shaven/sparkling: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - shaven

Synonyms - sparkling (spa)

Cytological map position - Probably most distal visible locus on chromosome 4

Function - Transcription factor

Keywords - Eye, CNS, PNS

Symbol - sv

FlyBase ID: FBgn0005561

Genetic map position - 4-

Classification - paired domain and homeodomain (partial)

Cellular location - nuclear

NCBI links: Entrez Gene

shaven orthologs: Biolitmine
Recent literature
Zandvakili, A., Campbell, I., Gutzwiller, L. M., Weirauch, M. T. and Gebelein, B. (2018). Degenerate Pax2 and Senseless binding motifs improve detection of low-affinity sites required for enhancer specificity. PLoS Genet 14(4): e1007289. PubMed ID: 29617378
Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF binding sites (TFBSs) from background sequences represents a significant challenge. This study shows that a Drosophila regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, a method was developed that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, it was found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs.

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.

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, lzts1. The strongest eye-specific allele of shaven, spapol, which is not transcribed in cone cell precursors, also enhances lzts1. 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 spapol 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 spapol 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 EGFRts 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 SMEmETSx6-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 (SMEmETS(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 Nts 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 (DlDN) 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 DlDN 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 EGFRDN or NECN, 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 yane2D/yanpokX8 discs also show ectopic expression of Shaven in undifferentiated cells. Similarly, in discs expressing SMEmETSx6-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 SMEmETS(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 (Nact), 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 Nact (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 Nact in the R3/R4 precursors fails to activate shaven expression in these cells. However, when Lz and Nact 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).

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).


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).
cDNA clone length - 3.8 kb

Genomic length - 24 kb

Exons - 13


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).

shaven/sparkling: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised: 10 Jan 98

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