The melted (melt) gene was identified by a single revertible P-element insertion (meltS144114) that results in abnormal morphology and mild loss of peripheral neurons (Salzberg, 1997). Use was made of plasmid-rescued fragments to clone two partially overlapping melt cDNAs. In addition, through database searches the HL03627 clone was identified as a candidate melt cDNA. The full-length sequence of the three clones was used to assemble a 2.903-kb melt cDNA that is incomplete at the 3'-end. BLAST searches with the sequence of Melt gave no significant results, except a limited homology to a predicted protein from C. elegans. In addition, Melt does not contain any functional domains or motifs. Early in embryogenesis (stage 5), melt RNA is expressed in 8 or 9 stripes and in the invaginating ventral furrow. During germ band extension, melt is expressed in discrete domains in each segment of the embryo. Later, this pattern is refined to several rows of ectodermal cells in the anterior of each segment. There are also low levels of expression in the brain and the gut. In conclusion, Melt is a novel protein of unknown function, which, on the basis of its expression pattern, may be required for ectodermal patterning (Prokopenko, 2000).
The suggestion that Melted is involved in ectodermal patterning should be re-explored in light of Teleman's demonstration (Teleman, 2005) of the involvement of Melted in the Tor pathway.
Melted was identified in a gain-of-function screen for genes affecting tissue growth during Drosophila development. EP31685 causes overgrowth when expressed in the posterior half of the wing with en-GAL4. When expressed ubiquitously with tubulin-GAL4, EP31685 caused a small but statistically significant increase in total body weight. EP31685 is located ~50 bp upstream of the annotated gene melted (melt, CG8624). Using a UAS-melt transgene, it was verified that the melted transcription unit is responsible for the tissue overgrowth phenotype (Teleman, 2005).
Deletions were prepared with P-element-mediated male recombination starting from EP31685 to generate melted mutants. meltΔ1 is a 22-kb deletion that removes the entire melted gene and two adjacent genes. The second allele, meltΔ2 is a 2.5-kb deletion that removes the first exon of CG8624. Flies homozygous mutant for meltΔ1 are semiviable (70% viable to adult) and fertile but are ~10% smaller than control flies. Though small in magnitude, the reduction in body size is statistically significant. Flies heterozygous mutant for meltΔ1/meltΔ2 are also significantly smaller than control flies. Two additional lines of evidence indicate that the growth defect is due to loss of melted and not the other genes deleted in meltΔ1: (1) the growth defect of the homozygous meltΔ1 deletion flies is fully rescued by ubiquitous expression of the UAS-melt transgene; (2) flies expressing UAS-RNAi constructs directed against two different regions of the melt transcript cause tissue undergrowth, resembling the meltΔ1 phenotype (Teleman, 2005).
It was next asked whether Melted might also affect fat metabolism. Drosophila store fat mainly as triglycerides. Total body triglyceride was measured for meltΔ1 mutant and control flies reared under identical controlled conditions. When normalized to total protein to take into account the 10% reduced body size of melted mutants, melt mutant flies had only 60% as much triglyceride as control flies. This reduction in fat content was statistically significant and was rescued by ubiquitous expression of a UAS-melt transgene. Further confirmation that the leanness of the mutant is due to reduced melted expression was obtained by ubiquitous expression of a melted UAS-RNAi construct in flies (25% leaner). Total body triglycerides of wandering third instar meltΔ1 mutant larvae were also 20% lower than controls, indicating that Melted regulates fat levels throughout development as well as in the adult (Teleman, 2005).
Fat levels can be controlled by humoral factors, including adipokinetic hormone (AKH), which induces mobilization of fat reserves. Insulin-like peptides (ILPs) also control fat metabolism in the fly. To determine whether elevated AKH or ILP-2, -3, and -5 expression could explain the leanness of melted mutants, transcript levels were tested by quantitative RT-PCR. Transcript levels were not significantly elevated. Because altered expression of the known humoral regulators did not provide an explanation for the mutant phenotype, it was asked if there was a defect in adipose tissue. The 'fat body' is the main fat-storage organ of the fly, containing over 80% of total body triglycerides. meltΔ1 mutant and control fat body tissue was isolated from larvae; 25% lower triglyceride levels were found in the mutant tissue. The total body leanness of meltΔ1 mutants was rescued by expressing melted specifically in the fat body with ppl-GAL4. In contrast, expression of melted in the nervous system with elav-GAL4 or in brain neurosecretory cells with dILP3-GAL4 does not rescue the mutant. This indicates that Melted activity is required in adipose tissue (Teleman, 2005).
