PAR-domain protein 1

Promoter

The pattern of Pdp1 mRNA accumulation in the developing embryo suggests that the transcriptional regulation of Pdp1 is likely to be controlled by multiple cis-acting elements distributed over several kilobases of DNA. Accordingly, gene fragments that contain enhancers regulating Pdp1 expression have been identified. Of the restriction fragments tested, two contain enhancer activity that closely mimics endogenous Pdp1 expression. A 4.4-kb restriction fragment was identified within the first intron that directs midgut and fat body expression in the embryo and larva. This enhancer fragment drives expression in the midgut of stage 11 and 12 embryos and in later stage 14 and 16 embryos in the posterior midgut (fourth lobe), the Malpighian tubules, and the fat body in a pattern that is similar to that of the endogenous gene. This embryonic pattern of expression is maintained in first- and third-instar larvae (Reddy, 2000).

Pdp1 protein is expressed in the segmented mesoderm of stage 11 embryos. At this stage, the more dorsal mesodermal cells are the precursor cells of the fat body and the more ventral cells are the progenitor/founder cells of the body wall muscles. Since Pdp1 is expressed in the developing embryonic fat body and body wall muscles in later embryos, it is important to determine whether Pdp1 is expressed in the precursors of these tissues. Thus it is particularly significant that the 4.4-kb enhancer drives expression in the segmented dorsal mesoderm of stage 11 embryos, since this enhancer expresses in the fat body and not the body wall muscles in older embryos. Thus, it can be concluded that Pdp1 is expressed in the fat body precursor cells and in the developing fat body as it differentiates (Reddy, 2000).

A second fragment observed to have enhancer activity is an 11-kb fragment located in the second intron. This enhancer fragment is expressed in the pharyngeal muscle and the body wall muscles in the developing embryo and larva. Expression in the body wall muscles is particularly strong in a subset of ventral and dorsal muscles; weaker and/or no detectable expression is found in other muscles. In the midgut of embryos, the 11-kb enhancer shows very weak expression in the anterior midgut and strong expression in a region of the posterior midgut and the hindgut. This embryonic pattern gives rise to enhancer-driven expression in the gastric caeca, posterior midgut, and hindgut of the first- and third-instar larva. Enhancer activity is not detected in the middle midgut of embryos; however, endogenous Pdp1 is expressed in the basophilic cells of the middle midgut in the embryo. These cells are the precursors to the larval copper cells. Furthermore, the 11-kb enhancer expresses strongly in the copper cells of the middle midgut in all larval stages. These results suggest that a separate unidentified enhancer regulates expression in the basophilic and copper cells of the middle midgut in the embryo (Reddy, 2000).

Pdp1 is expressed in the precursor cells of the fat body in stage 11 embryos. The 11-kb enhancer also drives expression in the segmented mesoderm of stage 11 embryos. A close comparison shows, however, two or three small clusters of mesodermal cells in each hemisegment expressing beta-galactosidase driven by the 11-kb enhancer that are slightly ventral to those expressed by the 4.4-kb enhancer. The position of these cells corresponds to those of the muscle progenitor/founder cells at this stage. Furthermore, since the 11-kb enhancer expresses in the body wall muscles of later embryos and does not express in the fat body, it is concluded that Pdp1 is expressed in muscle precursor cells that will eventually fuse to form the body wall muscles in later embryos (Reddy, 2000).

Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors

Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions, muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search were performed for direct Ladybird targets. The data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, bric-a-brac 2 gene was identified as a new component of identity code and inflated encoding αPS2-integrin playing a pivotal role in cell-cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells (Junion, 2007).

