The ventral veinless gene (vvl) encodes the previously identified Cf1a protein, a transcription factor containing a POU-domain. During embryonic development vvl function is required for the formation of the tracheal tree and in the patterning of the ventral ectoderm. During imaginal development vvl is required for cell proliferation and the differentiation of the wing veins. vvl expression is restricted to the regions where its function is required, and is dependent on the coordinate activities of signalling molecules such as decapentaplegic, wingless and hedgehog. vvl interacts with other genes involved in vein differentiation, including veinlet, thick veins, torpedo, decapentaplegic and Notch suggesting that vvl function may affect several cell-to-cell communication pathways. We propose that the gene vvl integrates information from different signalling molecules and regulates the expression of specific cell differentiation genes during tracheal development and vein differentiation.

Many genes involved in pattern formation during Drosophila development are required in cell communication (Woods and Bryant, 1992; Muskavitch, 1994; Klingensmith and Nusse, 1994). In general, their mutant alleles are associated with a variety of phenotypes, suggesting that similar mechanisms operate in different developmental processes. According to their mutant phenotypes and to their genetic and molecular interactions several of these genes can be grouped as belonging to particular functional pathways, many of which have been conserved in evolution. Well known events of pattern formation involving cell communication are those occurring during wing development. For example, the patterning of wing veins requires the coordinate differentiation of cells belonging to opposite wing surfaces (García-Bellido and de Celis, 1992) and the function of several genes involved in signal transduc- tion. Thus, wing vein formation is a model system in which to study how cells integrate different signals and differentiate in precise patterns.

Wing vein cells appear more compacted and pigmented than inter-vein cells. Vein histotype occurs on both surfaces of the wing, although it is more pronounced in one surface than the other. This difference results in dorsal and ventral veins. Genetic analysis indicates that during imaginal development proximodistal stripes of cells are specified as ‘vein competent cells’ (García-Bellido and de Celis, 1992; Sturtevant et al., 1993). In addition, mosaic analysis of mutations either removing veins or producing ectopic veins indicates that vein differentiation can also be affected after apposition of the dorsal and ventral wing surfaces (García-Bellido, 1977). These results suggest that cell-to-cell communication between dorsal and ventral cells is operative in the differentiation of both dorsal and ventral components of every vein.

The ventral veinless gene (vvl) is a candidate as a mediator of the coordination of vein differentiation between dorsal and ventral cells (Diaz-Benjumea and García-Bellido, 1990). vvl was defined by the phenotype of lack of ventral veins resulting from several mutations mapping in 65D2-3. Also the analysis of its genetic interactions with mutations producing ectopic bristles in veins suggested that vvl might be required for inductive interactions between the wing surfaces (Diaz- Benjumea and García-Bellido, 1990).

We have analysed the role of vvl during development, char- acterising the embryonic and imaginal phenotypes of lethal vvl alleles. We find that vvl is required in developmental processes as diverse as tracheal tree elongation during embryonic stages and cell proliferation and vein differentiation in the wing imaginal disc. We also find that vvl is allelic to the recently described gene drifter, which encodes the Cf1a transcription factor, a protein containing a POU-specific domain and a POU homeodomain (Johnson and Hirsh, 1990; Billin et al., 1991; Anderson et al., 1995). The vvl gene is expressed during embryonic development under the control of several signalling molecules including wingless (wg), hedgehog (hh) and decapentaplegic (dpp). During imaginal development, vvl is expressed in a dynamic pattern, and becomes restricted to vein territories during pupariation. vvl displays specific genetic interactions with other genes affecting vein differentiation, such as torpedo (top), thick veins (tkv) and Notch (N). Altogether these results indicate that the vvl/Cf1a transcription factor operates in different developmental processes linking several signalling pathways to cell differentiation.

Fly strains

We have used the following genetic variants described in (Lindsley and Zimm, 1992) unless otherwise stated. In the vvl locus the lethal alleles vvlZM of spontaneous origin; vvl6A3, X-ray induced; dfrE82 (Anderson et al., 1995); and the viable allele In(3LR)sep. We have used the following alleles: btlLG19 (Glazer and Shilo, 1991), tkv1, tkv7, tkv8, wgI2, hhIJ, dppshv1, top1, top4A, AxM1, Ax28 (de Celis and García- Bellido, 1994a) and ve1. As reporter lines we used a ctHZ-1 (Jack et al., 1991), and a wg-lacZ (Kassis et al., 1992). In experiments using the GAL4 system (Brand and Perrimon, 1993) we used the ptc-GAL4 (Hinz et al., 1994) and a UAS-dpp line (Capdevila and Guerrero, 1994). To identify homozygous mutant embryos we used two blue balancers of the third chromosome, TM3-ftz-lacZ and TM3-hb-lacZ.

In the clonal analysis we used the Minute mutation M(3)i55, the cell markers f36a and mwh1 and an f+ insertion in the 3L chromosome arm, P[f+]77C.

Preparation of embryonic and adult cuticle

For the analysis of embryonic cuticle, late embryos were removed from the chorion and vitelline membrane and mounted in a mixture of Hoyer’s medium (van der Meer, 1977) and lactic acid (1:1). Wings were removed and mounted in lactic acid/ethanol (1:1) for microscopic examination.