To better understand the metabolic impact of the loss of Melted function in the adipose tissue, microarray expression profiling of melted mutant versus control fat body was performed. Use was made of microarrays containing 11,445 cDNA clones (DGC1 and DGC2 collection). Allowing for a 1% false-positive rate, 315 genes were identified that were upregulated and 405 genes that were downregulated in the melted mutant. This represents 6% of all genes sampled and reflects substantial reprogramming of the transcriptional profile of the adipose tissue. Within this set, genes altered by at least 1.5-fold (249 genes) were considered, and grouped according to their functional annotation. 46% of the regulated genes are involved in metabolism. Interestingly, many of these are involved in lipid metabolism. Ten are cytochrome P450 enzymes involved in the oxidation of lipophilic molecules. The transcriptional regulation of some of these genes was confirmed by semiquantitative RT-PCR. In addition to the genes involved in fat metabolism, a significant proportion of the misregulated metabolic genes are involved in protein degradation (Teleman, 2005).
Several of the downregulated genes are involved in the accumulation of triglycerides. One of the most highly downregulated genes in the meltΔ1 mutant adipose tissue is the transcription factor sugarbabe (2.4-fold). sugarbabe was previously identified as a gene controlling the conversion of sugars to fats. It is the second most highly upregulated gene when flies were fed sugar but deprived of lipids, and it becomes expressed in the adipose tissue, gut, and Malphighian tubules. Acetyl-CoA sythase (AcCoAS) was also identified in the same screen as a gene upregulated on a sugar diet (7.3-fold), whereas in the meltΔ1 mutant adipose tissue it was downregulated (1.8-fold). The downregulation of both sugarbabe and AcCoAS suggests that meltΔ1 mutant adipose tissue might not be able to accumulate enough lipid. This is corroborated by the finding that glycerol kinase (Gyk) and phosphoenolpyruvate carboxykinase (PEPCK) are among the genes most downregulated in melted mutant adipose tissue. In order to generate triglycerides, both free fatty acids and 3-phosphoglycerol are required by the cell. In vertebrate brown adipose tissue, 3-phosphoglycerol is made by Gyk, whereas in white adipose tissue, it is made by PEPCK. PEPCK is rate limiting in that loss of function in the mouse leads to lipodystrophy, whereas overexpression in mouse adipose tissue leads to obesity. Therefore, the finding that both Gyk and PEPCK are downregulated in the meltΔ1 mutant fat body suggests that these animals are lean because they do not accumulate enough lipid in the adipose tissue. Consistent with what is known in vertebrates, increased PEPCK levels were detected by quantitative RT-PCR in other nonadipose tissues under starvation conditions and in the melted mutant; thus, the transcriptional changes in adipose and nonadipose tissues do not always correlate. Recently, adipose triglyceride lipase has been reported to catalyze the initial step in triglyceride hydrolysis in mice and inhibition of this enzyme markedly decreases total adipose acyl-hydrolase activity. BLAST searches have identified two Drosophila homologs of adipose triglyceride lipase: CG5295 and CG5560. Interestingly, CG5295 expression is markedly reduced in the meltΔ1 mutant fat body. This suggests that lipid hydrolysis might be downregulated in the mutant adipose tissue (Teleman, 2005).
To
test experimentally these predictions from expression data, measurements were made of
circulating lipids, which, in the fly, are mobilized from the fat body and
delivered to peripheral tissues as diacylglycerides (DAG) in the hemolymph.
Hemolymph DAG was
low in meltΔ1 mutant larvae compared
to controls. Thus, it is not
likely that the reduced triglycerides in the adipose tissue can be explained by
increased mobilization of fat in the form of circulating DAG. The level of
circulating blood sugar (trehalose + glucose) was also measured and it was found to be
not elevated in the meltΔ1 mutant.
