that act downstream of EGFR (Fuchs, 2012).

It was shown that loss of Mirr induces ectopic br expression in the dorsal midline follicle cells, but leads to a complete loss of br in the lateral cells, which form dorsal appendages. This region-specific effect can be now explained, and is fully consistent with our finding that Mirr represses the brE and activates brL regions, respectively. Previous studies suggest that Mirr functions as a dedicated repressor. Based on this theory, it is speculated that the activating effect of Mirr on the expression of the brL region is indirect and involves intermediate factors. In contrast, these results strongly suggest that Mirr represses the brE region directly (Fuchs, 2012).

In contrast to the brL region, which generates br expression in a two-domain pattern that is necessary for the formation of two eggshell appendages, the function of the brE region is unclear. At the same time, this regulatory region was instrumental in identification of a critical cis-element that controls the expression of pip, a gene which must be repressed in the dorsal follicle cells for proper induction of the DV polarity of the embryo. The regulatory regions of both br and pip contain a sequence essential for their transcriptional restriction to the ventral follicle cells. Moreover, the data suggest that the identified sequence is a direct sensor of Mirr, which is derepressed by EGFR. Thus, thus this study has upheld an earlier proposal that Mirr connects the EGFR-mediated patterning of the follicle cells to the DV patterning of the embryo. In the emerging transcriptional cascade, EGFR signaling down-regulates CIC, which derepresses Mirr, which in turn represses pip (Fuchs, 2012).

Previous studies have demonstrated that Mirr can repress pip, but suggested that this effect requires a relay mechanism. The current results, based on marked mirr overexpression clones, demonstrate that the effect is cell-autonomous. Other studies argue against Mirr-dependent pip repression, based on the fact that mirr mutant clones did not induce ectopic expression of pip. These results may be because of the fact that the mirr allele that was used is not a complete null and has residual activity sufficient for pip repression. It is argued that the current data, demonstrating pip derepression by deletion of a sequence that binds Mirr, provide a strong support for Mirr-dependent repression of pip. Thus, these findings close a long-standing gap in the chain of events that convert EGFR signaling to pipe repression, a key step in transmitting the DV polarity from the egg to the embryo (Fuchs, 2012).

EGFR-dependent patterning of the follicle cells and the resulting effects for patterning of the embryo represent canonical examples of inductive effects in development. Indeed, genetic connection between EGFR signaling and pipe repression are found in essentially all textbooks of development. However, as discussed above, the identity of transcription factors involved in pipe regulation remained controversial and the cis-regulatory sequences responsible for pipe repression were unknown. The current results, which established Mirr as a direct repressor or pipe and identified the regulatory element responding to Mirr, clearly change this status. Thus, the results provide a significant addition to a very important model of inductive signaling. The regulatory element of pipe was discovered using an approach that harnesses both conventional and modern techniques of gene regulation research and can be extended to other transcriptional targets of EGFR pathway in the follicle cells. Finally, it is noted that most of the available information on the transcriptional effects of EGFR signaling is related to gene activation (mediated by Pnt) or derepression (mediated by Cic). The current work reveals a mechanism for EGFR-dependent gene repression, mediated by Mirr. Given the central role played by the EGFR signaling in development, the identified regulatory sequences can shed light on other EGFR-dependent pattern formation events (Fuchs, 2012).

Molecular mechanisms of EGF signaling-dependent regulation of pipe, a gene crucial for dorsoventral axis formation in Drosophila

During Drosophila oogenesis the expression of the sulfotransferase Pipe in ventral follicle cells is crucial for dorsoventral axis formation. Pipe modifies proteins that are incorporated in the ventral eggshell and activate Toll signaling which in turn initiates embryonic dorsoventral patterning. Ventral pipe expression is the result of an oocyte-derived EGF signal which down-regulates pipe in dorsal follicle cells. The analysis of mutant follicle cell clones reveals that none of the transcription factors known to act downstream of EGF signaling in Drosophila is required or sufficient for pipe regulation. However, the pipe cis-regulatory region harbors a 31-bp element which is essential for pipe repression, and ovarian extracts contain a protein that binds this element. Thus, EGF signaling does not act by down-regulating an activator of pipe as previously suggested but rather by activating a repressor. Surprisingly, this repressor acts independent of the common co-repressors Groucho or CtBP (Technau, 2012).

The transcriptional regulation of pipe is crucial for establishing the dorsoventral axis of the embryo. pipe is down-regulated in dorsal follicle cells by Grk, a TGFα-like ligand which is localized close to germinal vesicle at a dorsal-anterior position of the oocyte. Local Grk secretion from the oocyte leads to an anteroposterior (AP) and dorsoventral (DV) gradient of Grk uptake in the follicular epithelium. Quantitative analyses have suggested that the resulting two-dimensional profile of EGF signaling is sufficient to explain the spatial pattern of pipe expression, which in turn determines the DV axis of the embryo. This is a remarkable result since the DV axis is established simultaneously along the entire length of the embryo in Drosophila. Thus, all positions along the AP axis of the embryo require precise DV patterning information prior to gastrulation. This places high demands on the accuracy of the system providing DV spatial information, i.e., on the transcriptional regulation of pipe. Although previous experiments have shown that some variation in width of the pipe domain is compatible with normal DV axis formation in the embryo, even slight variations along the AP axis cause severe embryonic defects (Technau, 2012).

Besides its accuracy with regard to the future embryonic axis, there is another remarkable feature of pipe regulation. The transcription of pipe shows a sharp on-off pattern in lateral regions of the follicular epithelium. Although it has been shown that EGF signaling extends even to the ventral side of the egg chamber the distribution of active MAPK indicates that the signaling levels are very low in the lateral regions of the follicular epithelium where the border of the pipe domain resides. This poses the additional question of how low levels of EGF signaling resulting in a shallow gradient of MAPK activation lead to a sharp transcriptional response. To address this point, a signaling relay had been suggested. However, later work has shown that EGF signaling controls pipe in cell-autonomous way. This might occur either through transcription factors which are a direct target of MAPK phosphorylation or through another tier of transcriptional regulation. According to the latter, alternative EGF signaling could either activate the transcription of a repressor or repress that of an essential activator (Technau, 2012).

Two experimental strategies were followed to approach these questions. (1) Clonal analyses was performed with mutants for transcription factors which have been implicated in EGF signaling in Drosophila or which have been suggested to be specifically involved in pipe regulation. (2) cis-regulatory sequences responsible for pipe regulation in the follicular epithelium were analyzed (Technau, 2012).

Using clonal analysis, the roles of the two Ets domain proteins, Pointed and Yan, which are targets of MAPK phosphorylation in many tissues, were investigated. pnt has been shown to play a role in follicle cell patterning, where it is required to establish the dorsal midline cell fate, which separates the two dorsal appendages. Eggs carrying pnt clones support embryonic development leading to larvae with no obvious DV patterning defects. This suggests that pnt has no major influence on pipe. However, previous studies did not analyze pipe expression and thus could not rule out subtle effects on pipe, e.g., on the precision of pipe repression in lateral regions. Clonal analysis shows that despite its effect on dorsal follicle cell patterning, pnt lacks any detectable influence on pipe. The same applies to the Ets domain protein Yan which normally acts as a repressor in conjunction with Pnt (Technau, 2012).

An influence on embryonic DV patterning had been proposed for the zinc finger transcription factor CF2. Follicle cell expression of antisense or sense CF2 constructs apparently resulted in DV patterning defects in the embryo, making CF2 a likely candidate for a transcription factor controlling pipe. In dorsal follicle cells, EGF signaling leads to cytoplasmic retention and degradation of CF2, while CF2 accumulates in the nuclei of lateral and ventral follicle cells. Accordingly, CF2 might be an activator of pipe, and the down-regulation of this factor would determine the lateral pipe border. This assumption, however, was never rigorously tested. Clonal analysis reveals that CF2 is not involved in pipe regulation. The same applies for the zinc finger transcription factor Ttk, which is expressed in the follicular epithelium and has been implicated in EGF signaling in other tissues (Technau, 2012).

The most likely candidate for EGF-mediated pipe regulation is the HMG-box protein Cic. cic mutant flies produce egg chambers with an anterior ring of dorsal follicle cells and lack of pipe expression. The expansion of dorsal follicle cells in cic mutant egg chamber is accompanied by ectopic expression of mirror in the anterior half of the follicular epithelium. Clonal analysis shows that cic represses mirror in a cell-autonomous manner. Cic function is down-regulated by EGF signaling through the prevention of nuclear accumulation of Cic in dorsal follicle. Thus, the dorsal follicle cell fate is established by EGF-dependent repression of repressor. Clonal analysis shows that cic is also required for pipe expression in a cell-autonomous manner. Thus, one could imagine that EGF signaling-dependent down-regulation of cic in dorsal follicle cells accounts for spatial regulation of pipe. Although this cannot be strictly excluded, the temporal and spatial profile of nuclear Cic accumulation are not in agreement with this suggestion. In particular, Cic is present uniformly in the nuclei of lateral follicle cells spanning the region where the sharp on-off boundary of pipe expression resides (Technau, 2012).

Since the clonal analysis of candidate genes presented in this paper did not lead to the identification of the crucial pipe regulators, promoter analysis of the pipe gene was carried out. The main result of this analysis is the finding that the spatial regulation of pipe is due to transcriptional repression rather than to the down-regulation of an activator. The cis-regulatory module (CRM) driving pipe expression consists of a repressor element of about 30 bp followed by approximately 100 bp which harbor essential activator binding sites. Ovarian extracts contain a protein which binds to the repressor element (Technau, 2012).

The constructs affecting the repressor element resulted in global de-repression along the entire AP axis of the egg chamber, suggesting that a single repressor binds to the element. However, the constructs affecting the activator binding sites showed region-specific effects. For example, partial pipe expression at the posterior of the egg chamber was observed for some constructs. Other constructs resulted in loss of medial expression while anterior and posterior expression was maintained. These findings indicate that the part of the pipe CRM which harbors the activator binding sites has a modular structure with separate binding sites and distinct transcription factors being responsible for the anterior, medial, and posterior subregions of pipe expression. Similar results have been described previously for the cis-regulatory region of the chorion gene s36 (Technau, 2012).

Regarding the function of the pipe repressor element, several alternatives can be envisaged. Transcriptional repressors have been subdivided into long-range and short-range repressors. Long-range repressors function over distances of at least 500 bp by inhibiting activators bound to CRMs or by directly blocking the basal transcription machinery. The factor binding to the pipe repressor element is unlikely to work as long-range repressor since the repressor element loses its function when it is separated from the activation domain. Both a distal and proximal shift of the element by 400 and 1,000 bp, respectively, led to complete de-repression of pipe. Thus, the pipe repressor appears to act in short-range manner (Technau, 2012).

Different modes of short-range repression have been described, which can be distinguished on the basis of the spatial organization of activator and repressor binding sites. In one scenario, repressors and activators directly compete for overlapping binding sites. In this case, the deletion of the common binding sites leads to a complete loss of expression. This mode of competitive binding can be excluded, due to the uniform expression in all follicle cells which arises as a consequence of the mutation or deletion of the repressor element in the pipe CRM (Technau, 2012).

Two other modes of short-range repression are known as quenching and direct repression. In the case of quenching, the repressors and activators bind simultaneously at independent binding sites and the repressors inhibit the interaction of the activators with the general transcription machinery. In contrast to this, direct or active repression involves repressors, which directly target the general transcription machinery. Both of these mechanisms could apply for the repression of pipe. However, it has been shown that in the case of direct repression, the repressor has to bind in close proximity. The distance of the identified pipe repressor element to the core promoter, however, exceeds 1,000 bp making direct repression an unlikely mechanism. In addition, positioning the element next to the transcription start site did not lead to repression (Technau, 2012).

For short-range repressors acting during embryogenesis, quenching has been reported to be the most prevalent mode. Quenching leads to the inhibition of every activator bound in a distance of up to 100 bp surrounding the repressor binding site. In addition, the specificity of the repression depends mainly on the position of the bound repressor and not on the type of activator. This fits to the results of the pipe promoter analysis, as extensive de-repression affecting all follicle cells was detected, although the observed partial expression patterns suggest that independent activators are required for different subdomains of the follicular epithelium (Technau, 2012).

Quenching requires the presence of co-repressors which mediate the interaction between the repressors and the activators bound to independent sites. Groucho and CtBP are among the most widely studied co-repressors. While Groucho mediates both long-range and short-range repression, CtBP locally interferes with neighboring activators. Surprisingly, neither Groucho nor CtBP are involved in pipe regulation. Thus, it is predicted that the molecular mechanisms of short-range repression functioning within the pipe CRM are different from those cases which have been studied most intensively in the early embryo (Technau, 2012).

In summary, this analysis of pipe regulation provides a solid basis for future studies on the molecular mechanisms of EGF signaling-dependent transcriptional repression. In addition, the systematic manipulation of the pipe CRM allows the generation of transgenes which change the expression pattern of pipe independent from EGF signaling. In the past, ectopic expression of pipe has been achieved only with the help of heat-shock constructs or the GAL4-UAS system. These experiments had the disadvantage that they did not reproduce endogenous levels of pipe expression. For example, no experiment has been reported so far which shows the consequences of uniform expression of endogenous pipe on the embryonic DV patterning. However, such an experiment would be of pivotal importance for understanding the self-organizing processes which occur downstream of pipe and lead to the formation of the embryonic nuclear dorsal gradient that establishes the pattern of cell-fates along the embryonic DV axis (Technau, 2012).

Transcriptional Regulation

Recent studies in vertebrates and Drosophila have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. This may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis. Ectopic Notch activation has a similar long-range effect on pip. These results suggest that Mirror and Notch induce secretion of diffusible morphogens; a TGF-beta (encoded by dpp) has been identified as one such molecule in the germarium. mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and mirr then represses pipe expression in all but the ventral follicle cells, connecting Egfr activation in the dorsal follicle cells to repression of pipe in the dorsal and lateral follicle cells. These results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signaling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation (Jordan, 2000).

In a number of developmental systems, regulation of fng by a homeobox gene has a role in establishing a domain in which Notch is activated. Thus the phenotypes observed in mirr and Notch (N) mutants during oogenesis have been compared. In oogenesis, Notch activity is required in the germarium and for the formation of the termini at stage 6. A test was performed to see whether Notch function is also required for dorsal-ventral patterning of follicle cells by analysing the eggs laid by Nts females at the restrictive temperature. The strongest phenotype observed in eggs laid by Nts females is similar to that observed in eggs laid by mirr loss-of-function females: a complete loss of the dorsal appendages. In addition, the ventral pipe expression domain is defective in Nts females and restricted due to expression of constitutively active Notch. Thus Notch, like Mirr, functions to restrict pipe expression to the ventral region and to organize dorsal structures; loss of either Mirr or Notch function affects follicle cells on both sides of the Mirr-Fng expression border (Jordan, 2000).

Activation of Notch at a fng expression border has been observed in wing and eye development. In the wing this border acts as an organizing center by producing a morphogen, Wingless, that acts on cells on both sides of the border. At stage 9 in oogenesis the mirr-fng expression border and a region of localized Notch activation are approximately 10 cell diameters from the ventral pip expression border. Nevertheless, reduction of mirr expression expands the pip domain laterally. If a Mirr-Fng border activates Notch locally to produce a morphogen that represses pip, a reduction of pip expression should be seen upon expansion of the mirr expression domain or ectopic activation of Notch. To examine this, mirr was expressed ectopically in anterior follicle cells. pip repression occurs 5-7 cell diameters beyond the mirr expression domain, showing that the effect of Mirr on pip is non-cell autonomous and supporting the idea that a Mirr-Fng border generates a pip-repressing agent. To further test the effect of ectopic Mirr expression, Mirr was expressed in the posterior follicle and the effect on pip and rho, which is normally expressed as two stripes on the dorsal region at stage 10, was tested. Such ectopic mirr expression induces a ring of rho expression and represses pip at a distance. Expression of constitutively active Notch in the posterior follicle cells also represses pip expression at a distance. These results suggest that Mirr and Notch induce secretion of a diffusible molecule that represses pip. Although it is not known what the Notch-dependent diffusible molecule is at stage 9, it was found that dpp is expressed in follicle cells in the mid-germarium near a stripe of cells showing localized Notch activity in a Notch-dependent manner. Furthermore, in follicle cell clones of MAD or MEDEA (downstream effectors of the Dpp pathway), encapsulation defects of 16-cell cysts are seen. This phenotype is similar to Notch- and mirr-mutant phenotypes in the germarium, suggesting that Dpp may be a morphogen induced by Notch activity in the germarium (Jordan, 2000).

