Lobe: Biological Overview | References
Gene name - Lobe
Cytological map position - 51A4-51A4
Function - signaling
Symbol - L
FlyBase ID: FBgn0267825
Genetic map position - 2R:10,368,708..10,384,817 [+]
Classification - PROLINE-RICH AKT SUBSTRATE, 40-KD; PRAS40 ortholog
Cellular location - cytoplasmic
|Recent literature||Maier, D., Nagel, A. C. and Preiss, A. (2019). Genetic interactions between Protein Kinase D and Lobe mutants during eye development of Drosophila melanogaster. Hereditas 156: 37. PubMed ID: 31889943
In Drosophila, the development of the fly eye involves the activity of several, interconnected pathways that first define the presumptive eye field within the eye anlagen, followed by establishment of the dorso-ventral boundary, and the regulation of growth and apoptosis. In Lobe (L) mutant flies, parts of the eye or even the complete eye are absent because the eye field has not been properly defined. This study has identified Protein Kinase D (PKD) as a strong modifier of the L mutant phenotype. PKD belongs to the PKC/CAMK class of Ser/Thr kinases that have been involved in diverse cellular processes including stress resistance and growth. Despite the many roles of PKD, Drosophila PKD null mutants are without apparent phenotype apart from sensitivity to oxidative stress. This study reports an involvement of PKD in eye development in the sensitized genetic background of Lobe. Absence of PKD strongly enhanced the dominant eye defects of heterozygous L (2) flies, and decreased their viability. Moreover, eye-specific overexpression of an activated isoform of PKD considerably ameliorated the dominant L (2) phenotype. This genetic interaction was not allele specific but similarly seen with three additional, weaker L alleles, demonstrating its specificity. It is proposed that PKD-mediated phosphorylation is involved in underlying processes causing the L phenotype, i.e. in the regulation of growth, the epidermal transformation of eye tissue and apoptosis, respectively.
The TOR and Jak/STAT signal pathways are highly conserved from Drosophila to mammals, but it is unclear whether they interact during development. The proline-rich Akt substrate of 40 kDa (PRAS40) mediates the TOR signal pathway through regulation of TORC1 activity, but its functions in TOR complex 1 (TORC1, a rapamycin-sensitive form of Tor in mice that consists of mTOR, raptor, and mLST8) proved in cultured cells are controversial. The Drosophila gene Lobe (L) encodes the PRAS40 ortholog required for eye cell survival. L mutants exhibit apoptosis and eye-reduction phenotypes. It is unknown whether L regulates eye development via regulation of TORC1 activity. This study found that reducing the L level, by hypomorphic L mutation or heterozygosity of the null L mutation, resulted in ectopic expression of unpaired (upd), which is known to act through the Jak/STAT signal pathway to promote proliferation during eye development. Unexpectedly, when L was reduced, decreasing Jak/STAT restored the eye size, whereas increasing Jak/STAT prevented eye formation. Ectopic Jak/STAT signaling and apoptosis are mutually dependent in L mutants, indicating that L reduction makes Jak/STAT signaling harmful to eye development. In addition, genetic data suggest that TORC1 signaling is downregulated upon L reduction, supporting the idea that L regulates eye development through regulation of TORC1 activity. Similar to L reduction, decreasing TORC1 signaling by dTOR overexpression results in ectopic upd expression and apoptosis. A novel finding from these data is that dysregulated TORC1 signaling regulates the expression of upd and the function of the Jak/STAT signal pathway in Drosophila eye development (Wang, 2009).
The target of rapamycin (TOR) and Jak/STAT signal pathways are highly conserved in animals and important in many developmental processes. Dysregulation of these pathways can lead to cancer formation. This study presents data showing that TOR regulates the function of Jak/STAT signaling during Drosophila eye development (Wang, 2009).
The gene unpaired (upd) encodes a ligand that activates Drosophila Jak/STAT signaling. It is expressed in the posterior margin of the dorsal/ventral (D/V) boundary, the posterior center (PC), in the larval eye imaginal disc at second and early third instar stages. Notch at the D/V boundary activates the transcription of eye gone (eyg), which activates upd expression at the PC. Expression of upd is also regulated by Hedgehog (Hh) signaling. The cells of Drosophila compound eyes are derived from the eye-antennal disc, which develops from ectoderm of the embryo and grows inside the larva. These cells proliferate rapidly during the first and second instar stage. In early third instar larvae, morphogenetic furrow (MF) that arise at the posterior margin progresses in a wave-like manner toward the anterior margin of the eye disc. Jak/STAT signaling is known to promote proliferation during eye development, and is required for MF initiation; a loss of Jak/STAT function results in reduced eyes. Therefore, Jak/STAT signaling is regulated by Notch/Eyg and the Hh signaling pathways, and plays positive roles in eye development (Wang, 2009).
TOR signaling is one of the downstream branches of insulin signal pathway. Insulin and insulin-like growth factor elicit a signal cascade involving phosphatidyl-inositol 3-kinase (PI3K) that stimulates PDK-mediated Akt phosphorylation. Phosphorylated Akt can activate TOR, which nucleates the TOR complex 1 (TORC1), allowing it to phosphorylate the downstream targets, the translational repressor eukaryotic initiation factor (4EBP) and the ribosomal protein S6 kinase (S6k). Phosphorylation of 4EBP and S6K promotes CAP-dependent translation and thereby increases protein synthesis. In addition, activation of TOR can also promote ribosome biogenesis via Myc. Loss of the Drosophila TOR (dTOR) function reduces eye size, indicating that TOR signaling is required for eye development (Wang, 2009).
PRAS40 mediates the insulin signal pathway from Akt to TORC1. Upon insulin stimulation, activated Akt phosphorylates PRAS40 and causes it to dissociate from TORC1, allowing TORC1 signaling to proceed. Thus, PRAS40 can apparently act as an inhibitor of TORC1 (Sancak, 2007; Vander Haar, 2007). However, it has been reported that PRAS40 is required for TORC1 activity (Fonseca, 2007), and thus the interactions of PRAS40 with TORC1, based on studies in cultured cells are controversial. The effect of PRAS40 on TORC1 signaling in vivo is still unclear (Wang, 2009).
The Drosophila Lobe (L) protein shares high sequence conservation with PRAS40 (Oshiro, 2007, Sancak, 2007; Vander Haar, 2007). L mutants have reduced adult eyes and exhibit ectopic apoptosis during eye development, indicating that L is required for eye development (Chern, 2002, Singh, 2005; Singh, 2006). But whether it regulates eye development via regulation of TORC1 activity is unknown (Wang, 2009).
This study identified a new L allele, Lfee. Quantitative RT-PCR and genetic analysis revealed that Lfee is a hypomorphic allele. The eye defect was mediated by ectopic Jak/STAT signaling and cell apoptosis. In L mutants, the ectopic Jak/STAT signaling had a negative effect on eye development, but not a positive one as previously reported. It was also found that TORC1 signaling was hypoactivated in L mutants, suggesting that, like PRAS40, L is required for TORC1 activity. This study suggests that hypoactivated TORC1 signaling in L mutants result in ectopic Jak/STAT signaling and apoptosis, impairing eye development (Wang, 2009).