Uncovering how the cell fate-specifying genes exert their functions and determine unique properties of cells in a tissue is central to understanding the basic rules governing normal and pathological development. To approach the cell fate determination process at a whole genome level a search was performed for transcriptional targets of the homeobox transcription factor Lb known to be evolutionarily conserved and required for specification of a subset of cardiac and muscular precursors. To this end the targeted expression profiling and the novel ChIP-on-chip method ChEST were combined. The data revealed an unexpectedly complex gene network operating downstream from lb, which appears to act not only by regulating components of the cell identity code but also as a modulator of pan-muscular gene expression at fiber-type level. Of note, the role of Drosophila lb in regulating segment border muscle (SBM) founder motility appears reminiscent of the role of its vertebrate ortholog Lbx1, known to control the migration of leg myoblasts (Vasyutina, 2005) in mouse embryos (Junion, 2007).

Earlier genetic studies revealed that within the same competence domain the cell fate specifying factors acted as repressors to down-regulate genes determining the identity of neighboring cells. Consistent with this finding, lb was found to repress msh and kruppel (kr) during diversification of lateral muscle precursors and even skipped (eve) within the heart primordium. This study found that additional identity code components are regulated negatively by lb. In the lateral muscle domain lb acts as a repressor of the MyoD ortholog nau and the NK homeobox gene slou, both known to be required for the specification of a subset of somatic muscles. This suggests that a particularly complex network of transcription factors (Ap, Msh, Kr, Nau, Slou) controls the specification of individual muscle fates in the lateral domain. Interestingly, none of these factors is coexpressed with lb in the SBM, which appears to be a functionally distinct muscle requiring a specific developmental program. Besides factors with well-documented roles in diversification of muscle fibers, the global approach identified a few novel potential players in the muscle identity network. Among those that are are expressed in somatic muscle precursors are the Pdp1 gene encoding Par domain factor and the CG32611 gene containing a zinc finger motif (Junion, 2007).

Interestingly, in the cardiac domain the data demonstrate that lb is able to positively regulate the expression of tin and the effector of RTK pathway pointed (pnt), both involved in cardiac cell fate specification. These findings are consistent with earlier observations that the forced lb expression leads to the ectopic tin-positive cells within the dorsal vessel. Also, during early cardiogenesis lb directly represses bric a brac 2 (bab2), which emerges as a novel component of the genetic cascade controlling the diversification of cardiac cells. Thus, the ability of Lb to act either as repressor or as activator suggests a context-dependent interaction with cofactors. Of note, several miroarray identified Lb targets have also been found in the RNAi-based screen for genes involved in heart morphogenesis (Junion, 2007).

The data indicate that lb exerts its muscle identity functions via regulation of pan-muscular genes that control cell movements, cell shapes and cell-cell interactions including myoblast fusion, myotube growth, and attachment events (Junion, 2007).

Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component

Many organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila, the master clock gene Clock promotes the transcription of several key target genes. Two of these gene products, Per and Tim, repress Clk-Cyc-mediated transcription. To recognize additional direct Clk target genes, a genome-wide approach was designed and clockwork orange (cwo) was identified as a new core clock component. cwo encodes a transcriptional repressor functioning downstream of Clk that synergizes with Per and inhibits Clk-mediated activation. Consistent with this function, the mRNA profiles of Clk direct target genes in cwo mutant flies manifest high trough values and low amplitude oscillations. Impaired activity of Cwo leads to an elevated trough of per, tim, vri, and Pdp1 mRNA at ZT3 (three hours into the morning) in cwo RNAi transgenic flies compared with those of wild-type flies. Because behavioral rhythmicity fails to persist in constant darkness (DD) with little or no effect on average mRNA levels in flies lacking cwo, transcriptional oscillation amplitude appears to be linked to rhythmicity. Moreover, the mutant flies are long period, consistent with the late repression indicated by the RNA profiles. These findings suggest that Cwo acts preferentially in the late night to help terminate Clk-Cyc-mediated transcription of direct target genes including cwo itself. The presence of mammalian homologs with circadian expression features (Dec1 and Dec2) suggests that a similar feedback mechanism exists in mammalian clocks (Kadener, 2007). To other studies similarly identified Clockwork orange an a transcriptional repressor that inhibits Clk-mediated activation (Matsumoto, 2007; Lim, 2007).

