InteractiveFly: GeneBrief

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


Gene name - rotund

Synonyms -

Cytological map position - 84D3

Function - transcription factor

Keywords - imaginal discs, leg, eye

Symbol - rn

FlyBase ID: FBgn0267337

Genetic map position - 3-47.6

Classification - zinc finger

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Li, Q., Barish, S., Okuwa, S. and Volkan, P. C. (2015). Examination of endogenous Rotund expression and function in developing Drosophila olfactory system using CRISPR-Cas9 mediated protein tagging. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26497147
Summary:
The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. This study added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila in order to investigate its sub-cellular localization and the genes that it regulates. Previously a reporter construct was used to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. This study shows that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, the expression pattern of the endogenous isoform-specific tags were mapped in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, a direct binding was shown of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.

BIOLOGICAL OVERVIEW

The Drosophila rotund (rn) gene is required in the wings, antenna, haltere, proboscis and legs. Previously identified in the rotund region was a member of the Rac family of GTPases, denoted the RacGAP84C or rotund racGAP gene (see Raymond, 2001). However, rotund racGAP is not responsible for the rotund phenotypes. The rotund gene has now been isolated. It is a member of the Krüppel family of zinc finger genes. The adjacent roughened eye locus specifically affects the eye and is genetically separable from rotund. However, roughened eye and rotund are tightly linked, and thanks to this connection, the roughened eye transcript was isolated. Intriguingly, roughened eye is part of the rotund gene but is represented by a different transcript. The rotund and roughened eye transcripts result from the utilization of two different promoters that direct expression in non-overlapping domains in the larval imaginal discs. The predicted Rotund and Roughened Eye proteins share the same C-terminal region, including the zinc finger domain, but differ in their N-terminal regions. Each cDNA can rescue only the corresponding mutation and show negative effects when expressed in each other's expression domain. These results indicate that in addition to the differential expression of rotund and roughened eye, their proteins have distinct activities. rotund and roughened eye act downstream of early patterning genes such as dachshund and appear to be involved in Notch signaling by regulating Delta, scabrous and Serrate (St Pierre, 2002).

The Drosophila rotund locus is recessive viable causing male and female sterility as well as defects in adult body structures (Cavener, 1986). These defects include those in the antennae, wing, haltere and proboscis as well as fusion of all five leg tarsi into one fused tarsal-like segment. Analysis of third instar larvae imaginal discs revealed localized cell death in the regions giving rise to the affected adult structures (Kerridge, 1988). The rn locus has previously been molecularly analyzed (Agnel, 1989) and a cDNA encoding a member of the Rac family of GTPase-activating proteins (GAP) was isolated from this genomic region (Agnel, 1992b). Since this gene was located in the rn genomic region it was denoted the rotund racGAP (rnracGAP), but molecular analysis of multiple rn alleles have indicated that the rnracGAP is not responsible for the rn phenotypes (Agnel, 1992a). In fact, all studies to date instead point to an uncharacterized larger transcript (Agnel, 1992a; Hoemann, 1996) as the likely candidate for the rn gene (St Pierre, 2002).

The closely linked roughened eye (roe) locus affects a late step in the development of the eye, and roe mutants display rough eye morphology and reduction of photoreceptors (Renfranz, 1989). The roe gene is genetically separable from rn, but the two genes show complex complementation (Brand, 1990; Kerridge, 1988; Ma, 1996). This finding previously led to the suggestion that rn and roe may be 'two classes of mutation of the same gene, each of them disrupting a subfunction' (Ma, 1996). To address the tight link between these two adjacent loci, the rn and roe genes have been isolated. Intriguingly, roe is part of the rn gene. Each cDNA can rescue only the corresponding mutation and misexpression in one another's domain of expression has negative effects. These results indicate that these two loci are genetically separable not only because of their differential expression but also because of distinct activities of the Rn and Roe proteins (St Pierre, 2002).