The spontaneous mutant fly, freaky eye (fee), is homozygously viable and has abnormal adult eyes. The eyes of most fee flies are smaller than those of wild type flies because of a nick at the anterior border of the eye. At the nicked region, extra hairs and/or rod-like tissues are usually present. Overgrowth of eye tissue occasionally occurs, resulting in eye enlargement. The eyes of fee flies were categorized into six classes depending on their size relative to the eyes of the wild type. The various eye-reduction phenotypes of fee flies were similar to those of L mutants. For example, the Lsi heterozygote exhibits slightly reduced eyes that are nicked near the anterior D/V boundary (Chern, 2002), similar to the major fee phenotype. In the Lsi homozygote, the ventral eye is absent, which is also reminiscent of the fee phenotype (Wang, 2009).
Whether fee is a mutant of L was investigated; the trans-heterozygotes for fee and the null mutant Lrev6-3 (Chern, 2002) had smaller eyes than fee flies. In addition, fee could to be recombined with Lrev6-3, suggesting that fee is allelic to L. Quantitative RT-PCR showed that the L mRNA levels were highly reduced in fee flies, suggesting that fee is a L hypomorphic mutant; therefore these were designated Lfee (Wang, 2009).
This study shows that reduction of L phenocopies overexpression of dTOR. Overexpression of dTOR has been reported to produce phenotypes similar to that of loss of dTOR, because excess dTOR may titrate cofactors and thereby decrease TOR activity. This suggests that TOR signaling is downregulated by L reduction. Consistent with this, genetic analysis of L mutants and TOR signal pathway component suggest TORC1 hypoactivity in L mutants. PRAS40 has been proposed to function in the assembly of TORC1 (Fonseca, 2007). It is possible that, in similar way to dTOR overexpression, reducing L impairs TORC1 assembly, thus decreasing TORC1 signaling. Reduction of L may disrupt eye development through downregulation of TORC1 signaling, supporting the idea that PRAS40 is required for TORC1 activity (Wang, 2009).
Drosophila eye development requires the TOR and Jak/STAT signal pathways, but it is not know whether an interaction between these two signal pathways occurs. Endogenous upd expression is present in the posterior center (PC) of the eye disc, but not in the interior eye disc. This study demonstrated that L reduction can induce ectopic upd expression in the interior eye disc, indicating that L is a negative regulator for upd expression. The data show that L reduction-mediated eye disruption is due to hypoactivation of TORC1 signaling, suggesting that hypoactivity of TORC1 is responsible for inducing upd expression (Wang, 2009).
Ectopic upd expression is induced by reduction of L (Lfee and Lrev/+), but not by its complete loss (Lrev homozygous clones), suggesting that different L levels may cause distinct effects. As PRAS40 acts to transmit the Akt signal to TORC1, complete loss, but not reduction, of L could result in an uncoupling between Akt and TORC1 (Vander Haar, 2007). This would release the Akt-mediated inhibition of TORC1, resulting in increased TORC1 activity. Thus, complete loss of L or PRAS40 may increase TORC1 activity. It is possible that the opposite functions of PRAS40 reported in cultured cells could be due to different PRAS40 levels remaining after knockdown. Whether complete loss of L function inhibits or promotes TORC1 signaling in Drosophila eyes remains to be investigated (Wang, 2009).
Mosaic analysis data showed that dTOR homozygote clones did not induce ectopic upd expression, suggesting that complete loss of dTOR function has a different effect from that of L reduction. Overexpression of dMyc can completely restore the eye size in the Lfee flies, but only partially represses the eye defect of dTOR overexpression. These data support the idea that L reduction may not equate to loss of dTOR. It was reasoned that as TOR is involved in TORC1 and TORC2, its loss should eliminate the functions of both TORC1 and TORC2. Because L participates only in TORC1 signaling, reduction of L would affect TORC1 signaling only. The regulation of TORC1 and TORC2 signaling by L needs further investigation (Wang, 2009).
It was found that suppressing apoptosis can decrease ectopic upd expression upon L reduction, suggesting that apoptosis is a cause of ectopic upd expression. It has been reported that apoptosis can activate ectopic upd expression and Jak/STAT signaling via Notch signaling in apoptosis-induced compensatory proliferation. However, ectopic upd expression on L reduction is not likely to be mediated by Notch activity, and no ectopic proliferation occurs. Thus, apoptosis due to L reduction is different from apoptosis-induced compensatory proliferation. Further, TOR hypoactivation may trigger ectopic upd expression independent of apoptosis; suppression of apoptosis did not eliminate all ectopic upd expression. Further investigation of how hypoactivated TORC1 regulates upd expression is needed (Wang, 2009).
The Drosophila Upd acts through Jak/STAT signaling to promote proliferation during eye development. However, this study found that on L reduction, decreasing Jak/STAT signaling could restore the eye defect, whereas increasing the upd expression level could completely abolish eye development. Thus, an unexpected finding was that ectopic Jak/STAT signaling in L mutants is harmful to eye development (Wang, 2009).
The fact that decreasing Jak/STAT signaling can reduce apoptosis in L mutants indicates that the induction of ectopic Jak/STAT signaling is required for apoptosis. It was reasoned that the apoptosis-promoting ability of Jak/STAT is possibly due to its repression of Serrate (Ser) expression (Flaherty, 2009). Ser expression is inhibited by L mutation (Chern, 2002), and loss of Ser function during eye development causes apoptosis (Singh, 2006). The current data showed that heterozygosity for Ser can reduce eye size in Lfee heterozygotes, but not in the wild type, suggesting that decreased Ser expression may play a role in eye reduction. Whether Ser repression mediates the apoptosis remains to be investigated. In addition, because inhibition of apoptosis does not strongly restore the L eye defect, but decreasing Jak/STAT activity fully restores it (comparing ey > p35 and Stat92Ets), there is the possibility that the ectopic Jak/STAT activity affects eye development via an apoptosis-independent mechanism. Thus, a novel finding from the data is that Jak/STAT signaling can negatively regulate eye development (Wang, 2009).
An important issue is the control over the positive and negative roles of Jak/STAT signaling during eye development. Overexpression of upd driven by ey-GAL4 in the wild type produces adult with enlarged eyes, but it eliminates eye formation in L mutants. Because L reduction exhibits hypoactivation of TORC1 signaling, it is speculated that TORC1 signaling plays a role in controlling the balance between the opposing functions of Jak/STAT signaling (Wang, 2009).
In summary, reduction of the Drosophila PRAS40 L results in hypoactivation of TORC1 signaling. This leads to apoptosis and ectopic Jak/STAT activation, both of contribute to disruption of eye development. The data indicate that TORC1 signaling is able to regulate the expression and functions of the Jak/STAT signal pathway during eye development. Further studies using L mutants may uncover the mechanisms by which L regulates TORC1 signaling, and how TOR controls the Jak/STAT signal pathways. Also noteworthy is the report that decreasing PRAS40 can increase apoptosis of tumor cells (Madhunapantula, 2007), and it is therefore of interest to investigate whether PRAS40 and TORC1 can regulate the Jak/STAT signal pathway in tumors (Wang, 2009).
PRAS40 has recently been identified as a protein that couples insulin/IGF signaling (IIS) to TORC1 activation in cell culture; however, the physiological function of PRAS40 is not known. This study investigate flies lacking PRAS40 (Lobe). Surprisingly, it was found, both biochemically and genetically, that PRAS40 couples IIS to TORC1 activation in a tissue-specific manner, regulating TORC1 activity in ovaries but not in other tissues of the animal. PRAS40 thereby regulates fertility but not growth of the fly, allowing distinct physiological functions of TORC1 to be uncoupled. The main function of PRAS40 in vivo is to regulate TORC1 activity, and not to act as a downstream target and effector of TORC1. Finally, this work sheds some light on the question of whether TORC1 activity is coupled to IIS in vivo (Pallares-Cartes, 2012).
