Institute for Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
* Present address: Howard Hughes Medical Institute, Department of Genetics, Yale University School of Medicine, New Haven, CN 06536, USA
Author for correspondence (e-mail: noll{at}molbio.unizh.ch)
Accepted 22 October 2001
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SUMMARY |
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Key words: Accessory gland, Cell differentiation, Cell proliferation, Drosophila, Pax genes, paired
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INTRODUCTION |
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The accessory glands are a pair of dead-end tubes that branch off the male genital tract at the anterior end of the ejaculatory duct. They arise from a special set of cells in the male primordium of the genital disc (Nöthiger et al., 1977) whose developmental fate is determined by the male sex determination pathway during the third larval instar (Chapman and Wolfner, 1988). Each accessory gland is composed of a single layer of secretory cells surrounded by a sheath of muscle cells that squeeze the gland and force the accumulated secretions into the ejaculatory duct during mating. The secretory cells consist of two morphologically distinct types of cells, the predominant main cells, which comprise about 1000 cells per lobe, and the 40-50 secondary cells (Bairati, 1968; Bertram et al., 1992). The main cells are flat, hexagonal, binucleate cells that surround the lumen of the glands. Interspersed between the main cells at the distal end of each lobe are the secondary cells, which are large, spherical, binucleate cells with large vacuoles. Each cell type produces and secretes a characteristic set of products, and thus may contribute to a subset of the responses elicited in mated females. Despite extensive studies on the functions of the accessory gland fluid, little is known about the regulation of its components and the molecular mechanisms that specify accessory gland development.
The Drosophila Pax gene paired (prd), initially characterized as a pair-rule segmentation gene required for the establishment of positional information along the anteroposterior axis in the Drosophila embryo (Nüsslein-Volhard and Wieschaus, 1980; Kilchherr et al., 1986), encodes a transcription factor whose N-terminal moiety contains two DNA-binding domains, a paired-domain and a prd-type homeodomain (Bopp et al., 1986; Treisman et al., 1991; Noll, 1993). In addition to its role in promoting proper segmentation of the larval cuticle (Nüsslein-Volhard and Wieschaus, 1980), prd is necessary for postembryonic viability and male fertility (Bertuccioli et al., 1996; Xue and Noll, 1996; Xue and Noll, 2000). Investigation of this male fertility function has revealed that prd is required for accessory gland formation (Bertuccioli et al., 1996; Xue and Noll, 2000). We demonstrate that this requirement consists of a dual role of prd in accessory gland development, an early function required for cell proliferation and a late differentiation function, regulating the expression of accessory gland products. While the early function depends on a domain or motif present in the C-terminal moiety of Prd, the late function crucially depends on the DNA-binding specificity of the N-terminal region of Prd. Both functions are essential for male fertility and require an enhancer located in the downstream cis-regulatory region of prd.
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MATERIALS AND METHODS |
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To generate prd-mf1 to prd-mf8, the 5.2 kb XbaI-SalI prd downstream fragment, produced by SalI- and partial XbaI-mediated digestion of prd-SN20, was first subcloned in the pSK plasmid, and the XbaI site close to the SalI site was destroyed by partial digestion with XbaI, blunt-ending and religation. Subsequently, the prd downstream fragments between XbaI and SalI, corresponding to prd-mf1 to prd-mf8, were removed from this plasmid and blunt-end ligated into the SmaI site of the pKSpL5 plasmid (Xue and Noll, 1996), from which they were again recovered as XbaI fragments and inserted, in the proper orientation, into the XbaI site of prd-mf0.
To obtain prd-mf9 and prd-mf10, the 1.6 kb EcoRI-SalI prd downstream fragment was cloned into the EcoRI site of the pKSpL2 plasmid (Gutjahr et al., 1994). From this plasmid, fragments were amplified by PCR by the use of the primers T7 (5'-AAT ACG ACT CAC TAT AG-3') and pmf9down (5'-CAT TGT GTG TGC GGC CGC GAC TCT AG-3'; underlined bases do not pair with the prd downstream sequence to generate a NotI site), or pmf10up (5'-CAC TAG TCG CGG GTC CAC ACA CAA T-3'; underlined bases do not pair with the prd downstream sequence to generate a SpeI site) and T3 (5'-ATT AAC CCT CAC TAA AG-3'), digested with SpeI and NotI, and inserted between the SpeI and NotI sites of prd-mf0.
All the rescue constructs were injected together with pUChs2-3 P-element helper plasmid (prepared by D. Rio and provided by E. Hafen) into w1118 embryos, and w+ transformants were selected (Rubin and Sprading, 1982).