Targets of Activity

Pdp1 is involved in regulating expression of the Tropomyosin I gene in somatic body-wall and pharyngeal muscles by binding to DNA sequences within the muscle activator that are required for activator function. Mutations that eliminate Pdp1 binding eliminate muscle activator function and severely reduce expression of a muscle activator plus Mef2 mini-enhancer. These and previous results suggest that Pdp1 may function as part of a larger protein/DNA complex, which interacts with Mef2 to regulate transcription of Drosophila muscle genes (S. C. Lin, 1997).

DEVELOPMENTAL BIOLOGY

Embryonic

Drosophila PDP1 mRNA can be detected as early as 3-6 hours. At 9-12 hours and continuing through hatching, seven different-sized RNAs are detected with the Pdp1 probe. By whole-mount in situ hybridization, PDP1 transcripts are first detectable at stage 9 in the developing posterior endoderm midgut primodium and, by stage 11, in both anterior and posterior endoderm midgut primordia and the cephalic mesoderm. The expression pattern is quite uniform and at a relatively high level in both midgut primordia. By stage 14, Pdp1 continues to be expressed, albeit unevenly, in the forming midgut endoderm and the cephalic mesoderm as it forms the pharyngeal muscle. Pdp1 is also expressed in the epithelial cells of the hindgut at this stage and in the Malpighian tubules in later stage embryos. Pdp1 expression can also be seen in the developing somatic body-wall muscles spanning the anterior-posterior and dorsal-ventral axes and in the fat body of stage 14 embryos. In cross sections of stage 13 embryos, PDP1 RNA can be detected in unfused somatic myoblasts that give rise to the body wall muscles. RNA expression is not detected in the muscle or fat body in twist mutant embryos since these embryos do not make mesoderm. PDP1 mRNA has not been detected in the cardial or pericardial cells of the dorsal vessel. PDP1 RNA is also expressed in the epidermis of stage 14 and stage 16 embryos, the latter also showing expression in mature muscle fibers and fat body. In late-stage embryos, a low level of Pdp1 expression is also seen in a small subset of cells in the central nervous system (S. C. Lin, 1997).

The Pdp1 gene is complex, containing at least four transcriptional start sites and producing at least six different mRNAs and Pdp1 isoforms. Five of the Pdp1 isoforms differ by the substitution or insertion of amino acids at or near the N-terminal of the protein. At least three of these alternately spliced transcripts are differentially expressed in different tissues of the developing embryo in which Pdp1 expression is correlated with the differentiation of different cell types. A sixth isoform is produced by splicing out part of the PAR and basic DNA binding domains, and DNA binding and transient transfection experiments suggest that it functions as a dominant negative inhibitor of transcription. Furthermore, two enhancers have been identified within the gene that express in the somatic mesodermal precursors to body wall muscles and fat body; together, they direct expression in other tissues that closely mimics the expression of the endogenous gene. These results show that Pdp1 is widely expressed, including in muscle, fat, and gut precursors, and is likely involved in the transcriptional control of different developmental pathways through the use of differentially expressed Pdp1 isoforms. Furthermore, the similarities between Pdp1 and the other PAR domain genes suggest that Pdp1 is the homolog of the vertebrate genes (Reddy, 2000).

