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Stepwise formation of a SMAD activity gradient during dorsal-ventral patterning of the Drosophila embryo

Genetic evidence suggests that the Drosophila ectoderm is patterned by a spatial gradient of bone morphogenetic protein (BMP). Patterns have been compared of two related cellular responses - signal-dependent phosphorylation of the BMP-regulated R-SMAD, MAD, and signal-dependent changes in levels and sub-cellular distribution of the co-SMAD Medea. Nuclear accumulation of Medea requires a BMP signal during blastoderm and gastrula stages. During this period, nuclear co-SMAD responses occur in three distinct patterns. At the end of blastoderm, a broad dorsal domain of weak SMAD response is detected. During early gastrulation, this domain narrows to a thin stripe of strong SMAD response at the dorsal midline. SMAD response levels continue to rise in the dorsal midline region during gastrulation, and flanking plateaus of weak responses are detected in dorsolateral cells. Thus, the thresholds for gene expression responses are implicit in the levels of SMAD responses during gastrulation. Both BMP ligands, DPP and Screw, are required for nuclear co-SMAD responses during these stages. The BMP antagonist Short gastrulation (Sog) is required to elevate peak responses at the dorsal midline as well as to depress responses in dorsolateral cells. The midline SMAD response gradient can form in embryos with reduced dpp gene dosage, but the peak level is reduced. These data support a model in which weak BMP activity during blastoderm defines the boundary between ventral neurogenic ectoderm and dorsal ectoderm. Subsequently, BMP activity creates a step gradient of SMAD responses that patterns the amnioserosa and dorsomedial ectoderm (Sutherland, 2003).

These in vivo studies validate the molecular model for signal-dependent nuclear accumulation of Medea. Nuclear accumulation of Medea requires both competence to oligomerize and MAD. Nuclear accumulation is signal dependent, requiring both BMP ligands, Dpp and Scw. Conversely, all cells accumulated nuclear Medea in the presence of constitutively active Tkv receptor. At these stages, any independent contribution from activin-like signals is below the detection limit (Sutherland, 2003).

Furthermore, levels of Medea determine the strength of BMP responses at these stages. Medea overexpression leads to expansion of the dorsal-most fate, with increased numbers of amnioserosa cells. Signal-dependence for nuclear accumulation is retained. Decreased Medea exacerbates loss of amnioserosa from reduced Dpp levels (Sutherland, 2003).

The intensity of Medea staining was surprisingly sensitive to signal activity. However, steady-state levels of Medea are unaffected by the level of BMP activity. The antibodies appear highly sensitive to a Medea conformation that is prevalent in the nucleus, most probably an active SMAD complex. This sensitivity makes nuclear Medea an excellent assay to distinguish spatial patterns of endogenous BMP activity (Sutherland, 2003).

In wild-type embryos, two transitions in the distribution of BMP activity are evident. Many cellular blastoderm embryos lack detectable levels of nuclear Medea, but a few have low levels of nuclear Medea in a broad dorsal domain, with little gradation. From the proportion of cellular blastoderm embryos with this pattern, the duration of nuclear Medea appears to be brief. These data parallel reports of broad, weak PMad staining during mid-cellularization, except that nuclear Medea is detected later and in a broader pattern. The time lag between the earliest reported detection of PMad and detection of nuclear Medea probably stems from a combination of technical differences and the time necessary for nuclear accumulation. In sum, initial BMP activity is weak and distributed broadly in dorsal regions. Low BMP activity at this phase is required to maintain the early phase of zen expression (Sutherland, 2003).

Onset of gastrulation is associated with a dramatic change in the domain of nuclear Medea, which narrows to a tight midline stripe of cells while staining levels intensify. PMad shows a similar transition to a narrower domain, but earlier. Thus, lateral SMAD responses became undetectable just as a steep activity gradient forms along the dorsal midline (Sutherland, 2003).

A third response pattern arises during mid-gastrulation: dorsolateral domains of cells exhibit low levels of nuclear Medea. Response levels remain high in the dorsal-most cells, even as they move laterally during gastrulation. Levels fall off rapidly over a few cells on either side, with a sharp transition to flanking plateaus of weak responses. The subcellular distribution of Medea is unchanging in ventral and ventrolateral cells. The full BMP response domain does not extend as far ventrally as it does during blastoderm, even though many dorsal cells move laterally during germband extension. Thus, the lateral-most cells with responses at blastoderm have decreased responses during gastrulation (Sutherland, 2003).

In sum, the dorsal midline stripe of SMAD responses corresponds to a steep BMP activity gradient, with thresholds that correlate with patterning markers. The edges of the Medea peak response correlates precisely with the position of dorsal cephalic markers during stage 8, the cycle 14 mitotic domains 1, 3 and 5. The second phase of zen expression occurs in cells with peak PMad responses at the end of stage 5. Flanking cells with lower PMad levels correlate with the broader expression domain for the BMP target genes tailup and u-shaped. The full Medea response domain correlates approximately with the expression domain for u-shaped and extends into the presumptive dorsomedial ectoderm. The sharp transitions in SMAD response levels predict expression boundaries for BMP-responsive genes (Sutherland, 2003).

Similarly, in the wing primordium, a BMP gradient creates sharp transitions in PMad levels, which match gene expression boundaries. However, BMP activity is modulated by different mechanisms in this tissue. dpp is expressed in a narrow stripe at the center, and ligand spreads to nearby cells over a period of hours. In contrast, the early embryonic BMP activity gradient forms rapidly, and is narrower than the expression domains for dpp and scw. Extracellular binding proteins form the embryonic BMP activity gradient (Sutherland, 2003).

The final width of the midline peak response is sensitive to gene dosage for both dpp and sog. It is broader when dpp dosage is increased, and narrower with only one copy of dpp. Similarly, the width of the stripe is broader, but more variable, when sog levels are reduced. The response domain is broadest in sog null embryos; however, the level of response is significantly reduced. This is distinct from the effect of increased dpp dosage, in which the response domain is broader, but normal SMAD response levels are achieved or exceeded (Sutherland, 2003).

The role of Sog as both a short-range inhibitor and a long-range potentiator of dorsal patterning has led to a proposal that Sog transports BMP ligands from lateral regions to the dorsal midline. Biochemical analyses suggest mechanisms for Sog-BMP binding and release. Computational analysis have defined conditions under which transport could occur with these mechanisms. The transition from weak, broad SMAD responses to narrow, strong responses is consistent with concentration of BMP activity at the dorsal midline, and the loss of this transition with loss of Sog is consistent with a Sog-dependent transport model. However, there are significant differences between the current results and the assumptions used to develop the computational model. These include the presence of a midline SMAD response in dpp–/+ embryos and the sensitivity to reduced sog dosage. It will be important to refine future computational models to fit the complete set of BMP response data (Sutherland, 2003).

Both BMP ligands, Dpp and Scw, are required to form the dorsal-midline gradient. However, scw mutant embryos retain a small amount of dorsal ectoderm, with concomitant expansion of ventral ectoderm. Surprisingly, the weak dorsolateral Medea response is lost in scw embryos. It is concluded that the full Medea response domain encompasses the cell fates that are lost in scw mutants, amnioserosa and dorsomedial ectoderm. It appears that dorsal cells can acquire a dorsolateral fate without gastrula BMP activity (Sutherland, 2003).

Mutants with expanded ventral ectoderm show reduced SMAD responses during the first phase of BMP activity. PMad was not detected in blastoderm tld embryos. Homozygotes for moderate dpp alleles have lower PMad levels during blastoderm. Conversely, sog embryos have a slightly expanded PMad response during blastoderm, and a slight expansion of dorsal ectoderm. Thus, BMP activity during blastoderm positions the boundary between dorsal and ventral ectoderm (Sutherland, 2003).

Mutations that shift the boundary between amnioserosa and dorsal ectoderm show altered SMAD responses in the third phase of BMP activity, the dorsal-midline gradient. dpp-/+ embryos have variable reductions in midline SMAD responses and in the number of amnioserosa cells. Strikingly, sog null embryos have little amnioserosa and a strong reduction in SMAD response levels during gastrulation. Thus, SMAD response levels during gastrulation are critical for amnioserosa specification (Sutherland, 2003).

Taken together, these data suggest a multi-step model for DV patterning of the embryonic ectoderm, incorporating aspects of the two previous models. In a previous gradient model, ectodermal fates are subdivided simultaneously by a continuous BMP gradient involving Dpp and Scw. In the successive cell-fate decision model, amnioserosa is specified by dorsal-midline Dpp+Scw activity, and the dorsal ectoderm by Dpp alone at stage 9 (Sutherland, 2003).

Instead, it is proposed that the blastoderm phase of weak BMP activity establishes a dorsal ectoderm domain. Mutations that shift the boundary between dorsal and ventral ectoderm also have altered SMAD responses at this stage. It is at this stage that SMADs compete with Brinker to regulate the first phase of zen expression. Furthermore, this early signal maintains BMP activity, for the late-blastoderm domain of dpp expression is set by competition between BMPs, Sog and Brinker. BMP activity subsequently maintains the dorsal boundary for brinker expression. Thus, BMP activity at blastoderm defines a dorsal domain where dpp is expressed and brinker is not (Sutherland, 2003).

