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

Gene name - ovo

Synonyms - shavenbaby

Cytological map position - 4E1

Function - Transcription factor

Keyword(s) - Ovary maturation, germ line sex determination, segment polarity

Symbol - ovo

FlyBase ID:FBgn0003028

Genetic map position - 1-10.2

Classification - C2H2 zinc fingers

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Hayashi, M., Shinozuka, Y., Shigenobu, S., Sato, M., Sugimoto, M., Ito, S., Abe, K. and Kobayashi, S. (2017).. Conserved role of Ovo in germline development in mouse and Drosophila. Sci Rep 7: 40056. PubMed ID: 28059165
Summary:
Ovo, which encodes a transcription factor with Zn-finger domains, is evolutionarily conserved among animals. In Drosophila, in addition to its zygotic function for egg production, maternal ovo activity is required in primordial germ cells (PGCs) for expression of germline genes such as vasa and nanos. This study found that maternal Ovo accumulates in PGC nuclei during embryogenesis. In these cells, ovo serves a dual function: activation of genes expressed predominantly in PGCs, and conversely suppression of somatic genes. Reduction of ovo activity in PGCs makes them unable to develop normally into germ cells of both sexes. In mice, knockout of the ovo ortholog, Ovol2, which is expressed in PGCs, decreases the number of PGCs during early embryogenesis. These data strongly suggest that ovo acts as part of an evolutionarily conserved mechanism that regulates germline development in animals.
Rizzo, N. P. and Bejsovec, A. (2017). SoxNeuro and shavenbaby act cooperatively to shape denticles in the embryonic epidermis of Drosophila. Development [Epub ahead of print]. PubMed ID: 28506986
Summary:
During development, extracellular signals are integrated by cells to induce the transcriptional circuitry that controls morphogenesis. In the fly epidermis, Wingless (Wg)/Wnt signaling directs cells to produce either a distinctly-shaped denticle or no denticle, resulting in a segmental pattern of denticle belts separated by smooth, or 'naked', cuticle. Naked cuticle results from Wg repression of shavenbaby (svb), which encodes a transcription factor required for denticle construction. This study has discovered that although the svb promoter responds differentially to altered Wg levels, Svb alone cannot produce the morphological diversity of denticles found in wild-type belts. Instead, a second Wg-responsive transcription factor, SoxNeuro (SoxN), cooperates with Svb to shape the denticles. Co-expressing ectopic SoxN with svb rescued diverse denticle morphologies. Conversely, removing SoxN activity eliminated the residual denticles found in svb mutant embryos. Furthermore, several known Svb target genes are also activated by SoxN, and two novel target genes of SoxN were discovered that are expressed in denticle-producing cells and that are regulated independently of Svb. Thus it is concluded that proper denticle morphogenesis requires transcriptional regulation by both SoxN and Svb.
BIOLOGICAL OVERVIEW

The ovo locus was once thought to be two separate genes: ovo and shavenbaby (svb). ovo is required in the female germ line for proper oogenesis. In the zygote, referred to often as shavenbaby, ovo is required for proper cuticular development. With the passage of time, and much research effort, it is now clear that ovo and shavenbaby are one and the same gene, one with a complex genetic function and an equally complex gene structure.

Ovo belongs to the ovarian tumor class of genes. Mutants of genes in this class present smaller than normal ovaries and egg chambers filled with an excess of undifferentiated germ cells. The initial observation that female germline cells defective in Sex lethal also form tumorous cysts led to the idea that this phenotype identifies loci involved in germline sex determination. This is supported by the fact that, among tumorous mutants (ovo, ovarian tumor, bag-of-marbles, sans fille, and Sex lethal,), only bam functions in the male germline. ovo is a key gene lying upstream of Sxl required for germline cells to respond to the somatic feminization signal (Mével, 1996 and references). Ovo is a transcription factor that is required in ovaries for the initiation of a pathway resulting in ovarian as distinct from testicular development.

Genes regulating expression of Sex-lethal in somatic tissues do not function in the germ line. Of the genes that control somatic sex determination Sex lethal is the only one required for germ line sex determination. The other four genes required for germ line sex determination (listed above) form a hierarchy, first involving initiation of ovary development, lead by ovo (Staab, 1995), and ultimately involved in Sex lethal expression in the female germ line. Of the genes listed above, only sans fille is known to be an RNA splicing factor (Oliver, 1993 and references).

What is the first somatic signal required for female germ-line sex determination? The signal is unknown, but without the functioning of somatic sex determination genes transformer, transformer-2 and doublesex, which control somatic sexual development, a somatic signal required for female germ-line sex determination does not take place. Mutants in these three genes cause partial sex transformation in the germline. tra, tra-2, and dsx activities are required for female-specific SXL pre-mRNA splicing in gonadal tissue. Males can be prepared from normal 2X females by mutation of the tra gene, and these 2X males have male-specific SXL pre-mRNA in their gonads (Oliver, 1993).

What are the genetic requirements for high ovo expression in gonads? In autosomes, the X to autosome ratio is sufficient for Sex lethal expression. Does a similar mechanism hold in the gonads? To study this question, expression of ovo was examined in gonads of mutant flies where the chromosomal sex does not match the somatic sexual identity. If a 2X karyotype is necessary and sufficient for high levels of ovo expression, then female somatic sex should have no effect on ovo expression. XY females can be produced by using a strain of flies bearing a transformer gene driver by a heat-shock promoter (trahs). In XY genetic males bearing the trahs transgene, Tra activity is sufficient to direct female somatic development in flies that would otherwise develop as males, but the germ line remains male. If a female somatic sexual identity is sufficient for a high level of ovo expression, then both XX females and XY trahs females should be expected to show similar ovo expression. In fact ovo expression is not activated to a high level by a female somatic identity, suggesting that either an XX karyotype or female germ-line identity is required for high ovo expression (Oliver, 1994).

It is presumed that ovo is part of a mechanism for counting the number of X chromosomes in the Drosophila germ line. Since Sxl, and snf are not required for germ cell viability and early germ line development, while ovo is required, it is suggested that ovo contributes to two functions: early germ line development, and later involvement in a hierarchy leading to sex specific splicing of Sex lethal pre-messenger RNA (Oliver, 1994).

Left out of the above list of ovarian tumor class genes is fused, a segment polarity gene that has been shown to have an ovarian tumor phenotype. Like bag-of-marbles, ovarian tumor, sans fille, Sex lethal and ovo, mutants in fused, a zygotic segment polarity gene, interfere with germ-line SXL pre-mRNA splicing. Might Fused act upstream of ovo regulating its expression in the gonads? This question awaits future investigation.

What about the role of ovo, known zygotically as shavenbaby, in cuticle development? shavenbaby mutation causes a defect in ventral denticle belts and dorsal hairs observed in cuticle preparations of late embryos. Both ventral denticle belts and dorsal hairs are eliminated or strongly reduced by svb mutations (Wieschaus, 1984). Might svb be activated in the segment polarity hierarchy, the same hierarchy that involves fused downstream of hedgehog and is involved in the transduction of hedgehog signals? This question can only be analyzed by further studies of ovo zygotic transcription.

OVO transcription factors function antagonistically in the Drosophila female germline

Ovo controls germline and epidermis differentiation in flies and mice. In the Drosophila germline, alternative Ovo-B and Ovo-A isoforms have a common DNA-binding domain, but different N-termini. These isoforms are transcription factors with opposite regulatory activities. Using yeast one-hybrid assays, a strong activation domain was identified within a common region and a counteracting repression domain within the Ovo-A-specific region. To identify and map Ovo transcriptional effector domains, the effect of fusion proteins on reporter gene expression was monitored. In flies, Ovo-B positively regulates the ovarian tumor promoter, while Ovo-A is a negative regulator of the ovarian tumor and ovo promoters. Ovo-B isoforms supply ovo+ function in the female germline and epidermis, while Ovo-A isoforms have dominant-negative activity in both tissues. Moreover, elevated expression of Ovo-A results in maternal-effect lethality while the absence of Ovo-A results in maternal-effect sterility. These data indicate that tight regulation of antagonistic Ovo-B and Ovo-A isoforms is critical for germline formation and differentiation (Andrews, 2000).