Immunochemistry and in situ hybridisation

We used antibody no. 55, which recognises the lumen of the tracheal tree (Reichman-Fried et al., 1994), and the antibody BP110 for staining of axons in the CNS. Embryos were stained according to the method of Reichman-Fried et al. (1994) using the Vectastain ABC kit. Whole-mount in situ hybridisations were done following the method of Tautz and Pfeifle (1989) with minor modifications. For double labelling experiments in the embryo, in situ hybridisation were performed after detection of β-gal with a specific antibody (Cappel). A btl probe was generated from a DNA clone provided by B. Shilo. As a probe for vvl we used a DNA fragment amplified with oligonucleotides designed according to the Cf1a sequence (Billin et al., 1991; Johnson and Hirsh, 1990). In situ hybridisation in imaginal discs were done as described by Cubas et al. (1991) and in pupal wings as described by Sturtevant et al. (1993).

Clonal analysis

Mitotic recombination was induced by X-rays at a dose of 1000 R in a Torrent 50 model, 100 kV and 15 mA, 2 mm aluminium filter. Irra- diated larvae were aged at 24-hour intervals after egg laying (AEL). Clones were initiated during 48-72 hours AEL and 72-96 hours AEL intervals. Irradiated larvae were of the genotype f36a; mwh P[f+]77C/vvlZM in the twin analysis, and mwh vvlZM/M(3)i55 in the M+ analysis. In the twin analysis all f clones are simultaneously vvl, and they have a mwh vvl+ twin. In the Minute analysis, only M+ clones would be mutant for vvl. A total of 23 and 57 large M+ clones induced in the ages 48-72 and 72-96 hours AEL respectively were analysed.

vvl is required for embryonic pattern

Strong vvl mutations are embryonic lethal. We have analysed vvl mutant embryos and have found that the width of the ventral abdominal denticle belts is reduced, a phenotype rem- iniscent of mutations in the spitz group of genes (Mayer and Nüsslein-Volhard, 1988; Fig. 1B). Other alterations in the denticle belts are also apparent, particularly in the third thoracic segment (Fig. 1D). As is the case for other mutations that produce a spitz-like phenotype, the regular spacing of commisures and connectives of the CNS of vvl embryos is perturbed (Fig. 1J).

Fig. 1.

vvl embryonic phenotypes. Cuticle phenotype of wild-type (A,C,E) and vvl mutant embryos (B,D,F). C and D show a detail of the third thoracic and first and second abdominal segments; E, F show a detail of filzkörpers). vvl mutant embryos show a reduction in the width of the denticle belts. Note also in D that denticles are also missing in the third thoracic segment. In wild-type embryos (E) each filzkörper connects the external openings to the tracheal trunk, while in vvl mutant embryos (F) the filzkörpers close over themselves suggesting a failure to connect with the tracheal trunk. vvl mutant embryos are defective in tracheal tree elongation. In wild-type embryos (G) the tracheal tree can be visualised with the antibody 55. In contrast, in vvl mutant embryos (H) the tracheal pits form abnormal cavities that remain disconnected. In vvl mutant embryos (J) the pattern of the embryonic CNS is distorted and some transverse commissures fail to be established. (I, wild-type pattern).

Fig. 1.

vvl embryonic phenotypes. Cuticle phenotype of wild-type (A,C,E) and vvl mutant embryos (B,D,F). C and D show a detail of the third thoracic and first and second abdominal segments; E, F show a detail of filzkörpers). vvl mutant embryos show a reduction in the width of the denticle belts. Note also in D that denticles are also missing in the third thoracic segment. In wild-type embryos (E) each filzkörper connects the external openings to the tracheal trunk, while in vvl mutant embryos (F) the filzkörpers close over themselves suggesting a failure to connect with the tracheal trunk. vvl mutant embryos are defective in tracheal tree elongation. In wild-type embryos (G) the tracheal tree can be visualised with the antibody 55. In contrast, in vvl mutant embryos (H) the tracheal pits form abnormal cavities that remain disconnected. In vvl mutant embryos (J) the pattern of the embryonic CNS is distorted and some transverse commissures fail to be established. (I, wild-type pattern).

Another feature of vvl mutant embryos is the abnormal shape of the filzkörpers, the large chambers housed in the posterior spiracles, which suggests that their connection with the tracheal trunk has failed (Fig. 1F). This phenotype prompted us to examine the morphology of the tracheal tree in vvl mutants. We have found that vvl mutant embryos lack the dorsal trunk and the branches of the tracheal system; instead the tracheal pits form abnormal cavities that remain disconnected (Fig. 1H).

vvl is the gene coding for Cf1a

The cytological position of vvl is 65D2-3 (Diaz-Benjumea and García-Bellido, 1990), which corresponds with that of the gene coding for the Cf1a transcription factor (Billin et al., 1991). Cf1a is a POU-domain protein that is expressed in the ventral midline and in the tracheal pits (Billin et al., 1991; Johnson and Hirsh, 1990; Treacy et al., 1991), the places where vvl gene activity is required according to its mutant phenotype. These observations suggested to us that vvl could be the gene coding for the Cf1a transcription factor. Anderson et al. (1995) have generated a mutation in the gene coding for the Cf1a factor and named it drifter (dfr). By means of a complementation test we have found that dfr and vvl mutations do not complement each other, indicating that they are allelic. We retain the ventral veinless (vvl) designation for the gene coding the Cf1a tran- scription factor by reason of precedent.

vvl tracheal expression is regulated by wg, hh and dpp

The expression pattern of the gene encoding Cf1a has already been described in embryos (Billin et al., 1991; Treacy et al., 1991; Anderson et al., 1995). It is expressed at germ band elongation in eleven pairs of clusters in the ectoderm that cor- respond to the tracheal placodes and the anterior spiracle pre- cursors. We have also identified weaker vvl expression in three additional pair of clusters in a similar dorsoventral position as the ones already described; one of them in a posterior position and the other two in a more anterior position (Fig. 2A). This metameric expression of vvl is likely to depend on the same positional cues in each parasegment, irrespective of the formation of tracheal placodes, positional cues that probably rely on the information provided by the segment polarity genes.