PRAS40 has been proposed to link IIS to TORC1 in cell culture. Two reports showed that PRAS40 binds the TORC1 complex thereby inhibiting its activity, and that phosphorylation of PRAS40 by Akt relieves this inhibition (Nascimento, 2010; Sancak, 2007; Vander Haar, 2007). Other studies, however, identified PRAS40 as a TORC1 substrate, suggesting that the apparent inhibitory effects of PRAS40 on the canonical TORC1 substrates 4EBP and S6K may reflect competition for substrate binding. This would place PRAS40 downstream, rather than upstream of TORC1. Indeed, as these studies point out, PRAS40 might function concomitantly as a TORC1 substrate and a TORC1 regulator, regulating mTORC1 activity via direct inhibition of substrate binding. These studies have led to several open questions: (1) does PRAS40 regulate TORC1 activity in vivo, as it does in cell culture? (2) does PRAS40 link IIS to TOR activation in vivo? and (3) is the main function of PRAS40 to act as a TOR substrate or as a TOR regulator? These two options can be distinguished in an animal context. If the main function of PRAS40 is to regulate TORC1 activity (i.e., it is genetically upstream of TORC1), then PRAS40 mutant phenotypes should be rescued by reducing activity of TORC1 or of a TORC1 target other than PRAS40. If, instead, PRAS40 functions mainly as a TOR substrate downstream of TORC1, then loss of PRAS40 cannot be rescued by manipulating TORC1. No animal models for PRAS40 loss of function have yet been reported to address these questions (Pallares-Cartes, 2012).
One physiological function of IIS and TORC1 of particular relevance to this present study is the regulation of fertility. In Drosophila, insulin-like peptides (DILPs) secreted by neurosecretory cells regulate the rate of germline stem cell division in the ovary. This links metabolic status to fertility, so that rich nutrient conditions cause high DILP secretion, leading to increased egg production. If IIS is abrogated in the ovary, as in the case of chico or InR mutants, egg production is completely blocked and the animals are sterile. The defect in chico mutant ovaries is ovary-autonomous because transplantation of chico mutant ovaries into wild-type hosts, containing normal levels of DILPS, does not rescue their defects. At the cellular level, IIS and TORC1 regulate almost all aspects of oogenesis including the rate of proliferation of ovarian somatic and germline cells, germline stem cell maintenance, vitellogenesis, and oocyte loss. Interestingly, the roles of IIS and TORC1 in regulating fertility are highly conserved throughout evolution, regulating similar processes in Caenorhabditis elegans and in mammals. As in flies, reduction of IIS via knockout of IGF-1 or IRS-2 causes infertility in mice. As in flies, normal TORC1 in mice prevents oocyte loss (Thomson, 2010) and hyperactivation of IIS or TORC1 leads to premature activation of all primordial follicles, resulting in premature follicular depletion (Reddy, 2010; Sun, 2010). In sum, IIS and TORC1 play critical roles in regulating fertility in an evolutionarily conserved manner (Pallares-Cartes, 2012).
This study presents a PRAS40 loss-of-function animal model. By generating PRAS40 knockout Drosophila, the in vivo function of PRAS40, as well as the connection between IIS and TORC1, were studied. PRAS40 is shown function to link IIS to TORC1 in the animal. Unexpectedly, however, it does so in a tissue-specific manner, influencing TORC1 activity predominantly in the fly ovary, but not in other tissues of the animal. As a result, PRAS40 regulates development of the ovary, but not growth or proliferation of somatic tissues, thereby influencing animal fertility but not animal growth. Because PRAS40 is present in all tissues of the fly, this indicates PRAS40 is a link between IIS and TORC1 that can be switched on and off in a tissue-specific manner. Furthermore, PRAS40 knockout phenotypes can be rescued by inhibiting TORC1 or by reducing S6K gene dosage, indicating that PRAS40 functions mainly as a TORC1 inhibitor in vivo. Finally, this work sheds light on the conundrum whether the IIS and TORC1 signaling pathways are linked under normal physiological conditions, showing that they are indeed linked, but only in particular tissues (Pallares-Cartes, 2012).
Both biochemically and genetically this study found that PRAS40 and IIS do not affect TORC1 activity in most tissues during growth of the fly. Removal of PRAS40 does not cause elevated TORC1 activity in larvae and, in agreement with previous studies, removal of chico does not lead to reduced TORC1 activity in the adult body or in larvae. Removal of PRAS40 does not cause any size abnormalities in the fly, which is a very sensitive readout for TORC1 activity during development. It was surprising to find, however, that in ovaries both IIS and PRAS40 do affect TORC1 activity. TORC1 activity drops in ovaries of chico− animals, and increases in ovaries of PRAS40− animals. Furthermore, in ovaries, PRAS40 links IIS to TORC1 in that removal of both chico and PRAS40 leads to renormalized TORC1 activity. These biochemical data are reflected by genetic epistasis data. Chico mutant flies are completely infertile, laying no eggs, and this phenotype is rescued by removal of PRAS40. These data indicate that under normal physiological conditions, IIS activates TORC1 in a tissue-specific manner (Pallares-Cartes, 2012).
Does PRAS40 also link IIS to TORC1 in the male germline? The fact that mutation of PRAS40 rescues the infertility of PDK14/5 mutant males, and that PRAS40 mutant testes are larger than control testes suggests that it does. PRAS40−, chico− mutant testes also appear mildly increased in size compared to chico− mutant testes, however, the result is not as clear cut as with ovaries, because chico mutant females are completely sterile whereas chico mutant males have only mildly reduced fertility. Further work will be required to look at this carefully (Pallares-Cartes, 2012).
All these data, indicating an ovary-specific link between IIS and TORC1 result from manipulations within physiological range. In contrast, overexpression of PRAS40 does cause reduced tissue growth as well as reduced TORC1 activity, indicating that PRAS40 can inhibit TORC1 in most tissues when overexpressed. Furthermore, in contrast to the tissue-specific link between IIS and TORC1 under normal physiological conditions, it was also observed that hyper-stimulation of IIS above physiological range does activate TORC1 in most tissues, for instance in tissue explants treated with insulin, or in animals overexpressing activated PI3K (Dp110-CAAX). This mechanism might be relevant for pathophysiological conditions with elevated IIS, such as in cancer cells. This may occur via elevated ATP production in the cell, inhibiting AMPK, because this activation was also observed in tissues simultaneously lacking PRAS40 and all Akt phosphorylation sites on Tsc1 and Tsc2 (Pallares-Cartes, 2012).
One open question is whether the main function of PRAS40 is to regulate TORC1 activity or whether it functions mainly as a downstream target and effector of TORC1. The data suggest the former is the case. If PRAS40 had effector functions downstream of TORC1, these functions would not be rescued by additional removal of other TORC1 substrates such as S6K. Instead, it was found that the elevated fertility of PRAS40 mutants is rescued by removal of one copy of S6K, suggesting that the phenotype found in PRAS40 mutants is due to elevated S6K activity (Pallares-Cartes, 2012).