The function of ovo in, and for, female germline development appears to be relatively straight forward. The data indicate that Ovo-B supplies the essential ovo + function in the female germline. This is fully consistent with the expression of Ovo-B isoforms early in oogenesis where ovo + activity is required. Genetic and molecular data indicate that ovo plus acts upstream of otu + and ultimately Sxl +. Ovo-B positively regulates otu transcription in the female germline, suggesting that part of the function of Ovo-B is to up-regulate Otu production. There are at least three known positive regulators of otu promoter activity: ovo; somatic signals, and stand still (stil). While it is not known how Ovo-B activates otu in conjunction with these other regulators, Stil is a germline restricted chromatin associated protein that is present at cytological sites of transcription. Thus, Stil and Ovo-B may act directly at the promoter. The somatic sex determination signals that influence otu expression are undefined. Whereas maternal Ovo-A is required for the germline of the progeny, it is extremely toxic when produced during early oogenesis (Andrews, 2000).

Consequently, there must be a tightly regulated way to produce Ovo-A late in oogenesis, and indeed, the major phase of Ovo-A protein production appears to be during terminal oogenesis. Perhaps late Ovo-A acts to shut down production from ovo-B, otu and ultimately Sxl at the end of oogenesis. This may occur too rapidly to be detected by reporter genes: differences in ovo-B, ovo-A, or otu reporter expression between females encoding both Ovo-B and Ovo-A, versus those that encode only Ovo-B, could not be detected. Nevertheless, it is quite clear that Ovo-A is able to repress target genes that are known to be part of the genetic hierarchy, including ovo (Andrews, 2000).

The data points to two significant roles for maternally deposited Ovo-A and Ovo-B. The maternal-effect-sterility phenotype seen when mothers lack Ovo-A, indicates that ovo is required maternally for germline formation or maintenance. Parsimony suggests that Ovo-A acts as a transcriptional repressor of genes that must be off in the embryonic germline. This is quite interesting in light of the limited transcriptional activity in the early germline, and the finding that premature transcription of at least some broad classes of genes is detrimental to germ cell migration and survival. Similar transcriptional repression controls germline determination in C. elegans. While it was not directly determined when the germline defect becomes apparent, the occasional observation of unilateral germ cell-less gonads is an argument for a sparse population of primordial germ cells. The few primordial germ cells that successfully migrate to a gonadal primordium can then fully populate an adult gonad (Andrews, 2000).

The maternal effect on somatic development is also a newly described ovo phenotype. In this case, a simple model is suggested; that maternal Ovo-B is required to activate target genes required for the soma. A maternal effect of Ovo-B cannot be tested for directly, because it is required for egg formation. The experimental evidence is that excessive Ovo-A proteins causes maternal-effect lethality that can be titrated by supplying additional copies of transgenes encoding Ovo-B. It can be suggested that maternal Ovo is not likely to play a direct role in somatic ovo expression required for cuticle development. These results show that ectopic expression of Ovo-A during the time that somatic ovo + is expressed results in naked cuticle, and reduced denticle belts were not observed in embryos that were overstocked with maternal Ovo-A product. A simple model is that Ovo-A repression is required for germline development and that Ovo-B activation is required for the soma. However, given the presence of both Ovo-B and Ovo-A in late oogenesis and in early embryos, it is likely that Ovo-B and Ovo-A compete for similar binding sites during these stages. This might result in cross-regulation of target genes by the opposing activities of Ovo-B and Ovo-A. Indeed, the maternal-effect lethality phenotype is best explained by interfering cross-regulation by Ovo-A. Likewise, the maternal-effect sterility phenotype could be due to absence of Ovo-A in the germline, excessive Ovo-B, or both (Andrews, 2000).

The Drosophila HMG-domain proteins SoxNeuro and Dichaete direct trichome formation via the activation of shavenbaby and the restriction of Wingless pathway activity

Trichomes are cytoplasmic extrusions of epidermal cells. The molecular mechanisms that govern the differentiation of trichome-producing cells are conserved across species as distantly related as mice and flies. Several signaling pathways converge onto the regulation of a conserved target gene, shavenbaby (svb, ovo), which, in turn, stimulates trichome formation. The Drosophila ventral epidermis consists of the segmental alternation of two cell types that produce either naked cuticle or trichomes called denticles. The binary choice to produce naked cuticle or denticles is affected by the transcriptional regulation of svb, which is sufficient to cell-autonomously direct denticle formation. The expression of svb is regulated by the opposing gradients of two signaling molecules - the epidermal growth factor receptor (Egfr) ligand Spitz (Spi), which activates svb expression, and Wingless (Wg), which represses it. It has remained unclear how these opposing signals are integrated to establish a distinct domain of svb expression. This study shows that the expression of the high mobility group (HMG)-domain protein SoxNeuro (SoxN) is activated by Spi, and repressed by Wg, signaling. SoxN is necessary and sufficient to cell-autonomously direct the expression of svb. The closely related protein Dichaete is co-regulated with SoxN and has a partially redundant function in the activation of svb expression. In addition, SoxN and Dichaete function upstream of Wg and antagonize Wg pathway activity. This suggests that the expression of svb in a discreet domain is resolved at the level of SoxN and Dichaete (Overton, 2007).

In the embryonic ventral epidermis of Drosophila, two alternative cell fates are specified: smooth cells and trichome-producing cells. These binary cell fates are distinguished by the expression of svb, the most-downstream effector of epidermal morphogenesis. svb is necessary and sufficient to cell-autonomously direct trichome formation. The expression of svb is regulated by the opposing gradients of two signaling molecules: Spi, which activates, and Wg, which represses, svb expression. svb is expressed in segmentally reiterated, epidermal stripes, which invariantly encompass six rows of cells. This raises the question of how is opposing extrinsic information integrated to establish a distinct domain of svb expression with a sharp posterior border (Overton, 2007)?

This study demonstrates that the HMG-domain proteins SoxN and Dichaete represent a molecular link between the expression of svb and the upstream Der- and Wg-signaling cascades. SoxN and Dichaete are expressed in the ventral epidermis at the time when epidermal cell fates are specified. The late phase of SoxN and Dichaete expression is stimulated by Der- and repressed by Wg-pathway activity. These regulatory mechanisms result in the expression of SoxN and Dichaete in those six rows of cells within each abdominal segment that differentiate to produce trichomes. SoxN and, to a lesser extent, Dichaete, are necessary and sufficient to activate the expression of svb. Furthermore, these results show that the well-described repression of svb by Wg is due to the repression of SoxN, which, in turn, results in the loss of svb activation. Likewise, the Spi-mediated activation of svb expression relies on the activation of SoxN, which, in turn, activates svb. This indicates that the competition of Der- and Wg-pathway activities for the specification of trichome-producing versus smooth cell fates is resolved at the level of SoxN and Dichaete (Overton, 2007).

These results do not provide much insight into the issue of how opposing extrinsic information is integrated such that a sharp posterior border of svb expression is achieved. Instead, they raise the question of how is a sharp posterior border of SoxN and Dichaete expression established/maintained? The findings suggest that this is achieved by a combination of negative- and positive-feedback loops. (1) Evidence is provided that SoxN and Dichaete negatively regulate Wg pathway activity. This negative-feedback loop provides a likely mechanism for the establishment and maintenance of a sharp posterior border of SoxN and Dichaete expression. The issue arises of how robust this system might be in the face of fluctuating levels of Wg pathway activity. The efficiency with which SoxN and Dichaete restrict Wg pathway activity will crucially rely on the levels of SoxN and Dichaete protein. In this context, it is noteworthy that the levels of SoxN protein, but not Dichaete, are several-fold higher in the two posterior-most rows of the SoxN stripe compared with the anterior four rows. The regulatory mechanisms that underlie the different levels of SoxN expression are currently unclear. (2) Evidence is provided that the maintenance of SoxN and Dichaete expression is supported by a positive-feedback loop: svb, the expression of which is activated by SoxN and Dichaete, is itself required for the maintenance of SoxN and Dichaete expression. Together, these mechanisms contribute to an invariant read-out of cell identity from opposing Der- and Wg-pathway activities (Overton, 2007).

