InteractiveFly: GeneBrief

ovo : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | 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 link: Entrez Gene
ovo orthologs: Biolitmine
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
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
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.
Tsai, A., Muthusamy, A. K., Alves, M. R., Lavis, L. D., Singer, R. H., Stern, D. L. and Crocker, J. (2017). Nuclear microenvironments modulate transcription from low-affinity enhancers. Elife 6. PubMed ID: 29095143
Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. This study reports that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.
Preger-Ben Noon, E., Sabaris, G., Ortiz, D. M., Sager, J., Liebowitz, A., Stern, D. L. and Frankel, N. (2018). Comprehensive analysis of a cis-regulatory region reveals pleiotropy in enhancer function. Cell Rep 22(11): 3021-3031. PubMed ID: 29539428
Developmental genes can have complex cis-regulatory regions with multiple enhancers. Early work revealed remarkable modularity of enhancers, whereby distinct DNA regions drive gene expression in defined spatiotemporal domains. Nevertheless, a few reports have shown that enhancers function in multiple developmental stages, implying that enhancers can be pleiotropic. This paper has studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. All seven shavenbaby enhancers drive expression in multiple tissues and developmental stages. This study explores how enhancer pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, revealing site pleiotropy, whereas for a second enhancer, these roles are encoded by distinct sites. Enhancer pleiotropy may be a common feature of cis-regulatory regions of developmental genes, and site pleiotropy may constrain enhancer evolution in some cases.
Kittelmann, S., Buffry, A. D., Franke, F. A., Almudi, I., Yoth, M., Sabaris, G., Couso, J. P., Nunes, M. D. S., Frankel, N., Gomez-Skarmeta, J. L., Pueyo-Marques, J., Arif, S. and McGregor, A. P. (2018). Gene regulatory network architecture in different developmental contexts influences the genetic basis of morphological evolution. PLoS Genet 14(5): e1007375. PubMed ID: 29723190
Convergent phenotypic evolution is often caused by recurrent changes at particular nodes in the underlying gene regulatory networks (GRNs). The genes at such evolutionary 'hotspots' are thought to maximally affect the phenotype with minimal pleiotropic consequences. This has led to the suggestion that if a GRN is understood in sufficient detail, the path of evolution may be predictable. The repeated evolutionary loss of larval trichomes among Drosophila species is caused by the loss of shavenbaby (svb) expression. svb is also required for development of leg trichomes, but the evolutionary gain of trichomes in the 'naked valley' on T2 femurs in Drosophila melanogaster is caused by reduced microRNA-92a (miR-92a) expression rather than changes in svb. The expression and function of components between the larval and leg trichome GRNs were compared to investigate why the genetic basis of trichome pattern evolution differs in these developmental contexts. Key differences were found between the two networks in both the genes employed, and in the regulation and function of common genes. These differences in the GRNs reveal why mutations in svb are unlikely to contribute to leg trichome evolution and how instead miR-92a represents the key evolutionary switch in this context. This work shows that variability in GRNs across different developmental contexts. Therefore, these findings have important implications for understanding the pathways and predictability of evolution.
Ray, S., Rosenberg, M. I., Chanut-Delalande, H., Decaras, A., Schwertner, B., Toubiana, W., Auman, T., Schnellhammer, I., Teuscher, M., Valenti, P., Khila, A., Klingler, M. and Payre, F. (2019). The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning. Elife 8. PubMed ID: 30896406
Small open reading frames (smORFs) encoding 'micropeptides' exhibit remarkable evolutionary complexity. Conserved peptides encoded by mille-pattes (mlpt)/polished rice (pri)/tarsal less (tal) are essential for embryo segmentation in Tribolium but, in Drosophila, function in terminal epidermal differentiation and patterning of adult legs. This study shows that a molecular complex identified in Drosophila epidermal differentiation, comprising Mlpt peptides, ubiquitin-ligase Ubr3 and transcription factor Shavenbaby (Svb), represents an ancient developmental module required for early insect embryo patterning. Loss of segmentation function for this module in flies evolved concomitantly with restriction of Svb expression in early Drosophila embryos. Consistent with this observation, artificially restoring early Svb expression in flies causes segmentation defects that depend on mlpt function, demonstrating enduring potency of an ancestral developmental switch despite evolving embryonic patterning modes. These results highlight the evolutionary plasticity of conserved molecular complexes under the constraints of essential genetic networks.
Tsai, A., Alves, M. R. and Crocker, J. (2019). Multi-enhancer transcriptional hubs confer phenotypic robustness. Elife 8. PubMed ID: 31294690
Previous work has shown in Drosophila melanogaster embryos that low-affinity Ultrabithorax (Ubx)-responsive shavenbaby (svb) enhancers drive expression using localized transcriptional environments and that active svb enhancers on different chromosomes tended to colocalize. This study tested the hypothesis that these multi-enhancer 'hubs' improve phenotypic resilience to stress by buffering against decreases in transcription factor concentrations and transcriptional output. Deleting a redundant enhancer from the svb locus led to reduced trichome numbers in embryos raised at elevated temperatures. Using high-resolution fluorescence microscopy, lower Ubx concentration and transcriptional output were observed in this deletion allele. Transcription sites of the full svb cis-regulatory region inserted into a different chromosome colocalized with the svb locus, increasing Ubx concentration, the transcriptional output of svb, and partially rescuing the phenotype. Thus, multiple enhancers could reinforce a local transcriptional hub to buffer against environmental stresses and genetic perturbations, providing a mechanism for phenotypical robustness.
Fuqua, T., Jordan, J., van Breugel, M. E., Halavatyi, A., Tischer, C., Polidoro, P., Abe, N., Tsai, A., Mann, R. S., Stern, D. L. and Crocker, J. (2020). Dense and pleiotropic regulatory information in a developmental enhancer. Nature 587(7833): 235-239. PubMed ID: 33057197
Changes in gene regulation underlie much of phenotypic evolution. However, understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations. Using an automated robotics pipeline, this study surveyed an unbiased mutation library for a developmental enhancer from the shavenbaby (svb; also known as ovo) gene in Drosophila melanogaster. Almost all mutations were found to alter gene expression, and parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.
Al Hayek, S., Alsawadi, A., Kambris, Z., Boquete, J. P., Bohere, J., Immarigeon, C., Ronsin, B., Plaza, S., Lemaitre, B., Payre, F. and Osman, D. (2020). Steroid-dependent switch of OvoL/Shavenbaby controls self-renewal versus differentiation of intestinal stem cells. Embo j: e104347. PubMed ID: 33372708
Adult stem cells must continuously fine-tune their behavior to regenerate damaged organs and avoid tumors. While several signaling pathways are well known to regulate somatic stem cells, the underlying mechanisms remain largely unexplored. This study demonstrates a cell-intrinsic role for the OvoL family transcription factor, Shavenbaby (Svb), in balancing self-renewal and differentiation of Drosophila intestinal stem cells. svb is a downstream target of Wnt and EGFR pathways, mediating their activity for stem cell survival and proliferation. This requires post-translational processing of Svb into a transcriptional activator, whose upregulation induces tumor-like stem cell hyperproliferation. In contrast, the unprocessed form of Svb acts as a repressor that imposes differentiation into enterocytes, and suppresses tumors induced by altered signaling. The switch between Svb repressor and activator is triggered in response to systemic steroid hormone, which is produced by ovaries. Therefore, the Svb axis allows intrinsic integration of local signaling cues and inter-organ communication to adjust stem cell proliferation versus differentiation, suggesting a broad role of OvoL/Svb in adult and cancer stem cells.
Soverna, A. F., Rodriguez, N. C., Korgaonkar, A., Hasson, E., Stern, D. L. and Frankel, N. (2020). Cis-regulatory variation in the shavenbaby gene underlies intraspecific phenotypic variation, mirroring interspecific divergence in the same trait. Evolution. PubMed ID: 33314059
Despite considerable progress in recent decades in dissecting the genetic causes of natural morphological variation, there is limited understanding of how variation within species ultimately contributes to species differences. Patterning was studyied of the non-sensory hairs, commonly known as "trichomes," on the dorsal cuticle of first-instar larvae of Drosophila. Most Drosophila species produce a dense lawn of dorsal trichomes, but a subset of these trichomes were lost in D. sechellia and D. ezoana due entirely to regulatory evolution of the shavenbaby (svb) gene. This study describes intraspecific variation in dorsal trichome patterns of first-instar larvae of D. virilis that is similar to the trichome pattern variation identified previously between species. A single large effect QTL, which includes svb, explains most of the trichome number difference between two D. virilis strains and that svb expression correlates with the trichome difference between strains. This QTL does not explain the entire difference between strains, implying that additional loci contribute to variation in trichome numbers. Thus, the genetic architecture of intraspecific variation exhibits similarities and differences with interspecific variation that may reflect differences in long-term and short-term evolutionary processes.
Li, X. C., Fuqua, T., van Breugel, M. E. and Crocker, J. (2023). Mutational scans reveal differential evolvability of Drosophila promoters and enhancers. Philos Trans R Soc Lond B Biol Sci 378(1877): 20220054. PubMed ID: 37004721
Rapid enhancer and slow promoter evolution have been demonstrated through comparative genomics. However, it is not clear how this information is encoded genetically and if this can be used to place evolution in a predictive context. Part of the challenge is that our understanding of the potential for regulatory evolution is biased primarily toward natural variation or limited experimental perturbations. To explore the evolutionary capacity of promoter variation, this study surveyed an unbiased mutation library for three promoters in Drosophila melanogaster. It was found that mutations in promoters had limited to no effect on spatial patterns of gene expression. Compared to developmental enhancers, promoters are more robust to mutations and have more access to mutations that can increase gene expression, suggesting that their low activity might be a result of selection. Consistent with these observations, increasing the promoter activity at the endogenous locus of shavenbaby led to increased transcription yet limited phenotypic changes. Taken together, developmental promoters may encode robust transcriptional outputs allowing evolvability through the integration of diverse developmental enhancers. This article is part of the theme issue 'Interdisciplinary approaches to predicting evolutionary biology'.
Benner, L., Muron, S., Oliver, B. (2023). Female germline expression of OVO transcription factor bridges Drosophila generations. bioRxiv, PubMed ID: 37662231
OVO is required for karyotypically female germ cell viability but has no known function in the male germline in Drosophila. ovo is autoregulated by two antagonistic isoforms, OVO-A and OVO-B. All ovo- alleles were created as partial revertants of the antimorphic ovo(D1) allele. Creation of new targeted alleles in an ovo+ background indicated that disrupting the germline-specific exon extension of ovo-B leads to an arrested egg chamber phenotype, rather than germ cell death. RNA-seq analysis, including >1K full length cDNAs, indicates that ovo utilizes a number of unannotated splice variations in the extended exon and a minor population of ovo-B transcripts utilizes an alternative splice. This indicates that classical ovo alleles such as ovoD1rv23, are not truly null for ovo, and are likely to be weak antimorphs. To generate bonafide nulls, the ovo-A and ovo-B promoters were deleted showing that only ovo-B is required for female germ cell viability and there is an early and polyphasic developmental requirement for ovo-B in the female germline. To visualize OVO expression and localization, endogenously tagged ovo was generated, and nuclear OVO was found in all differentiating female germ cells throughout oogenesis in adults. This study also found that OVO is maternally deposited into the embryo, where it showed nuclear localization in newly formed pole cells. Maternal OVO persisted in embryonic germ cells until zygotic OVO expression was detectable, suggesting that there is continuous nuclear OVO expression in the female germline in the transition from one generation to the next.

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).

Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis

To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life remains poorly understood. This study shows that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by the Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells (Bohere, 2018).

The results show that Shavenbaby is expressed and required for the maintenance of adult renal stem cells in flies, supporting the conclusion that the OvoL/Svb family of transcription factors plays a key and evolutionarily-conserved role in the behavior of progenitors/stem cells (Bohere, 2018).

The role of Svb in adult stem cell maintenance in flies requires both a fine control of its expression and of its transcriptional activity. Svb expression in RNSCs involves at least two separable enhancers, driving similar expression patterns. Svb was one of the first cases to reveal the functional importance of apparently redundant (or shadow) enhancers in the phenotypic robustness of regulatory networks across tissues and development stages. The data suggest that a similar cis-regulatory architecture is also underlying the control of adult stem cells (Bohere, 2018).

RNSCs maintenance further requires a proper post-translational maturation of the Svb protein, in response to Pri smORF peptides. During both embryonic and post-embryonic development, the main role of Pri peptides is to provide a temporal control of Svb activity, conveying systemic steroid signaling. It is therefore possible that Pri smORF peptides also connect genetic networks to hormonal control for the regulation of adult stem cells. Recent work has shown that various smORF peptides contribute to the regulation of developmental pathways, muscle formation and physiology, etc., and the current findings extend their influence to the control of adult stem cells. It has been proposed that the emerging field of smORF peptides may open innovative therapeutic strategies, for example peptidomimetic drugs, which might also be of interest for regenerative medicine (Bohere, 2018).

The current results establish that a main function of Svb in adult stem cells is mediated by a functional interplay with the Hippo pathway, well established for its roles in the control of adult stem cells. The results indicate that Svb behaves as a nuclear effector of Hippo, relying on a direct interaction with Yorkie in order to protect stem cells from apoptosis, at least in part through the regulation of DIAP1 expression. Analysis of genome-wide binding events further suggests that the Svb/Yki interaction is involved in the control of a broader set of Hippo-regulated genes, including during development. Since both Pri and Ubr3 are also essential for the survival of adult stem cells, it is interesting to note that Ubr3 protects the DIAP1 protein from degradation, and direct binding of Ubr3 on the activated form of DIAP1 is elicited in the presence of Pri peptides. Therefore, in addition to the control of DIAP1 expression (via Svb), Ubr3 and Pri could also stabilize the DIAP1 protein to protect stem cells from apoptosis. Although initially restricted to TEAD transcription factors, the number of Yorkie (YAP/TAZ) nuclear partners is rapidly growing. Interestingly, recent work has shown a direct interaction of YAP/TAZ with the pro-EMT factors Snail/Slug, in the control of stem cell renewal and differentiation. As previously reported for intestinal stem cells, this study shows that pro-EMT regulators are also required for preventing premature differentiation of renal stem cells. While pro-EMT and OvoL factors are often viewed as antagonistic factors, in vivo studies in Drosophila stem cells show that they both contribute to their maintenance, Svb/Yki preventing their apoptosis and EMT factors their differentiation. Since many studies have implicated the Hippo pathway, pro-EMT and OvoL/Svb factors in various tumors, new insights into their functional interactions in adult stem cells may provide additional knowledge directly relevant to understand their connections in human cancers (Bohere, 2018).


cis-Regulatory Sequences and Functions

ovo sequences (from the start of the ovarian transcript of ovo to 1.9 kb downstream of the start site) are sufficient for germ-line-specific sex-biased ovo transcription. In the germarium, staining for a reporter gene attached to these promoter sequences is seen in stem cells, cystoblasts and young cysts (Oliver, 1994).

The ovo+ and ovarian tumor+ (otu) genes function in the germline sex determination pathway in Drosophila, but the hierarchical relationship between them is unknown. Increased ovo+ copy number results in increased ovarian tumor expression in the female germline and increased ovo expression in the male germline. Males with two or three copies of ovo+ show increased staining activity in the apex of the testis. The zone of expression does not extend into the region of advanced primary spermatocytes, suggesting that the regulation of germline OVO mRNA abundance and the regulation of stage-specific expression are distinct. The correlation between ovo+ copy number and the degree of transactivation of a reporter, strongly suggests that ovo+ is autoregulated in the male germline, either directly or indirectly (Lu, 1998).

The bacterially expressed Ovo zinc-finger domain binds to multiple sites at or near the ovo and ovarian tumor promoters. This strongly suggests that Ovo is directly autoregulatory and that ovarian tumor is a direct downstream target of ovo in the germline sex determination hierarchy. Both positive and negative regulation by Ovo proteins appear likely, depending on promoter context and on the sex of the fly. The most striking observation is the presence in females of protected regions overlapping the ovo-B transcription initiation site, and near the major start sites for the otu promoter. In the case of the ovo-B promoter, the protected region is no more than about 10 base pairs from the three principle start sites and extends about 23 bp downstream of these start sites. Both appear to be high affinity sites based on DNase protection and gel shift assays. There are three binding sites located between the ovo-A and ovo-B transcription start sites. Additional binding sites are found upstream from the ovo-A promoter and downstream of the ovo-B promoter. Two sites between the ovo-A and the ovo-B promoters show high binding activity. Two Ovo-binding sites upstream of the otu promoters are found at positions 370-389 and 401-422 in a region required for otu+ function in vivo (Lu, 1998).

Sequence alignment reveal an 11-bp consensus sequence located centrally within each of the nine binding sites. The strong binding site at the ovo-B promoter has a direct repeat separated by a single G residue. The first A residue in this sequece is the Ovo-B transcripiton start site. The observation that two strong Ovo-binding sites are at the initiator of the TATA-less ovo-B and ovarian tumor promoters raises the possibility that Ovo proteins influence the nucleation of transcriptional pre-initiation complexes (Lu, 1998).

Core promoter sequences contribute to ovo-B regulation in the Drosophila melanogaster germline

Utilization of tightly linked ovo-A vs. ovo-B germline promoters results in the expression of OVO-A and OVO-B, C2H2 transcription factors with different N –termini, and different effects on target gene transcription and on female germline development. Two sex-determination signals, the X chromosome number within the germ cells and a female soma, differentially regulate ovo-B and ovo-A. Ovo regulates ovarian tumor transcription by binding the transcription start site. The regulation of the ovo-B promoter was explored using an extensive series of transgenic reporter gene constructs to delimit cis-regulatory sequences as assayed in wild-type and sex-transformed flies and flies with altered ovo dose. Minimum regulated expression of ovo-B requires a short region flanking the transcription start site, suggesting that the ovo-B core promoter bears regulatory information in addition to a "basal" activity. In support of this idea, the core promoter region binds distinct factors in ovary and testis extracts, but not in soma extracts, suggesting that regulatory complexes form at the start site. This idea is further supported by the evolutionarily conserved organization of Ovo binding sites at or near the start sites of ovo loci in other flies (Bielinska, 2005).

A reasonable understanding exists of the germline pathway centered on ovo. OVO-A and OVO-B functions are in a delicate balance in the female germline. OVO-B is absolutely required for oogenesis and is downregulated by OVO-A. An excess OVO-A results in defective oogenesis and subsequent embryogenesis, while too little results in defective germline function in progeny. Having the female soma repress ovo-A function in the germline may prevent damage to developing eggs, while the positive effect of a 2X karyotype may ensure that OVO-A protein is ultimately deposited in those eggs. OVO-B can have a positive effect on the ovo-B promoter following the deletion of some promoter-proximal sequences, but negative autoregulation occurs in all reporters. This difference between response to OVO-A vs. OVO-B does not appear to be due to different inherent strengths of the two transcription factors, since the otu promoter, a direct target of ovo, is strongly positively regulated by OVO-B in addition to being negatively regulated by OVO-A. Further, this difference in response dose not appear to be due to the ovo-B core promoter sequence, since in the otu sequence milieu, the ovo-B promoter is also strongly positively regulated by OVO-B. Thus, the ovo context is likely to specifically dampen the trans effect of OVO-B, but not OVO-A, on ovo-B promoter activity (Bielinska, 2005).

The ovo-B promoter encodes the OVO-B isoforms required and sufficient for female germline development and is regulated by the number of X chromosomes in the germline cells, and the sex of the surrounding soma positively regulates ovo-B, even though neither signal is absolutely required. For example, only 1X males fail to robustly express ovo-B in the germline, suggesting that both the intrinsic 2X signal and the extrinsic female somatic signal can upregulate ovo-B independently. Also it is known that somatic signaling is not required for ovo genetic function, because 2X males have germline cells, while 2X males lacking ovo do not. This dual regulatory input ensures that ovo-B is most highly expressed in the cells that require ovo activity—wild-type female germ cells. ovo-A expression is more dynamically regulated. The highest ovo-A promoter activity is in 2X males, followed by 2X females, 1X males, and 1X females. This pattern suggests that a 2X karyotype activates ovo-A, while a female soma inhibits ovo-A activity within the germline (Bielinska, 2005).

The combination of negative and positive autoregulation adds considerable complexity to the regulatory circuit. For example, the positive effect of a female soma on the expression of ovo-B in the working model could be due to repression of ovo-A expression by a female soma, followed by derepression of ovo-B because of lowered OVO-A levels, or a more direct positive effect of the female soma on ovo-B (Bielinska, 2005).