After cellularization is complete, a step gradient of BMP activity subdivides the dorsal region into amnioserosa, dorsomedial ectoderm and dorsolateral ectoderm. Peak activity levels determine the amount of amnioserosa. Flanking shoulders of weak activity specify the dorsomedial ectoderm. It is proposed that the dorsolateral ectoderm experiences a transient BMP response during late blastoderm, but little or no response during gastrulation. In sum, the dorsal-midline gradient of BMP activity specifies at least three cell fates (Sutherland, 2003).

BMP activity in the dorsal ectoderm does not end with germband extension. During stage 9, PMad is detected throughout the dorsal ectoderm and amnioserosa, and might finalize determination of dorsal ectoderm fates. Dpp expression within the dorsal ectoderm contributes to combinatoral regulation of gene expression patterns in subsets of dorsal ectodermal cells. However, the ventral boundary of dpp expression in the stage 9 dorsal ectoderm must be defined by earlier events (Sutherland, 2003).

The step gradient of SMAD responses is maintained during the morphogenetic movements of gastrulation and germband extension. The peak response is maintained only in cells that initially reside at the dorsal midline, even though ventral ectoderm moves to a dorsal position during stages 7 and 8. The BMP activity gradient is thought to form by diffusion in the perivitelline fluid; however, dorsal cells 'remember' their BMP exposure as they move laterally. It is probable that the ligand distribution is established prior to the time that peak SMAD responses are detected, and activity persists through cell biological mechanisms. For example, ligand may bind to the extracellular matrix, so that it remains associated with dorsal cells. Alternatively, receptor-ligand complexes may continue to signal following endocytosis. Understanding the intracellular modulation of BMP responses will be important to understand how extracellular morphogen gradients are translated into a stable pattern of cell fates (Sutherland, 2003).

Transgenic analysis of the Smad family of TGF-ß signal transducers

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).

hSma1 and Mad can transduce Dpp/BMP signals. hSmad4 and possibly Med can transduce signals for both TGF-ß subfamilies. hSmad4 forms complexes with hSmad1 and Med forms complexes with Mad. These relationships suggest that these Smads will produce similar phenotypes. One copy of UAS.Mad or UAS.hSmad1 does not generate many phenotypes. Strains containing two copies of these transgenes were then used. UAS.Mad and UAS.hSmad1 induced similar wing and leg phenotypes. For example, UAS.Mad/ptc.Gal4 genotypes have ectopic vein tissue between L3 and L5. This vein phenotype is consistent with two previous results: clonal analysis shows a role for Mad in vein formation and ptc.Gal4 expression in wing disc cells that eventually reside between L3 and L4. In UAS.Mad/ptc.Gal4 wings the distance between L3 and L5 appears reduced. This may be due to the smaller size of vein cells vs. intervein cells. A comparison of wing surface areas shows that UAS.Mad/ptc.Gal4 wings are 22% smaller than wild type. UAS.hSmad1/ptc.Gal4 wings have ectopic vein tissue in roughly the same region. UAS.hSmad1/ptc.Gal4 wings are 15% smaller than wild type. UAS.Mad and UAS.hSmad1 expression appears to mimic dpp's role in vein formation. UAS.Mad and UAS.hSmad1 expression does not appear to mimic dpp's other roles in wing development (cell proliferation and/or cell survival and anterior/posterior patterning) (Marquez, 2001).

UAS.Medea/ptc.Gal4 and UAS.hSmad4/ptc.Gal4 wings have ectopic vein tissue in the same region as UAS.Mad/ptc.Gal4 and UAS.hSmad1/ptc.Gal4 wings. This suggests that Med forms complexes with Mad during vein formation. UAS.Med/ptc.Gal4 wings are 11% larger and UAS.hSmad4/ptc.Gal4 wings are 6% larger than wild type. The larger, rather than smaller, size of UAS.Med/ptc.Gal4 and UAS.hSmad4/ptc.Gal4 wings suggests that multi-subfamily signaling Smads can influence wing size and vein formation (Marquez, 2001).

UAS.Mad/ptc.Gal4 flies have an ectopic leg on the ventral side of a normal limb. The ectopic leg has several segments and terminates in a set of tarsal claws. During leg development, wingless normally represses dpp expression on the ventral side of the limb. In limbs expressing ectopic dpp an additional leg develops on the ventral side of the limb. The similarity between phenotypes that result from ectopic dpp expression and UAS.Mad expression suggests that UAS.Mad is capable of simulating Dpp signals in leg patterning. UAS.hSmad1/ptc.Gal4 flies also have an ectopic leg on the ventral side of a normal limb. The ectopic leg consists of a single segment. UAS.Med/ptc.Gal4 and UAS.hSmad4/ptc.Gal4 flies have ectopic legs of a different type. Legs from these genotypes have short, abnormally wide tibia that lead to duplicated tarsi of one or more segments. The abnormal tibia of UAS.Med/ptc.Gal4 flies has an additional patterning defect: severe bristle overgrowth (Marquez, 2001).

Overall, these four Smads generated comparable vein and leg phenotypes. The phenotypes of phylogenetically related Smads show the greatest similarity. The phenotypes suggest that UAS.hSmad1 and UAS.hSmad4 can also simulate Dpp signaling in Drosophila limb development. These findings are consistent with cell culture studies noted above and further support the view that these hSmads transduce BMP signals during human development. The size of UAS.Med and UAS.hSmad4 wings suggests a role for these multi-subfamily signaling Smads not shared with the Dpp/BMP signaling Smads UAS.Mad and UAS.hSmad1 (Marquez, 2001).

dSmad2, hSmad2, and hSmad3 can transduce TGF-ß/Activin signals. hSmad4 and possibly Med can transduce signals for both TGF-ß subfamilies. hSmad4 forms complexes with hSmad2 and with hSmad3. These relationships suggest that these Smads will produce similar phenotypes (Marquez, 2001).

UAS.dSmad2, UAS.hSmad2, UAS.Med, and UAS.hSmad4 induce similar wing phenotypes. When expressed with A9.Gal4, each produces moderately large wings. A9.Gal4 is expressed throughout the wing disc. A comparison of wing surface areas reveals that UAS.dSmad2, UAS.hSmad2, and UAS.Med wings are ~22% larger than wild type. UAS.hSmad4 wings are 16% larger than wild type. It has been shown that dSmad2 transduces dActivin signals that modestly stimulate cell proliferation in wing development. The influence of dActivin signals on wing cell proliferation is much smaller than that of Dpp signals. Ectopic dActivin signaling results in an ~30% increase in wing size. The moderately large size of the common wing phenotype suggests that UAS.hSmad2, UAS.Med, and UAS.hSmad4 simulate dActivin-, rather than Dpp-, mediated cell proliferation (Marquez, 2001).

Consistent with their ptc.Gal4 phenotypes, UAS.Med/A9.Gal4 and UAS.hSmad4/A9.Gal4 wings also show ectopic veins. These wings have a distal crossvein between L2 and L3 and duplications of L2, L3, and L4 at the margin. The presence of wing size and vein phenotypes provides the first evidence that Med, like its counterpart hSmad4, can signal for both TGF-ß subfamilies. UAS.Med and UAS.hSmad4 influences vein formation like Dpp/BMP signaling Smads (UAS.Mad and UAS.hSmad1) and moderately increase wing size like TGF-ß/Activin signaling Smads (UAS.dSmad2 and UAS.hSmad2) (Marquez, 2001).

In contrast, UAS.hSmad3/A9.Gal4 wings are smaller than wild type (~32%). These wings have essentially wildtype venation although loops are occasionally present. The dramatic difference in wing size suggests that UAS.hSmad3 cannot participate in a dActivin pathway that stimulates cell proliferation. The UAS.hSmad3 wing phenotype suggests that UAS.hSmad3 can inhibit cell proliferation or stimulate apoptosis during wing development (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).

Neurophysiological defects and neuronal gene deregulation in Drosophila mir-124 mutants

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, its single mir-124 locus was deleted, and it was found to be dispensable for gross aspects of neural specification and differentiation. In contrast, a variety of mutant phenotypes were detected that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124-expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology (Sun, 2012).

microRNAs (miRNAs) are ~22 nucleotide (nt) regulatory RNAs that function primarily as post-transcriptional repressors. In animals, miRNAs have propensity to target mRNAs via 6-7 nt motifs complementary to their 5' ends, termed 'seed' regions. This limited pairing requirement has allowed most miRNAs to capture large target networks. Analysis of multigenome alignments indicates that typical human miRNAs have hundreds of conserved targets, and that a majority of protein-coding genes are under miRNA control. The extraordinary breadth of animal miRNA:target networks has been extensively validated by transcriptome and proteome studies (Sun, 2012).

miR-124 is strictly conserved in both primary sequence and spatial expression pattern, being restricted to the nervous system of diverse metazoans, including flies, nematodes Aplysia, and all vertebrates studied. Such conservation implies substantial functions of miR-124 in controlling neural gene expression. miR-124 has been a popular model for genomewide investigations of miRNA targeting principles. For example, studies of miR-124 yielded the first demonstration of the downregulation of hundreds of direct targets detected by transcriptome analysis, and that this activity was driven by the miRNA seed region. In addition, miR-124 provided one of the first illustrations of spatially anticorrelated expression of a miRNA and its targets and direct identification of Ago-bound target sites (Sun, 2012).