In Drosophila, SoxN and Dichaete are necessary and sufficient to activate the expression of svb, which in turn directly regulates the expression of genes involved in trichome morphogenesis. Is a function in hair formation of the Sox proteins conserved in other species, including vertebrates? A previous study has shown that the mouse Sox9 protein is required for the differentiation of hair-producing epidermal cells and acts genetically downstream of sonic hedgehog pathway activity (Vidal, 2005). This study did not address whether Sox9 regulates the expression of movo1 (Ovol1), the mouse ortholog of svb. Nevertheless, the demonstrated roles of SoxN, Dichaete and Sox9 raise the exciting question of do Sox proteins have an essential function in the activation of an epidermal differentiation program that is conserved across species as distantly related as mice and flies (Overton, 2007).

Pri sORF peptides induce selective proteasome-mediated protein processing

A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. This study shows that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. These data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators (Zanet, 2015).

Eukaryotic genomes encode many noncoding RNAs (ncRNAs) that lack the classical hallmarks of protein-coding genes. However, both ncRNAs and mRNAs often contain small open reading frames (sORFs), and there is growing evidence that they can produce peptides, from yeast to plants or humans. The polished rice or tarsal-less (pri) RNA contains four sORFs that encode highly related 11- to 32-amino acid peptides, required for embryonic development across insect species. In flies, pri is essential for the differentiation of epidermal outgrowths called trichomes. Trichome development is governed by the Shavenbaby (Svb) transcription factor; however, only in the presence of pri can Svb turn on the program of trichome development, i.e., activate expression of cellular effectors. Indeed, the Svb protein is translated as a large repressor, pri then induces truncation of its N-terminal region, which leads to a shorter activator (Kondo, 2010). Thereby, pri defines the developmental timing of epidermal differentiation, in a direct response to systemic ecdysone hormonal signaling (Chanut-Delalande, 2014). Although there is currently a clear framework for the developmental functions of pri, how these small peptides can trigger Svb processing is unknown (Zanet, 2015).

To identify factors required for Svb processing in response to pri, a genome-wide RNA interference (RNAi) screen was performed in a cell line coexpressing green fluorescent protein (GFP)-tagged Svb and pri. An automated assay was set up quantifying Svb processing for each of the Drosophila genes, with an inhibitory score reflecting the proportion of cells unable to cleave off the Svb N terminus. pri RNAi displayed the highest score, which validated this approach to identifying molecular players in Svb processing. Methods used to evaluate results from genome-wide screening all converged on a key role for the proteasome. For instance, COMPLEAT, a bioinformatic framework based on protein complex analysis, identified the proteasome in 66 out of the 71 top predictions. A survey of individual proteasome subunits indicated that both the 20S catalytic core and the 19S regulatory particles are required for Svb processing. Chemical proteasome inhibitors independently confirmed this conclusion, because they also prevented pri-induced Svb processing. These data thus provide compelling evidence that Svb processing results from a pri-dependent proteolysis by the proteasome (Zanet, 2015).

To investigate how pri regulates proteolysis of Svb, the protein region(s) in Svb were identified that are involved in pri-dependent processing. Systematic deletions demonstrated the importance of the Svb N terminus for pri response and restricted the minimal motif to the N-terminal 31 amino acids. Deletion of this motif within an otherwise full-length protein (Δ31) made Svb refractory to pri. Conversely, the Svb N terminus when fused to GFP (1s::GFP) was sufficient to transform this protein into a pri target and to make GFP sensitive to pri. Unlike Svb, however, 1s::GFP was completely degraded by the proteasome upon pri expression (Zanet, 2015).

Recent studies have shown that structural features of proteins influence their degradation by the proteasome: Whereas unstructured substrates, such as intrinsically disordered regions, favor degradation, tightly folded domains can resist proteasome progression. Analysis of Svb sequences predicted intrinsically disordered features throughout its N-terminal moiety, which is degraded. By contrast, the proteasome-resistant C-terminal moiety comprises two folded regions: the transcriptional activation and zinc finger domains. Within the transcriptional activation region, amino acids 532 to 701 protected Svb from complete degradation. Indeed, the C-terminally truncated mutants of 1 to 701 amino acids (and longer) were still processed, whereas mutants shortened by 1 to 532 amino acids (and shorter) were fully degraded. Whether other folded domains would also protect Svb from complete degradation was tested and it was found that attaching zinc fingers to short Svb mutants-otherwise degraded upon pri expression-was sufficient to restore processing. Likewise, the DNA binding domain of Gal4 protected against degradation, which indicated that even a heterologous protein domain with strong structure can protect Svb from full degradation in response to pri. Hence, distinct regions of Svb mediate its processing by the proteasome: the 31 N-terminal residues act as a pri-dependent degradation signal, or degron, and C-terminal domains act as stabilizing features that prevent complete degradation (Zanet, 2015).

Proteins are targeted to the proteasome by the covalent attachment of ubiquitin to Lys residues. The Svb N terminus is highly conserved from insects to human; it comprises two invariant Lys residues (K3 and K8) and a third one at a less constrained position (K28 in Drosophila). Individual Lys substitutions had only a weak effect or no effect, whereas simultaneous mutation of all three Lys (3Kmut) abolished Svb processing. Furthermore, strong pri-dependent ubiquitination of Svb was detected when the proteasome was inhibited. By contrast, this was no longer seen in the 3Kmut variant, which demonstrated the key role of these three Lys in ubiquitin-dependent Svb processing (Zanet, 2015).

Ubiquitin conjugation requires three enzymes (E1, E2, and E3); specificity is generally conferred by the E3 ubiquitin ligases that recognize and bind to substrates. A prominent hit from the RNAi screen was Ubr3 (7 hits out of the top 15), which encodes an E3. Ranking all Drosophila ubiquitin enzymes by their inhibitory score confirmed that Ubr3 was the major E3 required for Svb processing and identified UbcD6 (Rad6) as its associated E2, consistent with evidence that human Ubr3 also forms a complex with UbcD6. Like many proteasome factors, Ubr3 has a broad subcellular distribution in cytoplasm and nuclei, whereas Svb and UbcD6 are nuclear proteins. Svb processing still occurred normally when nuclear export was impaired, which indicated that the proteolytic activation of Svb takes place within the nucleus (Zanet, 2015).

Several additional lines of evidence support the conclusion that Ubr3 mediates the function of pri for Svb ubiquitination. First, Ubr3 coimmunoprecipitated with Svb in a pri-dependent manner and ubiquitinated Svb was found in a complex with Ubr3 upon proteasome inhibition. Second, the N terminus of Svb was sufficient for Ubr3 binding in response to pri. Note that a functional N-terminal degron in Svb was required for its interaction with Ubr3, because the ubiquitin-resistant 3Kmut variant no longer bound Ubr3. Third, in protein extracts from cells that do not express pri, addition of synthetic Pri peptide was sufficient to promote Ubr3-Svb interaction in vitro, in a dose-dependent manner. By contrast, a peptide of the same composition but in a 'scrambled' sequence lacked activity (Zanet, 2015).