Analysis of promoters active in the germline of Drosophila suggests that they are often more compact than many of the promoters studied in somatic cells. This may be the case for ovo-B. While the ovo-B core promoter alone is insufficient for transcription, transcriptional activity from ovo-B is remarkably resistant to deletions from either the 5' or the 3' direction. The lacZDeltaapDelta6 reporter has only 268 bp of ovo sequence but is expressed in the female germline. The overlap between the lacZDeltaapDelta6 and lacZDeltaapDelta8 reporters, both of which are expressed, is only 73 bp. This is unusually close to the transcription start site. The OVO binding site footprints overlap the transcriptional start sites of both otu and ovo-B , and there are proteins or complexes in gonad extracts that bind to this core sequence. It is therefore suggested that OVO alters the structure of the core promoter and promotes preinitiation complex formation. The highly conserved position of OVO binding sites at ovo-B in multiple species of flies supports the idea that OVO functions at the transcription start site. A recent study of human promoters suggests that the binding of transcription factors within 100 bp of the transcription start site may be more common than previously thought (Bielinska, 2005).

The importance of the core promoter raises some interesting questions about how ovo interprets the number of X chromosomes in the germline and the sex of the surrounding soma. For example, the Sex-lethal gene counts X chromosomes in the soma by binding several transcription factors, encoded on the X chromosome, to a region rich in the corresponding binding sites. The balance toward expression of Sxl is thus tipped by a graded occupancy at a complex cis-regulatory module. There does not appear to be an extended cis-regulatory module that is essential for the qualitative expression of ovo. Perhaps sex-determination signals indirectly regulate ovo. The molecular nature of the karyotype and somatic signals to the germline is a major unresolved problem in germline sex determination (Bielinska, 2005).

Regulation of Ovo in the germ line

In Drosophila, compatibility between the sexually differentiated state of the soma and the constitution of the sex chromosome in the germline is required for normal gametogenesis. In this study, important aspects of the soma-germline interactions controlling early oogenesis are defined. In particular, the sex-specific germline activity of the ovarian tumor (otu) promoter has been demonstrated to be dependent on somatic factors controlled by the somatic sex differentiation gene transformer. This regulation defines whether there is sufficient ovarian tumor expression in adult XX germ cells to support oogenesis. In addition, the ovarian tumor function required for female germline differentiation is dependent on the activity of another germline gene, ovo, whose regulation is transformer-independent. These and other data indicate that ovarian tumor plays a central role in coordinating regulatory inputs from the soma (as regulated by transformer) with those from the germline (involving ovo). transformer-dependent interactions influence whether XX germ cells require ovarian tumor or ovo functions to undergo early gametogenic differentiation. These results are incorporated into a model that hypothesizes that the functions of ovarian tumor and ovo are dependent on an early sex determination decision in the XX germline -- a decision that is at least partially controlled by somatic transformer activity (Hinson, 1999 and references).

With respect to interactions with the germline, transformer (tra) is the most extensively studied of the somatic sex regulatory genes. The masculinization of XX soma due to loss-of-function tra mutations causes germ cell aberrations during first instar larval stages and misregulates sex-specific germline gene expression in the embryo. Furthermore, when XY soma is feminized by ectopic tra expression (to form 'pseudofemales', the somatic components of the ovaries are sufficiently 'female' so that they can support the maturation of transplanted XX germ cells. The pseudofemale soma also appears to partially feminize the XY germline, since these cells now require the normally female-specific otu function for optimal proliferation. These observations indicate that tra controls a substantial portion of the somatic-germline interactions affecting early gametogenic differentiation (Hinson, 1999 and references).

In Drosophila, the sexual differentiation of the germline requires a complex interplay between cell autonomous factors controlled by the X:A ratio of the germ cells and sex-specific somatic functions. For example, certain allele combinations of transformer, transformer-2 and doublesex can cause chromosomally female (XX) flies to develop with most of their somatic tissues having a male identity, i.e., ‘XX pseudomales’. In these flies, oogenesis is aborted and there is even occasionally what appears to be early spermatogenic development. Since the germline expressions of these sex regulatory genes are not required for early stages of gametogenesis, the aberrant germline phenotypes must result from the male transformation of the soma (Hinson, 1999 and references).

It is not clear which germline genes are influenced by the proposed somatic interactions. Three possible candidates based on their early and sex-specific roles in female germline differentiation are ovarian tumor, ovo and Sex-lethal (Sxl). During oogenesis, the expression of otu is required in the germline at several stages, if not continually. The null mutant phenotype is characterized by the absence of egg chambers in an otherwise normal ovary, denoted as the quiescent phenotype, although substantial numbers of germ cells are still present in the germarium. Null and severe loss-of-function mutations can also produce 'ovarian tumors', a phenotype characterized by egg chambers containing hundreds of seemingly undifferentiated germ cells. Both the quiescent and tumorous cells are aborted at early oogenic stages, during the cystocyte divisions prior to cyst formation. Mutations in otu have no significant effect on spermatogenesis, although some aberrations in male courtship behavior have been reported. The ovo gene has been implicated in regulating sex determination and dosage compensation in the germline. This is based primarily on observations that ovo null XX germ cells are typically not found in the adult ovary, presumably because of reduced cell viability. In addition, certain ovo allele combinations produce tumorous germ cells that morphologically resemble primary spermatocytes. These phenotypes make ovo a candidate target for a somatic signal regulating early oogenesis, although the expression of ovo in adult germ cells does not appear to be responsive to somatic influences. ovo might directly regulate otu. The Ovo protein can bind to sites in the otu promoter, which displays sensitivity to changes in the dosage of ovo + function. It is not known when this putative regulation of otu occurs nor what role it plays in oogenesis (Hinson, 1999 and references).

The effects of an ovo null mutation on XX germ cells developing in pseudomale testes and female ovaries were examined. In females, ovo mutant XX germ cells typically arrest beginning at larval gonial stages. Occasionally, mutant germ cells survived to the adult stage. However, these cells generally failed to undergo gametogenic differentiation as seen by the absence of spectrosomes, fusomes or ring canals. It was reasoned that, if the requirement for ovo is solely dependent on the X:A ratio, then the phenotype of ovo mutant germ cells in pseudomales should be at least as severe. In this case, the ovo mutant XX pseudomale gonads should be either atrophic or contain a few clusters of mostly undifferentiated germ cells. There is an increase in the frequency of atrophic gonads (82%) compared to normal pseudomales (48%), many of the non-oogenic type. The non-oogenic gonads contained VASA-positive germ cells. This indicates that not only are a substantial fraction of the mutant germ cells viable in adults, but gametogenic differentiation occurs as well. The frequency of the non-oogenic gonads in ovo mutant pseudomales is essentially unchanged from that observed in normal pseudomales. This suggests that the observed increase in the atrophic category is due primarily to the loss of the oogenic class. Mutations in otu gave results similar to those described for ovo. This suggests that otu and ovo mutations specifically disrupt only those germ cells attempting female differentiation, rather than the indiscriminate elimination of the entire XX germline (Hinson, 1999).

Heat shock-otu can alter the XX pseudomale gonadal phenotype; to examine whether and to what degree otu expression could induce oogenic development in pseudomales, immunohistochemical studies were performed. When continually cultured at 20-25°C, hs-otu pseudomale gonads are as much as two to three times longer than normal. In addition, 88% of the hs-otu gonads examined show extensive Hu-li tai shao-labeling of ring canals (Hts is an adducin-like protein). These feminized gonads display a developmental progression of gametogenic stages. In section III of the gonad, the pseudomale germ cells have differentiated to postgermarial stages as defined by the expression of kelch. Kelch, an actin binding protein, is localized to female ring canals after the ring canal deposition of Hts and f-actin . Kelch is first detected in female ring canals in stage 1 egg chambers, but is not seen in all ring canals until stage 4. In hs-otu XX pseudomales, the germ cell clusters in section III contain thick ring canals, with virtually all of them showing Kelch deposition along the inner surface of the f-actin layer. In comparison, no Kelch-labeled ring canals are observed in XX pseudomales without hs-otu, indicating that oogenesis is not only less frequent, but also more limited. Taken together, these results indicate the masculinizing effect of male soma (or the absence of female soma) on XX germ cells can be partially, but consistently, overridden by the expression of otu from a heterologous promoter. The resulting fusome and ring canal development follows the same sequence of events as occurs in normal oogenesis. Therefore, pseudomale germ cells are competent to both initiate and undergo substantial oogenesis if provided with adequate levels of otu. Both ovo and Sxl were shown to be required for otu induced oogenic differentiation in XX pseudomales. However, an additional role for otu in some process affecting germline viability and/or proliferation can be identified that is separable from oogenic differentiation and independent of ovo and, possibly, Sxl functions (Hinson, 1999).

The finding that hs-otu can feminize XX pseudomale germ cells suggests oogenesis is blocked because of insufficient otu levels. Therefore, an examination was carried out to see whether tra-induced sexual transformation affects the level of otu gene expression. otu-lacZ is expressed in most, if not all, larval and pupal germ cells in both female and male gonads. Sex-specific regulation only becomes apparent in the adult testis where male germline expression become restricted to a few cells at the apical tip. As with otu, the ovo promoter is initially active in both male and female larval gonads. However, ovo-lacZ becomes sex-specific at an earlier stage, showing restricted expression in male gonads during the third instar larval and pupal periods. These results demonstrate that the otu and ovo promoters are under different regulatory control in the pre-adult germline. However, otu, but not ovo, promoter activity is influenced by tra-induced sexual transformation. These data demonstrate that the tra-induced sexual transformation specifically inhibits otu promoter activity. Also carried out was the reciprocal experiment, in which otu-lacZ activity was examined in XY germ cells developing in a female somatic background. XY pseudofemales produced by the ectopic expression of tra result in ovaries containing tumorous egg chambers. Because XY pseudofemale germ cells become sufficiently 'feminized' so that they acquire a need for otu function for optimal proliferation, it was anticipated they would also be permissive for otu promoter activity. This is in fact the case. Even in the absence of ovo function, XY pseudofemale germ cells consistently express otu-lacZ. This indicates that the feminizing effects of tra, but not ovo, are necessary for otu transcription. In comparison, the ovo promoter is not detectably active in XY pseudofemales, again illustrating differential regulation of ovo and otu (Hinson, 1999).

It is thought that during the pupal and adult stages, two critical events occur in the female germarium: (1) ovo activity allows XX germ cells to become receptive to the otu function controlling oogenic differentiation, and (2) tra-dependent somatic signals allow continued expression of otu in the female germline by maintaining otu promoter activity. The combination of these events constitutes a mechanism by which the otu gene serves to link the somatic sex differentiation pathway controlled by tra with a female germline developmental pathway controlled by ovo (Hinson, 1999).

Morphological evolution through multiple cis-regulatory mutations at a single gene

One central, and yet unsolved, question in evolutionary biology is the relationship between the genetic variants segregating within species and the causes of morphological differences between species. The classic neo-darwinian view postulates that species differences result from the accumulation of small-effect changes at multiple loci. However, many examples support the possible role of larger abrupt changes in the expression of developmental genes in morphological evolution. Although this evidence might be considered a challenge to a neo-darwinian micromutationist view of evolution, there are currently few examples of the actual genes causing morphological differences between species. This study examined the genetic basis of a trichome pattern difference between Drosophila species, previously shown to result from the evolution of a single gene, shavenbaby (svb), probably through cis-regulatory changes (Sucena, 2000). Three distinct svb enhancers were identified from D. melanogaster driving reporter gene expression in partly overlapping patterns that together recapitulate endogenous svb expression. All three homologous enhancers from D. sechellia drive expression in modified patterns, in a direction consistent with the evolved svb expression pattern. To test the influence of these enhancers on the actual phenotypic difference, interspecific genetic mapping was conducted at a resolution sufficient to recover multiple intragenic recombinants. This functional analysis revealed that independent genetic regions upstream of svb that overlap the three identified enhancers are collectively required to generate the D. sechellia trichome pattern. The results demonstrate that the accumulation of multiple small-effect changes at a single locus underlies the evolution of a morphological difference between species. These data support the view that alleles of large effect that distinguish species may sometimes reflect the accumulation of multiple mutations of small effect at select genes (McGregor, 2008).

Differences in larval trichome pattern between Drosophila species offer an attractive model of morphological evolution. Over the past 30 years, numerous studies have identified upstream patterning and downstream effector genes regulating trichome development in D. melanogaster. Questions about the evolution of trichome patterns can therefore be formulated explicitly within a developmental framework (McGregor, 2008).

Although the pattern of ventral trichomes has been conserved for more than 60 Myr, new dorsal trichome patterns have evolved repeatedly. In most species of the D. melanogaster subgroup, the dorsal and lateral surface displays stout trichomes on 1° and 3° cells and naked 2° cells, and a lawn of fine trichomes on 4° cells. D. sechellia has evolved a trichome pattern in which 4° trichomes were replaced by naked cuticle. Interspecific whole-genome genetic mapping demonstrated that the D. sechellia 'naked' phenotype is recessive to the 'hairy' phenotype of other species and mapped this evolutionary change to a single X-linked gene, shavenbaby/ovo (svb). Svb is required cell-autonomously for trichome formation and encodes a transcription factor regulating several classes of effector genes, which collectively build trichomes (McGregor, 2008).

In D. melanogaster, D. simulans and D. mauritiana, svb is expressed in 1° and 3° dorsal cells and 4° dorsal and lateral cells. In D. sechellia, svb is expressed in 1° and 3° dorsal cells but not in the 4° cells, where trichomes are absent. Together with previous genetic analyses, these expression patterns suggest that changes in the cis-regulatory region of svb underlie this evolved morphological pattern (McGregor, 2008).

This study sought to identify enhancers that drive svb expression. A systematic series of D. melanogaster reporter constructs, from 50 kilobases (kb) upstream to 20 kb downstream of the first exon of svb was used. Reporter expression was precisely mapped by double-staining for Miniature, the product of a cell-autonomous target of svb that accumulates in trichomes (McGregor, 2008).

Three genomic regions were found to drive expression in the epidermis, just before trichome differentiation. Each element contributes to both evolutionarily conserved and evolutionarily derived expression patterns. Dorsal expression of the 'proximal' enhancer started in stage 13 embryos, in 1° and 3° cells. Beginning at stage 15, expression was observed in some dorsal, but not dorsolateral, 4° cell. The 'medial' enhancer drove expression in the dorsal 4° cells at stage 13 and later expanded into dorsolateral 4° cells. The 'distal' enhancer drove expression in thoracic dorsal stripes and lateral 4° cells, starting at stage 14 and strengthening later. In ventral trichome-producing cells, the proximal and medial enhancers drove strong expression and the distal enhancer drove weak expression. The epidermal expression of svb therefore seems to be regulated in a complex manner by three separable cis-regulatory elements spread over 50 kb (McGregor, 2008).

To determine whether these enhancers have evolved in D. sechellia, orthologous D. sechellia regions (which differ by 3%-5% from the D. melanogaster sequences) were identified, and their activity was assayed as transgenes in D. melanogaster. The D. sechellia 'proximal' enhancer drove expression in 1° and 3° dorsal cells in a pattern similar to that of the D. melanogaster 'proximal' enhancer. However, unlike the D. melanogaster enhancer, expression from the D. sechellia proximal enhancer was never observed in dorsal 4° cells. Expression of the D. sechellia 'medial' enhancer was restricted to dorsal 4° cells. In contrast with the D. melanogaster medial enhancer, expression of the D. sechellia enhancer started later and did not extend to the lateral region. The D. sechellia 'distal' enhancer drove expression in thoracic stripes, in a similar manner to the D. melanogaster enhancer, but expression was observed only in restricted lateral spots. At the time of trichome formation, each D. sechellia enhancer drove a ventral expression pattern similar to that of its D. melanogaster counterpart (McGregor, 2008).

These results show that all three svb enhancers have evolved in D. sechellia and that these changes reflect a precise loss of expression in 4° cells. In addition, the D. sechellia medial and distal enhancers retain some activity in 4° cells, indicating that sites outside these regions might be required to repress this activity. Finally, minor changes were observed in the conserved 1° and 3° dorsal cells, and in ventral cells. These results suggest that evolution of the D. sechellia svb expression pattern was caused by multiple changes of limited effect rather than by drastic elimination of entire enhancers (McGregor, 2008).

To test the actual function of these enhancers within their native genomic locations for patterning trichomes, high-resolution interspecific recombination mapping was performed. A two-step screen was designed to maximize the probability of identifying recombinants within the svb gene. First, a screen was performed for recombinants between visible markers that flanked svb by about 1.2×106 base pairs (bp) and then these selected individuals were scored with molecular markers to identify 50 individuals with recombination breakpoints within the svb locus. This experiment provided a resolution of about one recombination breakpoint every 2kb (McGregor, 2008).

Recombinants that included the entire region upstream of the first svb exon from D. mauritiana produced trichome patterns indistinguishable from those of D. mauritiana. Conversely, chromosomes with the upstream svb region from D. sechellia produced a D. sechellia-like trichome pattern. These results demonstrate that the change(s) responsible for evolution of the D. sechellia phenotype are restricted to the genomic region that contains the three identified enhancers (McGregor, 2008).

If the D. sechellia trichome pattern resulted from the evolution of a single site, then only sechellia-like and mauritiana-like phenotypes would have been observed. Instead, three additional phenotypic classes were observed. First, recombinants that included only the proximal enhancer from D. mauritiana produced a few dorsal 4° trichomes (intermediate type 1). Second, a chromosome including the medial and proximal enhancers from D. mauritiana produced a dense pattern of 4° trichomes in the dorsal and dorsolateral region (intermediate type 2). Last, chromosomes that included only the distal enhancer from D. mauritiana produced a patch of dorsolateral and a few dorsal 4° trichomes (intermediate type 3). Backcrossing of all viable recombinant lines further ruled out any detectable influence of genomic regions outside svb on trichome patterns (McGregor, 2008).

These genetic results prove that at least three separate changes have evolved in the svb upstream region to cause trichome loss in D. sechellia. Furthermore, the recombination breakpoints localize functionally evolved sites to genomic positions containing enhancers defined by reporter constructs. The distal svb enhancer element includes CG12680, which has the potential to encode a short peptide. However, this gene is unlikely to contribute to the evolved difference because CG12680 is not expressed in embryos, and complementation assays implicate svb alone as the causal determinant. Finally, the recombinant intermediate phenotypes are similar to the expression patterns of the three individual enhancers. The combined results therefore imply that each enhancer contains at least one genetic change. These changes may have occurred sequentially by loss of expression from the distal, medial and proximal enhancers, or in any other order (McGregor, 2008).

Given that laboratory-induced mutations in dozens of genes alter trichome patterns, it is striking that multiple mutations at a single locus account for the entire evolved difference. Svb seems peculiar in the network of genetic interactions that establish the trichome pattern, because it sits at the nexus of the upstream patterning genes and the downstream effector genes. Although trichome pattern could be changed by altering any of several upstream genes, these changes would probably produce pleiotropic effects on other developmental processes. In contrast, none of the known downstream genes is sufficient on its own to prevent or promote trichome formation. Thus, changes at svb enhancers may provide the only available genetic mechanism to evolve trichome patterns without pleiotropic consequences (McGregor, 2008).

The results provide experimental evidence that the conflicting views of micromutationism and macromutationism can actually reflect observations of the same molecular mechanisms at different levels of resolution. Specifically, genes at integrative positions in developmental networks may be genetic 'hotspots' for evolutionary changes that differentiate species, although the individual mutations contributing to this change may be of smaller effect. Although results recently obtained from a broad range of species are consistent with this interpretation, only additional fine-scale functional analyses of morphological differences between species will allow a robust test of this hypothesis (McGregor, 2008).

Phenotypic robustness conferred by apparently redundant transcriptional enhancers

Genes include cis-regulatory regions that contain transcriptional enhancers. Recent reports have shown that developmental genes often possess multiple discrete enhancer modules that drive transcription in similar spatio-temporal patterns: primary enhancers located near the basal promoter and secondary, or ‘shadow’, enhancers located at more remote positions. It has been proposed that the seemingly redundant activity of primary and secondary enhancers contributes to phenotypic robustness. This hypothesis was tested by generating a deficiency that removes two newly discovered enhancers of shavenbaby (svb, a transcript of the ovo locus), a gene encoding a transcription factor that directs development of Drosophila larval trichomes. At optimal temperatures for embryonic development, this deficiency causes minor defects in trichome patterning. In embryos that develop at both low and high extreme temperatures, however, absence of these secondary enhancers leads to extensive loss of trichomes. These temperature-dependent defects can be rescued by a transgene carrying a secondary enhancer driving transcription of the svb cDNA. Finally, removal of one copy of wingless, a gene required for normal trichome patterning, causes a similar loss of trichomes only in flies lacking the secondary enhancers. These results support the hypothesis that secondary enhancers contribute to phenotypic robustness in the face of environmental and genetic variability (Frankel, 2010).