Why does PRAS40 regulate TORC1 activity in ovaries but not in other tissues of the animal? PRAS40 is expressed in all tissues that were tested. Therefore, the fact that removal of PRAS40 from larval tissues, for instance, has no effect on TORC1 activity must mean that larval PRAS40 protein is inactive. Data is presented suggesting that the state of phosphorylation of PRAS40 may be different in larval tissues compared to ovaries, providing a possible explanation for this inactivation. To date, a number of phosphorylations on PRAS40 have been reported, all of which are inhibitory in terms of TORC1 binding. These include phosphorylations by Akt, TORC1 itself, PIM1, and PKA. Intriguingly, this correlates with the observation that PRAS40 is highly phosphorylated in many cancers and that PRAS40 phosphorylation correlates with bad prognosis. The possibility is favored that PRAS40 phosphorylation on an inhibitory site could be regulated by a kinase that is absent in ovaries, or a phosphatase that is enriched in ovaries compared to other tissues. Future studies will shed light on this issue (Pallares-Cartes, 2012).
TORC1 has multiple physiological roles in various tissues. In Drosophila, TORC1 in the growing larva regulates both growth and metabolism of the animal whereas in the adult fly, it regulates mainly metabolic parameters. TORC1 in ovaries regulates fertility of the animal, whereas in the nervous system it regulates dendritic tiling. Therefore, unless TORC1 activity can be differentially regulated in various tissues, all these physiological functions would have to be controlled in a correlated fashion. Tissue-specific differential regulation of PRAS40 presents a mechanism that allows TORC1 activity to be uncoupled in a tissue-specific manner (Pallares-Cartes, 2012).
Organogenesis involves an initial surge of cell proliferation, leading to differentiation. This is followed by cell death in order to remove extra cells. During early development, there is little or no cell death. However, there is a lack of information concerning the genes required for survival during the early cell-proliferation phase. This study shows that Lobe (L) and the Notch (N) ligand Serrate (Ser), which are both involved in ventral eye growth, are required for cell survival in the early eye disc. The loss-of-ventral-eye phenotype in L or Ser mutants is due to the induction of cell death and the upregulation of secreted Wingless (Wg). This loss-of-ventral-eye phenotype can be rescued by (1) increasing the levels of cell death inhibitors, (2) reducing the levels of Hid-Reaper-Grim complex, or (3) reducing canonical Wg signaling components. Blocking Jun-N-terminal kinase (JNK) signaling, which can induce caspase-independent cell death, significantly rescued ventral eye loss in L or Ser mutants. However, blocking both caspase-dependent cell death and JNK signaling together showed stronger rescues of the L- or Ser-mutant eye at a 1.5-fold higher frequency. This suggests that L or Ser loss-of-function triggers both caspase-dependent and -independent cell death. These studies thus identify a mechanism responsible for cell survival in the early eye (Singh, 2006).
During development, cell survival, growth, proliferation and differentiation define the final shape and size of the organ. This study used the Drosophila eye to identify the genes required for cell survival and to sustain early growth. Previously, it was shown that ventral is the ground state of the entire early larval eye primordium (Singh, 2003), and that L/Ser are required for development of the ventral eye. During early eye development, little or no cell death is observed. At the mid-pupal stage, programmed cell death plays an important role in the selective removal of a large number of excess undifferentiated cells in the interommatidial space that fail to be recruited to the ommatidia. During this time window of pupal development, EGFR and Ras act as survival cues. Surprisingly, there is little information concerning genes responsible for cell survival during early larval eye imaginal disc development. These studies show that, during early eye development, L and Ser are required for the survival of ventral eye cells. It was found that one of the major reasons for the elimination of the ventral eye cells in L/Ser mutants is due to the induction of caspase-dependent cell death. L- and Ser-mutant phenotypes can be rescued by blocking inappropriate induction of (1) Wg, (2) JNK-signaling-mediated cell death and (3) caspase-dependent cell death (Singh, 2006).
In animal tissues, Wg is required to drive developmental patterning. Wg is produced in a restricted area and is distributed either by diffusion or by transport to generate a concentration gradient throughout the tissue to induce proper differentiation. In the developing wing imaginal disc, Wg has also been shown to promote growth. By contrast, it has been shown that abnormal expression of Wg or Dpp triggers aberrant differentiation signals that result in the induction of apoptotic cell death in the wing disc. However, it is difficult to directly extrapolate results from the wing disc to the eye disc because of organ-specific functions of Wg (Singh, 2006).
In the eye, Wg has complex functions at different stages of development: (1) prior to eye differentiation, Wg is involved in growth and in the establishment of the dorsal eye fate; (2) during eye differentiation, initiation of the morphogenetic furrow by hedgehog (hh) is restricted to the posterior margin by the presence of Wg, which represses hh and dpp at the lateral eye margins; and (3) in the pupal stage, Wg is responsible for inducing apoptosis by activating the expression of hid, rpr and grim in ommatidia at the periphery of the eye (Singh, 2006).
This study found that, during early eye development, L and Ser are required to repress Wg signaling in the ventral eye disc. Genetic-interaction studies demonstrate that Wg expression is ectopically induced in the L-mutant background. This paper proposes a model in which L and Ser downregulate the level of Wg activity and expression in the eye. Loss-of-function of L/Ser induce higher levels of Wg, is coincident with the elimination of the ventral eye pattern by ectopic induction of caspase-dependent cell death. Because blocking caspase-dependent cell death in L/Ser-mutant backgrounds results in striking but incomplete rescues of the loss-of-ventral-eye phenotypes, it suggests that L/Ser-mutant eye phenotypes are not solely due to the induction of caspase-dependent cell death. It is possible that ectopic upregulation of Wg signaling in the LOF of L/Ser causes abnormal induction of JNK signaling or that L/Ser LOF can induce JNK signaling independently. Upregulation of JNK signaling can also induce caspase-independent cell death. It is possible that the loss of L/Ser can result in cell death caused by both caspase-dependent and caspase-independent mechanism. This may be one of the underlying developmental mechanisms for the early cell-survival function of L and Ser. However, there can be several other interesting possibilities. Other studies have shown that JNK can be activated downstream of rpr, and that it affects the extent of rpr-induced cell death. Also, wg can be induced downstream of hid and diap1. Therefore, one can suggest an alternative model where a low level of apoptosis induced by hid, rpr and grim is augmented by a secondary activation of JNK and Wg, which ultimately results in eye ablation (Singh, 2006).
Many animal tissues counter cell death, induced in response to injury, by triggering compensatory cell proliferation in the neighboring cells. It has been reported in flies that apoptotic or dying cells actively signal to induce compensatory proliferation in neighboring cells to maintain tissue homeostasis. In cellular injury, Diap1 is inhibited, whereas the JNK pathway and Wg/Dpp are induced in the apoptotic cells. Secretion of these factors stimulates the growth or proliferation in competent neighboring cells and leads to compensatory proliferation. In other scenarios in which morphogenetic cell death occurs, altered levels of morphogenetic signals give rise to abnormal cell types, which are frequently removed by activating apoptotic signals. Morphogenetic cell death is one of the strategies employed by the developing field to correct its morphogen gradient by eliminating cells with abnormal levels of morphogen by inducing the JNK-signaling pathway (Singh, 2006).