Pdp1 RNA is expressed in the somatic mesoderm that gives rise to the body wall and pharyngeal muscles and fat body; the developing foregut, midgut, hindgut, and Malpighian tubules, and the epidermis. The synthesis and accumulation of mRNA do not necessarily ensure the synthesis of protein, however. Accordingly, it was of interest to determine whether Pdp1 protein correlates with the pattern of Pdp1 mRNA; an anti Pdp1-alpha antibody has been used in whole-mount embryo analysis to determine its expression pattern. Pdp1 protein is detected in stage 10 embryos in the cephalic mesoderm, the endoderm of the developing anterior and posterior midgut, and the segmented mesoderm of the extended germ band that will give rise to the fat body and somatic body wall muscles. The cephalic mesoderm staining gives rise to the pharyngeal muscles seen in the stage 14 embryos. The somatic mesoderm develops into the fat body and body wall muscles, and Pdp1 protein can be seen in their nuclei. Pdp1 expression in the developing gut includes the esophagus and the hindgut. Pdp1 is also expressed in Malpighian tubules. This pattern of Pdp1 protein expression coincides precisely with the expression of PDP1 mRNA. The expression of Pdp1 protein in the midgut is better resolved than in the RNA studies. Initially, Pdp1 is expressed uniformly throughout the midgut from stage 11 and up to early stage 13. The pattern of Pdp1 expression, however, very quickly becomes restricted in stage 14 embryos to the anterior portion of the midgut. It is this part of the midgut, which gives rise to developing gastric caeca, the large nuclei of the basophilic cells of the second (middle) midgut lobe, and the cells of the fourth (posterior) midgut lobe. The basophilic cells of the middle midgut differentiate to form the interstitial and copper cells of the late embryo and larva (Reddy, 2000).

The timing and pattern of Pdp1 protein in the different regions of the gut suggest that Pdp1 may play a fundamental role in their differentiation. Low amounts of Pdp1 protein are seen in the brain and nuclei of cells of the central nervous system (Reddy, 2000).

The Pdp1 gene encodes multiple proteins and raises the possibility that they may be differentially expressed and have unique functions. Since isoform-specific antibodies are not available, the alternately spliced exons were subloned and each was used in whole-mount in situ hybridization. The largest of the alternately spliced exon probes available (exon 3) is the 395-nt Pdp1-epsilon antisense probe. Using this exon probe, hybridization was detected in the central nervous system and the brain of stage 13-14 embryos. These results indicate that Pdp1-epsilon mRNA is expressed at a significantly lower level than total Pdp1 RNA. The alpha/beta/delta common second exon probe is 200 bp and relatively strong hybridization could be observed after 90 min of development. Indeed, the pattern of expression depicted by this exon 2 probe closely resembles the expression pattern observed for the entire Pdp1-alpha cDNA clone, which represents all Pdp1 mRNAs. Hybridization was observed in the developing anterior and posterior midgut rudiments of stage 11 embryos and the elongated midguts of stage 12 and 13 embryos, and later expression was seen in the gastric caeca and the anterior and posterior midgut. Hybridization could also be detected in the hindgut and Malpighian tubules and in the pharyngeal and body wall muscles. Hybridization was detected in the developing midgut of stage 11 and 12 embryos. By stage 13, Pdp1-gamma mRNA could be seen in the developing gastric caeca, middle midgut, Malpighian tubules, and fat body. In addition, barely detectable hybridization was occasionally observed in the pharyngeal muscles but not the body wall muscles. However, because the body wall muscles are adjacent and peripheral to the fat body and because the hybridization signal is low, it is not possible to rule out low-level expression in the body wall muscles. Expression of the Pdp1-phi (exon 4) mRNA in embryos could not be detected by PCR and thus was not hybridized to whole-mount embryos. Since the Pdp1-phi cDNA was isolated from an adult library, its expression may be limited to adult tissues (Reddy, 2000).