Regarding the function of the rnracGAP, both this work and previous studies argue against any involvement of rnracGAP in the rn or roe phenotypes (Agnel, 1989; Agnel, 1992a; Hoemann, 1996). In situ studies indicate that rnracGAP is only expressed at low levels in the imaginal discs during pupal stages (Agnel, 1989; Agnel, 1992a; Hoemann, 1996). In addition, there is no obvious difference in the severity of rn and roe phenotypes whether or not the rnracGAP is simultaneously removed. For instance, no significant difference was found in the severity of rn leg phenotypes in rn20/rn20 (that removes rn, roe and rnracGAP) compared to rn19/rn20 (rn19 does not remove rnracGAP). Similarly, roe3/rn20 (roe3 has a premature stop codon in the roe-specific exon) displays as severe an eye phenotype as rn20/rn20. Furthermore, rn and roe mutants can be rescued with the rn and roe cDNAs. Recent studies may indicate an involvement of rnracGAP specifically in male fertility, and high levels of rnracGAP expression have been observed in the adult testis (Agnel, 1989; Agnel, 1992a; Hoemann, 1996). The rn89 and rnGAL4#5 P-element insertions may provide useful starting materials for the generation of mutations specifically affecting the rnracGAP by local P-element mobilization (St Pierre, 2002).

Little is known about the genetic cascades within which roe and rn are acting. The results from eye-antennal imaginal discs indicate that roe acts at the morphogenetic furrow, as evident both from its expression and from the effects on Delta and Scabrous expression in roe mutants. Both Dl and sca play roles in spacing the array of ommatidial preclusters in the morphogenetic furrow, and it is interesting to note that the expression of roe at the furrow is not evenly distributed and appears stronger in clusters of cells. Genetic screens for modifiers of the Nspl mutation have identified roe as an enhancer, and sca and Dl as suppressors of the Nspl eye phenotype (Brand, 1990). Given the dynamics of N signaling, these results support models where Roe acts to either positively or negatively regulate Dl and Sca. A genetic interaction screen for enhancers of glass also identified roe (Ma, 1996), an interesting finding given that ectopic expression of roe using GMR-GAL4 leads to a glass-like phenotype with a loss of bristles and pigment cells (St Pierre, 2002).

In the leg, rn expression is the earliest marker known for tarsal development (Couso, 1998). rn is required for the development of this region and for its subsequent patterning, as observed by the loss of Ser expression. Thus, the transient expression of rn in the leg might reveal that the intercalation of the presumptive tarsal region between the distal tip and medial leg regions occurs during early third instar (St Pierre, 2002).

It is increasingly common, even in invertebrates, to find genes that utilize two or more promoters. Although this may lead to the generation of different proteins, it is often unclear whether the proteins have distinct activities. In fact, this issue is not easily resolved by traditional forward genetics and subsequent molecular analysis, since even if a locus can be genetically dissected into different subfunctions, this does not identify whether the different proteins have distinct activities. Perhaps the best way to test whether the variant proteins are interchangeable in vivo, is by cross-rescue in each others domain of expression. The rn gene is a clear example of a locus that utilizes both tissue-specific promoters and functionally distinct proteins to achieve functional diversity, a scenario likely to be observed more and more frequently in the post-genomic era (St Pierre, 2002).

Combinatorial rules of precursor specification underlying olfactory neuron diversity

Sensory neuron diversity ensures optimal detection of the external world and is a hallmark of sensory systems. An extreme example is the olfactory system, as individual olfactory receptor neurons (ORNs) adopt unique sensory identities by typically expressing a single receptor gene from a large genomic repertoire. In Drosophila, about 50 different ORN classes are generated from a field of precursor cells, giving rise to spatially restricted and distinct clusters of ORNs on the olfactory appendages. Developmental strategies spawning ORN diversity from an initially homogeneous population of precursors are largely unknown. This study has unraveled the nested and binary logic of the combinatorial code that patterns the decision landscape of precursor states underlying ORN diversity in the Drosophila olfactory system. The transcription factor Rotund (Rn) is a critical component of this code that is expressed in a subset of ORN precursors. Addition of Rn to preexisting transcription factors that assign zonal identities to precursors on the antenna subdivides each zone and almost exponentially increases ORN diversity by branching off novel precursor fates from default ones within each zone. In rn mutants, rn-positive ORN classes are converted to rn-negative ones in a zone-specific manner. This study provides a model describing how nested and binary changes in combinations of transcription factors could coordinate and pattern a large number of distinct precursor identities within a population to modulate the level of ORN diversity during development and evolution (Li, 2013).

