In wild-type embryos, Paired protein is expressed in several phases. Initial expression in broad domains evolves into a pair-rule pattern of eight stripes during cellularization. Subsequently, a segment-polarity-like pattern of fourteen stripes emerges. Later, at mid-embryogenesis, paired is expressed in specific regions of the head and in specific cells of the central nervous system (Gutjahr, 1993 and Baumgartner, 1991).

An influence of Paired protein on maxillary chemosensory ventral organs and dorsal-lateral cirri rows occurs when Paired is reduced by targeted ribozymes (Vanario-Alonso, 1995).

Effects of Mutation or Deletion

even-skipped (eve) function is crucially required for homeotic gene expression in mesoderm, whereas most other segmentation mutations have only minor effects on position and/or width of the homeotic expression domains in this germ layer. ftz-prd double mutant embryos completely lack mesodermal segmentation [Image]. Antp, Scr, Ubx and en are not expressed in the visceral mesoderm of these embryos. opa-prd double mutant embryos also appear to lack mesodermal segmentation (Tremml, 1989).

paired mutant males rescued by wild-type paired transgenes are all sterile, although the females are fertile. Since prd rescue has been achieved with a different transgene (Gutjahr, 1994) containing 10 kb of 5' and 5kb of 3' DNA, and these rescued flies were not male-sterile, it seems likely that rescue with a reduced transgene (which contains 4kb less 3' DNA) lacks a 3' enhancer required for fertility. While wild-type animals show strong Prd protein staining in the accessory glands, the accessory glands from rescued animals were missing. Thus there is a requirement for prd in accessory gland formation (Bertuccioli, 1996).

Although gastrulation is regarded as the stage during Drosophila development when the AP patterning system first influences morphological processes, transcription is regulated in complex patterns already at cycle 10. How soon this transcriptional complexity produces spatial differences in morphology has been unclear. Two new processes are described that establish visible morphological inhomogeneities before the onset of gastrulation. The first of these is the regulation of syncytial nuclear densities in the anterior end of the egg and represents the first zygotically driven AP asymmetry in the embryo. The second process is the generation of a fine-scale pattern in the actin/myosin array during cellularization. Three domains of different yolk stalk diameters as well as depths of cellularization along the AP axis are found. These domains are established under the control of the AP patterning system and require bicoid activity. The anterior-most domain is a region of large yolk stalk diameters and corresponds to the region of decreased nuclear densities observed during syncytial stages. The middle domain shows smaller yolk stalk diameters and more rapid cellularization. Its establishment requires wild-type paired activity and thus indirectly requires bicoid. It occurs in a region of the embryo that ultimately gives rise to the cephalic furrow and may account for the effect of paired on that structure during gastrulation. These results therefore suggest a link between cytoskeletal organization during cellularization and subsequent morphogenetic processes of gastrulation (Bakankenship, 2001).

To assay the degree of uniformity along the AP axis during cellularization, the furrow canals of wild-type embryos were visualized by staining for Myosin. By mid-cellularization, three domains of differing diameters of yolk stalks could be observed. The first domain is centered around the anterior pole of the embryo, and possesses the largest diameters. The second domain, immediately posterior to the first anterior domain, has the smallest diameters. This domain is centered around the location where the cephalic furrow will eventually form. The third domain lies posterior to this pre-CF domain and has furrow canals of intermediate diameter (Bakankenship, 2001).

These three domains also differ in the depth of the cellularization front. The anterior domain, which has large yolk stalk diameters, is the shallowest part of the embryo in its depth of cellularization. By the end of cellularization, the most anterior part of this domain can possess a depth of cellularization (15-20 µm) half that of the rest of the embryo (~35 µm on average). The region posterior to the pre-CF domain has an intermediate depth, whereas the pre-CF domain, which has the smallest yolk stalk diameters, has the greatest depth of cellularization. The regions of differing yolk stalk diameters correspond to the regions of varying depth during cellularization (Bakankenship, 2001).

