BIOLOGICAL OVERVIEW

Two signals are responsible for dorsoventral patterning in the egg and embryo (Schümpbach, 1994). The first signal requires both the ligand Gurken and the receptor Torpedo (EGF-R). Carried from the oocyte to follicle cells (maternal cells that surround the egg), it is a signal to enter a dorsal differentiation program. This signal delimits the ventral zone within the follicle cell epithelium. The formation of the ligand of the second signal (Spätzle) is thus spatially restricted to a zone in the ventral part of the egg. When fertilization takes place, Spätzle is activated and triggers the receptor Toll to activate the transcription factor Dorsal in the ventral cells of the developing embryo.

Gurken mRNA becomes localized to the anterior dorsal part of the oocyte, between the nucleus and the overlying oocyte membrane. A number of genes are required to localize GRK mRNA including cornichon, fs(1)K10, orb, squid, cappuccino and spire. Gurken is the ligand for the Torpedo/EGF receptor, located on the surface of follicle cells that envelope the oocyte. GRK triggers EGF-R/Torpedo signaling and the consequent modification of cell fate in the follicle cells.

One follicle cell gene regulated by Torpedo signaling is rhomboid, a gene involved in the specification of dorsal cell fate in dorsal follicle cells (Ruohola-Baker, 1993). Gurken signaling involves a feedback from follicle cells to the oocyte, resulting in a correct polarization of the oocyte's anterior-posterior microtubule cytoskeleton. The mechanism underlying this feedback is currently not known (Roth, 1995).

The establishment of dorsal cell fate in follicle cells is important in the later differential ability of ventral follicle cells to activate the second dorsal-ventral signal triggered upon fertilization, signaling through the Toll receptor to activate the Dorsal transcription factor in ventral cells.

Thus, through the action of Gurken, the dorsal-ventral polarity of the egg is established well in advance of fertilization and embryonic development. The interaction between the oocyte and its enveloping follicle cells is critical for the future development of the embryo.

In addition to its role in establishing dorsal-ventral polarity, the Gurken-EGF-R interaction has a prior role in the induction of posterior follicle cell fate and the specification of the egg's anterior-posterior polarity. Early Gurken is localized to the posterior pole of the oocyte. Gurken mutant egg chambers lack posterior follicle cells and show a mirror-image duplication of the AP axis of the oocyte. In addition, a large proportion of Egf-r mutants develop an AP phenotype very similar to that produced by grk mutation. It is believed that AP polarity arises from the movement of the oocyte to the posterior of the nurse cells within the egg chamber. The germinal vesicle (egg nucleus) and GRK mRNA both become localized to the posterior of the oocyte, leading to the polarized production of Gurken. EGF-R becomes activated in adjacent polar follicle cells to determine posterior follicle cell fate. The posterior follicle cells subsequently signal back to the oocyte to repolarize the oocyte microtubule cytoskeleton, probably by inducing the dissembly of the microtubule organizing center at the posterior of stage 6 oocytes. A new microtubule nucleating activity is generated at the anterior margin of the oocyte, and as a consequence the germinal vesicle moves to the anterior margin of the oocyte, ready for the subsequent involvement of Gurken in establishing dorsal cell fate (González-Reyes, 1995 and Roth, 1995).

decapentaplegic is required for patterning of anterior eggshell structures, reflecting expression of dpp in anterior somatic follicle cells. In grk mutant females, ectopic expression of dpp occurs at the posterior pole of 77% of the mutant egg chambers. sax mutant egg chambers exhibit similar posterior dpp expression. This suggests that SAX receptor function is required for acquisition of at least some posterior fate, and also suggests that dpp is the target of gurken-Egfr signaling (Twombly, 1996).