Neuronal diversity is a common characteristic of all sensory systems throughout the animal kingdom. Among these, the olfactory system demonstrates an extreme case in its diversity of ORN classes. In Drosophila, each of the 50 adult ORN classes is defined by the unique expression of typically a single olfactory receptor from a pool of around 80 genes. How this ORN diversity is generated from a field of homogeneous precursor cells during development remains elusive. Combinatorial control of transcription factors has been proposed as an important mechanism that complex systems utilize to create cellular diversity. This study demonstrates the nested and binary combinatorial rules by which transcription factors interact with each other to guide decisions regarding ORN precursor identities. The results suggest that nesting the regulatory relationship of transcription factor combinations allows the concurrent use of the same factors in parallel lineages to generate ORN diversity in a very efficient manner. Under this logic, binary lineage choices in precursor cells are made based on historical contingency, which could serve as an effective strategy for establishing cellular complexity in many other developing systems (Li, 2013).

In both vertebrates and invertebrates, each ORN class is spatially restricted to specific zones within the peripheral olfactory organs. In Drosophila, antennal ORNs are housed in three morphologically and topographically different types of sensilla occupying distinct zones, while maxillary palps have only a single type of sensilla. Each of the sensilla type zones on the antenna are subdivided into subzones that are defined by sensilla subtypes, which have similar morphology but differ in the set of olfactory receptors expressed in the ORNs they house. It has been shown that the decision for a given palp-specific olfactory receptor gene to be expressed in maxillary palp ORNs, but not in antennal ORNs, requires both positive and negative regulatory elements around that gene. For antennal ORNs, the proneural genes amos and ato and the prepatterning gene lz were found to assign sensilla type identities to the precursors and determine olfactory receptors expressed by the neurons housed in these sensilla. The loss of Amos or Ato leads to the complete loss of basiconic and trichoid or coeloconic sensilla types, respectively, and corresponding ORNs. Lz diversifies sensilla type identities within the Amos-expressing lineage, where high levels of Lz are associated with basiconic sensilla fates, versus low levels of Lz, which generates ORNs in trichoid sensilla. Hypomorphic alleles of lz result in basiconic-to-trichoid sensilla type conversions. Lz is also required for the expression of Amos, suggesting the existence of regulatory loops among transcription factors in the same network (Li, 2013).

The current results explain how the next level of diversification occurs following sensilla type specification in the antenna. Rn is expressed in a subset of antennal sensilla precursors and splits precursors of each zone into Rn-positive and Rn-negative subtypes. In rn mutants, ORN diversity decreases almost by half as ORN classes from rn-positive subtypes are switched to rn-negative identities within the same zone. The results suggest that Rn is required to branch off novel precursor identities from default ones, resulting in the generation of new ORN classes in a zone-specific manner. It should be noted that some rn-negative sensilla subtypes, for example at2 and ac3, neither decrease nor increase in their numbers in rn mutants, suggesting that there are additional factors driving the diversification of the ORN classes in these sensilla. Similarly, further diversification of rn-positive ORN precursors should also be under the control of additional factors, such as En, operating in concert with Rn function (Li, 2013).