Fig. 2.

Regulation of vvl expression in the tracheal precursors. (A) vvl is expressed at germ band extension in 14 pair of patches in the ectoderm, one in every parasegment; the expression of vvl is higher in the ten pairs that correspond to the tracheal placodes. (B) vvl expression in btl mutant embryos. (C) btl is expressed at germ band extension in vvl mutant as in wild- type embryos in ten pair of clusters that correspond to ten tracheal placodes. (D) vvl is expressed during germ band extension in a group of cells (purple) occupying the same position in all 14 parasegments between the wg stripes (brown) as visualised by anti-β- gal staining in a wg- lacZ line. (E) In wg mutant embryos vvl is expressed in a longitudinal stripe of cells, the tracheal pits are contiguous and in some cases a single tracheal pit differentiates along part of the vvl stripe (F). (G) vvl expression in the tracheal precursors of hh mutant embryos is absent or very much reduced. (H) Enlargement of vvl expression along the dorsoventral axis of each segment as a result of ectopic expression of dpp in the anterior compartment of each parasegment driven by the a ptc-GAL4 line.

Fig. 2.

Regulation of vvl expression in the tracheal precursors. (A) vvl is expressed at germ band extension in 14 pair of patches in the ectoderm, one in every parasegment; the expression of vvl is higher in the ten pairs that correspond to the tracheal placodes. (B) vvl expression in btl mutant embryos. (C) btl is expressed at germ band extension in vvl mutant as in wild- type embryos in ten pair of clusters that correspond to ten tracheal placodes. (D) vvl is expressed during germ band extension in a group of cells (purple) occupying the same position in all 14 parasegments between the wg stripes (brown) as visualised by anti-β- gal staining in a wg- lacZ line. (E) In wg mutant embryos vvl is expressed in a longitudinal stripe of cells, the tracheal pits are contiguous and in some cases a single tracheal pit differentiates along part of the vvl stripe (F). (G) vvl expression in the tracheal precursors of hh mutant embryos is absent or very much reduced. (H) Enlargement of vvl expression along the dorsoventral axis of each segment as a result of ectopic expression of dpp in the anterior compartment of each parasegment driven by the a ptc-GAL4 line.

The anteroposterior location of the vvl-expressing cells is between the wg stripes (Fig. 2D). Indeed, in wg mutant embryos vvl is not restricted to individual clusters; instead, it is expressed in a longitudinal stripe of cells (Fig. 2E). Consequently, the tracheal pits are not longer separated by non- invaginated cells, and in some cases a single tracheal pit differentiates along part of the vvl stripe (Fig. 2F). vvl expression partially overlaps with hh stripes. In hh mutant embryos, vvl expression in the tracheal precursors is variably reduced (Fig. 2G) or even absent in some cases, suggesting a positive role for HH protein in the activation of vvl. This positive role of HH protein in vvl expression has been confirmed by the finding that ectopic expression of hh induces an enlargement of the vvl domain (data not shown).

The dorsoventral position of vvl expressing cells makes it likely that dpp could act as one of the positive cues for its expression. The effect of dpp in the regulation of vvl expression was studied using the GAL4-UAS system (Brand and Perrimon, 1993). We find that expression of dpp driven by a patched (ptc)-GAL4 line produces an expansion of the vvl domain along the dorsoventral axis of each segment (Fig. 2H), emphasising the role of dpp in setting and limiting the domain of vvl expression along this axis.

In conclusion, wg, dpp and hh seem to be providing the positional cues that are used in each metamere to set the activation of vvl in the cells that will eventually give rise to the tracheal precursors.

Role of vvl in tracheal elongation

breathless (btl), the gene coding for a FGF receptor homologue and vvl are expressed in the tracheal placodes, and mutations of both genes abolish tracheal tree formation (Glazer and Shilo, 1991; Klambt et al., 1992). vvl expression in tracheal placodes occurs before they begin to invaginate to form the tracheal pits. both btl and vvl are not required for tracheal placode determination since tracheal pits form in vvl (Fig. 1H) and btl mutant embryos (Klambt et al., 1992). Both genes are also expressed in the ventral midline and are required for proper axon guidance. These observations raise the possibility that they could be part of a common mechanism required in many developmental events. In particular, since the btl gene codes for a receptor kinase (Glazer and Shilo, 1991), they could be part of the same signal transduction pathway. We have studied the requirement of one gene activity for the expression of the other. We observe that vvl is expressed in the tracheal placodes of btl mutant embryos (Fig. 2B) and that btl is expressed in the tracheal placodes of vvl mutant embryos (Fig. 2C). These results indicate that these genes are not required for each other to be expressed in the tracheal placodes, and suggest that they could act in parallel or be related posttranscriptionally.

Requirements of vvl during wing development and vein differentiation

Viable vvl combinations have a variable phenotype consisting in the absence of proximal stretches of veins LII and LIV (for nomenclature of wing veins see Fig. 3A). To analyse the phenotype of the lethal alleles we made mosaics by X-ray induced mitotic recombination.

Fig. 3.