These experiments, together with the expression
data, suggest that the leanness observed in the mutant is likely due to reduced
triglyceride accumulation in adipose tissue (Teleman, 2005).
Color vision in Drosophila relies on the comparison between two color-sensitive photoreceptors, R7 and R8. Two types of ommatidia in which R7 and R8 contain different rhodopsins are distributed stochastically in the retina and appear to discriminate short (p-subset) or long wavelengths (y-subset). The choice between p and y fates is made in R7, which then instructs R8 to follow the corresponding fate, thus leading to a tight coupling between rhodopsins expressed in R7 and R8. warts, encoding large tumor suppressor (Lats) and melted, encoding a PH-domain protein, play opposite roles in defining the yR8 or pR8 fates. By interacting antagonistically at the transcriptional level, they form a bistable loop that insures a robust commitment of R8 to a single fate, without allowing ambiguity. This represents an unexpected postmitotic role for genes controlling cell proliferation (warts and its partner hippo and salvador) and cell growth (melted) (Mikeladze-Dvali, 2005b).
The fly eye provides a powerful system to study cell-fate decisions: it develops from a flat epithelium into a complex three-dimensional structure of multiple cell types in less than a week. The adult eye allows the fly to perform various visual tasks, ranging from motion detection and the discrimination of colors to measuring the orientation of polarized light for navigation (Mikeladze-Dvali, 2005b).
In the fly compound eye, each of the 800 ommatidia is a single optical unit that contains 8 photoreceptor cells (PRs). The 8 PRs form widely expanded membrane structures, the rhabdomeres, which contain the photosensitive Rhodopsins (Rh). The rhabdomeres of the six outer PRs (R1-R6) form a trapezoid. R1-R6 all express the broad spectrum rhodopsin1 (rh1 or ninaE) and are morphologically and functionally invariant in all ~800 ommatidia (Mikeladze-Dvali, 2005b).
The center of the trapezoid is occupied by the two inner PRs, R7 and R8. The rhabdomeres of R7 are positioned on top of R8, so that they share the same optic path. Inner PRs are involved in color vision and can be viewed as equivalent to vertebrate cones. Each R7 and R8 expresses only one of the four rhodopsins, rh3, rh4, rh5, or rh6 in a highly regulated manner, defining three different subtypes of ommatidia: 'yellow' (y), 'pale' (p) (for their appearance under UV illumination), and the 'dorsal rim area' (DRA). Ommatidia in the DRA express rh3 in both R7 and R8 and are specified in a very restricted region by the gene homothorax. They are believed to function as polarized light detectors (Mikeladze-Dvali, 2005b).
In contrast, color vision depends on the y and p ommatidial subtypes that are randomly distributed through the main part of the retina, with a bias of y (~70%) over p subtype (~30%). In the p subtype, R7 expresses the UV-sensitive Rh3 and R8 the blue-sensitive Rh5. In the y subtype, R7 expresses a distinct UV-sensitive Rh4 while R8 expresses the green-sensitive Rh6. As in many other sensory systems, expression of a given Rhodopsin excludes all others to prevent sensory overlap. While the p subtype is better suited to discriminate among shorter wavelengths, the y subtype should discriminate amongst longer wavelengths (Mikeladze-Dvali, 2005b).
The choice between the p and y fate is first made in R7: once an R7 commits to the p fate and expresses rh3, it sends an instructive signal to the underlying R8, which then also commits to the p fate and expresses rh5. In the absence of the R7 signal (i.e., when R7 expresses rh4 or in a sevenless mutant), R8 commits to the y fate and expresses rh6. The stochastic choice appears to be made by each R7 independently of its neighbors, resulting in the biased random distribution of p and y ommatidia throughout the main part of the retina (for review see Mikeladze-Dvali, 2005a).
