bantam miRNA is expressed at all developmental stages, though at varying levels. To ask whether bantam expression is spatially regulated during development, an assay was developed based on the ability of miRNAs to inactivate genes by RNAi. A transgene was prepared expressing GFP under control of the tubulin promoter and two copies of a perfect bantam target were placed in the 3′UTR. A comparable construct without the bantam target sequences in the 3′UTR was used as a control. Where present, bantam miRNA should reduce expression of the transgene containing the target sequences by RNAi, providing an in vivo sensor for bantam activity. When expression of the two transgenes was compared using the same settings on the confocal microscope, it was apparent that the control transgene was expressed at much higher levels overall. The bantam sensor transgene showed a complex pattern of spatial modulation in the third instar wing disc, being higher in cells near the anteroposterior and dorsoventral boundaries and in patches in the dorsal thorax. The control transgene showed limited spatial modulation (Brennecke, 2003).
The difference in the overall levels of control and bantam sensor transgenes suggested that bantam miRNA is expressed broadly in the wing disc, lowering the expression of the specific sensor transgene. Indeed, removing bantam miRNA in clones of cells homozygous for the bantamΔ1 deletion increased expression of the bantam sensor. The level of sensor expression in the clones was considerably higher than the maximal endogenous level at the DV boundary, indicating that the miRNA is present throughout the disc, though at varying levels. The P element EP(3)3622 is inserted 2.7 kb from the hairpin and has been identified as a hypomorphic allele of bantam (Hipfner, 2002). Clones of cells homozygous mutant for EP(3)3622 also show upregulation of the bantam sensor, demonstrating that this insertion reduces bantam miRNA levels. In this case, the maximal level of sensor expression is similar to the level at the DV boundary. The level of sensor expression is lower in the twin spots, which express two copies of the endogenous bantam gene than in the surrounding cells, which have one wild-type and one mutant copy of the gene. This suggests that elevated bantam activity would reduce sensor expression. Indeed, clones overexpressing bantam do reduce sensor levels. The control sensor was not affected by overexpression of bantam. Taken together, these observations indicate that the sensor is capable of reflecting even subtle increases and decreases in bantam miRNA levels in vivo. In all cases, the effects on the sensor were cell autonomous. The sensor transgene method may provide a generally useful tool to visualize miRNA activity in vivo (Brennecke, 2003).
The bantam locus was identified by several EP element insertions clustered in a region of ~41 kb that lacks predicted genes. EP elements are transposable elements designed to allow inducible expression of sequences flanking the insertion site under control of the yeast transcription factor Gal4. Gal4-dependent expression of the EP elements inserted at the bantam locus causes tissue overgrowth due to an increase in cell number. Conversely, flies homozygous for the bantamΔ1 deletion, which removes ~21 kb flanking the insertion site of EP(3)3622 grow poorly and die as early pupae. Flies heterozygous for the bantamΔ1 deletion and three of the P element inserts survived and were morphologically normal but smaller than normal flies. These observations led to the conclusion that the bantam locus is involved in growth control during development (Hipfner, 2002). In an effort to molecularly define the bantam locus, transgenic flies were produced carrying fragments of genomic DNA overlapping the region where P element inserts clustered. Two fragments rescued the growth defects and pupal lethality of flies homozygous for the bantamΔ1 deletion. The 3.85 kb overlap of these transgenes defines the extent of essential sequences comprising the bantam locus. This region contains an EST, RE64518. Expression of RE64518 under Gal4 control failed to reproduce the overgrowth phenotype caused by the EP elements, indicating that RE64518 does not encode bantam function (Brennecke, 2003).