The cis-regulatory region of the svb gene integrates inputs from multiple gene regulatory networks to generate a complex pattern of expression in the embryonic epidermis of insect species. SVB protein then activates many downstream genes, ultimately resulting in trichome morphogenesis. Three enhancer modules located in a 50 kilobase (kb) region upstream of the svb transcription start site (called 7, E and A) together recapitulate the complete svb epidermal expression pattern. Partial loss of function of all three enhancers led to the evolutionary loss of the long, thin quaternary trichomes on first-instar larvae of Drosophila sechellia, a species that is closely related to Drosophila melanogaster. Evolution of svb expression patterns has probably also contributed to parallel loss of quaternary trichomes in the Drosophila virilis group, species of which are distantly related to D. melanogaster (Frankel, 2010).

It was noticed that a 41 kb region upstream of the three known svb enhancers displays high conservation among drosophilids, but contains only one small gene named SIP3. To test whether this region contained additional svb enhancers, reporter constructs were assayed encompassing the entire region. Two constructs drove expression in the dorso-lateral epidermis in patterns that reproduced part of the native svb expression pattern. To characterize the precise expression domains driven by these newly discovered enhancers, co-immunodetection was performed of the β-galactosidase reporter and of the Dusky-like protein, an early component of developing trichomes (Frankel, 2010).

The Z enhancer drove expression in many cells that produce quaternary trichomes. This expression overlaps the patterns driven by the three enhancers identified previously: 7, E and A. The DG2 enhancer drove expression in a more restricted region that overlaps the domain of expression driven by the E enhancer. Both Z and DG2 drive expression starting at stage 14 of embryogenesis, which is similar to the time when svb mRNA can be detected in epidermal cells (Frankel, 2010).

Given the redundant expression patterns of Z and DG2 with the three enhancers identified previously, further evidence was sought that Z and DG2 encode functional svb enhancers. It was reasoned that if the Z and DG2 enhancers contribute to trichome patterning, then they should have evolved in a similar way to the previously discovered 7, E and A enhancers; they should retain expression in species that also produce quaternary trichomes (such as Drosophila simulans), and show reduced expression in D. sechellia, which has lost quaternary trichomes. Therefore Z and DG2 enhancer constructs made with orthologous regions from D. simulans and D. sechellia were assayed. These regions were straightforward to identify because the genomes of these species are 3-5% divergent from D. melanogaster. The D. simulans Z and DG2 enhancers drove an expression pattern similar to that of the orthologous D. melanogaster enhancers, which indicates that Z and DG2 contribute to the production of quaternary trichomes both in D. melanogaster and in D. simulans. In contrast, the Z and DG2 enhancers from D. sechellia drove low levels of expression in only a few cells. The weak expression driven by the D. sechellia Z and DG2 constructs is consistent with the partial loss of expression driven by the D. sechellia A, E and 7 enhancers and with the loss of quaternary trichomes in this species (Frankel, 2010).

To further assess the functional importance of the Z and DG2 enhancers, a 32 kb chromosomal deficiency was generated on the X chromosome that removes both enhancers, called Df(X)svb108. As a control, strain C108 was used, which carries both of the parental transposable elements that were used to generate the deletion. Df(X)svb108 flies are viable and display no gross abnormalities. First-instar larvae were examined in detail and it was found that, when Df(X)svb108 embryos developed at the optimal temperature for development (25°C), larvae exhibited slightly fewer quaternary trichomes and a reduction in the size of the lateral sensory bristles. These results indicate that, under optimal conditions, Z and DG2 are functional enhancers of the svb gene that contribute to fine details of trichome patterning and perhaps to bristle morphogenesis. Despite this evidence that the Z and DG2 enhancers contribute to svb activity, their loss-of-function phenotype was considerably weaker than one would have expected, given the strong expression driven by these enhancers. It was reasoned that this resulted from the fact that the Z and DG2 enhancers drive overlapping expression with the enhancers 7, E and A, and that the latter three enhancers drive expression levels that are sufficient to generate most larval trichomes when embryos develop under optimal conditions (Frankel, 2010).

Therefore the hypothesis that Z and DG2 contribute to phenotypic robustness was considered. Natural populations experience repeated stresses over evolutionary time, including variable temperatures. Temperature influences membrane fluidity, enzymatic activity, protein folding, protein-protein interactions, and protein-DNA interactions. Organisms have evolved developmental mechanisms to buffer the phenotype in the face of temperature-induced cellular changes. It esd reasoned that sub-optimal temperatures might destabilize the transcriptional output of genes during embryogenesis and that secondary enhancers may confer a selective advantage by maintaining transcription above a required minimum threshold. Therefore the effect of Df(X)svb108 was tested in embryos that had developed at 17 and 32°C, temperatures close to the extremes at which Drosophila embryos survive. The number of quaternary trichomes were counted in the regions where Z and DG2 are expressed strongly. The svb gene is an ideal target for this analysis, because quantitative changes in SVB level influence trichome density, size and shape (Frankel, 2010).

Control embryos reared at all temperatures produced similar numbers of trichomes, implying that the number of trichomes is canalized against temperature variation. The number of trichomes on Df(X)svb108 larvae reared at 25° C was similar to the number on control C108 larvae at all temperatures. In contrast, Df(X)svb108 larvae displayed a highly significant decrease in trichome numbers when reared at extreme temperatures. The primary and tertiary trichomes look normal on Df(X)svb108 larvae at all temperatures, which is expected, because the Z and DG2 enhancers do not drive expression in cells producing primary and tertiary trichomes (Frankel, 2010).

In principle, the loss of trichomes observed on Df(X)svb108 larvae reared at extreme temperatures may have resulted from mechanisms acting independently of the Z and DG2 enhancers. If the effects observed with Df(X)svb108 resulted from loss of the Z and DG2 enhancers, then reintroducing a functional Z or DG2 enhancer into a Df(X)svb108 background should rescue some trichomes. This hypothesis was tested for the Z enhancer. A transgene was generated carrying the svb cDNA under the transcriptional control of the Z enhancer and introduced onto the third chromosome of Df(X)svb108 flies. At extreme temperatures, the Z::svb cDNA transgene completely rescued wild-type trichome numbers in the lateral patch. However, in the region dorsal to the lateral patch, the rescue is very weak or absent. This is consistent with the fact that Z drives expression at high levels in the lateral region, where rescue is observed, and only weakly in a small number of cells of the dorsal region. The loss of canalization in the dorsal region of Df(X)svb108 larvae may be caused by loss of DG2, which drives expression mainly in this dorsal region. These results demonstrate that Z contributes to phenotypic robustness. Moreover, the rescue of trichome numbers by a transgene introduced onto a different chromosome from the svb locus indicates that Z does not need to be in intimate contact with other svb enhancers or with the svb basal promoter to buffer svb function. Instead, it is propose that Z contributes to phenotypic robustness simply by boosting levels of svb transcription in the cells in which Z drives expression (Frankel, 2010).

These results indicate that the production of larval trichomes is normally canalized and that this is accomplished, at least in part, through transcriptional activation mediated by the svb secondary enhancers that are removed in Df(X)svb108 (Frankel, 2010).

The svb locus contains multiple enhancers with overlapping expression patterns. Similar patterns of overlapping enhancer activity have been found for the cis-regulatory regions of the Drosophila genes sog and for the cis-regulatory regions of the mouse genes sonic hedgehog. A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers. Moreover, it has been estimated that 50% of the target genes of the transcription factor Dorsal contain shadow enhancers. Therefore, the presence of additional enhancers in cis-regulatory regions may be a common signature of developmental regulators. This may explain why, in previous reports, animals carrying deletions of highly conserved enhancers have not displayed observable phenotypic defects when reared in standard laboratory conditions (Frankel, 2010).

Developmental buffering is likely to result from many molecular mechanisms. For example, deletion of the conserved miRNA miR7 in D. melanogaster has no obvious phenotypic effect in normal laboratory conditions, but it is required to canalize the expression of the gene atonal under fluctuating temperatures. Similarly, the results indicate that svb secondary enhancers have a minimal role at optimal conditions for development, but that they are essential to buffer the trichome phenotype under genetic or environmental variability. Secondary enhancers are likely to be evolutionarily maintained by selection for robustness against temperature fluctuation, genetic background effects and expression noise (Frankel, 2010).

Low affinity binding site clusters confer hox specificity and regulatory robustness

In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences.This study demonstrates that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) binds specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression (Crocker, 2015).

This study has demonstrated that the Hox protein Ubx regulates separate enhancers of the svb gene by binding, with its cofactors Exd and Hth, to clusters of low affinity binding sites. Combining in vitro and in vivo assays, experimental demonstration is provided of an affinity-specificity tradeoff for Hox proteins, such that enhancers that integrate Hox inputs to drive regionalized expression are unlikely to utilize high affinity Hox binding sites. Forced to utilize low affinity sites, enhancers have evolved to contain multiple binding sites to ensure regulatory robustness to genetic and environmental variations. Most individual Ubx-Exd sites have evolved rapidly, but evolution has conserved overall enhancer architecture, with clusters of low affinity sites (Crocker, 2015).

Homotypic clusters of transcription factor binding sites are pervasive in animal genomes and several models have been proposed to explain their existence. The current results provide experimental evidence that homotypic clusters of Hox binding sites can confer robustness to enhancers. This may reflect a more widespread phenomenon. Although many enhancers contain homotypic clusters with low affinity sites, previous studies have rarely detected changes in expression by deleting individual binding sites. However, these mutated enhancers have not been tested in variable environments. It is possible that many of these clustered sites confer regulatory robustness (Crocker, 2015).

It is useful to compare these results with previous studies that have demonstrated specific regulatory functions for homotypic clusters. For example, clustered binding sites in an enhancer of the Drosophila hunchback gene mediate cooperative DNA binding by Bicoid, which provides threshold-dependent enhancer activity. In other cases, clusters of homotypic binding sites act in a noncooperative manner to allow enhancers to respond in a graded fashion, for example to determine expression levels in response to transcription factor concentrations. It is worth noting that in these cases, where homotypic clusters mediate specific linear or nonlinear outputs, enhancers are bound by transcription factors that belong to small paralogous families: e.g., two paralogs for Msn2; three for p53; two for Dorsal; and five for NFκB. In contrast, there are 84 homeodomain-containing proteins encoded in the Drosophila genome, many with overlapping specificities. Therefore, in previously described examples of homotypic clusters, binding affinity may not be a strong constraint on specificity (Crocker, 2015).

For the Hox regulated svb enhancers, low affinity Ubx/AbdA-Exd binding sites enable specificity, while the clustering of low affinity sites confers phenotypic robustness. This is a fundamentally different constraint on clustered binding sites than observed in all previous examples. The affinity-specificity tradeoff, initially supported by computational analysis of in vitro data, was confirmed in vivo by progressively increasing the affinity of the Ubx-Exd binding sites. While replacement of low affinity sites with higher affinity sites always quantitatively altered enhancer activity, either positively or negatively, most higher affinity sites generated strong ectopic expression. This ectopic expression is driven, at least in part, by gaining the binding of additional Hox proteins, which are normally not involved in the regulation of these enhancers. Other studies have performed replacement of low affinity sites with higher affinity sites and, in some cases, they have also observed ectopic expression. These altered patterns of expression may reflect increased sensitivity of enhancers to the same transcription factor that binds to the wild-type enhancer. This study observed a similar effect for Ubx and AbdA-dependent upregulation of svb enhancers in the cells in which they are normally expressed. In addition, however, it was found that sites with higher affinity resulted in a reduced specificity, due to the binding of additional homeodomain proteins, such as Scr, to svb enhancers. Computational analyses suggest that this affinity-specificity tradeoff is a fundamental property of Hox proteins and would therefore influence the architecture of enhancers that must generate specific outputs in response to Hox factors. It is suggested that transcription factors that belong to other large paralog groups may exhibit a similar affinity-specificity tradeoff and that enhancers regulated by these factors may also exploit clusters of low affinity sites (Crocker, 2015).

The results help to explain previous difficulties with bioinformatic prediction of functional Hox binding sites, because low affinity sites are difficult to detect reliably. Indeed, the low affinity sites that implement Hox regulation within svb enhancers share little similarity with canonical Hox or Hox-Exd binding sites. Consequently, a very large number of seemingly disparate DNA sequences can confer low affinity binding for Hox proteins. If Hox-Exd sites are often clustered in the genome, then signals from genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) will reflect binding to the entire cluster (as was observed) and the signals associated with individual low affinity sites may be difficult to discern from noise. Identification of important low affinity sites will require a change in computational approaches to analyzing genome-wide data. Currently, it is de rigueur to apply an arbitrary threshold to genome-wide data and then to analyze only signals above this threshold. This approach is likely to bias detection toward high affinity sites, whose functions may be distinct from those of clusters of low affinity sites (Crocker, 2015).

These findings provide insight into how different Hox proteins regulate specific target genes to generate phenotypic diversity across the anterior-posterior axis. One unanswered question is how the many low affinity DNA sequences, which appear to share little in common, are bound by the same Hox-Exd complex with apparently similar affinity. It is possible that variations in DNA shape (deviations from the structure of canonical B-DNA) influence Hox-Exd binding to low affinity sites. It remains unclear if very different sequences can adopt similar shapes, or whether instead the Hox-Exd complex can recognize a range of shapes. Resolution of this question will require structural studies of Hox-Exd complexes bound to a range of low affinity DNA sequences and quantitative analysis of their binding dynamics in vivo (Crocker, 2015).

Targets of Activity

The Drosophila ovo locus codes for several tissue- and stage-specific proteins that all possess a common C-terminal array of four C2H2 zinc fingers. Three fingers conform to the motif framework and are evolutionarily conserved; the fourth diverges considerably. The ovo genetic function affects germ cell viability, sex identity and oogenesis, while the overlapping svb function is a key selector for epidermal structures under the control of wnt and EGF receptor signaling. Synthetic DNA oligomers bound by the OVO zinc finger array have been isolated from a highly complexity starting population, and a statistically significant 9 bp long DNA consensus sequence has been derived that is nearly identical to a consensus derived from several Drosophila genes known or suspected of being regulated by the ovo function in vivo. The DNA consensus recognized by Drosophila Ovo protein is atypical for zinc finger proteins in that it does not conform to many of the 'rules' for the interaction of amino acid contact residues and DNA bases. Additionally, these results suggest that only three of the Ovo zinc fingers contribute to DNA-binding specificity (Lee, 2000).

The function of four ovarian tumor genes (ovarian tumor, ovo, fused and snf) are required for the female-specific splicing of SXL pre-messenger RNA. A further ordering of the sex determination genes that function within the germ line can be inferred from the difference between the phenotypes produced by Sxl and snf mutations, versus otu and ovo mutations. Mutations of either otu or ovo result in the absence of 2X germ-line cells and sexual transformations of 2X germ-line cells. While Sxl or snf also result in sexual transformations, they do not have any effect on germ cell viability. A branched pathway provides the simple explanation for the differences found between these two groups of genes. Genes that function prior to the branch would be required for both germ cell viability and sex determation, whereas sex determination genes functioning after viability affects would be required only for sex determination roles (Oliver, 1993).

Three Ovo-binding sites exist in a compact regulatory region that controls germline expression of the otu gene. Interestingly, the strongest Ovo-binding site is very near the otu transcription start, where basal transcriptional complexes must function. Loss-of-function, gain-of-function and promoter swapping constructs demonstrate that Ovo binding near the transcription start site is required for Ovo-dependent otu transcription in vivo. These data unambiguously identify otu as a direct Ovo target gene and raise the tantalizing possibility that an Ovo site, at the location normally occupied by basal components, functions as part of a specialized core promoter (Lu, 2001). Increased ovo+ dose results in increased ovo mRNA and genetic activity. This important control means that an increased ovo+ copy number translates into increased functional Ovo protein. In those flies with increased Ovo activity, endogenous otu transcripts were present in greater quantity than wild type. Transgenes driven by the otu promoter respond positively to increased Ovo activity. This response is not limited to late stages. otu reporters respond to increased Ovo activity in larval gonads and in the stem cells and cystocytes of the adult ovary. Cells expressing otu reporters also express ovo reporters, suggesting that Ovo is at the scene of otu promoter activity. These data suggest that Ovo controls otu expression in early stages of oogenesis (Lu, 2001).

An extensive set of transgenes have been prepared with deleted and reconstituted Ovo-binding sites, which were tested in females with differing doses of ovo+ . Removal of Ovo-binding sites reduces or eliminates the response to ovo+ activity in trans, while reconstituting Ovo-binding sites confers activity. These data indicate that Ovo protein directly regulates otu transcription (Lu, 2001).

Surprisingly, Ovo functions very close to the transcription start site of otu. Ovo footprints within 20 bp of the transcription start sites of all but one of the reporter genes that respond to ovo dose in trans. Indeed, in the case of the ovo-B promoter, the transcription start site is in the middle of the region protected by Ovo. It is a reasonable assumption that RNA Polymerase II and basal transcription complex components also bind this region. For example, the TFIID complex protects about 60 bp, centered on the core promoter. Certainly, RNA Polymerase II must contact the +1 position in the ovo-B promoter that is covered by Ovo protein in vitro (Lu, 2001).

A standard model for transcriptional regulation holds that the binding of regulatory factors at control regions modulates the transcriptional activity of a variety of core promoters. In this model, core promoters (where the start site is +1 and the core promoter is from -35 to +35) can have different basal strengths, but they have little regulatory information. While in many cases different core promoters respond similarly to a given enhancer, there is some evidence supporting the idea that core promoters can bear important regulatory information. These data suggest that the ovo-B and otu core promoters have a regulatory function. Several possible mechanistic explanations for the promoter proximal binding of Ovo to these core promoters has been explored (Lu, 2001).

Binding of a regulatory protein to the transcription start site is unusual. There are only a few core-promoter binding proteins, such as AEF1 and YY1, that function in tissue or promoter-specific transcriptional control. Binding a short distance away from the transcription start is more common. Start sites are not often mapped to the base. Thus, a trivial explanation of the effect of promoter proximal binding of Ovo is that it binds near the start sites, but not at them. This is unlikely. For example, two groups have mapped the ovo-B transcription start site to the same location. Full-length cDNAs, showing evidence of 5' caps, also end at this site. The sequenced RACE product from the otu::lacZ swb transgene ends precisely at the same site. Similarly, the otu transcription start sites have been mapped by primer extension and by RACE. The otu start sites in reporter genes are within 20 bp upstream of the Ovo footprints, well within the region expected to bind basal factors. Thus, Ovo and basal transcription factors occupy the same region of the otu core promoter, concurrently or in series (Lu, 2001).

The concurrent occupancy model for Ovo function at the core promoter places Ovo in the basal transcriptional apparatus. Core promoters typically have binding sites for basal factors at characteristic locations. The best-studied site is the TATA element at about -30 to -25, but about half of Drosophila genes are TATA-less. In addition, Initiator elements (Inr) at the transcription start site, and downstream promoter elements (DPE) at about +28 to +34 have been described. The proteins that bind core promoter sites are components of the enormous pre-initiation complex, TFIID, which protects the entire 60 bp core promoter region. The combinatorial binding of TFIID components to characteristically spaced sequence elements provides enhanced specificity and binding strength. Ovo could function as a tissue-specific core element to augment TFIID binding, but this seems unlikely for three reasons. (1) The Ovo-binding site is slightly downstream of the otu start site, but overlaps the ovo-B initiation site. A more constrained position relative to the start site would be expected. (2) The promoter proximal Ovo binding sites at otu and ovo-B are in opposite orientation. Transcription is certainly directional. If Ovo serves to orient the complex at the transcription start site in a manner analogous to TATA, Inr and DPE elements, then directionality would be expected. To account for function in each orientation, Ovo would need a flexible domain between the DNA binding and complex contact domains, or a highly symmetrical structure outside the DNA-binding domain. (3) Tests were performed for dose-dependent genetic interactions between ovoD and mutations in the Drosophila TBP associated factors (TAFs) that are components of TFIID. Mutations in any of several TAFs fail to interact with ovoD. This is a circumstantial argument against an intimate relationship between Ovo and TFIID (Lu, 2001).

If Ovo and basal factors occupy the otu core promoter serially, orientation and spacing issues are less important. Ovo binding might alter the structure of the core promoter to make it more accessible to transcription initiation complexes. There is precedent for preconditioning a core promoter. For example, a bent configuration can enhance the binding of TBP to the TATA element. Similarly, RNA Polymerase II can initiate from a melted or negatively supercoiled core promoter in the absence of the normal stable of transcription factors. Thus, Ovo could precondition the core promoter to allow stronger and/or more precise subsequent binding by the transcriptional apparatus, by generating or stabilizing bends or single stranded regions (Lu, 2001).