Clonal analysis of the vvlZM allele. (A) Wild-type wing showing the position of longitudinal veins (LII to LV), marginal vein (LI), and anterior and posterior crossveins (cv-a, cv-p). (B) Plot of frequencies of territories occupied by dorsal vvlZMM+ clones induced at 72-96 hours AEL. Positions occupied 1 or 2 times are indicated by dots, 3 or 4 times by light stripes, 5 or 6 times by dark stripes and more than 6 times by squares. Control clones cover 2.7 and 1.4 inter-vein regions in average when induced at the Minute ages of 48-72 hours and 72-96 hours AEL respectively. By contrast vvl clones only cover 1.5 and 0.7 inter-vein regions at the same developmental ages. (C) Plot and phenotype of all dorsal clones vvlZMM+. The position of ectopic bristles is indicated by small dots, and the position of thick veins by dark rectangles. Note absence of vein LV and stretches of vein LIII. (D) Plot and phenotype of all ventral vvlZMM+ clones. Same symbols as in C. Note the absence of veins LII and proximal LIV. The distal portion of LIV is only affected when the mutant clone is simultaneously dorsal and ventral. (E) Twin analysis of vvlZM. Stripes mark the position of f36avvlZM mutant clones (48 clones, dorsal and ventral) and the empty circles indicate the position where each f36avvlZM and mwh vvl+ twins are adjacent. (F) Plot of mwh vvl+ clones without a f36avvlZM twin. The average size of vvl and controls clones in twin is 120 and 257 cells respectively, which represents a reduction of 0.56 in the size of vvl clones compared to the twin clones.

Fig. 3.

Clonal analysis of the vvlZM allele. (A) Wild-type wing showing the position of longitudinal veins (LII to LV), marginal vein (LI), and anterior and posterior crossveins (cv-a, cv-p). (B) Plot of frequencies of territories occupied by dorsal vvlZMM+ clones induced at 72-96 hours AEL. Positions occupied 1 or 2 times are indicated by dots, 3 or 4 times by light stripes, 5 or 6 times by dark stripes and more than 6 times by squares. Control clones cover 2.7 and 1.4 inter-vein regions in average when induced at the Minute ages of 48-72 hours and 72-96 hours AEL respectively. By contrast vvl clones only cover 1.5 and 0.7 inter-vein regions at the same developmental ages. (C) Plot and phenotype of all dorsal clones vvlZMM+. The position of ectopic bristles is indicated by small dots, and the position of thick veins by dark rectangles. Note absence of vein LV and stretches of vein LIII. (D) Plot and phenotype of all ventral vvlZMM+ clones. Same symbols as in C. Note the absence of veins LII and proximal LIV. The distal portion of LIV is only affected when the mutant clone is simultaneously dorsal and ventral. (E) Twin analysis of vvlZM. Stripes mark the position of f36avvlZM mutant clones (48 clones, dorsal and ventral) and the empty circles indicate the position where each f36avvlZM and mwh vvl+ twins are adjacent. (F) Plot of mwh vvl+ clones without a f36avvlZM twin. The average size of vvl and controls clones in twin is 120 and 257 cells respectively, which represents a reduction of 0.56 in the size of vvl clones compared to the twin clones.

Minute (M) analysis

We have studied three vvl lethal alleles, vvlZM, vvl6A3 and vvldfr with the Minute technique which allows the generation of clones that cover large wing territories (Morata and Ripoll, 1975). All three alleles result in similar phenotypes, although vvlZM cells grow to form larger clones. In what follows we describe the results of Minute analysis of the vvlZM allele. vvl clones are smaller than controls, suggesting that the proliferation of vvl mutant cells is impaired (see legend to Fig. 3). They appear all over the wing (Fig. 3C,D), in either wing surface and can extend over both wing surfaces. Interestingly, vvl mutant cells display higher viability in regions close to the wing margin (Fig. 3B). Large dorsoventral clones reduce the size of the mosaic wing (Fig. 4A,B). For clones occupying a large fraction of either the anterior (3 cases) or the posterior (4 cases) compartment, the average reduction in size of the mosaic compartment is between 20 and 30% relative to the same compartment of the contralateral wing. Furthermore, large vvl clones in one compartment also cause a reduction in the size of the adjacent compartment. Similar size reductions in both mosaic and adjacent compartments are also produced by mutant clones for vein (vn) (García-Bellido et al., 1994).

Fig. 4.

Phenotype of vvlZM clones in the wing. (A,B) Left and right wings of the same fly. The wing in B carries a large dorsoventral clone in the anterior compartment. Dashed and dotted lines mark the dorsal and ventral internal borders of the clone respectively. Note the reduction in its size relative to the contra-lateral wing (A). (C) Dorsal mwh vvlZM clone between veins LII and LIII. Note the loss of proximal vein LIII, the differentiation of a thicker LIII stretch distally, and the appearance of ectopic bristles. (D) Dorsal mwh vvlZM clone between veins LIV and LV. Note differentiation of thicker stretches of LIV, disappearance of LV, and differentiation of ectopic bristles in the region between veins LIV and LV. (E,F) Different focal planes of the same wing carrying a mwh vvlZM clone in the dorsal surface. Note that vein LV does not differentiate in the dorsal surface (E) but it is still present in the ventral surface as visualised by a stripe of pigmented trichomes in F. (G) Non- autonomous effects of a ventral mwh vvlZM clone on the differentiation of the LIV vein. This clone is close to the ventral LIV vein and as a result the vein does not differentiate. (H) Example of a clone f36avvlZM (dashed line) and its twin mwh vvl+ (dotted line). Note the relative position of the mutant and wild-type clones and their difference in size.

Fig. 4.