To test whether these cellularization phenomena are due simply to the position or geometry of the embryo (e.g. the curvature of the anterior part of the egg), or if these phenomena are being specified and positioned by the AP patterning system, the three domains were sought in embryos that lacked all positional information along this axis. In embryos derived from females triply mutant for bicoid nanos torso-like, yolk stalk diameters are uniform, even though other aspects of cellularization occur normally. The shallow cellularization front of the anterior domain, as well as the greater depth of the pre-CF, is also lost. Thus, these phenomena require the activity of the AP patterning system for their morphogenesis (Bakankenship, 2001).

These cellularization phenomena respond to specific levels of the bicoid gradient. Embryos that carry six copies of bicoid produce more Bicoid protein, shifting any given concentration of Bicoid to a position posterior to where it would be in a wild-type embryo. In embryos carrying six copies of bicoid, the pre-CF domain of small yolk stalk diameters and greater depth is shifted posteriorly, while the anterior domain of large yolk stalk diameters expands to cover close to half of the embryo. bicoid appears to be the important factor specifying these phenomena, since embryos derived from females mutant for bicoid lack the anterior and pre-CF domains (Bakankenship, 2001).

Since the pre-CF domain is a relatively narrow domain, it seemed unlikely that the broad Bicoid gradient would be directly specifying the pre-CF domain. Several pair-rule genes are expressed in approximately the right time and place to be mediating the formation of the pre-CF domain. While Even-skipped (Eve) is expressed in the posterior half of the pre-CF domain, Prd expression is directly centered on the pre-CF domain. Prd expression also has an unusual feature. When Prd expression is first detected during cycle 13, it is observed in a single, gap gene-like domain. Thus, Prd is expressed before the pre-CF domain is formed, and in the right location in the embryo. Moreover, the early expression of Prd in a single stripe, rather than in the stereotypical seven-stripe pattern, suggests that it could specify the pre-CF domain independent of other factors (Bakankenship, 2001).

Various patterning mutants were examined for a disruption of the pre-CF domain. prd homozygotes show a normal anterior domain of larger yolks, but there is very little difference in the yolk stalk diameters between the pre-CF domain and the posterior domain. The relatively subtle appearance of the pre-CF domain in different genetic backgrounds required the scoring of embryos in a blind test. On a classification scale of 0-5, with 0 indicating a complete absence of the pre-CF domain, prd homozygotes scored a 1.2, while their non-homozygous siblings scored a 3.3. These classifications are significant (P <0.001, n=61). Thus, prd is already active in the regulation of morphogenesis during the process of cellularization (Bakankenship, 2001).

The pre-CF domain is first visible when the cellularization front has reached about 25% of its depth. However, the larger yolk stalks of the anterior domain are visible throughout cellularization. The density of nuclei in the anterior is reduced by about 30% from the nuclear density that is found in the rest of the embryo. This anterior domain of lower nuclear density is centered around the anterior pole and extends to approximately two to three nuclei in front of the first stripe of Eve, or ~70% EL. Judging from the staining patterns of Eve and Prd, this is the same area of the embryo in which the anterior domain of large yolk stalk diameters meets the pre-CF domain. During cellularization the anterior nuclear domain is consistently observed in OreR embryos, with an average decrease in nuclear density of 27.6%. In this sample, the values for individual embryos ranged from 34% to 20%. This domain of lower nuclear densities is maintained and stays constant throughout the process of cellularization, and can still be observed in gastrulating embryos. This greater spacing of nuclei in the anterior would necessarily lead to a cellularization network with larger diameters. Additionally, by mid-cellularization, when a pre-CF domain is already visible, a slight clustering of nuclei in this region occurs (Bakankenship, 2001).

