BIOLOGICAL OVERVIEW

Shaven/Sparkling protein was first identified using a cross-reacting antibody. Antiserum directed against the Drosophila Pax protein Pox meso (Poxm) reacts not only with segmentally repeated mesodermal antigens but also with antigens of the developing peripheral and central nervous system, which are also expressed in a segmentally repeated pattern. Because the staining of the PNS and CNS remains unaltered for flies in which the poxm gene at 84F11-12 is deleted, the antigens revealed in the nervous system must be different from PoxM. A cDNA expression library (derived from 4- to 8-hr old embryos) was screened with the anti-PoxM anterserum, and a single cDNA was isolated, originating from a locus at 102F1-2. The cDNA detected a transcript expressed in the posterior portion of the eye disc. Of the six lethal loci and two recessive visibles generated 25 years ago and uncovered by a chromosome 4 terminal deficiency, only sparkling produces an eye phenotype. These results suggest that the cDNA is derived from the spa gene (Fu, 1997). Independently, sparkling was identified using polymerase chain reaction primers derived from the sequence of a Pax2/5/8 family member identified in the nematode C. elegans (Czerny, 1997). The sparkling gene proves to correspond to a classical mutation termed shaven, identified by Bridges in 1920 (FlyBase). Although the proper name for this gene is now shaven, and is is referred to as shaven or sparkling in the text that follows.

Delilah, prospero, and D-Pax2 constitute a gene regulatory network essential for the development of functional proprioceptors

Coordinated animal locomotion depends on the development of functional proprioceptors. While early cell-fate determination processes are well characterized, little is known about the terminal differentiation of cells within the proprioceptive lineage and the genetic networks that control them. This work describes a gene regulatory network consisting of three transcription factors-Prospero (Pros), D-Pax2, and Delilah (Dei)-that dictates two alternative differentiation programs within the proprioceptive lineage in Drosophila. D-Pax2 and Pros control the differentiation of cap versus scolopale cells in the chordotonal organ lineage by, respectively, activating and repressing the transcription of dei. Normally, D-Pax2 activates the expression of dei in the cap cell but is unable to do so in the scolopale cell where Pros is co-expressed. It was further shown that D-Pax2 and Pros exert their effects on dei transcription via a 262 bp chordotonal-specific enhancer in which two D-Pax2- and three Pros-binding sites were identified experimentally. When this enhancer was removed from the fly genome, the cap- and ligament-specific expression of dei was lost, resulting in loss of chordotonal organ functionality and defective larval locomotion. Thus, coordinated larval locomotion depends on the activity of a dei enhancer that integrates both activating and repressive inputs for the generation of a functional proprioceptive organ (Avetisyan, 2021).

A central question in developmental biology is how different cells that originate in the same lineage and develop within the same organ, acquire unique identities, properties and specialized morphologies. One of the common mechanisms involved in cell fate diversification within a cell lineage is asymmetric cell division in which cytoplasmic determinants of the mother cell differentially segregate into one of the two daughter cells. This asymmetry is then translated into differential gene expression and the activation of cell-type-specific gene regulatory networks (GRN) that dictate the differentiation programs of cells with unique properties. The transition from a primary cell fate to the characteristic phenotype of a fully differentiated cell involves complex GRNs in which numerous genes regulate each other's expression. Despite this complexity, genetic analyses in well-characterized developmental systems can often reveal elementary interactions in small GRNs which dictate a specific cell fate, or a specific feature of the differentiating cell (Avetisyan, 2021).

Many of the core components and the central processes underlying asymmetric cell divisions and primary cell fate decisions have been uncovered in studies performed on the central and peripheral nervous system (PNS) of Drosophila. The PNS of Drosophila contains two classes of multicellular sensory organs, external sensory organs and chordotonal organs (ChOs), whose lineages share a similar pattern of asymmetric cell divisions. In both types of organs, the neuron and support cells, which collectively comprise the sensory organ, arise from a single sensory organ precursor cell (SOP) through a sequence of precisely choreographed asymmetric cell divisions. Antagonistic interactions involving Notch and Numb are key regulators of the asymmetry generated between each two sibling cells within these lineages. Unlike the primary cell-fate specification, which has been extensively investigated, the process of terminal differentiation of the post-mitotic progeny is poorly understood. This study has used the larval lateral pentascolopidial ChO (LCh5) as a model system to study cell fate diversification within a sensory lineage (Avetisyan, 2021).