Indeed, retrotransposon targeting suggests that the ovo-B promoter has an unusual structure. The ovo-B promoter region, and the Ovo-binding sites in particular, are preferred targets for de-novo gypsy-transposon insertion. Transposable element targeting is believed to be sensitive to chromatin structure in many systems. It is thus possible that Ovo binding makes the chromatin especially available for gypsy insertion. Such accessibility could also promote the entry of transcriptional machinery. Finally, the presence of bound Ovo might even circumvent the need for TFIID. The YY-1 protein, also a C2H2-zinc-finger protein that binds core promoters, binds double-stranded DNA and a single-stranded bubble in the direction of transcription. YY-1 binding and RNA Polymerase II, but not TFIID, are sufficient for transcription from those core promoters in vitro. In summary, while there is no mechanistic understanding of Ovo function at the core promoter, it seems likely that Ovo and components of the machinery performing the work of transcription bind to the same sequence, but not at the same time (Lu, 2001).

Insulator and Ovo proteins determine the frequency and specificity of insertion of the gypsy retrotransposon in Drosophila melanogaster

The gypsy retrovirus of Drosophila is quite unique among retroviruses in that it shows a strong preference for integration into specific sites in the genome. In particular, gypsy integrates with a frequency of >10% into the regulatory region of the ovo gene. In vivo transgenic assays were used to dissect the role of Ovo proteins and the gypsy insulator during the process of gypsy site-specific integration. DNA containing binding sites for the Ovo protein are required to promote site-specific gypsy integration into the regulatory region of the ovo gene. Using a synthetic sequence, it was found that Ovo binding sites alone are also sufficient to promote gypsy site-specific integration into transgenes. These results indicate that Ovo proteins can determine the specificity of gypsy insertion. In addition, interactions between a gypsy provirus and the gypsy preintegration complex may also participate in the process leading to the selection of gypsy integration sites. Finally, the results suggest that the relative orientation of two integrated gypsy sequences has an important role in the enhancer-blocking activity of the gypsy insulator (Labrador, 2008).

Retroviral DNA integration into the host genome is an essential step for production and replication of viral RNA. It has been traditionally difficult to study the factors controlling selection of integration sites, since most retroviruses integrate throughout the genome with no apparent DNA sequence specificity. However, detailed analysis of multiple genomic integration sites in vivo has revealed that retroviruses have a strong preference for certain genomic regions. In particular, retroviruses integrate preferentially into actively transcribed DNA, which will thereafter facilitate transcription of the provirus. The distribution of retroviral integration sites along chromosomes suggests that open chromatin favors retroviral insertion, since integration events are favored in transcriptionally active chromatin and are rare in DNA sequences associated with heterochromatin. However, chromatin state or DNA accessibility could not be the only factor influencing integration, since different retroviruses manifest preferences for integration that are unlikely to be only the result of chromatin organization. For example, both HIV and murine leukemia virus (MLV) integrate in actively transcribing DNA, but HIV integrates with equal frequency throughout all transcribed DNA, whereas MLV integrates preferentially into transcription start sites (Labrador, 2008).

The mechanism of integration of retrotransposons is fundamentally identical to that of retroviruses. However, constraints imposed by small genome sizes have led some retrotransposons to the acquisition of mechanisms for site-specific integration. The best examples of site-specific integration are found in non-LTR retrotransposons such as the Drosophila telomeric elements HeT-A, TART, and TAHRE or rDNA elements such as the Drosophila R1 and R2 non-LTR retrotransposons. A number of examples involving integrases from yeast LTR retrotransposons have also shown that retroviral-like integrases have evolved to acquire strong site-specific integration properties. For example, the integration of Saccharomyces cerevisiae retrotransposons Ty1 and Ty3 is associated with RNA polymerase III transcription and the Tf1 retrotransposon from Schizosaccharomyces pombe integrates near RNA polymerase II promoters. In other examples, transposable elements are targeted to heterochromatic sites by tethering mechanisms involving interactions between the integrase and DNA binding proteins. Targeting of the Ty5 retrotransposon from S. cerevisiae to heterochromatin, for example, requires a six-amino-acid motif at the C terminus of the Ty5 integrase that interacts with the heterochromatin protein Sir4. It has also been proposed that the chromodomain (CHD domain) from certain transposable element chromointegrases targets the retrotransposon for insertion into sites bearing the specific epigenetic marks recognized by the CHD domain (Labrador, 2008).

In contrast to mammalian retroviruses, Drosophila retroviruses such as gypsy, ZAM, or Idefix, display a high rate of site-specific integration into certain regions of the genome. The mechanisms governing this specificity however are poorly understood, but the genetic tools available in Drosophila provide a unique opportunity to analyze retroviral site integration specificity in higher eukaryotes. In particular, gypsy insertions into the ovo locus occur in the germ line of ~10% of the female offspring from mothers carrying permissive mutations in the flamenco (flam) locus (Prud'homme, 1995; Dej, 1998). Gypsy integrations take place specifically into a sequence of ~1.3 kb spanning the 5' regulatory region of the ovo gene (Dej, 1998). The flam locus is located in the heterochromatin of the X chromosome (Prud'homme, 1995) and produces a long noncoding RNA that controls transcription of the gypsy retrovirus through the piwiRNA pathway (Labrador, 2008).

The process of gypsy transposition is maternally regulated, involving maternally inherited gypsy particles that originate in the developing oocyte of flam mutant females. These females fail to produce the flam RNA, allowing the transcription of euchromatic gypsy elements in the follicle cells surrounding the oocyte during oogenesis. Transcription of gypsy in follicle cells leads to the formation of virus particles that infect the oocyte and subsequently participate in the integration of gypsy in the germ line of the resulting embryo after fertilization (Song, 1994: Song, 1997). These integration events take place preferentially in the ovo gene, whose product is necessary for the development of the female germ line and the normal progression of oogenesis. The ovo gene encodes two isoform proteins, Ovo-A and Ovo-B, which have a common DNA-binding domain but different N-terminal domains. Ovo-B positively regulates the ovo promoter, whereas Ovo-A functions as a negative regulator of the ovo promoter. Adult females homozygous for a null mutation of the ovo gene do not develop germ line cells. The ovoD1 allele is caused by a point mutation that creates a new in-frame methionine codon in the 5' region of ovo, adding an extra amino terminus domain to Ovo-B that is normally only present in the wild-type Ovo-A protein. The ovoD1 allele is dominant negative and causes female sterility even when heterozygous. The sterility is due to the expression of OvoD1B protein, which is made at the same time of development as Ovo-B but has the repressor activity of Ovo-A; the presence of OvoD1B is sufficient to arrest oogenesis at stage 4 (Labrador, 2008).

Insertion of gypsy into the ovoD1 allele in a heterozygous female reverts to fertility by preventing the expression of the OvoD1B protein, although the reversion occurs only in those germ cells in which gypsy is inserted into the ovoD1 sequence. The ability of gypsy to integrate specifically into ovo sequences was analyzed by Dej (1998). These studies concluded that gypsy integrates in at least seven different target sites localized within a 200-bp sequence present in the promoter region of the ovo gene. Close analysis of these sites reveals a very relaxed consensus sequence consisting of six alternating pyrimidines and purines. The weak conservation of the observed target sequence suggests that gypsy site-specific integration is not due to a direct interaction of the gypsy integrase with these sequences. Instead, it has been proposed that Ovo proteins may mediate gypsy insertion specificity by promoting protein-protein interactions between Ovo or an associated protein and the gypsy preintegration complex (Labrador, 2001; Labrador, 2008 and references therein).

The gypsy retrovirus of Drosophila also exhibits the interesting property of blocking enhancers from activating promoters when gypsy is inserted between them. This property is referred to as insulator activity and resides in the Suppressor of Hairy wing [Su(Hw)] binding sites present in a 350-bp sequence located in the 5'-UTR of the gypsy retroviral genome. In addition, gypsy insulators are also able to buffer transgenes from position effects by preventing heterochromatin from spreading through the chromatin fiber. There is mounting evidence suggesting that gypsy insulators function by creating chromatin domains most probably defined by the interaction between adjacent insulator sites in chromosomes. Molecular evidence for such interactions has been obtained by measuring the distance between adjacent gypsy insertions in wild-type and in su(Hw) mutant cells (Gerasimova, 2000). These experiments revealed that the two gypsy sequences were significantly closer during interphase when the Su(Hw) protein was present. It has been proposed that such interactions might create chromatin domains by looping out the DNA contained between two interacting insulators. Additional evidence in support of this model has been provided by showing the presence of DNA loops attached at their base to the nuclear matrix by the gypsy insulator in the nucleus of Drosophila imaginal disc cells (Byrd; 2003). Interaction between gypsy insulators is also supported by data showing that two adjacent insulators were able to cancel each other, no longer exerting their enhancer blocking effect when located between the enhancer and the promoter of a reporter gene (Cai, 2001; Muravyova, 2001; Kuhn, 2003; Labrador, 2008 and references therein).

The molecular basis for interactions between individual insulators is not well understood but it has been suggested that Modifier of mdg4 [Mod(mdg4)] and CP190, both components of the insulator complex, might facilitate such interactions by mediating protein-protein contacts between the BTB domains present in the two proteins. These two properties of gypsy, site-specific integration and insulator activity, have been the subject of intense but unrelated studies during the past two decades. This study attempts to analyze these two properties simultaneously in an effort to understand how insulators might mediate genome organization and how this organization may influence retroviral selection of integration sites through the genome. An assay has been developed to show that the 5' regulatory region of ovo is able to recruit gypsy insertions independently of its position in the genome (Labrador, 2001). Genetic evidence has been provided suggesting that the Ovo protein is directly implicated in such recruitment. This study takes advantage of the ability of inducing two consecutive gypsy insertions into a yellow reporter gene to analyze the role that interactions between a gypsy provirus and the gypsy preintegration complex may play in the selection of retrovirus integration sites and the effect of the relative orientation of interacting proviruses on the enhancer-blocking activity of the gypsy retrovirus (Labrador, 2008).

The gypsy retrovirus of Drosophila may offer valuable clues as to how retroviruses develop strategies to specifically select integration sites into the genome. Results shown in this study suggest that interactions between the gypsy preintegration complex and, most likely, Ovo proteins are sufficient to promote site-specific integration of gypsy into the ovo locus of Drosophila. Alteration of Ovo binding sites from a wild-type ovo gene fragment abolishes the ability of gypsy to specifically integrate into adjacent sequences. In addition, a synthetic DNA sequence carrying eight Ovo binding sites flanked by random DNA sequences is sufficient to function as a highly specific target for integration of the gypsy retrovirus. Although direct interactions between Ovo proteins and the gypsy preintegration complex have not been substantiated, the data point to a mechanism by which Ovo proteins may tether the gypsy preintegration complexes to their binding sites (Labrador, 2008).

It is tempting to speculate that the gypsy integrase may actually interact with the Ovo proteins and that such interaction may target integration to genome sites enriched in Ovo. Evidence demonstrating tethering of integrases as a mechanism capable of targeting retroviruses to specific DNA binding sites has been shown in experiments using fusion proteins in which the DNA binding domain of phage lambda-repressor was fused to the integrase of the HIV retrovirus and successfully showed preferential integration into target DNA near lambda-repressor-binding sites. Similarly, experiments with yeast retrotransposons have shown that such interactions may occur between the retrotransposon integrase and proteins that target the integration to their cognate chromosomal DNA binding sites (Labrador, 2008).

It has been reasoned that retrotransposons in small genomes such as that of yeast may develop tethering mechanisms of site-specific integration by stimulating interactions between the integrase encoded by the retrotransposon and endogenous proteins, thus minimizing the chances of deleterious mutations induced by retrotransposon integration events. In larger genomes such mechanisms appear infrequently, probably due to the lack of selective pressure from the host genome. In humans for example, sequences related to interspersed retroviruses occupy >50% of the genome and only relatively low frequencies of integration events in specific target spots have been reported. In Drosophila only a number of specialized non-LTR retrotransposons have acquired specificity of integration associated with specialized chromosomal regions such as telomeres or ribosomal DNA. Nonvertebrate retroviruses such as ZAM, Idefix, and gypsy appear to be an exception when compared with their vertebrate counterparts, since some degree of sequence specificity and targeted site integration has been described in all three. The high rate of insertion of gypsy into the ovo gene and the role apparently played by the Ovo proteins provide an excellent tool to study the integration mechanism and how retroviruses may acquire integration site specificity in vivo (Labrador, 2008).

Interestingly, even though Ovo proteins appear to have a role in targeting gypsy to their binding sites, gypsy insertion sites do not necessarily occur into the Ovo binding sequences themselves. From a total of 85 sequenced insertion sites, only 13 (15%) occurred into the DNA fragment containing Ovo binding sites; the remaining integration sites fall within an interval of >1300 bp flanking the DNA containing the Ovo binding sites. The analysis of insertion sites suggests that the targeting and the integration mechanisms are uncoupled, with the precise integration sites distributed in a nonrandom manner. Results shown in this study confirm previous observations suggesting that gypsy has a preference for integration into YRYRYR sequences. However, a variety of other sequences appear to be able to function as integration sites. This disparity makes it difficult to draw a clear conclusion as to what is the mechanism ultimately involved in selecting target sites; however, it is tempting to speculate a role for nucleosome positioning as one of the factors determining the selection of insertion sites by the gypsy retrovirus (Labrador, 2008).

Several indirect lines of evidence suggest such a role. For example, integration frequencies are significantly higher between the promoter of the yellow gene and the Ovo binding sites, indicating a preference that probably reflects a difference in chromatin structure. Forty-two independent integration events occurred into a fragment of 441 bp located between Ovo binding sequences and the promoter of the yellow gene, whereas only 30 integrations occurred in a 869-bp DNA fragment located distal to the promoter and upstream of the Ovo binding sites. This asymmetry does not appear to reflect sequence differences or viability effects, suggesting an epigenetic basis for integration site selection. In addition, insertion sites found distally to the yellow promoter and upstream of the Ovo binding DNA sequences appear to be spatially distributed in four intervals >140 bp, whereas insertions proximal to the promoter are distributed in a random manner, with only one large gap of 145 bp and apparently lacking meaningful spacing intervals. A possible interpretation of these results is that gypsy integration preferentially occurs at specific points of either the nucleosome or the linker DNA. In the distal interval, one spacing >140 bp could reflect nucleosome positioning, whereas nucleosomes may be absent or not positioned in the promoter proximal region of the transgene (Labrador, 2008).

Results presented in this study also suggest that a preexisting gypsy insertion significantly increases the chances of new gypsy insertions into adjacent sequences by more than twofold. It is speculated that such enhancement of insertion frequency might be the result of a tethering mechanism mediated by protein-protein interactions between the gypsy element located in the chromosomal DNA and the gypsy preintegration complex during the normal process by which a new copy of the retrovirus is inserted into the chromosome. Since two copies of the gypsy insulator have been shown to be able to interact with each other, it is tempting to speculate that interactions between gypsy insulators are responsible for the increased frequency of gypsy insertions. Nevertheless, the possibility that gypsy sequences other than the insulator or proteins associated with the gypsy element itself are responsible for the observed interactions cannot be ruled out. If the high frequency of secondary insertions is due to interactions between gypsy insulator proteins present in the provirus and in the preintegration complex, the results would lend support to proposed models suggesting that individual insulators located in different regions of a chromosome can interact to form chromatin loops (Labrador, 2008).

The analysis of phenotypes resulting from double insertions allows further elaboration of this model and offers additional insights into the mechanisms by which insulators affect enhancer-promoter interactions. For example, it has been shown that a wing enhancer distal to two adjacent gypsy insertions is capable of bypassing the activity of the two insulators when the two copies of the gypsy provirus are inserted in opposite orientation. These results demonstrate that interactions such as the ones determined genetically in transgenes, involving pairs of 400-bp gypsy insulators, also occur between pairs of gypsy insulators embedded in the gypsy provirus and suggest that establishing such interactions is part of the normal life cycle strategy used by the retrovirus. Interestingly, when two gypsy insertions occur in the same orientation, distal enhancers are unable to bypass the two insulators and are blocked from activating the promoter, contrary to what it has been observed with direct repeats of insulator sequences. The main difference between the two sets of experiments is the presence of additional DNA sequences in the gypsy provirus. These sequences may be able to form a stem-loop structure when the two copies of gypsy are arranged in opposite, but not when they are in the same, orientation. A similar role has been suggested for the relative orientation of insulator sequences between interacting Mcp insulators. The stem-loop structure would allow interactions between insulator proteins present in the two copies of gypsy with opposite orientations but a direct tandem arrangement of the two copies of the provirus would preclude such interactions. These observations support the hypothesis that interactions between paired insulators are required to bypass insulator function and allow enhancer-promoter communication (Labrador, 2008).

Expression of the Drosophila secreted cuticle protein 73 (dsc73) requires Shavenbaby

Low stringency genomic library screens with genomic fragments from the sex determination gene doublesex identified the Drosophila secreted cuticle protein 73 (dsc73; doublesex cognate 73A or dsx-c73A) gene, which encodes an 852-residue protein with an N-terminal signal sequence. In embryos, dsc73 RNA and protein are expressed to high levels in the epidermal cells that secrete the larval cuticle as well as in other cuticle-secreting tissues such as the trachea and salivary duct. Embryonic expression of dsc73 requires Shavenbaby, a transcription factor regulating cuticle formation. Double-labeling experiments with alphaCrb and alphaSAS reveal that, as with chitin and other known cuticle proteins, Dsc73 is secreted apically. Zygotic loss of dsc73 results in larval lethality but loss does not result in overt patterning defects or overt morphological defects in the embryonic tissues in which it is expressed. Thus, dsc73 encodes a novel secreted protein, and it is conserved within the Drosophila group. dsc73 may serve as a useful embryonic marker for cuticular patterning (Andrew, 2008; Full text of article).

Genome-wide analyses of Shavenbaby target genes reveals distinct features of enhancer organization

Developmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes that remain poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known about how the ultimate effectors of cell differentiation are selected and controlled. This question is addressed during late Drosophila embryogenesis, when the finely tuned expression of the TF Ovo/Shavenbaby (Svb) triggers the morphological differentiation of epidermal trichomes. This study defined a sizeable set of genes downstream of Svb and used in vivo assays to delineate 14 enhancers driving their specific expression in trichome cells. Coupling computational modeling to functional dissection, the regulatory logic of these enhancers was investigated. Extending the repertoire of epidermal effectors using genome-wide approaches showed that the regulatory models learned from this first sample are representative of the whole set of trichome enhancers. These enhancers harbor remarkable features with respect to their functional architectures, including a weak or non-existent clustering of Svb binding sites. The in vivo function of each site relies on its intimate context, notably the flanking nucleotides. Two additional cis-regulatory motifs, present in a broad diversity of composition and positioning among trichome enhancers, critically contribute to enhancer activity. These results show that Svb directly regulates a large set of terminal effectors of the remodeling of epidermal cells. Further, these data reveal that trichome formation is underpinned by unexpectedly diverse modes of regulation, providing fresh insights into the functional architecture of enhancers governing a terminal differentiation program (Menoret, 2013).

The results identify a high-confidence set of more than 150 genes activated by Svb in trichome cells. 60 of these were confirmed, showing complete or partial down-regulation in the absence of active Svb protein. While most genes are expressed in all trichome cells, some are restricted to trichome subsets, suggesting that they can contribute to the diversity of trichome shape and organization observed along the body. Functional annotation (Gene Ontology and manual curation) indicates that Svb controls terminal players of trichome differentiation. In addition to novel factors of F-actin organization, extracellular matrix remodeling , cuticle formation and pigmentation, enzymes involved in oxidation-reduction, proteolysis and cell trafficking were identified, further extending the repertoire of cellular functions involved in the terminal differentiation of trichome cells. Hence, a major role of Svb in trichome formation is to directly activate the expression of a battery of cell morphogenesis effectors. In support of this, ChIP-seq peaks are present in >70% of these Svb-dependent effector genes. Experimental assays further validated 22 functional enhancers driving the expression of genes encoding factors involved in cytoskeletal or extracellular matrix reorganization, sugar binding, proteolysis and additional enzymes (Menoret, 2013).

Recent work has established that apparently redundant, or shadow, enhancers ensure robust expression of TFs. For example, the transcription of svb itself involves separate enhancers that buffer the trichome pattern against variations in the genetic background and external conditions. It has been proposed that shadow enhancers are required to drive acute expression of some key developmental regulators. This study defined within both shavenoid and miniature separable enhancers (sha1, sha3, Emin, EminB, EminC) that mediate Svb regulation. These data indicate that apparently redundant enhancers may not be limited to regulatory factors operating at high hierarchic positions in gene networks. Instead, evidence is provided that several 'blue collar' effector genes display a similar regulatory architecture, suggesting that multiple enhancers represent an overlooked feature of the successive tiers of gene networks (Menoret, 2013).

Early acting enhancers often comprise multiple BSs for a given TF. For example, conserved BS clusters have identified target enhancers of Dorsal or Bicoid and feature functional Twist-bound regions. Of note, most algorithms developed for enhancer detection extensively use motif clustering as an important predictor. This study found a clear enrichment in putative Svb BSs (OvoQ6 motif) in its downstream genes; however, only a small proportion of these motifs mediate in vivo regulation. There is very limited, if any, clustering of Svb BSs in ChIP peaks associated with Svb target genes, and even genome-wide. Within the trichome enhancers that were validated experimentally, 13 out of 22 display a single Svb site. Furthermore, for the enhancers tyn2, sha3 and dyl2, which contain two to three Svb BSs, the inactivation of individual sites has often limited consequences, as also reported for other TFs. Even if some sites have been missed by computational approaches, the presence of multiple BSs within a short region is not a deterministic feature of active Svb-dependent enhancers (Menoret, 2013).