Gurken is here shown to induce two different follicle cell fates because the follicle cells at the termini of the egg chamber differ in their competence to respond to Gurken from the main-body follicle cells in between. Anterior follicle cells are known to become subdivided into three distinct follicle cell types along the anterior-posterior axis: border cells, stretched follicle cells and centripetal follicle cells. The border cells are a group of 6-10 cells that delaminate from the follicular epithelium at the anterior tip of the egg chamber and migrate between the nurse cells to the anterior of the oocyte. At the same time, the adjacent stretched follicle cells spread to cover the nurse cells as the rest of the follicular epithelium moves posteriorly to envelop the oocyte. The centripetal follicle cells just posterior to the stretched follicle cells come to lie over the anterior of the oocyte after these movements are complete, and these cells then migrate between the oocyte and the nurse cells toward the center of the egg chamber during stage 10b (Gonzalez-Reyes, 1998).

It is argued that the terminal follicle cell populations (consisting of both anterior and posterior follicle cell populations) are equivalent prior to gurken signaling. To explain how Gurken can induce two different follicle cell fates, it has been proposed that the follicle cell layer is divided into two cell types during early oogenesis: the terminal follicle cells at each end of the egg chamber, which become posterior if they receive the Gurken signal and anterior if they do not, and the main-body follicle cells, which are induced to become dorsal rather than ventral. To determine whether the main-body follicle cells can adopt a posterior fate in response to Gurken, the original dicephalic mutation was analyzed. Unlike the spindle mutants, dicephalic alters the position of the oocyte without affecting Gurken signaling. This allows for a test of the fate of anterior, main-body and posterior follicle cells by allowing for the exposure of each of these populations to the Gurken signal. The induction of posterior follicle cell fate is followed directly, using a recently identified enhancer trap line that specifically labels these cells. When the oocyte is correctly positioned at the posterior of dicephalic mutant egg chambers, the enhancer trap line is strongly expressed in the posterior follicle cells just as it is in wild type. In contrast, no expression is observed when the oocyte lies in the middle of the mutant egg chamber. Thus, misplaced oocytes cannot induce main-body follicle cells to adopt a posterior fate, arguing that main-body and posterior follicle cells possess two different fates (Gonzalez-Reyes, 1998).

Although it has been clearly demonstrated that the posterior follicle cells adopt an anterior fate if they do not receive the Gurken signal, it has not been shown that the anterior terminal cells are competent to adopt a posterior fate. The dicephalic mutant egg chambers where the oocyte lies at the anterior of the cyst have been examined. In this case, the posterior marker is expressed in the anterior follicle cells of these egg chambers; this confirms that misplaced oocytes can still signal to induce posterior fate and confirms the equivalence of anterior and posterior follicle cells. Furthermore, these egg chambers develop completely normally although they have a reversed polarity with respect to the anterior-posterior axis of the whole ovariole. The terminal follicle cells at the opposite end of the oocyte become subdivided into the three anterior follicle cell types which undergo their normal migrations, whereas the 'anterior' cells in contact with the oocyte not only express the posterior marker, but also signal to polarize the oocyte cytoskeleton, since all of the maternal mRNAs tested localize to the appropriate position. These results demonstrate that the main-body follicle cells and the terminal follicle cells do indeed constitute two distinct populations that differ in their competence to respond to Gurken and prove that the terminal follicle cells at each end of the egg chamber are equivalent prior to Gurken signaling (Gonzalez-Reyes, 1998).

The Egfr, as receptor of the posterior Gurken signal, is required cell autonomously to repress anterior fate in all posterior follicle cells. Although the expression of several markers at the termini of developing egg chambers suggests the existence of populations of terminal follicle cells, it is not clear how many cells respond to Gurken directly by adopting a posterior rather than an anterior fate. To define this population, a mapping was performed to determine which cells revert to the default anterior fate when they cannot respond to Gurken because they lack its putative receptor. Small marked clones of cells were generated that are homozygous for top CO, a null allele of the Egfr, and their fate was followed by staining for the beta-gal activity of the L53b enhancer trap line, which labels all three subpopulations of anterior follicle cells from stage 9 onwards. When the clones are generated (at approximately stage 2 of oogenesis) and scored at stage 10, mutant cells that lie near the posterior of the oocyte are seen to always express L53b, whereas clones over the middle of the oocyte do not. Thus, removal of the Egfr causes a cell-autonomous transformation from posterior to anterior fate, indicating that Gurken signals directly to induce posterior fate in the whole terminal follicle cell population. With one exception, all Egfr- cells that fall within 10-11 cell diameters of the posterior end of the egg chamber express L53b, whereas mutant cells that fall anterior to this boundary do not. This analysis indicates that about 200 terminal follicle cells receive the Gurken signal directly, ruling out a model in which only the polar follicle cells (the most posterior cell population) are competent to respond to Gurken by becoming posterior. The cells that become anterior if they cannot respond to Gurken constitute the entire population of follicle cells that contact the oocyte during previtellogenic stages. Thus, all of the cells that receive the 'posteriorizing' Gurken signal are competent to respond to it (Gonzalez-Reyes, 1998).