The LCh5 organ is composed of five mechano-sensory units (scolopidia) that are attached to the cuticle via specialized epidermal attachment cells. Each of the five scolopidia originates in a single precursor cell that divides asymmetrically to generate five of the six cell types that construct the mature organ: the neuron, scolopale, ligament, cap, and cap-attachment cell. Three of the five cap-attachment cells are rapidly removed by apoptosis, leaving two cap-attachment cells that anchor the five cap cells to the epidermis. Later in development, following the migration of the LCh5 organ from the dorsal to the lateral region of the segment, a single ligament-attachment cell is recruited from the epidermis to anchor the five ligament cells to the cuticle. The mature LCh5 organ responds to mechanical stimuli generated by muscle contractions that lead to relative displacement of the attachment cells and the consequent shortening of the organ (Avetisyan, 2021).

Very little is known about the unique cell-type-specific differentiation programs that characterize each of the ChO cells, whose morphologies and mechanical properties differ dramatically from each other. To address this issue, focus was placed on the transcription factor Taxi wings/Delilah (Dei), an important regulator of cell adhesion (Egoz-Matia, 2011), which is expressed in the four accessory cell types (cap, ligament, cap-attachment and ligament-attachment) but is excluded from the neuron and the scolopale cell. Even though dei is expressed in all four accessory cells, its expression in these cells is differentially regulated. The transcription of dei in the ChO is controlled by two cis-regulatory modules (CRMs): The dei attachment enhancer, located ~2.5 Kb upstream of the dei transcription start site, drives expression in the cap-attachment and ligament-attachment cells (as well as tendon cells), whereas the deiChO-1353 enhancer, an intronic 1353 bp DNA fragment, drives dei expression specifically in the cap and ligament cells. The dei^attachment enhancer was shown to be activated by the transcription factor Stripe, which is considered a key regulator of tendon cell development and a known determinant of attachment cell identity. This study provides a high-resolution dissection of the deiChO-1353 enhancer and shows that it integrates both activating and repressive cues to drive dei expression in cap and ligament cells while suppressing it in scolopale cells. D-Pax2/Shaven (Sv), which is expressed in both branches of the cell lineage, is a positive regulator of dei, and Prospero (Pros) inhibits dei expression specifically in the scolopale cell. This small GRN is required for the realization of differentiation programs characterizing cap versus scolopale cell fates and is therefore essential for ChO functionality and coordinated larval locomotion (Avetisyan, 2021).

This work has identified a small GRN that governs the alternative differentiation programs of two cousins once removed cells within the ChO lineage - the cap cell and the scolopale cell. Pros and D-Pax2/Sv are direct regulators of dei that together dictate its expression in the cap cell and its repression in the scolopale cell. Both D-Pax2/Sv and Pros exert their effects on dei transcription via a 262 bp chordotonal-specific enhancer (deiChO-262) in which two D-Pax2/Sv and three Pros binding sites were identified (Avetisyan, 2021).

Following primary cell fate decisions within the ChO lineage, Pros expression becomes restricted to the scolopale cell, whereas D-Pax2/Sv expression becomes restricted to the scolopale and cap cells, similar to its behavior in the external sensory lineages. D-Pax2/Sv activates the expression of dei in the cap cell but is unable to do so in the scolopale cell where Pros is co-expressed. If D-Pax2/Sv activity is compromised, the cap cell fails to express dei and loses some of its differentiation markers, such as the expression of αTub85E. In contrast, if Pros activity is lost, dei is ectopically expressed in the scolopale cell that, as a consequence, acquires some cap cell features including the expression of αTub85E. The observed D-Pax2/Sv- and Pros-associated phenotypes do not reflect genuine cell fate transformations, suggesting that D-Pax2/Sv and Pros do not affect primary cell fate decisions within the ChO lineage. Rather, the observed phenotypes reflect a failure of the cap and scolopale cells to follow the cell type-specific differentiation programs responsible for bringing about their characteristic cellular phenotypes. The D-Pax2/Sv-deficient cap cells fail to express unique differentiation markers (such as αTub85E) and are therefore hardly detectable. It is also possible that the Sv/Pax2-deficient cap cells fail to survive. Thus, the possibility that some of the findings reflect more upstream roles of Sv/D-Pax2 in the specification of cap-cell identity cannot be excluded (Avetisyan, 2021).

