svb Directs m Expression in the Embryonic Epidermis

As a first step towards identifying players involved in denticle formation, we searched for genes expressed in epidermal cells by conducting a systematic survey of expression patterns available from the Berkeley Drosophila Genome Project [27] and the literature (Table S1). Among the approximately 400 genes reported to be transcribed in the epidermis, we found that only a small number (<10%) are expressed in a segmentally repeated pattern, at a time of epidermal cells morphological differentiation. We further analyzed the epidermal expression of these genes by in situ hybridization and tested for its dependence on svb (Table S1). We first focused on m [28], because its expression in stage-15 embryos strikingly resembles the trichome pattern, which is specified by svb at previous stages (Figure 1B).

We found that m expression in epidermal cells is abolished in svb mutant embryos (Figure1B), showing that svb is required for m transcription in the epidermis. Detailed examination (Figure 1C) 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 [13], able to counteract svb activity when its expression is artificially directed in the epidermis [13,14]. When expressed in ptc-expressing cells (see Figure 1A), OvoA leads to the replacement of denticle rows 2 and 3 by a stripe of naked cuticle interrupting each denticle belt (Figure 1C). 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, we examined the consequence of the ectopic expression of svb in smooth cells. Ectopic expression of svb in wg-expressing cells (Figure 1A) causes the formation of a supernumerary row of cuticular extensions and leads to an ectopic stripe of m expression (Figure 1C), showing that svb is sufficient to trigger m transcription in epidermal cells.

As the evolutionary loss of dorsal trichomes in D. sechellia embryos is the result of the restriction of svb expression to narrow stripes in dorsal epidermal cells [16], we asked whether m expression was modified accordingly in D. sechellia. When compared to D. melanogaster, m expression in D. sechellia embryos is indeed seen to be restricted to narrow bands in the dorsal region. These data thus bring independent support to the conclusion that m expression is controlled by svb in the embryonic epidermis and show that this regulation has been evolutionarily conserved.

All together, our results demonstrate that svb activity is necessary and sufficient to control m expression specifically in epidermal cells producing denticle and dorsal hairs.

Shavenbaby Directly Controls Miniature Transcription

To investigate whether svb directly regulates m expression, we first attempted to identify cis-regulatory elements directing m transcription in the embryonic epidermis.

We generated transgenic lines in which a reporter gene is placed under the control of a 6-kilobase (kb) genomic region encompassing the m transcriptional start site (6Kmin) (see Figure 2A). These 6Kmin reporter transgenes faithfully reproduce m expression in late embryos (Figure 2B and 2C) and display a similar dependence on svb activity (unpublished data). Deletion of most intronic sequences to a 3-kb fragment leads to a strong diminution of the expression (Figure 2D), showing that cis-regulatory elements required for m epidermal expression are located in intronic regions. We focused on a 0.4-kb region from the second m intron, which is highly conserved in Drosophilidae (Figure 2A). This fragment, when placed upstream of a minimal promoter, promotes a robust expression in the epidermis (Figure 2E), similar to that of the endogenous m gene (Figure 2B–2H). Expression of 0.4Kmin lines is lost in a svb mutant background (Figure 2F), whereas an additional stripe of expression appears when svb is ectopically expressed in smooth cells (Figure 2G). Therefore, this 0.4-kb region of the m gene behaves as a svb-responsive element, able to drive epidermal expression under the control of svb.

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Figure 2. An Evolutionarily Conserved Enhancer Directs the svb-Dependent Expression of m

(A) Evolutionary conservation of the m locus and summary of transgenic reporter constructs. Transcribed regions of m span more than 15kb and harbor an unrelated gene, CG9360, transcribed from the complementary stand. Histograms plot the level of sequence conservation between D. melanogaster and D. pseudoobscura (top) or D. virilis (bottom), as represented from the Vista Genome Browser package. Red, light blue, and dark blue peaks correspond to conserved sequences in non-coding regions, 3′ and 5′ UTR, and translated sequences, respectively. Genomic regions displaying high evolutionary conservation were fused with reporter LacZ genes, encoding either a cytoplasmic (6Kmin and 3Kmin) or nuclear (0.4Kmin) β-gal enzyme, and used to generate transgenic lines.