Dissection, immunostaining and X-Gal staining of accessory glands
Accessory glands were dissected (Xue and Noll, 2000) and stained with antisera directed against Prd (Gutjahr et al., 1993a), Acp26Aa (Monsma et al., 1990) and Gsb (Gutjahr et al., 1993b) as described elsewhere (Monsma et al., 1990). X-Gal staining was performed according to Bertram et al. (Bertram et al., 1992).
Fly stocks
Fly strains used in this work are: Df(2L)Prl, prd2.45 and prd-Gsb (Xue and Noll, 1996), prdRes (Bertuccioli et al., 1996), UAS-Prd (Jiao et al., 2001), UAS-Myc (Johnston et al., 1999), UAS-CycE (Neufeld et al., 1998), UAS-P35 (Hay et al., 1994), UAS-Dp110 (Leevers et al., 1996), Df(3L)th102, h kniri1 es/TM6C, Sb (Meier et al., 2000), Df(3L)H99, kniri1 pp/TM3, Sb (White et al., 1994) and sp-lacZ (D. Styger-Schmucki, PhD Thesis, University of Zürich, 1992).
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RESULTS AND DISCUSSION |
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These results demonstrate that Prd exhibits dynamic expression patterns in main and secondary cells of differentiated accessory glands that depend on age and mating activity in both secretory cell types. Similar age-dependent and mating-stimulated expression patterns in both secondary and main cells have been observed for several accessory gland proteins (DiBenedetto et al., 1990; Monsma et al., 1990) and accessory gland-specific enhancer trap lines (Bertram et al., 1992), which are probably regulated at the transcriptional level (Bertram et al., 1992). The fact that the expression of the Prd transcription factor correlates with that of these accessory gland products suggests that Prd is involved in their transcriptional regulation.
Delimiting the prd enhancers regulating accessory gland development and transcription in adult accessory glands
The prd-mf5 transgene is not only able to rescue accessory gland development (Fig. 1E,F), but also to express Prd in adult accessory glands with the same profile as endogenous Prd (Fig. 2; data not shown). In addition, it restores fertility (Fig. 2) in prd mutant males rescued by either prd-Gsb or prdRes transgenes. These results indicate that the 5.7 kb prd downstream sequences include all enhancers that are necessary for prd functions in accessory gland development and any possible later functions of prd required for fertility in differentiated glands of adult males. To map these enhancers, we constructed a series of prd transgenes derived from prd-mf5 by deleting different portions of the downstream sequences (Fig. 2). These transgenes were introduced into prd mutant males, rescued by either prd-Gsb or prdRes, and scored for their abilities to rescue accessory gland formation, drive Prd expression in adult accessory glands and restore fertility (Fig. 2). Expression of these transgenes in accessory glands was further tested and confirmed by examining their ability to express Gal4 and activate ß-gal expression from a UAS-lacZ transgene (data not shown). The prd-mf1, -mf2, -mf3 and -mf4 transgenes all lack the most distal 1.6 kb of the prd downstream region and are unable to perform any of these three functions (Fig. 2), which therefore strictly depend on enhancers partly or completely included in this 1.6 kb EcoRI-SalI fragment. By contrast, the prd-mf7 and prd-mf8 transgenes contain this fragment, and are able to execute all three functions (Fig. 2). It follows that the prd enhancers endowed with these functions are completely included in this region. To further delimit the enhancer region, prd-mf9 and prd-mf10 were constructed that subdivide this region into two halves of 0.8 kb (Fig. 2). While prd-mf9, which includes the proximal half, is again able to perform all three functions, prd-mf10 is unable to support accessory gland development (Fig. 2). Evidently, the 0.8 kb of the prd downstream region included in prd-mf9 harbor the prd male fertility enhancer (PMFE), which is necessary and sufficient for all prd functions required for accessory gland development and male fertility. Additional experiments would be required to elucidate whether this region contains a single or two separate enhancers responsible for the prd functions in accessory gland formation and its dynamic expression in adult accessory glands.
prd is required for cell proliferation during early accessory gland development
prd mutant males rescued by prd-Gsb or prdRes exhibit severely reduced (Fig. 1B) or no accessory glands (Fig. 1C), a phenotype that may result from an excess of apoptosis or a block in cell proliferation during early accessory gland development. To discriminate between these alternatives, we took advantage of a transgenic line, prd-mf9.7, that rescues the accessory glands of prdRes mutant males (Fig. 1C) completely with two copies of prd-mf9 (data not shown), but only partially with one copy (Fig. 4A), while restoration of fertility requires two copies. By contrast, most other prd-mf9 lines display a complete rescue with a single copy (data not shown). The weak rescue efficiency of the prd-mf9.7 line is presumably the result of a position effect on the prd-mf9 transgene causing its low expression (data not shown). The fact that, in addition to the expression of Prd, prd-mf9 drives Gal4 expression ubiquitously in developing accessory glands (data not shown) under the control of the same enhancer (Fig. 2) permits us to express any protein in developing accessory glands under the control of this enhancer by the use of the Gal4/UAS system and to subsequently test its ability to rescue the accessory gland phenotype of prd mutant males that carry one copy each of the prdRes and prd-mf9.7 transgenes.