Results from several developmental systems have led to the idea that the trio of a homeobox gene, FNG and Notch are fundamental to organogenesis. It is suggested that Mirr, Fng and Notch are part of a conserved mechanism for dividing epithelial cell layers into domains; it is thought that such a mechanism is not restricted to organogenesis. Furthermore, the data suggest that Mirr integrates the Egfr and Notch pathways in oogenesis: mirr transcription is induced by the Egfr pathway, and Mirr in turn spatially regulates fng expression leading to a Notch activation border. Finally, it is proposed that the link between Egfr pathway signaling in the dorsal follicle cells and the differentiation of the ventral follicle cells suggested by genetic studies is mediated by Mirr. The Egfr pathway induces mirr expression, which leads to creation of a Notch-Fng border in lateral follicle cells from which molecules are secreted that repress pipe expression. Pipe regulates the activity of a protease cascade that activates Toll and ultimately determines the dorsal-ventral pattern of the Drosophila embryo. These data show that expression of pip in the ventral follicle cells is not a direct consequence of a graded germline signal by Gurken, but depends on Mirr-dependent long-range signals from dorsal follicle cells. Mirr therefore connects the well-studied events in early and late Drosophila dorsal-ventral axis formation (Jordan, 2000).

The Mirror transcription factor links signalling pathways in Drosophila oogenesis

At stage 10 of oogenesis, mirror is expressed in anterior-dorsal follicle cells, and this is dependent upon the Gurken signal from the oocyte. The fringe gene is expressed in a complementary pattern in posterior-ventral follicle cells at the same stage. Ectopic expression of mirror represses fringe expression, thus linking the epidermal growth factor receptor (Egfr) signaling pathway to the Fringe signaling pathway via Mirror. The Egfr pathway also triggers the cascade that leads to dorsal-ventral axis determination in the embryo. twist was used as an embryonic marker for ventral cells. Ectopic expression of mirror in the follicle cells during oogenesis ultimately represses twist expression in the embryo, and leads to phenotypes similar to those that occur due to the ectopic expression of the activated form of Egfr. Thus, mirror also controls the Toll signaling pathway, leading to Dorsal nuclear transport. In summary, the Mirror homeodomain protein provides a link that coordinates the Gurken/Egfr signaling pathway (initiated in the oocyte) with the Fringe/Notch/Delta pathway (in follicle cells). This coordination is required for epithelial morphogenesis, and for producing the signal in ventral follicle cells that determines the dorsal/ventral axis of the embryo (Zhao, 2000).

One of the dorsal group of maternal genes, pipe, is negatively regulated by mirr since pipe expression is expanded in mirr minus clones. Since pipe is required for the activation of twi via Dorsal, it is likely that mirr affects twi expression by repressing the expression of pipe in dorsal follicle cells. The downstream targets of mirr, in addition to pipe, need to be further identified to understand how mirr executes these developmental decisions in response to Grk/Egfr signaling. It is possible that mirr is required for the repression of pipe and windbeutel (wind) along with fng in the anterior-dorsal follicle cells. The ventrally-localised activities of these genes then cooperatively generate an extracellular ventral signal. There is evidence that CF2, which is expressed in the ventral and posterior cells in a pattern similar to fringe, regulates pipe and wind, and is responsible for the ventral signal produced in follicle cells that determines the embryonic axis. One might speculate that mirr could repress CF2 in anterior/dorsal follicle cells. However, it is observed that CF2 transcripts are expressed in the mirr expressing cells, and the ventrally-localized protein distribution of CF2 is translationally regulated. Since mirr encodes a transcription factor it cannot be directly responsible for the lack of CF2 protein in anterior/dorsal follicle cells, but could indirectly cause a repression of its translation in the anterior-dorsal cells via another gene. Alternatively CF2 protein could be upstream of mirror expression and repress transcription in ventral cells (Zhao, 2000).

D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis

During Drosophila oogenesis, asymmetrically localized Gurken activates the EGF receptor (Egfr) and determines dorsal follicle cell fates. Using a mosaic follicle cell system a mutation has been identified in the Cbl gene that causes hyperactivation of the Egfr pathway. Cbl is required in ventral follicle cells to ensure that ventral patterning occurs correctly in the embryo. Cbl proteins are known to downregulate activated receptors. The abnormal Egfr activation is ligand dependent. These results show that the precise regulation of Egfr activity necessary to establish different follicle cell fates requires two levels of control. The localized ligand Gurken activates Egfr to different levels in different follicle cells. In addition, Egfr activity has to be repressed through the activity of Cbl to ensure the absence of signaling in the ventral most follicle cells (Pai, 2000).

To examine the Cbl expression pattern in the ovary, in situ hybridization experiments were performed. Cbl mRNA is detected both in nurse cells and in follicle cells that are associated with the oocyte. The high levels of expression in nurse cells may indicate a maternal contribution of Cbl to the embryo: this is consistent with its presence at the blastoderm stage. To test the requirement for Cbl in embryonic development, germline clones for Cbl were generated using heat shock Flipase and the ovoD1 system. A distinct head defect is observed in embryos lacking both maternally and zygotically contributed Cbl. Zygotic Cbl is able to rescue the maternal lack of Cbl. This results in normal embryos that hatch. A small percentage of embryos had dorsalization phenotypes, which is likely due to the simultaneous generation of follicle cell clones induced by the heat shock Flipase. Egfr signaling is also required for ventral ectoderm development during embryogenesis. However, there were no other cuticle phenotypes detected in Cbl germline clone embryos, which suggests that Egfr signaling in cuticle development is not severely affected by loss of Cbl. In particular, no obvious segmentation defects, that would have suggested hyperactivity of the torso pathway, were detected. Taken together, these results demonstrate that in some signaling pathways involving receptor tyrosine kinases, loss of Cbl has no visible phenotypes, but in processes that are very sensitive to levels of receptor activity, such as in the follicle cells, a mutation of Cbl has dramatic effects (Pai, 2000).

Ventral follicle cell clones mutant for Cbl produce a dorsalized embryo phenotype. Since restricted pipe expression in the follicle cells is of crucial importance for embryonic patterning, pipe expression was examined in mutant clones by RNA in situ hybridization on ovaries. pipe expression is detected in ventral follicle cells in wild-type stage 9 egg chambers and persists until stage 10B. When egg chambers were examined that contained follicle cell clones mutant for Cbl, pipe expression was found to be abolished in ventral mutant clones. Interestingly, there is a sharp boundary between cells with and without pipe expression. Since Egfr activity is elevated in Cbl mutant cells in a cell autonomous manner, the effects of Egfr signaling on pipe expression could be directly examined. The wild-type cells were marked with a c-myc antibody and simultaneously pipe in situ hybridization was carried out. There was an exact correspondence between the absence of pipe expression and the absence of c-myc staining on the ventral side of stage 9 or 10A egg chambers. This demonstrates that the ectopic Egfr activation in the mutant clones suppresses pipe expression in a cell autonomous fashion. Since pipe expression in ventral follicle cells is required for ventral cell fate determination in the embryo, these results also suggest that the elimination of pipe expression in mutant clones can account for the dorsalization phenotype of the resulting embryo. The cell by cell correspondence of mutant clones and pipe expression further implies that the Egfr pathway can downregulate pipe expression directly, rather than acting via a secondary, diffusible signal that would repress pipe at a distance (Pai, 2000).

Along the dorsoventral axis, there are four types of follicle cells that can be distinguished by their gene expression patterns as well as their imprints on the egg shell: the dorsal midline cells, which express argos and are located between cells that will give rise to two dorsal appendages; the dorsolateral cells, which express Broad-Complex and secrete the appendages; the ventral cells that express pipe; and the lateral cells located between ventral and dorsolateral cells. The different embryonic and egg shell phenotypes that were generated by follicle cell clones mutant for Cbl have demonstrated that all of the thresholds that define these cell populations are affected by Cbl. In follicle cells lacking Cbl, Egfr signaling is elevated. In ventral follicle cells, this level of elevation of Egfr signaling is sufficient to suppress pipe expression, and results in dorsalized embryos. However, this ectopic activation of Egfr in ventral mutant cells is not sufficient to induce dorsal appendage formation. A higher level of activity is required to reach the threshold necessary to produce dorsal appendages. Only Cbl mutant follicle cells in lateral regions close to dorsal appendages can produce ectopic dorsal appendages. Furthermore, dorsal midline cell fates, which initially have the highest level of Egfr activity, can only be induced in cells next to the dorsal midline, since the expansion of argos expression was observed in mutant cells adjacent to the dorsal midline cells, but not in ventral follicle cells. However, it is known that high levels of uniform Egfr activity can transform all follicle cells into dorsal midline or dorsolateral cell fates. Therefore, in the absence of Cbl, an underlying Egfr activity gradient exists in follicle cells along the dorsoventral axis, which needs to be regulated by the activity of Cbl (Pai, 2000).

A pivotal question of dorsoventral patterning in the egg chamber is how Gurken/Egfr signaling on the dorsal side of the follicular epithelium determines ventral follicle cell fates, which in turn establish the ventral cell fates in the embryo. The highly asymmetric distribution of Gurken along the dorsoventral axis had led to the model that the Egfr is only activated in a relatively small population of dorsal follicle cells along the dorsal midline. Genetic mosaic studies of the activity of pipe and wind in follicle cell epithelium have demonstrated that there is a ventral region in the follicle cell epithelium, comprising about one third of the circumference required for the establishment of the embryonic ventral cell fates. It had appeared therefore that there might be a considerable distance between the dorsal Egfr signaling and the ventral zone of pipe activity, suggesting the possibility that a secondary signal induced by Egfr signaling might be involved. This study of Cbl in axis formation has, however, shown that Egfr signaling itself appears to directly determine the size of the ventral pipe expressing region. From these results it appears that all follicle cells have some Gurken-dependent Egfr activity, but that this occurs at different levels along the dorsoventral axis. In this system, the essential role of Cbl is to control the level of Egfr signaling by destroying activated Egfr in order to keep the Egfr activity below certain thresholds, thus ensuring the full range of follicle cell fates. This conclusion is supported by the observation that ectopic kek expression depends on the presence of Gurken. In addition, the fact that in the Cbl mutant clones, pipe expression is repressed in a cell autonomous manner, shows that Egfr activity affects ventral patterning directly, and not at a distance. In the absence of Cbl, the low levels of Egfr signaling triggered by Gurken in ventral follicle cells are sufficient to repress pipe expression, which leads to the loss of embryonic ventral cell fates (Pai, 2000).

Two possible models have been suggested for the establishment of a gradient of Egfr activity by Gurken. One is that asymmetrically localized Gurken activates Rhomboid, which then processes and activates Spitz, another Egfr ligand. Activated Spitz or other unidentified ligands might diffuse away from the dorsal source and establish a gradient of Egfr activity. The alternative model is that Gurken distribution is broader than it appears, judging from antibody staining, and that some Gurken actually reaches the ventral side of the egg chamber and represses pipe expression directly. This could involve either diffusion of processed Gurken in the space between the follicle cells and the oocyte membrane, or it could involve diffusion of membrane tethered Gurken protein within the oocyte membrane. Mutant follicle cell clones for spitz cause only relatively minor defects in egg shell morphology, but not in embryonic development. This argues against Spitz acting as the global cell fate determinant. Therefore, the favored model is that Gurken is present in a graded distribution in the egg chamber and activates Egfr in ventral follicle cells. Similar to the results from the analysis of spitz and argos clones, defects in dorsal follicle cell patterning have no effects on embryonic patterning, since embryos develop normally and hatch out of eggs containing dorsal Cbl mutant clones. This further supports the model that the ventral region responsible for the ventral cell fate establishment in the embryo is directly defined by the Gurken signal, and that the second phase of Egfr signaling involving amplification through Rho, Spitz, and Argos only affects egg shell morphology, but not the dorsoventral axis of the embryo. Cbl activity is required in all follicle cells along the dorsoventral axis, and affects both patterning events. Its activity essentially acts as a sink that ensures that the full range of the Egfr activity gradient is reliably achieved and/or maintained in the follicle cell epithelium (Pai, 2000).

Spätzle regulates the shape of the Dorsal gradient in the Drosophila embryo

Dorsal-ventral polarity of the Drosophila embryo is established by a nuclear gradient of Dorsal protein, generated by successive gurken-Egfr and spätzle-Toll signaling. Overexpression of extracellular Spätzle dramatically reshapes the Dorsal gradient: the normal single peak is broadened and then refined to two distinct peaks of nuclear Dorsal, to produce two ventral furrows. This partial axis duplication, which mimics the ventralized phenotype caused by reduced gurken-Egfr signaling, arises from events in the perivitelline fluid of the embryo and occurs at the level of Spätzle processing or Toll activation. The production of two Dorsal peaks is addressed by a model that invokes the action of a diffusible inhibitor, which is proposed to normally regulate the slope of the Dorsal gradient (Morisato, 2001).

The shape of the Dorsal gradient is dramatically changed in embryos laid by females carrying mutations in the gurken-Egfr signaling pathway. Not only do these embryos expand Twist expression, as a consequence of a reduction in the dorsalizing signal that establishes egg chamber asymmetry, but they exhibit two distinct peaks within the Twist domain that give rise to two ventral furrows. In the experiments described here, this partial axis duplication is not evident during oogenesis, because pipe RNA was found to be expressed in a single broad domain in follicle cells. The production of two Dorsal peaks could be mimicked by injecting high levels of spz RNA into the pre-cellular embryo cytoplasm, suggesting that pattern refinement occurs during embryogenesis. It is suggested that while the size of the ventral domain is expanded in grk and Egfr ovarian egg chambers, the partial axis duplication observed in mutant embryos is caused by reactions occurring later in the embryo (Morisato, 2001).

It may have been easier to imagine how the selection of one or two gradient peaks would involve signaling within the follicular epithelium, because spatial information could then be stably maintained and transmitted by cells. The elaboration of the two dorsal appendages in the Drosophila eggshell results from a series of such intercellular signaling events. Activation of Egfr by Gurken stimulates transcriptional induction of Argos, a secreted Egfr inhibitor, which then downregulates Egfr activity in the initial central domain, leaving two lateral domains of signaling (Morisato, 2001).

In fact, the findings described in this paper argue that events involving the diffusion of an extracellular morphogen not only regulate the gradient slope, but perhaps unexpectedly, determine the position and number of maxima within the axis in response to the broad cues generated during oogenesis. Reaction-diffusion models have been applied to analyze the respective contributions of the gurken-Egfr and spätzle-Toll pathways in generating embryonic pattern. The current studies provide experimental support for this theoretical work, and present opportunities for understanding the underlying mechanisms (Morisato, 2001).

Formation and maintenance of the Dorsal gradient appear dynamic. The shape of the Dorsal gradient in the wild-type embryo does not change markedly after nuclear translocation is first detected. In embryos laid by grk females or embryos expressing high levels of Spätzle, however, the shape of the Dorsal gradient is subtly modified. In particular, the minimum lying between the two Dorsal peaks becomes deeper in older embryos. This observation suggests that signaling takes place over a period of time, and explains how an initial asymmetry, in the form of the broad stripe of pipe, might be gradually refined into a gradient of positional information (Morisato, 2001).

In embryos produced by grk females, it is inferred that Spätzle processing is occurring at wild-type levels, but the reaction is distributed over a broader domain. The ventral region becomes sufficiently expanded such that the difference between the diffusion rates of processed Spätzle and the inhibitor can reshape the ventral domain itself. In particular, rapid diffusion of the inhibitor results in a lower concentration at each border, compared with the center of the domain. This change in the ratio of processed Spätzle to inhibitor eventually produces a peak at each border of the expanded domain. By this reasoning, an expanded ventral domain never generates more than two peaks because there are never more than two borders (Morisato, 2001).