These results along with others suggest a two-step mechanism for ORN diversification: (1) successive restrictions on precursor differentiation potentials by spatiotemporal factors, such as proneural/prepatterning gene products and Rn, and (2) segregation of restricted fates through Notch-mediated asymmetric divisions and local transcription factor networks for directly turning on olfactory receptor expression. Hypothetically, the sensilla precursor differentiation potentials can be represented by distinct sets of olfactory receptor genes being organized into euchromatic regions in a lineage-specific manner. The aforementioned combinations of transcription factors may influence the dynamics of such epigenetic states, resulting in limited combinations of receptors transcriptionally accessible for later stages of ORN differentiation. Examples of chromatin modulation in OR expression have been demonstrated in both flies and mice. Once precursor potentials are set, the Notch signaling pathway could continue to bifurcate alternate sensory identities into ORNs generated through asymmetric precursor cell divisions. Transcription factor networks expressed later in development, including the well-characterized Acj6, Pdm3, and Scalloped, could then directly regulate olfactory receptor expression during these divisions based on their genomic accessibility, giving rise to terminally differentiated ORNs (Li, 2013).

In comparison with the Drosophila olfactory system, mammals exhibit remarkable organizational similarities in the olfactory circuitry, even though the numerical complexity far exceeds that of their insect counterparts. For example, the zonal pattern of olfactory receptor expression in the mammalian olfactory epithelium is analogous to the topographic segregation of sensilla type-dependent olfactory receptor expression in the antenna. A number of transcription factors were reported to regulate the zone-specific expression of a subset of olfactory receptors, yet no mutants resulting in ORN sensory conversion have been described. Despite the consensus on the stochastic nature of mammalian olfactory receptor expression within each zone, it would be interesting to see whether the zones are defined by a similar developmental strategy. The model presented in this study also provides an ancestral precursor decision landscape that reveals the interaction pattern among factors to maintain and modify phenotypic complexity and diversity within sensory neural circuits on evolutionary timescales. New regulatory nodes might be added to the combinatorial code at distinct stages of precursor cell development to change ORN specification programs. For example, addition of mir-279, a negative regulator of the transcription factor nerfin-1 expressed in maxillary palp ORN precursors, results in the elimination of CO2-sensory ORNs from specific maxillary palp sensilla (Hartl, 2011). Furthermore, olfactory receptor genes have been shown to be fast evolving across and within genomes. Incremental addition of individual regulatory modules to preexisting lineage-specific combinations operating in binary ON/OFF mode could facilitate the coordination of novel ORN fates with the evolution of receptor genes, which can be modified in response to changes in the quantity, quality, and context of the olfactory environment (Li, 2013).


REGULATION

Transcriptional Regulation

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-ß and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. This study revises understanding of limb development in flies and shows that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals (Campbell, 2002).

In addition to activating genes, EGFR signalling is required to repress genes in distal regions, and again different genes appear to be differentially sensitive, with some, such as B and rn, possibly being both activated and repressed above different thresholds. B, rn and dac are repressed in the center of wild-type discs, with dac being repressed over a wider region than B and rn. Lowering EGFR activity in Egfrts discs to a level sufficient only for loss of al, results in expression of B and rn in the center, but not dac. Raising the temperature still further results in extension of the dac domain to fill the center. Clonal analysis shows that Egfr acts autonomously to repress dac. Ectopic EGFR activity can also repress B, dac and rn but again predominantly in ventral regions (B is repressed mainly at later stages). Previous studies have shown that repression of dac in distal regions requires high levels of Wg and Dpp signalling, so all three pathways appear to be required to achieve this (Campbell, 2002).

Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development. Rotund in turn regulates wingless

Nab proteins form an evolutionarily conserved family of transcriptional co-regulators implicated in multiple developmental events in various organisms. They lack DNA-binding domains and act by associating with other transcription factors, but their precise roles in development are not known. This study analyzed the role of nab in Drosophila development. By employing genetic approaches it was found that nab is required for proximodistal patterning of the wing imaginal disc and also for determining specific neuronal fates in the embryonic CNS. Two partners of Nab were identified: the zinc-finger transcription factors Rotund and Squeeze. Nab is co-expressed with squeeze in a subset of neurons in the embryonic ventral nerve cord and with rotund in a circular domain of the distal-most area of the wing disc. These results indicate that Nab is a co-activator of Squeeze and is required to limit the number of neurons that express the LIM-homeodomain gene apterous and to specify Tv neuronal fate. Conversely, Nab is a co-repressor of Rotund in wing development and is required to limit the expression of wingless (wg) in the wing hinge, where wg plays a mitogenic role. Pull-down assays show that Nab binds directly to Rotund and Squeeze via its conserved C-terminal domain. Two mechanisms are described by which the activation of wg expression by Rotund in the wing hinge is repressed in the distal wing (Félix, 2007).