Phenotype of vvlZM clones in the wing. (A,B) Left and right wings of the same fly. The wing in B carries a large dorsoventral clone in the anterior compartment. Dashed and dotted lines mark the dorsal and ventral internal borders of the clone respectively. Note the reduction in its size relative to the contra-lateral wing (A). (C) Dorsal mwh vvlZM clone between veins LII and LIII. Note the loss of proximal vein LIII, the differentiation of a thicker LIII stretch distally, and the appearance of ectopic bristles. (D) Dorsal mwh vvlZM clone between veins LIV and LV. Note differentiation of thicker stretches of LIV, disappearance of LV, and differentiation of ectopic bristles in the region between veins LIV and LV. (E,F) Different focal planes of the same wing carrying a mwh vvlZM clone in the dorsal surface. Note that vein LV does not differentiate in the dorsal surface (E) but it is still present in the ventral surface as visualised by a stripe of pigmented trichomes in F. (G) Non- autonomous effects of a ventral mwh vvlZM clone on the differentiation of the LIV vein. This clone is close to the ventral LIV vein and as a result the vein does not differentiate. (H) Example of a clone f36avvlZM (dashed line) and its twin mwh vvl+ (dotted line). Note the relative position of the mutant and wild-type clones and their difference in size.

In addition, vvl clones affect the differentiation of wing structures. In wing proximal regions they cause the fusion of vein trunks (not show) and in the wing blade both dorsal and ventral clones autonomously fail to differentiate veins. We never see any effect of a clone on the vein of the opposite wing surface, i.e. dorsal clones do not affect ventral veins, and vice versa (Fig. 4). vvl dorsal clones in veins LIII and LIV cause both the absence of the vein and the differentiation of stretches of thick vein (Fig. 4) that in many cases result in the incorrect folding of the wing. Ventral clones in the posterior compartment close to vein LIV may cause the absence of this vein (Fig. 4), a non-autonomous effect that has also been observed in mutant clones for torpedo and lethal (1) pole hole (Diaz- Benjumea and Hafen, 1994). The veins in the anterior wing margin are not affected by vvl clones. Finally, vvl dorsal and ventral clones appearing between veins LIII and LV give rise to ectopic bristles (Fig. 3B,C). These ectopic bristles, appearing both on the remnant vein and in the inter-vein, have the typical features of wing margin bristles (Fig. 4). The different phenotypes observed in vvl clones suggest that the gene is required during wing development for other processes than just vein differentiation.

Twin analysis

The Minute analysis suggests that vvl might be required for cell proliferation in the wing. To evaluate the proliferation abilities of vvl mutant cells we have made a twin analysis, a technique that allows sister clones to be marked and thus generates a control clone for each mutant clone. Among clones induced at 48-72 hours AEL we find that the average number of cells of vvl mutant clones is reduced by more than 50% compared to their twin vvl+ clones, indicating that the proliferation or viability of vvl cells is impaired. A plot of the surviving vvl clones (Fig. 3E) shows that their viability is not homogeneous through the wing: vvl clones tend to be concentrated in regions close to either the anterior or posterior wing margin (Fig. 3E), while control clones occupy any region of the wing (Fig. 3F).

In summary, the analysis of vvl mutant cells shows that vvl is required both for cell grow in wing regions other than the wing margin and for differentiation of dorsal and ventral veins. The lack of effect of vvl clones in the differentiation of the veins on the opposite wing surface suggests that vvl is not involved in dorsal to ventral induction. The differentiation of ectopic bristles and the thickening of stretches of LIII and LIV veins, suggest that vvl is involved in the suppression of wing margin characteristics in internal regions of the wing blade.

Imaginal and pupal expression pattern

In third instar wing discs vvl is expressed in both dorsal and ventral regions of the presumptive wing blade and wing base, and in some regions of the presumptive hemi-thorax (Fig. 5A). Interestingly, a stripe of cells in the position of the presumptive wing margin does not express vvl. The correspondence of this region with the dorsoventral boundary of the disc was confirmed in double labelling experiments analysing vvl and cut (ct) expression. In mature wing discs, ct is expressed in a stripe including dorsal and ventral cells along the presumptive dorsoventral boundary (Jack et al., 1991; Blochinger et al., 1993). The region of the wing blade devoid of vvl expression includes the ct stripe and some dorsal and ventral adjacent cells (Fig. 5B). The vvl pattern of expression evolves during pupation. Around 0-4 hours APF, vvl expression disappears from the presumptive inter-vein territories and four stripes corresponding to the four longitudinal veins become apparent (Fig. 5C). This decay in the inter-vein territories is very clear at 24 hours APF, when vvl expression is restricted to both the dorsal and ventral components of every longitudinal and proximal vein (Fig. 5D). Again at this late stage there is no vvl expression in either the anterior or the posterior wing margin. The restriction of vvl expression to the veins occurs before the apposition of dorsal and ventral surfaces, suggesting that it is independent of dorsoventral interactions.