In mutants such as gurken in which the induction of posterior follicle cell fate is blocked, the terminal follicle cells at the posterior develop like their anterior counterparts by forming border, stretched and centripetal follicle cells. This raises the question of whether the anterior follicle cells are subdivided into three cell types after the decision between anterior and posterior is taken, or whether there is a symmetric prepattern in the terminal follicle cells at both ends of the egg chamber. The ability to generate small clones of anterior cells at the posterior by removing the Egfr makes it possible to distinguish between these possibilities. If the latter model is correct, isolated patches of anterior cells should still respond to the symmetric prepattern correctly and form the appropriate type of anterior cell, even though they are surrounded by posterior cells, whereas the former model predicts that these cells should be unable to interpret their position. To follow the fate of small patches of anterior cells at the posterior of the egg chamber, small Egfr- clones were generated, but in this case, clone generation took place in the presence of enhancer trap lines that are expressed specifically in each of the three anterior follicle cell types. Egfr- cells that fall within a region 8-11 cell diameters from the posterior pole show staining for a centripetal cell marker, whereas clones that fall either proximal or distal to this 3-cell-wide belt do not activate this marker. Thus, anterior cells at the posterior express the anterior BB127 centripetal cell marker autonomously in a region that is the exact posterior counterpart of the anterior centripetal follicle cell domain. Furthermore, clones of as few as 4 cells express BB127 if they fall within this region, indicating that anterior cells can correctly interpret their position with respect to the posterior pole, although all of the surrounding cells are posterior. The same conclusion applies to a border cell and a stretched cell marker. The results demonstrate that small posterior clones of anterior cells can interpret their position with respect to the posterior pole by adopting the appropriate anterior follicle cell fate: the most terminal Egfr- cells behave like border cells, the subterminal Egfr- cells behave like stretched follicle cells, and the least terminal like centripetal cells. Thus, the positional information that specifies the positions of these distinct cell types at the anterior pole is also present at the posterior, strongly suggesting that there is a symmetric prepattern within the terminal follicle cell population that is independent of the decision between anterior and posterior fate (Gonzalez-Reyes, 1998).

Notch is shown to be required for the correct subdivision of the terminal follicle cells. Although the phenotype of Notch and Delta mutants provided the first evidence that the posterior follicle cells play a role in the polarization of the oocyte, it is still not known at which step in anterior-posterior axis formation Delta/Notch signaling is required. To address this question, an examination was performed to determine whether the N ts mutant disrupts this pathway before or after the induction of the posterior follicle cells by the oocyte. Since the most sensitive assay for a failure in posterior follicle cell determination is transformation to anterior fate, the expression of the border cell enhancer trap line slbo 1 was examined in stage 10 N ts egg chambers that had been maintained at the restrictive temperature of 32°C. These conditions produce a penetrant oocyte polarization phenotype in which the germinal vesicle often remains at the posterior of the oocyte. Surprisingly, slbo is expressed in neither the anterior nor posterior follicle cells of these egg chambers. In addition, the anterior most follicle cells in N ts mutant egg chambers never round up or migrate between the nurse cells towards the oocyte, indicating that Notch activity is required for border cell development (Gonzalez-Reyes, 1998).