Precise temporal and spatial control of gene transcription is crucial for development. Sequence-specific DNA-binding factors and their association with a variety of modulator proteins, the co-factors, achieve this control. Co-factors do not bind DNA but act as adaptors between DNA-binding factors and other proteins. A number of transcription factors have been characterized, many of which act by recruiting multiprotein complexes with chromatin-modifying activities. By recruiting co-factors, a DNA-binding protein can act as co-activator or as co-repressor depending on the context. An example of a co-repressor is the retinoblastoma protein that converts the E2F transcription factor into a repressor of cell-cycle genes. The identification of co-factors and the determination of their precise roles are crucial for understanding the mechanisms that govern development (Félix, 2007).

Nab (NGFI-A-binding protein) proteins form an evolutionarily conserved family of transcriptional regulators. Nab was originally identified in mouse as a strong co-repressor by virtue of its capacity to interact directly with the Cys2-His2 zinc-finger transcription factor Egr1 (Krox24; NGFI-A) and inhibit its activity. Two Nab genes, Nab1 and Nab2, have been identified in vertebrates. Nab proteins do not bind DNA but they can repress or activate gene expression by interacting with Egr transcription factors. Nab proteins have two regions of strong homology: NCD1 and NCD2. The NCD1 domain interacts with the R1 domain of Egr1 (Svaren, 1998). The NCD2 domain is required for transcriptional regulation. Mice harboring targeted deletions of Nab1 and Nab2 have phenotypes very similar to Egr2 (Krox20)-deficient mice, suggesting that they act as co-activators of this gene. In zebrafish, egr2 controls expression of the Nab gene homologs in the r3 and r5 rhombomeres of the developing hindbrain. Egr2 has been implicated in determining the segmental identities of r3 and r5 by controlling the expression of several target genes as well as cell proliferation. Misexpression experiments suggest that Nab1/Nab2 antagonize Egr2 transcriptional activity by a negative-feedback regulatory loop. Nevertheless, Nab proteins might have additional functions as these experiments also led to alterations of the neural tube not found in Egr2-deficient embryos. Conversely, Egr2-deficient mice have a severe hindbrain segmentation defect that is not found in mice deficient in Nab1 and Nab2. Nab might also have Egr-independent functions in mice because, although epidermal hyperplasia has been observed in Nab1 Nab2 double mutant mice, this phenotype has not been observed in mice lacking any of the Egr proteins (Félix, 2007 and references therein).

In Drosophila, only one Nab gene has been identified; it is highly homologous to vertebrate Nab genes in the NCD1 and NCD2 domains. Drosophila nab mutants are early larval lethal. Detection of nab transcripts by in situ hybridization indicates expression in a subset of neuroblasts of the embryonic and larval CNS and weak expression in imaginal discs. The role of Nab in Drosophila development is not known and so far no binding partner has been identified. This report shows that nab is a component of the combinatorial code that determines the number of neurons that express the gene apterous (ap) in embryonic neural development, and that nab specifies the Tv neuronal fate in the ap thoracic cluster of neurons (Félix, 2007).