Although critical for the binding of Ubr3 to the Svb N terminus, Pri peptides are, however, not indispensable for Ubr3 activity. pri did not influence the binding of Ubr3 to Ape1 (Rrp1), a factor involved in DNA repair and regulated by Ubr3-dependent proteasome degradation. Also, the interaction of Ubr3 with DIAP1, which inhibits apoptosis, occurred with or without pri. Moreover, Pri peptides interacted with Ubr3, even in the absence of Svb. Finally, the isolated UBR-box of Ubr3 no longer required Pri peptides to bind Svb, which suggested that other Ubr3 motifs prevent Svb interaction in the absence of pri. It is therefore concluded that Pri peptides directly regulate the selectivity of Ubr3 for binding to the Svb N terminus and, thereby, trigger Svb ubiquitination and processing by the proteasome (Zanet, 2015).

Recently a Ubr3 loss-of-function allele was isolated, and its phenotype in the differentiation of epidermal cells was assayed. As observed for pri mutants, embryos lacking Ubr3 were unable to differentiate trichomes and to process Svb. Moreover, inactivation of either UbcD6 or Ubr3 prevented formation of adult trichomes in mosaic animals. When compared with their wild-type neighbors, Ubr3-null cells accumulated the repressor form of Svb, which demonstrated Ubr3's essential role for Svb processing in vivo (Zanet, 2015).

Taken together, thes data show that Pri peptides control the binding of the Ubr3 ubiquitin ligase to Svb and activate its processing by the proteasome. In the absence of Pri, Ubr3 nonetheless recognizes other substrates, which shows that a main role for Pri peptides is to modify the binding selectivity of Ubr3. This could potentially be achieved through a conformational change in Ubr3 protein, as proposed for Ubr1, that unmasked the recognition site for Svb upon Pri peptide binding to Ubr3 (Zanet, 2015).

Although recent work has uncovered thousands of novel sORF peptides, only a handful of their molecular targets have yet been identified. sORF peptides have recently been found to bind and regulate the Ca2+ uptake SERCA protein, the heterotrimeric guanine nucleotide-binding protein coupled signaling APJ (Apelin), and the DNA repair protein Ku. Protein-protein interactions often involve small protein regions, and artificial peptides that mimic these binding surfaces have been proven to be potent modulators of protein complexes. It is proposed that sORF-encoded peptides provide an unexplored reservoir of protein-binding interfaces, well suited to regulate the activity of a wide range of cellular factors (Zanet, 2015).


GENE STRUCTURE

At least three biologically functional transcripts are produced by the shavenbaby-ovo gene region. There are differences in the 5' untranslated region (Garfinkel, 1994). Transcripts use two alternative 5' exons (alternative transcription start sites) termed exon 1a and 1b, both functioning to produce maternal transcripts that code for Ovo-A and Ovo-B respectively. There is an indication that exon 1b is subject to alternative splicing (Garfinkel, 1994 and Mével-Ninio, 1995). It is suggested that 1a includes the start site for late ovarian and zygotic Ovo proteins. Exon 1b consists of the 3' UTR of the early ovarian OVO transcript (Mével-Ninio, 1996). The earliest ovarian Ovo protein and OVO transcript differs from late Ovo protein and OVO transcript and both of these differ from the Svb protein and SVB transcript in the absence of an extention to the second exon called 2b, coding for 170 amino acids present in Ovo protein but absent from Svb protein (Mével-Ninio, 1995). Svb and late ovarian Ovo proteins also contain an N-terminal sequence coded for by the 1a exon. The 1a start site, coding for the 3' end of the SVB transcript is derived from sequences upstream of the 1b ovo start site (Mével-Ninio, 1996).

Two major classes of germline OVO transcripts, distinguished from the zygotic Shaven transcript driven by an upstream promoter, are driven by the adjacent ovo-A and ovo-B promoters (Garfinkel, 1994; Mével-Ninio, 1995). Although, OVO-B and OVO-A transcripts differ only by their short first exons (77 and 126 n.t.) Ovo-B has 374 fewer amino acid residues than Ovo-A. OVO-B mRNAs encode only Ovo-B isoforms from an AUG initiation codon in exon 2, whereas OVO-A transcripts encode longer OVO-A isoforms from an AUG initiation codon in exon 1A, and potentially encode OVO-B from the downstream AUG (Andrews, 2000).

To make matters more complex, there is a short oocytic Ovo protein beginning at methionine 373, a full 1/3 of the protein length downstream of the starting methionine on exon 1b. This methionine is also found on exon 2a (downstream of exons 1a and 1b) and is used to generate a long oocytic protein. Distinct functions are predicted for the long and short oocytic proteins. ovo has a genetic fine structure generated by the complex transcripts of ovo gene (Mével-Ninio, 1996).

Characterization of ovo/svb genomic organization has indicated that regions specifically required for svb somatic functions remain to be identified. The Df(1)biD2 deletion, which disrupts svb without affecting ovo, shows that sequences distal to ovo promoters are required for svb function. A search was carried out for coding regions upstream of ovo and a single predicted ORF was found, located 15-kb upstream of ovo promoters. This novel exon (1s) corresponds to the 5'-end of an embryonic EST (LD 47350) that also contains ovo/svb exons 2a, 3 and 4, in-frame with ORF1s. Furthermore, when used for in situ hybridization, exon 1s displays a pattern identical, in somatic tissues, to that of an ovo/svb probe from exons 3 and 4. Therefore, these data indicate that LD 47350 corresponds to bona fide svb mRNA and that the novel exon contains the 5'-end of Svb ORF. Its conceptual translation predicts a 1354-aa protein corresponding to a further N-terminal extension of 129-aa, compared to OvoA. A P-element insertion has been isolated, located in the predicted first intron of svb. PL107 mutant embryos display a naked cuticle phenotype and complementation tests have established that PL107 specifically disrupts svb without altering ovo. Consistently, mature svb mRNA is absent from PL107 embryos whereas ovo is expressed normally in the gonads. Precise excisions of the PL107 element restore viability and suppress the mutant phenotypes, whereas a deletion resulting from imprecise excision complements neither svb, nor ovo mutations. This mutant allele (ovo/svbR9) appears as a molecular null and abolishes both svb and ovo transcripts. Taken together, these results provide the first molecular elucidation of the complex organization of the ovo/svb locus and the identification of corresponding products. These data demonstrate that svb corresponds to a somatic-specific promoter, located 15-kb upstream of the ovo germline promoters, and that it codes for a novel protein isoform containing an additional N-terminal region (Delon, 2003).


GENE STRUCTURE
Transcript length - 7.1 kb (SVB) and 5.0 kb (OVO)

Bases in 5' UTR - 1169 and 1193 for OVO

Exons - 4

Bases in 3' UTR - 780


PROTEIN STRUCTURE

Amino Acids - 1028 (Ovo) and 858 (Svb)

Structural Domains

The Ovo protein, in common with the Svb protein, contains four C2-H2 zinc finger motifs at amino acids 876 - 896, 904 - 924, 932 - 953 and 971 - 992 of the Ovo protein predicted sequence (Mével-Ninio, 1991). An intron interrupts the coding sequences for the third and four zinc fingers. The N-terminal putative activation domain, again in common with the Svb protein, contains regions with homopolymeric runs of glycine, serine, alanine and glutamine, characteristic of opa repeats. In addition, the putative activation domain contains acidic regions, (amino acids 180 - 200, 275 - 290 and 600 - 540), the final one of which is coded by the ovo-specific exon 2 extention (Garfinkel, 1994).


EVOLUTIONARY HOMOLOGS

Regulatory evolution of shavenbaby/ovo in Drosophila species underlies multiple cases of morphological parallelism

Cases of convergent evolution that involve changes in the same developmental pathway, called parallelism, provide evidence that a limited number of developmental changes are available to evolve a particular phenotype. In no case are the genetic changes underlying morphological convergence understood. However, morphological convergence is not generally assumed to imply developmental parallelism. A case of convergence of larval morphology has been investigated in insects -- the loss of particular trichomes, observed in one species of the Drosophila melanogaster species group, has independently evolved multiple times in the distantly related D. virilis species group. Genetic and gene expression data is presented showing that regulatory changes of the shavenbaby/ovo (svb/ovo) gene underlie all independent cases of this morphological convergence. These results indicate that some developmental regulators might preferentially accumulate evolutionary changes and that morphological parallelism might therefore be more common than previously appreciated (Sucena, 2003).