To determine whether Notch activity is required for the specification of other anterior follicle cell types, this experiment using enhancer trap line s to label all anterior follicle cells. N ts egg chambers lack both border and centripetal follicle cells, but these missing cells do not appear to be transformed into stretched follicle cells. Notch therefore seems to play a role in determining the size of the anterior terminal follicle cell population, as well as its subdivision into distinct cell types. The N ts mutant fails to disrupt the patterning of the posterior terminal follicle cells, confirming that Notch is not required for the determination of posterior identity. However, many fewer cells express a posterior marker than in the control egg chambers. This reduction in posterior follicle cell number indicates that Notch plays a role in specifying the size of the terminal follicle cell population that is competent to respond to Gurken and is consistent with the decrease in terminal follicle cell number observed at the anterior of these egg chambers (Gonzalez-Reyes, 1998).

These results suggest a three-step model for the anterior-posterior patterning of the follicular epithelium that subdivides this axis into at least five distinct cell types. Altogether, these observations support a stepwise model for the patterning of the follicle cell layer along the AP axis. In the first step, the follicle cell epithelium is divided into terminal and main-body follicle cell populations. There is no lineage restriction boundary between the posterior terminal follicle cells and the main-body follicle cells at a stage in development that is four cell divisions before stage 6, indicating that the distinction between these two cell types arises after stage 1. Because the terminal cells have to be specified before Gurken signaling occurs, this restricts the time at which this population is determined to between stages 2 and 5. Although the data do not suggest a mechanism for how these cells are specified, their position suggests a simple model in which they are induced by a 'terminalizing' signal that spreads from the two poles of the egg chamber. The most likely sources for such a signal are the two polar follicle cells at each end of the egg chamber, since these cells lie in the center of the terminal domain and adopt a terminal fate themselves. The next step in the patterning of the follicular epithelium is the formation of a symmetric prepattern within each terminal follicle cell population. How this prepattern is established is unknown, but the geometry of the egg chamber again suggests that it might involve signals that emanate from the poles. Indeed, it is possible that the terminal follicle cells are specified and patterned by the same process, since both events require Notch activity. For example, the 'terminalizing' signal could induce distinct terminal fates at different distances from the pole. The third step in the patterning of the follicle cell layer occurs when the oocyte induces one population of terminal follicle cells to adopt a posterior fate, thereby breaking the symmetry of the follicle cell layer. As a consequence, the symmetric prepattern in the terminal follicle cells is interpreted differently in the anterior and posterior populations. The anterior cells become subdivided into border, stretched and centripetal follicle cells, while the posterior cells may undergo a similar subdivision into posterior cell types. In this way, the sequential patterning of the terminal follicle cells gives rise to at least five different cell types along the anterior-posterior axis (Gonzalez-Reyes, 1998).

Determination of EGFR signaling output by opposing gradients of BMP and JAK/STAT activity

A relatively small number of signaling pathways drive a wide range of developmental decisions, but how this versatility in signaling outcome is generated is not clear. In the Drosophila follicular epithelium, localized epidermal growth factor receptor (EGFR) activation induces distinct cell fates depending on its location. Posterior follicle cells respond to EGFR activity by expressing the T-box transcription factors Midline and H15, while anterior cells respond by expressing the homeodomain transcription factor Mirror. This study shows that the choice between these alternative outputs of EGFR signaling is regulated by antiparallel gradients of JAK/STAT and BMP pathway activity and that mutual repression between Midline/H15 and Mirror generates a bistable switch that toggles between alternative EGFR signaling outcomes. JAK/STAT and BMP pathway input is integrated through their joint and opposing regulation of both sides of this switch. By converting this positional information into a binary decision between EGFR signaling outcomes, this regulatory network ultimately allows the same ligand-receptor pair to establish both the anterior-posterior (AP) and dorsal-ventral (DV) axes of the issue (Fregoso Lomas, 2016).