These findings highlight a paradoxical discrepancy between the enrichment of putative BSs accumulated in Svb downstream genes and the limited number of those acting as cis-regulatory elements. Is there a role for this evolutionary accumulation of Svb-like motifs in Svb targets? For example, these sites with presumably weaker affinity (at least in vivo) can increase the local concentration of the TF facilitating regulation through a few BSs stably bound in vivo, as it has been suggested on thermodynamics grounds or to explain the existence of thousands of binding events that are transcriptionally inactive (Menoret, 2013).

The motif bound by Svb in vivo is more constrained than the consensus defined from in vitro or one-hybrid approaches. This shows that slight sequence differences, not detected in vitro, can play a key role within genomic context], such as revealing the influence of co-factors (Menoret, 2013).

In addition, other motifs influence which Svb BSs are functional as regulatory elements, a notion well in line with recent results on the in vivo specificity of Hox factors. Statistical approaches identified a more widely spread 'blue' motif. Importantly, only half of the enhancers comprise blue motifs (WAGAAAGCSR), indicating that there are several ways to build Svb-responsive enhancers. Indeed, the systematic dissection of Emin disclosed an additional motif (TTATGCAA) ultra-conserved across Drosophilidae and contributing to its activity. This 'yellow' motif is retrieved in half of the trichome enhancers, with or without blue motifs. It is, however, barely specifically enriched in Svb-bound regions and therefore was not predicted by computational analyses (positives versus negative regions), showing the importance of unbiased functional dissection to disclose the full spectrum of cis-regulatory elements. Indeed, the disruption of either blue or yellow motifs strongly affects enhancer function in all tested cases, providing experimental evidence of their cis-regulatory activity (Menoret, 2013).

Trichome enhancers thus display various combinations of motifs, from those containing only Svb BSs (5/22), Svb plus yellow (4/22), Svb plus blue (6/22) or all three together (7/22). These different motif compositions do not appear to correlate with distinct subclasses of gene function (DM, unpublished data). Furthermore, multiple enhancers from the same gene can harbor distinct combinations, as exemplified by shavenoid and to a lesser extent by miniature. Several studies have shown that motif composition may correlate with a given spatio-temporal pattern - for example, for neurogenic or muscular gene regulatory networks (GRNs). Since most trichome enhancers are often active in the very same population of cells, with highly similar dynamics, it is surprising to observe such diversity in their motif compositions. There are four enhancers restricted to dorsal trichome cells, but again they accommodate different motif compositions, with EminB and 4702B, which contain blue motifs, versus cyrA and 31559, which do not. These data thus indicate that trichome enhancers display diverse distributions of functional motifs, supporting that distinct cis-regulatory architectures drive highly similar spatio-temporal expression (Menoret, 2013).

Although highly constrained sequences, such as the interferon-β enhanceosome, do not seem widely spread, developmental enhancers may yet require some 'grammar' for motif positioning - for example, with an optimal pair-wise spacing of motifs that could reflect the cooperative binding of TFs. For trichome enhancers no obvious bias was detected in the number or respective arrangement of the cis-regulatory motifs they rely on. Likewise, recent results from the analysis of Drosophila cardiac enhancers support that similar expression patterns can be generated from divergent compositions and positioning of motifs (Menoret, 2013).

That several different inputs lead to similar enhancer outputs does not, however, formally rule out the existence of constraints, even though they are not detected by 'horizontal' comparison of different enhancers within the same species. An independent way to evaluate this possibility is to look at the evolution of individual regulatory regions throughout species. Across Drosophilidae, trichome enhancers often display similar numbers and organization of cis-regulatory motifs. Furthermore, besides turnover of some motifs, svbF7, blue and yellow motifs are often embedded within short-sized islands of high evolutionary conservation, when compared to neighboring sequence. Similar strong evolutionary conservation was also noticed for the binding site of Twist and its partner TFs, although these studies did not examine evolution of the detailed pattern of motif positioning. These data therefore suggest that despite diverse arrangements of motifs, patterns of evolutionary conservation likely represent the signature of functional constraints that locally shape the architecture of individual enhancers (Menoret, 2013).

Posttranscriptional Regulation

Pri peptides are mediators of ecdysone for the temporal control of development

Animal development fundamentally relies on the precise control, in space and time, of genome expression. Whereas a wealth of information is available about spatial patterning, the mechanisms underlying temporal control remain poorly understood. This study shows that Pri peptides (see Tarsal-less), encoded by small open reading frames, are direct mediators of the steroid hormone ecdysone for the timing of developmental programs in Drosophila. A previously uncharacterized enzyme of ecdysone biosynthesis, Glutathione S transferase E14 (GstE14), was identified, and ecdysone was found to trigger pri expression to define the onset of epidermal trichome development, through post-translational control of the Shavenbaby transcription factor. Manipulating pri expression is sufficient to either put on hold or induce premature differentiation of trichomes. Furthermore, it was found that ecdysone-dependent regulation of pri is not restricted to epidermis and occurs over various tissues and times. Together, these findings provide a molecular framework to explain how systemic hormonal control coordinates specific programs of differentiation with developmental timing (Chanut-Delalande, 2014: 25344753).



During early nuclear cleavage stages, maternal transcripts are extremely abundant and uniformly distributed. Later, on, the hybridization signal decreases rapidly, except at the posterior pole. Staining in all embryos is specifically detected in pole cells, as soon as they form. The staining allows tracking of pole cell migration into the amnioproctodeal invagination, until stage 8. Subsequently, through successive stages until stage 17, there is no further staining of pole cells. In addition to pole cells, transcripts are detected at the late cellular blastoderm and early gastrulation stages in the head region, taking the form of two rings around the embryo. The more posterior ring is located just anterior to the position of the cephalic furrow. Throughout embryonic development, transcripts continue to be detected in the head, particularly in the region of the pharynx, the antennal region and the hypopharyngeal lobe. During germ band retraction (stage 12), an additional striped pattern appears which corresponds to the anterior part of each abdominal segment (Mével-Ninio, 1995). In the hypodermis along the ventral side of the embryo, trapezoidal zones stain in each segment, corresponding to the denticle belt primordia (Garfield, 1994). In older embryos (stages 14), this staining corresponds exactly with the regions of the denticle belt setae and dorsal hairs, whose number and size are affected in svb mutants. Finally, in the fully developed embryo (stage 17), staining is detected again in germline cells, in the gonads. With an OVO specific probe, a strong and uniformly distributed staining is observed in early embryos at nuclear cleavage stage (stage 2). This staining then decreases progressively, but never becomes localized to any region of the embryo, including pole cells. This hybridization signal most likely corresponds to maternal transcripts that are very abundant in mature oocytes. Other probes indicate that the pole cell transcripts are of maternal origin (Mével-Ninio, 1995).

Shavenbaby couples patterning to epidermal cell shape control

It is well established that developmental programs act during embryogenesis to determine animal morphogenesis. How these developmental cues produce specific cell shape during morphogenesis, however, has remained elusive. This question was addressed by studying the morphological differentiation of the Drosophila epidermis, governed by a well-known circuit of regulators leading to a stereotyped pattern of smooth cells and cells forming actin-rich extensions (trichomes). It was shown that the transcription factor Shavenbaby plays a pivotal role in the formation of trichomes and underlies all examined cases of the evolutionary diversification of their pattern. To gain insight into the mechanisms of morphological differentiation, attempts were made to identify shavenbaby's downstream targets. Shavenbaby controls epidermal cell shape, through the transcriptional activation of different classes of cellular effectors, directly contributing to the organization of actin filaments, regulation of the extracellular matrix, and modification of the cuticle. Individual inactivation of shavenbaby's targets produces distinct trichome defects and only their simultaneous inactivation prevent trichome formation. These data show that shavenbaby governs an evolutionarily conserved developmental module consisting of a set of genes collectively responsible for trichome formation, shedding new light on molecular mechanisms acting during morphogenesis and the way they can influence evolution of animal forms (Chanut-Delalande, 2006; full text of article).

As a first step towards identifying players involved in denticle formation, a search for genes expressed in epidermal cells was carried out by conducting a systematic survey of expression patterns available from the Berkeley Drosophila Genome Project. Among the approximately 400 genes reported to be transcribed in the epidermis, only a small number was found to be expressed in a segmentally repeated pattern, at a time of epidermal cells morphological differentiation. The epidermal expression of these genes was studied by in situ hybridization and its dependence on svb was examined. Focused on miniature m (Roch, 2003), because its expression in stage-15 embryos strikingly resembles the trichome pattern, which is specified by svb at previous stages (Chanut-Delalande, 2006).

m expression in epidermal cells is abolished in svb mutant embryos, showing that svb is required for m transcription in the epidermis. Detailed examination revealed a precise correlation between m expression and the denticle pattern in wild-type embryos, with a six to seven–cell-wide expression stripe in each segment that alternates with a band of m-negative cells. OvoA is a germinal isoform of the ovo/svb gene that acts as a transcriptional repressor, able to counteract svb activity when its expression is artificially directed in the epidermis. When expressed in ptc-expressing cells, OvoA leads to the replacement of denticle rows 2 and 3 by a stripe of naked cuticle interrupting each denticle belt. This OvoA expression also represses m transcription in the epidermal cells corresponding to denticle rows 2 and 3, providing evidence that svb activity is required cell autonomously for m expression in denticle cells. To test whether svb is sufficient to induce m expression in the epidermis, the consequence of the ectopic expression of svb in smooth cells was studied. Ectopic expression of svb in wg-expressing cells causes the formation of a supernumerary row of cuticular extensions and leads to an ectopic stripe of m expression, showing that svb is sufficient to trigger m transcription in epidermal cells (Chanut-Delalande, 2006).

One of the first recognizable signs of the morphological differentiation of epidermal cells is the formation of an apical bundle of microfilaments in denticle cells. These early steps of denticle formation depend on svb, which is necessary and sufficient to promote the formation of epidermal actin bundles. The current results show that svb controls the transcription of several genes involved in different steps of actin assembly/organization. First, it was found that svb directs the expression of shavenoid/kojak, a gene producing strong denticle defects when mutated and recently shown to encode a protein reported to associate with actin (Ren, 2006), but whose biochemical function is unknown. Second, Svb also directs the expression of singed and forked, coding respectively for the Drosophila putative homologs of Fascin and Espin, two proteins that crosslink parallel actin filaments and promote the formation of bundles of microfilaments. The Forked and Singed proteins sequentially accumulate in growing denticles, a situation reminiscent of that of wing hair formation, suggesting that these proteins play similar roles in the formation of adult and embryonic epidermal extensions. Accordingly, the inactivation of sn and f alters denticles, strongly suggesting that denticle formation indeed involves parallel actin bundles, as shown for wing hairs (Chanut-Delalande, 2006).

Several cytoskeletal regulators such as dAPC, Enabled, Diaphanous/Formin, and the Arp2/3 complex, accumulate in denticles, suggesting that they are involved in denticle formation, although their respective functions remains to be evaluated. Whereas svb does not control the expression of those ubiquitous actin-associated factors, it is possible that svb regulates their activity, or subcellular localization, indirectly. Consistent with this hypothesis, dAPC-2 is specifically relocalized in svb-induced ectopic epidermal extensions (Delon, 2003). In addition, svb directs the epidermal expression of Wasp, a key activator of the Arp2/3 actin nucleator complex, which is well known to trigger the formation/elongation of actin filaments. Moreover, it has been shown in vitro that Fascin switches the activity of the Arp2/3 complex from the formation of a mesh-like branched network to parallel microfilaments, therefore suggesting that svb targets can regulate both the formation of actin filaments and their reorganization, at least in part, through a tight control of the activity of the Arp2/3 complex during denticle formation (Chanut-Delalande, 2006).

Taken together, these results show that svb controls the expression of several cytoskeletal factors which, probably by modifying the activity of housekeeping actin-remodeling machinery, act together to trigger the formation of apical cell extensions. Whereas few molecules are sufficient to promote actin organization in vitro, these studies indicate that, in vivo, many players are required to make a simple cellular extension. Pursuing the identification of novel genes regulated by svb should provide a means of identifying additional factors required for actin remodeling in vivo (Chanut-Delalande, 2006).

A surprising outcome of these studies is that denticle formation requires a specific regulation of the membrane/cuticle interaction. svb directs the expression of m in trichome cells. Miniature is a single-pass membrane protein, with a short cytoplasmic tail and a large extracellular region that contains a conserved Zona Pellucida (ZP) domain (Roch, 2003). ZP domains were initially identified in the three major proteins of the zona pellucida, the extracellular envelope of mammalian oocytes, and they are thought to be components of apical matrices. Miniature is required for the correct formation of denticles, revealing a novel aspect of ZP protein function in the formation of polarized cellular extensions. The absence of m severely impairs the interaction between the plasma membrane and cuticle layers in denticle cells, a defect likely due to a disorganization of the extracellular matrix. In the embryonic epidermis, Miniature is required for the continuous membrane/cuticle interaction that is specific to denticles, whereas only the tips of microvilli contact cuticle in naked regions. The accumulation of Miniature at the base of denticles reveals the existence of a denticle-specific membrane subdomain, suggesting that additional membrane proteins might be involved in denticle formation. Two other ZP genes are regulated by svb and analysis of their individual role in denticle formation is under way. These findings shed light on the importance of membrane proteins and their interaction with extracellular matrices, an aspect of cell-shape control hardly accessible to cell-culture approaches. Future analysis of Miniature targeting to the denticle should help to understand the mechanisms required for localized cell-shape modification during morphogenesis (Chanut-Delalande, 2006).

svb also regulates the expression of yellow (y), a gene encoding an apically secreted protein that associates with cuticle and is required for the production of black pigments. While pigmentation per se is not related to cell morphogenesis, the role of Yellow in the catecholamine pathway remains elusive; it could be involved in denticle hardening, since y mutant larvae display defects, in the morphology of denticles, that have been proposed to account for their abnormal locomotor activity. In addition, svb could directly regulate the local protein composition of cuticle, as suggested by the identification of an additional target encoding a putative chitin-binding protein (Chanut-Delalande, 2006).

Experimental evidence suggests that svb is situated at the bottom of regulatory cascades determining trichome patterning and is in turn directly responsible for triggering the cellular program of denticle formation. First, svb remains the most-downstream regulator determining the pattern of denticles and dorsal hairs, despite the unprecedented extent of genetic screens based upon cuticle observation (which identified most members of the Wg and DER pathways). Second, among mutations producing trichome defects, svb mutants display the strongest phenotype, in which most denticles and dorsal hairs are replaced by naked cuticle. Third, svb directs the expression of genes involved in various aspects of denticle formation, including control of the cytoskeleton, membrane/matrix organization and cuticle differentiation. Finally, evidence is provided for a direct control of one of the targets (m). A 400-bp m enhancer has been defined reproducing the endogenous expression pattern of m in the epidermis and it is shown that the Svb transcription factor binds specifically to this evolutionarily conserved region. Substituting 2-bp in this cis-regulatory element preventing Svb binding is sufficient to abrogate its in vivo enhancer ability, thus suggesting that direct binding of Svb mediates the control of m epidermal expression. Several putative svb binding sites have been detected in evolutionarily conserved regions of other svb targets. Whether they are all required for svb transcriptional regulation remains to be tested. Further dissection of the m enhancer, as well as those of other svb targets, should lead to the definition of a functional cis-regulatory element responsible for svb control. This outcome should facilitate the identification by bioinformatic approaches of additional svb responsive enhancers and target genes (Chanut-Delalande, 2006).

It is proposed that svb directly controls the expression of a set of “effector” genes, all required for a concerted modification of cell shape and cuticle organization to achieve the formation of denticles. How many genes are regulated by svb to promote remodeling of epidermal cells? This analysis, which covers approximately 25% of the total number of Drosophila genes, has led to the identification of 11 downstream targets, suggesting that svb activates the expression of numerous additional genes to trigger the formation of embryonic epidermal cell extensions (Chanut-Delalande, 2006).

Several results suggest that this svb-controlled module is used in different developmental programs that produce cuticle extensions. Although signaling pathways act differently in ventral and dorsal embryonic regions, svb is also required to express the same target genes for the formation of denticles and dorsal hairs. These results show that the svb targets identified so far act collectively to promote the formation of various epidermal extensions, despite the fact that they display different shapes. In addition, svb mutations also affect the formation of adult wing hairs and antennae laterals (Delon, 2003), which are known to require the activity of several svb targets identified in the embryonic epidermis (m, sn, f, sha for adult wing hairs, and sn, f, sha for antennae laterals). How can the same set of svb-regulated effectors participate in the formation of epidermal extensions of diversified morphology? Additional cytoskeletal factors can be differentially expressed in distinct epidermal regions independently of svb. It is also possible that other regulators modulate the response to svb (cf. the weak expression of f and wsp in naked cells expressing ectopically svb, when compared to that of sn, m, sha and y). Finally, upstream signaling pathways certainly contribute to the sculpting of each kind of trichome through the regulation of the expression and/or activities of cellular effectors. Recent studies have shown that members of the planar polarity pathway (PCP) are indeed involved in defining denticle polarity in response to signaling pathways. Interestingly, one of the identified svb targets, shavenoid, has recently been reported to interact with PCP in the adult wing, raising the possibility that such a dialog also occurs during embryonic epidermal cell remodeling. svb thus appears to govern a morphological module responsible for the major switch from smooth surface to trichome, the precise shape of which is finely sculptured by independent intrinsic factors and activities (Chanut-Delalande, 2006).

The pattern of trichomes has been modified several times during the evolution of insects. Across the genus Drosophila, at least four independent evolutionary transitions have led to the loss (to various extents) of dorsal hairs. Nevertheless, evolution of the pattern of dorsal hairs and denticles results from the modification of svb expression in all studied cases. Although the expression of patterning genes is unchanged in all species examined, the difference in the dorsal hair pattern between D. melanogaster and D. sechellia is due only to the modification of shavenbaby's response to signaling pathways. This analysis further shows that the restriction of svb expression in D. sechellia embryos causes, in turn, the restriction of the dorsal expression of m, sn, f, sha, and wsp genes. Therefore, all these svb targets display a concerted modification of their expression in D. sechellia, bringing additional evidence that together they constitute a developmental module. Consistent with this interpretation, individual inactivation, or experimental modifications of the expression, of any of the svb target genes identified so far are not sufficient to modify the trichome pattern. These data indicate that denticle formation requires multiple factors that act collectively to remodel epidermal cell shape. This requirement for many genes to build a cuticular extension doubtless constitutes a developmental constraint, explaining why modifications of the expression of svb, the factor that governs this entire set of genes, are required for the trichome pattern to evolve (Chanut-Delalande, 2006).

Evolutionary modifications of cis-regulatory elements of lin-48, the putative svb homolog in worms, are responsible for the difference in the position of the excretory duct between C. elegans and C. briggsae. Although accumulated data thus demonstrate the particular role of svb genes in morphological diversification between relatively close species, how they are related to the evolution of animal forms across more distant phyla remains an open question. Several features of svb function have been conserved in mammals, including the role of one of its homologs, m-ovo1, in the differentiation of epidermal derivatives and its regulation by the Wnt pathway. Identification of genes regulated by svb in flies now opens the way to evaluate the contribution of the different parameters contributing to the role of svb in morphological evolution, from the modification of its response to signaling pathways to that of its cellular targets (Chanut-Delalande, 2006).

Larval and Adult

Ovo protein is detected in both male and female gonads. In the first larval instar ovaries all nuclei of germ cells are stained. This staining becomes restricted to a subpopulation of germ cells. In second and third larval instar male gonads, staining is restricted to the anterior part. Ovo protein is observed in the distal tip of the adult testis (Mével,Ninio, 1995). Expression of Ovo protein is detected in germinal stem cells of the germarium and later in nurse cells. High levels of transcripts are detected from stage 8 of oogenesis, and these ultimately accumulate in the growing oocyte (Mével-Ninio, 1995).

Most regulatory genes are employed multiple times to control different processes during development. The Drosophila Ovo/Shavenbaby (Svb) transcription factor is required both for germline and epidermal differentiation, two roles also found for its ortholog m-ovo1 in mice. In Drosophila, these two distinct functions are contributed by separate control regions directing the expression of Ovo/Svb in the germline (ovo) and soma (svb), respectively. Alternative splicing represents an additional level of the regulation of Ovo/Svb functional specificity. Characterization of the ovoD1rv23 mutation has revealed that the intragenic insertion of a novel retrotransposon, romano, inactivates ovo without altering svb. Evidence is provided that this insertion disrupts a germline-specific alternative exon, exon 2b, which encodes a 178-amino-acid internal extension (2B). While both isoforms, Ovo+2B and Ovo-2B, accumulate during oogenesis, only Ovo+2B is able to fulfill germinal ovo functions. Ovo-2B is unable, even when overexpressed, to fully rescue oogenic defects resulting from the absence of wild type ovo product. By contrast, either Ovo+2B or Ovo-2B germline protein can substitute for Svb in the epidermis. These results emphasize the specific features of splicing in the germline, and reveal its functional importance for the control of ovo/svb-dependent ovarian and epidermal differentiation (Salles, 2002).

The ovo/svb intron II is bordered by a single 3' splice acceptor site and two alternative 5' splice donor sites. Depending on which of the two 5' splice donors is used, ovo/svb mRNAs will or will not include the exon 2b, which codes for a 178-amino-acid peptide. The downstream splice donor site is specific to the germline, since exon 2b containing transcripts are found only in germinal cells. In somatic tissues, the splicing machinery always uses the upstream-most 5' splice site, thus generating transcripts lacking exon 2b. These data emphasize the specificity of the splicing machinery in the germline, as previously illustrated by the splicing of the P-element transposon. Splicing of the P-element third intron (IVS3) is active exclusively in germ cells, and repressed in somatic cells by the PSI factor, thus restricting P transposition to the germline (Salles, 2002).

Which candidate genes could be involved in the germline-specific splicing of ovo mRNAs? ovo belongs to the group of the so-called 'ovarian tumor genes,' in which mutant germ cells accumulate in excessive number and fail to form normal 16-cell cysts. Other genes of this class could be candidates for regulating ovo/svb pre-mRNA splicing, especially Sex-lethal (Sxl) and sans-fille (snf), which display splicing regulatory activities. The Sxl protein directs the female-specific processing of transformer RNA in the soma and acts to control the splicing of its own pre-mRNA. The snf gene encoding a component of U1 and U2 snRNPs is involved in somatic as well as germinal Sxl RNA splicing. Two lines of evidence argue, however, against either Sxl or snf being involved in regulating ovo splicing. (1) Previous genetic analyses have ruled out a role for snf and Sxl upstream of ovo in the cascade of genes controlling female germline differentiation, and (2) the splicing of ovo RNA is unaltered in females homozygous for the snf mutation. An alternative splicing that generates the inclusion of an internal protein extension has also been described in the case of another ovarian tumor gene, ovarian tumor (otu). Here, again, only the larger isoform can fully substitute for wild type otu function. This shows that alternative splicing is a major mechanism regulating the activity of genes controlling germline development, and opens the possibility that ovo and otu alternative splicing are controlled by the same factors (Salles, 2002).

The ovoD1rv23 mutation allowed the discovery of a novel retrotransposon, romano, which was most probably introduced very recently in the genome of Drosophila melanogaster. Indeed, romano sequences were not found in the sequences obtained from the Drosophila genome project. This recent introduction of romano represents a promising model to study how the spreading of this class of mobile genetic elements is controlled. The characterization of the ovoD1rv23 mutation revealed also that premRNA processing functions differentially in the germline and the soma. Insertion of the romano transposon into exon 2b causes premature transcription termination and polyadenylation of ovo mRNA, resulting in the production of truncated transcripts in ovaries. In somatic cells, however, presence of the romano sequences in the transcribed svb region does not alter the production of wild type svb mRNAs, as also attested by the absence of any cuticular phenotype in ovoD1rv23 homozygous embryos. This indicates that romano sequences directing transcription termination and polyadenylation in ovoD1rv23 ovaries are only recognized by RNA processing factors specific to the germline. Further studies on the functional organization of romano might allow the identification of such factors (Salles, 2002).

Analysis of Ovo isoforms shows that the 2B protein extension is necessary for the ovo germline function, since only Ovo+2B can fulfill normal germinal development. By contrast, the presence of the 2B-protein region has no noticeable influence on the somatic function, indicating that the germinal alternative splicing represents an important parameter of ovo/svb functional diversification. This is further supported by the evolutionary conservation of the 2b alternative splicing, and its regulatory function, in a distantly related diptera, Bactrocera oleae, a tephritid species thought to have separated from drosophilidae for more than 120 million years (Salles, 2002).

Rescue experiments with transgenes producing various amounts of the poorly active Ovo-2B product suggest that increasing Ovo activity is required during oogenesis. Only the early stages of germ cell division and differentiation can be achieved by endogenous levels of Ovo-2B. Overexpression of Ovo-2B allows further egg chamber development, but leads to oogenic arrest and apoptosis of germ cells. Full rescue of ovo mutations then requires higher ovo activity, only obtained with Ovo+2B. These results suggest that ovo might be required to activate an increasing number of target genes during oogenesis, or alternatively, some targets would require a higher threshold of ovo activity (Salles, 2002).

The use of Ovo-2B in rescue experiments also revealed novel features of ovo function during oogenesis. (1) Although ovoD1rv23 is considered as a null mutation, the results strongly suggest the existence of a residual antimorphic activity in the ovoD1rv23 females. This activity is most probably provided by the OvoD1-2B repressor isoform, synthesized from transcripts that have spliced out exon 2b and romano sequences. This hypothesis is further supported by experiments showing that the specific expression of the OvoD1-2B isoform causes dominant female sterility. (2) The rescue assays have created experimental conditions of unbalanced ratio of repressor and activator activity, allowing the detection of previously unnoticed phenotypes. Interestingly, some of the observed defects in ovoD1rv23 ovaries rescued by Ovo-2B resemble those resulting from otu hypomorphic mutations. These observations correlate with the recent findings that otu behaves as a direct target of Ovo transcription factors. However, increasing otu expression (hs-otu) is not sufficient to rescue all the ovarian defects resulting from ovo mutation. Therefore, a major challenge now resides in the identification of other ovo target genes. Since most of the available ovo mutations lead to either complete absence of germline, or early arrest of oogenesis, partial rescue of ovo function by Ovo-2B should provide a clue for the molecular identification of the specific target genes that mediate Ovo activity during the different steps of Drosophila oogenesis (Salles, 2002).

Maternal RNAs encoding transcription factors for germline-specific gene expression in Drosophila embryos

In early Drosophila embryos, germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm contains the maternal factors required for germline development. It has been proposed that germline-specific gene expression is initiated by the function of maternal factors that are enriched in the germ plasm. However, such factors have remained elusive. This paper describes a genome-wide survey of maternal transcripts that encode for transcription factors and are enriched in the germ plasm. Pole cells were isolated from blastodermal embryos by fluorescence-activated cell sorting (FACS) and then isolated cells were used in a microarray analysis. Among the 835 genes in the Gene Ontology (GO) category transcription regulator activity listed in FlyBase, 68 were found to be predominantly expressed in pole cells as compared to whole embryos. Since the early pole cells are known to be transcriptionally quiescent, the listed transcripts are predicted to be maternal in origin. In situ hybridization analysis revealed that 27 of the 68 transcripts were enriched in the germ plasm. Among the 27 transcripts, six were found to be required for germline-specific gene expression of vasa and/or nanos by knockdown experiments using RNA interference (RNAi). The identified transcripts encode a transcriptional activator (ovo), components of the transcriptional initiation complexes (Trf2, bip2 and Tif-IA), and the Ccr4-Not complex [CG31716 and l(2)NC136]. This study demonstrates that germ plasm contains maternal transcripts encoding transcriptional regulators for germline-specific gene expression in pole cells (Yatsu, 2008).

Small peptides switch the transcriptional activity of Shavenbaby during Drosophila embryogenesis

A substantial proportion of eukaryotic transcripts are considered to be noncoding RNAs because they contain only short open reading frames (sORFs). Recent findings suggest, however, that some sORFs encode small bioactive peptides. This study shows that peptides of 11 to 32 amino acids encoded by the polished rice (pri) sORF gene control epidermal differentiation in Drosophila by modifying the transcription factor Shavenbaby (Svb). Pri peptides trigger the amino-terminal truncation of the Svb protein, which converts Svb from a repressor to an activator. These results demonstrate that during Drosophila embryogenesis, Pri sORF peptides provide a strict temporal control to the transcriptional program of epidermal morphogenesis (Kondo, 2010).

Studies of eukaryotic genomes have revealed that a large proportion of genomic DNA produces atypical long transcripts, the functions of which are controversial. These transcripts contain only short open reading frames (sORFs, <100 codons) and thus are generally considered to be non-protein-coding RNAs (ncRNAs). However, there is growing evidence that the sORFs present in some ncRNAs are actually translated into small peptides, the abundance of which is probably greatly underestimated. Whereas sORF-encoded peptides may represent an overlooked repertoire of bioactive molecules, their functions and the mechanisms by which they operate are largely unknown (Kondo, 2010).

An evolutionarily conserved sORF gene, referred to as polished rice (pri) or tarsal-less (tal) has been identified in Drosophila and mille-pattes (mlpt) in Tribolium. pri mRNA is a polycistronic transcript that encodes four similar peptides, 11 to 32 amino acids in length, that play a redundant role in Drosophila embryogenesis. Embryos that lack pri display prominent defects, including the absence of trichomes and aberrant tracheal architecture. Reduced pri activity in imaginal development results in abnormal leg morphogenesis. Similarly, mlpt knockdown in Tribolium leads to appendage defects and the transformation of segmental identity (Kondo, 2010 and references therein).

To gain insight into the molecular function of Pri peptides, this study focused on their role in trichome formation during Drosophila embryogenesis. Epidermis differentiation results in a pattern of smooth cells and cells that form apical extensions, called trichomes (ventral denticles and dorsal hairs). Modifications of the trichome pattern that have been examined in insects (resulting from laboratory-induced mutations or evolutionary diversification) are so far all attributable to changes in expression of shavenbaby (svb). Indeed, svb encodes a transcription factor that directly regulates the expression of target effectors, which are collectively responsible for trichome formation. Although the absence of pri results in trichome loss, the expression of svb is not altered in pri mutants. Reciprocally, pri is expressed normally in svb mutants, showing that svb and pri act in parallel in trichome formation. Expression of Svb target genes, such as miniature and shavenoid (Chanut-Delalande, 2006), is lost in pri mutants, whereas the expression of other epidermal genes is unaffected. The activity of isolated Svb-responsive enhancers was also strongly reduced in pri mutants. Therefore, pri is specifically required for the transcription of Svb downstream targets in trichome cells (Kondo, 2010).

How can Pri peptides regulate the expression of Svb target genes without affecting svb expression? The svb locus encodes three overlapping protein isoforms: Svb and the germline-specific proteins OvoA and OvoB. Ovo/Svb proteins all share the same DNA-recognition and transcriptional-activation domains but differ in their N-termini. The shortest isoform, OvoB, is a transcriptional activator and induces trichomes when artificially expressed in the epidermis. OvoA contains an extended N-terminal region, which switches its function toward active transcriptional repression and thus dominantly inhibits trichome formation. Svb contains a further N-terminal extension, compared to OvoA, and promotes the formation of ectopic trichomes like OvoB. To evaluate the specificity of Pri/Svb interaction, the influence of pri on the different Ovo/Svb isoforms was examined with respect to trichome formation. In wild-type embryos, seven rows of ventral cells per segment express svb and form trichomes. Upon its ectopic expression in smooth cells, Svb [or Svb:green fluorescent protein (GFP)] induced supernumerary trichomes in control embryos but not in pri mutants. In contrast, OvoB (or OvoB:GFP) was insensitive to pri, with ectopic trichomes forming in both control and pri mutant embryos. In the latter case, only OvoB-induced ectopic trichomes was observed and no Svb-dependent endogenous trichomes. These results show that whereas pri has no effect on the shorter OvoB isoform, pri peptides specifically control the ability of Svb to induce trichomes (Kondo, 2010).

Whether Pri peptides affect the synthesis or trafficking of Ovo/Svb proteins was examined. Using transgenic C-terminal GFP-fusions (proven functional as described above), it was observed that pri does not influence the production of Ovo/Svb proteins or their import to the nucleus. However, it different patterns of their intranuclear distribution were observed. Regardless of pri activity, throughout embryogenesis OvoA accumulated in discrete foci, and OvoB was distributed diffusely in the nucleoplasm. During stages 11 and 12, before pri is expressed in the epidermis, Svb formed intranuclear foci, like OvoA. At the onset of pri epidermal expression (stage 13 onwards), the nuclear distribution of Svb became diffuse. Therefore, Svb distribution changes from a pattern similar to the OvoA repressor to that of the OvoB activator, and the timing of this conversion correlates with the expression of pri. This redistribution of Svb was abolished in pri mutants, in which Svb remained in nuclear foci throughout embryogenesis. Thus, pri participates in the conversion of nuclear distribution of Svb from punctate to diffuse (Kondo, 2010).

The nonpunctuated, diffuse nuclear distribution of Svb in epidermal cells correlates with its ability to induce trichomes, suggesting that Svb redistribution coincides with active transcription of its targets. This hypothesis was explored using assays in Drosophila Schneider cells (S2 cells), which are of embryonic origin. Similarly to observations in embryos, the nuclear pattern of Svb was converted from punctate to diffuse in a pri-dependent manner in S2 cells. The transcriptional activity of Svb/Ovo was quantified using the Enh-m enhancer, which is directly activated by Svb in vivo. OvoB strongly stimulated the transcription driven by Enh-m, and OvoA repressed transcription, both with or without pri. In contrast, Svb behaved like OvoA in the absence of pri, but similar to OvoB, activated Enh-m in the presence of Pri peptides. Inactivation of the Svb-binding site of Enh-m suppressed this activation, indicating that pri is required for the direct activation of Enh-m by Svb. These results demonstrate that Pri peptides switch the transcriptional activity of Svb from that of a repressor accumulated in nuclear foci to a nucleoplasmic activator (Kondo, 2010).

To explore the mechanisms by which Pri peptides trigger this switch in Svb intranuclear distribution, whether Pri requires de novo synthesis of the Svb protein was examined. Using a photoactivatable-GFP (PA-GFP), it was observed that photoactivated Svb:PA-GFP switched from foci to diffuse distribution after the induction of pri expression. Therefore, the same Svb molecules are relocated within the nucleus, suggesting that the action of Pri peptides relies on posttranslational modifications of Svb. Accordingly, Western blot analysis showed that whereas the size of OvoA and OvoB proteins (including that of their minor species) were not affected by pri, Svb exhibited a differential electrophoretic mobility in a pri-dependent manner. In the absence of pri, Svb appeared slightly larger than OvoA, as deduced from the cDNA sequences. Upon pri expression, the Svb protein displayed a faster mobility, corresponding to a truncation of approximately 50 kD, without apparent modification in the size of svb mRNA. An antibody raised against the N-terminal Svb-specific region (anti-Svb1s) recognized only the larger Svb protein but not the truncated product formed upon pri expression. This truncated Svb protein was detected by antibodies to Ovo and GFP, showing that it lacks the N-terminal region but retains an intact C terminus. To further characterize Svb truncation, the truncated Svb protein was purified, and its N-terminal end was microsequenced. The N terminus of truncated Svb matches the sequence AAGHGR, which is located 56 amino acids upstream of the OvoB-initiating methionine and within a protein region that shows strong evolutionary conservation in insects. The corresponding DNA sequence displays synonymous nucleotide substitutions across species and lacks canonical or alternative initiation codons, further supporting the view that Svb truncation results from a posttranslational cleavage. Hence, the pri-induced truncated form of Svb contains the DNA-binding and activation domains but not the repression domain, explaining why it acts as a transcriptional activator (Kondo, 2010).

Consistent with this idea, a pri-dependent truncation of the endogenous Svb protein was observed during embryogenesis. In wild-type embryos, anti-Svb1s detected a transient nuclear signal in trichome cells, at stages 11 and 12, that disappeared at later stages. The loss of the Svb N-terminal region coincided with the onset of pri expression in the epidermis. Indeed, pri is required for Svb truncation in vivo -- as revealed by the persistence of anti-Svb1s signal in pri mutants -- throughout embryogenesis. It is concluded that Pri peptides convert Svb from a transcriptional repressor to an activator via the truncation of its N-terminal region (Kondo, 2010).

This study has demonstrated that 11- to 32-amino acid peptides encoded by sORFs orchestrate epidermal differentiation through the control of Svb transcriptional activity. At stages 11 and 12, svb is already expressed and restricted to presumptive trichome cells, in which the full-length Svb repressor probably prevents the premature expression of cellular effectors. At stages 13 and 14, the expression of pri in epidermal cells then triggers N-terminal truncation of the Svb protein, probably through a proteolytic release of the repressor domain, causing a rapid conversion of Svb function toward activation. Thus, although svb expression defines the spatial pattern of trichomes, the action of Pri peptides defines the temporality of trichome formation (Kondo, 2010).

Besides the mechanisms of epidermal differentiation, these studies suggest broader functions for Pri peptides. Although pri is also required for tracheal morphogenesis, normal trachea were observed in svb mutant embryos, indicating that Pri peptides probably regulate additional developmental factors. Recent large-scale analyses indicate that thousands of unexplored transcripts are also probably encoding polypeptides of less than 100 amino acids in mice and humans. Future functional analyses should elucidate how small peptides encoded by transcripts improperly termed ncRNAs contribute to various biological processes including development and differentiation (Kondo, 2010).


Mutations in the genes ovo and/or otu can cause abnormal proliferation of XX germ cells (which lead to so-called ovarian tumors), or they can lead to the elimination of XX germ cells, such that adult females possess empty ovaries. Males carrying ovo or otu mutations are unaffected. To find out when this sexual dimorphism affects germ cells, the requirement of embryos and larvae for zygotic ovo and otu products has been analyzed. ovo is required for the survival of XX germ cells during larval stages, while XX germ cells lacking otu survive until metamorphosis. Furthermore, no sex-transformed mutant larval germ cells are found and no evidence is found for an early sex-specific vital process acting in the germ cells of the embryo (Staab, 1995).

Germ cell development in embryos was studied using a strategy that allowed the simultaneous labeling of pole cells with the determination of embryonic genotype. ovo- or otu- XX embryonic germ cells are indistinguishable in number and morphology from those present in wild-type siblings. The effects of the mutations are not consistently manifested in the female germline until pupariation, and there is no evidence that either gene is required for germ cell viability at earlier stages of development. The requirement for otu function in the pupal and adult ovary is supported by temperature-shift experiments using a heat-inducible otu gene construct. otu activity limited to prepupal stages is not sufficient to support oogenesis, while induction during the pupal and adult periods causes suppression of the otu mutant phenotype (Rodesch, 1995).

A class of ovo mutants exists that is homozygous- and hemizygous-lethal by virtue of a loss of the shavenbaby (svb) function. The shavenbaby phenotype was named because of the defect in ventral denticle belts and dorsal hairs observed in cuticle preparations of late embryos; it is a polyphasic lethal, and occasionally mutant males survive to adulthood. The shavenbaby escaper males (those that survive to adulthood) have thin bristles, rough eyes, and dusky wings, yet they are fertile (Garfinkel, 1992).

The three original ovo alleles are dominant mutations that act against the ovo+ gene product. Because ovoD alleles are dominant and because females heterozygous for deletions of the ovo region are viable and fertile, it is possible to isolate recessive ovo alleles by screening for loss of ovoD. Loss of ovo function results in the absence of eggs. The occurrence of shavenbaby mutations is characteristic of ovoD reversion. The alleles of shavenbaby and ovo can be arranged into three classes: ovo- svb+, ovo+ svb-, and ovo- svb-. The first type, class 1 alleles, include the original three dominant mutations and a recessive ovo mutation. Class 3 alleles include nearly all the reversion events (Oliver, 1987). In ovo mutants the somatically derived ovarian structures appear normal, but no egg chambers are visible in the adult ovary. In females heterozygous for ovoD2, oogenesis is blocked. These females have egg chambers with more than 15 nuclei, showing nurse cell morphology and/or tumorous egg chambers (Oliver, 1987).

Three dominant female-sterile ovoD mutations cause ovarian abnormalities that define an allelic series, with ovoD1 displaying the strongest phenotype and ovoD3 displaying the weakest. All three ovoD mutations are point mutations that create new in-frame methionine codons (AUG) in the 5' part of ovo. There are two types of overlapping ovo transcription units, ovoalpha and ovobeta. By using various ovo reporter genes, it was determined that the long Ovo isoforms starting at methionine M1, present in transcripts ovoalpha are expressed at low levels only in mature oocytes. Short Ovo isoforms are translated from methionine M373, the first in-frame start codon present in transcript ovobeta, and corresponding to the activity defined by recessive loss of function ovo mutations. The new AUGs generated in ovoD mutations all are located upstream of the M373 site. These results support the hypothesis that the new AUGs can substitute for M373 as translation starts and initiate the synthesis of Ovo proteins that have extra amino acids at their N termini. It is proposed that premature expression of long Ovo protein isoforms occurs in ovoD mutants and interferes with the wild-type Ovo function in controlling female germline differentiation. It is thought that the different Ovo isoforms serve different functions, with Ovoalpha being required early for oogenesis, while the Ovobeta isoform may provide maternal information to the embryo (Mével-Ninio, 1996).