Cell death caused by loss of L/Ser function results in the induction of both JNK- and Wg-signaling pathways. However, the outcome is different from that seen in compensatory proliferation or morphogen-gradient correction. Instead of compensatory growth in neighboring cells, LOF of L/Ser triggers ectopic signaling, which can be neither corrected nor compensated for. As a consequence, the affected tissue, in this case the ventral half of the eye discs, cannot be rescued. It results in the loss of the ventral or entire eye field, depending upon the domain of function of these survival factors. The results demonstrate that one of the essential roles of L and Ser is their requirement for the survival of early proliferating cells in the eye (Singh, 2006).
During development, N signaling is involved in many processes, including cell-fate commitment, cell-fate specification and cell adhesion. In the Drosophila eye, N signaling plays important roles in compound eye morphogenesis, such as DV patterning, cell proliferation and differentiation in the eye. However, N signaling has not been shown to promote cell survival during early eye development. These studies raise the possibility of the role of the Ser-N pathway in cell survival during early eye development. Earlier, the extremely reduced or complete loss of eye field in N mutant eye disc was interpreted as being caused by a loss of proliferation. The current data raises another possibility: that N signaling may be playing an important role in cell survival (Singh, 2006).
The compound eye of Drosophila shares similarities with the vertebrate eye. There is conservation at the level of genetic machinery, as well as in the processes of differentiation. Thus, the information generated in Drosophila can be extrapolated to higher organisms. Since Wnt signaling induces programmed cell death in patterning the vasculature of the vertebrate eye, it will be important to study what molecules prevent Wg signaling from inducing cell death during early eye development. Mutations in Jagged1, the human homolog of Ser, is known to cause autosomal-dominant developmental disorder, called Alagille's syndrome, which also affects eye development. Hence, it would be interesting to study what roles N pathway genes play in cell survival during early eye development and in the early onset of retinal diseases (Singh, 2006).
Dorsoventral (DV) patterning is essential for growth of the Drosophila eye. Recent studies suggest that ventral is the default state of the early eye, which depends on Lobe (L) function, and that the dorsal fate is established later by the expression of the dorsal selector gene pannier (pnr). However, the mechanisms of regulatory interactions between L and dorsal genes are not well understood. For studying the mechanisms of DV patterning in the early eye disc, a dominant modifier screen was performed to identify additional genes that interact with L. The criterion of the dominant interaction was either enhancement or suppression of the L ventral eye loss phenotype. Forty-eight modifiers were identified that corresponded to 16 genes, which included fringe (fng), a gene involved in ventral eye patterning, and members of both Hedgehog (Hh) and Decapentaplegic (Dpp) signaling pathways, which promote L function in the ventral eye. Interestingly, 29% of the modifiers (6 enhancers and 9 suppressors) identified either were known to interact genetically with pnr or were members of the Wingless (Wg) pathway, which acts downstream from pnr. The detailed analysis of genetic interactions revealed that pnr and L mutually antagonize each other during second instar of larval development to restrict their functional domains in the eye. This time window coincides with the emergence of pnr expression in the eye. These results suggest that L function is regulated by multiple signaling pathways and that the mutual antagonism between L and dorsal genes is crucial for balanced eye growth (Singh, 2005).
Axial patterning plays a crucial role in organizing growth and in differentiating developing fields. To understand how the DV pattern is established in the Drosophila eye, the genetic relationships between dorsal and ventral eye genes were analyzed. A group of new genes was identified that modifies the L mutant eye phenotype not only by misexpression but also by reduced gene function (Singh, 2005).
In the early eye disc, fng is preferentially expressed in the ventral eye. The DV domain specification by Fng is also important for growth of the eye disc as its ubiquitous overexpression in the eye disc blocks eye development. Even though L and fng play important roles during ventral eye growth and patterning, the developmental interaction between the two has been unknown. This study showed that overexpression of fng can partially compensate for the loss of L gene function in the eye. This suggests that fng works either downstream or parallel to L in the growth of the ventral eye. It is possible that L and fng interact through the induction of a common target, Ser, in the eye (Singh, 2005).
Like several other pathways, Wg signaling has multiple functions during eye development. This study identified Sgg, a serine/threonine kinase, as a modifier that suppresses the L mutant phenotype upon overexpression. Sgg is known to inhibit the Wg signaling pathway by downregulating Armadillo (Arm) via ubiquitin-mediated proteosomal degradation. Other components of the Wg signaling pathway such as pygo and dally were also identified as modifiers, which, upon overexpression in the eye, enhanced the L mutant phenotype. These results suggest that Wg signaling acts antagonistically to L function in the ventral eye. The genetic interaction of these EP lines with the L mutations represents specific enhancement rather than additive effects, since antagonists of Wg signaling were identified as suppressors, whereas members required for Wg signaling were identified as enhancers of the L mutant phenotype in the EP screen (Singh, 2005).
In this screen, it was found that the overexpression of Daughters against Dpp (Dad), an antagonist of Dpp signaling, enhances the L mutant phenotype, whereas EP insertions at hh and its receptor gene smo were identified as suppressors of the L mutant phenotype. The members of these two signaling pathways are known to be involved in eye growth and differentiation. These results raise another interesting possibility of the possible role of Hh and Dpp signaling pathways in early eye growth and patterning (Singh, 2005).
During Drosophila eye development, Hh controls progression of the furrow by inducing the expression of dpp and atonal (ato), a proneural gene responsible for R8 photoreceptor formation. In the eye, hh and dpp are involved in a positive feedback loop for the initiation and movement of the MF whereas Wg signaling acts antagonistically to Dpp signaling to block MF movement and progression. This antagonistic relation may be present even during early eye development since wg and dpp are localized to opposing regions of the undifferentiated younger eye primordia: dpp along the posterior margin and wg across the dorsal anterior region. These results suggest that the early function of Hh and Dpp signaling is to promote L-mediated ventral eye growth whereas Wg signaling acts as an antagonist (Singh, 2005).
BarH1 and BarH2 were identified as enhancers of the L mutant phenotype. BarH1 and BarH2 are a pair of homeobox proteins that express in a subset of photoreceptors and in the basal undifferentiated cells of the eye disc. B is required for the negative regulation of eye development by repressing the expression of the proneural gene ato. However, it is not known whether B plays a role in early eye growth, prior to retinal differentiation. Clonal analysis has not yet revealed evidence for B function in DV asymmetric eye patterning. However, the current data showed genetic interactions of L mutants with GOF and LOF mutants of B. The suppression of the L2/+ eye phenotype by a LOF mutation of B, which by itself has no defects in the eye, raises the possibility that B itself may not have DV asymmetric function but needs to be downregulated by L for normal growth of the early eye disc. This is also consistent with the dramatic eye reduction observed when ey-GAL4 drive B is overexpressed during early eye development. It has been shown that Wg and B have both positive and negative regulatory relationships in prepatterning of the notum. B expression is activated by Wg in the scutum whereas B represses Wg expression in the most anterior part of the notum. L mutants respond to GOF and LOF of both Wg signaling and B in a similar fashion, suggesting that Wg and B may be regulating each other positively during early eye development (Singh, 2005).
L is known to act downstream of N (Chern, 2002). In the eye, emc and h, the repressors of ato, are downregulated by N. During eye development, emc acts in collaboration with hairy (h) as the negative regulator of the morphogenetic furrow by repressing ato. Therefore, identification of emc as an antagonist of L-mediated early ventral eye growth seems possible. Interestingly, both emc and B have also been identified as modifiers of pnr (Singh, 2005).