(B and C) Compared to m mRNA (B), 6Kmin constructs reproduce endogenous m expression (C).

(D) Deletion of intronic sequences leads to a strong reduction of staining in 3Kmin constructs.

(E) The 0.4Kmin construct drives m-like epidermal expression at a high level. The white box indicates the ventral region selected for the close-up presented in panel (H).

(F and G) Like the endogenous gene, this enhancer is responsive to svb, since staining is absent in svb mutants (F) and additional stripes are produced after svb ectopic expression (arrowheads) (G).

(H) Close up of the ventral region (segments A3–A5) of a 0.4Kmin embryo, showing that the β-gal reporter (red) is co-expressed with endogenous Miniature protein (green) in epidermal cells.

(I and J) Electrophoresis mobility shift assays show the specific binding of the Svb protein to wild-type m enhancer. Introduction of 2 point mutations prevents in vitro binding (I) and leads to an inactive enhancer when assayed in vivo (J). Sequence of the Svb binding site (capital letters) and introduced mutations (red) are indicated.

https://doi.org/10.1371/journal.pbio.0040290.g002

We then assayed the ability of the Svb protein to bind in vitro to the corresponding DNA region. Gel retardation assays showed that the recombinant Svb protein binds specifically to the 0.4-kb m element through a single region, further refined to a 200-base pair (bp) fragment (Figure 2I). Sequence analysis revealed the presence of a nucleotide motif matching (7/7) the consensus binding site previously defined for Ovo proteins [29], which share the same DNA binding domain with Svb [12]. Additional DNA-binding experiments showed that Svb binds to this predicted site with a high affinity (estimated Kd = 2.5 ± −0.8 nM, unpublished data). The sequence of this Svb binding site on the m enhancer (at position chrX:11487686, on the genomic scaffold) is conserved in all sequenced Drosophila species. Introduction of point mutations that substitute two invariant nucleotides found in all Ovo binding sites [29] eliminates Svb binding in vitro (Figure 2I), showing that Svb interacts specifically with the m enhancer through this single binding-site. To test the consequence of preventing Svb binding in vivo, we introduced these two mutations into the 0.4-kb enhancer construct and generated corresponding transgenic lines (0.4KminKO). All the tested 0.4KminKO lines (12) lost m-like epidermal expression, with only some variable residual staining, variable from line to line (Figure 2J).

These results demonstrate that Svb binds to an evolutionarily conserved element of the m gene, which behaves as a svb-responsive enhancer in the embryonic epidermis. Mutations preventing Shavenbaby binding to this element abolish its enhancer activity, thus providing strong evidence that Svb exerts a direct control on m transcription.

Miniature Is Required for Denticle Formation

m encodes a membrane protein [30], which has recently been implicated in actin-based remodeling of wing cell shape during metamorphosis [28,31]. To examine whether m also plays a role in embryonic epidermal cells, we analyzed consequences of m inactivation on denticles.

Although the absence of m does not affect the denticle pattern, detailed examination shows that denticles are altered in m mutant embryos (Figure 3). In wild type, each denticle row has a characteristic morphology, but all denticles share a common organization resembling a rose thorn: a large base, followed by a median constriction forming a narrow neck, which supports the distal hook, which points either anteriorly or posteriorly (Figure 3). In larvae carrying the m¹ loss-of-function mutation, or the Df(1)m-MR deficiency that deletes the entire m gene [28], denticles are small and misshapen (Figure 3 and Table S2); the median constriction is absent, leading to poorly differentiated denticles that display an aberrant triangular shape. These mutant defects are rescued when m is re-expressed using the wild-type 0.4Kmin element as a GAL4 driver (Figure 3A). These results provide additional evidence that this m enhancer is fully functional in the epidermis and demonstrate that the observed denticle defects are due to the loss of m activity.

