lilli expression in the embryo was analyzed by in situ hybridization. High levels of lilli mRNA are found in the unfertilized egg, which is consistent with the maternal requirement for Lilli function. In the early embryo and until gastrulation, lilli is expressed uniformly. During later stages of embryogenesis, there is a slightly elevated expression in the nervous system. In order to determine the subcellular localization of Lilli, an HA-tagged minigene construct (UAS-lilli-HA) was constructed. UAS-lilli-HA rescues lilli lethality, suggesting that this construct encodes a functional Lilli protein. Staining salivary glands of larvae in which expression of the HA-tagged Lilli (Lilli-HA) was induced by heat shock, reveals that Lilli-HA is mainly localized in the nucleus. Nuclear localization of Lilli-HA was also observed in the eye imaginal disc. Therefore, it is believed that, like its mammalian homologs, Lilli also normally functions in the nucleus (Tang, 2001).
A genetic screen was carried out for dpp signaling pathway components that exploits transvection effects at the dpp locus. Transvection, or pairing-dependent intragenic complementation between two alleles of a gene, is seen at a number of loci. As a result of transvection, trans-heterozygous individuals of the genotype dppd-ho/dpphr4 display wild-type wings. The dppd-ho mutation is a small deletion in the 3' cis-regulatory region of dpp. dppd-ho homozygous flies have wings that are held out laterally from the body axis. The dpphr4 mutation is a missense mutation in the protein-coding region of dpp. When homozygous, the dpphr4 allele is embryonic lethal. When dppd-ho and dpphr4 are paired, the wild-type regulatory region of the dpphr4 allele appears to act in trans on the wild-type coding region of the dppd-ho allele to generate viable adults with wild-type wings (Su, 2001).
During transvection, the respective regions (regulatory and coding) must be in close physical proximity. A chromosomal rearrangement that physically moves a dpp allele to another part of the chromosome disrupts transvection. Rather than having wild-type wings, dppd-ho/dpphr4 flies with chromosomal rearrangements have heldout wings. Analyses of polytene chromosomes from rearrangement genotypes show asynapsis at the dpp locus. These rearrangements are referred to as normal dpp transvection-disruptors (normal DTDs). Trans-heterozygous dppd-ho/dpphr4 flies will also display a heldout phenotype if they contain a rearrangement with a breakpoint in a gene required for dpp function (e.g., Mad). This type of rearrangement, one that generates heldout phenotypes in trans-heterozygous flies without asynapsis at the dpp locus, is referred to as an exceptional DTD (Su, 2001).
To determine if a DTD is normal or exceptional, an unknown DTD is paired with a previously characterized normal DTD. If the unknown DTD is a normal DTD, trans-heterozygous flies will display wild-type wings. Two normal DTDs (even those with very different rearrangements) have the ability to arrange themselves in such a way that synapsis occurs at the dpp locus. If the unknown DTD is an exceptional DTD, trans-heterozygous flies will display heldout wings. The presence of a normal DTD cannot suppress a heldout phenotype that is due to a mutation in a gene required for dpp function. Mutations that act as exceptional DTDs are therefore candidates for components of the dpp signaling pathway (Su, 2001).