Embryos synthesizing high levels of precursor Spätzle increase the amount of processed Spätzle, thereby expanding the domain of high nuclear Dorsal. In contrast to embryos produced by grk females, where a wild-type level of processed Spätzle is distributed over a broader area, an increased level of processed Spätzle appears to generate a broader domain in these injected embryos. Pattern refinement is observed only at the highest levels of Spätzle production, perhaps because only in this situation can the minimum domain size be created (Morisato, 2001).

The complexity of the patterning process is underscored by the observation that partial axis duplication can be induced by both an increase and decrease in spz dosage, depending on the extent of pipe expression dictated by gurken-Egfr signaling. A deeper understanding of this dynamic behavior will probably require the application of mathematical approaches (Morisato, 2001).

Evidence is presented for the following model, which accounts for many of the observations described above. The initial shape of the gradient (at t0) is established by the proteolytic activation of Spätzle in a relatively broad domain, reflecting the ventral region of the egg chamber that expresses pipe RNA. It is proposed that the Spätzle processing reaction generates an inhibitor that negatively regulates the production of the ventral signal, possibly at the level of Easter protease activity or the interaction between processed Spätzle and Toll. Whereas processed C-terminal Spätzle is believed to bind to Toll quickly and show limited movement after cleavage, it is postulated that the hypothetical inhibitor undergoes broader diffusion. In the wild-type embryo, inhibitor action is responsible for establishing the region of high nuclear Dorsal, corresponding to the Twist domain, to be narrower than the ventral region of the egg chamber expressing pipe RNA. The final shape of the Dorsal gradient (at t1) is generated over time by the opposing effects of processed Spätzle and the inhibitor (Morisato, 2001).

Establishment of dorsal-ventral polarity of the Drosophila egg requires capicua action in ovarian follicle cells

The dorsal-ventral pattern of the Drosophila egg is established during oogenesis. Epidermal growth factor receptor (Egfr) signaling within the follicular epithelium is spatially regulated by the dorsally restricted distribution of its presumptive ligand, Gurken. As a consequence, pipe is transcribed in a broad ventral domain to initiate the Toll signaling pathway in the embryo, resulting in a gradient of Dorsal nuclear translocation. Expression of pipe RNA requires the action of fettucine (fet) in ovarian follicle cells. Loss of maternal fet activity produces a dorsalized eggshell and embryo. Although similar mutant phenotypes are observed with regulators of Egfr signaling, genetic analysis suggests that fet acts downstream of this event. The fet mutant phenotype is rescued by a transgene of capicua (cic), which encodes an HMG-box transcription factor. Cic protein is initially expressed uniformly in ovarian follicle cell nuclei, and is subsequently downregulated on the dorsal side. Earlier studies described a requirement for cic in repressing zygotic target genes of both the torso and Toll pathways in the embryo. cic controls dorsal-ventral patterning by regulating pipe expression in ovarian follicle cells, before its previously described role in interpreting the Dorsal gradient (Goff, 2001).

A class of dominant suppressors of a weakly ventralizing mutation (spzD1) were isolated in a dysgenic screen. These suppressors map to 92D and define a new locus that has been termed fettucine (fet). The fetE11 mutation is a representative allele caused by the insertion of a P element. In a subsequent screen, the EMS-induced alleles fetT6 and fetU6 were generated. While embryos laid by spzD1/+ females were ventralized and failed to hatch, about 10% of eggs laid by fet spzD1/++ females hatched. Flies carrying the fetE11 allele are viable as homozygotes and as transheterozygotes with fetU6 and fetT6, and these females exhibit a recessive maternal effect phenotype in which the eggshell and embryo are dorsalized. The fetU6 allele behaves genetically like a null allele and is larval lethal, while the fetT6 allele is a strong hypomorph, and homozygotes die as pharate adults (Goff, 2001).

The fet eggshell morphology is dorsalized, as assessed by a lateral shift of broadened dorsal appendages. In the strongest mutant combinations, ectopic dorsal appendage base material is secreted around the anterior circumference of the egg. Embryos produced by fet mutant females (referred to as fet embryos) exhibit an expansion of dorsal cell fates around the circumference of the embryo. These fet embryos fail to hatch. They secrete a cuticle which consistes entirely of dorsal epidermis lacking any structures derived from lateral or ventral regions. This cuticular phenotype is preceded at the cellular blastoderm stage by expanded expression of the dorsal marker zen around the circumference of the embryo at the expense of the expression of the ventrolateral and ventral markers sog and Twist (Goff, 2001).

Both the production of the Grk signal in the oocyte and initial activation of the Egfr in follicle cells appear unaffected in mutant fet ovaries. As a consequence of Egfr signaling, the follicular epithelium is normally partitioned into dorsal and ventral domains through the spatially restricted expression of mirror and fringe transcripts, respectively. Concomitant with this early response, Egfr modulates its own signaling during the course of oogenesis by inducing the expression of genes (e.g. rho, kek1 and Cbl) that encode regulators, which act at the level of receptor activation. At early stage 10, which corresponds to the period when Egfr activation leads to the transcription of target genes, the expression patterns of mirror, fringe, rho and kek1 in mutant fet ovaries are indistinguishable from wild type (Goff, 2001).

The dorsalized phenotype observed with the loss of fet activity in follicle cells is apparently caused by a requirement for fet in each branch of the Egfr pathway that separately patterns the embryo and eggshell. In establishing DV polarity of the embryo, fet is required as an essential transcriptional regulator of pipe RNA expression in the ventral follicle cells. By epistasis analysis, fet acts downstream of Egfr and causes a dorsalized phenotype even when Egfr signaling is reduced (Goff, 2001).

In establishing DV polarity of the eggshell, fet appears to be involved in refining Egfr activity later during oogenesis. Although mirror-lacZ is initially expressed correctly, the domain later expands around the anterior circumference of the egg chamber, suggesting a role for fet after the first round of Egfr signaling by Grk. This interpretation is supported by analysis of the double mutant. In contrast to the strictly linear relationship observed for establishing embryonic polarity, the eggshell phenotype produced by Egfr1; fet females shows contributions from each of the individual phenotypes. The distance between dorsal appendages is reduced, as observed for the ventralizing Egfr1 mutation, but the individual appendage structure is broadened, as seen for the dorsalizing fet mutation (Goff, 2001).

In the wild-type eggshell, dorsal appendage pattern is achieved through refinement of the Egfr activation profile by both positive and negative feedback regulation. Expression of rhomboid RNA is induced as a result of Egfr activation and positively regulates continued Egfr signaling. Induction of argos RNA expression leads to negative feedback on Egfr signaling at the dorsal midline, causing refinement of one domain of Egfr activation into two laterally symmetric domains that specify the placement of the paired dorsal appendages, This dorsal appendage pattern can be genetically altered in different ways. The K10 and squid mutant phenotype is characterized by an eggshell with a fused cylindrical dorsal appendage around the anterior circumference of the egg deposited around a dorsalized embryo. The fet and Cbl mutant eggshell phenotype appears distinct; rather than a single cylindrical structure, two laterally placed broadened dorsal appendages form, often associated with circumferential dorsal appendage base material. A change in either the strength or timing of Egfr signaling might translate into these different phenotypes (Goff, 2001).

In addition to the maternal effect eggshell and embryo phenotype, viable fet alleles exhibit wing phenotypes and strong fet alleles are lethal. This range of fet phenotypes, as compared with the cic1 phenotype, may be accounted for by the molecular nature of the mutations. The cic1 mutation is caused by a hobo mobile element insertion in the 5' untranslated region of the cic transcript, while the bwk8482 allele, which also has a maternal effect phenotype caused by a germline defect, is associated with a P-element insertion in the same region. These mobile elements may contain cryptic promoters that allow sufficient expression of the cic transcript to rescue the somatic but not the germline functions of this gene (Goff, 2001).

Mosaic analyses reveal the function of Drosophila Ras in embryonic dorsoventral patterning and dorsal follicle cell morphogenesis

In Drosophila, the Ras signal transduction pathway is the primary effector of receptor tyrosine kinases, which govern diverse developmental programs. During oogenesis, epidermal growth factor receptor signaling through the Ras pathway patterns the somatic follicular epithelium, establishing the dorsoventral asymmetry of eggshell and embryo. Analysis of follicle cell clones homozygous for a null allele of Ras demonstrates that Ras is required cell-autonomously to repress pipe transcription, the critical first step in embryonic dorsoventral patterning. The effects of aberrant pipe expression in Ras mosaic egg chambers can be ameliorated, however, by post-pipe patterning events, which salvage normal dorsoventral polarity in most embryos derived from egg chambers with dorsal Ras clones. The patterned follicular epithelium also determines the final shape of the eggshell, including the dorsal respiratory appendages, which are formed by the migration of two dorsolateral follicle cell populations. Confocal analyses of mosaic egg chambers demonstrate that Ras is required both cell- and non cell-autonomously for morphogenetic behaviors characteristic of dorsal follicle cell migration, and reveal a novel, Ras-dependent pattern of basal E-cadherin localization in dorsal midline follicle cells (James, 2002).

The mosaic analyses described here both clarify and contribute new information to the general understanding of dorsoventral patterning in Drosophila. In a simple model for embryonic dorsoventral patterning, Gurken/Egfr/Ras signaling in the egg chamber represses pipe transcription dorsally. pipe function in the ventral follicle cells is necessary to activate the serine protease cascade in the perivitelline space, which leads to activation of the Toll receptor in the embryonic plasma membrane by cleaved Spätzle (Spz) and, ultimately, a dorsoventral gradient of Dorsal nuclear translocation. Consistent with this hypothesis, loss of pipe function in the follicle cells leads to the absence of zygotic Twist and embryonic dorsalization, and ectopic expression of pipe throughout the follicular epithelium results in embryonic ventralization. In this work, however, no direct spatial relationship was found between dorsal Ras clones (and, therefore, ectopic pipe) in the egg chamber and ectopic Twist in the embryo (James, 2002).

These data are consistent with the hypothesis that additional patterning events modify the initial asymmetry determined by Egfr signaling and thereby establish the final Dorsal gradient. Additional patterning downstream of Egfr has also been suggested to explain another observation, that the embryonic phenotype resulting from maternal mutations in gurken or Egfr is not simply an expansion of ventral embryonic fates, as detected by Twist expression and formation of a larger ventral furrow. Rather, many embryos exhibit a pattern duplication characterized by two ventrolateral regions of maximum nuclear Dorsal and Twist, separated by a ventral minimum, which leads to the eventual invagination of two furrows. The result that Ras clones cell-autonomously derepress pipe suggests that such a refinement process occurs downstream of pipe (James, 2002 and references therein).

Consistent with these findings, the additional patterning events that govern the shape of the Dorsal gradient in wild-type embryos (and that can create two furrows in ventralizing mutants) do not occur in the follicular epithelium. Rather, the refinement process occurs at the level of Spätzle processing or Toll activation in the embryo. Furthermore, elegant mosaic analyses reveal that, while loss-of-function pipe or windbeutel (wind) clones on the ventral side of the egg chamber result in a local loss of ventral Twist expression in the embryo, ventral clones of pipe or wind also exert a non-autonomous effect on lateral embryonic fates. It has been hypothesized that an 8-12 cell wide ventral region in the egg chamber (defined by the spatial requirement for pipe) generates ventral information, perhaps imbedded in the vitelline membrane, which is then competent to establish the Dorsal gradient along the entire dorsoventral axis, presumably via outward diffusion of a ventral morphogen (James, 2002 and references therein).

In addition, other patterning mechanisms that influence the Dorsal gradient have been discovered recently. Nuclear translocation of Dorsal can be modified by maternal Sog and Dpp in a pathway parallel to Toll signaling. Also, the serine protease cascade upstream of Toll is subject to feedback inhibition, providing an additional level of regulation (James, 2002 and references therein).

This work, together with the discoveries discussed above, points to a model in which the initial asymmetry generated by restriction of pipe to the ventral-most 40% of the egg chamber leads to the cleavage of the zymogen Spz in the ventral-most 40% of the perivitelline space. The positive ventralizing activity of C-Spz (the active form of Spz) is opposed by the activity of a diffusible inhibitor, possibly N-Spz. In wild-type patterning, this inhibitor narrows the domain of ventralizing C-Spz activity, originally determined by pipe expression in follicle cells, from 40% of the perivitelline space to approximately 20%. This narrower region of ventralizing activity then activates Toll in a graded fashion, defining the final shape of the Dorsal gradient (James, 2002).

In Ras mosaic egg chambers, pipe is expressed ectopically in dorsal Ras clones, but is prevented from activating Twist because the size or width of the clone is too small to overcome the action of the inhibitor. Theoretically, this inhibitor could be present throughout the perivitelline space but overcome only by a large enough pool of C-Spz. The inhibitor could itself be a byproduct of Spz cleavage (N-Spz), emanating only from sources of ventralizing activity. If this mechanism is correct, then the data predict that, until the pool of C-Spz reaches a certain size, the inhibitor is more potent than C-Spz. It is hypothesized that only very large dorsal Ras clones could exceed the size or width threshold needed to overcome the inhibitor and cause ectopic expression of Twist in the embryo. In the rare cases in which embryos ectopically expressed Twist, the region of ectopic Twist was always found in contact with the normal ventral domain of Twist. It is hypothesized that these embryos result from egg chambers in which Ras clones overlap the edge of the normal pipe expression domain and alter the shape of the domain by creating a local bulge in pipe expression. The inhibitor downstream of pipe may then narrow that distorted ventral shape to produce a corresponding, though smaller, bulge in the Twist domain (James, 2002).

Support for this hypothesis comes from the analysis of egg chambers with ventral wind clones surrounding small 'islands'of wild-type cells in ventral-most positions. In these instances, if the wild-type 'island' is less than 4-6 cells wide, it is not able to induce Twist expression in the embryo. This situation is similar to the result that dorsal Ras clones fail to induce Twist. Together, these data suggest that ventral information from small patches of cells expressing pipe can be 'swamped out' by neighboring dorsally-fated cells, probably through the function of a diffusible inhibitor (James, 2002 and references therein).

The ventralizing activity of ectopic pipe expression in Ras mosaic egg chambers can be overcome by post-pipe patterning events. The existence of this compensatory mechanism demonstrates that embryonic dorsoventral patterning is a robust process. This resilience is generated by the action of additional rounds of pattern refinement, which may help to buffer these important early events of embryonic development from perturbations in signaling caused by genetic defects or environmental factors. Furthermore, the zygotic genes that orchestrate dorsoventral patterning (Dpp/BMP4 and Sog/chordin) are conserved between arthropods and chordates, and, extraordinarily, their biological function in dorsoventral patterning is also conserved. However, where flies use post-pipe pattern refinement to buffer the Dorsal gradient, which determines the Dpp/Sog pattern, chordates use a completely different developmental pathway to arrive at the BMP4/chordin patterning event. Consequently, the specific mechanism of dorsoventral 'buffering' in flies cannot be shared by chordates, suggesting that each group evolved unique mechanisms to buffer the conserved aspects of dorsoventral patterning. Understanding the degree of diversity among buffering systems will be useful in determining evolutionary relationships and will facilitate studies examining the evolution of developmental mechanisms (James, 2002).

To explain the differential phenotype of eggs laid by females transheterozygous for strong hypomorphic alleles of Ras, it is hypothesized that either eggshell patterning is more sensitive than embryonic patterning to reductions in Ras, or a Ras-independent Egfr effector pathway mediates embryonic patterning. The cell-autonomous derepression of pipe in Ras null clones demonstrates that Ras is required to initiate embryonic dorsoventral patterning and suggests that the first hypothesis is correct. Conversely, the result that embryos developing from Ras mosaic egg chambers are rarely abnormal supports the second hypothesis, with the added complexity that the Ras-independent effector pathway must also bypass pipe. As described above, however, the data suggest that very large Ras clones, or an entirely mutant epithelium, would induce ectopic Twist expression in the embryo and lead to severe embryonic dorsoventral patterning defects (James, 2002).