The patterning of dorsal trichomes is an interesting case of convergent morphological evolution, because four species from the D. virilis species group (D. ezoana, D. borealis eastern, D. lacicola and D. montana) also show evolutionary loss of the thin trichomes. All other species of the D. virilis species group examined (D. americana, D. borealis western, D. canadiana, D. flavomontana, D. kanekoi, D. littoralis, D. lummei, D. novamexicana and D. virilis) and the more distantly related D. arizonae, possess a lawn of trichomes similar to that observed in D. melanogaster. By mapping these phenotypes onto a recent molecular phylogeny of the D. virilis species group, it can be inferred that at least three evolutionary transitions are required to explain the current distribution of trichome loss. The convergence of trichome patterns in different fly lineages indicates that these changes might be driven by natural selection, although the selection pressure has not yet been identified. In addition, the evolutionary loss of trichomes in first-instar larvae mirrors an ontogenetic loss of the same trichomes in second-instar and third-instar larvae in all species examined, suggesting that these trichomes have a special function in first-instar larvae. In theory, many genes might have evolved to alter the patterning of larval trichomes. For example, the wingless (wg) and hedgehog (hh) pathways and the lines gene are involved in patterning the trichomes on the dorsal epidermis. It might therefore be interesting to test whether evolution of patterning genes involved in segmentation can account for the evolution of trichome patterns. However, it has been shown that six genes involved in segmentation (wg, gooseberry-distal, patched, engrailed, abdominal-A and hunchback) are expressed identically in 12 species of the D. virilis species group, indicating that differences in trichome patterning might have evolved by changes in genes downstream of the segmentation pathway (Sucena, 2003).

A full-genome genetic scan revealed that regulatory evolution at the svb gene accounts fully for the difference in trichome pattern between D. sechellia and other species of the D. melanogaster species group. The transcription factor Svb acts to switch cells between naked cuticle and the production of trichomes. In D. melanogaster embryos, svb is genetically required for trichome formation; when svb is expressed in a cell, that cell autonomously differentiates trichomes, whose morphology is determined by other patterning genes. Therefore svb integrates numerous sources of information (including the wg, hh, Epidermal growth factor receptor, homeotic and dorsal-ventral patterning systems) to specify the final pattern of trichomes (Sucena, 2003).

To determine the genetic nature of the phenotypic differences observed in the D. virilis species group, interspecific genetic crosses were performed. Although most D. virilis group species do not interbreed, interspecific hybrid larvae were obtained in three cases. First, D. borealis eastern females were crossed with D. montana males, both of which have a naked phenotype. Four larvae of unknown sex were recovered that all had the naked phenotype. This cross suggests that the naked phenotype is not caused by different autosomal recessive genes in each species. However, there are several possible genetic explanations for this result: the naked phenotype might be caused by a recessive X-linked gene (if all the larvae were male), by the same recessive X-linked gene in both species (if any of the larvae were female), by the same recessive autosomal gene in both species, or by a dominant allele in one or both of the species (Sucena, 2003).

Two further crosses that narrow down these possibilities were successfully performed. Larvae were obtained from a cross between D. virilis females and D. borealis eastern males. The anterior halves of these larvae were mounted to analyse the trichome pattern, and DNA was prepared from the posterior half for a polymerase chain reaction assay. Between D. virilis and D. borealis eastern, a restriction-site polymorphism was identified in the svb gene, which is X-linked in D. melanogaster and D. virilis. DNA analysis therefore allowed the determination of both larval sex and the origin of the svb allele(s). Two larvae carried only the D. virilis svb allele (males); two other larvae carried svb alleles from both species (females). All four larvae had a D. virilis-like trichome pattern, indicating that the D. virilis trichome pattern is dominant to the more naked D. borealis eastern pattern, just as it is in the D. melanogaster species group. In a reciprocal cross between D. borealis eastern females and D. virilis males, two larvae were obtained. Both carried only the D. borealis svb allele (males) and had the D. borealis eastern pattern of trichomes, confirming the X-linkage of the factor(s) responsible for the difference in trichome pattern. These species carry multiple X-chromosome inversions that are likely to be responsible for the observed absence of recombination between heterospecific X chromosomes, which prevented further genetic mapping of the factor(s) involved. Together, the interspecific genetic data show that the presence of trichomes is dominant to the absence of trichomes and that trichome pattern segregates with the X chromosome, which excludes about 80% of the genome but includes the svb gene (Sucena, 2003).

Given the central role of svb in patterning trichomes in D. melanogaster and its genetic co-segregation with trichome pattern in the D. virilis species group, tests were performed to see whether the evolution of divergent trichome patterns could be explained by the evolution of the svb expression pattern in embryos from nine species from the D. virilis species group. The embryonic pattern of svb expression was found to closely match the pattern of trichomes on the first-instar larval cuticle. In species with an evolutionary loss of dorsal trichomes, svb transcription is absent from corresponding cells that differentiate naked cuticle. This suggests a simple model in which the transcriptional enhancer promoting svb expression in the domain that produces thin trichomes is turned off in some lineages (Sucena, 2003).

When analyzed in detail, however, the patterning of trichomes indicates a more complicated history of svb regulatory evolution. In D. sechellia and D. ezoana, essentially all thin trichomes are lost from all except the most posterior segments. In contrast, in the D. montana subphylad, species fall along a continuum from very hairy (D. flavomontana) to almost completely naked (D. lacicola), with D. borealis eastern and D. montana having an intermediate naked phenotype. In the intermediate phenotypes, thin trichomes are absent to different degrees from the more anterior segments, whereas they are retained in the more posterior segments (Sucena, 2003).

These phenotypic differences might have been thought to imply that different genetic mechanisms were responsible for morphological evolution. However, a strict correlation is again observed between svb expression and the trichome patterns along the anterior-posterior axis, indicating that regulatory changes in svb expression are involved in all of these evolutionary events. This might be due either to different changes in the cis-regulatory region of svb or potentially to different trans-acting changes in the different lineages. The latter possibility would imply the evolution of additional genes on the X chromosome controlling svb expression. This was genetically proved not to occur between D. melanogaster and D. sechellia. However, rejecting this hypothesis for the D. virilis species group will require the identification and characterization of the appropriate regulatory regions of svb from these species (Sucena, 2003).

svb regulatory evolution was found to underlie all observed cases of convergent evolution of the larval trichome pattern. In the epidermis, svb works like a morphogenetic switch, making it an effective point in the developmental cascade to generate alternative trichome patterns. Alterations of broader-acting patterning genes might cause pleiotropic consequences, whereas changes to individual genes downstream of svb, presumably the cytoskeletal genes sculpting the apical extensions that form trichomes, might have no effect. In addition, the modular nature of svb regulatory regions permits alterations in part of the cuticular pattern without pleiotropic consequences (Sucena, 2003).

It is recognized that organs that are identical by descent (i.e., homologous), need not be constructed by identical genetic mechanisms. The results of this study indicate that changes in the same genetic mechanisms might result in morphological convergence. Therefore, observations of identical genetic mechanisms underlying similar morphologies do not necessarily imply homology: phylogenetic information must also be considered (Sucena, 2003).

The dual function of ovo/shavenbaby in germline and epidermis differentiation is conserved between Drosophila melanogaster and the olive fruit fly Bactrocera oleae

The olive fruit fly Bactrocera oleae (B. oleae) is a major olive damaging pest in the Mediterranean area. As a first molecular analysis of a developmental gene in this insect, the ovo/shavenbaby (ovo/svb) gene has been characterized. In Drosophila, ovo/svb encodes a family of transcription regulators with two distinct functions: ovo is required for female germline differentiation and svb controls morphogenesis of epidermal cells. This study reports the cloning and characterization of ovo/svb in B. oleae; the ovo genomic organization and complex pattern of germline transcription have been conserved between distantly related Dipterae. B. oleae svb embryonic expression precisely prefigures the pattern of larval trichomes, supporting the conclusion that regulatory changes in svb transcription underlie evolutionary diversification of trichome patterns seen among Dipterae (Khila, 2003).