Since the anterior domain of lower nuclear densities is present at the start of cycle 14, it was asked when this AP asymmetry arises. While both the sphere of nuclei that migrate to the surface of the embryo during cycle 9, and the cortical nuclei of cycle 10 are uniform along the AP axis, the first sign of increased nuclear spacing in the anterior can be observed in cycle 11 embryos. By cycle 12, a pronounced AP asymmetry is observed, which is maintained through the subsequent mitosis to the start of cellularization. The anterior domain of lower nuclear densities is also observable in living embryos. Nuclear counts on GFP-histone embryos demonstrate a similar ~30% reduction in nuclear density in vivo. These observations on living embryos also reveal that, except for when the nuclei are undergoing mitosis and the associated yolk contractions, the anterior nuclear domain is present throughout the rest of the cell cycle (Bakankenship, 2001).

Before cycle 14, cortical nuclei are associated with large actin caps. In embryos without actin caps, the regular spacing of nuclei is disrupted. One possible mechanism for the formation of the anterior domain of lower nuclear densities would use the regulation of the size of these actin caps. To see if asymmetries in actin cap size occur along the AP axis, wild-type embryos were stained with fluorescently labeled phalloidin to visualize F-actin. Measurements show that anterior actin caps are larger than caps in the rest of the embryo in cycle 11 and cycle 12, but not during cycle 10. This difference, although small, is highly reproducible from embryo to embryo (Bakankenship, 2001).

The exceptionally early appearance of the AP asymmetries in nuclear distributions led to an examination of whether the anterior domain of lower nuclear densities is specified by the AP patterning system. In embryos derived from bicoid nanos torso-like mutant females, nuclear spacing is uniform along the AP axis. In addition, the anterior nuclear domain can be expanded posteriorly in embryos carrying six copies of bicoid. Additionally, embryos from bicoid females are uniform in their nuclear densities, arguing that the anterior domain of lower nuclear densities is set up by the Bicoid gradient. Finally, the larger diameters of the actin caps in the anterior do not occur in embryos from bicoid nanos torso-like females (Bakankenship, 2001).

Since zygotic transcription only starts around cycle 10, the early appearance of the anterior domain by cycle 11 raised the question of whether zygotic transcription is required for this domain's formation. It is possible that bicoid might be regulating nuclear spacing through a post-transcriptional mechanism. To assay the effect of transcription on nuclear spacing, embryos were injected with alpha-amanitin to block RNA polymerase II. Consistent with earlier studies, such embryos develop to cycle 14 with normal gross morphology. However, when these embryos were examined with nuclear stains, they showed a total absence of asymmetric nuclear distributions. Embryos injected with alpha-amanitin fail to form the anterior domain of lower nuclear densities. It is concluded that bicoid regulates the zygotic transcription to bring about the formation of the anterior domain of lower nuclear densities (Bakankenship, 2001).

Two genes necessary for cephalic furrow formation have been identified. Embryos mutant for either eve or buttonhead (btd) lack cephalic furrows. Both eve and btd have a defect in the earliest phase of CF formation. At no point during development are the stereotypical cell shape changes of shortening along the apical-basal axis and widening of the cell observed in these mutants. Mutations in prd also cause a disruption in CF formation. At the onset of gastrulation, the cephalic furrow does not form, nor do initiator cells undergo their characteristic cell shape change. Because there is an absolute correlation between initiator cell behavior and the middle nucleus of the first stripe of Eve in wild-type embryos, the location where initiator cells should form in prd embryos can still be identified. In prd embryos these Eve-marked cells are indistinguishable from their neighbors at stages during gastrulation when the ventral furrow has formed and the CF would normally be visible in wild-type embryos. However, by mid-germband extension (GBE), prd embryos have formed a regular, CF-like invagination. This late fold is thought to arises through the same mechanisms that govern normal CF formation, but these processes are delayed relative to the development of wild-type embryos (in the stage 7 prd embryo there are no cell shape changes in the region where the CF would form, while the stage 6 wild-type embryo has an obvious CF). Initiator cells form in wild-type embryos at stage 6, at the onset of gastrulation. Imaging of prd embryos reveals initiator cell activities beginning at stage 7. At this stage, wild-type embryos have already begun to deform the yolk sack with a basal bulge of the epithelium. At the beginning of stage 8, or germband extension, wild-type embryos have a furrow that is many cells deep, while prd embryos have just begun to deform the yolk sack. It is only by mid-GBE that most, but not all, prd embryos have a regular CF stretching around the entire circumference of the embryo (Bakankenship, 2001).