Four genes required in R8 cells for ensuring the correct choice of y versus p cell fate have been identified. The warts (wts) gene, which encodes the Drosophila large tumor suppressor (also known as lats) and melted (melt) play a critical role in the specification of p and y R8 cells, without affecting the R7 choice. wts encodes a Ser/Thr kinase, while melt encodes a Pleckstrin Homology (PH) domain protein. wts is necessary and sufficient for R8 to adopt the y fate, while melt plays the opposite role and specifically induces the p fate in R8. wts and melt are expressed in a complementary manner in the yR8 and pR8 subsets, respectively. Evidence is presented that the two genes repress each other's transcription to form a bistable loop. melt seems to respond to the R7 signal, while wts appears to regulate the output of the loop. The tumor-suppressor genes hippo (hpo) and salvador (sav), which encode the two molecular partners of Wts/Lats, have phenotypes identical to wts. Interestingly, melt has been reported to regulate growth and fat metabolism in Drosophila. Thus, genes known to regulate both cell growth (melt) and proliferation (wts, sav, hpo) interact antagonistically during retinal patterning (Mikeladze-Dvali, 2005b).
To identify genes involved in the differentiation of p or y PR subsets, a Gal4 (pGawB) enhancer trap screen was performed in adult flies using GFP expression as a reporter. One insertion produced a strong GFP signal in inner PRs. Staining of sectioned adult eyes for the UAS-lacZ reporter gene revealed Gal4 expression in a large subset of R8 cells. Additional expression was found in DRA R7 and R8, as well as in outer PRs in the ventral half of the eye. Occasionally, weak expression was also found in some R7 cells, but not in any PR subset-specific pattern. Staining of the same enhancer trap (driving UAS-lacZnuc expression) with antibodies against β-Gal, Rh6 (α-Rh6), and Rh5 (α-Rh5) in whole-mounted retinas revealed that the reporter was specific to Rh6-positive R8 and was excluded from the Rh5-positive R8, indicating that the targeted gene is expressed in the yR8 subtype (Mikeladze-Dvali, 2005b).
The genomic DNA flanking the pGawB transposon, which is inserted upstream of the third exon of the gene warts (wts), was identified. An existing wts nuclear lacZ enhancer trap line P[lacZ,w+] was stained. lacZ expression in this line (wtsZn) was also specific to the y subset of R8 cells as well as the DRA and some ventral outer PRs, confirming the restricted expression pattern of wts (Mikeladze-Dvali, 2005b).
wts-Gal4 appears to be activated by a late eye-specific enhancer of wts, which first directs expression long after R8 has exited the cell cycle. wts therefore appears to play two distinct roles: a ubiquitous role in proliferating cells and a more restricted role in terminally differentiated PR (Mikeladze-Dvali, 2005b).
Flies with wts-Gal4 insertion were homozygous viable and did not exhibit any visible growth phenotype. However, it was noticed that heterozygous wts-Gal4 flies always exhibited a strong rh phenotype when present in combination with one specific UAS-lacZ reporter construct (P w[+mC] = UAS-lacZ.B Bg4-2-4b, FlyBase #1777). The y/p R8 ratio was dramatically affected: most R8 expressed rh5, while rh6 expression was almost completely lost, with wts-Gal4 expression reduced to the remaining rh6 expressing R8. However, specification of R7 and of outer PRs was unaffected. This phenotype was only observed with this specific UAS-lacZ transgene, and not with UAS-GFP or other UAS-lacZ transgenes. When homozygous (in the absence of wts-Gal4), this UAS-lacZ line manifested an even more severe R8 opsin phenotype: about 90% of R8 expressed rh5 at the expense of rh6. This suggested that this particular insertion disrupted a gene affecting the p/y choice in R8 (Mikeladze-Dvali, 2005b).
This UAS-lacZ P element was found to be inserted 21 bp upstream of the transcriptional start site of the gene melted (melt). The Melt protein has a C-terminal PH domain and is conserved from C. elegans to humans. Insertions in melt were initially identified in a screen for genes affecting peripheral nervous system development. Thus the role of melt in R8 subtype specification and its interaction with wts was examined (Mikeladze-Dvali, 2005b).
