In Dad enhancer trap lines, beta-galactosidase is expressed in a wide stripe that straddles the A/P compartment boundary of the imaginal discs, in contrast to Dpp, whose expression is confined to the anterior side. This pattern of expression suggests that Dad expression is positively regulated by the secreted Dpp molecule (Tsuneizumi, 1997).
Smad signal transducers are required for transforming growth factor-ß-mediated developmental events in many organisms including humans. However, the roles of individual human Smad genes (hSmads) in development are largely unknown. It was hypothesized that an hSmad performs developmental roles analogous to those of the most similar Drosophila Smad gene (dSmad). Six hSmad and four dSmad transgenes were expressed in Drosophila using the Gal4/UAS system and their phenotypes were compared. Phylogenetically related human and Drosophila Smads induce similar phenotypes supporting the hypothesis. In contrast, two nearly identical hSmads generate distinct phenotypes. When expressed in wing imaginal discs, hSmad2 induces oversize wings while hSmad3 induces cell death. This observation suggests that a very small number of amino acid differences, between Smads in the same species, confer distinct developmental roles. These observations also suggest new roles for the dSmads, Medea and Dad, in Drosophila Activin signaling (see Drosophila Activins Activin-ß and Activin Like Protein at 23B; the Drosophila Activin receptor is Baboon) and in potential interactions between these family members. Overall, the study demonstrates that transgenic methods in Drosophila can provide new information about non-Drosophila members of developmentally important multigene families (Marquez, 2001).
hSmad6, hSmad7, and possibly Dad can antagonize signals of both TGF-ß subfamilies. This relationship suggested that these Smads will produce similar phenotypes. The phenotypes generated by UAS.hSmad6, UAS.hSmad7, and UAS.Dad are comparable. UAS.hSmad6/ptc.Gal4 and UAS.hSmad7/ptc.Gal4 genotypes have truncated legs. These phenotypes could not be directly compared with those of UAS.Dad because UAS.Dad does not generate leg phenotypes. UAS.Dad is lethal with all but two Gal4 lines (A9.Gal4 and MS1096.Gal4). These lines have virtually no embryonic or leg disc expression. Alternatively, UAS.hSmad6 and UAS.hSmad7 do not generate wing phenotypes with A9.Gal4 or MS1096.Gal4. However, the tiny wings of UAS.Dad/MS1096.Gal4 flies and the truncated legs of UAS.hSmad6/ptc.Gal4 and UAS.hSmad7/ptc.Gal4 flies suggest that these Smads have similar abilities to inhibit limb growth. Perhaps the UAS.Dad/MS1096.Gal4 tiny wing phenotype results from Dad antagonizing both the Dpp and the dActivin cell proliferation pathways (Marquez, 2001).
The UAS.Dad/MS1096.Gal4 and UAS.Dad/A9.Gal4 tiny wings are also veinless. This phenotype is likely due to UAS.Dad antagonizing Dpp signals that promote vein formation via Mad and Med. Like hSmad6 and hSmad7 in cell culture, Dad may be capable of antagonizing signals from both TGF-ß subfamilies (Marquez, 2001).
The multi-subfamily signaling Smads, UAS.hSmad4 and UAS.Med, also generate truncated legs with several Gal4 lines. In fact, truncated legs on UAS.hSmad4/ptc.Gal4 and UAS.Med/ptc.Gal4 flies were noticed more frequently than duplicated legs. The truncated leg phenotypes of UAS.hSmad4/ptc.Gal4 and UAS.Med/ptc.Gal4 flies are similar to those of UAS.hSmad6/ptc.Gal4 and UAS.hSmad7/ptc.Gal4 flies. The common leg phenotype suggests that antagonist Smads (e.g., hSmad6) may interact with multi-subfamily signaling Smads (e.g., hSmad4) when expressed in Drosophila. Interactions between antagonist and multi-subfamily signaling Smads have been shown in Xenopus injection assays (Marquez, 2001).