Functional studies have connected vertebrate miR-124 to various aspects of neural specification or differentiation. Studies in chick ascribed miR-124 as a proneural factor that inhibits the anti-neural phosphatase SCP1. However, no substantial effect of miR-124 on chick neurogenesis was found in a parallel study, although miR-124 was observed to repress neural progenitor genes such as laminin gamma1 and integrin beta1. In the embryonic mammalian brain, miR-124 was reported to direct neural differentiation by targeting polypyrimidine tract binding protein 1 (PTBP1), a global repressor of alternative splicing in non-neural cells. In the adult mammalian brain, miR-124 promoted neural differentiation of the immediate progenitors, the transit-amplifying cells (TAs). Here, miR-124 was shown to directly target the transcription factor Sox9, which maintains TAs and is downregulated during neural differentiation. Other mammalian studies bolster the concept that miR-124 promotes neurogenesis or neural differentiation. One mechanism involves direct repression by miR-124 of Baf53a, a neural progenitor-specific chromatin regulator that must be exchanged for a neural-specific homolog to consolidate neural fate. However, complicating the picture is the recent report that Xenopus miR-124 represses neurogenesis by directly targeting the proneural bHLH factor NeuroD1 (Sun, 2012 and references therein).

All vertebrate miR-124 loss-of-function studies have relied on antisense strategies and have yet to be validated by bona fide mutant alleles. However, as the three vertebrate mir-124 loci are co-expressed in the nervous system, analysis of the null situation will require a triple knockout. So far, a mir-124 knockout has only been described in C. elegans, which harbors a single copy of this gene. Like most other miRNA mutants in this species, the loss of miR-124 did not cause obvious developmental, physiological or behavioral phenotypes. Nevertheless, comparison of gene expression in mir-124-expressing cells from wildtype and mir-124 mutant animals revealed strong enrichment in miR-124 target sites amongst upregulated transcripts, revealing the impact of miR-124 on neuronal gene expression (Clark, 2010). The broad, but phenotypically-tolerated, misregulation of miR-124 targets in this species is potentially consistent with the 'fine-tuning' model for miRNA regulation (Sun, 2012).

This study analyzed a knockout of the sole mir-124 gene in Drosophila. Although this mutant is viable and exhibits grossly normal patterning, numerous phenotypes were documented, including short lifespan, increased variation in the number of dendritic branches of sensory neurons, decreased locomotion and aberrant synaptic release at CNS motoneuron synapses. All of these phenotypes were rescued by a single copy of a 19 kilobase (kb) genomic transgene encompassing the mir-124 locus. A transcriptional reporter of mir-124 was generated that recapitulated the CNS expression of endogenous pri-mir-124, and this was used to purify mir-124-expressing cells from stage-matched wild-type and mir-124-mutant embryos. Transcriptome analysis revealed strong enrichment of direct miR-124 targets amongst genes upregulated in mir-124-mutant cells. The miR-124 target network included coordinate repression of multiple components in the retrograde BMP signaling pathway, whose activity controls synaptic release. Loss of miR-124 further correlated with increased activity of other neural miRNAs and the neural translational regulator Pumilio, and had the net effect of impairing transition from the neuroblast to neuronal gene expression signature. Altogether, it was demonstrated that endogenous miR-124 has substantial impact on CNS gene expression, which underlie its requirement for organismal behavior and physiology (Sun, 2012).

These studies of Drosophila mir-124 demonstrate that its loss is compatible with grossly normal neural development and differentiation, despite broad changes in gene expression and global upregulation of direct miR-124 targets. Nevertheless, many clear defects are detected in these mutants, including short lifespan of adult males, defective larval locomotion, and aberrant synaptic transmission. The latter phenotype is perhaps reminiscent of reports that inhibition of Aplysia miR-124 similarly results in an increase in evoked EPSP amplitude. These phenotypes were confirmed phenotypes to be due to miR-124 loss, as shown by their rescue by a mir-124 genomic transgene. Importantly, these phenotypes were obvious even under optimal culture conditions, demonstrating palpable requirements for this miRNA in the intact animal. It remains to be seen if synaptic overactivity in the mir-124 mutant can be directly linked to the behavioral defects observed at the organismal level. The electrophysiological defects in mir-124 mutants phenocopy activation of BMP signaling at the synapse, and miR-124 directly targets multiple components of this pathway. Still, it remains possible that the many other gene expression changes in mir-124 mutant neurons contribute to its loss of function phenotype. The detailed in vivo transcriptome-wide analysis of endogenous miR-124 targets sets the stage for future studies of how individual targets might affect different settings of miR-124 function (Sun, 2012).

Only a handful of other miRNA mutants are lethal or exhibit overt morphological defects, suggesting that many miRNAs serve as robustness factors. For example, a Drosophila mir-7 mutant exhibits minor cell specification defects, but these are enhanced by heat shock. In addition, the introduction of many C. elegans 'benign' miRNA mutants into genetically sensitized backgrounds uncovers a high frequency of phenotypes. Interestingly, miR-124 is not required for normal dendrite formation per se, but its absence caused a broader distribution of dendrite numbers on ddaD and ddaE neurons, i.e. a 'robustness' defect. It is speculated that environmental or genetic stress may reveal additional requirements for miR-124 in development and differentiation of the nervous system (Sun, 2012).

In light of the broad roles ascribed to endogenous miR-124 in neurogenesis, neural differentiation, and neural physiology (Gao, 2010), all from antisense strategies, the extensive negative data from the current Drosophila mir-124 knockout are equally compelling. While the relevant neural subpopulation may not have been examined, these studies indicate that miR-124 is not required for gross aspects of neurogenesis and differentiation in the embryonic and larval nervous system. Similarly, C. elegans deleted for mir-124, which is expressed mostly in ciliated sensory neurons, do not reveal obvious defects in neural development (Clark, 2010). Given that these invertebrate orthologs of miR-124 are identical in sequence to their vertebrate counterparts, and are highly and specifically expressed in their respective nervous systems, there is not strong reason a priori to suspect that miR-124 should not have comparable requirements amongst different animals. The analysis of vertebrate mir-124 knockouts is therefore highly anticipated (Sun, 2012).

The Drosophila system has been critical for elucidating fundamental features of miRNA target recognition in animals, and for studying specific miRNA-target interactions that mediate phenotype. However, it has been little-used to analyze the effects of miRNA-mediated gene regulation in the animal at the transcriptome-wide level. Perhaps the clearest example is the broad upregulation of maternal transcripts in early embryos lacking the mir-309 cluster. However, most miRNAs are tissue or cell-specific, and while it is much simpler to profile transcripts from whole flies, the inclusion of irrelevant cells can mask the action of the miRNA. For example, only 4/200 transcripts upregulated in mir-8 mutant pupae appeared to be direct conserved targets (Sun, 2012).

By purifying cognate miRNA-expressing cells from wild-type and miRNA-mutant backgrounds, this study succeeded in assessing transcriptome-wide effects of genetic removal of miR-124 with precision. The data provide a new perspective on the utilization of 'anti-targeting' in Drosophila. Previously, miR-124 was selected as a particularly compelling case in which its Drosophila targets were depleted for in situ terms related to nervous system development, and enriched for terms related to epidermal development. Since these tissues derive from a common developmental progenitor, the neuroectoderm, this led to a model in which miR-124 may solidify the neural fate by widespread suppression of epidermal genes that should be absent from neurons. This bioinformatic correlation has not been confirmed using an independently-derived set of miRNA targets (Sun, 2012).

Nevertheless, two observations suggest that the feature of mutual exclusion in the Drosophila miR-124 network is of subtle consequence. First, derepressed target genes were not enriched for epidermally-expressed genes. This is consistent with the view that on the transcriptome-wide level, the exclusion of epidermal genes from miR-124-expressing cells is primarily enforced by transcriptional mechanisms. Second, miR-124 targets were preferentially amongst the higher-expressed transcripts in miR-124+ cells, even in wild-type. Moreover, as well-conserved targets were expressed at overall higher absolute levels than poorly-conserved targets in miR-124+ cells, it is concluded that a dominant feature of the miR-124 target network has selected for substantial co-expression of the miRNA and its targets, perhaps to fine-tune their levels. This viewpoint is consistent with analyses of miR-124 targets in human, indicating a unifying theme for this particular miRNA across animals (Sun, 2012).

Early manifestations of the miRNA world emerged from pervasive control of the C. elegans heterochronic pathway and the D. melanogaster Notch pathway by miRNAs, and a few similar situations have been documented, i.e. direct targeting throughout the branched amino acid catabolism pathway by miR-277 or repression of multiple components of fatty acid metabolism by miR-33. Nevertheless, it is rare for such dedicated target networks to be seen amongst the miRNA oeuvre. Amongst the broad network of miR-124 targets, coordinate targeting of multiple components of the retrograde BMP signaling pathway is striking, including all three receptors (Sax/Tkv/Wit), the downstream transcription factor (Mad) and its cofactor (Medea). It was recently shown that misexpression of activated Sax and Tkv receptors in motoneurons increases evoked excitatory junctional potentials without affecting spontaneous activity, very similar to that of mir-124 mutants. This study extends this finding by analysis of activated Tkv alone. Therefore, deregulation of BMP signaling may contribute to the electrophysiological defects observed in mir-124 mutants (Sun, 2012).

Still, a 'one size fits all' description of miR-124 activity is not appropriate, since a number of functional miR-124 targets were observed whose predominant activities are in epidermal or other non-neural derivatives. Thus, the large miR-124 network accommodates a range of target properties. Derepression of a sufficient number of such non-neural transcripts may contribute collectively to the incomplete capacity of mir-124 mutant cells to transition from a neuroblast to neuronal gene expression signature (Sun, 2012).