Fig. 5.

vvl expression in the wing disc. (A) Mature third instar wing disc showing vvl expression in the presumptive dorsal and ventral wing blade, wing hinge and in the anterior region of the presumptive thorax. (B) Sagittal view of a third instar wing disc probed with vvl (purple) and stained with X-gal to reveal the expression of the gene cut (blue). Note the absence of vvl expression close to the presumptive wing margin (cut stripe) in both dorsal and ventral surfaces. (C) 0-4 hours APF wing disc, showing the disappearance of vvl expression from inter-vein territories. The wing margin is devoid of vvl expression. The presumptive dorsal wing surface is in focus. (D) 24-28 hours APF wing. vvl expression is restricted to both dorsal and ventral components of every longitudinal vein, with the exception of the LI, and to the venation of the wing base. (E) Expression pattern of wg in a wild-type disc as shown by anti-β-gal staining of discs carrying a P-lacZ insertion at the wg locus. (F) Expression pattern of wg in AxM1/Ax28 wing discs. Note the expansion of the wg stripe along the D/V boundary in both dorsal and ventral regions of the wing blade. (G) Expression pattern of vvl in AxM1/Ax28 wing discs. Note the correspondence between regions of ectopic wg expression (F) and the absence of vvl expression in the same regions along the wing margin.

Fig. 5.

vvl expression in the wing disc. (A) Mature third instar wing disc showing vvl expression in the presumptive dorsal and ventral wing blade, wing hinge and in the anterior region of the presumptive thorax. (B) Sagittal view of a third instar wing disc probed with vvl (purple) and stained with X-gal to reveal the expression of the gene cut (blue). Note the absence of vvl expression close to the presumptive wing margin (cut stripe) in both dorsal and ventral surfaces. (C) 0-4 hours APF wing disc, showing the disappearance of vvl expression from inter-vein territories. The wing margin is devoid of vvl expression. The presumptive dorsal wing surface is in focus. (D) 24-28 hours APF wing. vvl expression is restricted to both dorsal and ventral components of every longitudinal vein, with the exception of the LI, and to the venation of the wing base. (E) Expression pattern of wg in a wild-type disc as shown by anti-β-gal staining of discs carrying a P-lacZ insertion at the wg locus. (F) Expression pattern of wg in AxM1/Ax28 wing discs. Note the expansion of the wg stripe along the D/V boundary in both dorsal and ventral regions of the wing blade. (G) Expression pattern of vvl in AxM1/Ax28 wing discs. Note the correspondence between regions of ectopic wg expression (F) and the absence of vvl expression in the same regions along the wing margin.

vvl expression is excluded from the presumptive wing margin, suggesting that wg, which is expressed in wing discs in this region throughout the third larval instar (Couso et al., 1994; Phillips and Whittle, 1993) (Fig. 5E), is involved in vvl repression. In agreement with this, wg ectopic expression in the dorsal and ventral presumptive wing blade in Abruptex (Ax) mutant discs (Fig. 5F) correlates with the elimination of vvl in the same regions in these mutant discs (Fig. 5G). Interestingly, imaginal vvl expression is not modified in other Ax combinations that remove veins without modifying wg expression (data not shown).

Genetic interactions of vvl in wing vein differentiation

Genetic interactions between mutant alleles of different genes can be indicative of these genes belonging to the same functional pathway. It is known that vvl mutations interact with dppshv1, a viable mutation in the dpp gene that affects only vein differentiation (Diaz-Benjumea, 1988). We have studied the phenotype of genetic combinations between the viable vvlZM/In(3LR)sep heteroallelic combination and different viable mutations in the genes ve, top, rol, dpp, tkv and N. These genes can be grouped by their molecular nature and mutant phenotypes as belonging to three different pathways, the TOP tyrosine kinase pathway (ve, top and rol), the DPP pathway (dpp and tkv) and the N pathway (N).

The vvlZM/In(3LR)sep combination (vvl) has a wing phenotype that overlaps with wild type, i.e. a fraction of mutant flies have small gaps in the LIV vein (Fig. 6A). In combina- tion with dppshv1, both LII and LIV veins are affected (Fig 6B). As previously reported (Diaz-Benjumea and García-Bellido, 1990), the ectopic chaetae that appear in the veins in Hairy wing mutations are not affected in dppshv1; vvl combinations (Fig. 6C). Similarly, genetic combination with tkv viable mutations cause synergistic phenotypes of vein absence, mainly LII and LIV veins (Fig. 6H,L). Interestingly, the tkv phenotype of excess vein differentiation (Fig. 6K) is concomi- tantly suppressed in these combinations, suggesting that vvl is also required for the differentiation of the extra veins appearing in tkv flies.

Fig. 6.

Genetic interactions between vvl and mutations affecting vein differentiation. (A) vvlZM/In(3LR)sep. (B) dppshv1; vvlZM/In(3LR)sep. In dppshv1, distal stretches of vein LIV fail to differentiate. (C) Hw49c/+; dppshv; vvlZM/In(3LR)sep. The Hw mutation causes the differentiation of ectopic bristles in the veins. These bristles are also present in the putative position of the vein LIV that is absent in this mutant combination. (D) AxM1/+ vvlZM/In(3LR)sep. In AxM1/+ flies, the most distal portions of veins LIV and LV are absent (de Celis and García-Bellido, 1994a). In combination with vvl, the vein LIV is completely absent and the vein LII is affected in medial regions. (E) top1/top4A (F) top1/top4A; vvlZM/In(3LR)sep. top viable flies lack medial stretches of the LIV vein. In combinations with vvl, both the veins LII and LIV are completely removed. (G) tkv1/tkv7. (H) tkv1/tkv7; vvlZM/In(3LR)sep. The tkv1/tkv7 heteroallelic combination results in weak thickening of LII and LV veins, and in the lack of stretches of LIV. In combination with vvl the thickening of the veins is suppressed, and also veins LII and LIV are removed. (I) ve1/TM3, ve, In(3LR)sep. (J) ve1vvlZM/TM3, ve, In(3LR)sep. In homozygous ve flies, distal stretches of all longitudinal veins are absent. This phenotype is strongly enhanced in combinations between ve and vvl mutations. (K) tkv1/tkv8. (L) tkv1/tkv8; vvlZM/In(3LR)sep. Note the suppression of the extra-vein phenotype of this tkv heteroallelic combination, and the concomitant absence of LIV and LII veins. All wings are from females and pictures were taken at the same magnification. In addition to the vein phenotype, mutant combinations of tkv or dpp with vvl cause a reduction in the overall size of the wing.