Effects of Mutation or Deletion

Pdp1 is a regulator of larval growth, mitosis and endoreplication

PDP1 is a basic leucine zipper (bZip) transcription factor that is expressed at high levels in the muscle, epidermis, gut and fat body of the developing Drosophila embryo. Three mutant alleles of Pdp1 have been identified, each having a similar phenotype. This study describes in detail the Pdp1 mutant allele, Pdp1^p205, which is null for both Pdp1 RNA and protein. Interestingly, homozygous Pdp1^p205 embryos develop normally, hatch and become viable larvae. Analyses of Pdp1 null mutant embryos reveal that the overall muscle pattern is normal as is the patterning of the gut and fat body. Pdp1^p205 larvae also appear to have normal muscle and gut function and respond to ecdysone. These larvae, however, are severely growth delayed and arrested. Furthermore, although Pdp1 null larvae live a normal life span, they do not form pupae and thus do not give rise to eclosed flies. The stunted growth of Pdp1^p205 larvae is accompanied by defects in mitosis and endoreplication similar to that associated with nutritional deprivation. The cellular defects resulting from the Pdp1^p205 mutation are not cell autonomous. Moreover, PDP1 expression is sensitive to nutritional conditions, suggesting a link between nutrition, PDP1 isotype expression and growth. These results indicate that Pdp1 has a critical role in coordinating growth and DNA replication (Reddy, 2006).

Pdp1 is dispensable for embryogenesis despite the fact that Pdp1 is expressed in several tissues during embryogenesis. Tissues such as the gut, fat body, and muscle, which express PDP1 at high levels as they differentiate, have normal morphology in Pdp1^p205 embryos, and all appeared to function normally in Pdp1^p205 larvae. Although maternally supplied stores of mRNA and proteins can obfuscate loss-of-function embryonic phenotypes by compensating for loss of zygotic function, this does not seem to be the case here since maternally supplied Pdp1 messenger RNA or protein is not detected in early embryos. Therefore, either Pdp1 is not necessary for embryonic development, the effects of Pdp1 loss-of-function are subtle and not detected in the assays used, or Pdp1 function is being compensated for by another gene. This latter possibility is consistent with previously reported deletion analysis of PDP1 binding sites in the context of the larger muscle enhancer of the TmI gene (Reddy, 2006).

The Pdp1^p205 mutant embryos develop and hatch normally and become larvae that are able to crawl and eat, indicating normal muscle function and at least rudimentary gut function. However, these Pdp1^p205 larvae are severely growth delayed compared to their heterozygous counterparts. This Pdp1^p205 larval phenotype is very similar to the phenotype of amino acid starved larvae and larvae derived from dTOR and slimfast mutants, all of which affect larval growth through altered endoreplication. The growth of the larva is dependent upon its nutritional state. Wild-type larvae starved of nutrients (amino acids) have retarded growth that mimics the Pdp1 phenotype, although Pdp1^p205 larvae do crawl and eat. The coordination of growth with nutritional conditions is most readily observed in the endoreplicating cells of the larva. In larvae, the initial uptake of amino acids from an exogenous food source stimulates both endoreplicative and mitotic cell cycles. In addition, a constant influx of dietary amino acids is necessary to maintain endoreplication, as evidenced by complete loss of endoreplication 24 h after food withdrawal. Mitotic cells, in contrast, require only an initial pulse of amino acids to stimulate mitotic cycling since these cells continue cycling after food withdrawal. Thus, a fundamental difference between Pdp1^p205 larvae and amino acid starved larvae is that in Pdp1^p205 larvae, endoreplication does occur after hatching as assayed via BrdU incorporation. In wild-type and Pdp1^p205 larvae that are at 48 h AEL (late first instar for wild type), endoreplication is occurring in three locations within the gut during the 6-h BrdU incorporation time (Reddy, 2006).