This study shows that the choice between two alternative Grk/EGFR signaling outcomes in the follicular epithelium depends on positional input provided by Upd and Dpp. At the posterior, the presence of Upd allows Grk to induce Mid/H15 while, at the anterior, Grk together with Dpp positively regulates Mirr. In this context, Upd and Dpp serve to define the response to Grk/EGFR signaling, since they are not sufficient to induce Mid/H15 and Mirr, respectively, in the absence of Grk (Fregoso Lomas, 2016).

Mutual repression is demonstrated between Mid/H15 and Mirr that is proposed to generate a double-negative feedback circuit that toggles the system between anterior and posterior outcomes. Moreover, in addition to their mutual regulation, analysis of double-mutant clones reveals that Upd and Dpp each regulate both Mid/H15 and Mirr and, thus, each provides input to both sides of this circuit. Upd is required for the expression of Mid and H15 even in the absence of a functional mirr gene, demonstrating that Upd is required for Mid/H15 expression independent of its ability to repress Mirr. Similarly, Dpp signaling can repress Mid independently of its positive effect on Mirr. The choice of Grk/EGFR signaling outcome in this context thus depends not only on mutual repression between these alternate targets but also on their opposing regulation by Upd and Dpp (Fregoso Lomas, 2016).

It is proposed that these elements define a bistable network that controls the choice between two alternative outcomes of Grk/EGFR signaling. These outcomes are irreversible -- e.g., posterior EGFR signaling in later stages cannot induce Mirr unless Mid and H15 are absent - and mutually exclusive, and the factors described in this study include two key elements found in bistable networks: feedback and non-linearity. The feedback in this case is provided by the reciprocal repression between Mirr and Mid/H15, generating a double-negative feedback loop that reinforces the choice of signaling outcome. In addition, bistability requires non-linearity in the response of the circuit to its upstream regulators, which makes the switch more sensitive to graded inputs. It is proposed that, in the follicular epithelium, this is achieved by the joint opposing regulation of the feedback circuit by both Dpp and Upd; each activates one side of the switch while repressing the other, biasing the outcome in the same direction (Fregoso Lomas, 2016).

These alternative responses to Grk are separated in time, as the source of Grk moves from posterior to anterior during the course of development. An important element that determines the choice between them is the early pattern of Mirr expression. In early stages of oogenesis, Mirr is Grk independent and is restricted to the main body follicle cells, due to its repression in the terminal regions by Upd. These main-body follicle cells correspond to the future anterior region of the columnar epithelium, and it is proposed that this early expression of Mirr predisposes them to express Mirr instead of Mid/H15 when Grk adopts its final dorsal anterior localization. Such a role for the early phase of Mirr expression is also consistent with the DV asymmetry of the Mid expression domain; as Grk moves anteriorly, leaving the range of posterior Upd and entering this domain of early Mirr expression and Dpp pathway activity, only the peak dorsal levels of Grk are capable of inducing Mid (Fregoso Lomas, 2016).

These observations also provide an example of how antiparallel signaling gradients can be integrated during epithelial patterning. Tissue patterning by opposing morphogen gradients is observed in developmental contexts as diverse as the Drosophila blastoderm and vertebrate neural tube, where they engage downstream transcriptional networks whose dynamic properties generate reproducible gene expression boundaries. Mutual repression between downstream transcription factors helps define the position and boundaries of cell fate domains, but how the opposing gradients are integrated is not well understood. This study shows that, in the follicular epithelium, the opposing Upd and Dpp gradients are integrated at the level of the Mirr-Mid/H15 feedback circuit. This integration occurs not only at the level of the mutual repression between Mid/H15 and Mirr but also by the ability of each gradient to regulate both sides of this circuit (Fregoso Lomas, 2016).

Together, these elements define the framework of a regulatory network that integrates localized positional information to regulate a binary choice of EGFR signaling outcome. The results allow construction of a model that both accounts for how an individual cell responds to Grk/EGFR signaling and explains how these spatial inputs are integrated across the epithelium to generate a defined pattern of Mid/H15 and Mirr expression, ultimately defining the pattern of the eggshell. Mirr is required for the generation of the high- and low-Broad domains, and Mid/H15 expression is required to define the posterior limit of these domains. The ability of Dpp and Upd to influence the outcome of EGFR signaling allows a single signaling input, namely localized secretion of Grk by the oocyte, to generate multiple distinct outputs that are localized in space and time, thus establishing both the AP and DV polarity of the epithelium and generating a complex and reproducible pattern of cell fates (Fregoso Lomas, 2016).