One may speculate that dysfunction of miRNAs, which have large networks of targets, may trigger global changes in other modes of gene regulation. For example, overexpression of individual miRNAs or siRNAs can de-repress endogenous regulation via non-cognate miRNAs, possibly reflecting a titration mechanism. In addition to a global effect on neuroblast-to-neural transition, it was observed that genes downregulated upon in vivo loss of miR-124 were enriched for seeds of K box miRNAs and miR-10-5p. This is potentially consistent with a model in which absence of this abundant miRNA frees up AGO1 complexes to accept other neural miRNAs, yielding their overactivity. Another plausible mechanism might be that miR-124 represses a transcriptional repressor of these other miRNAs (Sun, 2012).

Pumilio binding sites were strongly associated with downregulated transcripts in mir-124 mutants. Pumilio is well-characterized as a neural RNA binding protein and translational regulator, and affects synaptic function and dendrite morphogenesis, which was also observed to be miR-124-regulated settings. Predictions of conserved miRNA binding sites (e.g., TargetScan or mirSVR) did not identify miR-124 target sites in the annotated pumilio 3' UTR or CDS; however modENCODE data revealed that pumilio transcription extends ~2 kb downstream of its annotated 3' end. The regulatory potential of such long pumilio 3' UTR isoforms remains to be studied. Other possibilities are that miR-124 regulates a transcriptional regulator of pumilio, or that Pumilio activity is altered in mir-124 mutants. Future studies should address the cross-talk of post-transcriptional regulation in neurons mediated by miR-124, neuronal miRNAs and Pumilio (Sun, 2012).

Bone morphogenetic protein- and mating-dependent secretory cell growth and migration in the Drosophila accessory gland

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).

Effects of Mutation or Deletion

Phenotypic analysis of embryos from homozygous Med 15 females reveals the direct role of Medea in patterning the embryonic dorsal-ventral axis. When Med 15 females are mated with wild-type males, all of their progeny die with a partially ventralized phenotype that is nearly identical to that of embryos lacking zerknullt activity. Consistent with their cuticular phenotypes, these embryos do not differentiate amnioserosa as assayed by beta-galactosidase staining of a Kruppel-lacZ transgene. The function of Medea in patterning the imaginal discs is revealed by analysis of transheterozygotes between Med 15 and any of the lethal Medea alleles. While some trans-heterozygotes die at the pharate adult stage, many escapers eclose with a range of phenotypes similar to those exhibited by dpp mutants defective in imaginal disc patterning. The observed defects include split nota, intercalary and terminal gaps in L4 wing veins, absent or gapped posterior crossveins, and foreshortened legs that lack tarsal claws and often lack several tarsal segments (Wisotzkey, 1998).

If Medea functions downstream of decapentaplegic, then reducing levels of Medea gene product should attenuate the Dpp signal. The earliest zygotic mutant phenotype for Medea is a variable failure of the second midgut constriction, the appearance of which is driven by visceral mesoderm. Loss of this constriction is observed in dpp, shv, Mad, tkv, sax and punt mutants, suggesting that Medea acts in the same pathway as other DPP signal transducers. Expression of two Dpp-responsive genes, labial (expressed in the endoderm), and the dpp gene itself, are interferred with in Medea mutants, since Dpp maintains its own expression. In concert with the variable effects of Medea allelic combinations on second constriction formation, a variable loss of labial expression is observed, with nearly total loss in the most severe cases. At this early stage of development, variability may be due to persistence of maternally-supplied Medea gene product. To determine whether Medea is required for dpp autoregulation, expression of a lacZ reporter, RD2, was examined driven by the visceral mesoderm regulatory region for dpp. Surprisingly, dpp reporter expression persists late in embryogenesis in Medea mutants, although at a slightly reduced level; likewise, dpp expression is detected in Medea3 homozygotes by in situ hybridization. Medea's minimal influence on midgut dpp autoregulation provided the first hint of a difference between Mad and Medea. To test whether Medea is necessary to transmit a Dpp signal, the GAL4/UAS system was used to drive the ectopic expression of dpp. With this expression system, ectopic expression of dpp throughout the visceral mesoderm leads to the loss of the anterior first midgut constriction, combined with exaggerated size of the dpp-induced second constriction. Decreased Medea function restores the first constriction in >90% of embryos of the appropriate genotype, even though this allelic combination causes only partial loss of the second constriction under endogenous dpp expression. In all embryos where the first constriction is restored, there is also significant rescue of the third constriction. Since a decrease in Medea function blocks the effects of ectopic Dpp, it is concluded that Medea is required downstream of dpp directly within the Dpp signal transduction pathway (Wisotzkey, 1998).

To test this possibility, the FLP/FRT system was used to induce germline clones at high frequency. From a population of over 100 females bearing Mad germline clones, and mated to Mad 12 /CyO males, only 4 eggs were produced; all were unfertilized and smaller than normal. The ovaries from ten of these females were examined. Five had no discernible ovarioles and five had ovaries that contained many degenerating egg chambers; of the discernible egg chambers, the most mature were at stage 10. In stark contrast to the Mad phenotype, females bearing mutant Medea germline clones produce fertilized eggs throughout their lifespan, although they decrease in fecundity with age. Thus, Mad is essential in the germline during oogenesis, whereas Medea function is dispensable. The absence of a requirement for Medea is unlikely to be a result of overlapping function for Mad and Medea. A ubiquitously expressed Mad rescue transgene (pUmMad) does not rescue Medea mutants, nor does it postpone the stage of lethality beyond the prepupal phase, even when the Mad transgene is present in two copies. Thus, increased levels of Mad do not significantly compensate for loss of Medea (Wisotzkey, 1998).

To test whether Medea might be essential for only a subset of Mad-dependent events, clonal analysis was used to compare null phenotypes in the absence of maternal contribution of wild-type protein. For this study, the Medea 8 allele was used, which gives a null phenotype, as assessed by stage of lethality and genetic interaction with dpp; it yields a highly unstable truncated protein that does not interact with MAD. For comparison, the Mad 12 null allele was used, which encodes a similar C-terminal truncation. Although fertilized eggs are recovered from Medea mutant germlines, oogenesis requires Dpp signaling. Preliminary studies suggest Mad may be required in the germline (Sekelsky, 1995).

The Med 15 and Med 17 alleles confer homozygous viability; therefore they must retain some Medea activity. However, Med 15 and Med 17 can enhance other dpp-pathway mutants as strongly as does a deficiency of the Medea locus. Specifically, Med 15 and Med 17 enhance zen f16 and dpp hr4 /+ as strongly as do null Medea alleles, and Med 15 enhances dpp hr56 as strongly as do null Medea alleles. These results are consistent with the hypothesis that the Med 15 and Med 17 compromise one of two independently mutable functions of Medea, while leaving a second function intact. To test this hypothesis, a determination was made whether a further reduction in maternal and zygotic Medea activity would worsen the dorsal-ventral patterning defects caused by Med 15. Specifically, the phenotypes of embryos from Med 15 females mated with wild-type males were compared to the phenotypes of embryos from Med 15 /Med 16 females crossed to Med 13 /TM3 males (Med 16 and Med 13 are null alleles of Medea). All of the embryos from this latter cross are thus derived from females that contain a two-fold reduction in the dose of Med 15: one fourth of the progeny embryos, of genotype Med 13 /Med 16 , completely lack zygotic Medea activity. This further reduction in Medea activity results in only a slight increase in the phenotypic severity of the mutant embryos: while all embryos from a Med 15 mother mated to wild-type males have phenotypes similar to those caused by a weak allele of dpp(dpp hr56), some embryos from the latter cross have phenotypes similar to those caused by a slightly stronger dpp allele, dpp hr4. Both of these phenotypes are much weaker than the phenotype caused by complete loss of Medea activity and support the hypothesis that Med 15 severely compromises, but does not totally eliminate, one of two independently mutable activities of the Medea protein (Hudson, 1998).

It is proposed that Medea 17 and especially Medea 15 are compromised in the dosage-sensitive specification of amnioserosa, but that both mutant proteins retain a separable function required for the specification of dorsolateral cell fates in the embryo. What could the two separately mutable activities of Medea represent? One possibility is that each activity represents a differential capacity to transduce a signal downstream of each of the two type I Dpp receptors, Tkv and Sax. Embryos that lack both maternal and zygotic tkv activity differentiate no dorsal structures, similar to the complete loss of Medea. In contrast, although the phenotypes of embryos completely lacking sax activity have not been reported because of a requirement for sax during oogenesis, existing mutations in sax result only in the loss of amnioserosa, similar to the phenotype caused by the Med 15 mutation. These parallels suggest that Med 15 and Med 17 mutants may be defective in the response to signals downstream of the Sax receptor, while still transducing signals from the Tkv receptor. In light of this proposal, it is noted that both Med 15 and Med 17 have amino acid substitutions in loop 3, an element of the Smad4 crystal structure that is implicated in productive heteromeric interactions with activated receptor-specific Smad proteins. The mutant Medea proteins might therefore have a diminished capacity to form particular heteromeric complexes with Mad in response to signaling by one receptor but not another. Alternatively, the mutant Medea proteins could have selective disruptions in interactions with other components of the signaling system, such as factors that may collaborate with Mad and Medea to regulate expression of specific target genes. Full evaluation of this proposal awaits biochemical characterization of signaling downstream of the Tkv and Sax receptors in vivo (Hudson, 1998).