A total of 44,000 dpphr4/dppd-ho flies were screened and 321 DTD mutations were isolated. Of these mutations, 30 were exceptional DTDs. All exceptional DTDs were cytologically mapped. If an exceptional DTD chromosome appeared cytologically normal, the DTD mutation was mapped by recombination. DTD46.4 is a recessive lethal strain obtained in the screen that has a T(2;3) 23C; 93F rearrangement. To determine which translocation breakpoint results in the recessive lethality, DTD46.4-bearing flies were mated to flies with deletions spanning one of the two breakpoints. DTD46.4 complemented Df(3R)e-N19, a deletion of 93B-94. DTD-46.4 failed to complement Df(2L)JS17, a deletion spanning cytological region 23C-D that includes Mad. Mad is known to act as a dpp transvection disrupter, so it was suspected that DTD46.4 might be a new allele of Mad. To test this hypothesis DTD46.4 was further characterized. Complementation tests were conducted with a number of deficiencies and other mutations in the 23C-D cytological region. The DTD46.4 chromosome failed to complement the deficiencies Df(2L)C144, Df(2L)DTD52xD51, and Df(2L)JS17 and an EMS-induced loss-of-function mutation l(2)a16. These five strains are referred to as the 23C complementation group. However, the DTD46.4 chromosome was viable over Mad6, Mad11, and Mad12 and the small deletion Df(2L)C28 that uncovers Mad. These results place the recessive lethality of DTD46.4 distal to Mad in 23C1-2. Polytene in situ hybridization studies utilizing a variety of probes have demonstrated that the Drosophila Genome Project P1 clones DS00906 and DS07149 span the 23C1-2 breakpoint (Su, 2001).
It was necessary to determine if the 23C1-2 breakpoint of DTD46.4 is also responsible for disrupting the dppd-ho/dpphr4 transvection-dependent phenotype. Df(2L)C144 and l(2)a16 were tested for the ability to disrupt this phenotype. Forty-six percent of dppd-ho Df(2L) c144 /dpphr4 flies had heldout wings; of these flies, 47% were severely heldout. Twenty percent of dppd-ho l(2)a16/dpphr4 flies had heldout wings; of these flies, 50% were severely heldout. These results are similar to those of DTD46.4. Twenty-six percent of dppd-ho DTD46.4/dpphr4 flies had heldout wings; of these flies, 53% were severely heldout. It was concluded that the site of DTD46.4 recessive lethality in 23C1-2 is also the site that disrupts the dppd-ho/dpphr4 transvection-dependent phenotype (Su, 2001).
Other studies had identified a new gene located in cytological region 23C1-2. This gene, lilliputian (lilli), was identified in two screens for Ras/Mitogen-activated protein kinase (MAPK) signal transduction pathway components. Complementation tests showed that both DTD46.4 and l(2)a16 failed to complement either lillis35 or lillixs407. It was concluded that members of the 23C1-2 complementation group are alleles of lilli. In addition, a screen for genes that interact with dRaf, another component of MAPK signaling pathways, identified a locus in 23C1-2. Loss-of-function mutations in Su(Raf)2A suppress gain-of-function dRaf phenotypes. It seems likely that Su(Raf)2A mutations are also allelic to DTD46.4 and lilli (Su, 2001 and references therein).
Four lilli alleles were tested for dominant maternal enhancement of dpp recessive embryonic lethality. Df(2L)JS17 was excluded because it uncovers Mad. The lilli alleles were tested with dppe87, dpphr56, dpphr4, and dpphr92. No genetic interactions were detected with the weak alleles dppe87 and dpphr56. However, all lilli alleles tested showed significant dominant maternal enhancement of the strong alleles dpphr4 and dpphr92. Modest dominant zygotic enhancement of dpphr4 was also detected. Thus, lilli alleles that disrupt a dpp transvection-dependent phenotype are also dominant enhancers of dpp recessive embryonic lethality (Su, 2001).
The same alleles of lilli were tested for genetic interactions with other genes that function in dpp signaling. lilli alleles do not enhance the recessive lethality of the loss-of-function mutations Mad12, Med1, sax1, tkv8, scwS12, or gbb1. However, lilli alleles show dominant maternal enhancement of the recessive lethality of scwE1. scwE1 is a gain-of-function allele that is itself a dominant zygotic enhancer of dpp recessive embryonic lethality (Su, 2001).