The switch between the differentiation programs of cap and scolopale identities cannot be simply explained by the nature of asymmetric cell divisions within the ChO lineage. The effects on the ChO lineage of major regulators of asymmetric cell division, such as Notch and Numb, and the expression pattern of cell differentiation determinants such as Pros and D-Pax2/Sv, were mainly postulated based on knowledge gained by analyzing external sensory lineages. According to the similarity between the lineages, the cap cell parallels the Notch-non-responsive hair (trichogen) cell, whereas the scolopale parallels the Notch- responder sheath (thecogen) cell. Thus, D-Pax2/Sv is expressed in one Notch responder and one non-responder cells in the lineage. The presence of Pros in the Notch-responder cell represses the cap-promoting activity of D-Pax2/Sv. Somewhat similar cousin-cousin cell transformation was found in external sensory organs in the adult where mutations in hamlet transform the sheath cell into a hair cell (parallel to scolopale-to-cap transformation). Ectopic expression of hamlet induced pros expression and repressed the hair shaft-promoting activity of D-Pax2/Sv (Avetisyan, 2021).

In the adult external sensory lineage, Pros was shown to be important for the specification of the pIIb precursor, which gives rise to the neuron and sheath cell (the scolopale counterpart). However, the absence of Pros from the pIIa precursor, which gives rise to the hair and socket cells (the cap and cap-attachment cells counterparts) was even more critical for proper development of this branch of the lineage. This phenomenon is somewhat conserved in the larval ChO. While Pros is required for proper differentiation of the scolopale cell, its absence from the cap cell is critical for adopting the correct differentiation programs within the lineage (Avetisyan, 2021).

Opposing effects of D-Pax2/Sv and Pros activities on cell differentiation have been also identified in the regulation of neuronal versus non-neuronal cell fate decisions in the developing eye, where they play a role in modulating the Notch and Ras signaling pathway. Interestingly, in the R7 equivalence group Pros and D-Pax2/Sv can only alter the cell-type-specific differentiation program of cells that already express the other gene. Similarly, in the ChO lineage, ectopic expression of Pros in the cap and ligament cells transforms the D-Pax2/Sv-positive cap cell toward a scolopale cell identity but does not affect the D-Pax2/Sv-negative ligament cell in a similar fashion, even though the ectopic expression of Pros does repress the transcription of dei in both cell types. Additionally, a loss of Pros activity in the scolopale cell can transform the identity of this cell toward a cap cell identity only in the presence of D-Pax2/Sv (Avetisyan, 2021).

This study has shown that the opposing influences of Pros and D-Pax2/Sv on dei expression is integrated by the deiChO-262 enhancer in both larval and adult ChO lineages. This is the first example of an enhancer that responds to these opposing signals to dictate cell-specific differentiation programs in a sensory lineage. While the identified enhancer is ChO-specific, it is plausible that other enhancers of sensory organ lineage-specific genes encode coupled Pros and D-Pax2/Sv binding sites. The expression of the dei gene in other (non-ChO) organs is regulated via different enhancers. Some of these enhancers are responsible for regulating dei expression in tissues where Pros and Pax2 play opposing roles, such as the eye and wing margin ES organs (the dei^wing+eye enhancer; Nachman , 2015). It is beyond the scope of this work, but in the future, it will be interesting to decipher whether these enhancers also serve as molecular platforms for integrating opposing effects of Pax2 and Pros (Avetisyan, 2021).