EGL-38 Pax regulates the ovo-related gene lin-48 during Caenorhabditis elegans organ development

The Pax gene egl-38 plays an important role in the development of several organs in C. elegans. egl-38 encodes a Pax transcription factor that is most similar to the mammalian Pax2/5/8 subclass of factors. To understand how a Pax transcription factor influences distinct developmental choices in different cells and tissue types, a second gene, lin-48, has been characterized. lin-48 functions with egl-38 in the development of one structure, the hindgut, but not in other tissues such as the egg-laying system. lin-48 encodes a C2H2 zinc-finger protein that is similar to the product of the Drosophila gene ovo and is expressed in the hindgut cells that develop abnormally in lin-48 mutants. Evidence is presented that lin-48 is a target for EGL-38 in hindgut cells. lin-48 requires egl-38 for its expression in the hindgut. Using deletion analysis, two elements in the lin-48 promoter have been identified that are necessary for lin-48 expression. EGL-38 binds with high affinity to one of these elements. In addition, genetic interactions have been observed between mutations in the lin-48 promoter and specific alleles of egl-38. These experiments demonstrate a functional link between Pax and Ovo transcription factors, and provide a model for how Pax transcription factors can regulate different target genes in different cells (Johnson, 2001).

Work with ovo genes in Drosophila and mouse has focused on their roles in fertility and epidermal development. Although lin-48 plays no apparent role in fertility or development of epidermis, ovo genes in mouse, Drosophila and C. elegans exhibit parallels in that they all play a role in the differentiation and maintenance of specific cell types. In addition, C. elegans and mouse ovo genes are similar in that they play a role in urogenital development. Mouse Ovo1 is important in development of the genital tract and kidney, and lin-48 plays a role in development of the hindgut (which develops into the C. elegans adult male cloaca) and potentially the excretory system. The experiments reported here indicate lin-48 is a direct target for EGL-38. A direct link between Pax factors and ovo genes has not been previously reported. However, genetic parallels in mammals indicate the potential for a conserved functional relationship between these classes of genes. In vertebrates, the Pax2 gene is essential for development of kidney, brain and ear, and the Pax8 gene plays a role in thyroid and kidney development. Mouse Ovo1 is expressed abundantly in the kidney, and is required for its normal differentiation. Thus, as in C. elegans, Ovo1 acts in a subset of the cells that require Pax2/5/8 factors. Future experiments will be required to test whether Ovo1 is a target for Pax2 or Pax8 during kidney development. Since all of the functions of the Drosophila Pax2/5/8 gene sparkling (shaven) have not been characterized, it is not known whether there are developmental functions shared by ovo and sparkling (Johnson, 2001).

Multiple regulatory changes contribute to the evolution of the Caenorhabditis lin-48 ovo gene

Recent work points to the importance of changes in gene expression patterns in species-specific differences. The evolution of the nematode lin-48 ovo gene has been studied. lin-48 is expressed in several cells in both Caenorhabditis elegans and C. briggsae, but acts in the excretory duct cell only in C. elegans. The differences result both from alterations in the cis-regulatory sequences and in proteins that mediate lin-48 expression. One factor that contributes to the species differences is the bZip protein CES-2. These results indicate the accumulation of several regulatory changes affecting one gene can contribute to evolutionary change (Wang, 2002).

A mutation analysis of the Ce-lin-48 upstream regulatory region identified an element essential for excretory duct cell expression that is conserved between C. elegans and C. briggsae. This site (termed lre2) binds the Pax protein EGL-38 and is required for expression in both the excretory duct cell and hindgut cells. However, this site cannot be sufficient for duct cell expression, because it is present in both C. elegans and C. briggsae genes. To identify additional sequences important for duct cell expression and responsible for the differences in the lin-48 cis-regulatory region, chimeric clones were made by swapping regions between Ce-lin-48 and Cb-lin-48, and these clones were tested for expression in C. elegans animals. The results indicated that the more proximal 1.5 kb of Ce-lin-48 upstream sequences also contain cis-regulatory sequences necessary for excretory duct cell expression that are absent from the C. briggsae gene. Further analysis of this region showed that it includes at least four independent sites (one distal and three proximal) that are each sufficient to increase expression levels. This conclusion results from the following observations: (1) either a more distal or more proximal portion of this region is sufficient for the expression; (2) deletions in the clone containing the most proximal 525-bp C. elegans sequence and the remaining sequence from C. briggsae revealed at least three elements that confer partial duct cell expression activity. Each tested subdivision of this region retained some activity. Although several distinct sites are present in the proximal region of lin-48, the sequence does not contain obvious EGL-38 recognition sites. This result suggests that a different factor or factors is also essential for excretory duct cell expression (Wang, 2002).

To identify additional factors important for Ce-lin-48 expression in excretory duct cell, MatInspector software was used to search for potential transcription factor-binding sites. This identified two sites with the potential to bind the C. elegans bZip protein CES-2. ces-2 was originally characterized for its role in mediating a developmental decision between survival and apoptosis. To test whether ces-2 plays a role in Ce-lin-48 excretory duct cell expression, the excretory duct morphology was examined in ces-2(n732) and ces-2(RNAi) animals and it was found to be similar to lin-48 mutants. The ces-1 gene has been shown to act downstream of ces-2 in regulating the cell death decision, but no effect on excretory duct morphogenesis was found in ces-1 mutants. Thus, the role of ces-2 in duct morphogenesis is distinct from its role in apoptosis (Wang, 2002).

To test the function of ces-2 in mediating lin-48 duct cell expression, lin-48::gfp expression was tested in ces-2(n732) and ces-2(RNAi) mutant animals, and it was found that lin-48::gfp expression is notably reduced in these animals. Finally, mutations disrupting the potential CES-2-binding sites in transgenes containing the 60-bp C. elegans proximal sequences eliminated the excretory duct cell expression. Taken together, these results indicate CES-2 mediates expression of lin-48 in the C. elegans excretory duct cell, and alteration of CES-2 regulatory sequences contribute to the differences between C. elegans and C. briggsae. Because mutations in ces-2 do not completely eliminate excretory duct expression, it suggests that other factors also contribute to the differences in lin-48 expression. This observation is consistent with the identification of multiple regulatory sites in the chimeric lin-48::gfp reporter transgenes (Wang, 2002).

Thus, using inter-specific gene expression assays, it was found that changes in cis-regulatory sequences and in trans-acting factors contribute to differences in Caenorhabditis lin-48 expression and function in the excretory duct cell. Two types of lin-48 cis-regulatory sequences are important for excretory duct cell expression: several sites are located in the proximal region that differ between C. elegans and C. briggsae, and the more distal lre2 that is conserved. Although both are necessary, the lre2 sequence from either C. elegans or C. briggsae can function with the C. elegans proximal sequences to mediate expression. These results provide experimental evidence that the modular architecture of cis-regulatory sequences allows for gene evolution. Specifically, evolutionary changes have affected the proximal sequences important only for duct cell expression, whereas lre2 (important for expression in two cell types) is unchanged. These results suggest that the bZip transcription factor CES-2 is one factor that acts through the proximal sequences that differ between C. elegans and C. briggsae. Further work is necessary to determine whether ces-2 function also differs between C. elegans and C. briggsae, or whether it is change of other cis-regulatory factors that prevents the expression of Ce-lin-48::gfp in the C. briggsae excretory duct (Wang, 2002).

Analysis of chimeric enhancers from Drosophila species suggests that cis-regulatory sequences are subject to complex stabilizing selection. These experiments show that conservation of a gene expression pattern between species results from multiple compensatory nucleotide changes. The experiments address the question of what molecular changes occur in the evolution of a gene expression pattern. A regulatory change could result from a sequence change in a single cis-regulatory element, or several sequence changes affecting multiple cis-regulatory elements. Evolution of gene expression by a single change might be associated with a dramatic phenotypic alteration fixed by strong selection, whereas the accumulation of multiple smaller changes might underlie a less stringent selection process or genetic drift. These small changes would act to stabilize an initial altered expression pattern. The results with Caenorhabditis lin-48 genes are consistent with the latter mechanism. It is speculated that the selective forces that influence cis-regulatory sequences and promote stabilization of gene expression can also act to stabilize gene expression differences between species (Wang, 2002).

Expression of murine novel zinc finger proteins highly homologous to Drosophila ovo gene product in testis

Two isoforms of cDNAs encoding novel zinc finger proteins have been cloned from mice. One form encodes a 274-amino acid protein containing an acidic amino acid and serine-rich domain and a zinc finger domain which shows high sequence homology to that of Drosophila Ovo protein. The other form encodes a 179-amino acid protein containing only the zinc finger domain. Expression of both proteins possessing an antigenic epitope in COS cells reveals that they are localized in the nucleus. The 1.3-kbp mRNAs are predominantly expressed in testis, and the expression increases from 3 weeks postnatal, implying that these proteins may play important roles in the development of the testes (Masu, 1998).

The ovo gene required for cuticle formation and oogenesis in flies is involved in hair formation and spermatogenesis in mice

movo1, a mouse gene, encodes a nuclear transcription factor that is highly similar to ovo, its fly counterpart, in its zinc-finger sequences. Encompassing roughly half of the encoded 30-kD protein, the zinc-finger domains of mouse Ovo share 73% identity with equivalent segments of Drosophila Ovo/Svb and 94% identity with human Ovo1 (hOvo1). On the basis of this similarity, this sequence are referred to as the mouse Ovo1a (mOvo1a). A second Ovo sequence has also been reported (Masu, 1998). The reported sequence shares 77% identity with mOvo1 within the zinc-finger domains, and 49% overall; its zinc-finger domains share 98% identity with a second human Ovo protein, hOvo2, reported as an expressed sequence tag (EST) in the database, here referred to as mOvo2. The zinc-finger region of mOvo1a is more similar to Drosophila Ovo than mOvo2; however, both mouse Ovo proteins diverge from Drosophila Ovo outside these domains. Curiously, Drosophila Ovo has a large amino-terminal segment not present in these mammalian Ovo proteins (Dai, 1998).

In mice, the gene is expressed in skin (where it localizes to the differentiating cells of epidermis and hair follicles) and in testes (where it is present in spermatocytes and spermatids). Using gene targeting, it has been shown that movo1 is required for proper development of both hair and sperm. movo1(-/-) mice are small, produce aberrant hairs, and display hypogenitalism, with a reduced ability to reproduce. These mice also develop abnormalities of the kidney, where movo1 is also expressed (Dai, 1998).

Scanning electron microscopy reveals structural abnormalities in the hairs of movo1 -/- mice even at early ages A number of hairs displayed kinks and/or intercellular splits within or along the hair shafts. Such splits are predominantly seen in guard hairs, the longest and straightest of the four hair types. Light microscopic examination of at least 50 plucked hairs from each type (~1000 hairs in total) confirms that 16% of the guard hairs exhibit some signs of separation or splitting. Occasional auchene or awl hairs are also defective. The abnormalities in the hairs seemed to arise from a structural weakness or subtle change in the intercellular interactions within the hair shaft. However, at the ultrastructural level, no obvious changes were seen within the cells of the inner root sheath, outer root sheath, cuticle, cortex, or medulla. The restriction of aberrations to late-stage differentiation within the hair shaft is consistent with movo1 gene expression in the precortical cells of wild-type follicles. Precisely how movo1 expression might influence these intercellular interactions in the hair shaft awaits the identification and characterization of the genes regulated by mOvo1 (Dai, 1998).

In situ hybridization reveal that movo1 RNAs in the wild-type kidney localize to the renal tubules of cortex and not to the glomeruli. In movo1 -/- kidneys, defects are detected as early as 6 days postnatally; small cysts appear within the developing cortex. The number and size of these epithelial cysts increases with age. In addition to these histological anomalies in the kidney, focal cystic dilation is sometimes observed in adult kidneys with accompanying signs of atrophy of surrounding renal tubules and glomeruli. Given their late onset, these changes are likely to be secondary consequences either from cyst formation within the kidney and/or from a defective urogenital system. Urogenital defects are particularly striking in female movo1 -/- mice. Approximately 60% of adult female movo1 -/- mice exhibit visibly moist skin in the external area surrounding the urogenital tract. This becomes so severe that in some cases, hair loss occurs in this region. When dissected and examined, these female mutants consistently show a markedly dilated uterus and cervix, with some degeneration of the epithelial lining of the lumen. In addition, the external vaginal opening is often constricted and in a few cases, completely fused. At present, it is not yet known how movo1 might function to control such processes. This said, it is curious that whereas hair defects are more prominent in males, urogenital defects occur predominantly in females. The possible relation between these sex-related alterations in mouse and the sex-related functions of ovo in flies awaits further investigation (Dai, 1998).

In situ hybridizations of wild-type mature testis detects movo1 RNAs in primary and secondary spermatocytes, but not in spermatogonia. These findings are interesting in that (1) Drosophila ovo/svb transcripts occur early, and not late, in male germ-cell development (Oliver, 1994 and Mevel-Ninio, 1995); and (2) movo2 transcripts are expressed in testis only 3 weeks postnatally, that is, at a time when spermatogonia have given rise to spermatocytes. Taken together, these findings suggest an evolutionary difference in transcriptional regulation of the ovo/svb class of genes in the male germ line, and a role for movo expression in late-stage differentiation of male germ cells. Morphological defects in movo1 mutant testes correlate with the timing of movo1 expression in wild-type mice. During the first few weeks postnatally, testes appeared normal in size and morphology. In contrast, by 4 weeks, testes are abnormally small: after correction for the overall reduction in body weight of the movo1 -/- male mice, mutant testes are only 15%-50% the weight of normal testes. Two types of testis-related morphological defects are detected. In the 1-month-old mutant testis, the diameters of seminiferous tubules and the numbers of cells within tubules are atypically small. Because testis size is not affected in younger animals, it is surmised that this reduction is a reflection of a failure of germ-cell maturation and/or survival. Consistent with this notion is a marked reduction in the number of mature spermatids that reached the lumen. Interestingly, a lack of vascularization is seen in the mutant testes, as is also the case for mutant kidneys. Additionally, seminiferous tubules of older mutant mice show signs of degeneration. Differentiating cells from the primary spermatocyte stage onward appear defective. In severely affected testes, only a few spermatogonia survive, and often Sertoli cells are the only remnants of the tubules. Because defects in germ-cell survival are not detected early in postnatal testis development, it is surmised that they developed over time, perhaps as a secondary consequence of defective sperm production. Despite defects in many seminiferous tubules, some produce sperm. Although sperm production is greatly diminished, male movo1 mutant mice are not completely sterile (Dai, 1998).

These findings reveal remarkable parallels between mice and flies in epidermal appendage formation and in germ-cell maturation. Furthermore, they uncover a phenotype similar to that of Bardet-Biedl syndrome, a human disorder that maps to the same locus as human ovo1. Fly larval epidermis is composed of a single layer of cells that secrete a proteinaceous, extracellular cuticle. It also makes appendages such as ventral denticles and dorsal hairs, presumably providing sensory and locomotor functions. Cuticular appendage morphogenesis entails the transient formation of filopodia-like protrusions supported by the epidermal cell's cytoskeleton; following cuticle secretion and hardening, these extensions retract, leaving a hardened, appendage-like structure at the fly's body surface. In contrast, higher vertebrates display a stratified epidermis whose layers differ in proliferative capacity and differentiation status. Within the innermost, basal epidermal layer, a small stem cell population gives rise to transiently dividing cells with a limited proliferative capacity. Periodically, one of these cells withdraws from the cell cycle and commits to terminal differentiation. As the cell moves outward, it changes its program of gene expression to tailor a tough, resilient cytoskeleton. In this way, the protective material shed from the skin surface is cellular rather than secreted. The major mammalian epidermal appendage is the hair follicle, which again is cellular, rather than extracellular. As distinct as epidermal morphogenesis may appear in these evolutionarily distant animals, some parallels still remain. Whether in fly or mouse, the epidermis must provide a protective armor to keep microorganisms out and essential bodily fluids in. In both species, the epidermis provides this function by producing a single-layered epithelium that can execute a differentiative process. Whether secretory or cellular, surface appendages in both species are produced by epidermis late in embryogenesis (Dai, 1998).

Even though the major structural genes of fly (cuticle) and mice (keratin) are not related, several of the genetic pathways that govern the patterning of appendages may be conserved, a notion underscored by the recent discovery that mutations in the human patched gene, first identified as a fly epidermal gene, are responsible for basal-cell carcinomas. Studies on movo1 coupled with prior studies on ovo/svb now suggest that genetic parallels between fly and mouse epidermis go beyond the patterning of their appendages to the genes that are involved in structural aspects of epidermal differentiation and/or appendage formation. Thus, the ovo/svb class of genes encodes proteins with four evolutionarily conserved Cys2-His2 zinc-finger motifs that are involved in regulating cuticle/denticle formation in flies and hair differentiation in mice, both processes that occur late in differentiation. When taken together with evidence that Ovo/Svb are nuclear DNA-binding proteins, it is anticipated that there will be downstream target genes for movo1-encoded proteins that are involved in terminal differentiation in hair, and possibly in epidermis, in which movo1 is also expressed (Dai, 1998).

Ovol2, a mammalian homolog of Drosophila ovo: gene structure, chromosomal mapping, and aberrant expression in blind-sterile mice

The ovo gene family consists of evolutionarily conserved genes including those cloned from Caenorhabditis elegans, Drosophila melanogaster, mouse, and human. Mouse Ovol2 (also known as movol2 or movo2) exists in multiple transcripts. These transcripts are produced by alternative promoter usage and alternative splicing and encode long and short OVOL2 protein isoforms. Mouse and human OVOL2 genes are expressed in overlapping tissues including testis, where Ovol2 expression is developmentally regulated and correlates with the meiotic/postmeiotic stages of spermatogenesis. Mouse Ovol2 maps to chromosome 2 in a region containing blind-sterile (bs), a spontaneous mutation that causes spermatogenic defects and germ cell loss. No mutation has been detected in the coding region of Ovol2 from bs mice, but Ovol2 transcription is dramatically reduced in testes from these mice, suggesting that Ovol2 is expressed in male germ cells (Li, 2002a).

The LEF1/beta-catenin complex activates movo1, a mouse homolog of Drosophila ovo required for epidermal appendage differentiation

Drosophila ovo/svb is required for epidermal cuticle/denticle differentiation and is genetically downstream of the wg signaling pathway. Similarly, a mouse homolog of ovo, movo1, is required for the proper formation of hair, a mammalian epidermal appendage. Evidence is provided that movo1 encodes a nuclear DNA binding protein (mOvo1a) that binds to DNA sequences similar to those bound by Ovo, further supporting the notion that mOvo1a and Ovo are genetically and biochemically homologous proteins. Additionally, the movo1 promoter is shown to be activated by the lymphoid enhancer factor 1 (LEF1)/beta-catenin complex, a transducer of wnt signaling. Collectively, these findings suggest that movo1 is a developmental target of wnt signaling during hair morphogenesis in mice, and that the wg/wnt-ovo link in epidermal appendage regulatory pathways has been conserved between mice and flies (Li, 2002b).

Ovol1 regulates meiotic pachytene progression during spermatogenesis by repressing Id2 expression

A targeted deletion of Ovol1 (previously known as movo1), encoding a member of the Ovo family of zinc-finger transcription factors, leads to germ cell degeneration and defective sperm production in adult mice. To explore the cellular and molecular mechanism of Ovol1 function, the mutant testis phenotype was examined during the first wave of spermatogenesis in juvenile mice. Consistent with the detection of Ovol1 transcripts in pachytene spermatocytes of the meiotic prophase, Ovol1-deficient germ cells are defective in progressing through the pachytene stage. The pachytene arrest is accompanied by an inefficient exit from proliferation, increased apoptosis and an abnormal nuclear localization of the G2-M cell cycle regulator cyclin B1, but is not associated with apparent chromosomal or recombination defects. Transcriptional profiling and northern blot analysis revealed reduced expression of pachytene markers in the mutant, providing molecular evidence that pachytene differentiation is defective. In addition, the expression of Id2 (inhibitor of differentiation 2), a known regulator of spermatogenesis, is upregulated in Ovol1-deficient pachytene spermatocytes and repressed by Ovol1 in reporter assays. Taken together, these studies demonstrate a role for Ovol1 in regulating pachytene progression of male germ cells, and identify Id2 as a Ovol1 target (Li, 2005).

Ovo1 links Wnt signaling with N-cadherin localization during neural crest migration

A fundamental issue in cell biology is how migratory cell behaviors are controlled by dynamically regulated cell adhesion. Vertebrate neural crest (NC) cells rapidly alter cadherin expression and localization at the cell surface during migration. Secreted Wnts induce some of these changes in NC adhesion and also promote specification of NC-derived pigment cells. This study shows that the zebrafish transcription factor Ovo1 is a Wnt target gene that controls migration of pigment precursors by regulating the intracellular movements of N-cadherin (Ncad). Ovo1 genetically interacts with Ncad and its depletion causes Ncad to accumulate inside cells. Ovo1-deficient embryos strongly upregulate factors involved in intracellular trafficking, including several rab GTPases, known to modulate cellular localization of cadherins. Surprisingly, NC cells express high levels of many of these rab genes in the early embryo, chemical inhibitors of Rab functions rescue NC development in Ovo1-deficient embryos and overexpression of a Rab-interacting protein leads to similar defects in NC migration. These results suggest that Ovo proteins link Wnt signaling to intracellular trafficking pathways that localize Ncad in NC cells and allow them to migrate. Similar processes probably occur in other cell types in which Wnt signaling promotes migration (Piloto, 2010).

OVO-like 1 regulates progenitor cell fate in human trophoblast development

Epithelial barrier integrity is dependent on progenitor cells that either divide to replenish themselves or differentiate into a specialized epithelium. This paradigm exists in human placenta, where cytotrophoblast cells either propagate or undergo a unique differentiation program: fusion into an overlying syncytiotrophoblast. Syncytiotrophoblast is the primary barrier regulating the exchange of nutrients and gases between maternal and fetal blood and is the principal site for synthesizing hormones vital for human pregnancy. How trophoblast cells regulate their differentiation into a syncytium is not well understood. This study shows that the transcription factor OVO-like 1 (OVOL1), a homolog of Drosophila ovo, regulates the transition from progenitor to differentiated trophoblast cells. OVOL1 is expressed in human placenta and was robustly induced following stimulation of trophoblast differentiation. Disruption of OVOL1 abrogated cytotrophoblast fusion and inhibited the expression of a broad set of genes required for trophoblast cell fusion and hormonogenesis. OVOL1 was required to suppress genes that maintain cytotrophoblast cells in a progenitor state, including MYC, ID1, TP63, and ASCL2, and bound specifically to regions upstream of each of these genes. These results reveal an important function of OVOL1 as a regulator of trophoblast progenitor cell fate during human trophoblast development (Renaud, 2015).


ovo: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 November 2003  

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