Chip

DEVELOPMENTAL BIOLOGY

Embryonic

Chip protein is maternally contributed during oogenesis. From the early cellular blastoderm stage through gastrulation to the end of embryogenesis, Chip protein is present in most, if not all nuclei, including the pole cell nuclei. Staining is undetectable or very weak in syncytial blastoderm nuclei until just before cellularization, although Chip must be present at low levels, because lack of Chip activity affects expression of segmentation genes at this stage (Morcillo, 1997).

Larval

Chip is present in the nuclei of larval tissue, including imaginal discs, fat body, and salivary gland. Chip associates with a very large number of specific sites on larval polytene chromsomes (Morcillo, 1997).

Intrinsic control of precise dendritic targeting by an ensemble of transcription factors

Proper information processing in neural circuits requires establishment of specific connections between pre- and postsynaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order, and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. A search was performed for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. Two POU-domain TFs, acj6 and drifter have been identified as essential players. After testing 13 additional candidates, four TFs were identified, (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip, that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit (Komiyama, 2007).

For technical simplicity, larval born GH146-Gal4-positive PNs, originating from three neuroblast lineages, anterodorsal (adPNs), lateral (lPNs), and ventral (vPNs), were studied. Out of ~25 classes defined by their glomerular targets, focus was placed on 17 classes whose target glomeruli are reliably recognized across different animals. The MARCM technique allows visualization and genetic manipulation of PNs in neuroblast and single-cell clones in otherwise heterozygous animals, so PN-intrinsic programs can be studied for dendritic targeting. GH146 is expressed only in postmitotic PNs (Komiyama, 2007).

acj6 and drifter have been identified as lineage-specific regulators of PN dendritic targeting. To identify additional transcription factors (TFs) that regulate dendritic targeting of different PN classes, candidates were tested that have been shown to regulate neuronal subtype specification and targeting specificity and have available loss-of-function mutants. The following was tested; (1) the expression of candidate genes in PNs at 18 hr after puparium formation (APF) when PN dendrites are in the process of completing their initial targeting, and/or (2) their requirement in PNs by examining dendritic targeting in homozygous mutant MARCM clones (Komiyama, 2007).

In addition to the eight genes described below, five other TFs were examined that were not pursued because of the lack of expression in GH146-PNs at 18 hr APF (aristaless and pdm-1) or the lack of targeting defects in homozygous mutant PNs (abrupt [ab^k02807], kruppel [Kr¹], and Dichaete [Dichaete⁸⁷]) (Komiyama, 2007).

LIM-HD factors and PN targeting: LIM-homeodomain (LIM-HD) TFs are involved in multiple events during neuronal development. Most functions of LIM-HD factors require the LIM domain-binding cofactor, which is represented in Drosophila by ubiquitously expressed Chip. Chip antibody revealed ubiquitous expression of Chip in cells around the antennal lobe (AL) including all GH146-PNs at 18 hr APF (Komiyama, 2007).

The requirement of Chip in PN dendritic targeting was tested. Wild-type adPNs, lPNs, and vPNs target stereotyped sets of glomeruli. PNs homozygous for a Chip null allele (Chip^e5.5) failed to target most of the correct glomeruli and occupied inappropriate glomeruli. Most adPN and lPN clones (12/13) also mistargeted a fraction of dendrites to the structure ventral to the AL, the suboesophaegeal ganglion (SOG). Thus, Chip is required for targeting specificity of most, if not all, PN classes studied here, and Chip-interacting proteins including LIM-HD factors likely play important roles in PN dendritic targeting (Komiyama, 2007).

Five LIM-HD factors have been characterized in Drosophila: apterous, arrowhead, islet, lim1, and lim3. apterous, arrowhead, or lim3 were not pursued because they are not expressed in GH146-PNs at 18 hr APF (apterous) or they do not have targeting defects in PNs homozygous for null alleles (lim3^37Bd6 and awh¹⁶) (Komiyama, 2007).

Islet antibody detected Islet expression in ~50% adPNs and most lPNs but not in vPNs at 18 hr APF and adult. isl^−/− adPNs failed to target many (but not all) of the normal target glomeruli, including VA1lm, VA3, and VM7. In addition, DA1, a lPN target, was often specifically mistargeted. Defects of isl^−/− lPNs were very similar to Chip^−/− lPN defects. A fraction of dendrites often mistargeted to the SOG. Within the AL, dendrites were diffusely spread, although DA1 and DL3 were always correctly innervated. Targeting of isl^−/− vPNs was normal, consistent with their lack of Islet expression (Komiyama, 2007).

Lim1 antibody revealed Lim1 expression in most or all vPNs, but not in adPNs or lPNs in adults. The expression pattern appears similar at 18 hr APF, although vPNs are difficult to identify unambiguously at early stages. lim1^−/− adPNs showed no defects, consistent with the lack of Lim1 expression. lim1^−/− lPNs rarely showed a cell number decrease, but in clones in which the cell number was normal, lim1^−/− lPNs targeted correct glomeruli. In contrast, lim1^−/− vPNs showed a specific targeting defect. Wild-type vPNs innervate DA1 and VA1lm densely because of the single vPNs that specifically innervate these glomeruli, in addition to the diffuse innervation all over the AL contributed by the pan-AL vPN. In lim1^−/− vPNs, DA1 innervation was greatly reduced and sometimes undetectable. Therefore, lim1 is required for dendritic targeting by a single vPN class, vDA1, despite its general expression in vPNs. lim1 might be redundant with other factors in non-DA1 vPNs. It was note that phenotypes of islet and lim1 combined are only a subset of the Chip phenotype. Additional Chip phenotype may be explained by non-Lim-HD molecules interacting with Chip (Komiyama, 2007).