This study has identified two D-Pax2/Sv and three Pros binding sites in the deiChO-262 enhancer. Apart from D-Pax2/Sv site 2, none of these sites match the published binding motifs for D-Pax2/Sv or Pros. These results agree with recent studies that showed that many transcription factors function in vivo through low-affinity or suboptimal binding sites that differ from their predicted binding motifs. It was suggested that low-affinity binding sites provide specificity for individual transcription factors belonging to large paralogous families, such as the homeodomain family of transcription factors, that share similar DNA-binding preferences. To compensate for their weak binding capabilities, low-affinity binding sites are often organized in homotypic clusters that can increase the cumulative binding affinity of an enhancer. The findings that the homeodomain transcription factor Pros functions through a cluster of low-affinity binding sites in deiChO-262, may represent another example for the suggested tradeoff between transcription factor binding affinity and specificity (Avetisyan, 2021).

It is not known how Pros opposes the effect of D-Pax2/Sv in the context of dei^ChO-262 to inhibit the expression of dei in scolopale cells. The results suggest that the inhibitory effect of Pros is not mediated through binding competition with D-Pax2/Sv at the D-Pax2/Sv high-affinity site (site 2), since this site does not overlap with a Pros-binding site. The D-Pax2/Sv low-affinity site does overlap with a Pros binding site and mutations in the Pros binding site affect D-Pax2/Sv binding in vitro, however, while being important for robust dei expression, this site is dispensable in the presence of the high-affinity site. It is possible that binding of Pros to the dei^ChO-262 enhancer targets this sequence to a repressed heterochromatin domain as was recently shown for other Pros target genes in differentiating neurons (Avetisyan, 2021).

How is dei regulated in other ChO cell types? dei is expressed in four out of six cell types comprising the ChO: the cap-attachment and ligament-attachment cells, in which dei transcription is activated by Sr via the dei^attachment regulatory module, and the cap and ligament cells in which the expression of dei is regulated via the deiChO-262 enhancer. This study now shows that D-Pax2/Sv activates dei transcription in the cap cell, and that Pros inhibits its expression in the scolopale cell. The identity of the positive regulator/s of dei in the ligament cell, whose cell-fate is determined by the glial identity genes gcm and repo, and the identity of the negative regulator/s of dei in the neuron remains unknown. Interestingly, the expression of dei was found to be altered in response to ectopic expression of gcm in the embryonic nervous system; its expression was upregulated at embryonic stage 11, but was repressed in embryonic stages 15-16. This observation points to GCM as a potential regulator of dei expression in the ligament cells. Another interesting candidate for repressing dei in the sensory neuron is the transcriptional repressor Lola. Lola has been identified as a putative direct regulator of dei in the Y1H screen and was shown to be required in post-mitotic neurons in the brain for preserving a fully differentiated state of the cells. The possible involvement of Gcm and Lola in the regulation of dei awaits further studies. The observed upregulation of the deiChO-262 reporter in the ligament cells of embryos with mutated Pros-binding sites may reflect an early role of Pros in the pIIb precursor before its restriction to the scolopale cell, which prevents dei expression in the ligament cell (Avetisyan, 2021).

Although the loss of dei in the genetic/cellular milieu of the ligament cell (unlike the cap cell), even when accompanied by ectopic expression of Pros, is not sufficient for transforming ligament cell properties towards those of scolopale cells, it is known that the expression of dei in the ligament cell is critical for its proper development. Ligament-specific knockdown of dei leads to failure of the ligament cells to acquire the right mechanical properties and leads to their dramatic over-elongation. By analysing the locomotion phenotypes of larvae homozygous for a dei null allele and the newly generated cap and ligament-specific deiΔChO allele, it was possible to show that the expression of dei in the cap and ligament cells is crucial for normal locomotion. Thus, it is concluded that the correct expression of dei within the ChO is critical for organ functionality. Surprisingly, the gross morphology of LCh5 of deiΔChO larvae appears normal. Yet, in a way that remains to be elucidated, the Dei-deficient cap and ligament cells fail to correctly transmit the cuticle deformations to the sensory neuron, most likely due to changes in their mechanical properties (Avetisyan, 2021).