Since endoreplication does occur, these larvae are not starving per se; they take up amino acids. Furthermore, they appeared to have normal gut functions, and adding amino acids to the diet of Pdp1^p205 larvae did not improve growth or viability. However, although endoreplication is occurring in first instar Pdp1^p205 larvae, it is progressing at a much slower rate since BrdU incorporation could not be readily detected after a 3-h period of incubation even though these were optimal conditions for wild-type larvae. Instead, a two- to three-fold longer incubation is required in Pdp1^p205 larvae to reach a comparable level of BrdU incorporation as in wild-type larvae. Thus, it is concluded that although larval cells are endoreplicating in young Pdp1^p205 larvae (48 h AEL), they are already doing so at a much slower rate, leading to a severely reduced number of endocycles. This is unlike older larvae where little or no endoreplication is observed. Since adding exogenous sucrose improved upon the phenotype, it is suspected that although Pdp1^p205 larvae may be getting sufficient nutrients in their diet, they may suffer from a metabolic defect that results in altered carbohydrate metabolism. Such larvae may not breakdown glucose or transport glucose efficiently and thus may grow slowly and experience developmental delays which may be partly overcome by the increasing the sugar (sucrose). It is known, for instance, that insulin signaling from the brain promotes mitosis and endoreplication and is particularly sensitive to carbohydrate levels. Therefore, it is possible that extra stimulation of the insulin-signaling pathway is able to partially bypass the Pdp1 null phenotype (Reddy, 2006).

In wild-type animals, DNA synthesis (as visualized by BrdU incorporation) is also occurring in the mitotic imaginal cells of the brain, gut, and discs that will give rise to adult organs. In young Pdp1^p205 mutant larvae (48 h AEL), BrdU incorporation into these cells is dramatically reduced and/or apparently absent in some cases. This lack of cell cycle progression carried over into older larvae (72 h AEL), a timeframe during which endoreplication had essentially ceased in the mutants even though midgut, salivary gland, and fat body cells are actively undergoing endoreplication in wild-type larvae. Also striking are the severely reduced levels of mitotic cell types including the imaginal cells of the midgut, brain neuroblasts, and imaginal disc cells. Thus, in comparison with endoreplicating cells, mitotically active cells in the larva seem to be especially sensitive to the loss of PDP1 function (Reddy, 2006).

Pdp1^p205 mutants also have abnormal circadian rhythm. It was shown previously that PDP1-ε is a positive regulator of Clock and is part of a second feedback loop in the circadian clock mechanism of cyclic output regulation. Thus, Pdp1^p205 mutants do not express TIM, PER, or PDF in the pacemaker cells of the brain. In mice, it has been shown that the circadian clock controls expression of cell cycle genes that regulate mitosis. Moreover, in vertebrates and Drosophila, hormonal levels regulated by circadian output control nutritional and behavioral responses. In Drosophila, the clock-regulated output gene takeout (to) has been shown to be directly linked to feeding and starvation and has been proposed to contribute to the metabolic and behavioral changes in response to the absence of food. This raises the intriguing possibility that Pdp1 might integrate circadian rhythm with the nutritional state of the organism. For instance, PDP1 might regulate clock output genes that determine cyclic feeding behavior or metabolism. PDP1 might also help regulate cyclic mitogenic and/or nutrient sensing pathways that coordinate growth with nutritional conditions. If true, then PDP1 itself might be regulated by nutritional conditions (Reddy, 2006).

Given that Pdp1^p205 larvae are sensitive to the nutritional environment, specifically sugar levels in the growth medium, the isotype profile was analyzed under starvation conditions in wild-type larvae. Indeed, upon starvation, the PDP1-φ isotype is upregulated. This is similar to the to gene which is also upregulated during starvation. Given the growth phenotype of Pdp1^p205 larvae and the PDP1 isotype switching that occurs under starvation conditions, it is conceivable that this sensitivity to nutritional conditions is fundamental and necessary to the function of the PDP1 protein. This is especially interesting since the PDP1-φ isoform is not expressed in embryos and may indicate that this isoform is necessary in larval/adult life for the purpose of sensing nutritional environment. In the embryo, such a sensor is unnecessary because the embryo has all nutritional requirements supplied by its yolk and therefore has no nutritional interaction with the environment. The nutritional regulation of PDP1 is also provocative given that the homologous vertebrate PAR proteins are involved in the circadian regulation of genes that are involved in metabolism. This link between Pdp1, vertebrate PAR proteins, and circadian rhythm is further reinforced by the observation that, in addition to the upregulation of the PDP1-φ isoform in starved larvae, typical observations show an upregulation of the PDP1-ε isoform as well. Since PDP1-ε is expressed in the clock cells where it regulates clk and other clock output genes, it is possible that nutritional entrainment of the molecular clock in Drosophila could occur through this isotype (Reddy, 2006).