The stage of lethality for the lilli loss-of-function mutation l(2)a16 was determined. lilli mutant individuals [l(2)a16/Df(2L)C144] were identified using the dominant visible marker Black cells (Bc). When l(2)a16/In(2LR)Gla Bc males were mated with Df(2L)C144/In(2LR)Gla Bc females, only Bc larvae were recovered. Bc is not visible in first instar larvae, suggesting that lilli mutants die as embryos or as first instar larvae. Examination of lilli mutant embryos revealed a partially ventralized phenotype. This phenotype is also seen in zygotic mutant embryos of dpphr56 and scwE1. Several of the hallmarks of this phenotype are a herniated head, internalized filzkorper, and disorganized/expanded denticle bands. Embryos derived from germline clones of weak Su(Raf)2A mutations (e.g., Su(Raf)2A161H1) also show this partially ventralized phenotype (Su, 2001).
Three results from genetic tests suggest that lilli is a strong candidate for a new component of the dpp signaling pathway. (1)lilli mutations enhance dpp heldout phenotypes and embryonic recessive lethality. The enhancement of dpp embryonic lethality by lilli mutations is not as strong as that of Mad or Med mutations. Mutations in Mad or Med enhance weak dpp alleles while lilli mutations do not. (2) lilli mutations enhance the recessive embryonic lethality of a gain-of-function allele of the TGF-ß family member scw. scw augments dpp signaling in embryonic dorsal-ventral patterning. To date, tests for interactions between scwE1 and other dpp pathway components such as Mad or Med have not been reported. lilli mutations do not enhance the recessive lethality of mutations in genes that encode Dpp signal transduction proteins (sax, tkv, Mad, or Med). (3) lilli homozygous mutant embryos have dorsal-ventral patterning defects similar to zygotic mutant embryos of dpp and scw. Utilizing these genetic criteria, lilli has as strong a connection to dpp signaling as Mad and Med (Su, 2001).
Phyllopod (Phyl) is one of the most downstream nuclear components identified in the Sevenless receptor tyrosine kinase-RAS1 signaling pathway. Using the eye-specific expression vector pGMR, which contains a multimerized binding site for the zinc-finger protein Glass placed upstream of the basal hsp70 promoter, Phyl was expressed in all cells posterior to the morphogenetic furrow during larval development, and in all cells except cone cells in the pupal eye. This results in a rough eye phenotype that was used to screen for dominant modifiers. The lilli gene corresponds to one of the complementation groups that strongly suppress the rough eye phenotype of GMR-phyl. Complementation analyses revealed that many lilli alleles have been identified as suppressors in a number of different GMR-based dominant modifier screens. For example, lilli alleles were isolated in a GMR-sina screen and in a GMR-YanACT screen. Mutations in lilli suppress the rough eye phenotypes generated by overexpression of either positive (Sina and Phyl) or negative (Ttk and Yan) components of the RAS1 signaling pathway under GMR control, as well as other GMR constructs from different signaling pathways. In addition, lilli mutants dominantly suppress the rough eye phenotypes of many sE transgenes, in which the sevenless enhancer is placed upstream of the hsp70 basal promoter. These observations suggest that lilli is required, either directly or indirectly, for proper transcription from the GMR and sE expression constructs. Further supporting this hypothesis, it was found that the levels of CAT activity from a GMR-CAT reporter construct are decreased by ~40% in third instar larvae heterozygous for lilli. Similar results were obtained when one copy of glass, a known activator of GMR transcription, is removed. These results suggest that lilli acts as a transcriptional regulator for GMR transgenes (Tang, 2001).
To gain insight into its role during embryogenesis, the phenotype of embryos mutant for lilli was examined. Most embryos lacking zygotic lilli fail to hatch and subsequently die, although a small percentage hatch and die as first or second instar larvae. Cuticle from the late embryos is normal, with three thoracic and eight abdominal segments. Loss-of-function lilli mutations are found to be allelic to a lethal P-element insertion, l(2)00632, that exhibits a pair-rule-like segmentation phenotype when the maternal component of the gene is removed. Since both RNA in situ hybridization and the l(2)00632 germline clone phenotype suggests that lilli transcript is maternally contributed, the DFS-FLP technique was used to produce germline clones (GLC) that result in lilli null embryos that lack both maternal and zygotic Lilli activity (Tang, 2001 and references therein).
