ovo

DEVELOPMENTAL BIOLOGY

Embryonic

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 ovo^D1rv23 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 ovo^D1rv23 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 ovo^D1rv23 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 ovo^D1rv23 homozygous embryos. This indicates that romano sequences directing transcription termination and polyadenylation in ovo^D1rv23 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 ovo^D1rv23 is considered as a null mutation, the results strongly suggest the existence of a residual antimorphic activity in the ovo^D1rv23 females. This activity is most probably provided by the Ovo^D1-2B 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 Ovo^D1-2B 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 ovo^D1rv23 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).

Effects of Mutation or Deletion

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).

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date revised: 10 April 2008

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