To verify that the miRNA produced by the predicted hairpin is the functional product of the bantam locus, a series of rescue assays and gain of function overgrowth assays were performed. bantamΔ1 homozygous mutant larvae generally lack some or all imaginal discs and show undergrowth of larval tissues, including the brain. These animals develop slowly, but survive larval development and die shortly after pupation, lacking evidence of imaginal structures. A 6.7 kb rescue fragment, UAS-A, is able to rescue growth of the imaginal discs and allowed the bantamΔ1 homozygous mutant animals to overcome pupal lethality, so that viable adults were produced. Although the construct is in a UAS vector, rescue was independent of GAL4, suggesting that the endogenous regulatory elements needed to produce the bantam miRNA are contained within this fragment. However, the surviving flies often had rough eyes, duplicated bristles, and missing halteres, suggesting that some regulatory elements may be missing. When provided with a weak ubiquitous source of GAL4, the smaller 584 nt fragment contained in UAS-C rescues the growth defect in the imaginal discs. Approximately half of the larvae formed morphologically normal pupae that expressed the GFP transgene from which the miRNA is excised. Many of these animals survive to adulthood. Thus, sequences contained within the 584 nt fragment are sufficient to provide bantam function when expressed. The UAS-B transgene contains the same DNA fragment as UAS-A, except that it lacks 81 nt containing the hairpin. UAS-B is unable to rescue the mutant phenotype, indicating that the deleted residues are essential for bantam function. When expressed under engrailed-GAL4 control, constructs UAS-A, UAS-C, and a shorter construct UAS-D (containing 100 nt including the hairpin) each produced overgrowth of the posterior compartment of the wing and of segments in the larval body. UAS-B did not produce any overexpression phenotype. Together these observations assign bantam function to the region containing the hairpin and suggest that the 21 nt miRNA is the bantam gene product (Brennecke, 2003).
In light of the observation that bantam acts cell autonomously to regulate sensor expression, it was asked whether bantam acts autonomously to control cell proliferation. FLP-induced mitotic recombination was used to produce clones of cells homozygous for the bantamΔ1 deletion and sister clones that were homozygous wild-type. Each pair of clones derives from a single cell division. Consequently, growth rates can be compared by measuring the areas of individual pairs of mutant and wild-type twin clones after a period of time. Clones were generated at the end of second instar and analyzed late in third instar. Mutant clones were on average 1/3 the size of the wild-type twins. Although a few relatively large bantam mutant clones were observed, mutant clones were typically very small. DAPI labeling did not reveal an observable difference in the size or spacing of nuclei in mutant and wild-type tissue, suggesting that cell size was unaffected by the deletion mutant. No obvious increase in apoptosis was observed in these clones. These observations suggest that bantam acts cell autonomously to control cell proliferation (Brennecke, 2003).
The bantam sensor transgene (expressing GFP under control of the tubulin promoter and two copies of the bantam target in the 3′UTR) provides a means to compare bantam activity and cell proliferation in vivo. Cell proliferation was visualized using BrdU incorporation to label cells undergoing DNA synthesis. A striking correlation was observed between bantam activity and cell proliferation in the developing larval brain. Proliferating cells had a lower level of sensor expression, indicating elevated bantam miRNA activity, compared to adjacent nonproliferating cells. This correlation was also observed in the wing disc. Elevated sensor levels coincide with the zone of nonproliferating cells adjacent to the dorsoventral boundary (ZNC), indicating that bantam miRNA levels are reduced in the ZNC (Brennecke, 2003).
To ask whether regulation of bantam miRNA contributes to the exit of ZNC cells from proliferation, bantam was expressed under ptc-Gal4 control. Restoring bantam expression was sufficient to direct cells in the nonproliferating zone to enter S phase. The ZNC depends on the activity of the secreted signaling protein Wingless and on Notch activity. Myc expression is downregulated in the ZNC by Wingless signaling. Forced expression of Myc in the ZNC can drive G1-arrested cells into S phase, but does not affect the G2-arrested cells. Although bantam can drive both G1/S and G2/M transitions when expressed in the wing disc (Hipfner, 2002), bantam expression does not affect Myc protein levels in the ZNC, suggesting that the myc transcript is not a target of bantam regulation (Brennecke, 2003).