The abnormalities in CF formation observed at the beginning of gastrulation in prd embryos are superficially similar to those observed in embryos mutant for eve or btd. In the absence of the activity of eve, btd or prd, the cell shape changes that occur in the row of cells that initiate CF formation at the beginning of gastrulation do not occur. In contrast to eve or btd, however, early activities of prd during cellularization have been identified that may account for the later differences observed in CF formation. Consistent with this view the analysis indicates that prd embryos often recover and form a fairly regular CF by mid-germband extension, unlike the severe disruption of the CF in eve and btd embryos. While the start of gastrulation occurs normally in prd embryos, CF formation and initiator cell behavior is delayed. It is proposed that this delay is due not to a function of prd during gastrulation in the specification of initiator cells, as has been proposed proposed for eve and btd function. These latter mutants completely block initiator cell and CF formation. Instead, prd function is necessary for the formation of the pre-CF domain, and it is suggested that prd functions in CF formation only through this disruption of the pre-CF domain. The recovery of the CF observed in prd embryos may be a reflection that cellularization throughout the embryo has finally reached the point that the pre-CF domain reaches at the very beginning of gastrulation, and so CF formation, although delayed, may be correctly initiated. The advanced rate of cellularization in the pre-CF domain may reflect a required premature closing of the base of the initiator cells so that cell volumes may be maintained during the severe cell shape changes of gastrulation, or that cytoskeletal components involved in cellularization must be freed for initiator cell movements. Because little deformation, or loss of volume, occurs in the presumptive initiator cells of early prd embryos, the latter of these two possibilities is favored. Thus, the subtle regulation of one stage of development can have profound effects upon a later, seemingly discrete, process of development (Bakankenship, 2001).

The cellularization front that arises during early cycle 14 is rich in actin and myosin, and is thought to provide a contractile force that orients and drives the process of cellularization. This network, as well as cellularization itself, proceeds through a two-phase process. The first phase is a basal movement of the cellularization front towards the interior of the embryo. During this phase, the furrow canals stay constant in their small size, and the actin/myosin array has a hexagonal shape. The second phase is a lateral movement of the cellularization front, which creates a pinching off at the bases of cells in the newly forming cell sheet. The morphology of the cellularization front is not uniform along the AP axis. The pre-CF domain is distinguished from other regions of the embryo by its small yolk stalk diameters and greater depth of cellularization (Bakankenship, 2001).

These results suggest the following model for the generation of AP asymmetries during cellularization. In the pre-CF domain, the Bicoid gradient directs the correct localization of the early gap gene-like expression domain of prd, which, in turn, directs a greater local contraction of the cellularization network. When cellularization is in its first phase of inward directed movement, the greater contraction of the pre-CF domain leads to a greater advance inwards of the cellularization front, thus creating the greater depth of the pre-CF domain. Then, when cellularization shifts to the second phase of a lateral, pinching-off movement, the greater contractility of the pre-CF domain leads to a greater widening of the furrow canals, which creates the smaller yolk stalk diameters observed in the pre-CF domain. The creation of this domain of advanced cellularization may be necessary for the initiator cell shape change required for cephalic furrow formation (Bakankenship, 2001).