Indeed, in five of 26 cases, Ras follicle cell clones did result in ectopic Twist in the embryo, presumably because those clones were large enough to overcome the post-pipe patterning events that dampen the ventralizing effects of smaller clones. Furthermore, ectopic expression of pipe throughout the follicular epithelium is indeed sufficient to cause embryonic ventralization. Therefore, the lack of embryonic defects resulting from most Ras mutant follicle cell clones, which are too small to overcome post-pipe patterning processes, is misleading with respect to the question of whether Ras is required generally for embryonic dorsoventral patterning. Given these considerations, the results are more consistent with the first hypothesis, that is, Ras is required for dorsoventral patterning of the embryo, and eggshell patterning is more sensitive than embryonic patterning to reductions in Ras (James, 2002).

Why would eggshell patterning be more sensitive than embryonic patterning? Furthermore, why would that sensitivity be specific to reductions in Ras and not Gurken or Egfr? Two hypotheses can explain why eggshell patterning would be acutely sensitive, specifically to reductions in Ras. (1) Dorsoventral patterning of the eggshell requires amplification and modulation of Egfr activity, achieved by autocrine signaling involving Spitz, Rhomboid, Vein, and Argos, to define two dorsolateral populations of follicle cells. These post-Gurken patterning events are eggshell-specific, while pipe repression is achieved solely by Gurken signaling. Furthermore, since the results support the hypothesis that low lateral levels of Gurken suffice to repress pipe, it is likely that pipe repression requires only low-level signaling. The hypomorphic Ras mutations primarily affect the transcription of Ras, which, theoretically, reduces the number of Ras molecules rather than the effectiveness of each molecule. Therefore, the hypomorphic mutant may produce enough Ras molecules to transduce the initial Gurken signal, repress pipe and pattern the embryo, but too few Ras molecules to transduce the high levels of Egfr signaling required for eggshell patterning. Thus, the number of Ras molecules may be limiting for eggshell patterning, which requires an intense surge of Ras signaling, but not limiting for pipe repression, which may be accomplished with only a trickle of signaling that is easily transduced by a small number of Ras molecules (James, 2002).

(2) A second hypothesis can also explain why eggshell patterning would be more sensitive than embryonic patterning specifically to reductions in Ras. The data demonstrate that Ras mutant cells fail to initiate or accomplish dorsal follicle cell morphogenesis. These phenotypes, along with the pipe-lacZ results, suggest that Ras is required to establish dorsal follicle cell fate, a prerequisite for morphogenesis. The data are also consistent with an additional requirement for Ras specifically regulating morphogenesis. Gurken and Egfr, however, may be directly required only for patterning and may therefore influence morphogenesis only indirectly. In support of this hypothesis, the Nrk receptor tyrosine kinase, identified as an Enhancer of Ras in eggshell development, may provide an independent Ras pathway input specific for morphogenesis. Thus, a Ras mutation that produces weak embryonic defects may dramatically disrupt eggshell structures by affecting both patterning and morphogenesis. This synergism may compromise dorsal appendage morphology enough to resemble the eggshell phenotype of a severe gurken or Egfr allele, which affects eggshell and embryo equally (James, 2002).

In addition to extending understanding of embryonic dorsoventral patterning and defining the relative contribution of Ras signaling toward establishing eggshell and embryonic fates, mosaic analyses have revealed the requirement for Ras during dorsal appendage morphogenesis. How might Ras regulate dorsal follicle cell migration? By transducing signals for dorsal follicle cell fate, Ras may affect transcription of genes involved in morphogenesis. Notably, Broad-Complex (BR-C), Mirror, Bunched, and Fos/Kayak respond to Egfr signaling and likely affect transcription of genes involved in dorsal appendage formation. Alternatively, Ras activity may directly affect key cytoskeletal or adhesion molecules that play active roles during the morphogenetic process. Since many of the events of dorsal follicle cell morphogenesis occur quite rapidly -- stage 11, for example, encompasses profound morphogenetic changes, but is completed in less than 30 minutes, and since MAP kinase activity is dynamic during the early stages of morphogenesis, it is suspected that Ras signaling directly modulates the activity of migration molecules. Most likely, Ras functions in dorsal follicle cell migration through some combination of direct cytoplasmic effects and transcriptional regulation (James, 2002).

To understand the role of Ras in cell migration, other migration events controlled by Ras signaling have to be taken into consideration. In Drosophila, disruptions in Ras signaling can hinder the migration of a subset of follicle cells called border cells. Significantly, border cell fate remains properly specified in these experiments, revealing migration-specific functions for Ras. Importantly, the movement of dorsal follicle cells differs significantly from that of border cells, which navigate through germline cells as a small epithelial patch. Dorsal appendage formation involves the coordinated morphogenetic movements of an epithelial sheet. These differences demonstrate that each migration event offers a unique opportunity to study the role of Ras during developmentally regulated cell migration in Drosophila (James, 2002).

What are the effectors of Ras during dorsal follicle cell migration? One possibility is E-cadherin, since Ras is required for the basal localization of this molecule in midline follicle cells. Importantly, the levels of apicolaterally-localized E-Cad appear normal in Ras clones. This result suggests that Ras signaling does not regulate the canonical adherens-junction function of E-Cad, which provides integrity to the epithelium during morphogenesis. Instead, Ras affects the basal localization of E-Cad on the dorsal midline, which may anchor the midline cells or otherwise influence the mechanical movements of the dorsal appendage primordia (James, 2002).

The loss of basal E-Cad in a Ras clone on the midline was surprising because the dorsal midline is thought to be a region of significantly diminished Egfr activity. Why, then, would Ras be required there for the localization of an adhesion molecule? Perhaps Ras signaling is actually active on the dorsal midline between stages 10B and 12. Alternatively, a history of high and then low Egfr/Ras signaling may be required for basal E-Cad protein localization in midline cells. Further exploration of the precise regulation of E-Cad during dorsal follicle cell morphogenesis is needed to elucidate the relationship between Ras signaling and E-Cad localization, and to address whether basal localization of E-Cad in anterior and midline cells is required for the proper morphogenesis of dorsal appendages (James, 2002).

In addition to E-Cad, Ras may regulate other adhesion, signaling, or cytoskeletal molecules to permit or instruct dorsal follicle cell migration. Known cellular effectors of Ras in mammalian cells include c-Raf, RalGDS, and PI 3 kinase. To identify molecules that interact with Ras in Drosophila follicle cells, dominant enhancers of a weak Ras eggshell phenotype have been sought. Interestingly, two enhancers, dock and Tec29A, encode signaling molecules that directly regulate cytoskeletal function. Studies that separate cell fate specification from morphogenetic events are needed to determine whether Ras actively controls the morphogenesis of dorsal follicle cells (James, 2002).

In conclusion, the finding that Ras is required for pipe repression argues against the hypothesis that a Ras-independent pathway transduces Egfr signals to pattern the embryo. This result contributes to the growing body of evidence that, in Drosophila, Egfr signaling is transmitted to the nucleus primarily by the Ras pathway rather than by alternative effector molecules. Furthermore, the data demonstrate that dorsoventral patterning is buffered by post-pipe patterning events that define the final shape of the embryonic dorsoventral gradient. Additionally, it has been shown that Ras is required for dorsal appendage patterning and morphogenesis as well as for the proper subcellular localization of E-cadherin, a major epithelial adhesion protein. Ras signaling is linked to cell migration in many developmental and disease contexts, providing justification for further dissection of Ras pathway function during dorsal appendage morphogenesis in Drosophila (James, 2002).

Regulation of subcellular location

Drosophila embryonic dorsal-ventral polarity originates in the ovarian follicle through the restriction of pipe gene expression to a ventral subpopulation of follicle cells. Pipe, a homolog of vertebrate glycosaminoglycan-modifying enzymes, directs the ventral activation of an extracellular serine proteolytic cascade that defines the ventral side of the embryo. When pipe is expressed uniformly in the follicle cell layer, a strong ventralization of the resulting embryos is observed. This ventralization is dependent on the other members of the dorsal group of genes controlling dorsal-ventral polarity, but not on the state of the epidermal growth factor receptor signal transduction pathway which defines egg chamber polarity. Pipe protein expressed in vertebrate tissue culture cells localizes to the endoplasmic reticulum. Strikingly, coexpression of the dorsal group gene windbeutel in those cells directs Pipe to the Golgi. Similarly, Pipe protein exhibits an altered subcellular localization in the follicle cells of females mutant for windbeutel. Thus, Windbeutel protein enables the correct subcellular distribution of Pipe to facilitate its pattern-forming activity (Sen, 2000).

Consistent with its predicted function in the sulfation of glycoprotein-associated oligosaccharides and its type II transmembrane structure, Pipe functions as a resident of the Golgi apparatus. The most compelling evidence of Golgi localization comes from several observations of Pipe expressed (together with Windbeutel) in COS7 cells. (1) Pipe is present in a perinuclear distribution characteristic of Golgi; (2) Pipe co-localizes with Sialyltransferase, a well-characterized resident of the Golgi; (3) the distribution of Pipe is altered dramatically in cells incubated with brefeldin A, an agent known to result in the depolymerization of the Golgi apparatus. Studies of the expression of Pipe in Drosophila follicle cells are similarly consistent with the notion that Pipe acts in the Golgi. Here, Pipe protein exhibits a punctate distribution similar to that seen for another known Golgi enzyme, N-acetyl-glucosaminyltransferase I. Flag-tagged Pipe assessed by immuno-EM has been detected in a structure morphologically similar to one previously identified as Golgi (Sen, 2000).

Although the precise function and catalytic target(s) of Pipe remain elusive, the demonstration that Pipe expressed in COS7 cells (with Windbeutel) resides in the Golgi strongly supports the suggestion that Pipe is involved in the modification of glycosaminoglycans or other oligosaccharides, thereby facilitating the activation of the serine proteolytic cascade activated on the ventral side of the egg that gives rise to active Toll ligand. Unexpectedly, a rather specific role has been found for the Windbeutel protein in directing the correct subcellular targeting of Pipe. This is consistent with the observation that the role of Pipe in dorsal-ventral patterning is dependent on Windbeutel activity. wind is expressed in both tissues in which Pipe expression has been detected. Pipe expressed in follicle cells lacking Windbeutel appears to be mislocalized in the ER, presumably rendering Pipe unable to modify its oligosaccharide target, which is likely to be synthesized in the Golgi. Strikingly, in a heterospecific expression system, COS7 vertebrate tissue culture cells, Windbeutel expression appears to be sufficient to direct Pipe to the Golgi (Sen, 2000).

The mechanisms operating in the ER to ensure the fidelity of proteins traversing the secretory pathway have been collectively termed 'quality control'. These events comprise a stringent selection process in which only proteins that undergo proper folding and maturation are transported to their target compartments. Those quality control mechanisms which apply to all proteins expressed in the ER have been termed 'primary quality control'. 'Secondary quality control' comprises a rapidly growing list of protein-specific factors influencing folding, maturation, and assembly of proteins in the ER as well as factors that act to facilitate or inhibit forward transport of mature proteins in the secretory pathway. Accessory factors which facilitate movement of proteins out of the ER have been categorized as 'outfitters', which establish or maintain a secretion-competent conformation of the transported protein; 'escorts', which additionally accompany their cognate transported proteins to the Golgi; and 'guides', which mediate the selective uptake of transported proteins into transport vesicles. The observations presented here are most consistent with the notion that Windbeutel functions as either an 'outfitter' or an 'escort' for Pipe transport to the Golgi. The reported structural similarity between Windbeutel/ERp29 and PDI may point toward a role for Windbeutel as a folding catalyst or chaperone for Pipe folding, a likely prerequisite for the migration of Pipe from the ER to Golgi. PDI and its homologs have been demonstrated to possess chaperone-like activities. In some respects, the relationship between Windbeutel and Pipe is reminiscent of that between receptor associated protein (RAP) and low-density lipoprotein (LDL) receptor. A physical interaction between RAP and LDL receptor prevents aggregation and premature ligand binding in the ER, with RAP escorting LDL receptor to the Golgi. Here, the complex dissociates and RAP is retrieved to the ER by the KDEL receptor. It is considered likely that in the highly synthetic environment of the follicle cell it is important that inappropriate interaction between Pipe and its target(s) be prevented, allowing only spatially and temporally appropriate oligosaccharide modification by Pipe. Ongoing studies will address the precise mechanism of Pipe regulation by Windbeutel and identify the specific targets of Pipe action during Drosophila dorsal-ventral pattern formation (Sen, 2000).

slalom transports the sulfate donor required for sulfotransferase activity of Pipe

Sulfation of all macromolecules entering the secretory pathway in higher organisms occurs in the Golgi and requires the high-energy sulfate donor adenosine 3'-phosphate 5'-phosphosulfate. A gene has been identified that encodes a transmembrane protein required to transport adenosine 3'-phosphate 5'-phosphosulfate from the cytosol into the Golgi lumen. Mutations in this gene, which has been called slalom, display defects in Wg and Hh signaling; these defects are likely due to the lack of sulfation of glycosaminoglycans (GAGs) by the sulfotransferase sulfateless. Analysis of mosaic mutant ovaries shows that sll function is also essential for dorsal-ventral axis determination, suggesting that sll transports the sulfate donor required for sulfotransferase activity of the dorsal-ventral determinant Pipe (Lüders, 2003).

GAGs have also been proposed to play a role in the determination of the dorsal-ventral (D/V) axis of the Drosophila embryo. The D/V polarity of the embryo is established during oogenesis by asymmetric expression of the key D/V determinant pipe (pip) in the follicle cell epithelium. pip expression in the ventral follicle cell layer is necessary and sufficient to trigger a serine-protease cascade in the perivitelline space; this leads to the generation of an active ligand for the transmembrane receptor Toll (Tl). Activation of Tl on the ventral side of the embryo results in a gradient of nuclear localization of the transcription factor Dorsal, which patterns the D/V axis. Based on sequence similarity to a family of vertebrate enzymes and its localization in the Golgi apparatus, pip has been hypothesized to encode a heparan sulfate 2-O-sulfotransferase. However, neither the enzymatic activity nor the substrate specificity of pip have been demonstrated directly (Lüders, 2003).

Sulfation of secreted molecules occurs in the Golgi and requires the high-energy sulfate donor adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to be present within that organelle. In Drosophila, PAPS is synthesized in the cytoplasm by PAPS-synthetase, which incorporates both ATP-sulfurylase and adenosine 5'-phosphosulfate-kinase (APS-kinase) activity. PAPS must be transported into the Golgi thereafter to serve as a substrate for sulfotransferases. This study reports the molecular identification and functional characterization of a PAPS transporter. Mutations in this gene, called slalom (sll), are associated with defects in multiple signaling pathways, including Wg and Hh signaling. A phenotypic analysis suggests that the effects of sll on signal transduction are caused by its requirement for GAG modification. Evidence is presented that sll is also required to supply PAPS to the machinery initiating the establishment of embryonic D/V polarity, supporting the view that Pipe protein is a sulfotransferase (Lüders, 2003).

A hydrophobicity analysis of the putative Sll protein predicts a hydrophobic polypeptide with at least 10 transmembrane regions, a structural characteristic of many nucleotide-sugar transporters. A BLAST search of non-redundant protein databases with the Sll sequence revealed that Sll is conserved throughout the animal kingdom, as well as in plants, and shares nearly 40% of amino acid sequence identity with predicted mouse and human proteins of unknown function. Sll is also similar to mammalian proteins that have been classified as nucleotide-sugar transporters on the basis of their homology to the human UDP-galactose transporter hUGT. However, Sll itself has no significant sequence similarity to hUGT. While these data suggest that Sll encodes a transmembrane transporter, they leave open what substrate Sll may be transporting (Lüders, 2003).