lilli GLC embryos (lilliXS575 and lilliXS407) exhibit pair-rule segmentation defects more severe than those previously reported for lilli l(2)00632. Two classes of phenotypes have been observed. Approximately half of the embryos were missing odd numbered segments, with the remaining denticle belts often fused. The other class of lilli GLC embryos fail to secrete cuticle properly. These two phenotypic classes appear to reflect variation inherent to the lilli loss-of-function phenotype, rather than partial rescue by a paternal copy of lilli, as they are similarly observed whether wild-type or heterozygous lilli males are used. To further characterize these segmentation defects, an examination was made of the expression of the Engrailed (En) protein, which is present in 14 stripes along the anterior-posterior axis of wild-type embryos and marks the parasegment boundaries. In lilli GLC embryos, the even-numbered En stripes are missing. Similar defects in Wingless expression are also observed. Together, these results show that lilli is required for the establishment of odd-numbered segments in the embryo. The activity of other known pair-rule genes is not only required for segmental patterning, but also for germband extension. Similarly, it was found that germband extension is affected by loss of lilli. About 90 minutes after onset of gastrulation, germband extension in wild type reaches 60% of dorsal egg length. In contrast, the germband never extends beyond 25% of dorsal egg length in lilli GLC embryos. These results suggest that lilli is required for the convergent extension movements during germband extension, consistent with its function as a maternally provided pair-rule gene (Tang, 2001).
The significant percentage of embryos that fail to properly secrete cuticle suggests that lilli GLC embryos had defects in addition to the patterning defects described above. Cytoskeletal architecture integrity during cellularization was examined. In wild-type embryos, early in cellularization the distribution of actin filaments changes from an apical cortical cap to an apical internuclear position. Following this initial phase of cellularization, nuclei elongate and microtubules form characteristic arrays described as perinuclear inverted baskets, while actin filaments maintain a contractile regular network of hexagonal units surrounding the microtubular arrays. Toward the end of cellularization, the individual units of the actin network contract and the resulting cells retain thin connections, called yolk stalks, to the center of the embryo. lilli GLC embryos exhibit specific defects in the maintenance of the actin network during cellularization. The initial phase of cellularization occurs normally. However, during the second phase of cellularization, specific defects in the maintenance of the contractile actin network are observed: the actin network begins to contract and the furrow tips move basally. The actin filaments become unevenly distributed between nuclei, ranging from abnormally large bundles to regions where the actin network is thin or absent, resulting in multinucleated cells. The microtubular baskets surrounding each nucleus appear largely normal, even in regions where actin filaments are unevenly distributed. At the end of cellularization, the yolk stalks are irregular in shape and size, and large connections between cortical cells and the central yolk cell are frequently seen. Despite these defects, video-timelapse analysis has revealed that the timing of cellularization and membrane formation is unimpaired in lilli GLC embryos and does result in an epithelial monolayer of cells with proper apical-basal polarity (Tang, 2001).
In addition to the failure in maintaining the actin network, lilli GLC embryos exhibit defects in transport of organelles during cellularization. In wild-type embryos, lipid droplets move along microtubules in a bi-directional fashion and accumulate basally during cycle 14, near the plus ends of microtubules. As the cortical cytoplasm becomes depleted of lipid droplets, it appears transparent. In lilli GLC embryos, this cortical clearing is perturbed, resulting in a 'halo' of non-cleared cytoplasm around the central yolk. Living lilli GLC embryos have an abnormal distribution of lipid droplets during cellularization and about 80% of the embryos fail to separate from the central yolk sac shortly after cellularization. To determine whether this failure to clear is caused by a general breakdown of cytoplasmic transport, the transport of yolk vesicles and the integrity of the microtubule network was examined. The distribution of yolk vesicles can be observed in fixed embryos following extraction of neutral lipid from the lipid droplets. In lilli GLC embryos, yolk vesicle movement is normal during cellularization, and the general distribution of microtubular arrays is largely unaffected. Thus, lilli does not induce general breakdown of microtubule-based transport, but rather is required specifically for the microtubule-based basal transport of lipid droplets (Tang, 2001).