Some of the genes that were identified as L modifiers, such as B, emc, and smo, have been well characterized, but their roles in early eye disc growth and/or DV asymmetric function have not been studied. Genes were identified involved in cell survival and growth such as disc over grown (dco), a member of the serine/threonine protein kinases family, and genes involved in vesicular trafficking, including RhoGAP68F, an ion transport such as nrv 1, and the acetyl transferase nej. It is possible that potential DV asymmetric function of these genes might have been missed by LOF analysis because of functional redundancy or these genes may be modifying the early growth function of L in the eye. More in-depth studies will be necessary to explore these possibilities. However, it is important to note that both GOF and LOF of these genes exhibit specific genetic interactions with L mutant backgrounds. In addition to the well-characterized genes, a few novel genes like EP1229 and EP1595 were identified whose functions are not known. These genes were not listed in this study as the specificity of their genetic interaction with L was not tested by using LOF mutations (Singh, 2005).
The results demonstrate that the level of pnr gene function is a crucial factor for DV patterning of the eye as increased levels of pnr gene function enhance the L mutant phenotype of ventral eye loss to no eye, whereas reduction of pnr gene function rescued the loss of the ventral eye phenotype of the L mutant. Further, the phenotypes of LOF clones of L where only the ventral cells are lost can be rescued by reducing the levels of pnr gene function. These results suggest that pnr acts antagonistically to the ventral eye growth function of L. However, it was also found that the antagonism of pnr and L is mutual. This conclusion is based on the fact that the gain-of-function phenotype of pnr in the eye is significantly enhanced when L function is reduced. These conclusions were also validated by showing that the dorsal eye enlargements associated with LOF clones of pnr can be prevented by reducing the levels of L gene function. These results suggest that optimal levels of pnr and L are necessary for DV patterning and growth of the eye. It was also found that the downstream dorsal eye selectors, Iro-C members (ara, caup, mirr) are involved in a mutually antagonistic relationship with L. These studies demonstrate that the antagonism of L holds true for key components involved in dorsal fate selection during early eye development (Singh, 2005).
The time window of the second instar of larval development was identified as the periord during which mutual antagonistic interaction of L and pnr is required for DV patterning and growth in the eye. Previously, it was shown that the pnr function in eye development is critically required during the second instar larval stage. This time window is coincident with the one that is required for the antagonism of pnr and L as shown in this study, suggesting that a major function of L in early eye development is to establish the DV domains by negatively regulating the dorsal selectors. These studies also support the physiological relevance of this mutually antagonistic interaction in DV patterning (Singh, 2005).
It is not known how L antagonizes Pnr function. One possibility is that L may be required for restricting the pnr expression domain to the dorsal margin of the eye disc. It was difficult to check whether L is cell-autonomously required for pnr repression because LOF clones of L result in the elimination of the entire or ventral eye, depending on the time when the clones are generated. Alternatively, the effect of a L mutation on pnr expression was studied. Interestingly, pnr expression, which is restricted to the dorsal eye margin in wild type eye discs, shows a nearly twofold expansion in L2/+ mutant discs. It remains to be studied whether L is required for the repression of pnr expression or for the inhibition of growth of pnr-expressing cells. On the basis of these data it is suggested that during early DV patterning, the onset of pnr expression might restrict the functional domain of L and Ser to the ventral eye. It is possible that pnr may also suppress L gene function via the Wg signaling pathway (Singh, 2005).
The results support the view that various developmental pathways cross-talk with each other to define the final form of a developing eye field. Such genes are likely to interact with both pnr and L. It is interesting to note that several pnr-interacting genes were identied as L modifiers in the screen. This illustrates the importance of the interaction of L and pnr pathways and also the efficacy of the screen. Further study of new modifiers of L may provide important clues to the mechanism of pnr-L interactions in the control of growth and/or DV patterning of the eye. Since the compound eye of Drosophila shares some similarities with the vertebrate eye and genetic machinery is highly conserved, it would be interesting to see if these antagonistic interactions between the dorsal eye selectors and the ventral eye genes play roles in the DV patterning and growth of vertebrate eyes (Singh, 2005).
Notch (N) activation at the dorsoventral (DV) boundary of the Drosophila eye is required for early eye primordium growth. Despite the apparent DV mirror symmetry, some mutations cause a preferential loss of the ventral domain, suggesting that the growth of individual domains is asymmetrically regulated. The Lobe (L) gene is required non-autonomously for ventral growth but not dorsal growth; it mediates the proliferative effect of midline N signaling in a ventral-specific manner. L encodes a novel protein with a conserved domain. Loss of L suppresses the overproliferation phenotype of constitutive N activation in the ventral, but not in the dorsal eye, and gain of L rescues ventral tissue loss in N mutant background. Furthermore, L is necessary and sufficient for the ventral expression of a N ligand, Serrate (Ser), which affects ventral growth. These data suggest that the control of ventral Ser expression by L represents a molecular mechanism that governs asymmetrical eye growth (Chern, 2002).
The L gene was first reported in 1925 by Morgan and has been commonly used as a second chromosome dominant marker. However, mechanisms that underlie its growth defect are little understood. With variable severities and penetrances, all L alleles examined (L1, L2, L4, L5 and Lsi) as heterozygotes exhibit a nick near the anterior midline of the eye, and the overall size of the eye is slightly reduced. As homozygotes, eye size is greatly reduced, primarily in the ventral domain. Lsi allele typifies the observed phenotypes with the highest penetrance. Homozygous Lsi animals show a preferential loss of the ventral eye with complete penetrance, and ~70% of Lsi/+ animals have an anterior nick in one or both eyes. The preferential loss of the ventral eye is also apparent in the eye imaginal disc morphology. Importantly, since homozygous Lsi mutants are viable and its half-eye mutant phenotype is indistinguishable from that of Lsi over deficiency chromosomes, this suggests that Lsi is a strong eye-specific allele that minimally affects the development of other tissues (Chern, 2002).
To assess the extent of ventral eye loss in Lsi homozygotes, an enhancer trap line, mirrB1-12 (mirr-lacZ), which has a dorsal-specific expression of white (w) reporter gene, was used. In w-; Lsi/Lsi; mirrB1-12/+ flies, the overall eye size is reduced, and all but one or two rows of remaining ommatidia are w+, suggesting that most, if not all, of the ommatidia are dorsal. The dorsal polarity was confirmed in adult-eye sections. Eye imaginal discs from staged w-; Lsi/Lsi; mirrB1-12/+ larvae showed ventral domain reduction starting at early second instar, indicating that L functions are required for early eye development (Chern, 2002).
Clones of L- cells were generated by mitotic recombination, using a loss-of-function allele, Lrev6-3. Homozygous Lrev6-3 embryos with no detectable L protein expression fail to complete germ-band retraction and show no cuticle formation. Lrev6-3 clones cause distinct eye phenotypes depending on the time of clone induction and the location of the clones. In one scheme, clones were induced at the first instar stage. In the resulting third instar eye discs that contained ventral Lrev6-3 clones, the ventral eye disc is greatly reduced, and to a large extent the Lrev6-3/+ and +/+ tissue disappear together with the Lrev6-3/Lrev6-3 tissue. By contrast, dorsal clones of considerable size do not cause obvious size reduction in the dorsal eye, nor do the mutant clones significantly affect the ensuing photoreceptor differentiation and polarity determination. Consistent with the eye disc phenotype, adult mosaic eyes have a relatively normal appearing dorsal domain, while most of the ventral region is replaced by the cuticle. In related experiments, mitotic recombination induced at late second and third instar stages generated multiple clones in both dorsal and ventral domains but did not result in any obvious eye defects, suggesting an early, transient requirement of L. All together, studies of Lsi phenotype and Lrev6-3 clonal phenotype indicate that L is non-autonomously required for the ventral eye growth but not so in the dorsal, and its functions are required during early stages of eye development (Chern, 2002).