Interestingly, the overall size of sugar chains attached to the glypican Dally does not appear to be affected in sll mutants. Consistent with this result, it has been observed that the overall level of HS, which is reduced to trace amounts in the sgl mutant that affects GAG biosynthesis, is not markedly changed in sfl mutants, which should affect sulfate modification but not synthesis of GAG chains. However, it cannot be excluded that residual sulfation of GAGs occurs in the cell culture assay owing to incomplete inhibition of the PAPS transport activity of sll by dsRNA interference. However, the phenotypic analysis of sll suggests that sll function is essential for at least two sulfotransferases, sfl and pip, and that the GAG chains present in sll mutants are not able to fulfill their normal function owing to altered sulfation patterns (Lüders, 2003).

The signal determining the D/V axis of the Drosophila embryo is produced by a proteinase cascade active in the perivitelline space of the egg chamber during oogenesis. Activation of this cascade requires localized expression of pip in the ventral follicle cells of the ovarian egg chamber in which the egg is constructed, but the mechanism leading to activation remains enigmatic. pip has significant similarity to a family of mammalian heparan sulfate 2-O-sulfotransferases; however, the enzymatic activity of pip has not been demonstrated directly. The data show that transport of the sulfate donor PAPS into the Golgi is essential for establishment of the D/V axis. The requirement of PAPS for D/V patterning strongly supports the notion that sulfotransferase activity is an essential feature of pip function (Lüders, 2003).

This view is also supported by the specific expression patterns of pip and sll during development. In the embryo, both genes are highly expressed in the developing salivary glands. Another gene involved in PAPS biosynthesis, PAPS synthetase, is also highly expressed in salivary glands, suggesting that these genes act together in a common pathway providing the sulfate donor for the modification of macromolecules produced in the salivary glands in late embryos and larvae (Lüders, 2003).

Interestingly, the defects in D/V polarity associated with sll mutants are not observed in mutants of the UDP-glucose dehydrogenase sgl or the UDP-glucuronic acid transporter frc. The activity of both genes is required for the formation of GAGs, such as heparan sulfate, chondroitin sulfate or dermatan sulfate, and levels of these GAGs are dramatically reduced in sgl mutants. These observations support the possibility that GAGs may not play a role in D/V axis determination and that, despite its homology to heparan sulfate 2-O-sulfotransferases, pip may be required for sulfation of a non-GAG substrate. This view is also consistent with the highly restricted expression pattern of pip, which suggests that pip is not generally involved in GAG modification (Lüders, 2003).

The central position of sll in sulfate metabolism along the secretory pathway makes it an interesting tool for the identification of developmental pathways sensitive to sulfate modification. The results demonstrate that several cell-cell communication pathways are critically dependent on the sulfation of macromolecules, and highlight the importance of sulfation during pattern formation and development (Lüders, 2003).

Protein interactions downstream of Pipe

Three serine protease zymogens -- Gastrulation defective (GD), Snake (Snk) and Easter (Ea) -- and a nerve growth factor-like growth factor ligand precursor, Spaetzle, are required for specification of dorsal-ventral cell fate during Drosophila embryogenesis. The proteases have been proposed to function in a sequential activation cascade within the extracellular compartment called the perivitelline space. Biochemical interactions between these four proteins have been examined using a heterologous co-expression system. The results indicate that the three proteases do function in a sequential activation cascade, that GD becomes active and initiates the cascade and that interaction between GD and Snk is sufficient for GD to cleave itself autoproteolytically. The proteolytically active form of Ea cleaves GD at a different position, revealing biochemical feedback in the pathway. Both GD and Snk bind to heparin-Sepharose, providing a link between the pipe-defined ventral prepattern and the protease cascade. These results suggest a model of the cascade in which initiation is by relief from inhibition, and spatial regulation of activity is due to interaction with sulfated proteoglycans (Dissing, 2001).

nudel, a somatically required dorsal group gene, encodes a 320 kDa mosaic protein with a centrally located serine protease catalytic domain. It has been suggested that nudel might activate a dorsal-ventral proteolytic cascade. This possibility was investigated using the assay system by co-expressing either the full-length form of Nudel, or a constitutively active Nudel serine protease catalytic chain (ndlDeltan) in different combinations with the four proteins described here. In all experiments conducted thus far, no reproducible effect of Nudel or nudDeltan upon any of the other proteins nor an effect of any of the other proteins upon Nudel-specific polypeptides was observed. Consequently there is no evidence for direct biochemical interaction of Nudel with GD, Snk, Ea or Spz and therefore no direct role for Nudel within the protease cascade can be ascribed. Nudel apparently does not directly activate the cascade but rather is required earlier for proper establishment or maintenance of the ventral prepattern (Dissing, 2001).

GD appears to play a critical role in the proteolytic cascade since it can initiate the cascade yet does not appear to require classical zymogen activation in order to do so. The data suggest that exposure to the zymogen form of Snk is sufficient for GD to become active and activate Snk, triggering the cascade, and for GD to generate lower molecular weight polypeptides. With respect to a mechanism of activation, it is interesting to note that GD bears some similarity to mammalian complement factors C2 and B. These proteases have novel activation mechanisms requiring complex formation and a conformational change as a prerequisite to activation. An alternative explanation for how GD functions is that it has some intrinsic activity as a zymogen. Upon binding to and activating Snk, it then proteolytically processes itself to generate lower molecular weight inactive forms (Dissing, 2001).

Activated Ea can proteolytically process GD, suggesting that a second form of feedback occurs within the cascade. Ea cleaves GD at a novel position to generate a GD polypeptide that is slightly larger than the predominant band generated by GD itself. The significance of processing by Ea is not altogether clear from the data. However, it is reasonable to assume that cleavage by Ea is a way to modify GD's biochemical properties. It is proposed that Ea may feed back negatively on the precursor form and/or the active form of GD. This would provide a means of down-regulating the protease cascade to prevent amplification from 'running away', resulting in overproduction of the ventralizing signal (Dissing, 2001).

Since GD can autoactivate the cascade, a requirement for an upstream protease to activate the cascade need not be postulated. Rather, the data might argue that a mechanism exists to prevent GD from becoming activated too early in embryogenesis before the Toll receptor is completely expressed on the plasma membrane. This idea has been incorporated into a biochemical model of the cascade. The existence of an inhibitory factor that prevents GD from activating Snk is proposed. The inhibitory factor must itself be inactivated in a spatially or temporally regulated way for proper activation of the cascade. However, the net result must be to permit the activation of GD within the ventral perivitelline space at the proper time for accurate elaboration of the ventralizing signal. This region of the perivitelline space may correspond to the ventral stripe prepattern described from gd mRNA injection experiments (Dissing, 2001).

The ability of both GD and Snk to bind to heparin-Sepharose suggests that their activities may be regulated in vivo by sulfated proteoglycans. Since pipe expression in somatic follicle cells is ventrally restricted and the gene encodes a heparan sulfate 2-O-sulfotransferase, sulfate modification of an as yet unknown proteoglycan may provide the ventral cue in the egg. GD and Snk may interact directly with this sulfated proteoglycan and this interaction may provide the ventral restriction to activation of the cascade (Dissing, 2001).

It has been suggested that the protease cascade may enable an initial asymmetry in the form of a ventral stripe prepattern to be converted into a graded distribution of processed Spz ligand. The potential for both (1) amplification with subsequent steps and (2) feedback after activation could enable the cascade to self-regulate the shape of the Dorsal protein gradient. This property could provide plasticity in the patterning process and a means of compensating for minor variation in the size and shape of individual embryos. Such a mechanism would also be sufficiently adaptable that it could be conserved evolutionarily. The data suggest some remarkable similarities between the dorsal-ventral protease cascade and the classical complement and blood coagulation pathways (Dissing, 2001).

Pipe is a candidate for defining an asymmetric cue directing D/V pattern during embryogenesis. To test whether gastrulation-defective (gd) RNA ventralization requires pipe, gd RNA was injected into embryos from pip664/pip664 females. At low gd RNA concentrations, no effect was seen. However, at 100 µg/ml, small ectopic patches of ventral denticles were observed. Between 100-500 µg/ml localized expression of Twist appeared close to the injection site. When hybridized in situ for rho, either one or two radial stripes spanning the D/V axis were generated. In wt embryos a third rho stripe appears in the future amnioserosa very shortly after the two lateral stripes appear. When two rho stripes were observed in embyros from pipe-null mothers, the distance between them was the same as the distance between the lateral rho stripes and the amnioserosal rho stripe. All injected embryos stained for Twist show only one patch of Twist expression near the injection site. When it appears, the second rho stripe correlates with amnioserosal rho expression. It is concluded that high gd RNA concentrations in a pipe-null background induces an ectopic axis with the ventral pole defined by the point of injection. Embryos from snk229-, ea1- and spz197-null females were microinjected with 100 µg/ml gd RNA and remained dorsalized, indicating that this effect requires their activities (DeLotto, 2001).

The data presented here suggest that the shape of the Dorsal gradient is not directly determined by asymmetric cues in the eggshell but rather arises within the perivitelline space as a consequence of self-regulatory properties of the protease cascade triggered by Gd. Localized phenotypic rescue is consistent with the idea that Gd is membrane bound. Since Gd can produce an ectopic axis in a pipe-null background, pipe activity is not required for binding. Binding of Gd to a surface within the PVS is therefore independent of the spatial control of activation that is normally ventrally restricted and requires Pipe (DeLotto, 2001).

The spatial activation of Gd must be regulated subsequent to and independent of binding. This could be explained via interaction of Gd with a Pipe-modified proteoglycan. Since pipe is a heparan sulfate 2-O-sulfotransferase and its expression is restricted to the ventral third of the somatic follicle cell, it presumably modifies a somatically expressed proteoglycan. Ventrally restricted modification by Pipe of this proteoglycan could control Gd activation. It is known that complement and coagulation pathways can be both positively and negatively regulated by interaction with heparan sulfate proteoglycans (DeLotto, 2001).

A ventrally localized protease in the Drosophila egg controls embryo dorsoventral polarity

Drosophila embryo dorsoventral (DV) polarity is defined by serine protease activity in the perivitelline space (PVS) between the embryonic membrane and the inner layer of the eggshell. Gastrulation Defective (GD) cleaves and activates Snake (Snk). Activated Snk cleaves and activates Easter (Ea), exclusively on the ventral side of the embryo. Activated Ea then processes Spatzle (Spz) into the activating ligand for Toll, a transmembrane receptor that is distributed throughout the embryonic plasma membrane. Ventral activation of Toll depends upon the activity of the Pipe sulfotransferase in the ventral region of the follicular epithelium that surrounds the developing oocyte. Pipe transfers sulfate residues to several protein components of the inner vitelline membrane layer of the eggshell. This study shows that GD protein becomes localized in the ventral PVS in a Pipe-dependent process. Moreover, ventrally concentrated GD acts to promote the cleavage of Ea by Snk through an extracatalytic mechanism that is distinct from GD's proteolytic activation of Snk (see Figure 4: Model for ventral processing of Ea by Snk). Together, these observations illuminate the mechanism through which spatially restricted sulfotransferase activity in the developing egg chamber leads to localization of serine protease activity and ultimately to spatially specific activation of the Toll receptor in the Drosophila embryo (Cho, 2012).

The results demonstrate that GD provides two essential functions in the dorsoventral (DV) pathway: activation of the Snk protease and ventrally localized Ea cleavage. These findings support a model in which processing of GD generates a fragment with a C-terminal protease domain and N-terminal sequences that interact with the sulfated ventral cue to localize GD to the ventral region of the PVS. GD may bind directly to carbohydrates associated with vitelline membrane protein that have been sulfated as a result of Pipe enzymatic action in ventral cells of the follicle layer. Consistent with this possibility, GD protein has been shown to bind to heparin and to anionic components of a highly purified Drosophila eggshell matrix preparation (Cho, 2012).

It is proposed that ventrally localized GD binds to both Ea and Snk and plays a direct role in promoting an interaction between them. It is possible that a single GD molecule binds either to Ea or to Snk and that ventral localization of GD acts to concentrate GD-bound Ea and Snk and bring them into proximity. Alternatively, GD bound only to Snk or Ea may undergo a conformational change when it interacts with the Pipe-sulfated ventral cue that results in an enhancement of Snk proteolytic activity or an increased susceptibility of Ea to cleavage by Snk. Lastly, GD bound simultaneously to both Ea and Snk may respond to the ventral cue by undergoing a conformational change that brings Snk and Ea into productive juxtaposition and results in Ea cleavage. A mechanism in which GD interacts with Pipe and the sulfated ventral cue to promote productive interaction between Ea and Snk can also explain the results of RNA injection studies. Injection of very high levels of in vitro synthesized RNA encoding the GD zymogen could lateralize or reorient the polarity of embryos produced by pipe mutant females. Those results were interpreted to indicate that Pipe is normally required for activation of GD on the ventral side of the embryo, but that at high concentrations, GD could become enzymatically active by an alternative Pipe-independent mechanism. The current results suggest instead that high concentrations of GD can promote interactions between Easter and Snake, or conformational changes in those proteins that lead to Snake-mediated cleavage of Easter, even in the absence of Pipe and the ventral cue. In conclusion, by demonstrating that the GD serine protease is localized within the ventral PVS in a Pipe-dependent manner and that the interaction of GD with the Pipe-sulfated ventral cue enables it to bring about the ventrally restricted processing of Easter, the work reported in this study explains how ventrally localized sulfotransferase activity in the follicle cell layer leads to spatially localized activation of the Toll receptor and to the formation of the Drosophila embryonic DV axis (Cho, 2012).


Ovaries and Embryos

RNA in situ hybridization to stage 10 follicles with probes specific for pipe-ST2 indicate that it is strongly expressed in the follicle cells comprising the ventral one-third to one-half of the DV circumference of the egg chamber. In contrast, probes derived from sequences specific to pipe-ST1 indicate that it is comparatively poorly expressed in the egg chamber. Using probes specific for both pipe-ST isoforms, whole-mount RNA in situ hybridization to early embryos was carried out. Starting at stage 12 and continuing through development, both isoforms show strong expression in the salivary glands. In addition, at stage 14, half of the embryos initiate pipe-ST2 expression in a tissue that is inferred to be the somatic component of the embryonic ovary, based on comparison with the expression pattern of other clones reported to be expressed in that tissue. On Northern blots, a probe capable of recognizing both isoforms hybridizes to two embryonic RNA species of 1.7 and 1.5 kb but only the 1.7 kb band in ovarian RNA. On probing the same blot with pipe-ST1-specific exon-derived probes, only the 1.5 kb embryonic band is detected, while a pipe-ST2-specific probe detects only the 1.7 kb band in both embryos and ovaries. Thus, it is considered likely that the pipe-ST2 isoform corresponds to an ovary-specific form of pipe (Sen, 1998).

Effects of Mutation or Deletion

Twelve maternal effect loci are required for the production of Drosophila embryos with a correct dorsoventral axis. Analysis of mosaic females indicates that the expression of the genes nudel, pipe, and windbeutel is required in the somatic tissue, presumably in the follicle cells that surround the oocyte. Thus, information coming from outside the egg cell influences dorsoventral pattern formation during embryogenesis. In transplantation experiments, the perivitelline fluid from the compartment surrounding the embryo can restore dorsoventral pattern to embryos from females mutant for nudel, pipe, or windbeutel. The positioning of the transplanted pervitelline fluid also determines the polarity of the restored dorsoventral axis. It is proposed that the polarizing activity, normally present at the ventral side of the egg, is a ligand for the Toll receptor. Presumably, local activation of the Toll protein by the ligand initiates the formation of the nuclear concentration gradient of the Dorsal protein, thereby determining dorsoventral pattern (Stein, 1991).

Establishment of dorsoventral polarity within the Drosophila embryo requires extraembryonic positional information generated during oogenesis. The genes windbeutel, pipe, and nudel are required within the somatic follicle cells of the ovary for production of this spatial cue. Using a novel follicle cell marker system, the effect of mutant follicle cell clones on the embryonic dorsoventral pattern was directly evaluated. No spatially localized requirement for nudel activity is found. In contrast, windbeutel and pipe are required only within a restricted ventral region of the follicular epithelium. This ventral region can determine lateral embryonic cell fates nonautonomously, indicating that spatial information originating ventrally is subsequently refined, perhaps via diffusion, to yield the gradient of positional information that determines the embryonic dorsoventral pattern (Nilson, 1998).