Combinatorial signaling in the specification of unique cell fates

How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Together with the transcription factor Lozenge, the nuclear effectors of the Egfr and Notch signaling pathways directly regulate shaven transcription in cone cells of the Drosophila eye disc. Moreover, the specificity of shaven expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required to regulate target genes and ultimately specify distinct cell fates within this group. It is expected that similar mechanisms may specify pattern formation in vertebrate developmental systems that involve intercellular communication (Flores, 2000).

shaven is represented by at least two classes of mutant alleles: shaven (sv) and sparkling (spa). spa mutants show cone cell defects resulting from mutations in the fourth intron of the gene, which have led to the identification of a 926 bp SpeI fragment within this intron that includes the eye-specific enhancer (Flores, 2000).

spa alleles give an enhancement of lozenge eye phenotypes. Two new spa alleles were isolated as enhancers of the temperature-sensitive lz allele, lz^ts1. The strongest eye-specific allele of shaven, spa^pol, which is not transcribed in cone cell precursors, also enhances lz^ts1. Shaven is not expressed in cone cell precursors of lz mutants, which suggests that Lz regulates shaven expression. There are three Lz/Runt domain (RD) binding sites (5'-RACCRCA-3', where R = purine) in the shaven eye-specific enhancer (RDI-RDIII). To determine whether these sites are required for proper shaven expression, a series of smaller enhancer fragments derived from the SpeI fragment was combined with the shaven promoter and the transcribed region from which introns 1-8 had been removed. This combination was tested as transgenes for the ability to rescue spa^pol mutants. There is no loss in rescue efficiency if the truncation does not eliminate any of the three RD binding sites. However, if RDI is deleted, the rescue efficiency and Shaven expression in cone cell precursors are considerably reduced, and rescue cannot be improved by two copies of the transgene. Similarly, when both RDII and RDIII are removed, the rescue efficiency and expression in cone cell precursors are clearly reduced, but rescue to wild type is achieved with two copies of the transgene. These experiments suggest that the RD binding sites are essential for the control of shaven transcription and that omission of RDI has more severe effects than that of RDII and RDIII (Flores, 2000).

Electrophoretic mobility-shift assays (EMSA) demonstrate that in vitro translated Lz can bind specifically to each of the RD binding sites in the minimal eye-specific enhancer (SME). As an in vivo correlate to these experiments, the three RD sites were mutated in the context of a transgenic shaven rescue construct. Mutation of all three RD binding sites (mRDx3) causes a failure to rescue the spa^pol eye phenotype and Shaven expression in cone cell precursors. Together, the in vitro and in vivo data demonstrate that Lz directly regulates shaven transcription through the RD binding sites in the SME (Flores, 2000).

A construct expressing lacZ under the control of the SME and the hsp70 promoter (SME-lacZ) faithfully reproduces the wild-type Shaven expression pattern in cone cell precursors. Mutation of all three RD binding sites in SME-lacZ results in the loss of this expression, further indicating that Lz acts directly through the SME. For the remainder of the analysis, both endogenous Shaven expression as well as SME-lacZ expression were examined. In all genetic backgrounds tested, the results obtained in both assays were identical. This suggests that the SME is sufficient for transcriptional regulation of shaven in cone cell precursors, and that SME-lacZ faithfully reflects this regulation (Flores, 2000).