Since R7 and R8 in a given ommatidium share the same optic path, their fates must be tightly regulated. The decision of a given ommatidium to become y or p is initially made by R7. Once R7 has chosen its fate, it imposes it onto the underlying R8. To coordinate opsin expression between R7 and R8, R8 has to respond to the R7 signal with high fidelity (Mikeladze-Dvali, 2005b).
This study shows that wts and melt act in R8 to prevent an ambiguous response to the instructive R7 signal. wts and melt play opposite roles in the specification of R8 subtypes. In the absence of wts, the yR8 subtype is completely misspecified into pR8. By contrast, in melt mutants, the pR8 subtype is lost with expansion of yR8. Overexpression of wts or melt leads to the transformation of all R8 into the y or p fate, respectively. The complementary expression patterns of the two genes in y or p R8 subtypes are set up in response to the pR7 signal. Therefore, wts and melt appear to interpret the signal from R7, and mutations in wts and melt render R8 insensitive to this signal without influencing R7 or outer PR (Mikeladze-Dvali, 2005b).
The decision to express wts or melt in R8 is determined by R7, but the two genes repress each other's transcription. Thus, wts and melt act in a loop of negative crossregulation. However, if R7 imposes its fate upon R8, what then is the role of this crossregulation? It is suggested that the bistable loop allows only an unambiguous readout while R7 provides an asymmetric bias of this choice (Mikeladze-Dvali, 2005b).
In a negative bistable crossregulatory loop, the input signal biasing cell-fate choice might act at any level. Similarly, any member of the loop can serve as the output. For instance, wts could positively regulate rh6 expression (yR8 fate), while melt could activate rh5 (pR8 fate). Double misexpression and double loss-of-function experiments suggest that wts is the output regulator of the loop. When both wts and melt are ectopically expressed, all R8 acquire the y fate, i.e., the fate imposed by wts. In melt, wts double mutants, all R8 acquire the p fate. These phenotypes resemble the single gain- or loss-of-function phenotypes of wts, which appears to be necessary and sufficient for rh6 expression. In contrast, while melt is sufficient to induce rh5 in yR8, rh5 remains expressed in the absence of melt in the double mutant. This argues that melt is not necessary for the pR8 fate (rh5). In melt, wts double-mutant eyes, rh5 does not depend on instruction from pR7, which confirms that rh5 expression is a consequence of the absence of wts (a derepression rather than activation by the pR7 signal) (Mikeladze-Dvali, 2005b).
The following model is proposed: in the absence of an instructive pR7 signal, i.e., in y ommatidia, the loop is biased in favor of wts expression, which represses melt. In p ommatidia, the R7 signal either induces melt expression in R8 or represses expression of wts in R8. In either case, the balance of the loop is shifted, leading to upregulation of melt and complete suppression of wts expression. This system is able to amplify a weak or transient signal to ensure that the cell-fate decision is made unambiguously (Mikeladze-Dvali, 2005b).
There are clearly a number of examples of bistable loop that often reinforce stochastic decisions or transient differentiation stimuli. Bistable systems require positive feedback loops as proposed for the BMP signaling during dorso-ventral patterning in Drosophila or double-negative feedback loops as in the case of the wts-melt loop. The left-right choice by chemosensory ASE neurons in C. elegans is a similar example where a negative bistable loop is involved in making an unambiguous cell-fate decision. This loop includes two transcription factors and two microRNAs. In the left ASE, this loop is strongly biased toward Na+-sensitive fate and in the right ASE, toward Cl− sensitivity (Johnston, 2005
The bistable loop is specific to those R8 that are involved in color vision: in DRA ommatidia, melt misexpression does not lead to wts downregulation. This is not surprising since R7 and R8 in DRA are specified independently by positional information and do not appear to communicate (Mikeladze-Dvali, 2005b).
The transcriptional regulation of wts and melt expression is surprising, since kinases and PH domain proteins are usually regulated by changes in their activity or subcellular localization. For instance, Wts/Lats kinase activity is regulated through phosphorylation by Hpo in the presence of Sav. However, the nature of the signal that triggers activation of the Wts/Hpo/Sav proliferation control pathway has remained elusive. Thus, identification of the signal from pR7 to R8 could provide important insights into the mechanism by which this tumor-suppressor complex is regulated to control proliferation and cell death (Mikeladze-Dvali, 2005b).