In certain respects, the anterior domain of large yolk stalk diameters and shallow cellularization appears to be the opposite of the pre-CF domain, and thus might be produced by a downregulation of the same contractile mechanisms that are proposed to operate in the pre-CF domain. However, the formation of the larger yolk stalk diameters in the anterior domain clearly involves a different mechanism. The anterior cellularization domain is pre-figured by an anterior domain of lower nuclear densities, while the pre-CF domain initiates in a region where nuclear densities are uniform. The greater spacing of nuclei in the anterior necessarily causes the formation of actin/myosin arrays of greater diameter during cellularization. A model for the formation of the anterior domain is favored in which the bicoid gradient, by cycle 11, has regulated the transcription of a set of zygotic genes, which in turn regulate the size of the actin caps overlaying the nuclei. This regulation of the actin caps results in larger caps in the anterior that necessitates a greater spacing of nuclei. By cycle 14, when cellularization is initiated, the greater spacing of nuclei dictates the generation of a cellularization network in the anterior in which the hexagonal components are larger in diameter. The initiation of large hexagons would thus lead to larger yolk stalks. It may be that the larger hexagons of the cellularization network contract less efficiently, thus generating the shallower depth of cellularization that is observed for the anterior domain (Bakankenship, 2001).

The concept of a mid-blastula transition (MBT) has generally referred to a time when the genome of an embryo begins to exert an influence on development, presumably through zygotic transcription. The best-defined MBT is for Xenopus, where the MBT was characterized by a cessation of synchronous mitotic cycles, the start of zygotic transcription, and a change in the morphology of blastula cells (i.e., the acquisition of cell motility). Various aspects of this characterization have since been called into question. There is low level zygotic transcription before the MBT, and it appears as though cell motility may not be a function of zygotic transcription, but of slower mitosis. Although these discrepancies call into question the usefulness of the MBT as a concept, the idea of an MBT remains an attractive model for explaining the changing morphology of the Drosophila embryo (Bakankenship, 2001).

In flies, the MBT has traditionally been discussed in terms of cycle 14 development. It is at this point that the fly embryo ceases its synchronous syncytial divisions and several morphogenetic processes require zygotic transcription for their genesis. At a superficial level, the first observable defects in embryos deficient for zygotic transcription is at cycle 14, when alpha-amanitin-injected embryos show defects in the processes of lipid droplet clearing and cellularization. However, as in Xenopus, defining a precise MBT presents some difficulties. Although cycle 14 marks a major increase in transcriptional efficiency, the start of zygotic transcription occurs in different nuclear cycles for different genes. In general, for the early acting genes involved in patterning, transcription begins around cycle 10, although some genes are transcribed as early as cycle 8, and mutations in specific segmentation genes show subtle disruption as early as cycle 10. What is striking in the results reported for Bicoid and Prd is the correspondence between spatial patterns of expression and regional alterations in morphology. These results show that the genome of the embryo is active in the spatial regulation of morphogenesis at a much earlier time than previously described. The genome directs reproducible asymmetries in morphology by cycle 11. The generation of an anterior domain of lower nuclear densities argues against a single discrete MBT at cycle 14. These results further suggest a more refined view of the Drosophila MBT in which there is a gradual shifting in the guidance of development from maternal to zygotic gene products, one that stretches from as early as cycle 8 until the cessation of mitosis and formation of a cellularized embryo at cycle 14 (Bakankenship, 2001).

paired executes a dual role in the development of male accessory glands, the organ homologous to the human prostate. An early function is necessary to promote cell proliferation, whereas a late function, which regulates the expression of accessory gland products such as the sex peptide and Acp26Aa protein, is essential for maturation and differentiation of accessory glands. The late function exhibits in main and secondary secretory cells of accessory glands dynamic patterns of Paired expression that depend in both cell types on the mating activity of adult males, possibly because Paired expression is regulated by negative feedback. The early Paired function depends on domains or motifs in its C-terminal moiety and the late function on the DNA-binding specificity of its N-terminal paired-domain and/or homeodomain. Both Paired functions are absolutely required for male fertility, and both depend on an enhancer located within 0.8 kb of the downstream region of paired (Xue, 2002).