Fig. 6.

Genetic interactions between vvl and mutations affecting vein differentiation. (A) vvlZM/In(3LR)sep. (B) dppshv1; vvlZM/In(3LR)sep. In dppshv1, distal stretches of vein LIV fail to differentiate. (C) Hw49c/+; dppshv; vvlZM/In(3LR)sep. The Hw mutation causes the differentiation of ectopic bristles in the veins. These bristles are also present in the putative position of the vein LIV that is absent in this mutant combination. (D) AxM1/+ vvlZM/In(3LR)sep. In AxM1/+ flies, the most distal portions of veins LIV and LV are absent (de Celis and García-Bellido, 1994a). In combination with vvl, the vein LIV is completely absent and the vein LII is affected in medial regions. (E) top1/top4A (F) top1/top4A; vvlZM/In(3LR)sep. top viable flies lack medial stretches of the LIV vein. In combinations with vvl, both the veins LII and LIV are completely removed. (G) tkv1/tkv7. (H) tkv1/tkv7; vvlZM/In(3LR)sep. The tkv1/tkv7 heteroallelic combination results in weak thickening of LII and LV veins, and in the lack of stretches of LIV. In combination with vvl the thickening of the veins is suppressed, and also veins LII and LIV are removed. (I) ve1/TM3, ve, In(3LR)sep. (J) ve1vvlZM/TM3, ve, In(3LR)sep. In homozygous ve flies, distal stretches of all longitudinal veins are absent. This phenotype is strongly enhanced in combinations between ve and vvl mutations. (K) tkv1/tkv8. (L) tkv1/tkv8; vvlZM/In(3LR)sep. Note the suppression of the extra-vein phenotype of this tkv heteroallelic combination, and the concomitant absence of LIV and LII veins. All wings are from females and pictures were taken at the same magnification. In addition to the vein phenotype, mutant combinations of tkv or dpp with vvl cause a reduction in the overall size of the wing.

Flies double mutant for vvl and viable heteroallelic combi- nations of both top (Fig. 6F) and rol (data not show) display synergistic phenotypes of absence of veins, mainly veins LII and LIV and also vein LIII although to a lesser extent. Also, all longitudinal veins except LIII disappear in combinations with ve (Fig. 6J). Previous analysis suggests that localised expression of ve in the presumptive veins (Sturtevant et al., 1993) is necessary to obtain the level of top signal required for vein differentiation (Sturtevant et al., 1994). The observation that ve is expressed in the presumptive veins before the restriction of vvl to the same regions, suggests that an increase in top signalling might be a requisite for the maintenance of vvl expression in the veins.

Finally, the vvlZM/In(3LR)sep combination also increases the phenotype of lack of veins associated with Ax (Fig. 6D), gain- of-function mutations in the N gene (de Celis and García- Bellido, 1994a; Palka et al 1990). It has been proposed that N is responsible for the restriction of vein differentiation in vein presumptive regions (de Celis and García-Bellido, 1994b). Interactions between N and vvl mutations suggest that the restriction of vein differentiation somehow involves the modulation by N of vvl expression or function in vein presumptive territories.

In summary, interactions of vvl with the TOP, DPP and N pathways indicate that vvl is a key gene responsible for vein differentiation and that these signalling pathways are involved in the regulation of vvl expression or in the modulation of its activity.

Analysis of vvl mutant phenotypes has revealed that the gene is required in embryogenesis and wing development. We have also shown that vvl is the gene coding for the Cf1a transcription factor. Localised expression of vvl is the result of a complex regulation directed by different signalling molecules. For instance vvl appears to be expressed in the tracheal primordia as the result of wg, hh and dpp signalling pathways, and wg is also responsible for the exclusion of vvl from the presumptive wing margin during imaginal development. vvl is expressed in the cells fated to be tracheal placodes and wing veins before they undergo differentiation and its absence prevents tracheal elongation and vein differentiation. Since vvl codes for a transcription factor it is likely that it is responsible for the coordinate expression of genes required to confer a particular program of differentiation. Thus, vvl appears to be expressed as a result of the activity of multiple transduction pathways in a set of cells where in turn it directs the formation of specific structures.

vvl and tracheal elongation

Tracheal tree formation occurs by cell migration and cell fusion from the tracheal placodes (Manning and Krasnow, 1993). Migration of cells from the tracheal placodes is dependent on the activity of the btl gene, the Drosophila homologue of the FGF receptor gene. In btl mutant embryos, these cells lose their ability to migrate and thus they lack one of the characteristics of their differentiation (Glazer and Shilo, 1991; Klambt et al., 1992; Reichman-Fried et al., 1994). We have shown that the same tracheal phenotype occurs in vvl mutant embryos, suggesting that vvl and btl may act through similar downstream cell differentiation genes. However, vvl is expressed in btl embryos and btl is expressed in vvl mutant embryos, indicating that vvl is not required for btl expression and also that vvl is not expressed as a result of Btl receptor acti- vation. These observations are consistent with the two genes functioning in parallel pathways. Alternatively, vvl could act upstream of btl, being required in the activation of the Btl receptor.