Salivary gland formation in the Drosophila embryo is linked to the expression of the homeotic gene Sex combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when Scr is expressed everywhere, salivary glands form in new places. However, not every cell that expresses Scr is recruited to a salivary gland fate. Along the anterior-posterior axis, the posteriorly expressed proteins encoded by the teashirt (tsh) and Abdominal-B (Abd-B) genes block Scr activation of salivary gland genes. Along the dorsal-ventral axis, the secreted signaling molecule encoded by decapentaplegic prevents activation of salivary gland genes by Scr in dorsal regions of parasegment 2. Five downstream components in the Dpp signaling cascade required to block salivary gland gene activation have been identified: two known receptors (the type I receptor encoded by the thick veins gene and the type II receptor encoded by the punt gene); two of the four known Drosophila members of the Smad family of proteins which transduce signals from the receptors to the nucleus [Mothers against dpp (Mad) and Medea (Med)], and a large zinc-finger transcription factor encoded by the schnurri (shn) gene. The expression patterns of d-CrebA and Trachealess were examined in embryos missing zygotic function of schnurri. In embryos homozygous for shn, a dorsal expansion of salivary gland protein expression is observed. The presence of amnioserosa, an extreme dorsal cell type, suggests that embryos lacking zygotic shn function are not ventralized, as are embryos missing maternal and zygotic function of tkv, pt, Mad, or Med or missing zygotic function of dpp. These results reveal how anterior-posterior and dorsal-ventral patterning information is integrated at the level of organ-specific gene expression (Henderson, 1999).

To determine roles for Medea during larval development, clones mutant for Medea have been examined in the eye. Dpp has an important role in the initiation and progression of the morphogenetic furrow. Mad clones in the posterior of the eye result in the loss of eye structures, which are instead replaced by head cuticular structures. These clones showed the ectopic expression of wingless (wg), a gene that is normally repressed by dpp signaling and is required at the lateral margins of the eye disk to regulate the proper timing of furrow initiation and progression. Hence clones of Mad mutant cells were unable to transduce the Dpp signal and are unable to initiate the morphogenetic furrow. Clones of the strong Medea alleles, Med 1 and Med 26 were induced, and these clones gave very similar phenotypes to Mad clones, such as loss of eye tissue. Such clones are observed only at the margins of the eye, most commonly the posterior margin, where the furrow initiates. This indicates that Medea has overlapping functions with Mad in dpp signaling during furrow initiation. Clonal analysis with Medea has also revealed abnormalities in other tissues, in keeping with its involvement in dpp signaling. For example, partial duplications of the leg are observed, a phenotype reported in the dorsal regions of the leg for clones of the Dpp receptor, punt. These analyses strongly suggest a closely related function for Medea and dpp during imaginal disc development (Das, 1998).

In order to directly address the role of Medea in the Dpp pathway, the ability of Medea mutants to suppress ectopic signaling from Dpp receptors was tested. A constitutively activated Dpp Type I receptor, Saxophone (Sax*), was generated by the substitution of a single amino acid (Q263D) near the GS box of the intracellular domain. The activated receptor is expressed in a spatially controlled manner using the GAL4-UAS system. Under engrailedGAL4(enGAL4) control, UAS-Sax* produces a phenotype in the wing, characterized by posterior defects, such as overgrowth and ectopic venation. Removal of a single copy of a gene that is required for Sax signaling, for example Mad, suppresses this phenotype. This same suppression is observed when a single copy of Medea is removed from enGal4,UAS-Sax* transgenic flies. Since two type I receptors have been identified for Dpp, the ability of Medea to suppress signaling from the other activated receptor, Thick veins (Tkv*) was tested. Interestingly, Medea does not show the same ability to suppress a Tkv* phenotype, typified by ectopic vein material and severe blistering. In fact, a subset of Medea alleles showed very low levels of suppression, and to a much lesser extent than Mad. One explanation for the differential ability of Medea mutants to suppress the Sax* and Tkv* phenotypes is that the two activated receptors achieve different levels of signaling. In addition, Medea may not be a limiting component in Tkv* signaling. Therefore, the removal of one copy of Medea may be insufficient to affect the high levels of Tkv* signaling, but may be enough to influence the weaker Sax* signal (Das, 1998).

Since Mad and Medea are separately mutable, it is expected that they function non-redundantly and cannot substitute for each other. Consistent with this model, ubiquitous expression of Mad (Ubi-Mad) cannot rescue Medea lethality. To further examine the relationship between these two Smads, a sensitized assay system was used. This assay utilizes the dominant maternal effect lethality of Mad and Medea with dpp. The extent of this lethality depends on the strength of the Mad or Medea allele and the dpp allele with which it is crossed. Crossing a strong, hypomorphic dpp allele, dpp hr27, to the strongest available alleles of Mad, (Mad 12) or Medea (Med 1), results in 100% lethality of both dpp classes among the progeny. As expected, Ubi-Mad can rescue the maternal effect lethality of Mad 12, and Ubi-Medea rescues that of Med 1. The effects of introducing Ubi-Medea or Ubi-Mad from Mad/+ or Medea/+ females, respectively, were examined. Interestingly, a Ubi-Medea transgene can reduce the maternal effect lethality of Mad 12 /+ females with dpp from 100% to 12%, while Ubi-Mad reduces that of Med 1/+ females to 68%. To assay whether this rescue is simply due to increased levels of dpp pathway components, a Ubi-tkv line was usedin the same assay system. While this line is able to rescue a tkv mutant, it cannot rescue the maternal effect lethality associated with Mad or Medea. The lower extent of Ubi-Mad rescue of Medea maternal effect lethality, may be due to the fact that Med 1 may be an antimorphic allele. Alternatively, this may be indicative of an important aspect of Smad function. While it is clear that Mad and Medea cannot substitute for each other, the genetic data argue that a reduction in one class of Smads can be at least partially compensated by augmenting the dosage of the other Smad class. This compensation may be a Smad-specific feature, as elevated levels of tkv do not yield the same results. The simplest explanation for these genetic observations is that increased levels of one class of Smads may enhance the ability of the other class to signal (Das, 1998).

The role of Dpp signaling in maintaining the Drosophila wing anteroposterior compartment boundary: A role for Mad in A cells in maintaining the A/P boundary

The subdivision of the developing Drosophila wing into anterior (A) and posterior (P) compartments is important for its development. The activities of the selector genes engrailed and invected in posterior cells and the transduction of the Hedgehog signal in anterior cells are required for maintaining the A/P boundary. Based on a previous study, it has been proposed that the signaling molecule Decapentaplegic (Dpp) is also important for this function by signaling from anterior to posterior cells. However, it has not been known whether and in which cells Dpp signal transduction is required for maintaining the A/P boundary. The role of the Dpp signal transduction pathway and the epistatic relationship of Dpp and Hedgehog signaling in maintaining the A/P boundary has been analyzed by clonal analysis. A transcriptional response to Dpp involving the T-box protein Optomotor-blind is required to maintain the A/P boundary. Further, Dpp signal transduction is required in anterior cells, but not in posterior cells, indicating that anterior to posterior signaling by Dpp is not important for maintaining the A/P boundary. Finally, evidence is provided that Dpp signaling acts downstream of or in parallel with Hedgehog signaling to maintain the A/P boundary. It is proposed that Dpp signaling is required for anterior cells to interpret the Hedgehog signal in order to specify segregation properties important for maintaining the A/P boundary (Shen, 2005).

For many years, it was thought that En and Inv regulated the segregation of A and P cells by specifying a P-type cell segregation in a cell-autonomous fashion. Recent work has challenged this view by showing that a unidirectional Hh-mediated signal from P to A cells is required to specify the A-type segregation behavior of A cells and that the role of En and Inv is mainly to control Hh signaling. Based on the findings that A cells signal back to P cells via Dpp and that wings from flies hypomorphic for dpp have a distorted A/P boundary, it has been proposed that A to P signaling by Dpp might also be important to maintain the A/P boundary. However, whether Dpp signal transduction is required for the maintenance of the A/P boundary and in which cells the Dpp signal is required remained unknown. By analyzing clones mutant for tkv, mad, and omb, several independent lines of evidence are provided that Dpp signal transduction is required to maintain the A/P boundary and that it is only required in A cells, but not in P cells. Thus, the results do not support the hypothesis that A to P signaling by Dpp is required to maintain the A/P boundary. Instead, the results suggest that Dpp signaling within Dpp-producing A cells is required to maintain the A/P boundary (Shen, 2005).

Through analysis of mutant clones located at the A/P boundary lacking the activity of the type I Dpp receptor Tkv, evidence is provided that the reception of the Dpp signal in A cells is required to maintain the A/P boundary. When generated in the P compartment, a few tkvbsk clones displace the A/P boundary to a small extent: this is attributed to the unusual round shape of these clones. However, the majority of P tkvbsk clones do not displace the A/P boundary, suggesting that the reception of the Dpp signal is not required in P cells to maintain the A/P boundary. In contrast, mutant clones generated in the A compartment at the A/P boundary displace the position of the A/P boundary toward P, indicating that the reception of the Dpp signal is required in A cells to maintain the A/P boundary (Shen, 2005).