Clones homozygous for mutant wind and pipe alleles induce locally dorsalized embryonic phenotypes. Since ventral clones induced dorsalization, while dorsal and lateral clones have no effect, these results indicate that the wind and pipe genes are required only on the ventral side of the follicular epithelium for establishment of the embryonic dorsoventral pattern. This ventrally restricted requirement therefore reveals an existing asymmetry, or prepattern, within the follicle cell epithelium. By comparison of the size of wind and pipe clones and their effects on twist expression, a ventral region approximately 8-12 follicle cells wide in which these genes are required has been identified. This region corresponds to approximately 20%-30% of the follicle cells around the circumference at the middle of an average egg. Although these results demonstrate a localized requirement for wind and pipe, they do not imply that their expression or activity is spatially restricted. Rather, these results identify a functionally distinct region within the ventral follicular epithelium that is required to establish the embryonic Dorsal gradient. A likely interpretation is that this region defines a spatially localized process that ultimately generates active Toll ligand. Interestingly, this area corresponds well to the width and position of the twist expression domain (Nilson, 1998).

During oogenesis, an initial spatial asymmetry within the germline specifies the dorsoventral pattern of the follicular epithelium, which in turn is required for establishment of the embryonic dorsoventral pattern. However, it is unclear how much of the patterning information required to generate the Dorsal gradient is already determined during oogenesis. For example, the shape of the gradient may be already encoded in the graded distribution of some positional cue in the vitelline membrane, so that the profile of protease cascade activity and shape of the Dorsal gradient would directly reflect the result of a patterning process within the follicular epithelium. Alternatively, a pattern within the follicular epithelium may provide only an initial dorsoventral asymmetry, which would then be refined in a later step to yield a gradient of positional information (Nilson, 1998).

This analysis of embryonic dorsoventral patterning in mosaic ovaries reveals that wind and pipe are required in the ventral region of the follicular epithelium defined above to initiate a nonautonomous patterning process that generates the Dorsal gradient. Ventral clones mutant for either wind or pipe eliminate twist expression in the corresponding region of the embryo but also cause loss of more lateral pattern elements. In addition, lateral clones do not cause alterations in short gastrulation expression, even though these clones overlie the regions of the embryo where sog is expressed. In principle, these results can be interpreted in two ways. Either the ventral region could act as a localized source for diffusion of some activity, or the ventral follicle cells could participate in a mechanism of sequential induction of adjacent cell fates. In the diffusion model, the ventral region would act as a source of some patterning activity that diffuses to form a gradient. Such a process could represent diffusion of Toll ligand, or ligand-producing activity, to yield a gradient of receptor activation. A gradient could also arise via diffusion of signal downstream of the receptor within the syncitial embryo. The loss of twist expression, and the coincident ectopic sog expression, observed at the border of ventral clones, would then be best explained as a depletion of this diffusible activity from the wild-type region into the region of the clone. In wild-type embryos, this diffusion process would result in sog expression at the lateral boundaries of the ventral region. A sequential induction scenario would require interactions between follicle cells to generate distinct cell fates, such that the ventral follicle cells, where wind and pipe are required, correspond to the ventral-most embryonic fates and in turn induce more lateral fates in neighboring follicle cells. Such a model can also account for the ectopic sog expression patterns observed at clone borders. Note that in this model the follicular epithelium could contain a gradient of positional information that directly specifies the Dorsal gradient. However, the nonautonomous effect of wind and pipe clones on sog expression eliminates the possibility that the Dorsal gradient is specified by a corresponding gradient of wind and pipe activity within the follicular epithelium. Data that argue against the sequential induction model are cases in which wild-type follicle cells within this ventral region are flanked by mutant cells. Either model predicts that these cells should be able to generate a pattern, but in fact, they are unable to induce twist expression. This observation is most readily explained by the diffusion model, with depletion of activity by diffusion from these relatively narrow regions of wild-type cells resulting in their inability to specify the ventral-most part of the Dorsal gradient. In the sequential induction model, more complicated explanations would have to be invoked to explain why such an "island" of wild-type follicle cells would not induce ventral and lateral cell fates in adjacent cells. The favored hypothesis is that the ventral region described here defines a source for a diffusion gradient. As noted above, this diffusible activity could represent any of the components of the Toll signaling pathway, and this diffusion could occur upstream or downstream of Toll (Nilson, 1998).

Subtle differences between the effects of pipe and wind clones on the embryonic dorsoventral pattern have been observed. Clones mutant for pipe appear to have a more autonomous effect than wind clones on twist expression in the embryo. This difference could reflect the differential diffusibility of these two gene products or their substrates. Alternatively, these results could suggest that an activity diffusing from the ventral region is less diffusible in the absence of pipe than in the absence of wind. In either case, the fact that wind and pipe clones yield slightly different phenotypes may indicate that the two genes do not function in a simple linear pathway (Nilson, 1998).

Dorsal-ventral specification of the Drosophila embryo is mediated by signaling pathways that have been very well described in genetic terms. However, little is known about the physiology of Drosophila development. By imaging patterns of free cytosolic calcium in Drosophila embryos, it has been found that several calcium gradients are generated along the dorsal-ventral axis. The most pronounced gradient is formed during stage 5, in which calcium levels are high dorsally. Manipulation of the stage 5 calcium gradient affects specification of the amnioserosa, the dorsal-most region of the embryo. This calcium gradient is inhibited in pipe, Toll, and dorsal mutants, but is unaltered in decapentaplegic or punt mutants, suggesting that the stage 5 calcium gradient is formed by a suppression of ventral calcium concentrations. It is concluded that calcium plays a role in specification of the dorsal embryonic region (Creton, 2000).

During early development (0 -2.5 h), calcium concentrations were elevated in the ventral region of the embryo and oscillate along with the cell cycle. Early Drosophila development is characterized by nuclear division without cell division, indicating that these calcium oscillations are associated with the embryo's nuclear cycle. The lowest levels of calcium are observed at the end of stage 4 (2.5 h). These low calcium concentrations represent the 'resting level' of calcium and average 72 nM. Cell formation (stage 5) lasts for about an hour and calcium levels increase during this time. This calcium increase is most pronounced at the dorsal region, thus creating a stage 5 calcium gradient with high calcium dorsally. The calcium concentrations in the dorsal region average 107 nM. The calcium gradient remains visible until the end of stage 6 (3 h 35 min). Calcium levels increase further in late embryonic development. These calcium elevations seem to be associated with gross morphological changes such as germ band extension and stomodeal invagination. Calcium levels reach a maximum of 137 nM during germ band extension (4 h 15 min). At 5.5 h, calcium gradients reverse for a second time to give high calcium concentrations ventrally. Thus, a total of three calcium gradients was observed along the dorsoventral axis during the first 6 h of development (Creton, 2000).

Injected BAPTA-type calcium buffers are known to permanently suppress calcium-dependent development by repeatedly carrying calcium from a source (such as a leaky region of the plasma membrane) to a calcium sink (such as the subsurface ER) into which this calcium is released. Such 'shuttle buffers' thereby suppress the development of high calcium zones. To determine if BAPTA injection affects specification of the amnioserosa, the dorsal-most region of the embryo was used, from which a Drosophila line was generated in which the amnioserosa element of the Kruppel upstream region drives the expression of lacZ. The Kr-lacZconstruct is exclusively expressed in the amnioserosa cells of stage 14 embryos. Injection of dibromo-BAPTA during stage 5 strongly inhibits Kr-lacZ expression. Thus the dorsal calcium zone is needed for the development of dorsal-specific gene expression. Kr-lacZ expression is fully restored or 'rescued' by raising the injectate's calcium to the micromolar level. This rescue further confirms that the observed inhibition is a direct effect of suppressing the dorsal calcium zone (Creton, 2000).

Embryos injected with dibromo-BAPTA at a final concentration of 3 mM show severe developmental abnormalities. The head fold and dorsal folds are mostly reduced or missing. In some cases, a head fold is formed that is symmetric along the dorsal-ventral axis. Cuticles are not formed in most of the embryos. Embryos that form cuticles show a reduced number of denticle belts, which are localized at their normal ventral position and do not seem to be extended or reduced along the dorsoventral axis. A normal mouth, present in treated embryos, includes mouth hooks, H-pieces, and ventral arms. However, the dorsal bridge and dorsal arms are often absent. This latter defect indicates a mild ventralization of the embryo. The lack of cuticle formation in the majority of the embryos may be expected, since calcium is a widely used messenger that probably affects many aspects of development. Nonetheless, the signs of ventralization further support the concept that a high calcium zone is needed for dorsal development, The expression of Kr-lacZ is significantly inhibited in treated morphologically normal stage 14 embryos; i.e., only half (49%) of the stage 14 embryos express Kr-lacZ. This experiment indicates that low concentrations of dibromo-BAPTA can inhibit specification of the amnioserosa without affecting the overall morphology of the stage 14 embryo (Creton, 2000).

Dorsalized pipe mutant embryos show highly elevated levels of calcium during stage 5 in the dorsal as well as the ventral regions. The stage 5 calcium gradient is lost in these embryos. Pipe is a key regulator of dorsoventral specification during oogenesis. Thus, the formation of the stage 5 calcium gradient is far downstream of this event. Calcium patterns were subsequently imaged in the Toll10B mutants. Toll10B mutant embryos are ventralized due to the overactive plasma membrane receptor Toll. These embryos do not show the typical dorsal calcium elevation during stage 5 and the stage 5 calcium gradient is lost. This indicates that the stage 5 calcium gradient is formed downstream of Toll. The dorsalized dl2 embryos show highly elevated levels of calcium during stage 5 in the dorsal as well as the ventral region. The stage 5 calcium gradient is thus lost in these embryos. This shows that the formation of the calcium gradient is downstream of dl. Other experiments show that the calcium pattern is upstream or independent of the Dpp signaling pathway (Creton, 2000).

The analysis of calcium patterns in mutant embryos shows that the formation of the stage 5 calcium gradient is downstream of dl and suggests that the Dl protein plays a role in formation of this calcium gradient by inhibiting ventral calcium elevations. At present it is not clear by which mechanisms Dl inhibits calcium concentrations in the ventral region. Possibly, nuclear Dl affects transcription of genes coding for calcium channels or calcium pumps. This modulation of gene expression may be a direct effect of Dl, or may be mediated by other proteins such as Twist or Snail. It is also not clear which mechanisms are responsible for the observed calcium elevation in the dorsal region during stage 5. It is speculated that this calcium increase may be caused by formation of the cleavage furrows, which activate stretch-sensitive calcium channels. This would cause a general calcium increase during stage 5, which would subsequently be inhibited on the ventral side by the Dl protein (Creton, 2000).

Drosophila Pipe protein activity in the ovary and the embryonic salivary gland does not require heparan sulfate glycosaminoglycans

The Drosophila pipe gene encodes ten related proteins that exhibit amino acid sequence similarity to vertebrate heparan sulfate 2-O-sulfotransferase. One of the Pipe isoforms, which is expressed in the ventral follicular epithelium, is a key determinant of embryonic dorsoventral polarity, suggesting that Pipe-mediated sulfation of a heparan sulfate proteoglycan provides a spatial cue for dorsoventral axis formation. Several approaches were used to investigate this possibility. The nucleotide alterations were determined in 11 different pipe alleles. Ten of the mutations specifically affect the pipe isoform that is expressed in the ovary. Among these ten mutations, two alter an amino acid in the putative binding site for 3'-phosphoadenosine 5'-phosphosulfate, the universal sulfate donor. Using Alcian Blue, a histochemical stain that detects sulfated glycans, a novel, pipe-dependent macromolecule was observed in the embryonic salivary glands. Genes known to participate in the formation of heparan sulfate in Drosophila are not required for the production of this material. To investigate whether a heparan sulfate proteoglycan is involved in pipe function in dorsoventral patterning, females carrying follicle cell clones mutant for heparan sulfate synthesis-related genes were generated. Embryos from follicles with mutant clones did not exhibit a dorsalized phenotype. Taken together, these data provide evidence that Pipe acts as a sulfotransferase, but argue against the hypothesis that the target of Pipe is a heparan sulfate glycosaminoglycan (Zhu, 2005).

Although the pipe locus encodes ten different protein isoforms, this analysis of pipe mutant alleles indicates that the Pipe-ST2 isoform is uniquely required for embryonic DV patterning. Ten out of the 11 EMS-generated alleles characterized at the molecular level specifically affect the Pipe-ST2 isoform. Females homozygous for these mutations produce dorsalized embryos, implying that the function of Pipe-ST2 in the follicle cells is essential for the establishment of the DV axis in the embryo. All of the identified mutations that affect only the Pipe-ST2 isoform are viable in trans to a deficiency. The pipe3 mutation, which affects all Pipe isoforms, is semi-lethal, suggesting a distinct requirement for other Pipe isoforms. The hypomorphic pipe7 mutation is associated with the relatively conservative change of valine to isoleucine within a domain that is predicted to be the binding site for the 5' phosphosulfate of PAPS. This result is consistent with the prediction that the pipe7 mutation alters the affinity of the mutant Pipe protein for PAPS and this isoform alone is essential for embryonic DV polarity (Zhu, 2005).

The finding that the pipe7 mutant phenotype is significantly enhanced by sodium chlorate treatment strongly supports the identification of Pipe as a sulfotransferase. This identification is further bolstered by the demonstration that the presence of a Pipe-dependent Alcian Blue-stained material in the embryonic salivary glands requires the function of two other genes essential for the sulfotransferase reaction: slalom, which encodes the Drosophila PAPS Golgi transporter; and papss, the PAPS synthetase gene. The finding that embryos mutant for pipe, slalom or papss all lack Alcian Blue staining in their salivary glands is strong evidence that the stained material represents a sulfated macromolecule (Zhu, 2005).

The original molecular identification of Pipe as a putative sulfotransferase was made on the basis of its similarity to HS2ST. Consequently, it has been assumed that heparan sulfate is the likely substrate of Pipe activity. It was reasoned that if Pipe acts as a heparan sulfate sulfotransferase, then the presence of the Alcian Blue-stained material in the embryonic salivary glands would be dependent upon the activity of genes whose products have been demonstrated to participate in heparan sulfate synthesis and modification in Drosophila. In contrast to this expectation, Alcian Blue staining was found to be present in the salivary glands of embryos mutant for sgl, sfl or frc (Zhu, 2005).

A similar strategy was used to investigate the possibility that heparan sulfate is the target of Pipe activity in the ovary. It was anticipated that genes encoding products involved in the sulfotransferase reaction, or in the synthesis of the Pipe substrate, would be required in the ventral follicle cells. Females carrying follicle cell clones mutant for pipe or slalom produce embryos with a dorsalized phenotype. By contrast, embryos derived from females carrying ventral clones of follicle cells mutant for sgl, sfl or frc exhibited normal DV polarity. This suggests that like the Alcian Blue-stained material in the embryonic salivary glands, the target of Pipe function in the ovary does not correspond to heparan sulfate (Zhu, 2005).

Surprisingly, females carrying papss mutant follicle cell clones did not produce dorsalized embryos. Although this result could be interpreted as an argument against Pipe acting as a sulfotransferase in the ovary, it is not believed that this is the explanation. Because PAPS, the product of PAPS synthetase activity, is a small molecule (507 Da), it may be able to pass through the gap junctions that exist between the oocyte and follicle cell layer. Gap junctions are known to allow passage of molecules of approximately 1 kDa in mass, which would permit passage of PAPS from a wild-type oocyte into mutant follicle cells. Another gene whose mutant alleles may behave nonautonomously for the same reason is sgl, which encodes UDP-glucose dehydrogenase. The product of Sugarless activity, UDP-glucuronic acid, is also a small molecule (577 Da) that may be capable of passing through gap junctions. Although the result for sgl mutant follicle cell clones may therefore be inconclusive, neither sfl nor frc mutations would be expected to exhibit nonautonomous behavior. sfl encodes N-deacetylase/N-sulfotransferase, a Golgi resident enzyme of Type II transmembrane topology. The product of Sfl activity, sulfated heparan sulfate, is too large to move between cells through gap junctions. The product of frc mediates the uptake into the Golgi of nucleotide sugars required for GAG synthesis and thus could not be rescued nonautonomously. Therefore, the finding that females carrying ventral follicle cell clones of sfl or frc did not give rise to dorsalized embryos provides the strongest evidence that heparan sulfate plays no role in the function of Pipe in embryonic DV patterning (Zhu, 2005).