The Drosophila accessory gland is a secretory organ of the male reproductive system and a functional homolog of the human prostate. It secretes a complex mixture of proteins, lipids and carbohydrates that are transferred, together with sperm produced by the testes, to females during copulation. Accessory gland secretions (or seminal fluid) induce a number of physiological and behavioral responses in mated females, including increased oviposition, reduced sexual receptivity, diminished attractiveness to males and shortened life expectancy. In addition, components of the seminal fluid are absolutely required for sperm fertility and essential for the storage of sperm in the female genital tract. 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 whose developmental fate is determined by the male sex determination pathway during the third larval instar. 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'. 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 (Xue, 2002 and references therein).

The mouse homolog of the Prd protein, Pax3, when expressed under the control of the complete cis-regulatory region of prd, is able to rescue this 'cuticular' function of prd, yet not its embryonic lethality. Therefore, Prd has a 'viability' function that is separable from its cuticular function. The prd transgene prd-SN20, a genomic fragment extending from 9.8 kb upstream to 5.7 kb downstream of the transcribed region of prd, rescues prd null mutants to fertile wild-type adults and hence includes the enhancers of all prd functions. Two additional prd transgenes are also able to rescue prd mutants to viable adults: prdRes, which lacks the distal 5.2 kb of the downstream region of prd-SN20, and prd-Gsb, in which the coding region of prd-SN20 has been replaced by that of gsb. However, in both these cases all rescued males are sterile, while rescued females are fully fertile. It follows that the wild-type prd gene includes, in addition to its cuticular and viability functions, functions required for male fertility. The sterile prd males rescued by prd-Gsb or prdRes possess severely reduced or no accessory glands, which is the primary cause of the sterility. Since prd males rescued by prd-SN20 have accessory glands of normal size, the 5.2 kb downstream sequences of prd-SN20, which are missing in prdRes, might include the enhancers that are essential for accessory gland formation and the male fertility function of prd. To test this conjecture, prd-mf5, a prd-Gal4 transgene was constructed consisting of the prd promoter, prd transcribed region, and 5.7 kb adjacent downstream sequences placed upstream of the hsp70 basal promoter and the yeast Gal4-coding region. This transgene is expected to function both as a Prd rescue construct for functions mediated by downstream enhancers of prd and as a Gal4 reporter construct, because it drives the expression of Prd as well as Gal4 proteins under the control of the same cis-regulatory region. Indeed, prd-mf5 rescues both the accessory gland phenotype and the male fertility of prd mutant males rescued to adulthood by either prd-Gsb or prdRes. It follows that the enhancer(s) required for prd functions in accessory gland formation and male fertility is located within the 5.7 kb downstream region of prd (Xue, 2002).

In addition to its requirement for accessory gland development, Prd is expressed in the differentiated glands of adult males. To examine the expression pattern of Prd in adult accessory glands more closely, genital tracts were dissected from virgin males 1 day, 5 days and 10 days after eclosion, and stained for Prd protein by the use of a Prd antiserum. Prd is initially expressed at high levels in all secretory accessory gland cells of newly eclosed flies, but levels are gradually reduced in virgin males as a function of increasing age -- rapidly in main cells and slowly in secondary cells. In 10-day-old virgin males, Prd protein remains detectable only in secondary cells, the few scattered cells in the distal region of the glands (Xue, 2002).

To determine the effect of mating on Prd expression in accessory glands, these glands were dissected from 10-day old males that had been allowed to mate after 5 days. Such males display enhanced Prd levels in both main and secondary cells throughout the entire glands. Similar patterns were observed in glands of 13-day-old males mated only after 10 days. Therefore, the elevated Prd levels result from an increase in synthesis rather than a slower decay of the Prd protein after mating. It is possible that mating induces factor(s), for example a hormonal response, that regulate prd positively. Alternatively, Prd expression might be regulated by negative feedback that inhibits Prd synthesis in the presence of high concentrations of accessory gland fluid or at least one of its products. Accumulation of these secreted factors in the absence of mating would thus downregulate Prd protein, whereas a reduction in concentration as a result of mating would in turn relieve the inhibition of Prd synthesis (Xue, 2002).