Taken together, the data suggest that PDP1 in the larva may be acting as a transcriptional regulator of growth and proliferation through sensing the nutritional environment. Indeed, it has been shown recently that VRI, which together with PDP1 regulates the Clock gene, is required for normal cell growth and proliferation. VRI, however, appears to act cell autonomously, whereas PDP1 does not. The pleiotropic phenotype exhibited by Pdp1^p205 larvae suggests that the growth defects are not cell autonomous since there is no detectable PDP1 expression in some of the affected tissues. Mosaic analysis confirms that the effects of the Pdp1^p205 mutation on cell growth and proliferation are non-autonomous. A possible model to explain how PDP1 could function to integrate the nutritional state with the regulation of growth and proliferation suggests that Pdp1 exerts its effects through a diffusible factor(s). There are two organs in which PDP1 is expressed that are known to emit diffusible factors that affect mitosis, endoreplication, or both. The fat body is known to secrete mitogenic factors required for proliferation of cells in the imaginal disc. It has long been known that growing imaginal discs in culture require medium conditioned with fat body or fat body extracts. The Imaginal Disc Growth Factors (IDGF) is a group of recently discovered peptides that are required for proliferation in the imaginal tissues and are expressed in the fat body (see Igdf1 and Igdf3). There is no evidence that these factors impact endoreplication directly in the larva, but they do cooperate with insulin-like growth factors to promote endoreplication. The fat body can, however, regulate growth and endoreplication. Reducing amino acid uptake by the fat body or inhibiting dTOR signaling in the fat body inhibits growth and endoreplication in peripheral tissues perhaps through the regulation of transport protein genes in the fat body such as slimfast, which when inhibited leads to larval growth defects and reduced endoreplication. Perhaps PDP1 is involved in regulating fat body functions associated with either IDGF, other mitogen expression, transport, or fat body response to amino acid levels (Reddy, 2006).

PDP1 might exert its effect on insulin growth factor expression or its pathway(s) through signaling from the fat body (see The Insulin receptor signaling pathway). Insulin-like growth factors are expressed in neurosecretory cells in the brain, and the insulin-signaling pathway is implicated in regulating both proliferation and endoreplication. Ectopically expressing either the insulin receptor (InR) or Drosophila S6 kinase (dS6K), two proteins in this pathway, is able to relieve endoreplication defects seen in starved larvae. Mutations in several factors in this pathway, such as dS6K, InR, InsP3 and dTOR, have larval growth defects. Insulin signaling is also implicated in the proliferation of the imaginal disc cells. The most downstream factors in this pathway regulate the S-phase promoting factor E2F and ectopically expressing E2F is also able to rescue endoreplication defects seen in starved larvae or larval growth defective mutants (Reddy, 2006 and references therein).

Since the effects of the Pdp1 mutation are pleiotropic, even though PDP1 is not expressed in all affected tissues, a working model places PDP1 downstream of nutritional signals and upstream of mitogenic signals. In this model, a nutritional signal could be received by either the fat body or the brain and, either directly or indirectly, affect PDP1 regulation of the production of further signaling molecules. PDP1 could either be required for proper organ function or could regulate factors involved in nutritional homeostasis and signaling. In the case of the fat body, PDP1 could modulate the production of IDGFs or other circulating mitogenic factors, while in the brain, PDP1 could conceivably modulate IGF production. Furthermore, incorporating data from PDP1 regulation of the circadian clock, it is possible that PDP1 could serve as a link between nutritional state, growth, and circadian rhythm (Reddy, 2006).

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date revised: 30 May 2006
  

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