\
Thus phylogenetically related Smad family members (Mad/hSmad1, dSmad2/hSmad2, Med/hSmad4, and Dad/hSmad6/hSmad7) induce similar phenotypes. This result supports the hypothesis that an hSmad performs roles in human development analogous to the ones their dSmad counterpart plays in Drosophila development. It is suggested that the developmental roles of hSmads can now be more profitably investigated using clues from dSmads. For example, tinman is a Mad/Med target gene for Dpp signals during the subdivision of the embryonic mesoderm. On the basis of these results, the highly conserved human homologs of tinman are candidate targets of hSmad1 and hSmad4 in human mesodermal cells (Marquez, 2001).
Many of the phenotypes observed reinforce known roles for dSmads. For example, the moderately large wing phenotype seen with UAS.dSmad2 is consistent with a role in a dActivin pathway that stimulates cell proliferation in wing development. However, other phenotypes suggest new roles for dSmads. For example, moderately large wings generated with several Gal4 lines suggest that Media participates in dActivin signaling. The tiny wings generated with MS1096.Gal4 suggest that Dad may have the ability to antagonize both Dpp and dActivin signaling. In addition, the common truncated leg phenotype generated by Medea, hSmad6, and hSmad7 suggests that Med may interact with antagonist Smads such as Dad. These potential roles for Med and Dad are consistent with activities already shown for their human counterparts. For example, hSmad4, hSmad6, and hSmad7 can influence signals from both TGF-ß subfamilies in cell culture and hSmad4 can interact with hSmad6 in Xenopus injection assays (Marquez, 2001).
In summary, this analysis of hSmad and dSmad transgenes supports the hypothesis that phylogenetically related Smads fulfill developmental roles that are conserved between humans and Drosophila. The results also suggest a number of new hypotheses regarding roles for human and Drosophila Smads in pattern formation, cell proliferation, and cell death. The data suggest that a small number of amino acid differences between two very similar Smads in the same species can confer distinct activities. Overall, this study demonstrates that transgenic methods in Drosophila can provide new information about mammalian members of developmentally important multigene families (Marquez, 2001).
The paired male accessory glands of Drosophila melanogaster enhance sperm function, stimulate egg production, and reduce female receptivity to other males by releasing a complex mixture of glycoproteins from a secretory epithelium into seminal fluid. A small subpopulation of about 40 specialized secretory cells, called secondary cells, resides at the distal tip of each gland. These cells grow via mechanisms promoted by mating. If aging males mate repeatedly, a subset of these cells delaminates from and migrates along the apical surface of the glandular epithelium toward the proximal end of the gland. Remarkably, these secretory cells can transfer to females with sperm during mating. The frequency of this event increases with age, so that more than 50% of triple-mated, 18-d-old males transfer secondary cells to females. Bone morphogenetic protein signaling specifically in secondary cells is needed to drive all of these processes and is required for the accessory gland to produce its normal effects on female postmating behavior in multiply mated males. It is concluded that secondary cells are secretory cells with unusual migratory properties that can allow them to be transferred to females, and that these properties are a consequence of signaling that is required for secondary cells to maintain their normal reproductive functions as males age and mate (Leiblich, 2012).
The secondary cells of the male fly accessory gland
selectively grow during aging in adults, a process enhanced by
repeated mating. These cells exhibit a range of behaviors, induced
by mating, that are atypical of secretory cells in glands,
including active delamination and migration. Although migrating
cells were initially observed in less than 5% of repeatedly mated
males, introducing a delay between two previous matings and
dissecting the resulting 18-d-old males revealed migrating cells in
all animals, suggesting that this process is common in aged,
mated animals (Leiblich, 2012).
The growth, delamination and migratory activities of secondary
cells all require cell-autonomous BMP signaling. One or
more of these BMP-regulated processes modulates long-term,
postmating behavior in females, particularly when males are
repeatedly mated over short periods of time, requiring rapid
replenishment of luminal content in the accessory gland. Although
the numbers of vacuoles in secondary cells with high
levels of BMP signaling seem more variable than controls, vacuole
number in Dad-expressing secondary cells appears relatively
normal, suggesting that reduced BMP signaling does not simply
block the general secretory machinery. However, reduced signaling
presumably affects the synthesis or function of one or
more secondary cell products, leading either to direct effects in
mated females or to indirect effects through modulation of main
cell function or products in males (Leiblich, 2012).