It is known that N activation at the DV boundary is vital for eye disc growth. Since L is required specifically for ventral growth, it raises the possibility that L may mediate the proliferative effect of midline N signaling in the ventral eye. The Gal4-UAS system was used to test this hypothesis. Overexpression of a constitutively active N (Nintra) by the dpp-Gal4 driver, which drives expression along the posterior edge of the eye disc, causes gross overgrowth of the eye in both dorsal and ventral domains. Reducing L gene dose strongly suppresses the ventral overgrowth but has much less of an effect on dorsal overgrowth. This ventral-specific suppression of N gain-of-function phenotype suggests that L acts downstream of N (Chern, 2002).
In contrast to N-induced overgrowth, eliminating N signaling by expressing a dominant-negative form of N (NDN) using the eyeless (ey)-Gal4 driver consistently results in small-eye or no-eye. ey-Gal4 drives Gal4 expression in early eye discs and anterior to the furrow in the third instar discs. Co-expression of L and NDN partially suppresses this NDN overexpression phenotype in the ventral domain: ventral eye was selectively restored in close to 20% of ey-Gal4/UAS-NDN UAS-L animals. The size of the restored ventral eye was either smaller or equal to the reduced dorsal eye, and in no instances was ventral tissue detected without the presence of at least some dorsal tissue. The presence of residual dorsal eye indicates that NDN overexpression may not completely eliminate endogenous N functions. It also suggests that N activity, even at a low level, is a prerequisite for L to induce ventral proliferation (Chern, 2002).
Given that the requirement of L functions is early and transient, the suppression by L of NDN phenotype may be specific to undifferentiated cells. This is indeed the case. NDN was overexpressed using GMR-Gal4 that induces Gal4 expression in all cells posterior to the furrow. GMR-Gal4/UAS-NDN animals show a rough eye phenotype with a relatively normal eye size, and the eye roughness is not suppressed by overexpression of L in GMR-Gal4/UAS-NDN UAS-L animals (Chern, 2002).
If L and N act in the same pathway, transheterozygous mutations of these two genes may result in enhanced phenotypes. Lsi/+ flies have nicks at the anterior edge of the eye, but the defect is not so severe to result in half-eyes. Loss of one copy of N does not cause visible eye defects. Transheterozygote N264-47/+; Lsi/+ adults, however, had half-eyes in one or both eyes with approximately 50% penetrance. Similar enhancement is observed of L phenotype by mutations in Enhancer of split, a major downstream effector of N signaling. In summary, genetic interactions between L and N support the hypothesis that L mediates the proliferative effect of N signaling specifically in the ventral domain (Chern, 2002).
It is likely that L interacts with other ventral-specific genes, and one candidate gene is Ser, a N ligand, whose expression is ventrally enriched in the wild-type first instar eye disc. Ser is required for eye development; Ser loss-of-function mutants have small eyes. To understand possible regulatory relations between L and Ser, Ser expression was examined in first instar eye discs from Lsi homozygotes. In these eye discs, ventral Ser expression is greatly reduced, but notably the expression along the DV boundary is not affected. A Ser-lacZ enhancer trap line and an anti-Ser antibody were both used to detect Ser expression in these Lsi homozygotes eye discs, and similar Ser expression patterns were observed. This observation suggests that L might promote ventral Ser expression except in regions near the DV boundary. In addition, since the Lsi homozygote eventually loses its ventral eye, loss of ventral Ser expression in these mutants suggests that Ser may positively regulate ventral eye growth (Chern, 2002).
L null clones were induced to further test the hypothesis that L is a region-specific, positive regulator of Ser expression in first instar eye discs. Lrev6-3 clones alter Ser expression in a position-dependent way. Ventral L- clones away from the DV boundary show decreased Ser expression within the clone, but clones near the posterior DV boundary have no such effects. The effect of L- cells on Ser expression appear to be restricted within the clone since Ser expression outside of the clone is not visibly affected. To test if L positively regulates Ser expression, the flp-out system was used to generate clones of cells that overexpress L. In dorsal and ventral domains of first instar eye discs, L flp-out clones either induce or upregulate Ser expression, respectively. In summary, L is crucial in maintaining ventral Ser expression levels (Chern, 2002).
To understand the role of Ser in eye growth, Ser-null (Serrev2-11) mutant clones were examined in eye discs and adult eyes. No obvious defects were found in the size of the eye disc or photoreceptor differentiation. This lack of Ser-null clone phenotype implies that either the Ser protein is diffusible, or Ser is functionally redundant. In order to remove more of the wild-type Ser functions, flp-out clones expressing a diffusible, truncated form of Ser (SerDN) were generated. SerDN consists of Ser extracellular domain but lacks the transmembrane domain and is capable of antagonizing wild-type Ser functions (Chern, 2002).
Eye discs that contain SerDN flp-out clones are variably reduced. Three kinds of phenotypes are observed and might be attributed to slight variations in the timing of clone inductions and the location of the clones:
Taken together with the regulatory relations between L and Ser, these results suggest that loss of ventral Ser expression probably contributes to L ventral eye loss (Chern, 2002).
The putative L protein is 562 amino acids long and contains a poly-glutamine rich region and a C terminus that shares significant sequence similarities with novel insect, mouse and human proteins of unknown functions. A polyclonal antibody detected a single band of ~60 kDa on a Western blot prepared from third instar eye imaginal discs. Furthermore, this antibody revealed no detectable level of L protein expression in null animals. To examine L expression pattern, L transcript and protein were detected by mRNA in situ hybridization and by the polyclonal antibody. L is ubiquitously expressed in first instar eye discs. In the third instar disc, the RNA transcripts are detected in the antenna disc and in undifferentiated cells anterior to the furrow in the eye disc. By comparison, the protein can be readily detected in the antenna and anterior to the furrow, but a much lower level of expression is also present posterior to the furrow (Chern, 2002).
The putative L protein contains a poly-glutamine rich region. The C terminus covering 67 amino acids is conserved in homologs from various species; bee (BI509118) (43% identical; 63% positive), mouse (AK003638) and human (BC007416) (37% identical; 53% positive). In the last 30 amino acids, 56% are identical. The conserved L sequence suggests that its mammalian counterparts may play similar functions in mediating N signaling, although the precise function of the conserved domain is not known. The striking domain specificity of L-mediated growth may be the result of various mechanisms. It is possible that signaling molecules present in the ventral domain are different from the ones present in the dorsal domain. Signaling molecules may be selectively active in one domain but not the other, as in the case of dpp signaling in the wing disc. L may cooperate with other ventral-specific genes to transduce the N signaling, or the expression of L may be transiently ventral specific in the early eye disc. More than one mechanism may contribute to the domain specificity of L functions (Chern, 2002).