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Figure 3. m Impinges on Denticle Formation

(A) Consequences of alterations of m function and expression on denticle formation. The m¹ mutation leads to morphological alteration of denticles, with a characteristic abnormal triangular shape. A similar phenotype is observed with Df(1)m-MR, a deficiency in which the entire m locus is deleted. Overexpression of m, by crossing the Ptc-Gal4 driver line to EP345-m, does not modify the cuticular phenotype in the corresponding regions (arrowheads). Re-expression of wild-type m products driven by the 0.4Kmin-gal4 transgenic lines, but not 0.4KminKO-Gal4 lines, rescues the characteristic denticles defects of m¹ embryos. All pictures correspond to the A4 segment.

(B) High magnification views of wild-type (wt) and m¹ denticles (fourth rows of A4), as observed in either light (left) or scanning electron microscopy (right). Whereas the absence of m does not affect the denticle height (h), the width (w) is reduced, producing denticles of smaller area that display an abnormal shape lacking the median constriction characteristic of wild-type denticles. See Table S2 for quantification of these defects.

https://doi.org/10.1371/journal.pbio.0040290.g003

To further investigate the role of m in denticle formation, we tested the influence of its ectopic expression in wild-type embryos. We used both an EP (target P-element) insertion located 2 kb upstream of the m start site, or the min cDNA placed under the control of UAS sequences (Gal4 binding sites), which both allow a strong m ectopic expression upon crossing with Gal4 drivers (unpublished data). None of the drivers we tested led to cuticular defects. For example, overexpression of m with the ptc-gal4 driver, both in denticle rows 2 and 3 and in a two-cell-wide stripe in naked cells (see Figure 1), does not affect cuticle organization (Figure 3A). Therefore, m expression is required, but not sufficient, for the correct formation of denticles.

These results show that m is directly involved in a specific aspect of denticle formation. All together, these data further demonstrate that svb directly impinges on epidermal cell morphogenesis through the direct control of m transcription.

Mutations Altering Denticles Allow the Identification of Additional svb Targets

The inactivation of m is not sufficient to mimic the svb phenotype, indicating that svb must regulate additional targets to achieve denticle formation. Mutations known so far to prevent denticle formation affect either svb or upstream regulators, therefore suggesting that inactivation of a given svb downstream target produces denticle malformation rather than prevents denticle formation. To test this hypothesis, we searched extensively for genes affecting denticle morphology (Table S1) and examined whether they are regulated by svb.

A dozen genes reported to affect denticle formation lead, when inactivated, to general cuticle defects, which are not, however, specific to trichomes (Table S1 and references therein). These genes (e.g., the Halloween family that encodes members of the ecdysteroid pathway) are either not expressed in the epidermis (e.g., are restricted to the ring gland) or display ubiquitous expression in epidermal cells (Table S1 and references therein).

Mutations corresponding to seven genes affect denticles, without altering either the general cuticle organization or embryonic patterning (Table S1). While the expression of fritz, hem, tricornered, and Stubble are ubiquitous or undetectable in the embryonic epidermis, forked (f), singed (sn), and shavenoid/kojak (sha) display a segmental pattern of expression (Table S1). Mutations in both sn and f lead to similar defects [22], with thin, crooked abnormal denticles, whereas sha mutants display a distinct phenotype [25,32] with small, misshapen, multiply split denticles (Figure 4A and Table S2). Detailed analysis of mRNA distribution revealed that sn, f, and sha are specifically expressed in denticle cells (Figure 4B). The epidermal expression of their transcript is strongly reduced in svb mutants (Figure 4B), revealing that svb is necessary for sn, f, and sha expression in denticle cells. In addition, svb is sufficient to promote sn and sha expression in the epidermis, since their mRNA is robustly expressed in additional stripes following ectopic expression of svb (Figure 4B). The svb-driven ectopic expression of f is barely detectable, indicating that additional factors are required to promote f expression in naked cells. Taken together, these data show that the transcription of genes previously implicated in denticle formation is specifically activated by svb.