The possible role of otu in the determination of the sexual identity of germ cells has not been extensively explored. Some otu alleles produce a phenotype known as ovarian tumors: ovarioles are filled with numerous poorly differentiated germ cells. These mutant germ cells have a morphology similar to primary spermatocytes and they express male germ line-specific reporter genes. This indicates that they are engaged along the male pathway of germ line differentiation. Consistent with this conclusion, the splicing of Sex-lethal pre-mRNAs was found to occur in the male-specific mode in otu-transformed germ cells. The position of the otu locus in the regulatory cascade of germ line sex determination has been studied by using mutations that constitutively express the feminizing activity of the Sxl gene. The sexual transformation of the germ cells observed with several combinations of otu alleles can be reversed by constitutive expression of Sxl. This shows that otu acts upstream of Sxl in the process of germ line sex determination. Other phenotypes of otu mutations were not rescued by constitutive expression of Sxl, suggesting that several functions of otu are likely to be independent of sex determination.

Finally, the gene dosage of otu modifies the phenotype of ovaries heterozygous for the dominant alleles of ovo. One dose of otu+ enhances the ovoD ovarian phenotypes, while three doses partially suppress these phenotypes. Synergistic interaction between ovoD1 and otu alleles leads to the occasional transformation of chromosomally female germ cells into early spermatocytes. These interactions are similar to those observed between ovoD and one allele of the sans fille locus. Altogether, these results imply that the otu locus acts, along with ovo, snf, and Sxl, in a pathway (or parallel pathways) required for proper sex determination of the female germ line (Pauli, 1993).

otu and ovo genes are required cell autonomously in the female germline for germ cell proliferation and differentiation. Mutations in otu and ovo cause a range of ovarian defects, including agametic ovaries and tumorous egg cysts, but do not affect spermatogenesis. XY germ cells do not require otu when developing in testis, but become dependent on otu function for proliferation when placed in an ovary. This soma-induced requirement can be satisfied by the induced expression of the 98 x 10(3) M(r) OTU product, one of two isoforms produced by differential RNA splicing. These results indicate that the female somatic gonad can induce XY germ cells to become 'female-like' because they require an oogenesis-specific gene. In contrast, the requirement for ovo is dependent on a cell autonomous signal derived from the X:A ratio. It is proposed that differential regulation of the otu and ovo genes provides a mechanism for the female germline to incorporate both somatic and cell autonomous inputs required for oogenesis (Nagoshi, 1995).

A new Drosophila gene, stand still (stil), is required in the female germline for proper survival, sex determination and differentiation. Three strong loss-of-function alleles were isolated. The strongest phenotype exhibited by ovaries dissected from adult females is the complete absence of germ cells. In other ovaries, the few surviving germ cells frequently show a morphology typical of primary spermatocytes. stil is not required either for fly viability or for male germline development. When cloned, the gene was found to encode a novel protein. stil is strongly expressed in the female germ cells. Using P[stil+] transgenes, it has been shown that stil and another closely linked gene are involved in the modification of the ovarian phenotypes of the dominant alleles of ovo caused by the heterozygosity of region 49 A-D. The similarity of the mutant phenotypes of stil to that of otu and ovo suggests that the three genes function in common or in parallel pathways necessary in the female germline for survival, sex determination and differentiation. It is unknown whether the strong expression of stil in the female germline is regulated, directly or indirectly, by the X:A ratio or whether stil expression might be controlled by the inductive sex-determining somatic signals (Pennetta, 1997).

Promoters active in the germline produce OVO-A and OVO-B mRNAs encoding isoforms of a putative transcription factor. The isoforms have a common C2H2 zinc-finger domain but different N-termini that include potential effector domains. Single point mutations in three dominant-negative ovoD mutations result in new in-frame initiation codons in OVO-B mRNAs and amino acid substitutions within charged regions of OVO-A proteins. Three lines of evidence suggest that the dominant activity is due to the new initiation codons in OVO-B mRNAs and not the amino acid substitutions in OVO-A. (1) A fourth ovoD allele has been made by inserting a new in-frame AUG. This ovoD4 allele encodes a nearly full-length OVO-A isoform from OVO-B mRNAs. (2) Engineered stop codons in ovoD1 downstream of the new AUG abolish dominant negative activity. (3) A substantial deletion of an OVO-A region encoding a highly charged amino acid domain fully rescues loss-of-function ovo alleles. These data suggest that ovoD mutations result in inappropriate expression of OVO-A in the female germline (Andrews, 1998).

An extreme morphological difference between Drosophila sechellia and related species of the pattern of hairs on first-instar larvae is reported. On the dorsum of most species, the posterior region of the anterior compartment of most segments is covered by a carpet of fine hairs. In D. sechellia, these hairs have been lost and replaced with naked cuticle. Genetic mapping experiments and interspecific complementation tests indicate that this difference is caused, in its entirety, by evolution at the ovo/shaven-baby locus. The pattern of expression of the ovo/shaven-baby transcript is correlated with this morphological change. The altered dorsal cuticle pattern is probably caused by evolution of the cis-regulatory region of ovo/shaven-baby in the D. sechellia lineage (Sucena, 2000).

All species of the D. melanogaster species subgroup, except D. sechellia, possess a dorsal pattern of denticles and hairs similar to that described previously for D. melanogaster. The precise pattern of hairs and denticles varies between segments, and the following description focuses on the abdominal segments. The most anterior cells of the anterior compartment produce naked cuticle. More posteriorly, there are two to three rows of short and thick denticles and then six to eight rows of fine hairs. On the lateral surface of the larvae, fine hairs are also found in the same anterior-posterior domain of each segment as the dorsal hairs. In the posterior compartment, cells of the anterior row secrete naked cuticle, and cells of the posterior row produce large thick denticles. The dorsal cuticle of D. sechellia first-instar larvae differs from the above description primarily by the absence of the lawn of fine hairs both dorsally and laterally. There are other minor variations in trichome patterning between species of the D. melanogaster species subgroup, but they have not been characterized in detail. Phylogenetic analysis suggests that the naked cuticle phenotype has evolved within the D. sechellia lineage (Sucena, 2000).

Examination of the distribution of svb transcripts indicates that cell-specific loss of svb transcription is correlated with the absence of hairs in D. sechellia. In D. melanogaster the svb transcript is detected at lower levels in the cells that differentiate the fine hairs of the dorsum, in contrast to the stronger expression detected in the rows of more robust denticles on both the dorsum and ventrum. In D. sechellia, the svb transcript is detected at high levels in the cells forming the ventral denticle rows and in other trichome-forming cells, including the dorsal denticle rows. The svb transcript was not detected in the dorsal cells that differentiate naked cuticle (Sucena, 2000).

The regulatory nature of the change between species is supported by several observations. (1) Independent regulatory elements drive the ovo and svb functions. The ovo function is required for development of the female germline, and ovo mutants can be rescued by genomic DNA spanning the known exons of ovo. The svb function is required for the differentiation of denticles and hairs in the larvae. Some svb mutants, including breakpoints 5' of the known exons of ovo/svb, are not rescued by the ovo transgene, suggesting that regulatory elements 5' of the known exons are required for svb function. (2) The pattern of naked cuticle and denticles in D. sechellia is not identical to the svb loss-of-function phenotype and instead appears to represent the loss of a subset of the hairs lost in svb mutants. This suggests that the transition to the D. sechellia phenotype was caused by cis-regulatory evolution of part of svb function. Analysis of the distribution of ovo/svb transcripts in embryos of D. melanogaster and D. sechellia suggests that this change occurred at the level of transcriptional control of ovo/svb (Sucena, 2000).

Models of adaptation suggest that single mutational events causing dramatic phenotypic alterations are more likely to be fixed by strong selection and at the beginning of a bout of adaptation. This contrasts with the traditional Darwinian view that large differences arise from the accumulation of many small changes. Although these experiments indicate that a major difference in larval hair patterning is caused by evolution of a single gene, this large change may have resulted from the accumulation of multiple mutations of smaller effect within the cis-regulatory region of ovo/svb. In either case, the results are surprising, but for different reasons. If this morphological transition is caused by a single mutational change, then mutations of relatively large effect must be recognized as contributors to species differences. If, however, the transition is caused by multiple mutations at ovo/svb, then why all of the mutations occur at a single locus and are not distributed among multiple loci must be explained. The latter result would support the idea that a limited number of genes may be available to generate evolution of at least some morphological features. Resolution of this problem requires experiments to identify the individual mutations at ovo/svb that have generated these differences. Given the complexity of the ovo/svb locus and particularly the currently unknown structure of the svb regulatory regions, these experiments will not be trivial, but they are tractable in Drosophila, and they are currently under way (Sucena, 2000).

Ovo/Shavenbaby transcription factor specifies actin remodelling during epidermal differentiation in Drosophila

In Drosophila, differentiation of the epidermis results in a stereotyped array of cells with F-actin-based extensions at their apical face. Ovo/Shavenbaby (Svb) has been identified as a transcription factor that governs changes in epidermal cell shape. Svb is required for the formation of apical extensions and cells deficient in svb differentiate a smooth surface. In both the embryo and the adult, Svb is shown to be necessary and sufficient for the cells to grow extensions; the tight regulation of ovo/svb activity is critical for morphogenesis to occur correctly. Svb triggers early F-actin redistribution and is able to initiate the entire process of cytoskeletal remodelling, thereby defining it as a major regulator of epidermal differentiation (Delon, 2003).

In the germline, alternative ovo promoters control the synthesis of OvoA and OvoB, two protein isoforms with identical DNA-binding domains and different N-termini, which endow OvoA and OvoB with antagonistic transcriptional activities. The somatic svb function, distinct from both OvoA and OvoB, corresponds to a separate promoter that generates mRNA that differs from ovo transcripts only by its first exon. The predicted Svb protein is thus identical to OvoA, except for an additional N-terminal extension. Svb acts in a similar way to the OvoB transcriptional activator. Indeed, either Svb or OvoB can promote formation of embryonic denticles or wing hairs, whereas the OvoA repressor behaves as a dominant-negative form in the soma. Compared to Svb, OvoB misexpression produces even stronger phenotypes, probably reflecting the activity of the 2b extension, a germline-specific protein domain believed to increase protein stability. Together, these data indicate that Svb acts as a transcriptional activator in the soma. Therefore, it is proposed that the alternative use of three ovo/svb promoters and AUGs leads to progressive addition of N-terminal extensions to the same activator protein module (OvoB), which turns it first into a repressor (OvoA) and then again into an activator (Svb). While it does not present similarity with known protein domains, the region specific to the predicted Svb protein does, however, display strong sequence conservation between distant insect species. How this protein region could be able to restore activator function remains to be elucidated (Delon, 2003).

In addition to the production of different isoforms, one critical feature of ovo/svb resides in the exquisite regulation of its expression. Embryonic epidermal morphogenesis depends on the transcriptional status of svb, which is controlled by Wg and Egfr signalling pathways. Spatial control of svb expression is thus decisive for proper epidermal differentiation in the embryo. Confirming this conclusion, the evolution of svb expression has been shown to account for the morphological diversity in trichome patterns observed between insect species. This analysis of Svb function during adult morphogenesis now provides evidence that svb expression must also be tightly regulated in quantitative and temporal terms (Delon, 2003).

Svb thus appears to be a major player during epidermal differentiation, specifying in a cell-autonomous fashion, from the embryo to the adult, cytoskeletal reorganization. Interestingly, a mouse ortholog, m-ovo1, has also been demonstrated to play a role in epidermal differentiation. Although epidermal differentiation in fly and mammals involves different morphological processes, molecular analysis pleads in favour of the evolutionary conservation of several mechanisms. In addition to the dependence on ovo/svb, as in Drosophila, criss-cross interplay between Wnt and hedgehog pathways organizes vertebrate epidermal differentiation, including formation of hairs and feathers. ß-Catenin-dependent regulation of ovo/svb transcription by the Wnt pathway is also conserved in mammals. Furthermore, recent findings have shown the importance of F-actin dynamics for epidermal morphogenesis in mice. A major challenge remains in the elucidation of the mechanisms whereby regulation of svb activity results in cytoskeleton reorganization in these different model systems (Delon, 2003).

During embryonic epidermal morphogenesis, Wg activity results in cells adopting a smooth apical surface, suggesting a direct effect of the Wg pathway on cytoskeletal remodelling. Consistent with this interpretation, dAPC-1 and dAPC-2, two paralogous proteins that display overlapping roles in the down regulation of Wg signalling through shuttling between nucleus and cytoplasm, associate with the cytoskeleton in different cell contexts. In the epidermis, dAPC-2 (E-APC) localizes at the basis of actin bundles that support cell extensions. Since dAPC2 mutant embryos lack ventral denticles, dAPC2 has been suggested to be a direct effector of Wg on cytoskeleton dynamics. However, svb is shown to be the determinant of cytoskeletal reorganization that leads to F-actin bundling during epidermal morphogenesis. Furthermore, Svb expression is necessary and sufficient (regardless to Wg activity) to localize dAPC-2 protein at the base of the apical actin-rich extensions. This shows that dAPC-2 is not primarily directed to actin bundles through Wg signalling. It also strongly suggests that the loss of denticles in dAPC2 embryos results from overactivity of the Wg pathway, which represses svb transcription. This interpretation is further supported by the fact that, as in case of ectopic activation of Wg signalling, dAPC2 mutations do not prevent formation of dorsal trichomes, which is dependent upon svb activity. Thus, dAPC2 and svb display two distinct functional interactions during denticle morphogenesis: (1) cytoplasmic dAPC2 acts to inhibit the signalling activity of ß-catenin that represses svb transcription; (2) when svb is expressed in epidermal cells, svb activity triggers F-actin bundling and redirects a pool of dAPC2 protein to the base of microfilament bundles. These data demonstrate that, rather than acting directly on F-actin dynamics, the Wg pathway acts through a ß-catenin/TCF-dependent signalling pathway culminating, in the nucleus, in the regulation of svb transcription during epidermal morphogenesis. A growing accumulation of evidence also supports the theory that APC proteins have Wnt independent roles in cytoskeletal regulation during the Drosophila development, such as spindle anchoring in syncytial embryos, cell adhesion, and larval brain development. Further elucidation of a putative Wg-independent function of dAPC2 in F-actin dynamics during epidermal differentiation now awaits uncoupling of its signalling activity from its association with the cytoskeleton (Delon, 2003).

In addition to its role in embryogenesis, svb is also involved in the formation of the various apical cell extensions that develop during the differentiation of adult epidermis. Inactivation of svb, through specific mutations or misexpression of the OvoA repressor that acts as a dominant-negative form, prevents hair and trichome formation, resulting in cells displaying an abnormal smooth apical surface. Moreover, the level of svb activity must be finely regulated to achieve a correct epidermal differentiation, since the reduction to half the dose of svb (hemizygous conditions) leads to shorter and sparser wing hairs whereas increasing svb activity results in longer wing hairs. That increased, or longer, svb expression increases in turn the length of wing hairs suggests that svb is not only required for the initiation, but also that it is involved in the growth of wing prehairs. Furthermore, when svb overexpression is induced at early stages of wing development, it results in the formation of numerous ectopic prehairs per cell. It has recently been shown that mutations that lead to the formation of giant wing cells, with a high polyploidy presumably resulting from cell fusion or abnormal divisions, display multiple hairs. In the case of svb, multiple hairs are seen even in small clones of cells (2/4) overexpressing svb, thus suggesting that the formation of ectopic hairs in these cells occurred via a different mechanism. Aside from their role in planar cell polarity (PCP), which determines that prehairs point toward the distal part of the wing, members of the frizzled (fz) pathway participate in the specification of the site of actin bundling in developing wing cells. Activation of the Fz cascade results in the polarized localization of Fz itself, Dsh and the unconventional cadherin Starry night/Flamingo (Stan/Fmi). Inactivation of PCP members abolish polarized accumulation of Fz, Dsh and Stan/Fmi, leading to the formation of randomly oriented, centrally positioned F-actin prehairs. Interestingly, mutations of dsh, as well as a reduced activity of the downstream member rhoA, led to some duplicated prehairs, with a random orientation. By contrast, svb gain-of-function does not alter prehair polarity and svb-induced ectopic prehairs retain a distal location and polarity. Accordingly, neither inactivation nor overactivity of svb disrupts these early phases of PCP, since the polarized localization of Fmi at the distal edge is not affected by alterations of svb. A model is proposed in which the Fz cascade acts first to establish spatial coordinates and asymmetrical organization of the cytoskeleton. The cellular location where svb promotes cell extension is determined by polarity cues. When svb overactivity is induced early, presumably before the definitive restriction of F-actin bundling to a single point, it leads to the production of numerous prehairs. Then, when the sophisticated mechanisms of single-hair restriction are set up, svb overactivity induces longer prehairs. Further analysis of the relationships between svb and downstream members of the PCP pathway is underway (Delon, 2003).

Drosophila wing hair formation constitutes a powerful genetic system in which to analyse actin-based morphogenesis and a major challenge is to understand what triggers the growth of F-actin prehairs. Although loss-of-function mutations in dozens of genes lead to duplicated and/or split hair phenotypes, no genes other than svb have been identified genetically as being required for growth of wing hairs (aside from shavenoid, a mutation not yet molecularly characterized). Several conclusions can thus be drawn from these data. (1) Most genes identified so far are likely to act in the spatial and temporal restriction of the initiation of actin bundling in the PCP pathway. The complex interactions among components of the PCP pathway make each member indispensable, thus facilitating its phenotypic identification. (2) Actin reorganization during wing hair formation appears to involve players different from those identified for the motility of Acanthamoeba or bacterial pathogens. While dominant-negative forms of Cdc42 and Rac alter wing hair formation, these findings have not been supported by genetic analysis. Cells deprived of the three Drosophila Rac proteins (Rac1, 2 and Mtl) form wild-type hairs, and those mutant for cdc42 die. In addition, mediators of the action of Cdc42 on actin reorganization, such as Scar or Arp2/3 subunits are not required for wing hair formation. This favors the hypothesis that actin dynamics occurring during relatively slow developmental processes are based on hitherto unknown mechanisms. (3) The rarity of mutations that prevent wing hair formation may be due to the fact that this process requires the action of numerous genes, the absence of any one being either dispensable, or resulting in subtle phenotypes that can only be detected at a sophisticated level of analysis. Interestingly, although Profilin and Slingshot are two proteins that are essential for the control of actin polymerization/depolymerization, mutations in both genes alter the final shape of trichomes but do not prevent their formation (Delon, 2003).

Several lines of evidence suggest that svb can coordinate the expression of the set of genes needed to achieve the formation of actin-based apical cell extensions. svb is required for hair formation in the various tissues analysed and the extension growth is dependent on the level of svb activity. In addition, ectopic expression of svb can trigger the formation of apical extensions in cells that normally present a smooth surface in both the embryo and adult. This would imply that, in spite of their differences in morphology, the different extensions affected by svb mutations are formed, at least in part, via similar mechanisms. Supporting this prediction, svb is involved both in arista lateral and bristle formation, two structures that display ultra-structural similarities, with separate bundles of parallel microfilaments supporting the cell extension. Moreover, recent work on arista morphogenesis has demonstrated that formation of laterals requires many players involved in wing hair formation, including components of the Fz pathway. Finally, that several mutations affecting either adult trichome or bristle morphology also alter the shape of embryonic trichomes further supports the idea that many functional links exists between these processes. In conclusion, this study provides the demonstration of the coordinating role of the Svb transcription factor in the formation of F-Actin-rich apical extensions during development. Its analysis might provide an invaluable entry point to an understanding of F-actin dynamics during developmental morphogenesis (Delon, 2003).

Planar polarization of the denticle field in the Drosophila embryo: roles for Myosin II (zipper) and fringe

Epithelial planar cell polarity (PCP) allows epithelial cells to coordinate their development to that of the tissue in which they reside. The mechanisms that impart PCP as well as effectors that execute the polarizing instructions are being sought in many tissues. The epidermal epithelium of Drosophila embryos exhibits PCP. Cells of the prospective denticle field, but not the adjacent smooth field, align precisely. This requires Myosin II (zipper) function, and it was found that Myosin II is enriched in a bipolar manner, across the parasegment, on both smooth and denticle field cells during denticle field alignment. This implies that actomyosin contractility, in combination with denticle-field-specific effectors, helps execute the cell rearrangements involved. In addition to this parasegment-wide polarity, prospective denticle field cells express an asymmetry, uniquely recognizing one cell edge over others as these cells uniquely position their actin-based protrusions (ABPs; which comprise each denticle) at their posterior edge. Cells of the prospective smooth field appear to be lacking proper effectors to elicit this unipolar response. Lastly, fringe function was identified as a necessary effector for high fidelity placement of ABPs and it was shown that Myosin II (zipper) activity is necessary for ABP placement and shaping as well (Walters, 2006).

Since the prospective denticle field is clearly polarized, it was wondered if smooth field cells were similarly polarized but simply did not express a marker, such as the ABPs, that revealed that polarity. To test this, the formation of ABPs among prospective smooth cells was induced by ectopically expressing the transcription factor svb/ovo in small groups of cells in the ventral epidermis and these cells were marked by co-expression of GFP. Expression of svb/ovo is necessary and sufficient to induce the formation of ABPs, so by expressing svb/ovo in the smooth field, the localization of these protrusions can be visualized within the cell. When svb/ovo is induced in the smooth field, the ectopic ABPs did not preferentially localize to the posterior edge of cells. Instead, they showed a stochastic dispersal around the apical surface of the cell Both anti-phosphotyrosine and phalloidin stains label these misplaced ABPs, indicating that phospho-epitopes as well as actin are present. Out of the thirty-eight ectopic denticles scored, 63% were mis-positioned (nine on an anterior edge, fifteen placed centrally) and only 37% were localized to the posterior edge of the cell (Walters, 2006).

It is also possible that the stochastic ABP localization observed in the svb/ovo-positive cells was not due to the lack of polarity effectors in the smooth field, but instead a simple issue of developmental timing. Since a heat-shock-driven recombinase was used to induce svb/ovo expression, precise control over the timing of the recombination event or svb/ovo induction was not occuring. Attempts were made to remedy this concern by ectopically expressing svb/ovo using Ptc-GAL4. Since patched is expressed well before cell fate specification, any ectopic ABPs that are present should have had ample time to localize to the posterior cell edge. However, even when svb/ovo is expressed at this early time point, ABPs in the smooth field showed no preference for the posterior edge of cells and remained stochastically positioned. These data strongly suggest that smooth cells do not possess a latent ability to place ABPs with the unipolar asymmetry seen among prospective denticle field cells. At the minimum, an effector of unipolar asymmetry must be active only among prospective denticle field cells. Alternatively, unipolar asymmetry is established only late and is restricted to the prospective denticle field (Walters, 2006).

crinkled reveals a new role for Wingless signaling in Drosophila denticle formation

The specification of the body plan in vertebrates and invertebrates is controlled by a variety of cell signaling pathways, but how signaling output is translated into morphogenesis is an ongoing question. This study describes genetic interactions between the Wingless (Wg) signaling pathway and a nonmuscle myosin heavy chain, encoded by the crinkled (ck) locus in Drosophila. In a screen for mutations that modify wg loss-of-function phenotypes, multiple independent alleles of ck were isolated. These ck mutations dramatically alter the morphology of the hook-shaped denticles that decorate the ventral surface of the wg mutant larval cuticle. In an otherwise wild-type background, ck mutations do not significantly alter denticle morphology, suggesting a specific interaction with Wg-mediated aspects of epidermal patterning. This study shows that changing the level of Wg activity changes the structure of actin bundles during denticle formation in ck mutants. It was further found that regulation of the Wg target gene, shaven-baby (svb), and of its transcriptional targets, miniature (m) and forked (f), modulates this ck-dependent process. It is concluded that Ck acts in concert with Wg targets to orchestrate the proper shaping of denticles in the Drosophila embryonic epidermis (Bejsovec, 2012).

The ventral epidermis of Drosophila embryos is a well-established system for studying cell fate specification. At the end of embryogenesis, epidermal cells secrete a patterned array of cuticular structures that reflect the cell identities acquired in the epidermis at earlier stages of development. On the ventral surface of the larval abdomen, eight segmental belts of hook-shaped denticles alternate with expanses of flat, or naked, cuticle. Each belt contains roughly six rows of denticles, with each row displaying a characteristic size, shape and polarity. These distinct morphologies indicate unique positional values, at least some of which are imparted by signal transduction from the highly conserved Wg/Wnt growth factor pathway. During early embryogenesis, a cascade of transcription factors leads to activation of wg gene expression in segmental stripes that lie within the zone of cells that will secrete naked cuticle. Ectopic overexpression of wg across the segment, or hyperactivation of downstream components in the Wg signaling pathway, eliminates the denticle belts. Conversely, loss of wg activity causes all ventral epidermal cells to secrete denticles. The diversity of denticles is also reduced in wg null mutants, with most resembling the large denticles typical of the fifth row of the wild-type belt. Thus Wg signaling controls not only the segmental specification of naked cuticle expanses, but also generates the diversity of cell fates that give rise to the uniquely shaped denticles within the denticle belt (Bejsovec, 2012).

Denticles are formed by bundles of actin that accumulate apically and push out the apical membrane as they elongate. Incipient denticles first can be visualized as apical actin condensations in the ventral epidermal cells of stage 13 embryos, at roughly 10 hours after egg-laying (AEL). These actin condensations form preferentially along the posterior edge of the columnar epithelial cells, and over the next 2 hours become increasingly more organized and begin to elongate; during this elongation phase, microtubules become enriched at the base of the denticle and also within the core of the growing denticle. The mechanism by which the distinctive shapes of the denticles are specified is not well understood, but it requires Wg signaling between 4 and 6 hours AEL (Bejsovec, 2012).

This early phase of Wg activity stabilizes expression of engrailed (en) and its target, hedgehog (hh), in the adjacent row of cells. Wg and Hh signaling together control the expression of Serrate and rhomboid, which activate the Notch and EGF pathways, respectively, in defined rows within the segment; these gene activities are required to specify the diverse denticle types characteristic of a wild-type denticle belt (Bejsovec, 2012).

The organization of the actin-based denticle precursors and their transition to cuticular elements is directed by a set of structural proteins whose expression is controlled by the Wg-regulated transcription factor, Shaven-baby (Svb). Wg signaling represses svb, restricting its ventral expression to the domain of cells fated to secrete denticles. Ectopic svb expression in the naked region of the embryonic epidermis drives formation of apical actin extensions and subsequent production of ectopic denticles. A number of downstream targets of Svb have been identified; these include genes such as singed (sn) and forked, which encode known actin-remodeling proteins, and miniature, which encodes a membrane-anchored extracellular protein thought to mediate interaction between the cell membrane and the cuticle. However, the question remains as to how these structural proteins are deployed to form the distinct morphologies characteristic of each row of denticles. This study shows that the cytoplasmic myosin, Crinkled, interacts genetically with the Wg signaling pathway and plays a role in organizing the final shapes of the denticles during epidermal development (Bejsovec, 2012).

A genetic screen for modifiers of wg mutant phenotypes revealed an unexpected interaction between Wg signaling and the cytoplasmic myosinVIIA homolog, Ck, in shaping the denticles at late stages of embryonic development. Like other myosins, Ck/myosinVIIA has a typical actin-binding/ATPase head domain that mediates movement along actin filaments. However, the carboxy-terminus of Ck/myosinVIIA is unique in containing an SH3 domain and two FERM domains, which are shared by band 4.1, ezrin, radixin, moesin -- a family of proteins that link the actin cytoskeleton to membrane spanning proteins. These motifs are consistent with a role near the plasma membrane, possibly interacting with cell-surface receptors and/or adherens junctions. This raises the possibility that Ck may be involved in the association between the actin bundles of the incipient denticles and the apical membrane, where it could link the actin cytoskeleton to extracellular components of the cuticle through transmembrane proteins such as Miniature. Either loss or gain of function for the Wg target gene svb alters denticle morphology in the ck mutant background. Therefore, it is proposed that Ck myosin may help to distribute the products of some Svb target genes, such as Miniature, and thus facilitate the final morphology of the developing denticle. Genetic data suggest that wild-type Ck provides a buffering mechanism for the incorrect Svb target levels that accumulate in a wg null mutant (Bejsovec, 2012).

Wg signaling is most commonly associated with specifying the naked cuticle cell fate, but its other role in generating diverse denticle morphologies allowed requirements for Ck function in this process to be detected. The morphogenesis role requires lower levels of Wg signaling, as evidenced by weak mutations such as wgPE2 that can generate diversity but cannot specify naked cuticle cell fate. The finding that levels of svb and its target genes influence denticle morphology suggests that Wg signaling may generate denticle diversity in conjunction with Notch and EGF signaling by producing subtly graded differences in svb expression that are below the limits of current detection methods. Temperature shift experiments suggest that this is a continuing, independent role for Wg signaling, as it is detected after the point at which Wg input regulates the pattern of Serrate and rhomboid expression. It is proposed that late Wg signaling helps titrate the synthesis of svb target gene products to optimal levels required for shaping the denticles. The ck mutant provides a sensitized background that may allow further investigation of this possibility. The enhancement of denticle morphology defects along the dorsolateral edges of the denticle field also suggests input from dorsoventral patterning pathways, such as Dpp signaling (Bejsovec, 2012).

MyosinVIIA in humans is known to play a crucial role in hearing (reviewed by Hasson, 1999; Maniak, 2001; Petit, 2001; Dror, 2009). Stereocilia on the hair cells of the inner ear transduce the mechanical stimulation of sound waves into electrical impulses. Stereocilia are stabilized by bundles of actin filaments and microtubules, much like the denticles and bristles that decorate the fly epidermis. Mutations in the human myosinVIIA are associated with Usher syndrome, the most common hereditary deafness/blindness disorder, which results in disorganized stereocilia that cannot transduce sound. The precise role of myosinVIIA in organizing and maintaining these structures is as yet unknown. However, ck mutants in the fly also are deaf, and show morphological changes in the auditory sensory structures (Todi, 2005), suggesting that the fly is a powerful model system for exploring this aspect of myosinVIIA function. Indeed, the connection between Ck and Miniature may also be relevant to human hearing disorders. Mutations in α-tectorin, a human protein that shares functional domains with Miniature and organizes extracellular matrix in the cochlea, are associated with hereditary hearing loss (Bejsovec, 2012).


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).


Search PubMed for articles about Drosophila ovo

Andrews, J., Levenson, I. and Oliver, B. (1998). New AUG initiation codons in a long 5' UTR create four dominant negative alleles of the Drosophila C2H2 zinc-finger gene ovo. Dev. Genes Evol. 207(7): 482-7. PubMed Citation: 10648246

Andrew, D. J. and Baker, B. S. (2008). Expression of the Drosophila secreted cuticle protein 73 (dsc73) requires Shavenbaby. Dev. Dyn. 237(4): 1198-206. PubMed Citation: 18351665

Andrews, J., et al. (2000). OVO transcription factors function antagonistically in the Drosophila female germline. Development 127: 881-892. PubMed Citation: 10648246

Bejsovec, A, and Chao, A. T. (2012). crinkled reveals a new role for Wingless signaling in Drosophila denticle formation. Development 139(4): 690-8. PubMed Citation: 22219350

Bielinska, B., Lu, J., Sturgill, D. and Oliver, B. (2005). Core promoter sequences contribute to ovo-B regulation in the Drosophila melanogaster germline. Genetics 169(1): 161-72. 15371353

Bohere, J., Mancheno-Ferris, A., Al Hayek, S., Zanet, J., Valenti, P., Akino, K., Yamabe, Y., Inagaki, S., Chanut-Delalande, H., Plaza, S., Kageyama, Y., Osman, D., Polesello, C. and Payre, F. (2018). Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis. Nat Commun 9(1): 5123. PubMed ID: 12925706

Cai, H. N., and Shen, P. (2001). Effects of cis arrangement of chromatin insulators on enhancer-blocking activity. Science 291: 493-495. PubMed Citation: 11161205

Chanut-Delalande, H., et al. (2006). Shavenbaby couples patterning to epidermal cell shape control. PLoS Biol. 4(9): e290. Medline abstract: 16933974

Chanut-Delalande, H., Hashimoto, Y., Pelissier-Monier, A., Spokony, R., Dib, A., Kondo, T., Bohere, J., Niimi, K., Latapie, Y., Inagaki, S., Dubois, L., Valenti, P., Polesello, C., Kobayashi, S., Moussian, B., White, K. P., Plaza, S., Kageyama, Y. and Payre, F. (2014). Pri peptides are mediators of ecdysone for the temporal control of development. Nat Cell Biol 16: 1035-1044. PubMed ID: 25344753

Crocker, J., Abe, N., Rinaldi, L., McGregor, A. P., Frankel, N., Wang, S., Alsawadi, A., Valenti, P., Plaza, S., Payre, F., Mann, R. S. and Stern, D. L. (2015). Low affinity binding site clusters confer hox specificity and regulatory robustness. Cell 160: 191-203. PubMed ID: 25557079

Dai, X., et al. (1998). The ovo gene required for cuticle formation and oogenesis in flies is involved in hair formation and spermatogenesis in mice. Genes Dev. 12(21): 3452-63. PubMed Citation: 9808631

Dej, K. J., Gerasimova, T., Corces, V. G. and Boeke, J. D. (1998) A hotspot for the Drosophila gypsy retroelement in the ovo locus. Nucleic Acids Res. 26: 4019-4025. PubMed Citation: 9705514

Delon, I., Chanut-Delalande, H. and Payre, F. (2003). The Ovo/Shavenbaby transcription factor specifies actin remodelling during epidermal differentiation in Drosophila. Mech. Dev. 120: 747-758. 12915226

Frankel, N., et al. (2010). Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature 466(7305): 490-3. PubMed Citation: 20512118

Garfinkel, M. D., Lohe, A. R. and Mahowald, A. P. (1992). Molecular genetics of the Drosophila melanogaster ovo locus, a gene required for sex determination of germline cells. Genetics 130: 791-803. PubMed Citation: 1349870

Garfinkel, M. D., et al. (1994). Multiple products from the shavenbaby-ovo gene region of Drosophila melanogaster: relationship to genetic complexity. Mol. Cell. Biol. 14: 6809-6818. PubMed Citation: 7935398

Gerasimova, T. I., Byrd, K. and Corces, V. G. (2000). A chromatin insulator determines the nuclear localization of DNA. Mol. Cell 6: 1025-1035. PubMed Citation: 11106742

Hinson, S. and Nagoshi, R. N. (1999). Regulatory and functional interactions between the somatic sex regulatory gene transformer and the germline genes ovo and ovarian tumor. Development 126: 861-871. PubMed Citation: 9927588

Johnson, A. D., et al. (2001). EGL-38 Pax regulates the ovo-related gene lin-48 during Caenorhabditis elegans organ development. Development 128: 2857-2865. 11532910

Khila, A., El-Haidani, A., Vincent, A., Payre, F., Ibn-Souda, S. (2003). The dual function of ovo/shavenbaby in germline and epidermis differentiation is conserved between Drosophila melanogaster and the olive fruit fly Bactrocera oleae. Insect Biochem. Mol. Biol. 33(7): 691-9. 12826096

Kondo, T., Plaza, S., Zanet, J., Benrabah, E., Valenti, P., Hashimoto, Y., Kobayashi, S., Payre, F. and Kageyama, Y. (2010). Small peptides switch the transcriptional activity of Shavenbaby during Drosophila embryogenesis. Science 329: 336-339. PubMed ID: 20647469

Kuhn, E. J., et al. (2003). A test of insulator interactions in Drosophila. EMBO J. 22: 2463-2471. PubMed Citation: 12743040

Labrador, M. and Corces, V. G. (2001). Protein determinants of insertional specificity for the Drosophila gypsy retrovirus. Genetics 158: 1101-1110. PubMed Citation: 11454759

Labrador, M., Sha, K., Li, A., Corces, V. G. (2008). Insulator and Ovo proteins determine the frequency and specificity of insertion of the gypsy retrotransposon in Drosophila melanogaster. Genetics 180: 1367-1378. PubMed Citation: 18791225

Lee, S. and Garfinkel, M. D. (2000). Characterization of Drosophila OVO protein DNA binding specificity using random DNA oligomer selection suggests zinc finger degeneration. Nucleic Acids Res. 28(3): 826-34. 10637336

Li, B. et al. (2002a). Ovol2, a mammalian homolog of Drosophila ovo: gene structure, chromosomal mapping, and aberrant expression in blind-sterile mice. Genomics 80: 319-325. 12213202

Li, B., et al. (2002b). The LEF1/beta-catenin complex activates movo1, a mouse homolog of Drosophila ovo required for epidermal appendage differentiation. Proc. Natl Acad. Sci. 99: 6064-6069. 11983900

Li, B., et al. (2005). Ovol1 regulates meiotic pachytene progression during spermatogenesis by repressing Id2 expression. Development 132(6): 1463-73. 15716349

Lu, J., Andrews, J., Pauli, D. and Oliver, B. (1998). Drosophila OVO zinc-finger protein regulates ovo and ovarian tumor target promoters. Dev. Genes Evol. 208(4): 213-22. PubMed Citation: 9634487

Lu, J. and Oliver, B. (2001). Drosophila OVO regulates ovarian tumor transcription by binding unusually near the transcription start site. Development 128: 1671-1686. 11290304

Masu, Y., et al. (1998). Expression of murine novel zinc finger proteins highly homologous to Drosophila ovo gene product in testis. FEBS Lett. 421(3): 224-8. PubMed Citation: 9468311

Mével-Ninio, M., Terracol, R. and Kafatos, F. C. (1991). The ovo gene of Drosophila encodes a zinc finger protein required for female germ line development. EMBO J 10: 2259-66. PubMed Citation: 1712294

Mével-Ninio, M., et al. (1995). ovo, a Drosophila gene required for ovarian development, is specifically expressed in the germline and shares most of its coding sequences with shavenbaby, a gene involved in embryo patterning. Mech Dev 49: 83-95. PubMed Citation: 7748792

Mével-Ninio, M., et al. (1996). The three dominant female-sterile mutations of the Drosophila ovo gene are point mutations that create new translation-initiator AUG codons. Dev. Biol 122: 4131-38. PubMed Citation: 9012532

McGregor, A. P., et al. (2007). Morphological evolution through multiple cis-regulatory mutations at a single gene. Nature 448: 587-590. PubMed Citation: 17632547

Menoret, D., Santolini, M., Fernandes, I., Spokony, R., Zanet, J., Gonzalez, I., Latapie, Y., Ferrer, P., Rouault, H., White, K. P., Besse, P., Hakim, V., Aerts, S., Payre, F. and Plaza, S. (2013). Genome-wide analyses of Shavenbaby target genes reveals distinct features of enhancer organization. Genome Biol 14: R86. PubMed ID: 23972280

Muravyova, E., et al. (2001). Loss of insulator activity by paired Su(Hw) chromatin insulators. Science 291: 495-498. PubMed Citation: 11161206

Nagoshi, R. N., et al. (1995). The somatic sex determines the requirement for ovarian tumor gene activity in the proliferation of the Drosophila germline. Development 121: 579-587. PubMed Citation: 7768194

Oliver, B., Perrimon, N. and Mahowald, A. P. (1987). The ovo locus is required for sex-specific germ line maintenance in Drosophila. Genes Dev 1: 913-23. 88112833

Oliver, B., Kim, K.-J. and Baker, B. S. (1993). Sex-lethal, master and slave: a hierarchy of germ-line sex determination in Drosophila. Development 119: 897-908. PubMed Citation: 8187645

Oliver, B., et al. (1994). Function of Drosophila ovo+ in germ-line sex determination depends on X-chromosome number. Development 120: 3185-3195. PubMed Citation: 7720561

Overton, P. M., Chia, W. and Buescher, M (2007). The Drosophila HMG-domain proteins SoxNeuro and Dichaete direct trichome formation via the activation of shavenbaby and the restriction of Wingless pathway activity. Development 134(15): 2807-13 . PubMed citation; Online text

Pauli, D., Oliver, B. and Mahowald, A. P. (1993). The role of the ovarian tumor locus in Drosophila melanogaster germ line sex determination. Development 119: 123-34. PubMed Citation: 8275850

Pennetta, G. and Pauli, D. (1997). stand still, a Drosophila gene involved in the female germline for proper survival, sex determination and differentiation. Genetics 145 (4): 975-987. PubMed Citation: 9093851

Piloto, S. and Schilling, T. F. (2010). Ovo1 links Wnt signaling with N-cadherin localization during neural crest migration. Development 137(12): 1981-90. PubMed Citation: 20463035

Prud'homme, N., et al. (1995). Flamenco, a gene controlling the gypsy retrovirus of Drosophila melanogaster. Genetics 139: 697-711. PubMed Citation: 7713426

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Renaud, S. J., Chakraborty, D., Mason, C. W., Rumi, M. A., Vivian, J. L. and Soares, M. J. (2015). OVO-like 1 regulates progenitor cell fate in human trophoblast development. Proc Natl Acad Sci U S A 112: E6175-6184. PubMed ID: 26504231

Roch, F., Alonso, C. R. and Akam, M. (2003). Drosophila miniature and dusky encode ZP proteins required for cytoskeletal reorganisation during wing morphogenesis. J Cell Sci. 116: 1199-1207. Medline abstract: 12615963

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