In early larval development, the wing fate is established in the distal-most region of the wing disc by a combination of two factors: activation of the gene vestigial (vg) and repression of the gene teashirt (tsh). Later, in early third instar larvae, wingless (wg) is activated in a ring of cells (the inner ring, IR) that borders the vg expression domain in the presumptive wing region. It has been suggested that activation of the IR involves a signal from the vg-expressing cells to the adjacent cells. Interpretation of this signal by the adjacent cells requires the transcription factors encoded by rotund (rn) and nubbin (nub). Expression of wg in the IR plays a mitogenic role; hence, as a consequence of wg expression, cells proliferate and the IR moves away from the vg border. At a distance from the source of the signal that drives the initial activation, wg IR expression is maintained by an autoregulatory loop that involves homothorax (hth). It is thought that an additional mechanism distally represses wg IR expression and, in so doing, controls cell proliferation in the wing hinge. In this report, it is shown that during imaginal disc development, nab is strongly expressed in the wing presumptive domain under the control of vg, and that nab is required in proximodistal axis development to control the expression of wg in the wing hinge (Félix, 2007).

Two putative partners of Nab have been identified: Rn and Squeeze (Sqz). These proteins are members of the Krüppel family of zinc-finger proteins. Pull-down assays show that that Nab interacts with both proteins via a conserved C-terminal domain, and evidence is presented that Nab acts as co-activator of Sqz in embryo development and as co-repressor of Rn in wing development. Finally, it is proposed that there are two mechanisms to repress the activation of wg expression by Rn in the wing pouch: the first involves Nab as a co-repressor of Rn; the second involves Sqz as a competitor of Rn for binding to specific DNA target sites (Félix, 2007).

Antibody against Nab revealed a low level of expression in all imaginal discs. In late third instar wing discs, Nab was strongly expressed in a circular domain that delimits the expression of wg in the inner ring. Nab expression was first detected in early third instar larvae, in a group of cells of the distal-most wing, and was maintained throughout the remainder of the larval and pupal stages. There was a low level of expression in the rest of the wing disc, except in the hinge where there was no detectable expression. In the eye disc, Nab was detected in a stripe corresponding to the morphogenetic furrow (Félix, 2007).

It was asked whether, as with other genes involved in proximodistal patterning, nab expression in the wing is dependent upon vg. No expression of nab was detected in the distal wing of vg83b27r wing discs. However, nab is ectopically expressed in clones of vg-expressing cells. Together, these results indicate that the expression of nab in the wing depends on vg. In wild-type discs and vg ectopic-expressing clones, the domain of nab expression is broader than that of vg, pointing to the nonautonomous control of nab expression. A similar mechanism has been proposed for other genes, such as rn and nub, whose expression depends on vg. Expression of vg in the wing starts in second instar larvae, whereas nab expression is first detected at early third instar. This suggests that some other mechanism controls the initiation of nab expression (Félix, 2007).

The nabSH143 allele is a P(lacW) insertion in the first exon. Most larvae homozygous for this allele die in first instar. Thus, to analyze the role of nab in development of the wing nabSH143 homozygous mutant clones were generated by mitotic recombination using the FLP/FRT mitotic recombination system. In the wing, these clones activated wg ectopically. However, it was noted that not all the clones activated wg. It is therefore possible that there is functional redundancy between Nab and other proteins (Félix, 2007).

Two enhancers drive the expression of wg in the wing: the wing margin enhancer, which is activated by the Notch signaling pathway, and the spade (spd) enhancer, which drives wg expression in the inner ring. Previous results suggest that activation by the latter depends on a nonautonomous signal coming from the vg-expressing cells. nab co-expresses with wg in the wing margin and abuts on wg expression in the inner ring. It was therefore assumed that Nab should repress activation of the inner ring enhancer derepressed in nab clones. To obtain independent evidence that the inner ring enhancer is being activated, tests were performed to see whether other genes activated in the wing margin were activated in the nab clones. To this end, cut (ct) was analyzed, and no ectopic expression was detected. It has been reported that wg expression can be detected in the wing after induction of cell death. To detect cell death in the nab clones, use was maed of an antibody that recognizes the activated form of Caspase 3, but no cell death was detected. These results, together with the pattern of expression, strongly suggest that the inner ring enhancer is being activated in the nab clones and, therefore, that in normal development Nab acts as a repressor of the wg inner ring enhancer in the distal wing. To confirm this hypothesis, nab was expressed ectopically in the inner ring domain using the nubGAL4 driver, which is expressed in a circular domain that includes the inner ring. In nubGAL4>UASnab larvae, expression of wg in the inner ring was lost, whereas its expression in the wing margin was not affected. Clones of nab-expressing cells were generated, and it was found that wg expression was cell-autonomously lost in these clones, whereas wg expression in the wing margin was not affected. In the light of these results, it is proposed that the function of nab in wing development is to delimit, distally, the domain of wg expression in the inner ring by inhibiting the mechanism of inner ring activation (Félix, 2007).