In EGFR^ts third-instar larvae raised at 29°C for 36 hr prior to dissection, Shaven expression is lost in cone cell precursors. To restrict the loss of Egfr function to the undifferentiated cells posterior to the furrow and cells that acquire their fates during the second phase of morphogenesis, a lz-Gal4 driver was used to express a dominant-negative form of Egfr. In these discs, Shaven expression is lost from cone cell precursors, while neuronal patterning in the precluster is maintained. Shaven expression was further examined in mutants of genes encoding the nuclear components of the Egfr signaling pathway, the repressor Yan and the activator PntP2. Shaven expression is also lost in discs in which lz-Gal4 drives the expression of a nonphosphorylatable form of Yan refractory to the Egfr signal. Similarly, in the hypomorphic pnt¹²³⁰ mutant, a modest reduction of Shaven expression occurs in cone cell precursors, while a stronger reduction is observed upon expression of a dominant-negative form of PntP2. These experiments together suggest that the Egfr signaling pathway activates shaven expression in cone cell precursors by relieving Yan-mediated repression and stimulating PntP2 activation (Flores, 2000).

The above genetic analysis does not address whether the effects of Egfr signaling on shaven transcription are direct or indirect. Therefore, in vitro mutagenesis was used to examine potential direct effects. Six ETS domain consensus binding sites were found in the SME. EMSAs show that two of these sites (1 and 6) are bound by both Yan and PntP2. Yan also binds to two additional sites (2 and 4). All six ETS sites were mutated to 5'-TTAA/T-3' in the context of SME-lacZ, and the resulting SME^mETSx6-lacZ construct was transformed into flies. In these transgenic flies, ß-galactosidase expression is lost from cone cell precursors. Since PntP2 was found to bind only to Ets sites 1 and 6, a SME-lacZ construct in which only these sites were mutated (SME^mETS(1,6)-lacZ) was transformed into flies. ß-galactosidase expression in cone cells is completely eliminated. These in vitro and in vivo results together demonstrate that PntP2 directly controls shaven expression in cone cell precursors by binding to ETS domain sites in the SME (Flores, 2000).

In N^ts third-instar larvae raised at 29°C for 20 hr prior to dissection, Shaven expression is eliminated from cone cell precursors. Similarly, expression of a dominant-negative form of N under lz-Gal4 control causes a loss of Shaven expression in cone cell precursors without perturbing neuronal development. Shaven expression is also reduced in discs mutant for Delta (Dl), which encodes a N ligand. Moreover, expression of a dominant-negative form of Dl (Dl^DN) under lz-Gal4 control causes a loss of Shaven expression in cone cell precursors, while neuronal patterning occurs in a wild-type fashion. A further reduction in Shaven expression is seen when Dl^DN is driven by GMR-Gal4. A loss of Shaven expression is also seen upon ectopic expression of Hairless (H), a direct antagonist of Su(H) function. These results together suggest that N/Dl signaling via Su(H) is required for proper shaven expression in cone cell precursors. This is an inductive rather than lateral inhibitory function of the N signaling pathway in cone cell development that has not been previously analyzed with molecular markers. A reporter gene under the transcriptional control of Su(H) binding sites is expressed in cone cell precursors, which demonstrates that Su(H) is activated by the N pathway in cone cells (Flores, 2000).

The Su(H) binding sites in the SME were altered to determine whether the N pathway directly regulates shaven transcription. The SME contains eight putative Su(H) binding sites. EMSAs show that the Su(H) consensus binding sequence is not strictly followed, since three sites with one mismatch can bind Su(H). Su(H) binding is eliminated when the central 5'-GRG-3' sequence is mutated to 5'-CCC-3' in all eight sites. A construct containing these mutations in the context of SME-lacZ was transformed into flies. In these transgenic flies, ß-galactosidase expression is lost in cone cell precursors. These in vitro and in vivo results together demonstrate that Su(H) directly controls shaven expression in cone cell precursors by binding to the SME (Flores, 2000).

Mutating Su(H) and ETS binding sites eliminates expression of the target gene in the cone cells, which demonstrates a direct role for these pathways in transcriptional activation of shaven. Clonal analysis was undertaken to establish the requirement of the Notch and Egfr pathways in shaven expression. Unfortunately, these pathways are necessary for proliferation and have many layers of function. Therefore a flip-out strategy was used to inhibit N and Egfr function in GFP-labeled single-cell clones. This was best achieved in clones induced by GMR-flp. The GMR enhancer is only active behind the furrow and only a single cell division takes place in this population of cells. As a result, the clone size is very small. In a wild-type background, single cells marked with GFP express Shaven. However, when these single cells also express EGFR^DN or N^ECN, they do not express Shaven. Thus, cone cells need functional Notch and Egfr receptors in order to express Shaven (Flores, 2000).