lilli is required for the expression of serendipity alpha, a zygotic regulator of the actin cytoskeleton. Pan-genomic zygotic screens for genes that are required for proper function of the actin network during cellularization have identified three genes: nullo, Sry alpha and brinker, a target of the Dpp pathway. The cellularization phenotypes of lilli GLC embryos are similar to those observed for mutations in the blastoderm-specific genes nullo and Sry alpha. In contrast, mutations in bnk disrupt the timing of microfilament rearrangement during cellularization. Antibodies against the Nullo and Bnk proteins were used to examine their distribution in lilli GLC embryos. In wild-type embryos, Nullo and Bnk proteins colocalize with filamentous actin at the leading edge of the invaginating furrows at mid-cellularization. In lilli GLC embryos, Nullo and Bnk are expressed and localized normally, although the vesicular Nullo staining in the basal periplasm is somewhat less pronounced. In grazing sections, the alterations observed in Nullo and Bnk distribution likely reflect the disruptions of the actin network. Thus, it is concluded that the cellularization defects in lilli GLC embryos cannot be attributed to lack of Nullo or Bnk expression (Tang, 2001).
Sry alpha mRNA is normally expressed at low and uniform levels at cycle 13, and is then concentrated in two broad bands prior to its down regulation late in cycle 14. Interestingly, expression of the Sry alpha gene during cellularization could not be detected in lilli GLC embryos. Expression of a Sry-lacZ transgene is likewise abolished in lilli GLC embryos, indicating that the defect in Sry alpha expression is at the transcriptional level. Since the mutant phenotype of Sry alpha is very similar to that of lilli GLC embryos, it seems likely that the cellularization defects observed in lilli GLC embryos are caused, at least in part, by a strong reduction in Sry alpha expression (Tang, 2001).
In contrast to its absolute requirement during embryogenesis lilli is dispensable for the specification of the endogenous R7. However, it is essential for the formation of ectopic R7 cells induced by the activation of either Sev, Ras, Raf or MAPK in the cone cell precursors. Interestingly, while the ectopic R7 cells caused by constitutive activation of Raf are suppressed by removal of one copy of lilli, the multiple-R7 phenotype caused by constitutive activation of Sev, Ras, or MAPK is only suppressed by complete removal of Lilli function. To test whether Lilli is required only for the formation of ectopic R7 cells or whether it also performs a partially redundant function in the endogenous R7 cell, clones of lilli mutant cells were generated in a background containing the hypomorphic rafHM7 allele. The rafHM7 mutation causes a reduction of raf transcript levels and leads to the loss of R7, as well as some R1-R6 cells, in a subset of ommatidia. Indeed, the number of both R7 and R1-R6 cells in rafHM7 mutants is further reduced significantly within the lilli mutant clones. This result suggests that Lilli functions not only in the formation of ectopic R7 cells but also plays a partially redundant role downstream of Raf in the specification of the endogenous photoreceptor cells (Wittwer, 2001).