The mammalian Nab partner Egr1 contains an inhibitory domain called R1. When this domain is deleted the transcriptional activity of Egr1 increases 15-fold. It has been shown that the R1 domain mediates a functional interaction between Nab and Egr1. Since no R1 domain has been identified in the fly genome and all the previously identified partners of Nab are Krüppel-type zinc-finger transcription factors, transcription factors of the Krüppel family expressed in the wing were examined as potential Nab partners in the fly. The gene rn encodes a Krüppel-like zinc-finger protein that in the wing is expressed in a circular domain slightly broader than the nab domain. The wg inner ring enhancer is active only in the cells that express rn and that do not express nab. Previous studies have shown that Rn is required for activation of the wingless spd enhancer. The results so far suggest that Rn could be a partner of Nab in the wing: first, nab is expressed in the rn-expressing cells that do not express wg; second, nab loss-of-function clones contain ectopic Wg; and third, nab misexpression represses the wg inner ring enhancer (Félix, 2007).

rn was also expressed in leg discs in a broad ring that corresponded to three tarsal segments (T2-4). In rn mutant legs, the T2-4 tarsal segments were deleted. It would therefore be expected that if Rn were a partner of Nab, ectopic expression of nab in the leg would generate the same phenotype as the lack of Rn. This proved to be the case when nab was misexpressed in the rn expression domain under the control of the rnGal4 driver. The phenotype of these flies was indistinguishable from the rn mutant phenotype in both legs and wings. The specificity of this interaction was examined by rescuing the phenotype caused by nab misexpression by co-expressing rn (rnGal4>UASrn+UASnab), as well as by misexpressing nab in a broader domain using Distal-less Gal4 (DllGal4), which is expressed from mid-tibia to distal leg (DllGal4>UASrn). In the first experiment, the phenotype was markedly reduced in both wing and leg, indicating that adding more rn antagonizes the inhibitory effect of nab misexpression. In the second experiment, although nab was misexpressed in a broader domain of the leg, the phenotype was unaltered and was restricted to the area where rn was expressed. Taken together, these results support a role for Rn as a potential partner of Nab and that Nab acts as co-repressor of Rn function in the cells where both are expressed. The rn mutant phenotype in the wing is caused by the loss of wg expression in the inner ring. Whether wg expression was affected in rnGal4 UASnab and rnGal4 UASnab UASrn wings was examined. In the first case, the inner ring was found to be absent, whereas in the second it was partially restored. In summary, these results indicate that Nab functions in wing development by antagonizing the transcriptional activation function of Rn (Félix, 2007).