NineTeen Complex-subunit Salsa is required for efficient splicing of a subset of introns and dorsal-ventral patterning

The NineTeen Complex (NTC), also known as Pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for splicing. During Drosophila midblastula transition, splicing is particularly sensitive to mutations in NTC-subunit Fandango, which suggests differential requirements of NTC during development. This study shows that NTC-subunit Salsa, the Drosophila orthologue of human RNA helicase Aquarius (CG31368), is rate-limiting for splicing of a subset of small first introns during oogenesis, including the first intron of gurken. Germ line depletion of Salsa and splice site mutations within gurken first intron both impair adult female fertility and oocyte dorsal-ventral patterning due to an abnormal expression of Gurken. Supporting causality, the fertility and dorsal-ventral patterning defects observed after Salsa depletion could be suppressed by the expression of a gurken construct without its first intron. Altogether these results suggest that one of the key rate-limiting functions of Salsa during oogenesis is to ensure the correct expression and efficient splicing of the first intron of gurken mRNA. Retention of gurken first intron compromises the function of this gene most likely because it undermines the correct structure and function of the transcript 5'UTR (Rathore, 2020).

The spliceosome is a highly dynamic molecular machine, composed of five small nuclear ribonucleoproteins (snRNPs) that sequentially associate to the precursor mRNA (pre-mRNA) during the splicing reaction. Each snRNP (U1, U2, U4, U5, and U6) contains a U-rich snRNA and a unique group of proteins. Although spliceosome assembly is ordered (U1 > U2 > U4/U5/U6 > NineTeen Complex), the splicing reaction is without an apprarent irreversible and/or rate-limiting step, with commitment to splicing progressively increased as snRNPs and NTC bind to the pre-mRNA (Rathore, 2020).

The spliceosomal NineTeen Complex (NTC), also known as Pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for efficient pre-mRNA splicing (Hogg, 2010; Chanarat, 2013). NTC composition is dynamic and comprises a subset of conserved core subunits and many transiently associated ones. NTC associates with the spliceosome during its activation and just before the first transesterification. Interestingly, NTC also has a significant role in the crosstalk between transcription, cotranscriptional processing of the nascent RNA, and DNA repair, as distinct NTC subunits have been reported to be important for transcriptional elongation and genomic stability. Human NTC-subunits PRP19, XAB2, and CDC5L are important for transcriptional elongation, transcription-coupled DNA repair, and activation of the ATM-related (ATR)-dependent DNA damage checkpoint. RNA Polymerase II (RNA Pol II) also promotes cotranscriptional splicing activation through the recruitment of NTC (Rathore, 2020 and references therein).

Human Aquarius (AQR) (also known as intron-binding protein 160, IBP160) is an ATP-dependent RNA helicase that associates with NTC during spliceosome activation and formation of the activated B complex (BACT). AQR binds to introns independently of sequence, but usually upstream of the branch-site (BS) and close to the associated U2 snRNP SF3a and SF3b proteins, being essential for intron-binding complex formation and efficient splicing. AQR has also been suggested to be important for deposition of the exon junction complex (EJC) during the splicing reaction and formation of intron-encoded snoRNAs, suggesting it regulates the cross-talk between splicing and other RNA processing events (Rathore, 2020).

Splicing during Drosophila early embryonic development is notably sensitive to mutations in NTC-subunit Fandango (Guilgur, 2014), suggesting differential requirements of NTC during development (Martinho, 2015). To test this possibility, it was decided to investigate the role of other NTC-subunits during Drosophila oogenesis and early embryonic development. Focused of initial work was placed on uncharacterized gene CG31368, which encodes the Drosophila ortholog of human Aquarius. Since there is already a nonrelated Drosophila protease named aquarius (CG14061), CG31368 was renamed salsa. The working hypothesis is that salsa, similar to its Caenorhabditis elegans ortholog emb-4, is likely to have important developmental functions (Rathore, 2020).

During Drosophila oogenesis, gurken mRNA localizes to the posterior cortex of the developing oocyte and Gurken signal is restricted to the underlying posterior follicle cells. In response to a signal from the posterior follicle cells, there is a considerable reorganization of the cytoskeleton and a microtubule-dependent migration of the oocyte nucleus to the anterior cortex. The anteriorly localized nucleus defines the dorsal-anterior region and provides the first detectable dorsal-ventral (D/V) asymmetry of the oocyte, with the expression of both gurken mRNA and protein restricted to the cytoplasmic perinuclear region of the oocyte (Rathore, 2020).

gurken mRNA is transcribed in the supporting nurse cells and actively transported to the dorsal-anterior region of the oocyte by a dynein-mediated transport. The oocyte dorsal-anterior localization of gurken mRNA relies on multiple elements localized to the transcript 5' UTR, 3' UTR and open-reading frame. Although this localization is crucial for its efficient translation, the precise contribution of each element for RNA localization is still a matter of debate (Rathore, 2020).

D/V patterning of the developing Drosophila egg is dependent on the dorsal-anterior localization of Gurken during mid-oogenesis. Gurken is the ligand for the Epidermal growth factor receptor (Egfr) that locates to the apical surface of follicle cells that surround the developing oocyte. Activation of Egfr modifies the cell fate of the dorsal follicle cells and restricts the formation of Spätzle ligand to the ventral region of the oocyte, which is essential for normal morphogenesis of the eggshell dorsal appendages (Rathore, 2020).

This study found that Salsa, the Drosophila ortholog of AQR, is rate-limiting for efficient splicing of a subset of small first introns, including the first intron of gurken. Consistent with the functional relevance of gurken splicing defects, mutations within the splice sites of the first intron of gurken impair the function of this gene. Female germline depletion of Salsa and splice mutations within gurken first intron were both associated to a decrease in female fertility, significant D/V patterning defects of the eggshell and abnormal expression of Gurken during oogenesis. Supporting causality, expression of a gurken construct without its first intron suppressed the female fertility and D/V patterning defects observed after Salsa depletion. Altogether these results suggest that one of the key rate-limiting functions of Salsa during oogenesis is to ensure the correct expression and efficient splicing of the first intron of gurken mRNA (Rathore, 2020).

Oocyte dorsal-anterior localization of gurken mRNA relies on multiple elements localized to the transcript 5'UTR, 3'UTR and open-reading frame, yet the relative importance of each element for mRNA localization is still unclear. The 5' and 3'-UTRs of gurken were reported to be required for dorsal-anterior localization of gurken transcript. Furthermore, and using a genomic gurken construct with a lacZ reporter inserted within the gene open-reading frame, it was shown that whereas gurken 5'UTR is required for transcript oocyte accumulation, its coding region and 3'UTR are necessary for its posterior and dorsal-anterior localization. Nevertheless, it was recently reported, using an oocyte injection assay, that a small stem-loop located within the open-reading frame was necessary and sufficient for gurken transcript localization (Rathore, 2020).

The results show that efficient splicing of the first intron of gurken is required for mRNA dorsal-anterior localization and dorsal-ventral patterning. This is most likely because retention of the first intron impairs the secondary RNA structure of gurken 5'UTR, and the function of a closely located RNA element important for its localization. Splicing of the first intron of gurken is also likely to facilitate Gurken protein expression, as deletion of the first intron of gurken suppresses the dorsalization phenotype associated with increased copy number of gurken gene without affecting the levels of gurken mRNA. The results therefore fully support the role of gurken 5'UTR in mRNA localization within the oocyte, and strongly suggest that Salsa-dependent splicing of the first intron of gurken mRNA is important for the correct expression and function of this gene (Rathore, 2020).

The precise function of human Aquarius in splicing is still poorly understood. This RNA helicase is recruited to the spliceosome as a pentameric complex known as intronbinding complex (IBC), which also contains hSyf1 (also known as Xab2), hIsy1, CypE, and CCDC16 (De, 2015). Coimmunoprecipitation experiments suggest a large interaction interface between IBC and U2 snRNP, within the activated spliceosome (Bact stage) and just before the first splicing reaction. Although Aquarius ability to bind and hydrolyze ATP is important for spliceosome activation and splicing efficiency, the role of its RNA unwinding activity is less clear (Rathore, 2020).

This work has identified a small subset of introns whose splicing is particularly sensitive to depletion of Salsa (the Drosophila ortholog of human Aquarius). The fact that splicing was only affected in a small number of introns is consistent with the observation that immunodepletion of human Aquarius from nuclear extracts only weakly impaired splicing in vitro. This suggests that although this RNA helicase is apparently not critical for overall splicing, during female gametogenesis there is a subset of introns whose efficient removal relies on the function of this enzyme (Rathore, 2020).

Analysis of the introns whose splicing was sensitive to Salsa depletion showed a clear bias for small first introns with weak 3'splice sites, independently of their distance to the transcription start site (TSS), 5'splice site strength and GC content. The bias for small introns suggests that Salsa is mostly rate-limiting when introns are recognized by intron definition, where the initial pairing between U1 and U2 snRNPs occurs across the intron. Furthermore, the bias for introns with weak 3'splice sites is in accordance with the extensive interaction between IBC and U2 snRNP in the activated spliceosome, and implies that depletion of Salsa is likely to impair, at least in a subset of introns, U2 snRNP function during splicing. The absence of any detectable bias for short distances between the TSS and 5'splice site, when evaluating affected and control first introns, or any bias for weak 5'splice site strength, suggests that Salsa is not likely rate-limiting for Cap-Binding Complex-mediated splicing (Rathore, 2020).

Drosophila first introns are more likely to be cotranscriptionally retained than internal and terminal introns. This is not consistent with the kinetic competition model, where the fastest processes are the ones most likely to occur, suggesting additional constraints to first intron splicing. Although the precise nature of such constraints is still poorly understood, binding of transcriptional initiation factors to the 5'splice site-associated U1snRNP potentially restricts splicing efficiency, as it might impair the initial pairing between U1 and U2 snRNPs. The current working hypothesis is that Salsa is required for splicing of small first introns with weak 3'splice site because this enzyme facilitates U2 snRNP function, minimizing the interference effect of transcriptional initiation factors on splicing. Future work will help define the function of this RNA helicase and its contribution for differential gene expression during development (Rathore, 2020).

GENE STRUCTURE

Bases in 5' UTR -370

Bases in 3' UTR - 454

PROTEIN STRUCTURE

Amino Acids - 294

Structural Domains

The Gurken protein has an N-terminal signal peptide involved in the secretion of Gurken and a single EGF repeat. The protein has a potential transmembrane domain and a cytoplasmic region. In addition there are two potential N-glycosylation sites. The region between the signal sequence and the EGF repeat contains PEST sequences tagging the protein for rapid turnover. There is a set of six conserved cysteine residues (Neuman-Silberberg, 1993).

GRK is 24% identical to Spitz protein; 22% identical to Neu differentiation factor, and 28% identical to TGF-alpha. The EGF-like region is 35% identical to repeats found in EGF, Notch and Xotch, the Xenopus homolog of Notch (Neuman-Silberberg, 1993).

The EGF-like domain in Vein is 43 amino acids long and has the six invariant cysteines and highly conserved glycine and arginine residues characteristic of the motif. The cysteines are thought to form disulfide bonds, thereby producing a looped structure. The EGF motif in VN is between 30% and 44% identical to other EGF-like ligands. It shares 37% identity with Spitz and 33% identity with Gurken. Amino-terminal to the EGF domain of VN is an Ig-like domain of the C2 type that includes nonimmunological proteins (Schnepp, 1996).

date revised: 20 December 2019

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