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 and accessory gland-specific enhancer trap lines, which are probably regulated at the transcriptional level. 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 (Xue, 2002).

The prd-mf5 transgene is not only able to rescue accessory gland development, but also to express Prd in adult accessory glands with the same profile as endogenous Prd. In addition, it restores fertility 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, a series of prd transgenes derived from prd-mf5 was constructed by deleting different portions of the downstream sequences. 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. 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. 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, 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. 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. 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. Evidently, the 0.8 kb of the prd downstream region included in prd-mf9 harbors 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 (Xue, 2002).

prd mutant males rescued by prd-Gsb or prdRes exhibit severely reduced or no accessory glands, 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, advantage was taken of a transgenic line, prd-mf9.7, that rescues the accessory glands of prdRes mutant males completely with two copies of prd-mf9, but only partially with one copy, while restoration of fertility requires two copies. By contrast, most other prd-mf9 lines display a complete rescue with a single copy. 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. The fact that, in addition to the expression of Prd, prd-mf9 drives Gal4 expression ubiquitously in developing accessory glands under the control of the same enhancer permits the expression of any protein in developing accessory glands under the control of this enhancer by the use of the Gal4/UAS system and the subsequent testing of 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 (Xue, 2002).

As expected, one copy of UAS-Prd rescues the accessory glands to nearly wild-type size. In addition, overexpression of Myc or CycE, both of which are required for promoting cell proliferation, rescues the accessory glands to a large extent and restores male fertility. The rescue by CycE or Myc completely depends on the low level of Prd expression from prd-mf9.7 in developing accessory glands. This is evident from the complete absence of accessory glands in prd mutant males that are rescued by prdRes and carry a prd3.1-Gal4 transgene driving UAS-CycE or UAS-dMyc expression in accessory glands under control of the prd downstream region. An inhibition of the cell cycle, presumably in G1, rather than induced apoptosis, is the primary cause for the reduction or loss of accessory glands in prd mutant males, and prd is required for promoting cell proliferation during early accessory gland development (Xue, 2002).

In adult accessory glands, prd exhibits a dynamic expression profile that depends on aging and mating activity. 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. 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. 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, since the accessory glands of prd mutant males rescued by two copies of prd-Gsb are severely reduced, 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, 2002).

The sterility of prd mutant males, whose accessory glands have been rescued to normal size by prd-GsbN+PrdC, might result from a failure to express certain accessory gland factors required for sperm fertility. To test this supposition, the expression of Acp26Aa, Gsb and sex peptide (SP) was examined in the accessory glands of wild-type and prd mutant males rescued by prd-GsbN+PrdC. While the Acp26Aa protein is important for enhanced female oviposition during the first day after copulation, SP is a key component of accessory gland secretions responsible for increased oviposition and reduced sexual receptivity in mated females. The function of Gsb in adult accessory glands is not known. In wild-type accessory glands, Acp26Aa is expressed in all secretory cells, while Gsb is expressed only in secondary cells and SP only in main cells as assayed by the expression of a lacZ reporter gene under control of the sp enhancer. In prd mutant males rescued by prd-GsbN+PrdC, the accessory glands fail to express Acp26Aa, Gsb and SP, which suggests that prd is indeed also required in late accessory gland development to regulate the expression of at least these three accessory gland products (Xue, 2002).

Although Prd and Gsb share a highly conserved N-terminal moiety, including two DNA-binding domains, a paired-domain and a prd-type homeodomain, 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 (Xue, 2002).

It is concluded 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 (Xue, 2002).

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, overexpression of Pax3 in cultured cells produces foci of transformed cells that are able to develop tumors in nude mice. Moreover, a Pax3 gain-of-function mutation produces alveolar rhabdomyosarcoma, a highly proliferative cancer. In addition to its role in regulating cell proliferation, Pax3 acts upstream of MyoD and can induce muscle differentiation. 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 (Xue, 2002).

Because gene networks have been conserved during evolution, 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 (Xue, 2002).


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paired continued: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 3 July 2014

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