Lilli has a partially redundant function downstream of Ras/MAPK signaling in cell fate specification. Loss-of-function mutations in lilli were identified as dominant suppressors of the specification of supernumerary R7 photoreceptor cells in response to constitutive activation of Raf in the developing eye (Dickson, 1996). However, without constitutive activity of the Ras/MAPK pathway, the normal number of photoreceptor cells is specified in each ommatidium in the complete absence of Lilli function. Given the fact that lilli encodes a putative transcription factor, a possible explanation for why lilli mutants specifically suppress the differentiation of extra R7 photoreceptor cells is that lilli may be required for the expression of the sE-RaftorY99 transgene. Indeed, lilli mutations affect transcript levels of transgenes containing a hsp70 basal promoter (Wittwer, 2001 and Tang, 2001). Although the effect of lilli on hsp70 transcription may explain some of the genetic interactions, it is not sufficient to explain the specific interaction with the Raf/MAPK pathway for the following reasons. (1) In the original screen, two different RaftorY9 transgenes were used. One contained the sev enhancer fused to the hsp70 promoter (sE), the other the sev enhancer fused to the sev promoter (sEsP). lilli mutations suppress both. (2) lilli mutations fail to suppress the rough eye phenotype caused by the ectopic expression of rough under the control of the sev enhancer and heat shock promoter. (3) Heterozygosity for lilli does not suppress the multiple R7 phenotype of SevS11, a transgene encoding a truncated version of the Sev receptor also expressed under the control of the sev enhancer and the hsp70 promoter (sE). The formation of multiple R7 cells caused by sE-SevS11 or sEsP-RasV12 is only suppressed in homozygous lilli clones. If lilli controlled expression of the sev enhancer, one would expect a dominant suppression of all sev enhancer driven transgenes. (4) Finally, complete loss of Lilli function in clones decreases the number of photoreceptor cells recruited in the background of rafHM7, a hypomorphic mutation of raf. Therefore, it is concluded that Lilli has a specific function in regulating the efficiency of signal transduction downstream of Raf. As a putative transcription factor, Lilli may regulate the expression levels of one or multiple components of the Ras/MAPK signaling pathway that become rate-limiting when Ras/MAPK signaling is too high, in cells where it is normally low (as in the case of ectopic activation of Raf in the eye), or when signaling is reduced (Wittwer, 2001).
Although clones homozygous for lilli alleles in the adult eye were wild-type with respect to photoreceptor cell differentiation and arrangement, it was noted that in clones of strong alleles (i.e. lilli4U5 and lilli15D1) the size of the photoreceptor cells is reduced in comparison to the heterozygous cells adjacent to the clone. Analysis of mosaic ommatidia shows that this phenotype is cell autonomous. A reduction of cell size in mutant tissue is typical for components of the PI3K/PKB and Ras/MAPK pathways. In addition to reducing cell size, loss of Lilli function may also decrease cell number. Clones mutant for chico, which display similar cell size defects as lilli, grow more slowly and cannot compete with faster growing wild-type cells. In contrast, two experiments indicate that growth of lilli mutant tissue is not impaired during early larval development: (1) when sister clones, one homozygous for lilli, the other homozygous for chico, were generated, it was found that lilli mutant cells compete successfully with heterozygous tissue while chico mutant cells are outcompeted. (2) The size of lilli clones is similar to that of their wild-type sister clones in the wing imaginal disc and the eye imaginal disc anterior to the furrow. Posterior of the furrow however, lilli mutant clones are significantly smaller than their wild-type sister clones. Cells posterior to the morphogenetic furrow undergo a final round of cell division and are then integrated into the ommatidial clusters. The reduced clone size could thus be the result of a failure of some lilli mutant cells to undergo this last division or to a slight increase in apoptosis in lilli clones posterior to the morphogenetic furrow. Using TUNEL staining to detect cells undergoing apoptosis, and anti-phosphohistone H3 staining to detect mitotic cells, no significant difference between mutant and control clones could be detected. The relatively small difference in clone size may make it difficult to observe significant differences in cell death or mitosis with methods that detect cells undergoing cell death or cell division, respectively, only at the time of the experiment. Therefore, at present it is not known whether the reduction in clone size posterior to the morphogenetic furrow is due to an increase in apoptosis or an inhibition of the cell cycle or a combination of both (Wittwer, 2001).
Consistent with the reduced clone size in the eye disc posterior to the morphogenetic furrow it was found that the selective removal of Lilli function in eye and head precursor cells by the ey-Flp system results in flies with reduced eye and head size. The reduction in eye size is caused by a reduction in both the number and size of ommatidia (24% and 18%, respectively, for lilli4U5). The degree of head size reduction allows for the definition of a class of strong alleles (e.g. lilli4U5 , lilli15D1 ) and a class of weak alleles (e.g. lilli3E8, lilliP1, lilliP4). The small head phenotype of lilli mutant tissue is similar to that observed with components of the PI3K/PKB pathway. Lilli partially suppresses the overgrowth phenotype caused by the loss of Pten function (Wittwer, 2001).
Since loss of Lilli function affects cell size and head size, tests were performed for genetic interactions between lilli and components of the PI3K/PKB pathway. Pten acts as a negative regulator in the PI3K/PKB pathway by dephosphorylating the second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3; PIP3]. Tissues mutant for Pten show hyperplastic and hypertrophic growth: Pten mutant cells are larger and proliferate at a higher rate than wild-type cells. Removal of Pten function from the eye imaginal disc tissue using the ey-Flp system results in an increase in eye and head size. The increase in eye size is due to an increase in cell number and cell size as indicated by the increase in number and size of ommatidia. To test whether the Pten large-head phenotype is modified by the removal of Lilli function, Pten;lilli double mutant eyes were generated. Indeed, eyes double mutant for Pten and lilli are considerably smaller than Pten mutant eyes due to a reduction in cell number and cell size. Loss of Lilli function, however, does not completely suppress the Pten phenotype. Although these results suggest that Lilli and Pten cooperate in the control of cell and organ growth, the absence of a clear-cut epistasis between the two mutants indicates that Lilli does not act downstream of Pten in a simple linear pathway but it rather acts in a parallel pathway required for growth (Wittwer, 2001).
Although Tang (2001) also observed that retinal cells lacking lilli are significantly smaller than wild-type cells, that study interpreted results somewhat differently from Wittwer (2001). Despite the small size of individual lilli mutant adult cells, relatively large clones of lilli mutant cells can be generated, suggesting that lilli may affect cell size without changing the overall rate of growth. This was tested by comparing the size of individual lilli mutant clones to their wild-type twinspots in third instar eye and wing imaginal discs. lilli clones induced either at 24-36 hours or 36-48 hours after egg deposition (AED) are indistinguishable in size and number of cells from their wild-type twinspots. Of 75 individual clones examined at 96 hours after induction, the average area of lilli mutant clones was 1440 pixels, compared to 1400 pixels for corresponding wild-type twinspots. Thus, lilli mutant cells grew at 1.03 times the rate of wild-type cells, indicating that lilli is not required for normal rates of cell growth (Tang, 2001).
Interestingly, despite an approximately 50% reduction in the size of lilli photoreceptor cells and wing margin bristles in the adult, other cell types in the adult eye and wing are unaffected. For example, the surface of eyes containing lilli mutant clones appears normal by SEM analysis, suggesting that loss of lilli does not reduce the size of cone cells. The size of lilli cells in developing wing and eye imaginal discs appears normal as well. This was confirmed by FACS analysis of dissociated wing discs, which has revealed no significant difference in size between lilli and wild-type cells. In addition, unlike mutations in components of the PI3K pathway, lilli mutant cells display a normal cell cycle profile. To test whether lilli is required for PI3K-mediated growth, cells doubly mutant for lilli and Pten, an inhibitor of this pathway, were examined. Mutations in Pten increase cell size and advance G1/S progression; these effects are prevented by mutations in downstream components such as torso (tor). In contrast, loss of lilli does not prevent the cell enlargement or cell cycle changes caused by Pten mutation, indicating that lilli is not an essential element of the PI3K pathway. Interestingly, it was noticed that ommatidia containing the enlarged lilli;Pten photoreceptor cells are severely disorganized, and contain malformed rhabdomeres characteristic of cytoskeletal defects. Together, these results indicate that lilli affects the cell size through a growth-independent and PI3K-independent mechanism. It is suggested that mutations in lilli may affect final cell size by disrupting the morphological changes that cells such as rhabdomeres and bristles, which are the specializations of photoreceptor and trochogen cells respectively, undergo during pupal development (Tang, 2001).
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date revised: 4 March 2001
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