Nab binds directly to Rn and Sqz via a conserved C-terminal domain

In order to analyze the molecular role of Nab as a co-factor of Sqz and Rn GST pull-down assays were performed. The complete nab cDNA was cloned in a glutathione S-transferase (GST) vector and incubated with radioactively labeled Rn or Sqz. Nab-GST, but not GST alone, readily retained [35S]methionine-labeled Rn or Sqz. Rn and Sqz share a C-terminal domain of 32 amino acids with a homology greater than 80%. To further test whether this domain mediates the interaction with Nab, the pull-down assays were repeated with an [35S]Rn in which the C-terminal domain was deleted. This deletion removes the region from amino acid 894 to the C-terminus (943) of the protein (RnΔ894). The ability of Nab-GST to retain the [35S]RnΔ894 was notably reduced. It is concluded that this conserved domain mediates the direct interaction of Nab with Rn and Sqz. To further test whether the C-terminal domain is sufficient to mediate this interaction, the Nab-GST was incubated with a 32 amino acid peptide containing just the sequence of the C-terminal domain. Nab-GST did not retain the peptide, indicating that the C-terminal domain is not sufficient to mediate Nab-Rn interaction. Since no other conserved domains have been identified between Rn and Sqz besides the zinc-finger and C-terminal domains, it is considered that either secondary structure or an additional modification of the protein is required for binding Nab. In order to provide an in vivo functional test of this hypothesis, the rnΔ894 fragment was cloned into the pUAST vector and clones of cells misexpressing UASrnΔ894 were generated (Act>Gal4>UASrnΔ894). These clones activated the expression of wg throughout the wing pouch. As a control experiment, the wild-type version of rn (Act>Gal4>UASrn) was misexpressed. These clones only activated wg expression in the wing hinge, outside of the nab expression domain (Félix, 2007).

Sqz competes with Rn in wing disc development

sqz expression was examined in the wing disc. Because of the high degree of sequence homology between rn and sqz and to avoid interference with the rn mRNA present in the wing, in situ hybridization assay was performed in rn mutant discs. sqz expression was detected by in situ hybridization in rn20 wing discs in a circular pattern that faded off laterally and whose proximal limit coincided with the limit of vg expression; this corresponded to the distal-most wing fold. To determine whether sqz plays a role in wing development the phenotype was analyzed of sqz mutant clones induced by mitotic recombination. These clones had no adult phenotype, nor did they alter the expression of wg. Since Sqz and Rn share zinc-finger and the C-terminal domains and differ in their N-terminal domains, the roles of Sqz and Nab might be functionally redundant, both repressing Rn activity but by different mechanisms: Nab would repress Rn activity by direct binding to Rn protein as a co-repressor, whereas Sqz would compete for binding to the same DNA targets. To test this hypothesis, the effect was analyzed of misexpressing sqz in the rn expression domain. rnGal4/UASsqz UASGFP flies had small deletions of the wing hinge and shortened legs, a phenotype that resembles the nab misexpression and rn mutant phenotypes. In agreement with these results, wg expression in the inner ring was downregulated in rnGal4/UASsqz wing discs. An alternative explanation for these results is that sqz activates nab expression, but no nab misexpression was seen in this experiment. It is suggested that there must be some functional redundancy, irrespective of whether Nab and Sqz play similar roles in the wing by repressing Rn activity, and this would account for the low penetrance of the nab mutant clones. Because nab and sqz map on different chromosome arms it was not possible to generate double-mutant clones. Therefore nabSH143 homozygous clones were generated in a sqzlacZ/+ background. In this situation, the frequency of clones misexpressing wg increased by 38%). It was also noted that the clones that showed wg misexpression were preferentially located in the lateral-most regions of the wing, which correspond to the regions with the lowest levels of sqz expression. Taken together, these observations support the hypothesis that Nab and Sqz play similar roles in wing development: Nab as a co-repressor of Rn via its conserved C-terminal domain, and Sqz by competing with Rn for binding to its DNA targets. This function of Sqz would differ from its above-proposed role as a transcriptional activator in CNS development, and would not require Nab (Félix, 2007).

This study presented evidence that Nab is a co-activator of Sqz. This protein has been implicated in two aspects of embryonic ventral nerve cord development: first, in a Notch-dependent lateral inhibition mechanism that specifies the number of cells that express ap in the ap thoracic neuronal cluster; and second, in the specification of the Tv neuronal fate. nab and sqz are co-expressed in a subset of neurons, including several of the ap cluster, as well as the Tv neuron. nab loss-of-function embryos reproduce all the phenotypes of sqz loss-of-function embryos: additional cells express ap in the cluster and the Tv neuronal fate is lost. In addition, in both nab and sqz mutants an increased number of cells in the clusters express dimm. These findings indicate that Nab is required for all identified Sqz functions in embryonic development. