InteractiveFly: GeneBrief

lilliputian : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Mutations in lilliputian (lilli) have been identified in three separate studies: (1) lilli is a dominant suppressor of the rough eye phenotype caused by constitutive activation of Raf during eye development (Wittwer, 2001); (2) lilli mutation strongly suppresses the rough eye phenotype of ectopically expressed phyllopod (Tang, 2001), and (3) lilli was identified in a screen for genes that enhance the embryonic lethal phenotype of dpp alleles (Su, 2001). Lilli plays a partially redundant function downstream of Raf in cell fate specification in the developing eye. Complete loss of Lilli function leads to a reduction in cell and organ size (Wittwer, 2001). Embryos lacking maternal lilli expression show specific defects in the establishment of a functional cytoskeleton during cellularization, and exhibit a pair-rule segmentation phenotype. These mutant phenotypes correlate with markedly reduced expression of the early zygotic genes serendipity alpha, fushi tarazu and huckebein, which are essential for cellularization and embryonic patterning (Tang, 2001). lilli mutations disrupt the transvection-dependent dpp phenotype and are also dominant maternal enhancers of recessive embryonic lethal alleles of dpp and screw. lilli zygotic mutant embryos exhibit a partially ventralized phenotype similar to dpp embryonic lethal mutations (Su, 2001). The lilli gene encodes a nuclear protein related to the AF4/FMR2 family. In humans, mutations affecting the genes of this family are associated with specific diseases. Loss of FMR2 gene transcription causes mental retardation. Translocations between MLL (a human trithorax-related gene) and AF4 or AF5q31 are involved in acute lymphoblastic leukemia (Wittwer, 2001, Tang, 2001 and Su, 2001).

lilli functions as a maternally provided pair-rule gene that is essential for proper cellularization, gastrulation and segmentation during embryogenesis. Two classes of phenotypes have been observed. Approximately half of the embryos are seen to be 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, since they are similarly observed whether wild-type or heterozygous lilli males are used. Four lines of evidence support the idea that lilli functions during embryogenesis as a pair-rule gene: lilli germ-line clone (GLC) embryos fail to establish even-number Engrailed stripes; they subsequently lack odd-number segments; they exhibit defects in germband extension, and these phenotypes are not seen in homozygous lilli embryos lacking zygotic Lilli function. Thus, Lilli is required maternally for normal segmentation during embryogenesis (Tang, 2001).

What is the mode of action of lilli in segmentation? Unlike zygotic pair-rule genes that are expressed in the trunk region of the embryo in seven stripes, the expression pattern of lilli transcript is not segmental. Thus, lilli may play a role in the expression, localization or activity of other pair-rule genes. lilli probably does not act through the major gap genes knirps, Krüppel, or giant, because the striped pattern of the primary pair-rule genes eve, h and run are unaffected in lilli GLC embryos. One function of lilli is to regulate ftz transcription, since expression of both endogenous ftz mRNA and a ftz-lacZ transgene are markedly reduced in lilli GLC embryos. Although a low level of ftz mRNA remains in lilli GLC embryos, these transcripts never resolve into seven stripes. It is possible that lilli directly regulates ftz transcription in combination with other transcription and regulatory factors, or that lilli is required for the function or expression of such molecules. A candidate for such a factor is the product of the ftz-f1 gene, which is required maternally for ftz expression (Tang, 2001).

Lilli has a partially redundant function downstream of Ras/MAPK signaling in cell fate specification in the Drosophila eye. 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. 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).

The ability of lilli loss-of-function mutations to suppress MAPK signaling gain-of-function phenotypes and to enhance dpp loss-of-function phenotypes is very intriguing. lilli is one of the first genes involved in MAPK and TGF-ß signaling pathways in a developmental system. lilli encodes a transcription factor. This fact suggests one hypothesis for lilli's role in MAPK signaling and another hypothesis for a role in Dpp signaling. For MAPK signaling, lilli may be a transcriptional effector of MAPK signal transduction pathways. This hypothesis fits the observation that lilli loss-of-function mutations suppress MAPK signaling gain-of-function phenotypes. For Dpp signaling, lilli may be a maternally supplied transcriptional activator of dpp and/or scw during dorsal-ventral patterning. This hypothesis fits three observations: (1) lilli loss-of-function mutations maternally enhance the recessive lethality of several dorsal-ventral patterning mutations; (2) lilli mutant phenotypes mimic the mutant phenotypes of dorsal-ventral patterning mutations; (3) lilli mutations do not enhance, either maternally or zygotically, the embryonic lethality of genes that encode Dpp signal transduction proteins. Alternatively, lilli could participate in a signaling pathway parallel to the Dpp pathway that is also required for the expression of Dpp target genes (Su, 2001).

To test the hypothesis that lilli is a maternal activator of dpp and/or scw in dorsal-ventral patterning one would examine dpp and scw expression in embryos derived from lilli mutant germline clones. The prediction is that there would be reduced dpp and/or scw expression in these embryos during dorsal-ventral patterning. At this time, maternal activators of zygotic dorsal-ventral patterning genes such as dpp and scw, as opposed to well-known repressors such as Dorsal, are unknown. It is tempting to speculate that a maternal MAPK signal induces lilli to activate dpp in embryonic dorsal-ventral patterning (Su, 2001).

Determining a role for lilli in dpp signaling in adult wings, where lilli mutations enhance the heldout phenotype, is more problematic. There is no a priori reason to believe that lilli plays the same role in dpp signaling during dorsal-ventral patterning and adult appendage formation but it seems a logical place to begin. Thus it is possible that lilli activates dpp expression in wing imaginal discs. This hypothesis fits a report of dpp transcriptional regulation by the heldout cis-regulatory region. Two consensus HMGI binding sites (A/TA/TCAAG) are identified as dTcf (Pangolin) binding sites in the heldout region. The expression of reporter genes containing the dpp heldout region is disrupted when these putative dTcf sites are mutagenized. In addition, dominant negative forms of dTcf expressed in wing imaginal discs eliminate dpp expression. Thus dTcf is required for dpp expression by the heldout cis-regulatory region. However, these data do not preclude the possibility that the HMGI binding sites are actually the target of another HMGI domain protein, such as Lilli. To determine which HMGI domain protein is actually responsible for dpp expression from heldout regulatory sequences, one would examine dpp expression in wing imaginal discs bearing dTcf or lilli somatic clones (Su, 2001).

In addition to specific involvement of Lilli in MAPK and Dpp pathways, Lilli also functions in growth control. Growth is controlled by many different signals and during development it is tightly linked to pattern formation. In Drosophila, the insulin receptor controls growth via the PI3K/PKB pathway without affecting pattern formation. The EGF receptor also controls growth most likely via the Ras/MAPK pathway. Little is known about the nuclear factors that integrate the signals from these two pathways. Could the nuclear factor Lilli be a point of convergence of these two pathways for growth control? Although the cell size phenotype observed in lilli mutants is similar to that of mutants in either of the two pathways, mutations in lilli also display some unexpected phenotypes that are difficult to reconcile with Lilli functioning exclusively as a target of the two pathways. (1) lilli mutant cells do not have a growth disadvantage during early imaginal disc development. (2) The partial suppression of the overgrowth phenotype of Pten mutants by lilli mutants suggests a complex epistatic relationship between lilli and the PI3K/PKB pathway. (3) Both Lilli overexpression and lilli loss-of-function mutations reduce cell size. This is in contrast to components of the PI3K/PKB and Ras/MAPK signaling pathways. Overexpression of PI3K or PKB increases cell size, whereas loss-of-function mutations decrease cell size. Similarly, loss- and gain-of-function mutations of Ras have opposite effects on cell size. Lilli may activate the transcription of genes involved in cell growth and differentiation (Wittwer, 2001).

A fourth function for Lilli is suggested by the fact that significant percentage of embryos fail to properly secrete cuticle. Thus lilli GLC embryos have defects in addition to the patterning defects described above. Cytoskeletal architecture integrity during cellularization was examined. lilli germ-line clone (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, as the actin network begin to contract and the furrow tips move basally. The actin filaments becomes unevenly distributed between nuclei, ranging from abnormally large bundles to regions where the actin network is thin or absent, resulting in multinucleated cells. In addition to the failure in maintaining the actin network, lilli GLC embryos exhibit defects in transport of organelles during cellularization. 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. lilli does not induce general breakdown of microtubule-based transport, but rather is required specifically for the microtubule-based basal transport of lipid droplets. lilli is also required for the expression of serendipity alpha, a zygotic regulator of the actin cytoskeleton. The cellularization phenotypes of lilli GLC embryos are similar to those observed for mutations in the blastoderm-specific genes nullo and Sry alpha. Tang (2001) relates the insulin pathway-like phenotypes of lilli in the eye disc to the defects in cytoskeleton observed during embryonic development. Tang argues 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 cytoskeletal based 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).

How does the function of Lilli in Drosophila correlate with the phenotypes observed in disease conditions associated with mutations in the human homologs AF4, AF5q31 and FMR2? In the case of acute lymphoblastic leukemia, two related genes, AF4 and AF5q31, are involved in translocations to the MLL gene (also known as ALL-1, htrx), which encodes a human homolog of the Drosophila protein Trithorax. Lilli function is essential for increased activity in the Ras/MAPK pathway. Activating mutations in Ras are frequently found in lymphomas but normally absent in leukemias associated with MLL rearrangements. It is possible that expression of the MLL/AF4 fusion gene circumvents the need for Ras activation in transformation. Recently, the targeted inactivation of the Af4 gene in mice has been reported (Isnard, 2000). Af4 mutant mice display an altered development of lymphoid cells and, on a mixed 129/Balb/c background, a subset show a significant reduction in body weight at birth. The reduction in weight may suggest that AF4 and Lilli control cellular growth in mice and Drosophila, respectively (Wittwer, 2001).

In summary, Lilli appears to function in MAPK and Dpp signaling pathways; it appears to affect growth, a function associated with the insulin pathway, and also affects the cytoskeleton early in development. Detailed studies of Lilli function in Drosophila will likely shed light on the wild-type function of human FMR2/LAF4 family members. For example, the functional conservation of dpp signaling pathway components suggests that human homologs of Lilli's transcriptional targets are likely to be targets of human FMR2/LAF4 family members. Given that mutations in these human genes lead to mental retardation or childhood cancer and that information on human developmental genes is difficult to gather directly, studies of Lilli are an important weapon in efforts to combat these human syndromes (Su, 2001).

REGULATION

Targets of Activity

Given the potential role of lilli as a transcriptional regulator, an examination was performed to see whether the pair-rule phenotype in lilli germ-line clone (GLC) embryos corresponds to changes in the expression of early patterning genes. Examined was the spatiotemporal pattern of mRNA expression for several of these genes: the maternal coordinator gene bicoid; the gap gene hunchback; the pair-rule genes fushi tarazu, even-skipped, hairy, and runt; the segment polarity genes engrailed and wingless and the terminal gap genes tailless and huckebein. The expression patterns of bcd, hb, eve, h and run mRNA appear relatively normal. In contrast, levels of ftz mRNA were significantly lower in lilli GLC embryos than wild-type embryos at the end of cellularization. ftz expression appears normal prior to mid-cellularization, after which its distribution becomes diffuse and uniform, and it rarely accumulates in stripes. Using ftz-lacZ and hb-lacZ transgenes to determine the level of such regulation, it was found that expression of the ftz-lacZ transgene is markedly reduced in lilli GLC embryos, while that of the hb-lacZ transgene remains unimpaired. This suggests that Lilli regulates ftz gene expression at the transcriptional level. Since both ftz and lilli are required for even-numbered En stripes and odd-numbered segment formation, this disruption of ftz expression may account for the pair-rule phenotype observed in lilli GLC embryos (Tang, 2001).

In addition, hkb mRNA is either reduced or absent at the embryonic termini, while the expression of tll mRNA is largely unaffected in lilli GLC embryos. hkb establishes the anterior and posterior borders of the ventral furrow during gastrulation, and lack of hkb expression causes the mesoderm and ventral domain to extend to the poles; this domain is marked by expression of snail. Accordingly, a small extension was found of the ventral domain that expresses sna and undergoes ventral furrow formation (Tang, 2001).

lilliputian, the sole Drosophila member of the FMR2/AF4 (Fragile X Mental Retardation/Acute Lymphoblastic Leukemia) family of transcription factors, is widely expressed with roles in segmentation, cellularization, and gastrulation during early embryogenesis with additional distinct roles at later stages of embryonic and postembryonic development. This study identified lilli in a genetic screen based on the suppression of a lethal phenotype that is associated with ectopic expression of the transcription factor encoded by the segmentation gene runt in the blastoderm embryo. In contrast to other factors identified by this screen, lilli appears to have no role in mediating either the establishment or maintenance of engrailed (en) repression by Runt. Instead, it was found that Lilli plays a critical role in the Runt-dependent activation of the pair-rule segmentation gene fushi–tarazu (ftz). The requirement for lilli is distinct from and temporally precedes the Runt-dependent activation of ftz that is mediated by the orphan nuclear receptor protein Ftz-F1. A role is described for lilli in the activation of Sex-lethal (Sxl), an early target of Runt in the sex determination pathway. However, lilli is not required for all targets that are activated by Runt and appears to have no role in activation of sloppy paired (slp1). Based on these results it is suggested that Lilli plays an architectural role in facilitating transcriptional activation that depends both on the target gene and the developmental context (Vanderzwan-Butler, 2006).

This study uncovered Lilli's role in Runt-dependent transcriptional regulation based on the identification of lilli mutations as dose-dependent suppressors of the lethality produced by threshold levels of NGT-driven Runt expression. In contrast to all of the other Runt-interacting genes and deficiency intervals identified as suppressors in this genetic screen, a reduction in maternal lilli dosage has no effect on either the establishment or maintenance of Runt-dependent en repression. The target of Runt that provides the most dramatic and clearest evidence for a functional interaction between runt and lilli is the pair-rule gene ftz. It is notable that the ftz expression is not discernibly altered by the relatively low levels of NGT-driven Runt used in the genetic screen. This raises a question regarding the basis for lilli acting as a dose-dependent suppressor of the lethality associated with ectopic Runt expression. One explanation is that there are subtle changes in ftz expression at the threshold levels of NGT-driven Runt used in the viability assays that contribute to lethality. A second possibility is that there are other targets of Runt and Lilli that contribute to the lethality associated with ectopic Runt expression. Although Sxl would seem to be one obvious candidate for such a target, the developmental window for Sxl activation occurs prior to the stage during which the NGT-drivers are useful for manipulating gene expression. Indeed, it has not been possible to detect activation of Sxl by NGT-driven Runt, even at levels that are tenfold higher than the levels used in the genetic screen. Finally, it is possible that the effect on viability is in part due to a non-specific effect of Lilli on GAL4-dependent activation of Runt. There is some evidence for non-specificity, especially with Df(2L)C144; however, there is also a clear suppression of lethality with other lilli alleles that do not show a comparable reduction in NGT-driven UAS-lacZ expression. Thus it seems likely that the identification of lilli is due to a combination of specific and non-specific effects on the lethality produced by NGT-driven Runt expression. If this interpretation is correct, then it also seems likely that other deficiency intervals that were eliminated from further consideration due to apparent non-specific effects may in fact have specific and interesting effects that would be revealed by more directly assaying the effects of these mutations on the responses of different targets to NGT-driven Runt expression (Vanderzwan-Butler, 2006).

These observations confirm and extend findings regarding a role for Lilli in the transcriptional activation of the pair-rule gene ftz (Tang, 2001). Lilli does not appear to have any role in regulating other pair-rule genes, and the effects of eliminating maternal Lilli on segment-polarity gene expression have been interpreted as an indirect effect due to the loss of Ftz (Tang, 2001). Thus ftz appears to stand out as the sole gene in the segmentation pathway that shares a requirement for both Lilli and Runt. The previous work from Tang did identify two other targets for Lilli in the early Drosophila embryo, serendipity-α (sry-α) and huckebein (hkb). There is no evidence that either of these genes is regulated by Runt. Thus, just as there are targets of Runt in the segmentation pathway whose regulation is Lilli-independent, there are also targets of Lilli that do not involve interactions with Runt (Vanderzwan-Butler, 2006).

This work adds Sxl as an additional candidate target for Lilli. The elimination of maternal Lilli interferes with the activation of the Sxl_PE:lacZ reporter gene in all somatic cells of the female embryo. This global effect stands in contrast to the more localized role of Runt which is only required for Sxl activation in cells within the pre-segmental region of female embryos. The failure in Sxl activation observed in the absence of maternal Lilli is similar to the phenotype of embryos that are mutant for either sisA or sisB. Indeed the possibility is considered that the primary defect in lilli germline clone embryos was the failure to activate either of these two X-chromosome linked numerators. Expression of sisA was found to be normal in lilli germline clone embryos (VanderZwan, 2003). The low level of sisB expression in wild-type embryos made it difficult to unambiguously determine whether lilli was important for sisB activation. To further investigate the role of Lilli in Sxl activation the expression of the Sxl_GOF:lacZ reporter gene was examined. Both Runt and SisB contribute to the ectopic expression of this reporter in male embryos. The elimination of maternal Lilli has a more severe effect on the expression of the Sxl_GOF:lacZ reporter than is observed in embryos that are mutant for either runt or sisB (VanderZwan, 2003). The most straightforward interpretation of these results is that Lilli is directly involved in the transcriptional activation of the early embryonic Sxl promoter, in this case in cooperation with the four different X-linked factors that are responsible for the sex-specific expression of Sxl in female embryos (Vanderzwan-Butler, 2006).

The inclusion of Sxl gives four putative direct targets of Lilli in the Drosophila embryo. These four genes, Sxl, ftz, sry-α and hkb are normally activated at very early stages, and in all four cases this activation is reduced, if not abrogated, in the absence of maternally provided lilli. The notion that Lilli functions primarily in activation is consistent with observations on the properties of the mammalian homologs FMR2 and LAF4 (Hillman, 2001). However, early activation is clearly not the sole identifying characteristic of Lilli's targets. Indeed, for three of these targets, there are other genes in the same developmental pathway that are activated at the same time that do not require Lilli. In the cellularization pathway, Lilli is required for expression of sry-α but has no role in the activation of bottleneck (bnk) or nullo (Tang, 2001). In the segmentation pathway, the gap gene hkb is expressed in the anterior and posterior poles in response to signaling by the terminal pathway. Elimination of maternal Lilli greatly reduces hkb expression, but has no obvious effect on tailless (tll), another gap gene that is activated at the same stage in response to the terminal signaling pathway (Tang, 2001). Finally, the requirement for maternally provided Lilli that is observed for ftz is not shared with the pair-rule segmentation genes eve, hairy and runt (Tang, 2001). This last observation is perhaps most important as the wealth of information that exists on pair-rule gene regulation provides a useful framework for further considering the potential attributes of Lilli-dependent targets (Vanderzwan-Butler, 2006).

Elimination of maternal Lilli reduces, but does not eliminate ftz expression. The reduced expression that remains is similar to what is obtained in embryos that lack Runt, and is presumed to be in response to other activating factors. The complications presented by these other factors are bypassed in experiments in which Runt is over-expressed, either by heat-shock or by NGT-driven expression. Indeed, the inability of ftz to respond to ectopic Runt in the absence of maternal Lilli provides very compelling evidence for the importance of Lilli in ftz activation (Vanderzwan-Butler, 2006).

Lilli is acutely required for mediating Runt-dependent activation of ftz during the blastoderm stage, and this requirement precedes the temporal requirements for Ftz-F1. Ftz-F1 was initially identified as a factor that interacts with sequences within the ftz 'zebra element', a 669-bp, promoter proximal element that drives early expression in response to gap and pair-rule gene transcription factors. However, subsequent studies revealed that Ftz-F1 plays a more significant role in mediating Ftz-dependent auto-regulation by the so-called 'upstream element' during the early stages of germ-band extension. The earlier requirement for Lilli strongly suggests it contributes to the early 'zebra element'-dependent activation of ftz in response to activating inputs from Runt (Vanderzwan-Butler, 2006).

What is the role of Lilli in mediating Runt-dependent activation? Directed yeast two-hybrid assays fail to detect direct interactions between the full length Runt and Lilli proteins. This observation suggests that other factors contribute to the functional interactions described above. A notable conserved feature that Lilli shares with its mammalian homologs is an HMG-box. This structural DNA-binding motif interacts with the minor groove of DNA and modulates DNA structure by bending. These properties have been interpreted to reflect an architectural role for HMG-box proteins in facilitating the formation of higher order chromatin structures that contribute to the regulation of gene expression. It seems likely that chromatin architecture is important for ftz zebra element function. Although the 'zebra element' was one of the very first cis-regulatory elements in the segmentation pathway to be described, there is not yet a clear understanding of the rules that govern its activity. This is in contrast to the relatively simple combinatorial rules that have been elucidated for several of the stripe-specific elements of the pair-rule genes eve, hairy, and runt. It is proposed that the difficulty in identifying discrete regulatory modules within the zebra element reflects the importance of chromatin architecture in conferring high-fidelity regulation of the zebra element in response to inputs from Runt and other gap and pair-rule transcription factors (Vanderzwan-Butler, 2006).

It is interesting to note that the cis-regulatory element responsible for the initial activation of Sxl shares several properties with the ftz zebra element. The minimal DNA element necessary to faithfully recapitulate the strong, early sex-specific activation of the Sxl promoter is 1.7 kb in size. As found for the ftz zebra element, smaller reporter gene constructs do not function properly, although sub-elements that confer sex-specific regulation, and augment this activation have been identified. The on/off regulation of Sxl is in response to a twofold difference in the activity of four different DNA-binding transcription factors. It is easy to imagine that chromatin architecture may be critical in sensing this twofold difference in a robust and reproducible manner. There is one further similarity shared by Sxl and ftz that is intriguing. The initial Lilli-dependent activation of both genes is followed by a second phase of gene expression that involves distinct cis-regulatory components. In the case of ftz, the switch is from regulation by the zebra element to regulation by the upstream element, whereas for Sxl the switch is from expression at the Sxl_Pe promoter to expression at Sxl_M a different promoter that is activated in all somatic cells of both male and female embryos . Perhaps the unique requirements for Lilli reflect architecture-dependent regulatory elements that retain the ability to be rapidly re-organized in a developmentally dynamic manner. Further studies on the mechanisms by which Lilli participates in the activation of ftz and Sxl in the early Drosophila embryo should provide new insights on the role of chromatin architecture in developmentally regulated gene expression (Vanderzwan-Butler, 2006).

Drosophila lilliputian is required for proneural gene expression in retinal development

Proper neurogenesis in the developing Drosophila retina requires the regulated expression of the basic helix-loop-helix (bHLH) proneural transcription factors Atonal (Ato) and Daughterless (Da). Factors that control the timing and spatial expression of these bHLH proneural genes in the retina are required for the proper formation and function of the adult eye and nervous system. This study reports that lilliputian (lilli), the Drosophila homolog of the FMR2/AF4 family of proteins, regulates the transcription of ato and da in the developing fly retina. lilli controls ato expression at multiple enhancer elements. lilli was found to contributes to ato auto-regulation in the morphogenetic furrow by first regulating the expression of da prior to ato. FMR2 regulates the ato and da homologs MATH5 and TCF12 in human cells, suggesting a conservation of this regulation from flies to humans. It is concluded that lilli is part of the genetic program that regulates the expression of proneural genes in the developing retina (Distefano, 2012).

This study has shown that lilli, the Drosophila homolog of the FMR2/AF4 family of proteins, regulates the transcription of the bHLH proneural genes atonal and daughterless in the developing retina and antenna of flies. It was further shown that this transcriptional regulation is conserved from flies to human cells. The data suggest that lilli regulates ato differentially at the 5' and 3' enhancer elements. The 3' cis-regulatory element is a 1.2-kb region of DNA located approximately 3.1 kb downstream of the ato transcription unit, and controls the early phase of ato expression in the developing retina. At this element, lilli appears to regulate transcription of ato at multiple sites (Distefano, 2012).

While analysis of transcription driven by a 5.8-kb element described previously is significantly reduced to less than 25% of controls, transcription driven by a minimal 348-bp enhancer element found within the larger 5.8-kb 3' enhancer is only reduced to 55% of controls. An important question remains for lilli-mediated ato transcription: is lilli function required to directly induce ato expression, or rather to maintain expression once previously induced? Given its function as a transcriptional activator, lilli function may be directly required to turn on ato expression at the 348-bp enhancer element in the developing retina. However, lilli may also be required to maintain ato expression once activated by other factors (such as Sine oculis or Eyeless/ Pax6) at this enhancer element. Alternately, other cis-regulatory elements along the 5.8- kb enhancer region may require lilli function to modulate ato expression after activation. Further experimentation will be required to answer these questions (Distefano, 2012).

The data also show that lilli-mediated regulation of ato expression at the 5' enhancer region requires the expression of the Da protein. The ato protein auto-regulates its expression at the 50 enhancer region to sustain ato gene expression in the intermediate groups and single R8 photoreceptors. It is hypothesized that the lilli protein regulates ato expression indirectly, by affecting ato auto-regulation at the 5' enhancer element. This hypothesis on multiple observations. (1) ato protein expression and ato transcription from the 5' enhancer element is decreased, but not absent within lilli mutant clones, consistent with an indirect effect on ato transcription. (2) Da protein expression and da transcription is also decreased, but not absent within lilli mutant clones in the region corresponding to the 5' enhancer expression. (3) By replacing Da expression within lilli mutant clones, ato transcription directed by the 5' enhancer element can be fully rescued, but not ato transcription directed by the 3' enhancer element. This is also true for ato protein expression in these clones (Distefano, 2012).

Thus, the lilli protein must first induce da transcription in cells in the intermediate groups and R8s prior to ato expression within these cells. Then, Ato-Da heterodimers can form, and maintain the activation of ato expression within the intermediate groups and R8 cells. If lilli function is compromised, da expression decreases, as does ato's ability to auto-regulate (Distefano, 2012).

This study has shown that lilli is required for the proper expression of hairy, another bHLH factor that is expressed anterior to the furrow. Hairy is an inhibitory factor to furrow progression. Interestingly, ato and da expression does not expand anteriorly in lilli clones, suggesting that loss of hairy anterior to the furrow is not sufficient to remove all of hairy function in these clones. Still, it is interesting that this analysis has identified a third bHLH factor regulated by lilli, and may suggest that lilli protein functions broadly to regulate the expression of multiple bHLH factors in different tissues (Distefano, 2012).

lilli is homologous to the AF4/FMR2 family of nuclear proteins in humans. This family includes FMR2, LAF4, AF4, and AF5Q31. Members of this family are implicated in acute lymphoblastic leukemia and FRAXE nonsyndromic Fragile X mental retardation (Distefano, 2012).

This study has shown that FMR2 also regulates the expression of the proneural genes MATH5 and TCF12 in HEK293 cells, showing that the observations made in the fly retina are conserved in human cell. Patients with FRAXE exhibit various developmental and morphological problems, including mental retardation, delays in speech development, attention deficit disorder, hyperactivity, and impaired motor coordination. While the etiology of these symptoms remains unknown, defects in neurogenesis, and/or the regulation of critical transcription factors such as the human homologues of ato and Da may be related to these symptoms. Further, recent evidence has shown that ato also functions as a tumor suppressor gene. This may provide an additional link between the misregulation of bHLH protein in lilli mutants and the leukemia observed in AF4 mutants. Further research is required to determine the significance of this connection (Distefano, 2012).

DEVELOPMENTAL BIOLOGY

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).

EFFECTS OF MUTATION

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 dpp^d-ho/dpp^hr4 display wild-type wings. The dpp^d-ho mutation is a small deletion in the 3' cis-regulatory region of dpp. dpp^d-ho homozygous flies have wings that are held out laterally from the body axis. The dpp^hr4 mutation is a missense mutation in the protein-coding region of dpp. When homozygous, the dpp^hr4 allele is embryonic lethal. When dpp^d-ho and dpp^hr4 are paired, the wild-type regulatory region of the dpp^hr4 allele appears to act in trans on the wild-type coding region of the dpp^d-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, dpp^d-ho/dpp^hr4 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 dpp^d-ho/dpp^hr4 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 dpp^hr4/dpp^d-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 dpp^d-ho/dpp^hr4 transvection-dependent phenotype. Df(2L)C144 and l(2)a16 were tested for the ability to disrupt this phenotype. Forty-six percent of dpp^d-ho Df(2L) c144 /dpp^hr4 flies had heldout wings; of these flies, 47% were severely heldout. Twenty percent of dpp^d-ho l(2)a16/dpp^hr4 flies had heldout wings; of these flies, 50% were severely heldout. These results are similar to those of DTD46.4. Twenty-six percent of dpp^d-ho DTD46.4/dpp^hr4 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 dpp^d-ho/dpp^hr4 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 lilli^s35 or lilli^xs407. 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 dpp^e87, dpp^hr56, dpp^hr4, and dpp^hr92. No genetic interactions were detected with the weak alleles dpp^e87 and dpp^hr56. However, all lilli alleles tested showed significant dominant maternal enhancement of the strong alleles dpp^hr4 and dpp^hr92. Modest dominant zygotic enhancement of dpp^hr4 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, scw^S12, or gbb¹. However, lilli alleles show dominant maternal enhancement of the recessive lethality of scw^E1. scw^E1 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 dpp^hr56 and scw^E1. 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)2A^161H1) 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 scw^E1 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-Yan^ACT 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 (lilli^XS575 and lilli^XS407) 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 raf^HM7 allele. The raf^HM7 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 raf^HM7 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 Raf^torY9 transgenes were used. One contained the sev enhancer fused to the hsp70 promoter (sE), the other the sev enhancer fused to the sev promoter (sE^sP). 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 Sev^S11, 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-Sev^S11 or sEsP-Ras^V12 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 raf^HM7, 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. lilli^4U5 and lilli^15D1) 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 lilli^4U5). The degree of head size reduction allows for the definition of a class of strong alleles (e.g. lilli^4U5 , lilli^15D1 ) and a class of weak alleles (e.g. lilli^3E8, lilli^P1, lilli^P4). 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).

EVOLUTIONARY HOMOLOGS

Five folate-sensitive fragile sites have been characterized at the molecular level (FRAXA, FRAXE, FRAXF, FRA16A and FRA11B). Three of them (FRAXA, FRAXE and FRA11B) are associated with clinical problems, and two of the genes (FMR1 in FRAXA and CBL2 in FRA11B) have been identified. All of these fragile sites are associated with (CCG)n/(CGG)n triplet expansions that are hypermethylated beyond a critical size. FRAXE is a rare folate sensitive fragile site only recently recognized. Its cytogenetic expression was found to involve the amplification of a (CCG)n repeat adjacent to a CpG island. Normal alleles vary from 6 to 25 copies. Expansions of greater than 200 copies were found in FRAXE expressing males and their FRAXE associated CpG island was fully methylated. An association of FRAXE expression with concurrent methylation of the CpG island and mild non-specific mental handicap in males has been reported by several groups. The cloning and characterization of a gene (FMR2) adjacent to FRAXE is reported. Elements of FMR2 were initially identified from sequences deleted from a developmentally delayed boy. Loss of FMR2 expression is correlated with (CCG)n expansion at FRAXE, demonstrating that this is a gene associated with the CpG island adjacent to FRAXE and contributes to FRAXE-associated mild mental retardation (Gecz, 1996).

Five folate-sensitive fragile sites have been identified at the molecular level to date. Each is characterized by an expanded and methylated trinucleotide repeat CGG (CCG). Of the three X chromosome sites, FRAXA, FRAXE and FRAXF, the former two are associated with mental retardation in their expanded forms. FRAXA expansion results in fragile X syndrome due to down regulation of expression of the FMR1 gene, which carries the hypermutable CGG repeat in the 5' untranslated portion of its first exon. Mild mental retardation without consistent physical findings has been found associated with expanded CCG repeats at FRAXE. A large gene (FMR2) has been identified, transcribed distally from the CpG island at FRAXE, and down-regulated by repeat expansion and methylation. The gene is novel, expressed in adult brain and placenta, and shows similarity with another human protein, MLLT2, expressed from a gene at chromosome 4q21 involved in translocations found in acute lymphoblastic leukaemia (ALL) cells. Identification of this gene will facilitate further studies to determine the role of its product in FRAXE associated mental deficiency (Gu, 1996).

A novel human gene, LAF-4, was isolated from a subtracted cDNA library that showed strong sequence similarity to AF-4, a gene that is translocated in t(4;11)(q21;q23) acute lymphoblastic leukemias (ALLs). In t(4;11) ALL, the AF-4 gene at 4q21 is translocated into the MLL locus at 11q23, resulting in the expression of an MLL/AF-4 fusion protein that is the presumptive oncoprotein. AF-4 and LAF-4 are homologous throughout their coding regions, yet neither protein is related to previously cloned genes. Human LAF-4 readily hybridizes with genes in mouse and chicken, thus showing that this gene family has been highly conserved during vertebrate evolution. In mouse tissues, LAF-4 mRNA was found to be present at highest levels in lymphoid tissues, present at lower levels in brain and lung, and absent from other tissues. In human and mouse lymphoid cell lines, LAF-4 expression is highest in pre-B cells, intermediate in mature B cells, and absent in plasma cells, thus pointing to a potential regulatory role for LAF-4 in lymphoid development. Antibodies to LAF-4 showed it to be a nuclear protein that shows an uneven, granular immunofluorescence pattern. In vitro-translated LAF-4 is able to bind strongly to double-stranded DNA cellulose. Furthermore, both LAF-4 and AF-4 have domains that activate transcription strongly when fused to the GAL4 DNA-binding domain. Interestingly, the AF-4 transactivation domain is retained in the MLL/AF-4 fusion protein; thus, it may contribute to the transforming potential of the oncoprotein. Therefore, the cloning of LAF-4 has defined a new family of potential regulatory proteins that may function in lymphoid development and oncogenesis (Ma, 1996).

FMR2 is the gene associated with FRAXE mental retardation. It is expressed as an 8.7-kb transcript in placenta and adult brain. A fetal-specific FMR2 transcript of approximately 12 kb was detected in fetal brain and at a lower level in fetal lung and kidney. FMR2 is a large gene composed of 22 exons spanning at least 500 kb on Xq28. Alternative splicing involving exons 2, 3, 5, 7, and 21 is not tissue specific as tested on mRNA from human fetal and infant brain. FMR2 is translated into a 1311-amino-acid nuclear protein with putative transcription transactivation potential. Subcellular localization studies with green fluorescent protein as a reporter show that both nuclear addresses found in the FMR2 sequence are functional and direct the FMR2 protein into the nucleus. FMR2 together with AF4 and LAF4 forms a new family of nuclear proteins with DNA-binding capacity and transcription transactivation potential. BLAST searches of the dbEST database have revealed the presence of at least two other groups of nonoverlapping ESTs showing high similarity to the FMR2-related family of proteins. One of them, represented by the EST W26686, maps to chromosome 5q31. Amino acid similarity among the proteins encoded by members of the gene family is high in the NH2 terminus, low in the middle, and high again in the COOH end. Available information from members of the family shows that genomic organization is conserved. This FMR2-related gene family encodes nuclear proteins with involvement in mental retardation (FMR2), cancer (AF4), and lymphocyte differentiation (LAF4) or with unknown function (Gecz, 1997).

Acute leukemia with t(4;11)(q21,q23) translocation results from the in-frame fusion of the MLL to the AF4/FEL gene. AF4 transcripts are present in a variety of hematopoietic and nonhematopoietic human cells. To further study the wild-type and leukemia fusion AF4, glutathione S-transferase (GST)-fusion proteins were used as immunogens to produce rabbit polyclonal antibodies that are specific for normal and chimeric AF4 proteins. Using Western blotting analysis, it was demonstrated that the AF4 gene encodes proteins with apparent molecular weight of 125 and 145 kD. A 45-kD protein coprecipitates with AF4 protein in immunoprecipitation. Also, the anticipated MLL-AF4-encoded 240-kD protein is detected in all cell lines with t(4;11) translocations; fusion proteins are present in lesser quantity than the wild-type AF4. The proteins recognized by the antibodies are of the predicted sizes of the AF4 and MLL-AF4-encoded proteins. The MLL-AF4 fusion protein has a similar subcellular distribution as AF4. Both t(4;11) and non-t(4;11) leukemic cells show a similar pattern of punctate nuclear staining in all cell lines tested. AF4 antibodies should be useful for further elucidation of the function of AF4 in normal cellular physiology, as well as the function of MLL-AF4 in leukemogenesis. The antibodies should also be helpful for the diagnosis of the MLL-AF4 fusion proteins in t(4;11) leukemias (Li, 1998).

The expression of the FRAXE fragile site on the human X chromosome is associated with the expansion of a CCG repeat at the 5' end of the FMR2 gene. The repeat expansion results in transcriptional silencing of the gene and this event has been found to be associated with mild mental handicap in families. The gene is particularly abundantly expressed in the hippocampus and amygdala. The expression pattern of the homologous gene is demonstrated in adult mouse brain and early mouse embryos. High levels of fmr2 mRNA have been noted in the hippocampus, the piriform cortex, Purkinje cells and the cingulate gyrus. Expression of fmr2 occurs on, or before, day 7 in the embryo and reaches its highest levels at 10.5-11.5 days. A more detailed analysis shows that the fmr2 expression in the embryo at 11 days is more specific and evident in the roof of the hind brain and the lateral ventricle of the brain. The coding sequence of the mouse fmr2 gene shows very high conservation with 88% amino acid identity to the human FMR2 sequence (Chakrabarti, 1999).

The transcriptional silencing of the FMR2 gene has been implicated in FRAXE mental retardation. FRAXE individuals have been shown to exhibit learning deficits, including speech delay, reading and writing problems. FMR2 encodes a large protein of 1311 amino acids and is a member of a gene family encoding proline-serine-rich proteins that have properties of nuclear transcription factors. To characterize the expression of the fragile X mental retardation 2 (FMR2) protein, polyclonal antibodies were raised against two regions of the human FMR2 protein and used in immunofluorescence experiments on mouse brain cryosections. The FMR2 protein is localized in neurons of the neocortex, Purkinje cells of the cerebellum and the granule cell layer of the hippocampus. FMR2 staining is shown to colocalize with the nuclear stain DAPI confirming that FMR2 is a nuclear protein. The localization of FMR2 protein to the mammalian hippocampus and other brain structures involved with cognitive function is consistent with the learning deficits seen in FRAXE individuals (Miller, 2000).

Some chromosomal translocations in acute leukemias involve the fusion of the trithorax-related protein Mll (also called HRX, All1 or Htrx) with a variety of heterologous proteins. In acute lymphoblastic leukemia associated with the t(4;11)(q21;q23) translocation, the 4q21 gene that fuses with Mll is AF4. To gain insight into the potential role of AF4 in leukemogenesis and development, this gene was inactivated by homologous recombination in mice. As expected from the tissue distribution of the AF4 transcript, development of both B and T cells is affected in AF4 mutant mice. A severe reduction of the thymic double positive CD4/CD8 [CD4(+)/CD8(+)] population was observed; in addition most double- and single-positive cells express lower levels of CD4 and CD8 coreceptors. Most importantly, the reconstitution of the double-positive compartment by expansion of the double-negative cell compartment is severely impaired in these mutant mice. In the bone marrow pre-B and mature B-cell numbers are reduced. These results demonstrate that the function of the mAF4 gene is critical for normal lymphocyte development. This raises the possibility that the disruption of the normal AF4 gene or its association with Mll function by translocation may orient the oncogenic process toward the lymphoid lineage. This represents the first functional study using a knock-out strategy on one of the Mll partner genes in translocation-associated leukemias (Isnard, 2000).

REFERENCES

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