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Figure 4. svb Directs the Expression of Genes Encoding Actin-Remodeling Proteins Required for Denticle Formation

(A) Cuticle preparations showing denticle morphology in f^36a sn³ sha¹ and wsp³ mutants. All views correspond to the same region, i.e., the ventral-most region of the A4 segment. Close-ups are scanning electron microscopy magnification of a representative denticle from the fourth row of denticles.

(B) mRNA expression of forked, singed, shavenoid/kojak, and wasp in the ventral embryonic abdomen (A2–A6) of wild-type, svb mutants, and embryos expressing svb in wg cells, as observed from in situ hybridization. Arrowheads point to wg cells. Anterior is to the left in all pictures.

https://doi.org/10.1371/journal.pbio.0040290.g004

sn, f, and sha encode actin-associated factors [32,33], and a recent study has shown that other components of the actin cytoskeleton (Diaphanous, Enabled, and the members of Arp2/3 complex) accumulate in forming denticles [23]. This prompted us to analyze the embryonic expression of these genes, as well as additional actors in actin remodeling (chickadee, gelsolin, and twinstar). All exhibit ubiquitous expression in embryos, which is not influenced by svb activity (Table S1). However, we found that wasp (wsp), which encodes an activator of the Arp2/3 actin nucleation complex [34], is specifically expressed in denticle cells, in a svb-dependent manner (Figure 4B). The epidermal expression of wsp is strongly reduced in svb mutants and ectopic expression of svb leads to additional bands of wsp mRNA in naked cells, albeit at a much weaker level than in denticle cells (Figure 4B). Cuticle analysis revealed that zygotic wsp mutations [35] lead to the alteration of denticles, which are abnormally thin and often display bent extremities (Figure 4A). These defects are likely due to the lack of activation of the Arp2/3 complex, since the zygotic inactivation of two ubiquitous Arp2/3 subunits (ArcP34 and ArcP41) causes denticle alterations similar to those observed in wsp mutants (unpublished data).

Consistent with the idea that actin reorganization is required for denticle formation, our results show that Svb activates the expression of a subset of genes controlling actin dynamics, whose individual inactivation produces defects in denticle morphology.

svb-Regulated Genes Functionally Collaborate in Denticle Formation

Inactivation of each svb target we identified leads to specific defects in the morphology of denticles, but is not sufficient to inhibit their formation. This suggests either that these genes are involved in nonessential aspects of denticle formation (e.g., fine shaping) or, alternatively, that denticle formation relies on the collective activity of numerous effectors, none of them being absolutely indispensable. To discriminate between these possibilities, we analyzed embryos carrying combinations of mutations in different svb targets.

The phenotype of sn, f double mutants is slightly aggravated when compared to that of sn or f mutants (Table S2). When m mutation is combined with either sn or f, denticles display both the crooked extremities found in sn and f, and the defects characteristic of m. In addition, the inactivation of m and f (or sn) causes denticle splitting, as revealed by a significant increase in the number of abnormal denticles per row (Table S2). Simultaneous inactivation of all three genes leads to small and highly misshapen denticles that are more affected than in double mutants (Figure 5A and Table S2). Adding a fourth mutation, wsp or sha, further impairs the formation of denticles, leading in many cases to their replacement by naked regions (Figure 5A). The cumulated inactivation of svb targets thus progressively leads to tiny and highly malformed denticles, ending in the inhibition of denticle formation (Figure 5A). A similar effect is observed in the dorsal region, where combining mutations in svb targets ultimately prevents dorsal hair formation, in addition to producing cumulative morphological defects (Figure 5B). These results demonstrate that the morphological differentiation of the embryonic epidermis requires the concerted action of multiple genes, all playing a direct role in the formation both of denticles and dorsal hairs.