bantam mutant animals are smaller than wild type, due to a reduction in cell number but not cell size, and do not have significant disruptions in patterning. Conversely, overexpression of the bantam product using the EP element EP(3)3622 causes overgrowth of wing and eye tissue (see Abdelilah-Seyfried, 2000). Overexpression in clones of cells results in an increased rate of cell proliferation and a matched increase in cellular growth rate, such that the resulting tissue is composed of more cells of a size comparable to wild type. These effects are strikingly similar to those associated with alterations in the activity of the cyclinD-cdk4 complex. However, epistasis and genetic interaction analyses indicate that bantam and cyclinD-cdk4 operate independently. Thus, the bantam locus represents a novel regulator of tissue growth (Hipfner, 2002).
bantam gene function appears to be important for regulation of tissue growth rates. Several EP elements inserted in this locus, most notably EP(3)3622, are capable of promoting substantial tissue overgrowth in the eye and wing in a GAL4-dependent manner. Conversely, ban mutations decrease tissue growth. Mutant phenotypes range from decreased body size to lethality. The strongest available allele is a small deletion that does not remove any known genes. This allele is pupal lethal and causes the absence of detectable imaginal discs. The simplest explanation for the reciprocal nature of gain-of-function and loss-of-function phenotypes is that EP(3)3622 is driving expression of the same transcription unit that is affected by ban mutations. This is further supported by the specific and reciprocal nature of the genetic interaction of gain and loss of ban function with the expanded tumor suppressor gene in the eye. However, this remains to be confirmed by molecular characterization of the locus. Growth regulation appears to be a primary function of ban, since EP(3)3622 expression does not cause significant patterning alterations, and ban mutant flies, although small, are proportioned normally (Hipfner, 2002).
Two of the ban EP insertions were identified initially in a genetic interaction screen as suppressors of the phenotype caused by overexpression of the expanded tumor suppressor gene. When misexpressed in the eye under the control of the sevGAL4 driver, expanded causes a reduction in eye size relative to wild type and external roughening and blistering. Coexpression of EP(3)3622 almost completely suppresses this phenotype, restoring the eye to nearly wild-type size and appearance. Reducing ban function has the opposite effect. Introducing one copy of the banDelta allele noticeably reduces the overall eye size and increases the blistering in the central and anterior regions of the eye. In contrast, alterations in cycD-cdk4 activity does not alter the expanded overexpression phenotype. Coexpression of cycD-cdk4 with expanded increases the overall size of the eye, but has little effect on the roughness and blistering. Removing one copy of cdk4 has no effect. The lack of a strong genetic interaction between expanded and cycD-cdk4 provides additional evidence that bantam is acting independently of this complex to promote coordinated cell growth and cell cycle progression (Hipfner, 2002).
The results suggest that ban regulates tissue growth by a mechanism that involves coordinated stimulation of cell growth and cell division. ban alters tissue growth through effects on cell number rather than cell size. Decreased ban function causes a reduction in cell number in the adult wing, but the surviving cells are of wild-type size, suggesting a coordinated decrease in the rate of cell growth and division. Activation of EP(3)3622 has the opposite effect on cell number, causing an increase in the rate at which imaginal disc cells proliferate. Despite this increased proliferation rate, cell sizes are little changed. These observations suggest that the rate of increase in cell division is coordinated with the rate of increase of cell mass when ban is overexpressed. The effects of ban on growth and fertility are remarkably similar to those of cycD-cdk4. However, no evidence was found of a direct connection between cycD-cdk4 and ban. It seems unlikely that ban regulates growth by controlling the activity of cycD-cdk4, because ban-driven overgrowth is unaffected in the absence of cdk4. Similarly, cycD-cdk4-driven growth is unaffected by reduction of ban, indicating that ban is unlikely to be a downstream effector. The view is favored that ban and cycD-cdk4 act independently. The similarity in their growth phenotypes suggests that they may have some targets in common. However, as attested to by the differences in their interactions with expanded, they clearly can act differently as well (Hipfner, 2002).