The results described so far suggest that shaven expression is limited to cells which (1) express Lz; (2) receive a sufficiently strong Egfr signal to both alleviate Yan-imposed repression and stimulate PntP2 activation, and (3) receive a N signal able to stimulate Su(H) activation. The tripartite control of shaven expression in the cone cell precursors requires that they receive all three inputs at the proper time in their development. Lz expression in cone cell precursors has been documented. Consistent with their reception of the Egfr signal, activated MAPK is detected in cone cell precursors at the time when they initiate Shaven expression. Dl is expressed in developing photoreceptor clusters at the time when the cone cell precursors express Shaven. Thus, the neuronal clusters signal through an inductive Dl/N pathway to activate shaven expression in the neighboring cone cell precursors. These results suggest that, in addition to expressing Lz, the cone cell precursors receive the Egfr and N signals at the time of fate acquisition and Shaven expression. Presumably, at least one of these three activation mechanisms is lacking in cells that do not express shaven. This hypothesis was tested through genetic manipulation of the system (Flores, 2000).

Undifferentiated cells immediately posterior to the furrow receive the N signal and express Lz, but they do not express Shaven. It is hypothesized that the absence of Shaven expression in these cells is caused by a lack of the Egfr signal. This hypothesis is consistent with the observation that Egfr signaling causes these cells to differentiate. Indeed, Shaven is ectopically expressed in undifferentiated cells that express an activated form of Egfr. Loss-of-function yan^e2D/yan^pokX8 discs also show ectopic expression of Shaven in undifferentiated cells. Similarly, in discs expressing SME^mETSx6-lacZ, in which the six ETS sites in the SME are mutated, ß-galactosidase is also expressed in undifferentiated cells. Presumably, relief of Yan repression is sufficient to activate some shaven in undifferentiated cells. In SME^mETS(1,6)-lacZ,where the Pnt binding sites are eliminated but two of the Yan binding sites are still intact, there is no expression of ß-galactosidase in the undifferentiated cells. These results suggest that while the undifferentiated cells posterior to the furrow express Lz and receive the N signal, they fail to express Shaven because they do not receive the Egfr signal and are therefore unable to relieve the Yan-imposed repression of shaven (Flores, 2000).

The R7 precursors express Lz and receive RTK signals, yet they do not express Shaven. It is hypothesized that this is due to the lack of the N signal at the time of R7 determination. Indeed, expression of an activated form of N (N^act), leads to ectopic Shaven expression in R7 precursors, which suggests that Shaven is not normally expressed in R7 because this cell does not receive the N signal. These results are consistent with the previous observation that the R7 cell loses its neuronal characteristics upon expression of N^act (Flores, 2000).

Thus far, this study has focused on cells that express Lz. However, the regulation of shaven expression can also be tested in cells that lack Lz, such as the R3/R4 precursors. These cells receive the Egfr signal but receive the N signal after their initial fate specification, during ommatidial rotation. Ectopic expression of either Lz or N^act in the R3/R4 precursors fails to activate shaven expression in these cells. However, when Lz and N^act are coexpressed in the R3/R4 precursors, Shaven is expressed in these cells. These results demonstrate that the lack of both N signaling and Lz during the proper time window prevents R3/R4 cells from expressing shaven (Flores, 2000).

Engrailed and Hedgehog make the range of Wingless asymmetric in Drosophila embryos

Sparkling is a target of Wingless and Egfr pathways during segmentation. The spread of Wingless within the embryonic epidermis of Drosophila was examined. Using two assays for Wingless activity (specification of naked cuticle and repression of rhomboid transcription), it was found that Wingless acts at a different range in the anterior and posterior directions. This asymmetry follows in part from differential distribution of the Wingless protein. Transport or stability is reduced within engrailed-expressing cells, and farther posteriorward Wingless movement is blocked at the presumptive segment boundary and perhaps beyond. The role of hedgehog in the formation of this barrier is demonstrated (Sanson, 1999).