Unexpectedly, some secondary cells are transferred to females
after multiple matings, particularly in aged flies, raising the
possibility that these delaminating cells continue to function
together with sperm even outside the male. Transfer is not essential
for these cells to mediate their BMP-regulated effects in
females, because not all mated females receive these cells.
However, it is possible that transfer could contribute to changes
in accessory gland function as the glandular epithelium undergoes
BMP-dependent structural alterations during aging and
mating. A recent study from Minami (2012) indicates that
secondary cells are required for normal male fecundity and
effects on female postmating behaviors. The current work now clearly
demonstrates that BMP-mediated events in secondary cells are
involved in maintaining these latter functions specifically
during adulthood (Leiblich, 2012).
The data highlight some surprising parallels between the accessory
gland and the prostate, in addition to those previously
reported. Like the prostate, the structure of the accessory
gland epithelium changes significantly with age. Furthermore,
BMP signaling is implicated in normal prostate development and in the progression of prostate cancer. Importantly, prostate cells have been identified in human semen and the
phenotype of these cells may be altered in prostate cancer. Although many of these cells are likely to have sloughed off from the epithelium, the current study raises the possibility that some
actively delaminate into seminal fluid (Leiblich, 2012).
The secondary cells of the accessory gland
require BMP signaling to regulate the synthesis or function of
one or more important components of the seminal fluid as flies
age and mate. However, this signaling simultaneously drives cell
loss and changes in the morphology and function of the epithelium,
which appears to lack regenerative capacity in flies. The
prostate gland of most human males over 50 y of age is hyperplastic, and it is tempting to speculate that this reflects a regenerative response to similar events in this organ. A more
detailed analysis of secondary cell biology should help to further
elucidate the processes that underlie functional changes in the
accessory gland epithelium and test whether these are shared by
male reproductive glands in other organisms (Leiblich, 2012).
spinster (spin), which encodes a multipass transmembrane protein, has been identified in a genetic screen for genes that control synapse development. spin mutant synapses reveal a 200% increase in bouton number and a deficit in presynaptic release. spin is expressed in both nerve and muscle and is required both pre- and postsynaptically for normal synaptic growth. Spin has been localized to a late endosomal compartment and evidence is presented for altered endosomal/lysosomal function in spin mutants. Evidence is presented that synaptic overgrowth in spin is caused by enhanced/misregulated TGF-ß signaling. TGF-ß receptor mutants show dose-dependent suppression of synaptic overgrowth in spin. Furthermore, mutations in Dad, an inhibitory Smad, cause synapse overgrowth. A model is presented for synaptic growth control with implications for the etiology of lysosomal storage and neurodegenerative disease (Sweeney, 2002).
It was hypothesized that enhanced or unregulated growth factor signaling is the cause of overgrowth in spin, and therefore, whether enhanced TGF-ß signaling is sufficient to cause synaptic overgrowth was tested. If TGF-ß signaling is sufficient to cause enhanced synaptic growth, then a mutation in a negative regulator of TGF-ß signaling is predicted to cause an increase in bouton number. Daughters against DPP (Dad) encodes an inhibitory Smad that negatively regulates TGF-ß signaling in Drosophila and other systems. Synapse morphology was examined in a strong loss-of-function Dad mutation that is viable to third instar larvae. Dad mutant synapses reveal a dramatically altered morphology with increased numbers of clearly distinct, small boutons that sprout from what appears to be the normal synaptic process. This is a highly penetrant phenotype and is observed at muscles 6/7 and muscle 4. Quantification of total synaptic bouton number demonstrates a significant increase in bouton numbers that is nearly equivalent to that observed in the spin mutant. These data demonstrate that enhanced TGF-ß signaling can cause synaptic overgrowth (Sweeney, 2002).
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date revised: 15 February 2011
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