The ability of wts to indirectly regulate transcription of other genes (here melt) is less surprising. wts, sav, and hpo have been reported to negatively regulate the transcription of Cyclin E and DIAP1, leading to a decrease in cell cycle progression and to an increase in cell death. The same (unknown) transcription factor required downstream of wts could therefore also play a role in repressing melt and rh5, and possibly in activating rh6 (Mikeladze-Dvali, 2005b).
Cbk1, the Lats/Wts homolog in S. cerevisiae has been shown to regulate a broad range of daughter specific genes during budding. The asymmetric gene expression between mother and daughter cells is due to Cbk1-dependent activation and nuclear localization of the transcription factor Ace2 in daughter cells. Cbk1 kinase activity requires another gene, Mob2. Recently, a member of the Mob family in Drosophila, Mats, has been shown to bind and synergistically interact with Wts/Lats to control proliferation and apoptosis. Although Melt is not known to regulate the transcription of other target genes, it can affect subcellular localization of FOXO and the TSC1/TSC2 complex to regulate fat metabolism. However, the members of the TOR or InR do not seem to play a role in the specification of R8 subtypes (Mikeladze-Dvali, 2005b).
Wts, together with the Ser/Thr kinase Hpo and the adaptor protein Sav, acts as a potent tumor suppressor. All three genes play a critical role for the establishment of the R8 subtypes. The function described in this study for hpo/sav/wts represents an unexpected new role unrelated to their tumor-suppression function: R8 PRs have exited the cell cycle for at least 4 days when they choose to express a particular rhodopsin, and these cells are not prone to die (PRs are particularly difficult to kill through induction of the cell death pathway). Furthermore, there is no detectable difference in cell size or shape between y and p R8, which specifically express or exclude wts or melt expression. However, it is interesting to note that p and y inner photoreceptors are morphologically distinguishable in Calliphora blowflies. Perhaps Wts and Melt represent an evolutionary remnant of a system in large flies where subtypes required different morphologies. Therefore, specification of the correct R8 fate utilizes two signaling cassettes used for different purposes earlier in development, after these cassettes are no longer in use in these highly differentiated PR cells (Mikeladze-Dvali, 2005b).
Lats1, the human ortholog of Wts, is able to rescue the lethality of wts in flies. Canine Lats1 splice variant is specifically expressed in the retina. Moreover, a gene responsible for an autosomal dominant cone dystrophy (involving impaired color vision, sensitivity to light, and gradual loss of visual activity) has been mapped close to the Lats1 locus. Thus, it might be expected that the hpo/sav/wts pathway functions in the human retina as well. Although, melt knockout mice are viable and fertile, it will be interesting to test whether they are defective in cone differentiation or vision (Mikeladze-Dvali, 2005b).
Johnston, R. J., et al. (2005). MicroRNAs acting in a double-negative feedback loop to control a neuronal cell fate decision, Proc. Natl. Acad. Sci. 102: 12449-12454. 16099833
Mikeladze-Dvali, T., Desplan, C and Pistillo, D. (2005a). Flipping coins in the fly retina. Curr. Top. Dev. Biol. 69: pp. 1-14. 16243594
Mikeladze-Dvali, T., et al. (2005b). The growth regulators warts/lats and melted interact in a bistable loop to specify opposite fates in Drosophila R8 photoreceptors. Cell 122: 775-787. 16143107
Prokopenko, S. N., He, Y., Lu, Y. and Bellen, H. J. (2000). Mutations affecting the development of the peripheral nervous system in Drosophila: a molecular screen for novel proteins. Genetics 156(4): 1691-715. 11102367
Salzberg, A., et al. (1997). P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: mutations affecting embryonic PNS development. Genetics 147(4): 1723-41. 9409832
Teleman, A. A., Chen, Y. W. and Cohen, S. M. (2005). Drosophila Melted modulates FOXO and TOR activity. Dev Cell 9(2): 271-81. 16054033
date revised: 20 December 2005
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