Besides btl and vvl, other genes are also required for proper tracheal elongation. In particular, tkv, the gene coding for a putative dpp receptor, is required for tracheal migration along the dorsoventral axis (Affolter, et al., 1994). Indeed, our exper- iments show that dpp promotes vvl expression; however, while vvl mutations completely abolish tracheal migration, tkv mutations only do so in particular branches (Affolter et al., 1994), implying that the cell motility functions impaired in btl or vvl mutants are still functional, at least in some cells, in tkv mutant embryos. Moreover, activation of vvl by ectopic dpp signalling occurs in spite of the localised expression of tkv. Therefore, the role of dpp in vvl activation might be mediated by another receptor, unless the early maternal component or undetected zygotic expression of tkv could have a role in this process. The tkv zygotic tracheal phenotype may indicate a different role for tkv expression in the tracheal placodes, for instance in setting the direction of migration of particular branches.

vvl and vein differentiation

Analysis of viable vvl combinations showed that vvl is required for ventral vein differentiation (Diaz-Benjumea and García- Bellido, 1990). However stronger vvl alleles in mosaics remove both dorsal and ventral veins. These requirements are ‘wing surface autonomous’ and are consistent with the expression pattern of the gene: vvl expression becomes restricted to the future veins before dorsal and ventral wing surfaces make contact, and it is expressed in both dorsal and ventral components of every longitudinal vein. The expression of other genes, such as ve (Sturtevant et al., 1993), in vein territories occurs before vvl is restricted to the veins, suggesting that vein presumptive regions are established independently of vvl function. Therefore, it is likely that, similarly to what happens during trachea development, vvl is required in vein formation to define or to implement a ‘vein differentiation program’. This could be accomplished if vvl is regulating the expression of down- stream genes required for vein differentiation. Candidates for vvl subordinate genes are those encoding cell adhesion molecules such as integrins and Laminin A, whose expression is regulated differentially in vein and interveins territories after pupariation (Fristrom et al., 1993).

The function of vvl in vein differentiation is highlighted by the genetic interactions observed between vvl and mutations in the genes ve, top, tkv and N. The ve gene is expressed in vein territories in late third instar discs, and it has been proposed that the VE protein increases TOP signalling (Sturtevant et al., 1994). The interaction of vvl mutations with both ve and top mutations suggests that the maintenance of vvl expression in the veins depends on ve-mediated increase in TOP signalling. It is more difficult to interpret the interactions between vvl and dpp and its putative receptor TKV, because different tkv heteroallelic combinations cause different phenotypes of both excess and lack of veins. However, it is clear that the ectopic veins caused by some tkv viable combinations depend on vvl function. The synergistic effects of vvl with both dpp and tkv in the elimination of veins, suggest that dpp/tkv and vvl may interact in vein differentiation late in pupal development. Finally, Notch is known to be required both for the establish- ment of vein regions and for their differentiation (de Celis and García-Bellido, 1994). The interaction between vvl and N gain- of-function alleles suggests that either directly or indirectly N is regulating the restriction of vvl expression to the veins.

Therefore, we propose that vvl could act as a link between genes defining the positioning of the veins (such as ve and N), genes modulating vein differentiation during pupariation (such as tkv and N), and presumptive genes implicated in final vein histotypic differentiation.

vvl functions in cell growth and wing margin formation

In addition to its role in vein differentiation, vvl is also required for the normal growth of the wing. Thus, large vvl clones reduce the size of the region they occupy, and therefore the size of the whole wing. Similar reductions in wing size have also been found associated with vn mutant clones (García- Bellido et al., 1994). Clonal restrictions are associated with the adult veins (González-Gaitan et al., 1994) and they can influence the growth of inter-vein territories (García-Bellido and de Celis, 1992). In this context, the reduction in size in both vn and vvl mosaic territories may reflect the requirement of these genes in the control of intercalar cell proliferation between vein presumptive regions. The pattern of vvl expression in the wing disc can be correlated with the viability of vvl mutant cells. Thus vvl cells in a Minute background tend to grow close to the presumptive wing margin, a region where vvl is not expressed, and the same vvl allele behaves as a cell lethal in non-M clones in regions away from the D/V boundary.

Finally the appearance of ectopic bristles and thicker veins in some internal regions of the wing blade in vvl+ clones suggests that vvl function is preventing the generation of structures typical of the wing margin in internal regions of the wing. The pattern of vvl expression in the presumptive wing margin is complementary to that of wg and, as is the case during tracheal placode establishment, wg represses vvl expression in the presumptive wing margin. It is then possible that some of the patterning effects of wg in the development of the structures typical of the wing margin (Phillips and Whittle, 1993; Couso et al., 1994) are mediated by wg repression of vvl.

We thank M. Ashburner, in whose laboratory part of this work was done. We also thank A. García-Bellido, C. S. Goodman, P. Martin, J. Capdevila and I. Guerrero, M. Reichman-Fried and B. Shilo, K. Basler and the Indiana Stock Center for providing flies and materials, S. Romaní for help in analysing nervous system phenotypes, N. Martín for technical assistance and J. Collet, S. Collier, D. Gubb, I. Guerrero and E. Sánchez-Herrero for critical comments on the man- uscript. J. F. d.C. is a postdoctoral fellow of EMBO in M. Ashburner’s laboratory. M. Ll is supported by a fellowship from the Generalitat de Catalunya. This work was supported by the Dirección General de Investigación Científica y Técnica and the Fundación Ramón Areces.

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