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We conclude that an inhibition of the cell cycle, presumably in G1 (Neufeld et al., 1998; Johnston et al., 1999), rather than induced apoptosis is the primary cause for the reduction or loss of accessory glands in prd mutant males, and that prd is required for promoting cell proliferation during early accessory gland development.
prd is essential for accessory gland maturation
In adult accessory glands, prd exhibits a dynamic expression profile that depends on aging and mating activity (Fig. 3). This suggests that prd might be required for the regulation of accessory gland products. In support of this hypothesis, prd mutant males rescued to adulthood by two copies of a particular prd transgene are sterile even though the size of their accessory glands appears normal (Fig. 5D-F). This transgene, prd-GsbN+PrdC, expresses a chimeric protein consisting of the N-terminal half of Gsb and the C-terminal region of Prd under the control of the complete prd cis-regulatory region (Xue et al., 2001). This finding suggests that development of accessory glands to normal size does not strictly depend on the binding specificities of the paired-domain and homeodomain in the N-terminal moiety of Prd when compared with those in the homologous half of Gsb. Moreover, as the accessory glands of prd mutant males rescued by two copies of prd-Gsb are severely reduced (Fig. 1B), their development to normal size requires functions in the C-terminal region of Prd having the N-terminal moiety of the protein derived from Gsb. These C-terminal functions reside partially, though not exclusively, in the PRD transactivation domain of Prd (Xue et al., 2001).
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Although Prd and Gsb share a highly conserved N-terminal moiety, including two DNA-binding domains, a paired-domain and a prd-type homeodomain (Bopp et al., 1986; Baumgartner et al., 1987; Treisman et al., 1991), the N-terminal region of Gsb is apparently unable to substitute for this particular function of Prd. It seems therefore probable that the enhancers of Acp26Aa, gsb, sp, and perhaps of other genes specifically expressed in adult accessory glands include DNA-binding sites recognized by one or both DNA-binding domains of Prd, but not by those of Gsb, whose expression depends on Prd. Preliminary experiments suggest that the enhancer of the sp gene includes DNA-binding sites recognized by the paired-domain of Prd but not that of Gsb. It is possible, however, that other genes whose expression in accessory glands depends on Prd are regulated more directly by the Gsb transcription factor.
We conclude that prd performs a dual role in accessory gland development, an early function promoting cell proliferation that is required for accessory gland formation and a late function promoting cell differentiation that is essential for accessory gland maturation. The early function demands a domain or motifs present in the C-terminal region of Prd, whereas the late function depends on the DNA-binding specificity of at least one of the two N-terminal DNA-binding domains of Prd. Both functions are essential for male fertility.
Interestingly, Pax3, which encodes a vertebrate homolog of Prd, also seems to be necessary for both cell proliferation and differentiation. While splotch mutations in Pax3 of mice lead to the absence of limb muscles and a reduction in trunk muscle mass (Franz et al., 1993; Tajbakhsh et al., 1997), overexpression of Pax3 in cultured cells produces foci of transformed cells that are able to develop tumors in nude mice (Maulbecker and Gruss, 1993). Moreover, a Pax3 gain-of-function mutation produces alveolar rhabdomyosarcoma, a highly proliferative cancer (Shapiro et al., 1993). In addition to its role in regulating cell proliferation, Pax3 acts upstream of MyoD and can induce muscle differentiation (Maroto et al., 1997). The fact that both Prd and its vertebrate homolog Pax3 play pivotal roles in the regulation of cell proliferation and differentiation might reflect an evolutionary mechanism important during the evolution of Pax genes as well as many other genes encoding transcription factors (Noll, 1993).
Because gene networks have been conserved during evolution (Noll 1993), it is reasonable to expect that many of the factors present in seminal fluid whose synthesis depends on the late male fertility function of prd are also synthesized in the human prostate and required for sperm fertility, a proposition now testable on the basis of the results reported here.
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ACKNOWLEDGMENTS |
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