Previous experiments have shown that growth of neighboring, symmetrical domains may be independently controlled. In Drosophila wing discs, increased expression of hedgehog along the anteroposterior boundary causes anterior wing overgrowth but has no effects on the posterior wing; ubiquitous overexpression of Ser increases the ventral wing tissue but not the dorsal. Additionally, there are other Drosophila eye mutants that show preferential reductions of the ventral eye, such as wg mutants and dpp mutants. However, dominant mutation Rough eye suppresses these 'furrow stop' mutant phenotypes of dpp and wg but not the L phenotype, suggesting fundamental differences in nature and function between L and furrow stop mutants (Chern, 2002).
Clonal study shows that L null clones have striking domineering non-autonomous effects, such that the viability of wild-type tissue immediately adjacent to L mutant clones are severely reduced. Nevertheless, this non-autonomous deleterious effect is limited to the ventral domain, since clones abutting the DV boundary do not seem to affect dorsal cell viability. This domineering non-autonomous effect may be the result of interspersed L null clones disrupting the physical integrity of the imaginal disc epithelium, causing the disc to fall apart. It is also possible that L is redundant in the dorsal domain, thus loss of L can be compensated by another dorsal-specific gene. Another possibility that is favored incites a failure of the clone cells to send out a locally acting growth signal. Since the data indicate that L is a regulator of Ser expression, could Ser be this local-acting, diffusible factor (Chern, 2002)?
Homozygous Ser mutants have small eyes, indicating that Ser is required for proper eye growth. However, removing Ser in clones of cells does not result in mutant eye phenotypes. These observations are consistent with Ser being a diffusible factor, but other possibilities exist (Chern, 2002).
First, there may be other functionally redundant Ser-like molecules, but no candidates have yet been identified. Second, in Ser-expressing cells, Ser may autonomously induce the expression of diffusible signaling molecules that act non-autonomously. Ligand and receptor interactions within the membrane of the same cell have been demonstrated in the case of N and Dl. N and Dl can physically associate within the membrane of a single cell, and the expression level of Dl in a cell can modulate its own N response. In this manner, Ser-N interaction may lead to the expression of diffusible factors that rescue clones of Ser- cells. Third, the ability of adjacent wild-type cells to rescue Ser- cells suggests that Ser protein may be diffusible. This is consistent with low levels of Ser expression observed in L- clones; and in anti-Ser antibody staining, intense, dotty cytoplasmic staining, possibly secretory vesicles were consistently observed. However, the presence of secreted Ser has yet to be confirmed, even though diffusible Dl has been detected in Drosophila extract (Chern, 2002).
The domain-specificity of L phenotype indicates that the eye disc is partitioned, and the growth of individual domain is differentially regulated. Loss of the ventral eye in L mutants does not seem to affect DV boundary formation or the associated midline N activation, because disruptions of either of these events would result in abnormal dorsal growth. Additional data suggest that L does not affect the initial DV domain specification: (1) L is functionally downstream of N activation; (2) L mutation does not affect Ser expression at the DV boundary; and (3) domain-specific expression patterns of dpp, fng and wg are not affected in the first instar L mutant eye discs (Chern, 2002).
Consistent with this model, it is proposed that in the seemingly homogenous Ser-expressing, first instar ventral domain, there are actually two distinct groups of Ser-expressing cells: ventral midline cells abutting the dorsal midline cells, and the rest of the ventral cells. Their putative functions are different and their Ser expression is independently regulated. In the ventral midline cells, Ser is involved in setting up the DV boundary, and its expression is regulated by the Ser-N-Dl positive-feedback loop. The midline Ser expression can be further modified by Fng and Hedgehog, both of which can induce Ser expression only near the DV boundary but not elsewhere in the eye field, emphasizing again the distinctiveness of these midline cells (Chern, 2002).
By comparison, in the rest of the ventral domain, Ser is directly involved in controlling local growth. Loss of Ser in the ventral domain causes ventral-specific growth defects similar to the loss of L. Ser expression in the ventral domain may not be sustained by the Ser-N-Dl loop, since ventral Fng inhibits potential Ser-N interaction which is necessary to initiate the positive feedback loop. Instead, ventral Ser expression is regulated by L (Chern, 2002).
The data suggest the eye primordium is partitioned into dorsal, midline and ventral domains with different gene expression and growth properties. It highlights the importance of local cellular context in interpreting signals released from the domain boundaries and shows that the growth of symmetrical domains may be asymmetrically regulated. The model may also be applicable to the development of other imaginal discs as well as other developmental systems (Chern, 2002).
Search PubMed for articles about Drosophila Lobe
Chern, J. J., and and Choi, K. W. (2002). Lobe mediates Notch signaling to control domain-specific growth in the Drosophila eye disc. Development 129: 4005-4013. PubMed ID: 12163404
Flaherty, M. S., Zavadil, J., Ekas, L. A. and Bach, E. A. (2009). Genome-wide expression profiling in the Drosophila eye reveals unexpected repression of notch signaling by the JAK/STAT pathway. Dev. Dyn. 238: 2235-2253. PubMed ID: 19504457
Fonseca, B. D., et al. (2007). PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex. J. Biol. Chem. 282: 24514-24524. PubMed ID: 17604271
Madhunapantula, S. V., Sharma, A. and Robertson, G. P. (2007). PRAS40 deregulates apoptosis in malignant melanoma. Cancer Res. 67: 3626-3636. PubMed ID: 17440074
Oshiro, N., et al. (2007). The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1. J. Biol. Chem. 282: 20329-20339. PubMed ID: 17517883
Pallares-Cartes, C., Cakan-Akdogan, G. and Teleman, A. A. (2012). Tissue-specific coupling between insulin/IGF and TORC1 signaling via PRAS40 in Drosophila. Dev. Cell 22(1): 172-82. PubMed ID: 22264732
Reddy, P., Zheng, W. and Liu, K. (2010). Mechanisms maintaining the dormancy and survival of mammalian primordial follicles. Trends Endocrinol. Metab. 21: 96-103. PubMed ID: 19913438
Sancak, Y., et al. (2007). PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol. Cell 25: 903-915. PubMed ID: 17386266
Singh, A. and Choi, K. W. (2003). Initial state of the Drosophila eye before dorsoventral specification is equivalent to ventral. Development 130: 6351-6360. PubMed ID: 14623824
Singh, A., Chan, J., Chern, J. J., Choi, K. W. (2005). Genetic interaction of Lobe with its modifiers in dorsoventral patterning and growth of the Drosophila eye. Genetics 171(1): 169-183. PubMed ID: 15976174
Singh, A., Shi, X., Choi, K.W. (2006). Lobe and Serrate are required for cell survival during early eye development in Drosophila. Development 133(23): 4771-4781. PubMed ID: 17090721
Sun, P., et al. (2010). TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation. Development 137: 2461-2469. PubMed ID: 20573703
Thomson, T. C., Fitzpatrick, K. E. and Johnson, J. (2010). Intrinsic and extrinsic mechanisms of oocyte loss. Mol. Hum. Reprod. 16: 916-927. PubMed ID: 20651035
Vander Haar, R., et al. (2007). Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 9: 316-323. PubMed ID: 17277771
Wang, Y. H. and Huang, M. L. (2009). Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development. Mech. Dev. 126(10): 781-790. PubMed ID: 19733656
date revised: 22 April 2010
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