How does the reception of the Dpp signal control cell segregation at the A/P boundary? Although the molecular basis is unknown, a cell's segregation behavior presumably depends on its cytoskeletal or surface properties (cell affinity). Members of the TGFβ superfamily have been observed in other systems to be able to activate regulators of the actin cytoskeleton independently of Mad/Smad transcription factors, raising the possibility that Dpp reception could control cell segregation by directly altering structural components of the responding cells. Alternatively, Dpp could control the segregation of cells by regulating the transcription of one or several target genes. To distinguish between these possibilities, the role of downstream components of the Dpp signal transduction pathway were analyzed. Three independent lines of evidence is provided that a transcriptional response to the Dpp signal is required to maintain the A/P boundary. (1) The segregation behaviors of madbsk and tkvbsk clones are indistinguishable. Like tkvbsk clones, A madbsk clones displace the A/P boundary toward P, indicating a role for the transcription factor Mad in A cells to maintain the A/P boundary. (2) madbrk clones respect the A/P boundary, indicating that repression of brk transcription by Mad is important for normal A/P cell segregation. (3) A omb clones displace the A/P boundary toward P. The frequency and extent of the boundary displacement of A omb, tkvbsk, and madbsk clones is comparable, suggesting that the Dpp target gene omb is the main mediator of this aspect of the Dpp signal. In contrast to omb clones, most A clones mutant for the Dpp target gene sal do not displace the A/P boundary, indicating that sal does not play an important role in maintaining the A/P boundary. Together, these data suggest that the transduction of the Dpp signal controlling the maintenance of the A/P boundary bifurcates at the level of the Dpp target genes (Shen, 2005).

Cells of tkvbsk, madbsk, and omb clones displacing the A/P boundary do not appear to intermingle well with P cells. In fact, within the entire wing disc pouch, these mutant clones have a round shape and smooth borders, suggesting that these mutant cells in general do not intermingle freely with wild-type cells. Similar clone shapes have been reported upon mutation or misexpression of several genes, including mutants in the Dpp target gene sal and misexpression of a constitutively active form of Tkv. The round shapes and smooth borders of clones have been attributed to differences in the affinity of clone cells for their neighbors, suggesting that Tkv, Mad, and the Dpp target genes omb and sal may affect some aspects of wing pouch cell affinity. Therefore, the inability of A tkvbsk, madbsk, and omb clones displacing the A/P boundary to intermingle well with P cells is attributed to this particular role (Shen, 2005).

Taken together, this analysis indicates two roles for Dpp signal transduction: (1) it provides some aspects of the cell affinity of both A and P wing pouch cells; (2) it is required in A cells to specify an A cell affinity important for maintaining the A/P boundary. These two roles of Dpp signal transduction could either be related or distinct. The finding that the Dpp target gene sal is required for the first role, but not the second, provides a first indication that these two roles are implemented by partially distinct molecular mechanisms (Shen, 2005).

How might Omb regulate the segregation behavior of cells at the A/P boundary? Recent work has shown that Omb has at least two roles during the patterning of the Drosophila wing. First, Omb is required for the expression of two Dpp target genes sal and vestigial (vg) (del Alamo Rodriguez, 2004). Since sal mutant clones do respect the A/P boundary, the role of Omb in maintaining the A/P boundary cannot depend on sal induction. Since Vg is required for wing cell proliferation, its role in maintaining the A/P boundary cannot be tested. Second, Omb is involved in shaping the expression pattern of tkv along the A/P axis of the wing disc (del Alamo Rodriguez, 2004). The expression of tkv is reduced in Dpp-producing A cells along the A/P boundary. This reduction of tkv expression is mediated by the transcription factor Master of thickveins (Mtv, also known as Brakeless and Scribbler, which is expressed in these cells in response to the Hh signal. Since both tkv and mtv are upregulated in omb mutant clones, it has been proposed that Omb is required for Mtv to repress tkv (del Alamo Rodriguez, 2004). However, reduction of tkv transcription in A cells does not seem to be important for the segregation of cells at the A/P boundary, because A clones either mutant for mtv, in which tkv levels are increased, or overexpressing tkv, respect the A/P boundary. Thus, neither the role of Omb in repressing tkv nor in activating sal transcription appears to be important for Omb's function in maintaining the A/P boundary. Therefore, other target genes of Omb must exist that mediate Omb's function in maintaining the A/P boundary (Shen, 2005).

Anterior cells at the A/P boundary have been shown to require Hh signal transduction to segregate from P cells. Evidence is provided that A cells in addition need to transduce the Dpp signal for normal segregation. What is the epistatic relationship between Hh and Dpp signaling? The activity of the Hh transduction pathway is not affected in either tkvbsk or madbsk clones as monitored by the expression of the Hh target gene ptc, indicating that Hh signal transduction does not require Dpp signal transduction components for its activity. However, the Dpp target gene omb appears to be important for A cells to interpret the Hh signal because the ability of Ci to specify A-type segregation properties depends, in part, on the activity of Omb. Thus, Dpp signaling acts either downstream of or in parallel with Hh signaling in maintaining the A/P boundary (Shen, 2005).

Previously, three transcription factors, a transcriptional activator form of Ci (hereafter referred to as Ci[act]), En, and Inv, have been shown to be required for the segregation of A and P cells. Evidence exists for the involvement of a fourth transcription factor, the T-box protein Omb. Omb is further shown to act downstream of or in parallel with Ci. How could these four transcription factors regulate the segregation of A and P cells? In a simple model, Ci[act], En, Inv, and Omb could regulate the segregation of A and P cells by controlling the transcription of the same set of target genes that may encode cell affinity molecules or regulate the activity of cell affinity molecules. Omb is activated in both A and P cells in a broad domain centered around the A/P boundary by Dpp signaling where Omb may upregulate the expression of this putative target gene(s). The activity of Ci[act] is restricted to Hh-responding A cells along the A/P boundary. In these A cells, the target gene(s) would be further induced. En and Inv expressions are mainly confined to P cells in which they are known to act as repressors of transcription. Thus, En and Inv would repress the putative target gene(s) in P cells. The abrupt difference in the expression of putative target gene(s) would contribute to the segregation of A and P cells. Anterior clones (but not P clones) of cells lacking Omb would displace the A/P boundary because normally the putative target gene would be highly expressed in A cells, but not in P cells, where it would be repressed by En and Inv. Omb may therefore provide a basal affinity to cells in the center of the wing disc that is modified by Ci[act], En, and Inv to create a sharp difference of this affinity in cells on both sides of the A/P boundary. In an alternative model, Omb, Ci[act], En, and Inv would regulate distinct sets of genes. To distinguish among these models, it will be necessary to identify the Ci[act], En, Inv, and Omb target genes mediating cell segregation (Shen, 2005).

The precise position and shape of the Dpp organizer along the A side of the A/P boundary are important for normal growth and patterning of the wing. It has been proposed that the segregation of cells at the A/P boundary contributes to maintain this precise position and shape of the Dpp organizer in the growing wing disc epithelium. It is intriguing to notice that the Dpp-organizing activity itself plays a role in the segregation of A and P cells, suggesting that the Dpp-organizing activity contributes to maintain its own position. It will be interesting to investigate whether other organizers associated with compartment boundaries have similar functions (Shen, 2005).

BMP signaling is required for controlling somatic stem cell self-renewal in the Drosophila ovary: Med is required for somatic stem cell survival in a BMP-independent pathway

BMP signaling is essential for promoting self-renewal of mouse embryonic stem cells and Drosophila germline stem cells and for repressing stem cell proliferation in the mouse intestine and skin. However, it remains unknown whether BMP signaling can promote self-renewal of adult somatic stem cells. In this study, BMP signaling is shown to be necessary and sufficient for promoting self-renewal and proliferation of somatic stem cells (SSCs) in the Drosophila ovary. BMP signaling is required in SSCs to directly control their maintenance and division, but is dispensable for proliferation of their differentiated progeny. Furthermore, BMP signaling is required to control SSC self-renewal, but not survival. Moreover, constitutive BMP signaling prolongs the SSC lifespan. Therefore, this study clearly demonstrates that BMP signaling directly promotes SSC self-renewal and proliferation in the Drosophila ovary. This work further suggests that BMP signaling could promote self-renewal of adult stem cells in other systems (Kirilly, 2005)

This study shows that SSCs in the adult ovary are capable of responding to BMP signaling. Genetic mosaic analyses demonstrate that known BMP downstream components are also required for SSC self-renewal, but not survival. Hyperactive BMP signaling enhances SSC self-renewal capacity. Glass bottom boat (Gbb) is essential for controlling SSC maintenance, at least in the GSC niche. Furthermore, BMP signaling appears to be specific to stem cells, since follicle cells mutant for BMP-specific downstream components proliferate and differentiate normally. In addition to participation in BMP signaling, Medea (Med) is likely involved in other TGF-β-like pathway(s) to control proliferation and size of differentiated follicle cells. The results from this study led to the proposal of a working model that Gbb perhaps as well as Dpp from neighboring somatic cells function as stem cell growth factors in vivo for promoting self-renewal of ovarian SSCs (Kirilly, 2005).