Although sgl mutations may behave nonautonomously in the ovary, this explanation cannot be invoked to explain the lack of effect of sgl mutations on the Alcian Blue staining in the embryonic salivary glands. Because these embryos are both maternally and zygotically mutant for sgl, there would be no wild-type cells present to supply UDP-glucuronic acid to the sgl mutant cells. By contrast, even though a role for papss could not be demonstrated in the ovary because of the possibility of nonautonomous rescue, its function was clearly necessary for the formation of the Pipe-dependent Alcian Blue-stained material in the embryonic salivary glands (Zhu, 2005).

In addition to heparan sulfate, the ability of the Alcian Blue-stained material to form in the absence of sgl activity also rules out the possibility that Pipe is involved in the sulfation of dermatan/chondroitin sulfate, at least in that tissue. This is because UDP-glucuronic acid, the product of Sugarless activity, is required not only for the synthesis of heparan sulfate, but also for the synthesis of dermatan/chondroitin sulfate polysaccharide chains. Two other pieces of evidence also argue against a role for Pipe in the sulfation of either heparan sulfate or dermatan/chondroitin sulfate GAGs. (1) Expression in the follicle cell layer of cDNAs corresponding to hamster HS2ST and the human dermatan/chondroitin sulfate 2-O-sulfotransferase failed to rescue the dorsalized phenotypes of the progeny of pipe/pipe mutant females. The Drosophila genome contains another gene, CG10234, that encodes a protein that is much more similar to vertebrate HS2ST than are the Pipe isoforms; the product of this gene is likely to represent the bona fide Drosophila heparan sulfate 2-O sulfotransferase. (2) It has not been possible to detect heparan sulfate sulfotransferase or dermatan/chondroitin sulfate sulfotransferase activity in vitro using Pipe-ST2 protein expressed in cell culture. Although the data argue against a role for Pipe in the sulfation of uronic acid residues in heparan sulfate, the similarity of the Pipe isoforms to heparan sulfate 2-O sulfotransferase and dermatan/chondroitin sulfate 2-O-sulfotransferase suggests that Pipe acts on the 2-O position of a monosaccharide component of an as yet unidentified glycoprotein or glycolipid (Zhu, 2005).

The existence of multiple Pipe isoforms is an intriguing feature of the pipe gene in Drosophila melanogaster. Blast analysis of the D. pseudobscura genome indicates that multiple isoforms of Pipe exist in that species as well. By contrast, only a single Pipe isoform is encoded in the mosquito and flour beetle genomes. Similarly, only a single Pipe isoform was detected in a database of silk moth ESTs. In each of these three organisms, the single Pipe isoform exhibits strong sequence similarity to Drosophila Pipe-ST2. It therefore appears likely that only the Pipe-ST2 isoform was present in the common ancestor of true flies, mosquitoes, moths and beetles. This suggests that the ancestral role of the pipe gene was to act during oogenesis to regulate embryonic DV patterning. Multiple Pipe isoforms were probably generated via genomic duplication in Drosophila, where they appear to be required for salivary gland development and/or function. Lack of Pipe activity in the salivary gland may lead to a disruption of the feeding behavior of the larvae, which in turn reduces their growth rate and viability. The generation and expression of multiple protein isoforms may be a mechanism to produce extremely high levels of Pipe protein, if each isoform has a similar enzymatic specificity. Alternatively, each isoform may have a distinct substrate specificity that contributes uniquely to salivary gland development and/or function (Zhu, 2005).

The elucidation of Pipe-ST2 function is crucial to understanding the spatial regulation of the serine protease cascade whose ventrally restricted activity defines embryonic DV polarity. The simplest model of Pipe action posits that Pipe-ST2 functions as a sulfotransferase, and that the target of Pipe must be sulfated in order to exert its function. Although the target of Pipe may be present throughout the follicle cell layer, it would be sulfated only in the ventral follicle cells and following its secretion it would be deposited into the ventral side of the egg. There, it would assemble or activate the dorsal group serine protease cascade, leading to ventrally restricted processing of the Spätzle ligand. Although the specific targets of Pipe action in the follicle cell layer and the salivary gland may not be the same molecule, the general class of glycan on which Pipe acts in the two tissues is likely to be related. Current efforts are directed towards identifying these molecules and defining their roles in DV patterning and salivary gland function (Zhu, 2005).

Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array

Dorsal-ventral (DV) patterning of the Drosophila embryo is initiated by Dorsal, a sequence-specific transcription factor distributed in a broad nuclear gradient in the precellular embryo. Previous studies have identified as many as 70 protein-coding genes and one microRNA (miRNA) gene that are directly or indirectly regulated by this gradient. A gene regulation network, or circuit diagram, including the functional interconnections among 40 Dorsal (Dl) target genes and 20 associated tissue-specific enhancers, has been determined for the initial stages of gastrulation. This study attempts to extend this analysis by identifying additional DV patterning genes using a recently developed whole-genome tiling array. This analysis led to the identification of another 30 protein-coding genes, including the Drosophila homolog of Idax, an inhibitor of Wnt signaling. In addition, remote 5' exons were identified for at least 10 of the ~100 protein-coding genes that were missed in earlier annotations. As many as nine intergenic uncharacterized transcription units (TUs) were identified, including two that contain known microRNAs, miR-1 and -9a. The potential functions of these recently identified genes are discussed and it is suggested that intronic enhancers are a common feature of the DV gene network (Biemar, 2006).

The Dl nuclear gradient differentially regulates a variety of target genes in a concentration-dependent manner. The gradient generates as many as five different thresholds of gene activity, which define distinct cell types within the presumptive mesoderm, neuroectoderm, and dorsal ectoderm. Total RNA was extracted from embryos produced by three different maternal mutants: pipe/pipe, Tollrm9/Tollrm10, and Toll10B. pipe/pipe mutants completely lack Dl nuclear protein and, as a result, overexpress genes that are normally repressed by Dl and restricted to the dorsal ectoderm. For example, the decapentaplegic (dpp) TU is strongly "lit up" by total RNA extracted from pipe/pipe mutant embryos. The intron-exon structure of the transcribed region is clearly delineated by the hybridization signal, most likely because the processed mRNA sequences are more stable than the intronic sequences present in the primary transcript. There is little or no signal detected with RNAs extracted from Tollrm9/Tollrm10 (neuroectoderm) and Toll10B (mesoderm) mutants. Instead, these other mutants overexpress different subsets of the Dl target genes. For example, Tollrm9/Tollrm10 mutants contain low levels of Dl protein in all nuclei in ventral, lateral, and dorsal regions. These low levels are sufficient to activate target genes such as intermediate neuroblasts defective (ind), ventral neuroblasts defective (vnd), rhomboid (rho), and short gastrulation (sog) but insufficient to activate snail (sna). In contrast, Toll10B mutants overexpress genes (e.g., sna) normally activated by peak levels of the Dl gradient in ventral regions constituting the presumptive mesoderm (Biemar, 2006).

To identify potential Dl targets, ranking scores were assigned for the six possible comparisons of the various mutant backgrounds, pipe vs. Tollrm9/Tollrm10, pipe vs. Toll10B, Tollrm9/Tollrm10 vs. Toll10B, Tollrm9/Tollrm10 vs. pipe, Toll10B vs. Tollrm9/Tollrm10, and Toll10B vs. pipe, using the TiMAT software package. As a first approximation, only hits with a median fold difference of 1.5 and above were considered. For further analysis, the top 100 TUs were selected for each of the comparisons, with the exception of Tollrm9/Tollrm10 vs. pipe for which the TiMAT analysis returned only 43 hits that meet the cutoff. To refine the search for TUs specifically expressed in the mesoderm, where levels of nuclear Dl are highest, only those present in the Toll10B vs. Tollrm9/Tollrm10 and Toll10B vs. pipe, but not pipe vs. Tollrm9/Tollrm10 comparisons were selected. For TUs induced by intermediate and low levels of nuclear Dl in the neuroectoderm, those present in both the Tollrm9/Tollrm10 vs. Toll10B and Tollrm9/Tollrm10 vs. pipe, but not pipe vs. Toll10B comparisons were selected. For TUs restricted to the dorsal ectoderm, only those present in the pipe vs. Tollrm9/Tollrm10 and pipe vs. Toll10B, but not Tollrm9/Tollrm10 vs. Toll10B, were selected. Finally, the TUs corresponding to annotated genes already identified in the previous screen were eliminated to focus on annotated genes not previously considered as potential Dorsal targets, as well as transcribed fragments (transfrags) not previously characterized. Using these criteria, 45 previously annotated protein-coding genes were identified, along with 23 uncharacterized transfrags. Of the 45 protein-coding genes, 29 exhibited localized patterns of gene expression across the DV axis, whereas the remaining 16 were not tested (Biemar, 2006).

The previous microarray screen relied on high cutoff values for the identification of authentic DV genes. For example, only genes exhibiting 6-fold up-regulation in pipe/pipe mutant embryos were tested by in situ hybridization for localized expression in the dorsal ectoderm. Many other genes displayed >2-fold up-regulation but were not explicitly tested for localized expression. The whole-genome tiling array permitted the use of much lower cutoff values. For example, CG13800, which was identified by conventional microarray screens, falls just below the original cutoff value but displays 5-fold up-regulation in pipe/pipe mutants in the analysis. In situ hybridization assays reveal localized expression in the dorsal ectoderm. This pattern is greatly expanded in embryos derived from pipe/pipe mutant females, as expected for a gene that is either directly or indirectly repressed by the Dl gradient. Genes exhibiting even lower cutoff values were also found to display localized expression. Among these genes is a Wnt homologue, Wnt2, which is augmented only 2.25-fold in mutant embryos lacking the Dl nuclear gradient (Biemar, 2006).

The 4-fold cutoff value used in the previous screen for candidate protein-coding genes expressed in the neuroectoderm also excluded genes expressed in this tissue. The Trim9 gene exhibits just a 2-fold increase in mutant embryos derived from Tollrm9/Tollrm10 females. Nonetheless, in situ hybridization assays reveal localized expression in the neuroectoderm of WT embryos. As expected, expression is expanded in Tollrm9/Tollrm10 mutant embryos. Another gene, CG9973, displays just 1.8-fold up-regulation but is selectively expressed in the neuroectoderm. CG9973 encodes a putative protein related to Idax, an inhibitor of the Wnt signaling pathway. Idax inhibits signaling by interacting with the PDZ domain of Dishevelled (Dsh), a critical mediator of the pathway. A Wnt2 homologue is selectively expressed in the dorsal ectoderm. Recent studies identified a second Wnt gene, WntD, which is expressed in the mesoderm. Thus, the CG9973/Idax inhibitor might be important for excluding Wnt signaling from the neuroectoderm. Such a function is suggested by the analysis of Idax activity in vertebrate embryos (Biemar, 2006).

Additional genes were also identified that are specifically expressed in the mesoderm. Among these is CG9005, which encodes an unknown protein that is highly conserved in different animals, including frogs, chicks, mice, rats, and humans. It displays <2-fold up-regulation in Toll10B embryos but is selectively expressed in the ventral mesoderm of WT embryos. Expression is expanded in embryos derived from Toll10B mutant females (Biemar, 2006).

Other protein-coding genes were missed in the previous screen because they were not represented on the Drosophila Genome Array used at the time. These include, for instance, CG8147 in the dorsal ectoderm and CG32372 in the mesoderm (Biemar, 2006).

An interesting example of the use of tiling arrays to identify tissue-specific isoforms is seen for the bunched (bun) TU. bun encodes a putative sequence-specific transcription factor related to mammalian TSC-22, which is activated by TGFβ signaling. It was shown to inhibit Notch signaling in the follicular epithelium of the Drosophila egg chamber. Three transcripts are expressed from alternative promoters in bun, but it appears that only the short isoform (bun-RC) is specifically expressed in the dorsal ectoderm. A number of bun exons are ubiquitously transcribed at low levels in the mesoderm, neuroectoderm, and dorsal ectoderm. However, the 3'-most exons are selectively up-regulated in pipe/pipe mutants. It is conceivable that Dpp signaling augments the expression of this isoform, which in turn, participates in the patterning of the dorsal ectoderm (Biemar, 2006).

In addition to protein-coding genes, the tiling array also identified uncharacterized TUs not previously annotated. Some of them are associated with ESTs, providing independent evidence for transcriptional activity in these regions. For 14 of these transfrags (61%), visual inspection of neighboring loci using the Integrated Genome Browser suggested coordinate expression of a neighboring protein-coding region (i.e., overexpressed in the same mutant background). The N-Cadherin gene (CadN) has a complex intron-exon structure consisting of ~20 different exons. The strongest hybridization signals are detected within the limits of exons, but an unexpected signal was detected ~10 kb upstream of the 5'-most exon. It is specifically expressed in the mesoderm, suggesting that it represents a previously unidentified 5' exon of the CadN gene. Support for this contention stems from two lines of evidence: (1) in situ hybridization using a probe against the 5' exon detects transcription in the presumptive mesoderm, the initial site of CadN expression; (2) using primers anchored in the 5' transfrag as well as the first exon of CadN, confirmation was obtained by RT-PCR that the recently identified TU is part of the CadN transcript. This recently identified 5' exon appears to contribute to the 5' leader of the CadN mRNA. It is possible that this extended leader sequence influences translational efficiency as seen in yeast. Because there seems to be a considerable lag between the time when CadN is first transcribed and the first appearance of the protein, it is suggested that this extended leader sequence might inhibit translation. An interesting possibility is that it does so through short upstream ORFs, as has been shown for several oncogenes in vertebrates (Biemar, 2006).

A 5' exon was also identified for crossveinless-2 (cv-2), a component of the Dpp bone morphogenetic protein (BMP) signaling pathway. cv-2 binds BMPs and functions as both an activator and inhibitor of BMP signaling. It is specifically required in the developing wing disk to generate peak Dpp signaling in the presumptive crossveins. cv-2 is also expressed in the dorsal ectoderm of early embryos, but its role during embryonic development has not been investigated. The whole-genome tiling array identified a 5' exon located ~10 kb 5' of the transcription start site of the cv-2 TU. Using RT-PCR and in situ hybridization assays, it was confirmed that the exon is part of the cv-2 transcript. It is possible that the exon resides near an embryonic promoter that is inactive in the developing wing discs. Future studies will determine whether this 5' exon influences the timing or levels of Cv-2 protein synthesis (Biemar, 2006).

In addition to the identification of 10 5' exons associated with previously annotated genes such as CadN and cv-2, three other transfrags appear to correspond to 3' exons, and nine of the RNAs seem to arise from autonomous TUs. Three of these represent annotated computational RNA (CR) genes: CR32777, CR31972, and CR32957. CR32777 corresponds to roX1, which is ubiquitously expressed at the blastoderm stage, hence it represents a false positive. The other two potential noncoding RNAs were recently identified independently in two other studies, and although the expression of CR32957 could not be detected by in situ hybridization, CR31972 transcripts are detected in the mesoderm. There is no evidence that these transcripts are processed into miRNAs, but noncoding genes corresponding to known miRNA loci were also identified in the screen. Transfrag 22 corresponds to the miR-9a primary transcript (pri-mir9a) and is detected in both the dorsal- and neuroectoderm. Expression of pri-mir9a is ubiquitous in embryos derived from pipe/pipe or Tollrm9/Tollrm10 females. Transfrag 8 corresponds to pri-mir1, which is present in the mesoderm (Biemar, 2006).

A third noncoding transcript (Transfrag 12) maps next to a known miRNA, miR-184. It is selectively expressed in the mesoderm and overexpressed in Toll10B mutants. The mesodermal expression of miR-184 has been reported. It is possible that Transfrag 12 corresponds to pri-mir-184, and that secondary structures in the miRNA region preclude detection on the array. This is seen for several other miRNA precursors expressed at various stages during embryogenesis. Alternatively, Transfrag 12 might represent the fragment resulting from Drosha cleavage of the pri-mir-184 to produce the miR-184 precursor hairpin (pre-miR-184). A similar situation has been observed for the iab4 locus. Like miR-1, miR-184 is selectively expressed in the ventral mesoderm. It will be interesting to determine whether the two miRNAs jointly regulate some of the same target mRNAs (Biemar, 2006).