The imaginal discs are patterned while they grow. The secreted signaling proteins Decapentaplegic (Dpp) and Wingless pattern the wing and leg discs along their main axes. Dpp and Wingless signaling are also required in some way for disc growth. The parts of the discs that produce the appendages are very small in flies lacking either signal. Cells unable to transduce the Dpp or Wingless signals display cell-autonomous defects in proliferation and are lost from the disc. To date it has not been reported whether loss of cells under these conditions is due to reduced proliferation or to reduced survival. However, recent studies suggest that Dpp signaling may directly influence cell proliferation in the wing disc. Wingless signaling has been shown in one situation to repress growth at late stages of wing development, in part by negative regulation of dmyc expression. If Dpp and Wingless act directly to regulate tissue growth, it would be expected that they coordinately regulate cell growth and cell division rates. It will be of interest to learn whether ban and/or cycD-cdk4 mediate the growth effects of these signaling molecules (Hipfner, 2002).
Altering cell division rates does not alter compartment size, but can increase or decrease the number of cells per compartment. This is consistent with the effects of Minute mutations that vary the proportion of a compartment that can be contributed by the progeny of a single cell, without affecting compartment size or shape. However, it is possible to alter the size of one compartment relative to another by manipulating activity of the insulin/PI3K pathway. PI3K-induced overgrowth requires that the pathway be activated in all cells of the compartment. Clones of overgrowing cells do not affect the size of the compartment. Thus a mechanism must exist that allows a population of cells to measure the size of the compartment. Interestingly, it has been found that altering the size of the compartment feeds back by an unknown mechanism to alter the shape of the Dpp morphogen gradient (Hipfner, 2002).
Overexpression of ban with enGAL4 promotes significant overgrowth of the posterior compartment. Posterior compartment overgrowth is compensated for by a nonautonomous reduction in the final size of the anterior compartment in most cases. This compensation suggests that total disc size may also be regulated to some extent during development. Only in the case of the strongest EP element, EP(3)3622, are total disc and wing size increased (Hipfner, 2002).
These observations suggest that there may be multiple layers of size control operating during imaginal disc development. Morphogen gradients influence tissue growth. Tissue growth rates influence compartment size and morphogen gradient shape. Finally, size compensation mechanisms exist to control both compartment and disc size. At present, little is known about the size-sensing mechanisms, except that they can be overridden by stimulating cell and tissue growth rates by various experimental means. Identifying how size is measured during tissue growth poses a significant challenge (Hipfner, 2002).
Abdelilah-Seyfried, S., et al. (2000). A gain-of-function screen for genes that affect the development of the Drosophila adult external sensory organ. Genetics 155: 733-752. 10835395
Bilen, J., et al. (2006). MicroRNA pathways modulate polyglutamine-induced neurodegeneration. Molec. Cell 24: 157-163. Medline abstract: 17018300
Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B. and Cohen, S. M. (2003). bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113: 25-36. 12679032
Forstemann, K., Tomari, Y., Du, T., Vagin, VV., Denli, A. M., Bratu, D. P., Klattenhoff, C., Theurkauf, W. E. and Zamore, P. D. (2005). Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol. 3(7): e236. 15918770
Hipfner, D. R., Weigmann, K., and Cohen, S. M. (2002). The bantam gene regulates Drosophila growth. Genetics 161: 1527-1537. 12196398
Llave, C., Xie, Z., Kasschau, K. D., and Carrington, J. C. (2002). Cleavage of scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297: 2053-2056. 12242443
Nolo, R., Morrison, C. M., Tao, C., Zhang, X. and Halder, G. (2006). The bantam microRNA is a target of the hippo tumor-suppressor pathway. Curr. Biol. 16(19): 1895-904. Medline abstract: 16949821
Saito, K., Ishizuka, A., Siomi, H., Siomi, M. C. (2005). processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells.PLoS Biol. 3(7): e235. 15918769
Thompson, B. J. and Cohen, S. M. (2006). The Hippo pathway regulates the bantam microRNA to control cell proliferation and apoptosis in Drosophila. Cell 126(4): 767-74. Medline abstract: 16923395
date revised: 20 July 2007
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