Effects of Mutation or Deletion

Dorso-ventral axis formation in the Drosophila wing requires the localized accumulation of the Apterous LIM/homeodomain protein in dorsal cells. dLdb/Chip encodes a LIM-binding cofactor that controls Ap activity. Both lack and excess of dLdb/Chip function cause the same phenotype as apterous (ap) lack of function; i.e. dorsal to ventral transformations, generation of new wing margins, and wing outgrowths. These results indicate that the normal function of Ap in dorso-ventral compartmentalization requires the correct amount of the Chip co-factor, and suggest that the Ap and Chip proteins form a multimeric functional complex. In support of this model, it has been shown that the dLdb/Chip excess-of-function phenotypes can be rescued by ap overexpression (Fernández-Fúnez, 1998).

Chip mutations behave as strong enhancers of wing phenotypes produced by hypomorphic ap mutations. This synergistic interaction suggests that Chip and ap have related functions. To investigate further the function of Chip during wing development, genetic mosaics were generated by induced mitotic recombination using the Minute technique. Clones of Chip mutant cells in the wing ventral compartment show a wild-type phenotype and appear with normal frequencies, indicating that Chip is not required in this compartment. In contrast, Chip clones in the dorsal compartment are associated with wing outgrowths and ectopic wing margins. Cells within these clones have a ventral identity revealed by the morphology of the wing margin bristles they differentiate. Normal cells abutting the mutant clones are induced to form the dorsal structures characteristic of the wing margin. The ectopic margins can be visualized in undifferentiated imaginal discs with the use of molecular markers that label the wing margin. The largest wing outgrowths correspond to clones far from the normal wing margins. These clones cause the outgrowth of wild-type tissue, with the mutant clones located at the tip of the outgrowth . Thus, although Chip is expressed in all wing cells, Chip mutations produce specific phenotypes that are indistinguishable from ap phenotypes in clones. One possibility is that normal Chip function is required for ap expression. To test this possibility, ap expression was monitored in Chip mutant clones induced in wing imaginal discs. Ap protein is shown to accumulate normally in Chip mutant cells. Thus Chip does not regulate ap expression but it does show genetic interactions with ap, and Chip also produces the same mutant phenotypes in genetic mosaics. Taken together, these results are consistent with the hypothesis that Chip encodes a co-factor required for ap function as a dorsal selector gene (Fernández-Fúnez, 1998).

If Ap and Chip physically interact forming a functional complex, their stoichiometry may be important for the formation of the complex and for dorso-ventral patterning. To test whether the levels of Chip expression are important for dorso-ventral patterning, Chip was overexpressed in various patterns. Overexpression of Chip using a decapentaplegic GAL4 driver results in wing outgrowths, the creation of an ectopic wing margin on the dorsal compartment and a cut in the wing. These phenotypes are also evident in imaginal discs using wingless-lacZ (wglacZ) expression as a marker of the wing margin. Because lack of ap function in clones also causes wing outgrowths and ectopic wing margins, the distribution of Ap protein was examined in these wing imaginal discs. Chip overexpression does not alter the distribution of Ap protein; these results indicate that the phenotypes produced by Chip overexpression are not caused by Chip repressing ap. Interestingly, overexpression of ap using the drivers dppGAL4 or apGAL4 described above does not result in wing abnormalities in the dorsal compartment. Thus overexpression of Chip results in the same phenotype as its lack of function, i.e. transformation of dorsal into ventral cells, and, as a consequence, wing margin formation and outgrowth. These observations suggest that the relative amounts of Ap and Chip are important for dorso-ventral patterning (Fernández-Fúnez, 1998).

If the relative amounts of Ap and Chip are critical for dorso-ventral patterning, then it should be possible to rescue the excess of Chip phenotype by overexpressing ap. The proposed domain structure of the LDB/NLI family of proteins provides a conceptual framework to understand the phenotypes produced by altering the dosage of Chip and ap. The presence of homodimerization and LIM-interacting domains in LDB/NLI proteins suggests that LDB and LIM domain proteins may form tetrameric complexes. These complexes would be formed by two LDB molecules interacting through the N-terminal homodimerization domain; in addition, each LDB molecule would interact with a LIM domain through the C-terminal LIM-interacting domain. The occurrence of these complexes has been demonstrated between murine LDB and the hamster LIM/homeodomain protein LMX1. In the case of Chip-Ap, this tetrameric complex may be the functional complex carrying out the dorso-ventral patterning functions. This model predicts that Chip overexpression would lead to the formation of non-functional complexes, and it also predicts that functional Chip-Ap complexes would be reconstituted by overexpressing ap in addition to Chip. To test this model, a GAL4 insertion in ap was used. Expression from this GAL4 driver faithfully reproduces ap expression in the wing imaginal disc. When Chip is expressed from the ap promoter, the wing is reduced or eliminated depending on the UAS:Chip transgenic line used. Most lines reduce the wings, whereas the strongest line completely eliminates them. The reduced wing phenotype can be completely rescued by ap overexpression. These results provide further evidence for the idea that the stoichiometry of Ap and Chip is critical for dorso-ventral patterning (Fernández-Fúnez, 1998).

Cuticle of embryos lacking maternal Chip exhibit normal dorsal-ventral polarity, but have severe segmentation defects. Most cuticles have a single fused, irregularly shaped patch of ventral denticles. A few rare embryos display a pair-rule-like phenotype, with approximately half the normal number of segments (Morcillo, 1997).

The mechanisms that allow enhancers to activate promoters from thousands of base pairs away are disrupted by the Drosophila Suppressor of Hairy-wing protein (Su[Hw]). Su[Hw] binds a DNA sequence in the gypsy retrotransposon and prevents activation of promoter-enhancers that are distal to a gypsy insertion in a gene without affecting promoter-enhancers that are proximal to gypsy (next to the structural gene). Several observations indicate that SUHW does not affect enhancer-binding activators. Instead, SUHW may interfere with factors that structurally facilitate interactions between an enhancer and promoter. To identify putative enhancer facilitators, a screen for mutations that reduce activity of the remote wing margin enhancer in the cut gene was performed. Mutations in scalloped, mastermind, and a previously unknown gene, Chip, were isolated. A TEA DNA-binding domain in the Scalloped protein binds the wing margin enhancer. Interactions among scalloped, mastermind and Chip mutations indicate that Mastermind and Chip act synergistically with Scalloped to regulate the wing margin enhancer. Chip is essential and also affects expression of a gypsy insertion in Ultrabithorax. Relative to mutations in either scalloped or mastermind, a Chip mutation hypersensitizes the wing margin enhancer in cut to gypsy insertions. Therefore, Chip might encode a target of su(Hw) enhancer-blocking activity (Morcillo, 1996).

Chip may encode an enhancer-facilitator, acting to facilitate the activity of distal enhancers. The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila Suppressor of Hairy-wing protein [su(Hw)] that binds to gypsy retrovirus insertions between enhancers and promoters. su(Hw) bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. Chip is needed for the wing margin enhancer of cut. The Chip mutation dominantly enhances the mutant phenotypes displayed by partially suppressing gypsy insertions in both cut and Ultrabithorax and is a homozygous larval lethal, indicating that Chip regulates multiple genes. Chip is normally required for the wing margin enhancer function of cut because Chip mutations enhance the cut wing phenotype of a cut mutation. Heterozygotes for Chip display cut wing phenotypes when either scalloped or mastermind (mam) is also a heterozygous mutant. Both Sc and Mam are known to regulate the cut distal enhancer, but in contrast to sd and mam mutants, Chip mutants display stronger genetic interactions with gypsy insertions than with wing margin enhancer deletions. Thus, in a heterozygous Chip mutant, a heterozygous gypsy insertion in cut displays a cut wing phenotype, whereas a heterozygous enhancer deletion does not. Dependence on the nature of the heterozygous lesion in the regulatory region strongly suggests that Chip directly regulates cut. More strikingly, it indicates that in a Chip heterozygote, a gypsy insertion is more deleterious to enhancer function than deletion of the enhancer. The simplest explanation is that su(Hw) bound to a gypsy insertion in one cut allele acts in a transvection-like manner (interchromosomally) to block the wing enhancer in the wild-type cut allele on a second chromosome. This implicates Chip in enhancer-promoter communication (Morcillo, 1997 and references).

LIM-homeodomain transcription factors are expressed in subsets of neurons and are required for correct axon guidance and neurotransmitter identity. The LIM-homeodomain family member Apterous requires the LIM-binding protein Chip to execute patterned outgrowth of the Drosophila wing. To determine whether Chip is a general cofactor for diverse LIM-homeodomain functions in vivo, its role in the embryonic nervous system was studied. Loss-of-function Chip mutations cause defects in neurotransmitter production that mimic apterous and islet mutants. Chip is also required cell-autonomously by Apterous-expressing neurons for proper axon guidance, and requires both a homodimerization domain and a LIM interaction domain to function appropriately. Using a Chip/Apterous chimeric molecule lacking domains normally required for their interaction, the complex was reconstituted and the axon guidance defects of apterous mutants, of Chip mutants and of embryos doubly mutant for both apterous and Chip were rescued. These results indicate that Chip participates in a range of developmental programs controlled by LIM-homeodomain proteins and that a tetrameric complex comprising two Apterous molecules bridged by a Chip homodimer is the functional unit through which Apterous acts during neuronal differentiation (van Meyel, 2000).

Chip is expressed in most, if not all, embryonic and larval tissues. In wild-type embryos, strong, nuclear Chip expression is found throughout the developing VNC with no apparent subclasses of neurons excluded. A substantial fraction of embryonic Chip is contributed maternally during oogenesis, and this maternally derived expression is required for early embryonic segmentation. To estimate the relative contribution of zygotic and maternally derived Chip to the embryonic VNC, homozygous embryos mutant for a Chip null allele were examined. Derived from an intercross of heterozygous parents, mutants are expected to retain half the maternal and not any zygotic Chip expression. Little reduction of staining in mutant embryos is observed, relative to Chip/+ heterozygotes. Thus it appears a substantial fraction of Chip in the VNC is provided maternally. Co-labelling embryos with anti-Ap and anti-Chip antibodies reveals that Chip expression overlaps with all the Ap neurons of the developing VNC (van Meyel, 2000).

If Chip were required for Ap function, elimination of Chip might be expected to result in an ap-like phenotype. The requirement of maternally supplied Chip in segmentation precluded an examination of the effects of eliminating both maternal and zygotic Chip on neuronal development. Thus, neurotransmitter expression and axon guidance were examined in Chip mutants in which half of the maternal and all of the zygotic Chip expression were absent. In each thoracic hemisegment of the VNC, ap is expressed in a lateral cluster of four neurons, one of which is the Tv neuroendocrine cell that expresses the neurotransmitter dFMRFa. In wild-type embryos, there are a total of six Tv cells, one in each thoracic hemisegment. In ap mutants, the Tv neurons are present, but half of all Tv neurons stochastically fail to express dFMRFa. This regulation of dFMRFa by ap is transcriptional, since expression of a fusion transgene comprising a 446 bp Tv neuron-specific enhancer of the dFMRFa gene driving beta-galactosidase (Tv-lacZ) is similarly reduced in ap mutants. Ap binds in vitro to each of three sequences within the enhancer, and mutagenesis of these sites has confirmed that these sequences are important for Tv-lacZ expression in vivo (van Meyel, 2000).

To determine whether reduction of Chip results in an ap-like reduction in transcriptional activation of dFMRFa, expression of the Tv-lacZ reporter transgene was assayed in wild-type, ap and Chip mutant embryos. Both ap and Chip mutant embryos show decreased Tv-lacZ activity in Tv neurons relative to wild-type controls, implicating Chip in the establishment of this Ap-regulated aspect of neuronal differentiation. The reduction of Tv-lacZ activity is less severe in Chip null mutants than ap null mutants, probably because of the maternally supplied Chip remaining in Chip mutants. In embryos homozygous for an antimorphic Chip mutation, Tv-lacZ expression is reduced further than Chip null mutants but not to the level of ap mutants (van Meyel, 2000).

Like Ap, the LIM HD protein Isl also regulates neurotransmitter identity of embryonic neurons. There are three dopaminergic cells per segment of the VNC, one unpaired midline cell and a pair of dorsal lateral cells, all of which express Isl protein and thus represent a subset of the isl interneurons. isl mutants show loss of expression of tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of dopamine. To test the role of Chip in the expression of TH, late-stage wild-type and Chip mutant embryos were stained with anti-TH antibodies. Homozygous Chip mutant embryos retain TH expression in the ventral unpaired midline cells, but few of the dorsal lateral cells express TH, and in those that do, TH levels are significantly reduced relative to wild-type. In embryos homozygous for a Chip antimorph, TH expression is greatly diminished in both the ventral midline and dorsal lateral dopaminergic neurons. While it is clear that the paired dorsal TH cells are more sensitive to the reduction in Chip dosage than the unpaired ventral cells, the effects of the antimorphic Chip allele suggest that TH production in the latter cells is also dependent on Chip. From these results, together with the above results on the expression of FMRFamide, it is concluded that Chip is required for both Ap- and Islet-regulated neurotransmitter production in the CNS (van Meyel, 2000).

tailup, a LIM-HD gene, and Iro-C cooperate in Drosophila dorsal mesothorax specification

The LIM-HD gene tailup has been categorised as a prepattern gene that antagonises the formation of sensory bristles on the notum of Drosophila by downregulating the expression of the proneural achaete-scute genes. tup has an earlier function in the development of the imaginal wing disc; namely, the specification of the notum territory. Absence of tup function causes cells of this anlage to upregulate different wing-hinge genes and to lose expression of some notum genes. Consistently, these cells differentiate hinge structures or modified notum cuticle. The LIM-HD co-factors Chip and Sequence-specific single-stranded DNA-binding protein (Ssdp) are also necessary for notum specification. This suggests that Tup acts in this process in a complex with Chip and Ssdp. Overexpression of tup, together with araucan, a `pronotum' gene of the iroquois complex (Iro-C), synergistically reinforces the weak capacity of either gene, when overexpressed singly, to induce ectopic notum-like development. Whereas the Iro-C genes are activated in the notum anlage by EGFR signalling, tup is positively regulated by Dpp signalling. These data support a model in which the EGFR and Dpp signalling pathways, with their respective downstream Iro-C and tup genes, converge and cooperate to commit cells to the notum developmental fate (de Navascues, 2007).

Tup has been categorised as a prepattern factor that controls the expression of the proneural achaete-scute genes in the third instar wing disc. This study shows that tup functions earlier in the development of the dorsal mesothorax. Loss of tup causes a range of phenotypes, which taken together indicate interference with the assignment of cells to form notum. Thus, depending on the time of induction of the clones and their location multiple effects are observed; the formation of notum-like cuticle with altered cell-cell adhesion properties, the generation of ectopic wing-hinge structures including tegulae, sclerites or sensilla typical of the proximal wing, or even the loss of the entire heminotum. Consistent with these adult phenotypes, in third instar wing discs tup mutant cells can upregulate genes typically expressed at high levels in the wing-hinge territory of the disc, such as zfh2, msh, sal and the lacZ insertion line l(2)09261. Concomitantly, notum-expressed genes such as eyg, ush and pnr are generally repressed, although in some cases tup cells may abnormally express notum and hinge genes together. These data indicate that notum tup cells undergo transformation towards either an altered notum fate or a hinge fate. Moreover, the activation of hinge markers in wild-type cells surrounding some tup clones might reflect the presence of ectopic notum/hinge borders, which are known to promote non-autonomous effects (de Navascues, 2007).

Unequivocal notum-to-hinge transformations are consistently observed in clones induced during the first larval instar. In later-induced clones, this phenotype becomes less manifest and the modified notum cuticle phenotype becomes prevalent. Accordingly, the upregulation of hinge marker genes and the converse downregulation of notum genes in the notum territory are most consistently observed in first instar-induced clones. This suggests that the requirement for the 'pronotum' function of tup progressively decreases as development advances. Lesions associated with tup clones can appear anywhere within the notum, although each particular phenotype shows a degree of topographic specificity. Interestingly, the activation of hinge genes and the repression of notum genes are best shown in early-induced clones located in the presumptive medial notum. Probably, these clones, which are normally large, do not yield adult structures, since the expected large regions of mutant cuticle have not been recovered. The clones might give rise to flies lacking part or most of a heminotum. The dynamic expression pattern of tup fits well with the spatial distribution of these phenotypes and the early requirement for tup function for the development of the notum. Indeed, tup is expressed very early in the wing disc, when it has less than 100 cells, and the expression occurs within the region that will form the notum. It is concluded that, similar to other LIM-HD factors such as Ap and the vertebrate Tup homologue Isl1, Tup is required for the proper specification of not only cell types, but also developing territories (de Navascues, 2007).

Tup is known to bind the co-factor Chip. Since, in dorsal compartment specification, Chip functions in a 2Ap-2Chip-2Sspd hexamer, it was asked whether a similar 2Tup-2Chip-2Sspd complex might mediate Tup function in notum specification. The results support this interpretation. The loss of either Chip or Ssdp upregulates hinge genes (zfh2, msh), represses a notum marker (eyg), and induces cuticular defects similar to those associated with tup clones. Moreover, an excess of Chip would be expected to titrate Tup and/or Ssdp in incomplete complexes and mimic the loss-of-function phenotype of notum-to-hinge transformation, as was experimentally observed (de Navascues, 2007).

By contrast, during the later process of sensory organ formation, Tup appears to act by sequestering both Chip and Pnr, thus preventing activation of the proneural genes achaete-scute. This negative function of Tup does not seem relevant for notum specification, where both Tup and Chip work as positive effectors. Moreover, the Tup homeodomain is dispensable for titrating Chip and Pnr, but this is not the case for its 'pronotum' function. Interestingly, a missense mutation within the LIM-interacting interacting domain of Chip (Chip^E) severely reduces its ability to interact with Tup and suppresses the negative regulation by Tup of bristle formation. However, homozygous Chip^E flies have no defects in notum specification. This suggests that a residual interaction between Chip^E and Tup might persist, as additionally suggested by the suppression of the extra bristles present in Chip^E individuals by UAS-tup overexpression. A weak interaction between Tup and Chip, which might only permit the formation of low levels of hexameric complex, might still allow proper notum specification. This suggestion agrees with the fact that tup^d03613, a strong hypomorphic allele (as substantiated by its embryonic lethality over the null tup^ex4, allows proper notum formation in homozygosis (de Navascues, 2007).

Similarly to tup, Iro-C also has a 'pronotum' function. However, their roles are not entirely equivalent. Anywhere within the notum territory, loss of Iro-C during first or second instar induces a clear switch to hinge fate. By contrast, loss of tup causes an assortment of different combinations of derepressed hinge genes and repressed notum genes. Moreover, many tup clones induced during the second larval instar, and even some induced in the first, can develop recognisable notum cuticle. Thus, it is proposed that tup reinforces/stabilises the commitment of cells to develop as notum, a commitment imposed mainly by Iro-C. This reinforcement or stabilisation might be most necessary in the proximal part of the disc, where expression of ara/caup ceases after the second instar, but that of tup persists. This might account for the derepression of hinge genes being most manifest in this region. Depending on the location and time of Tup deprival, its loss may be inconsequential or lead to a partial or even a complete loss of notum commitment. Such diversity of consequences led to an exploration of whether tup might act on target genes by affecting chromatin remodelling. However, no genetic interactions have been found with Polycomb (Pc, Scr+Pcl+esc) or trithorax (trx, osa, brm, Trl, lawc) group genes (de Navascues, 2007).

In contrast to the absolute requirement for Iro-C for notum specification, overexpression of UAS-ara can impose a notum fate only on the wing anlage, and only when provided early in the development of the disc. An extra notum with mirror-image disposition versus the extant notum is generated at the expense of the wing, a phenotype identical to that resulting from early deprivation of Wg function. Since UAS-ara overexpression can interfere with wg expression, Wg deprival probably explains the formation of the extra notum. Thus, by itself, overexpression of UASara probably lacks a genuine potential for imposing the notum fate. Similar notum duplications arise upon early and strong overexpression of UAS-tup (MD638, dpp-Gal4 and ptc-Gal4 drivers) and, again, they probably result from inhibition of Wg activity. Consistent with this interpretation, weaker and later expression of either UAS-tup or UAS-ara (C765 driver) has little or no capacity to promote notum fate. However, when coexpressed, these transgenes are effective in imposing the notum fate and this should not be attributed to Wg depletion. Indeed, the transformation consists of an expansion of the notum tissue, rather than a notum duplication. Moreover, as detected by the onset of the ectopic expression of notum markers (eyg, DC-lacZ), the transformation occurs in late third instar discs (J.deN., unpublished) that have a nearly wild-type morphology and a distinguishable wing pouch. This indicates that these markers are activated in territories previously specified as wing, hinge or pleura, and subsequently forced to acquire notum identity. Moreover, overexpression of the Wg pathway antagonists UAS-Axin or UAS-dTCF^DN (dTCF or pan with the same driver failed to transform wing towards notum. Finally, the activation of eyg and the formation of notum tissue in the sternopleurite, a derivative of the leg disc, also attest to the capacity of tup plus ara to commit cells to develop as notum (de Navascues, 2007).

It is well established that signalling by the EGFR pathway is essential for notum development. Its inhibition prevents activation of Iro-C and the growth of the notum territory. By contrast, Dpp negatively regulates Iro-C and restricts its domain of expression at both its distal and proximal borders. The data indicate a novel function of Dpp in notum development; namely, the activation or maintenance of tup expression in second and third instar discs. In the notum region of the early disc, Dpp signalling occurs at low levels, but the results suggest that these are sufficient for activating tup. Expression of tup is largely independent on EGFR signalling. Thus, EGFR and Dpp signalling seem to cooperate in specifying notum identity to the cells of the proximal part of the disc by activating their respective 'pronotum' downstream genes, Iro-C and tup (de Navascues, 2007).

Chip is required for posteclosion behavior in Drosophila

Neurons acquire their molecular, neurochemical, and connectional features during development as a result of complex regulatory mechanisms. This study shows that a ubiquitous, multifunctional protein cofactor, Chip, plays a critical role in a set of neurons in Drosophila that control the well described posteclosion behavior. Newly eclosed flies normally expand their wings and display tanning and hardening of their cuticle. Using multiple approaches to interfere with Chip function, it was found that these processes do not occur without normal activity of this protein. Furthermore, the nature of the deficit was identified to be an absence of Bursicon in the hemolymph of newly eclosed flies, whereas the responsivity to Bursicon in these flies remains normal. Chip interacts with transcription factors of the LIM-HD (LIM-homeodomain) family, and one member, dIslet, was identified as a potential partner of Chip in this process. These findings provide the first evidence of transcriptional mechanisms involved in the development of the neuronal circuit that regulates posteclosion behavior in Drosophila (Hari, 2008).

This study used a selective overexpression strategy to identify a novel function of Chip in a set of neurons that control a stereotyped behavioral program. Chip is a widely expressed multidomain cofactor molecule that interacts with many transcription factors. It can function both as a transcriptional coactivator and a bridging factor between proteins that bind to distal enhancers and the core transcription machinery. Identifying specific functions of such a protein is confounded by the superposition of a multitude of effects. The mutations in Chip cause early lethality precluding the examination of later functions. Because the molecule exists as a part of multiple complexes, even simultaneously within the same cell, altering the level of one class of interactors can potentially disrupt several functions. This study has elucidated a highly specific role of Chip in a particular class of neurons in Drosophila and implicated a known LIM-HD partner of Chip, Islet, in this function (Hari, 2008).

It is proposed that the defect is attributable to a failure in the release rather than in the production or the responsiveness to the neurohormone Bursicon. How might Bursicon release be controlled as a result of Chip function in development? The hemolymph transfer experiments provide a unique insight into this puzzle. The literature describes a model wherein posteclosion wing expansion requires a combination of a neural signal from the brain as well as Bursicon release. The results extend the understanding of how this interplay of activity and secreted factors is set up in development. It appears that, several days before eclosion, Chip is able to regulate an as-yet-unidentified event in the CCAP neurons, such that the hemolymph contains adequate levels of Bursicon after eclosion. The CCAP-expressing neurons are divided into at least two interacting subpopulations, only one of which secretes Bursicon. The other subpopulation does not secrete Bursicon but is implicated in regulating its release. Chip may therefore mediate the formation of proper connectivity among CCAP neurons, which eventually ensures timely Bursicon release several days later. Supporting this scenario, Chip has been reported to regulate axon pathfinding and proper innervation of targets in other systems. The data are suggestive of Chip requirement in the early period of puparium formation, which fits well with a report that CCAP neurons undergo extensive remodeling in this period of metamorphosis. The importance of this connectivity is underscored by the identification of several other genes in a gain-of-function screen, which displayed a simultaneous disruption of both posteclosion wing expansion and the pattern of CCAP neuron innervation. Therefore, thes findings motivate an examination of Chip function in regulating the connectivity of CCAP neurons, a role that directly links this key aspect of neuronal development with the control of posteclosion behavior in Drosophila (Hari, 2008).

Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila

Cell growth arrest and autophagy are required for autophagic cell death in Drosophila. Maintenance of growth by expression of either activated Ras, Dp110, or Akt is sufficient to inhibit autophagy and cell death in Drosophila salivary glands, but the mechanism that controls growth arrest is unknown. Although the Warts (Wts) tumor suppressor is a critical regulator of tissue growth in animals, it is not clear how this signaling pathway controls cell growth. This study shows that genes in the Wts pathway are required for salivary gland degradation and that wts mutants have defects in cell growth arrest, caspase activity, and autophagy. Expression of Atg1, a regulator of autophagy, in salivary glands is sufficient to rescue wts mutant salivary gland destruction. Surprisingly, expression of Yorkie (Yki) and Scalloped (Sd) in salivary glands fails to phenocopy wts mutants. By contrast, misexpression of the Yki target bantam is able to inhibit salivary gland cell death, even though mutations in bantam fail to suppress the wts mutant salivary gland-persistence phenotype. Significantly, wts mutant salivary glands possess altered phosphoinositide signaling, and decreased function of the class I PI3K-pathway genes chico and TOR suppressed wts defects in cell death. Although it has been shown that salivary gland degradation requires genes in the Wts pathway, this study provides the first evidence that Wts influences autophagy. These data indicate that the Wts-pathway components Yki, Sd, and bantam fail to function in salivary glands and that Wts regulates salivary gland cell death in a PI3K-dependent manner (Dutta, 2008).

Wts was identified as a protein that is expressed during autophagic cell death of Drosophila larval salivary glands with a high-throughput proteomics approach. This was surprising, given that wts RNA was not detected with DNA microarrays. Therefore, this study investigated whether Wts is present in salivary glands, and it was determined to be constitutively expressed at stages before and after the rise in ecdysone that triggers autophagic cell death. Animals that are homozygous for the hypomorphic wts^P2 allele, which is caused by a P element insertion, are defective in salivary gland cell death (Martin, 2007). Significantly two forms of Hpo are expressed during stages preceding salivary gland cell death, suggesting that phosphorylated Hpo is present in these cells and that this signaling pathway is activated (Dutta, 2008).

These studies indicate that Wts and other core components of this tumor-suppressor pathway are required for autophagic cell death of Drosophila salivary glands. wts is required for cell growth arrest and for proper regulation of caspases and autophagy, which contribute to the destruction of salivary glands. Although it is well known that cell division, cell growth, and cell death are important regulators of tissue and tumor size, it has been unclear whether a mechanistic relationship exists between cell growth and control of cell death (Dutta, 2008).

It is possible that wts and associated downstream growth-regulatory mechanisms could suppress cell death in other animals and cell types. Autophagic cell-death morphology has been reported in diverse taxa, but little is known about the mechanisms that control this form of cell death, and this lack of understanding is probably related to the limited investigation of physiologically relevant models of this process (Dutta, 2008).

This study used steroid-activated autophagic cell death of salivary glands as a system to study the relationship between cell growth and cell death. It is logical that cell growth influences cell death in salivary glands, given that autophagy is known to be regulated by class I PI3K signaling, which contributes to the death of these cells (Berry, 2007). It is unclear whether growth arrest is a determinant of autophagic cell death in other cell types and animals, and this question is important to resolve because of the importance of growth and autophagy in multiple disorders, including cancer. wts mutant salivary gland cells fail to arrest growth at the onset of puparium formation, and this suppresses the induction of autophagy. The inhibitor of apoptosis DIAP1 influences salivary gland cell death and is one of the best-characterized target genes of the Wts signaling pathway, but DIAP1 levels are not altered in wts mutant salivary glands. Significantly, the data provide the first evidence that Wts regulates autophagy and support previous studies indicating that caspases and autophagy function in an additive manner during autophagic cell death. Given the importance of both the Wts pathway and autophagy in human health, it is critical to determine whether this relationship exists in other cells (Dutta, 2008).

Cell growth and division are often considered to be synonymous, even though they are controlled by independent mechanisms. The Wts signaling pathway must influence cell growth, but most studies have emphasized the influence of this pathway on cell division and death. bantam is the only previously studied gene that is regulated by the Wts pathway and that is known to regulate cell growth. However, the mechanism of bantam action remains obscure. The current studies suggest the possibility that Wts may regulate growth via different mechanisms and that the nature of this regulation may depend on cell context. It is premature to conclude that bantam regulates a completely novel cell growth program, but the fact that misexpression of bantam stimulates cell growth in the absence of changes in a phosphoinositide marker and that chico and TOR fail to suppress the bantam-induced salivary gland-persistence phenotype minimally suggests that this microRNA regulates genes downstream of TOR. Significant progress has been made in the identification of microRNA targets, and future studies should resolve the mechanism underlying bantam regulation of cell growth (Dutta, 2008).

Recent studies of Wts signaling in Drosophila have identified a linear pathway that terminates with Yki and Sd regulation of effector genes that influence cell growth, cell division, and cell death. These studies indicate that the Wts pathway may not always regulate downstream effector genes via Yki and Sd, given that Yki expression was not able to phenocopy the wts mutant salivary gland destruction and expression of Sd induced premature degradation of salivary glands. Although bantam expression is sufficient to induce growth and inhibit cell death in salivary glands, bantam function is not required for the wts mutant phenotype. wts mutant salivary glands possess altered markers of PI3K signaling, and their defect in cell death is suppressed by chico and TOR. Combined, these results indicate that Wts regulates cell growth and cell death via a PI3K-dependent, and Yki- and Sd-independent, mechanism. Future studies will determine whether Wts regulates cell growth in a PI3K-dependent manner in other cells and animals (Dutta, 2008).

The transcriptional co-factor Chip acts with LIM-homeodomain proteins to set the boundary of the eye field in Drosophila

Development involves the establishment of boundaries between fields specified to differentiate into distinct tissues. The Drosophila larval eye-antennal imaginal disc must be subdivided into regions that differentiate into the adult eye, antenna and head cuticle. The transcriptional co-factor Chip is required for cells at the ventral eye-antennal disc border to take on a head cuticle fate; clones of Chip mutant cells in this region instead form outgrowths that differentiate into ectopic eye tissue. Chip acts independently of the transcription factor Homothorax, which was previously shown to promote head cuticle development in the same region. Chip and its vertebrate CLIM homologues have been shown to form complexes with LIM-homeodomain transcription factors, and the domain of Chip that mediates these interactions is required for its ability to suppress the eye fate. Two LIM-homeodomain proteins, Arrowhead and Lim1, are shown to be expressed in the region of the eye-antennal disc affected in Chip mutants, and both require Chip for their ability to suppress photoreceptor differentiation when misexpressed in the eye field. Loss-of-function studies support the model that Arrowhead and Lim1 act redundantly, using Chip as a co-factor, to prevent retinal differentiation in regions of the eye disc destined to become ventral head tissue (Roignant, 2009).

Regionalization of the eye-antennal disc is a progressive process in which selector genes and signaling pathways specify the fates of different head structures. Clones of eye-antennal disc cells induced during the second larval instar can contribute to multiple organs, indicating that these cells retain developmental plasticity at this stage. The anteroposterior boundary of the wing disc is established much earlier; expression of the selector gene engrailed (en) specifically in the posterior cells during embryogenesis generates an affinity border that keeps the two compartments clonally separated. By contrast, the eye selector gene ey is uniformly expressed throughout the early eye-antennal disc, and only retracts to the eye field in the second instar. It was initially proposed that localized Notch signaling controls this retraction, as expression of dominant-negative forms of Notch in the eye disc abolishes ey expression and leads to antennal duplications. However, a later study demonstrated that loss of Notch function does not affect ey expression directly, but reduces cell proliferation in the retinal field, preventing the initiation of eya expression. This study shows that Chip and Lim1 are both necessary to repress ey expression in the anterior of the antennal disc. Additional factors probably help to restrict ey expression to the eye disc, because ey expression does not extend throughout the normal Lim1 expression domain in Lim1 or Chip mutant clones in the antennal disc (Roignant, 2009).

Since Lim1 mutant clones always misexpress Ey, but rarely misexpress Eya and never differentiate ectopic photoreceptors, additional proteins must interact with Chip to repress retinal differentiation. Awh is a good candidate because it is expressed at the ventral margin of the eye-antennal disc, its misexpression in the retina represses photoreceptor differentiation in a Chip-dependent manner, and loss of both Lim1 and Awh leads to ectopic photoreceptor differentiation in the ventral eye-antennal disc. Since ectopic photoreceptors differentiate only in the absence of both Lim1 and Awh, whereas Ey expansion is observed in Lim1 single mutants, Awh must control the expression of target genes other than ey. It may negatively regulate other genes involved in retinal determination, such as eya, or positively regulate genes important for head capsule development, such as Deformed and odd-paired (Roignant, 2009).

Like Chip, Hth is required to prevent retinal differentiation at the ventral eye-antennal disc boundary. Investigation of the relationship between Chip and Hth indicates that Chip is not required for Hth expression or activity. The ability of Hth to repress photoreceptor differentiation in Chip mutant clones rules out the possibility that Chip acts as a co-factor for Hth or an essential downstream mediator of its effects. The normal expression of Hth and its target gene wg in Chip mutant clones also make it unlikely that Chip controls the expression of Hth or its co-factor Exd. However, the possibility that Hth and Chip act in parallel poses the paradox that misexpressed Hth is sufficient to repress photoreceptor development in the eye field in the absence of Chip, but endogenous Hth is insufficient to do so in the head field. It is possible that Hth expression levels in the head field early in development are too low to repress the eye fate in the absence of Chip. Consistent with this hypothesis, it was found that overexpression of Hth in Chip mutant cells prevents ectopic photoreceptor differentiation. Similarly, overexpression of Awh or Lim1 prevents ectopic photoreceptor differentiation in hth mutant cells, suggesting that endogenous levels of these LIM-HD proteins are not sufficient to compensate for the absence of Hth. The two classes of transcription factors may normally act on different sets of target genes, but show some cross-regulatory ability when overexpressed (Roignant, 2009).

The boundary between the eye and the dorsal head appears to be established differently from the boundary in the ventral region. The LIM-HD gene tup is expressed at the dorsal eye-antennal disc boundary, in a pattern resembling the mirror image of the Awh pattern, and is capable of repressing photoreceptor development in a Chip-dependent manner. However, loss of Chip in this region does not lead to ectopic eye formation, although it can cause overgrowth and mispatterning of the head. In the absence of Chip, the GATA transcription factor Pannier (Pnr) and its target gene wg may be sufficient to maintain dorsal head fate. The ventral margin of the eye-antennal disc may be particularly susceptible to ectopic photoreceptor differentiation because of the high level of Dpp signaling there. A 5' enhancer element has been shown to direct dpp expression specifically in the ventral marginal peripodial epithelium of the eye-antennal disc. The ability of Dpp and Ey to synergize to drive retinal differentiation therefore makes it critical to repress Ey in this region, which is fated to form head capsule (Roignant, 2009).

In addition, this domain of Dpp overlaps with Wg present at the anterior lateral margin of the eye disc; the combination of these two growth factors induces proximodistal growth of the leg. One function of Chip and its partner proteins might thus be to repress the outgrowth that would otherwise be triggered by the combination of Dpp and Wg. Unlike growth of the wild-type eye disc, growth of Chip mutant regions appears to be Notch-independent, as they do not contain a fng expression boundary and do not show activation of the Notch target genes E(spl)mβ or eyg. Notch has been thought to trigger growth by inducing the expression of the JAK/STAT ligand Unpaired (Upd); however, a recent report describes an earlier function for Upd upstream of Notch, raising the possibility that upd expression is activated independently of Notch in Chip mutant clones. As hth mutant clones, or clones lacking the Odd skipped family member Bowl, frequently show ectopic ventral photoreceptor differentiation but rarely induce outgrowths like those seen in Chip mutants, the functions of Chip in growth and differentiation are likely to be separable (Roignant, 2009).

LIM-HD proteins also set developmental boundaries in other imaginal discs, acting in concert with other classes of transcription factors. In the wing disc, Tup specifies the notum in collaboration with homeodomain transcription factors of the Iroquois complex, and Ap specifies the dorsal compartment. Ap interacts with the homeodomain protein Bar and Lim1 with Aristaless to establish specific tarsal segments within the leg disc. LIM-HD proteins have also been implicated in vertebrate eye development, although those that have been studied appear to play positive roles. The Ap homologue Lhx2 is expressed within the mouse retinal field at the neural plate stage, and contributes to the expression of Pax6, Six3 and Rx. Lmx1b, the homologue of CG32105, is required for the development of anterior eye structures such as the cornea and iris, and is mutated in human patients with nail-patella syndrome, often characterized by glaucoma. Within the retina, loss of Lim1 results in mispositioning of horizontal cells within the amacrine cell laye. Drosophila Lim3 shows photoreceptor-specific expression, and might therefore have a positive function in eye development (Roignant, 2009).

In the central nervous system, LIM-HD proteins act combinatorially to specify different neuronal cell fates. In both Drosophila and vertebrates, combinations of Islet and Lhx3/4/Lim3 proteins regulate motoneuron specification and pathfinding. The ability of Chip to interact with LIM-HD proteins and other transcription factors as well as to dimerize enables it to form heteromeric transcription factor complexes. In the wing disc, the active complex is a tetramer containing two subunits each of Chip and Ap, whereas in motoneuron development the Chip homologue NLI can form either a tetramer with Lhx3 or a hexamer containing both Isl1 and Lhx3. The finding that Lim1 and Awh act redundantly to prevent eye development in the ventral head primordium, whereas Chip is absolutely required, seems most consistent with regulation of distinct subsets of target genes by independent Chip-Awh and Chip-Lim1 complexes; however, a contribution from a complex containing all three proteins, or even additional transcription factors, cannot be ruled out. The role of the Chip co-factor may be to coordinate multiple transcriptional regulatory complexes to restrict developmental fates within the eye-antennal imaginal disc, allowing it to give rise to the head cuticle as well as distinct external sensory structures (Roignant, 2009).

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date revised: 25 June 2011 

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