gbb and dpp are expressed in cap cells, inner germarial sheath (IGS) cells, and follicle cells. SSCs are located in the middle of the germarium and are likely exposed to both BMPs, since both Dpp and Gbb are diffusible molecules. gbb mutants exhibit severe SSC/follicle cell proliferation defects and SSC loss. Furthermore, SSCs mutant for BMP downstream components such as tkv, punt, and mad are lost faster and divide slower than wild-type ones. Although dpp mutants show much weaker mutant defects, it is still possible that it plays as important a role as does gbb, since only weak dpp mutations could be used for studying the regulation of adult SSCs due to its stringent requirements during early development. Therefore, these findings support the idea that Gbb, perhaps together with Dpp, controls SSC self-renewal and division. Studies on GSCs in the Drosophila ovary have shown that BMPs control GSC self-renewal by directly repressing transcription of differentiation-promoting genes such as bam. Possibly, BMP signaling also represses differentiation-promoting genes and thereby maintains SSC self-renewal. Meanwhile, BMP signaling could also positively regulate other genes that are important for maintaining the undifferentiated state of SSCs. This study also shows that BMP signaling also promotes SSC division. It has been shown that BMP signaling promotes GSC division. In order to better understand how BMP signaling controls SSC self-renewal and division, it is critical to identify the BMP target genes in SSCs, that are either repressed or activated by BMP signaling (Kirilly, 2005)

This study also shows that tkv is a major type I BMP receptor for controlling SSC self-renewal in the Drosophila ovary. The SSCs mutant for sax4, a null allele of sax, behave close to normal wild-type ones, while the SSCs mutant for a strong tkv allele, tkv8, are lost rapidly, indicating that Tkv is a major functional receptor to control SSC self-renewal. Given the evidence that gbb signaling is essential for maintaining SSCs, this study strongly supports the idea that Gbb signals mainly through Tkv to control SSC self-renewal in the Drosophila ovary. A recent study on Drosophila spermatogenesis also suggests that Gbb signaling primarily functions through Tkv, but not Sax. In the Drosophila testis, gbb and tkv are both essential for maintaining GSCs, but sax is not. Although one study on dominant-negative tkv and sax receptors suggests that dpp and gbb signal preferentially through tkv and sax, respectively, another more recent study has shown that both dpp and gbb use tkv, but not sax, control the process of vein promotion during pupal development and disc proliferation and vein specification during larval development. Taken together, the results from this study and the previous studies indicate that Gbb can use Tkv as a major receptor for its signal transduction in Drosophila (Kirilly, 2005).

Although Gbb/BMP signaling plays a critical role in controlling SSC self-renewal and division, it appears that it is dispensable for SSC survival, follicle cell proliferation, and cell size control. For example, expression of the baculovirus antiapoptotic gene p35 could not rescue the mutant punt SSC loss; the follicle cell clones mutant for strong tkv and mad alleles, tkv8 and mad12, proliferate normally, and the sizes of the mutant follicle cells are quite normal. In contrast, p35 expression can rescue the Med26 SSC loss to the levels of the mutant punt, tkv, and mad mutant SSC loss. The partial rescue indicates that Med is required for SSC survival in a BMP-independent pathway. The Med mutant follicle cell clones proliferate slower than wild-type, and the size of follicle cells is also smaller than that of wild-type, suggesting that Med is required for follicle cell proliferation and growth. Since BMP signaling is not involved in the control of SSC survival, follicle cell proliferation, and growth, these findings further suggest that Med must participate in other TGF-β-like pathways controlling these processes. In mammalian systems, SMAD4 has been shown to be a common SMAD for all TGF-β-like signaling pathways, including TGF-β, Activin, and BMP. A likely candidate TGF-β-like signaling pathway includes Activin and TGF-β. Activin and TGF-β molecules exist in Drosophila. Activin-like signaling has been shown to be involved in regulating growth control and neuronal remodeling. However, the role of TGF-β signaling in Drosophila remains a mystery. It could not be completely ruled out, however, that Med is involved in other signaling pathways unrelated to TGF-β-like pathways to control SSC survival, follicle cell proliferation, and growth. In the future, it is very important to figure out which pathway Med takes part in for controlling SSC survival, follicle cell proliferation, and growth control (Kirilly, 2005).

DPP signaling controls development of the lamina glia required for retinal axon targeting in the visual system of Drosophila: A requirement for medea for expression of gcm

The Drosophila visual system consists of the compound eyes and the optic ganglia in the brain. Among the eight photoreceptor (R) neurons, axons from the R1-R6 neurons stop between two layers of glial cells in the lamina, the most superficial ganglion in the optic lobe. Although it has been suggested that the lamina glia serve as intermediate targets of R axons, little is known about the mechanisms by which these cells develop. DPP signaling has been shown to play a key role in this process. dpp is expressed at the margin of the lamina target region, where glial precursors reside. The generation of clones mutant for Medea, the DPP signal transducer, or inhibition of DPP signaling in this region results in defects in R neuron projection patterns and in the lamina morphology; these defects are caused by defects in the differentiation of the lamina glial cells. glial cells missing is expressed shortly after glia precursors start to differentiate and migrate. Its expression depends on DPP; gcm is reduced or absent in dpp mutants or Medea clones, and ectopic activation of DPP signaling induces ectopic expression of gcm and Repo. In addition, R axon projections and lamina glia development are impaired by the expression of a dominant-negative form of gcm, suggesting that gcm indeed controls the differentiation of lamina glial cells. These results suggest that DPP signaling mediates the maturation of the lamina glia required for the correct R axon projection pattern by controlling the expression of gcm (Yoshida, 2005).

dpp is expressed in the dorsal and ventral margins of the posterior region of the optic lobe, adjacent to the cells expressing wg, which induces dpp expression. Glial cells in the lamina target region arise from these regions and migrate into the lamina target region as they contact R axons. Axons from R1-R6 neurons stop between two rows of glial cell layers, the epithelial and marginal layers, and form the lamina plexus. The third row of glial cells, the medulla glia, is located just beneath the marginal glia. The homeodomain protein Repo is expressed in these glial cells (Yoshida, 2005).

The expression pattern of dpp-lacZ, an enhancer-trap allele of dpp, was compared with the expression pattern of Repo. At a stage prior to glia differentiation and migration, expression of the dpp reporter is detected in the dorsal and ventral margins of the lamina target region. dpp continues to be expressed at the margins of the lamina target region throughout the third larval instar (Yoshida, 2005).

wg at the posterior-most domain induces the expression of dpp and omb. Some wg-expressing cells extend projections towards the lamina target region. These cells extend scaffold axons along which the lamina glia migrate. Thus, it was possible that the wg signal is involved in the migration and/or differentiation of lamina glia. However, partial elimination of Wg activity with a wgts allele does not cause a specific defect in glia migration. Therefore, wg may play a role in organizing domains in the visual cortex by activating/repressing various genes, rather than contributing to the generation of specific cell types (Yoshida, 2005).

Medea is required for lamina glia development. Medea encodes a co-SMAD and mediates a range of DPP/BMP/TGFß signaling events. In addition to dpp, four related genes -- glass bottom boat (gbb), screw, activin and activin2 -- have been identified in Drosophila. GBB signals through TKV/Saxophone (SAX) and Wishful Thinking (WIT) type I and type II receptors, respectively. Activin uses Baboon as a type I receptor, and Punt and WIT as type II receptors. Brains mutant for gbb and wit were examined, but no defects in lamina glia development were observed. It is concluded that it is highly likely that dpp is the ligand responsible for lamina glia development. However, the possibility that one or more of the DPP-related ligands acts redundantly in this process cannot be excluded (Yoshida, 2005).

In the embryo, gcm initiates the specification of glial cells from neural cells of various lineages. gcm expression is strictly controlled to ensure the correct separation of glial versus neuronal cell fate. Analysis of the cis-regulatory elements of gcm suggests that gcm expression depends on multiple regulatory elements to allow the control of lineage-specific transcription and autoregulation. The analysis carried out in this study suggests that a different situation exists in the optic lobe; gcm is expressed in the glia and the lamina neuronal cells, and is required for the differentiation of these cell types. In addition, differentiation is controlled differently in the lamina and in the glia. In the lamina, gcm expression seems to be controlled by hh, and in the glia, by dpp. These results suggest that gcm is controlled and functioning in a different manner in the optic lobe. Uncovering the mechanisms of the control and function of gcm would probably prove an intriguing focus for future research (Yoshida, 2005).

DPP and its vertebrate homolog BMP play crucial roles in many aspects of development by controlling patterning, cell growth and differentiation. This analysis reveals a role for DPP signaling in lamina glia differentiation in the Drosophila visual system. DPP has also been reported to function in several aspects of visual center development; for instance, DPP signaling has been shown to be involved in the proliferation and migration of the subretinal glia in eye disc development, which plays an important role in the R axon navigation. In addition, defects have been reported in the medulla neuropile in dpp mutant animals, suggesting a role for dpp in neuronal fate specification. Furthermore, tkv is expressed in lamina precursor cells just ahead of the lamina furrow, where these cells meet R axons and start to differentiate. Although this possibility is one of the things that prompted an examination of the role of DPP signaling in lamina development, no defects were uncovered when Mad or Medea clones were generated in the OPC or the lamina. Moreover, dpp appears to be expressed in the inner proliferation center (IPC), which will form the lobula, in addition to its expression in the dorsal and ventral marginal domains. Thus, dpp may be required for some aspects of lobula development. Unfortunately, this cannot be easily addressed at this moment because of a lack of appropriate markers. Further study of the requirements for dpp in the lamina, the medulla, the lobula and other cell types could lead to a more comprehensive understanding of how DPP signaling controls differentiation and other events during development of the visual system (Yoshida, 2005).