The identity of the last three transfrags is less clear. Visual inspection using the Integrated Genome Browser suggests expression of Transfrag 10 in the mesoderm, Transfrag 21 in the neuroectoderm, and Transfrag 11 in both the dorsal ectoderm and neuroectoderm. However, in situ hybridization assays confirm the predicted expression pattern only for Transfrag 11. Computational analyses designed to estimate the likelihood of translation suggest a protein-coding potential for Transfrag 10 [Likelihood Ratio Test (LRT) P < 0.001] and possibly Transfrag 11 (LRT P < 0.01), whereas Transfrag 21 could not be analyzed because of lack of conservation in other Drosophila species (Biemar, 2006).

This work has attempted to identify nonprotein coding genes involved in patterning the DV axis of the Drosophila embryo using an unbiased approach to survey the entire genome. This study, along with earlier analyses, identified as many as 100 protein-coding genes and five to seven noncoding genes that are differentially expressed across the DV axis of the early Drosophila embryo. Roughly half of the noncoding RNAs correspond to miRNAs, although <1% of the annotated genes in the Drosophila genome encode miRNAs. Future studies will determine how these RNAs impinge on the DV regulatory network (Biemar, 2006).

Recent studies have identified large numbers of noncoding transcripts in the mouse and human genomes. If the present study is predictive, less than one-fourth of the transcripts correspond to novel noncoding RNAs of unknown function, akin to CR31972 and Transfrag 11 expressed in the mesoderm and ectoderm, respectively. Most of the noncoding transcripts are likely to derive from intronic sequences because of the occurrence of cryptic remote 5' exons as seen for the CadN and cv-2 genes. At least 10% of the DV protein-coding genes were found to contain such exons. As a result, these genes contain large tracts of intronic sequences that might encompass regulatory DNAs such as tissue-specific enhancers. The FGF8-related gene, thisbe (ths), represents such a case. A neurogenic-specific enhancer that was initially thought to reside 5' of the TU actually maps within a large intron because of the occurrence of a remote 5' exon. It is suggested that such exons are responsible for the evolutionary "bundling" of genes and their associated regulatory DNAs. Gene duplication events are more likely to retain this linkage when regulatory DNAs map within the TU. In contrast, enhancers mapping in flanking regions can be uncoupled from their normal target gene by chromosomal rearrangements (Biemar, 2006).

Distinct functional specificities are associated with protein isoforms encoded by the Drosophila dorsal-ventral patterning gene pipe

Spatially regulated transcription of the pipe gene in ventral cells of the Drosophila ovary follicle cell epithelium is a key event that specifies progeny embryo dorsal-ventral (DV) polarity. pipe encodes ten putative protein isoforms, all of which exhibit similarity to vertebrate glycosaminoglycan-modifying enzymes. Expression of one of the isoforms, Pipe-ST2, in follicle cells has been shown to be essential for DV patterning. pipe is also expressed in the embryonic salivary gland and its expression there is required for normal viability. In addition to Pipe-ST2, seven of the other Pipe isoforms are expressed in the ovary, whereas all Pipe isoforms are abundantly expressed in the embryo. Of the ten isoforms, only Pipe-ST2 can restore ventral and lateral pattern elements to the progeny of otherwise pipe-null mutant females. By contrast, three Pipe isoforms, but not Pipe-ST2, support the production of a novel pipe-dependent epitope present in the embryonic salivary gland. These data indicate that differences in functional specificity, and presumably enzymatic specificity, are associated with several of the Pipe isoforms. In addition, it was shown that uniform expression of the Pipe-ST2 isoform in the follicle cell layer of females otherwise lacking pipe expression leads to the formation of embryos with a DV axis that is appropriately oriented with respect to the intrinsic polarity of the eggshell. This suggests the existence of a second mechanism that polarizes the Drosophila embryo, in addition to the ventrally restricted transcription of the pipe gene (Zhang, 2009).

pipe is a highly conserved gene in the insects. In insects other than the cyclorrhaphan (true) flies, only a single Pipe isoform is normally present. Moreover, the individual Pipe isoforms that are predicted to be expressed in mosquito (Anopheles gambiae), silkworm (Bombyx mori), honeybee (Apis mellifera) and flour beetle (Tribolium casteneum) exhibit greatest sequence similarity to the Pipe-ST2 isoform, among the multiple isoforms expressed by Drosophila melanogaster. By contrast, the genomes of all Drosophila species that have been sequenced (D. ananassae, D. erecta, D. grimshawi, D. mojavensis, D. persimilis, D. pseudoobscura, D. sechellia, D. simulans, D. virilis, D. willistoni and D. yakuba) predict multiple Pipe protein isoforms. Taken together, these observations suggest that an insect ancestral to the holometabolous Diptera, Coleoptera, Hymenoptera and Leptidoptera expressed a single Pipe isoform, the role of which was to regulate the formation of the embryonic DV axis. Pipe protein may also regulate DV axis formation in more basal genera of insects and in other arthropods, as the genome of the water flea (Daphnia pulex), a crustacean, apparently encodes an orthologous protein that is more similar to Pipe-ST2 than to HS2ST or D/C2ST (Zhang, 2009).

The existence of multiple protein isoforms in the genomes of the various Drosophila species indicates that several duplications of pipe exons have occurred during the evolution of the members of the dipteran suborder Brachycera, perhaps in the line leading to the family Drosophilidae. Some of the isoforms might have arisen as a means of modulating the effects of the essential Pipe-ST2 isoform during oogenesis, perhaps by modifying the carbohydrate target of Pipe-ST2 to make it a more avid substrate. This pre-modification of the Pipe target would not be essential for Pipe-ST2 action, but would make the developmental process more robust and reproducible from egg chamber to egg chamber, an adaptation to the rapid oogenesis in the Drosophilid species. Alternatively, the multiple Pipe protein isoforms in the Drosophilids might have arisen to provide a function that is totally unrelated to embryonic DV patterning. It has been shown that the absence of expression of all isoforms of Pipe leads to the formation of structurally aberrant salivary glands and to a decrease in fly viability and growth. Although these effects are correlative, it is reasonable to assume that the decrease in viability and growth results from an alteration in feeding behavior that is attributable to a perturbation of the development or function of the salivary gland owing to the absence of all Pipe catalytic activity. As such, the presence of multiple isoforms of the Pipe protein in the Drosophilids might represent an adaptation that is specifically related to the feeding behavior and physiology of those species. Ultimately, an understanding of the basis for the multiple Pipe proteins and their functions will require a precise determination of the distinct catalytic activities of the various proteins, a goal that will first require the identification of the class of molecule(s) on which these enzymes act (Zhang, 2009).

The production of appropriately polarized, partially rescued embryos by females expressing Pipe-ST2 in an otherwise pipe-null background strongly suggests the existence of a second signal that can impart polarity, even in a situation in which Pipe-ST2 activity is not ventrally restricted. A model has been proposed in which maternally directed activation of the Decapentaplegic (Dpp) pathway facilitates degradation of the Cactus protein on the dorsal side of embryos in a Toll-independent manner, thus representing a second mechanism for polarizing the DV axis of the embryo. Although this pathway could, in principle, correspond to the second polarizing input, this explanation is not favored. Previous findings are difficult to reconcile with the existence of mutant gain-of-function alleles of easter and Toll that cause uniform activation of Toll around the DV circumference of the embryo, and the consequent production of apolar lateralized or ventralized embryos. The production of such apolar embryos would not be expected were the Dpp pathway acting independently of Toll signaling to establish embryonic DV axis formation. Based on the existence of these unusual apolar alleles of easter and Toll, a model is favored in which the pipe-independent signal contributes to the polar activation at a step upstream of Easter-mediated cleavage of Spätzle (Zhang, 2009).

Two possible explanations are proposed for the second polarizing influence. According to the current model, Pipe protein action in ventral follicle cells leads to the sulfation of a secreted glycoprotein that is deposited ventrally in the egg, where it mediates the activation of the dorsal group serine protease cascade. If any of the genes that encode proteins involved in the synthesis of the direct carbohydrate target of Pipe or its predicted carrier protein were expressed preferentially in ventral follicle cells, then the ventrally enriched production of the Pipe substrate could account for the residual polarity of progeny embryos under conditions of uniform Pipe-ST2 activity. The ability of Pipe-ST2 to invert the polarity of progeny embryos when expressed ectopically in dorsal follicle cells would result from the ability of Pipe-mediated sulfation to overcome the polarizing effects of ventral enrichment of the Pipe target (Zhang, 2009).

An alternative basis for residual polarity in the presence of uniform Pipe-ST2 activity might be the existence of an inhibitor of Gd or Snake that is enriched dorsally within the perivitelline space, or of a ventrally enriched, positively acting regulator of the processing or activity of Gd, Snake or Easter, distinct from Pipe-ST2. Although a serpin inhibitor of Easter, Spn27A, has been shown to operate in the perivitelline space, this molecule is uniformly distributed. Moreover, available data suggest that Spn27A acts to restrict Easter activity to the ventral side of the embryo following its activation, and does not have any intrinsic ability to orient the activity of Easter (Zhang, 2009).

The geometry of egg formation might also introduce an asymmetry that could influence the effects of Pipe-ST2 along the embryonic DV axis. Over the course of oogenesis, the egg chamber and egg assume characteristic shapes that are polarized along the DV axis. The morphogenetic processes that govern egg formation and the physical properties of the mature egg might influence the distribution of the modified target of Pipe or the spatial constraints of serine protease activation and in this way impart a weakly polarizing influence that is detectable when pipe expression is uniform in the developing egg chamber (Zhang, 2009).

The extent to which this second polarizing input is required for the formation of embryonic DV polarity in wild-type embryos is difficult to assess. The second input cannot impart DV polarity to embryos in the absence of Pipe-ST2 activity. This suggests that the role of the second input might be to buffer the effects of rare perturbations in the normal ventral pattern of pipe expression. The power of this second input to influence the DV axis is demonstrated by the generation of viable stocks in which Pipe-ST2 is expressed uniformly along the DV axis under the control of 55B-Gal4 in otherwise pipe-null mutant backgrounds that lack endogenous ventral expression of Pipe-ST2 (Zhang, 2009).

In conclusion, these investigations of the functions of the various Pipe protein isoforms has demonstrated the existence of additional layers of complexity in the mechanism generating embryonic DV polarity, including the presence of a previously unappreciated mechanism that reinforces the effects of the ventral transcription of the pipe gene. The elucidation of the structure of the substrate of Pipe-ST2 and its function in regulating the dorsal group serine protease cascade is likely to provide insights into this alternate polarizing input and into the specific roles of the sulfotransferases encoded by the pipe locus (Zhang, 2009).


Biemar, F., et al. (2006). Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array. Proc. Natl. Acad. Sci. 103(34): 12763-8. Medline abstract: 16908844

Cho, Y. S., Stevens, L. M., Sieverman, K. J., Nguyen, J. and Stein, D. (2012). A ventrally localized protease in the Drosophila egg controls embryo dorsoventral polarity. Curr Biol 22: 1013-1018. PubMed ID: 22578419

Creton, R., Kreiling, J. A. and Jaffe, L. F. (2000). Presence and roles of calcium gradients along the dorsal-ventral axis in Drosophila embryos. Dev. Biol. 217: 375-385. 10625561

DeLotto, L. (2001). Gastrulation defective, a complement factor C2/B-like protease, interprets a ventral prepattern in Drosophila. EMBO Reports 2: 721-726. 11493599

Fuchs, A., et al. (2012). Transcriptional interpretation of the EGF receptor signaling gradient. Proc. Natl. Acad. Sci. 109(5): 1572-7. PubMed Citation: 22307613

Goff, D. J., Nilson, L. A. and Morisato, D. (2001). Establishment of dorsal-ventral polarity of the Drosophila egg requires capicua action in ovarian follicle cells. Development 128: 4553-4562. 11714680

Hong, C. C. and Hashimoto, C. (1995). An unusual mosaic protein with a protease domain, encoded by the nudel gene, is involved in defining embryonic dorsoventral polarity in Drosophila. Cell 82: 785-794.

James, K. E., Dorman, J. B. and Berg, C. A. (2002). Mosaic analyses reveal the function of Drosophila Ras in embryonic dorsoventral patterning and dorsal follicle cell morphogenesis. Development 129: 2209-2222. 11959829

Jordan, K. C., et al. (2000). The homeobox gene mirror links EGF signaling to embryonic dorso-ventral axis formation through Notch activation. Nat. Genet. 24(4): 429-33. PubMed Citation: 10742112

Kobayashi, M., et al. (1996). Purification and characterization of heparan sulfate 2-sulfotransferase from cultured Chinese hamster ovary cells. J. Biol. Chem. 271(13): 7645-53. PubMed Citation: 8631801

Kobayashi, M., et al. (1997). Molecular cloning and expression of Chinese hamster ovary cell heparan-sulfate 2-sulfotransferase. J. Biol. Chem. 272(21): 13980-5. PubMed Citation: 8631801

Konsolaki, M. and Schupbach, T. (1998). windbeutel, a gene required for dorsoventral patterning in Drosophila, encodes a protein that has homologies to vertebrate proteins of the endoplasmic reticulum. Genes Dev, 12(1): 120-31. PubMed Citation: 9420336

Lüders, F., et al. (2003). slalom encodes an adenosine 3'-phosphate 5'-phosphosulfate transporter essential for development in Drosophila. EMBO J. 22: 3635-3644. 12853478

Morisato, D. (2001). Spätzle regulates the shape of the Dorsal gradient in the Drosophila embryo. Development 128: 2309-2319. 11493550

Nilson, L. A. and Schupbach, T. (1998). Localized requirements for windbeutel and pipe reveal a dorsoventral prepattern within the follicular epithelium of the Drosophila ovary. Cell 93(2): 253-62. PubMed Citation: 9568717

Pai, L.-M., Barcelo, G. and Schüpbach, T. (2000). D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis. Cell 103: 51-61. PubMed Citation: 11051547

Sen, J., Goltz, J. S., Stevens, L. and Stein, D. (1998). Spatially restricted expression of pipe in the Drosophila egg chamber defines embryonic dorsal-ventral polarity. Cell 95(4): 471-81. PubMed Citation: 9827800

Sen, J., et al. (2000). Windbeutel is required for function and correct subcellular localization of the Drosophila patterning protein Pipe. Development 127: 5541-5550. PubMed Citation: 11076773

Spradling, A.C. (1993). Developmental genetics of oogenesis. In The Development of Drosophila melanogaster, M. Bate and A. Martinez-Arias, eds. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp. 1-70.

Stein, D., Roth, S., Vogelsang, E. and Nusslein-Volhard, C. (1991). The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal. Cell 65(5): 725-35. PubMed Citation: 1904007

Technau, M., Knispel, M. and Roth, S. (2012). Molecular mechanisms of EGF signaling-dependent regulation of pipe, a gene crucial for dorsoventral axis formation in Drosophila. Dev. Genes Evol. 222(1): 1-17. PubMed Citation: 22198544

Zhao, D., Woolner, S. and Bownes, M. (2000). The Mirror transcription factor links signalling pathways in Drosophila oogenesis. Dev. Genes Evol. 210: 449-457. 11180850

Zhang, Z., Zhu, X., Stevens, L. M. and Stein, D. (2009). Distinct functional specificities are associated with protein isoforms encoded by the Drosophila dorsal-ventral patterning gene pipe. Development 136(16): 2779-89. PubMed Citation: 19633171

Zhu, X., Sen, J., Stevens, L., Goltz, J. S. and Stein, D. (2005). Drosophila Pipe protein activity in the ovary and the embryonic salivary gland does not require heparan sulfate glycosaminoglycans. Development 132(17): 3813-22. 16049108

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

date revised: 20 April 2013

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