It is proposed that asymmetric Wingless distribution ensures the establishment of well-differentiated cell fates on either side of the engrailed domain. Anteriorly, at the wingless source, rhomboid expression is repressed. In contrast, reduced Wingless movement and/or stability within the engrailed domain allows nascent posterior rhomboid expression. Around this time (stage 11), a barrier to Wingless that requires hedgehog signaling forms at the posterior of the engrailed domain and ensures that Wingless does not foray across and repress rhomboid. rhomboid then activates the Egfr pathway within its expression domain and in adjacent cells. It may be that rhomboid itself contributes to barrier formation and thus builds a line of defense against invasion by its repressor. In addition, activation of the EGF pathway by rhomboid would antagonize any Wingless leaking through. Denticle formation requires transcription of sparkling/shavenbaby, which is under positive regulation by the Egfr pathway and negative regulation by the wingless pathway. Activated EGFR and the absence of Wingless posterior to the engrailed domain allow shavenbaby expression and hence denticle formation. At the anterior side, converse conditions exist, since Wingless is present at high levels and the Egfr pathway is inactive. Therefore, it is proposed that polarization of Wingless transport by engrailed and hedgehog guarantees the naked fate anterior to the engrailed domain and the denticle fate posteriorly, and thus establishes the anteroposterior polarity of each segment (Sanson, 1999 and references).

GENE STRUCTURE

spa has conserved a pattern of differential splicing and protein isoforms, reminiscent of that observed for Pax8. In the 3' portion of the spa transcript, alternative splice products are generated in which exon 11 or exons 10 and 11 are skipped, producing in-frame deletions of the Ser/Thr-rich region that precedes the C-terminal transactivation and inhibitory domains. In another splice variant, an alternative 3' acceptor site is used in intron 11, preceding the most frequently used site by 17 nucleotides and thus generating a frameshift and premature termination at the end of exon 12. Hence, the resulting Spa protein lacks the conserved transactivation and inhibition domains as do the Pax 8 isoforms of the human Pax8c,d splice variants. The splicing process of SPA transcripts is also affected by differential poly(A) addition (Fu, 1997).

Genomic length - 24 kb

Exons - 13

PROTEIN STRUCTURE

Amino Acids - 844

Structural Domains

Spa protein includes a paired domain of 128 amino acids. The paired domain is preceded by a relatively long amino-terminal peptide of 174 amino acids, whereas all known paired domains are located much closer to the amino terminus. The paired domain of Spa is clearly the closest Drosophila relative of the vertebrate Pax2, Pax5, and Pax8 paired domains, displaying 88% identity and 91% similarity to the paired domains of mouse and human Pax2 (relatively). The closest Drosophila relative of the Spa paired domain, the Eyeless paired domain, exhibits only 73% identity. Additional domains of the Spa protein possess homologous counterparts in the vertebrate Pax2, Pax5, and Pax8 proteins. Thus, the octapeptide, present in most Pax proteins, is extended to a nonapeptide identical to that of Pax2 and closely linked to a highly charged dodecapeptide conserved in Pax2, Pax5 and Pax8. In addition, the amino-terminal portion of a paired-type homeodomain, characteristic of Pax2, Pax5 and Pax8, has been conserved in Spa, although its conservation extends over only 14 of the 31 amino acids found in Pax2, Pax5 and Pax8. Most interestingly, a transactivation domain and its inhibitory domain found at the C-terminus of Pax2, Pax5 and Pax8, but in no other Pax proteins, have also been conserved at the C-terminus of Spa. Spa and Pax2, Pax5 and Pax8 include three Ser/Thr-rich regions at roughly equivalent positions that also may serve as transactivation domains. Pax2, Pax5 and Pax8 and Spa have also conserved precisely the locations of introns within the paired domain and between the transactivation and inhibitory domain (Fu, 1997).

date revised: 10 Jan 98

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