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Figure 5. svb Downstream Targets Act Collectively in the Formation of Both Denticle and Dorsal Hairs

(A) Denticle defects resulting from the combinations of individual mutations of sn³, f^36a, m¹, sha¹, and wsp³ (views of the ventral region of the A4 segment). Embryos triple mutant for m, sn, and f display an aggravated phenotype with respect to each simple mutant or double mutant. This leads to poorly differentiated denticles, which display an extremely thin tip and a small triangular base. In addition, the lateral spacing of mutant denticles is reduced, a consequence of denticle splitting with two tiny extensions side by side. The combined inactivation of sn, f, m, and wsp further increases the severity of mutant phenotypes, producing very small and highly abnormal denticles. Similarly, in embryos simultaneously lacking sn, f, m, and sha, the few remaining extensions are atrophic, and numerous denticles are replaced by naked cuticle.

(B) svb downstream targets are required for dorsal hair formation. The dorsal region of a wild-type abdominal segment displays a stereotyped arrangement of epidermal extensions presenting a specific morphology: a row of large trichomes pointing anteriorly, a stripe of naked cuticle, three rows of extensions of intermediate size, and several rows of thin hairs. The cumulated inactivation of svb targets profoundly impairs dorsal hair formation. Hairs that display the superimposition of single mutant phenotypes are thickened (m), crooked (sn, f) and split (f, wsp, sha). In sn³, f^36a, m¹ triple mutants, dorsal hairs are severely reduced in size and, in several cases, the formation of dorsal hair is abrogated, leaving abnormal naked regions, as best seen in the first row of trichomes. These phenotypes are even more pronounced following the combination with wsp mutation, and culminate in embryos lacking m, sn, f, and sha, where most dorsal hairs are absent and replaced by naked cuticle. In some cases, atrophic dorsal hairs are seen as duplicated spots, revealing hair splitting as observed in sha and f embryos. All pictures correspond to the A4 segment.

https://doi.org/10.1371/journal.pbio.0040290.g005

To better understand the role of svb targets, we analyzed the subcellular distribution of Singed, Forked, and Miniature proteins at the onset of denticle formation. Forked, Singed, and Miniature are sequentially accumulated in denticle cells (Figure S1) and, consistent with their bundling activity, both Singed and Forked co-localize with F-actin in growing denticles (Figure 6A). Forked accumulates in nascent denticles, from the earliest steps of F-actin reorganization, whereas Singed appears slightly later, when the apical bundle is already formed and is continuing to grow. Miniature appears concomitantly with Singed and is initially detected in the cytoplasm of denticle cells (Figure S1). In later embryos (stage 15 and 16), Miniature accumulates in a restricted apical domain that underlines the base of each denticle (Figure 6A). Since Miniature is a membrane protein, this observation suggests the existence of a denticle-specific membrane subdomain. We therefore analyzed mutant defects at the ultrastructural level. In stage 16 wild-type embryos, the apical surface of epidermal cells organizes a dense array of microvilli that contact the cuticle only at their distal tips (Figure 6B). In contrast, there is a continuous and tight interaction between plasma membrane and cuticle layers in the denticle region (Figure 6B). Although naked cells are unaffected, denticle cells do not contact cuticle correctly in m mutant embryos (Figure 6B). The apical membrane is disorganized, with an abnormal gap between the denticle membrane and cuticle layers, indicating that m is required for the membrane/cuticle interaction that is specific to denticle cells. These defects are not observed in sn, f mutants (Figure 6B), confirming that they are specific to the lack of m activity.

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Figure 6. svb Target Genes Are Involved in Separate Features of Denticle Edification

(A) Subcellular localization of Singed, Forked, and Miniature in the epidermis of stage 15 wild-type embryos. Distribution of α-catenin, a component of adherens junctions that underlines the cell contour, was observed in embryos expressing α-catenin-GFP driven by E22C-Gal4. Red indicates F-actin, and green indicates α-catenin, Singed, Forked, and Miniature.

(B) Transmission electron microscopy analysis of denticle cells from wild-type (wt), m¹, and sn³, f^36a double mutant embryos. As in smooth cells, the flat region of the apical cell face organizes microvilli that contact cuticle layers only at the apex. In contrast, the plasma membrane is in close contact with cuticle along the entire wild-type denticle contour. Although the m¹ mutation alters this membrane/cuticle contact, no defects are visible in sn³, f^36a embryos. Close-up pictures show a region of the growing extension. Scale bar represents 0.25 μm.

https://doi.org/10.1371/journal.pbio.0040290.g006

Therefore, these data reveal that denticle formation requires, besides actin remodeling, a specific modification of the membrane/cuticle interaction that is directly dependent on the localized activity of Miniature. All together, these results show that svb directs the expression of cellular effectors involved in different steps and aspects of cell shape control, which are collectively responsible for denticle formation.

svb Controls Denticle Pigmentation in the Embryonic Epidermis through the Regulation of yellow Transcription

In addition to the reorganization of cell shape, denticle formation implies pigmentation of cuticle extensions. We noticed that the supernumerary cuticular extensions formed after ectopic expression of svb are as darkly pigmented as normal denticles (Figure 7A), thus suggesting that svb also controls the expression of pigmentation genes in the ventral epidermis.

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Figure 7. svb Controls Denticle Pigmentation through the Regulation of y Transcription

(A) Cuticle preparations (A3–A4 segments) showing the effect of Ptc-Gal4–driven ectopic expression of svb. Ectopic cuticular extensions (arrowheads) are as densely pigmented as normal denticles, as demonstrated by bright-field observation (right).

(B) Summary of the cuticular pigmentation pathway, showing genes that are putatively implicated in the transformation of tyrosine to pigmented compounds. Of the ten genes thought to be involved in pigment production we tested, only four (red) are expressed in the embryonic epidermis at the time of cuticle formation.

(C) In situ hybridization showing the expression of y mRNA in the epidermis in late embryos. In wild type, y mRNA accumulates in three stripes per segment. Due to deep morphological folds at this stage, it is hard to define accurately the number of cell rows that express y in each segment. y transcription is controlled by svb, since staining is strongly decreased in svb mutant and an ectopic stripe of y mRNA (arrowheads) results from svb ectopic expression in wg cells.

https://doi.org/10.1371/journal.pbio.0040290.g007

Cuticle pigmentation results from the activity of the catecholamine biosynthetic pathway, which transforms tyrosine into DOPA derivatives that are responsible for both sclerotization and coloration of the cuticle (reviewed in [24]). We examined the embryonic expression of ten genes putatively involved in pigmentation [24]; among them, four are expressed in the epidermis: pale (ple), dopadecarboxylase (Ddc), alpha-methotrexate deficient (amd), and yellow (y) (Figure 7B). While the expression of ple, Ddc, and amd are unaffected in svb mutants, we found that y is regulated by svb. Although y expression is strongly up-regulated in a subset of denticle cells in late wild-type embryos , this expression is undetectable in svb mutants (Figure 7C). Furthermore, ectopic expression of svb turns on y transcription in additional cell stripes (Figure 7C), showing that svb regulates y expression in epidermal cells.

Therefore, in addition to cytoskeletal organization, svb also controls cuticle pigmentation through the up-regulation of y expression in denticle cells.

svb Controls Several Aspects of Actin Organization

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 [22,23]. These early steps of denticle formation depend on svb, which is necessary and sufficient to promote the formation of epidermal actin bundles [14]. Our results show that svb controls the transcription of several genes involved in different steps of actin assembly/organization. First, we 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 [32], 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 [36,37]. The Forked and Singed proteins sequentially accumulate in growing denticles, a situation reminiscent of that of wing hair formation [38], 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.

Several cytoskeletal regulators such as dAPC, Enabled, Diaphanous/Formin, and the Arp2/3 complex, accumulate in denticles [14,23], 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, we have previously shown that dAPC-2 is specifically relocalized in svb-induced ectopic epidermal extensions [14]. In addition, we show here that 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 [39], 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.

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, our 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.

Denticle Formation Requires a Specific Membrane/Cuticle Interaction

A surprising outcome of our studies is that denticle formation requires a specific regulation of the membrane/cuticle interaction. We show that 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 [28]. ZP domains were initially identified in the three major proteins of the zona pellucida, the extracellular envelope of mammalian oocytes (see [30] for a recent review), and they are thought to be components of apical matrices [40]. We show that 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 [5]. 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. We found that two other ZP genes are regulated by svb (Table S1) and analysis of their individual role in denticle formation is under way. Our 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.

svb also regulates the expression of y, a gene encoding an apically secreted protein that associates with cuticle and is required for the production of black pigments [24]. 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 [41]. 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 (Table S1).

Svb Governs a Morphological Module

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 (Figure 8). 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, we show that 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, we provide evidence for a direct control of one of the targets (m). We have defined a 400-bp m enhancer reproducing the endogenous expression pattern of m in the epidermis and show 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.

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Figure 8. Model of svb Regulation and Activity during Denticle Formation

During epidermal differentiation, regulatory regions governing svb transcription integrate outputs from many signaling pathways (Wg, Hh, and DER) and positional cues to define the precise subset of epidermal cells that express svb. The Shavenbaby transcription factor triggers in turn the expression of different classes of genes encoding cellular effectors. They are directly involved in distinct aspects of trichome formation, including the reorganization of actin (singed, forked, wasp, and shavenoid), extracellular matrix (m) and cuticle (y), likely through modifying the activity of ubiquitous cellular machineries. Additional cytoskeletal factors or regulators (independent of svb) might be required for the fine sculpturing of each kind of trichome, characteristic of a given body region. Modifications of svb cis-regulatory regions thus provide a rich source of plasticity to evolve the trichome pattern and generate morphological diversification throughout species.

https://doi.org/10.1371/journal.pbio.0040290.g008

We propose 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 (Figure 8). How many genes are regulated by svb to promote remodeling of epidermal cells? Our 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.

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, we show that 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 [14], 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 [23]. 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.

Evolutionary Perspectives

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 [17–19]. Although the expression of patterning genes is unchanged in all species examined [42], 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 [16]. Our 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 (Figure S2). 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, we show that 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.

Evolutionary modifications of cis-regulatory elements of lin-48, the putative svb homolog in worms, were responsible for the difference in the position of the excretory duct between Caenorhabditis elegans and C. briggsae [43]. 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 [44] and its regulation by the Wnt pathway [45]. Our 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.

Fly strains and transgenic constructs.

We used UAS-svb; UAS-ovoA; Ptc-Gal4; Wg-Gal4; svb¹; btd¹, svb¹; svbR9; w,m¹; sn³; f³; f⁵; f^36a; sha¹; sha^V15; sn³,f^36a; m¹,f^36a; m¹,sn³; m¹,sn³, f^36a; wsp¹; wsp³; m¹,sn³, f^36a; m¹,sn³, f^36a;sha¹; m¹,sn³,f^36a;sha^V15; m¹,sn³, f^36a;wsp³; w, Df(1)m-MR; P(EP)EP345, and a w D. sechellia stock from the D. Stern Laboratory. Stocks with multiple mutations were obtained by recombination, and mutant chromosomes were kept over balancers carrying Kr-Gal4>>UAS-GFP transgenes [46]. Transgenic lines were generated using standard P-element transformation. Based on the 2003 genome release, the 6kmin construct corresponds to genomic position chrX:11585555–11591094 and 11596605–11597139, 3kmin to chrX:11585555–11588410; positions, both inserted into pCasPER β-gal. Wild-type, or site-directed mutagenized, 0.4Kmin enhancer was amplified from genomic regions (11487355–11487753) and cloned into the pCβ vector (carrying a nuclear-LacZ reporter) and in the pGal4 vector for rescue experiments. A full-length m cDNA inserted into the pUASt vector was used to generate pUAS-min transgenic lines. Details of cloning strategy are available upon request.

Embryo staining.

Antisense probes derived from cDNAs or genomic fragments were synthesized in vitro and processed for in situ hybridization following standard procedures. To identify svb mutant embryos, we used either a svb mutant chromosome carrying the btd1 mutation (leading to head defects; a gift from E. Wieschaus), or embryos sorted using the GFP balancer. For high-resolution imaging, ventral epidermis was hand-dissected. Immunostaining was performed according to [14], with anti-Miniature [28] at 1/400, anti-Forked [37] at 1/300, anti-Singed (Development Studies Hybridoma Bank, Iowa City, Iowa, United States) at 1/2, anti β-galactosidase (Cappel, Solon, Ohio, United States), Alexafluor488-labeled antibodies (Molecular Probes, Eugene, Oregon, United States) and TRITC-phalloidin (Sigma, St. Louis, Missouri, United States). Embryos were mounted in Vectashield (Vector Laboratories, Burlingame, California, United States) and photographed with a Leica TSP2 confocal microscope (Wetzlar, Germany).

Cuticle preparation and electronic microscopy.

Live mutant embryos (lacking GFP fluorescence from balancers) were hand-selected and cuticles prepared in hoyers/lactic acid (1/1). Abdominal segments 3 and 4 were imaged using a Zeiss Axioplan microscope equipped with phase contrast (Zeiss, Oberkochen, Germany). All high-magnification pictures correspond to the midline region of the fourth abdominal segment. For scanning electron microscopy, stage 16 embryos were fixed 15 min in heptane saturated in glutaraldehyde, devitellinized by hand and, after several water washes, dehydrated through an ethanol series. They were then dried using CO₂ in a BOMAR SPC-900/EX critical-point dryer (Bomar, Tacoma, Washington, United States), sputtered (MED 020, Bal-Tek, Balzers, Liechtenstein) with a 20-nm gold palladium coat and examined with a Philips XL30 field emission microscope. For transmission electron microscopy, stage 16 embryos, fixed and devitellinized as above, were post-fixed for 1 h in para-formaldehyde 3%, glutaraldehyde 0.5% in PBS and then in 1% osmium-tetroxide for 1h. After dehydration, samples were embedded in Epon. Sections were stained with uranyl acetate and photographed with a Leo 510 (Zeiss) microscope.

Electrophoretic mobility shift assay and in vitro DNA-binding.

DNA binding assays were performed as previously described [29]. Wild-type or site-directed mutagenized m DNA probes (position 11487606–11487768) were synthesized by PCR, end-labeled and incubated with recombinant proteins, produced in bacteria, and purified according to the manufacturer's specifications (Pharmacia, Uppsala, Sweden). We used GST-SVB, a fusion protein with the 450 C-terminal aa of Svb encompassing the DNA binding domain, and GST alone as control. Electrophoresis gels were analyzed using a Bio imaging analyzer (Fuji, Tokyo, Japan).

We thank J. Smith, P. Blader, A. McGregor, D. Stern, I. Delon, L. Waltzer, and other members of our laboratory for discussions and critical reading of the manuscript. We are grateful to Y. Latapie and P. Valenti for their help during the latest stages of this work. We are grateful to G. Guild, D. Stern, E. Wieschaus, the Bloomington and National Institute of Genetics (NIG-Fly) fly stock centers, and Developmental Studies Hybridoma Bank (DSHB) for providing us with fly stocks and antibodies. We very much thank M. Affolter, in whose laboratory part of this work was done, and Daniel Mathys and Ursula Sauder (Zentrum Mikroskopie der Universität Basel) for the skilful electron microscopy pictures.