DNA-binding domain mutations in SMAD genes yield dominant-negative proteins or a neomorphic protein that can activate WG target genes in Drosophila

Mutations in SMAD tumor suppressor genes are involved in approximately 140,000 new cancers in the USA each year. How the absence of a functional SMAD protein leads to a tumor is unknown. However, clinical and biochemical studies suggest that all SMAD mutations are loss-of-function mutations. One prediction of this hypothesis is that all SMAD mutations cause tumors via a single mechanism. To test this hypothesis, five tumor-derived alleles of human SMAD genes and five mutant alleles of Drosophila SMAD genes were expressed in flies. All of the DNA-binding domain mutations conferred gain-of-function activity, thereby falsifying the hypothesis. Furthermore, two types of gain-of-function mutation were identified -- dominant negative and neomorphic. In numerous assays, the neomorphic allele SMAD4100T appears to be capable of activating the expression of Wingless target genes. These results imply that SMAD4100T may induce tumor formation by a fundamentally different mechanism from other SMAD mutations, perhaps via the ectopic expression of WNT target genes -- an oncogenic mechanism associated with mutations in Adenomatous Polyposis Coli. These results are likely to have clinical implications, because gain-of-function mutations may cause tumors when heterozygous, and the life expectancy of individuals with SMAD4100T is likely to be different from those with other SMAD mutations. From a larger perspective, this study shows that the genetic characterization of missense mutations, particularly in modular proteins, requires experimental verification (Takaesu, 2005).

This study establishes guidelines for interpreting data from transgenic analyses of human tumor alleles. In principle, studies of this type can be applied to mutant alleles of any well-conserved tumor suppressor gene or oncogene. Furthermore, the unexpected finding that all of the tested mutations in the DNA-binding domain of SMAD genes are gain of function, whereas all of the tested mutations in the multimerization domain are loss of function, indicates that missense mutations in modular proteins should be experimentally characterized rather than defaulted to the loss-of-function category (Takaesu, 2005).

For SMAD tumor suppressor genes, identification of two gain-of-function alleles of SMAD4 (dominant negative and neomorphic) falsifies the prevailing hypothesis that all SMAD tumor mutations are loss-of-function mutations. Instead, the data support an alternative hypothesis: that there are multiple classes of SMAD mutation and that each class is associated with a different mechanism of tumorigenesis (Takaesu, 2005).

This alternative hypothesis may also apply to other TGFß signaling pathway components with tumor-associated mutations. For example, mutations in TGFß receptors are found in tumors from the same tissues that exhibit SMAD mutations (e.g. pancreas). However, missense mutations in TGFß receptors conferring gain-of-function activity have not been identified in tumors, because the most common mutational assays are loss of expression and polyA tract sequencing (Takaesu, 2005).

The data for SMAD4100T are unprecedented in studies of TGFß signaling in flies. This suggests that SMAD4100T may induce tumors in humans by an unexpected method. The fact that SMAD4100T expression mimics activated WG signaling and suppresses an antagonist of WG signaling further suggests that SMAD4100T utilizes a mechanism of tumorigenesis associated with loss-of-function mutations in Adenomatous Polyposis Coli (APC) (Takaesu, 2005).

In vertebrates and flies, APC serves as a component of the highly conserved WG/int-1 (WNT) signal transduction pathway. Like ZW3 and its homolog Glycogen Synthase Kinase-3ß (GSK3ß), APC functions as a WNT antagonist via participation in a cytoplasmic retention complex that prevents Armadillo (or its vertebrate homolog ß-catenin) from accumulating in the nucleus in the absence of WNT signals. Studies in flies have shown that homozygous null clones bearing mutations in any member of the retention complex (ZW3, Drosophila APC and Drosophila Axin) lead to the same phenotype: ectopic anterior margin bristles on the wing blade as a result of the unregulated expression of WG target genes such as AC. First identified in the rare inherited cancer Familial Adenomatous Polyposis, homozygous mutations in APC are now found in roughly 85% of all colon tumors. In studies of mice engineered to homozygose APC null mutations only in cells of their intestinal epithelium, the immediate consequence of APC loss was ectopic expression of WNT target genes via constitutively nuclear ß-catenin (Takaesu, 2005).

Given their roles in their respective signal transduction pathways, loss-of-function mutations in APC cause tumors by a fundamentally different mechanism than loss-of-function SMAD mutations. Specifically, inactive APC proteins cannot block a mitogenic WNT signal (an oncogenic mechanism), whereas inactive SMAD proteins cannot transduce an anti-mitotic TGFß signal (a tumor suppressor mechanism). Interestingly, one study of SMAD4100T in mammalian cells suggested that this allele could employ an oncogenic mechanism of tumorigenesis, a proposal consistent with the data (Takaesu, 2005).

An examination of the primary difference between the phenotypes of SMAD4100T and zw3, Apc and Axin mutant clones may shed light on SMAD4100T-associated tumor formation. The primary difference is the location of ectopic bristles on the wing blade. In wings expressing SMAD4100T, ectopic mechanosensory bristles are derived from a cell fate transformation of mechanosensory receptors (campaniform sensilla). Cells extruding sensilla and those extruding bristles are derived from Sensory Organ Precursor cells. Alternatively, zw3, Apc and Axin mutant clones generate cell fate transformations anywhere on the wing blade -- regardless of cell lineage. This discrepancy suggests that SMAD4100T is not as potent an activator of WNT target genes as zw3, Apc or Axin mutations. SMAD4100T may only activate WNT target genes in cells predisposed to tumorigenesis, perhaps by pre-existing mutations (Takaesu, 2005).

This possibility is supported by studies of wings with an increasing dosage of UAS.SMAD4100T. When wild-type WG signaling is present, increasing the UAS.SMAD4100T copy number did not increase the frequency of sensillum to bristle transformation. However, in wings with reduced WG signaling due to the activity of UAS.DN-TCF, increasing the copy number of UAS.SMAD4100T quantitatively increased the rescue of WG-dependent functions outside of sensilla (e.g., in wing outgrowth and anterior margin formation) (Takaesu, 2005).

The transformation of campaniform sensilla to mechanosensory bristles has been reported previously. Several transheterozygous mutant genotypes of ash2, a member of the Trithorax group of transcriptional regulators, generate this phenotype. Although the ASH2 protein contains a zinc finger motif, its function has not yet been demonstrated biochemically. Studies of its yeast homolog suggest that ASH2 may function in chromatin remodeling and transcriptional activation as part of a complex containing histone methyltransferases. The similarity of SMAD4100T and ash2 mutant phenotypes, and the ability of SMAD4100T to suppress DN-TCF phenotypes, suggest that SMAD4100T contributes to the activation of WNT target genes downstream of APC, perhaps by participating in transcription factor complexes (Takaesu, 2005).

Three previous reports have shown physical interactions between the wild-type SMAD proteins and transcriptional effectors of WNT signaling (ß-catenin and TCF). One study used Xenopus embryos to demonstrate that SMAD4/ß-catenin/TCF complexes activate the transcription of the WNT target gene twin. Recently, SMAD1/ß-catenin/TCF complexes were detected in renal medullary dysplasia in ALK3 transgenic mice, and in human dysplastic renal tissue. The possibility that SMAD4100T cooperates with ARM and/or TCF to activate the transcription of achaete is currently being investigated (Takaesu, 2005).

At this time, therapeutic research on SMAD-associated tumors is guided by the current hypothesis that all SMAD mutations lead to tumors via a loss of a TGFß-encoded anti-mitogenic signal. As a result, effort is focused on restoring the wild-type function in tumors by gene replacement. However, this study has shown that two SMAD4 tumor alleles are gain-of-function mutations. One important feature of gain-of-function mutations is that they exert their effect even in the presence of a wild-type allele on the homologous chromosome. Thus, it seems unlikely that gene replacement will be successful in inhibiting tumorigenesis in cells with SMAD4 gain-of-function mutations (Takaesu, 2005).

In individuals with APC mutant colon tumors (those with unregulated Wingless target gene expression such as Familial Adenomatous Polyposis), the transition from adenomatous polyps to carcinoma will take roughly ten years. Alternatively, for TGFß receptor mutant colon tumors [those unable to respond to a TGFß-encoded anti-mitogenic signal such as Hereditary Non-Polyposis Colorectal Cancer (HNPCC)], progress from adenomatous polyps to carcinoma takes less than three years. Given these data, if SMAD4100T induces tumorigenesis by an 'APC-like' mechanism while SMAD4 dominant-negative and loss-of-function alleles stimulate tumorigenesis by an 'HNPCC-like' mechanism, then the prognosis for cancer patients with a SMAD4100T mutation is distinctly different from that of patients with other SMAD mutations (Takaesu, 2005).

This raises several issues for future investigation. (1) How many different mutations in SMAD4 can generate an 'APC-like' gain-of-function allele? To date, three SMAD4 missense mutations near codon 100 have been identified in colon tumors [Y95N, C115R and N118K]. All of these mutations (including R100T) occur in the L2/L4 double-loop region identified in the crystal structure of the SMAD3 MH1 bound to DNA. This loop occurs at the surface of the molecule and is important for macromolecular interactions. Are these also gain-of-function mutations? (2) What is the relative frequency of 'APC-like' gain-of-function SMAD4 alleles versus 'HNPCC-like' loss-of-function alleles in tumors from various tissues? (3) Can an accurate and efficient diagnostic test be developed to distinguish between 'APC-like' and 'HNPCC-like' alleles in tumors with a SMAD4 mutation? Answering these questions will require a continued collaboration between model organism geneticists and oncologists (Takaesu, 2005).

Medea: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

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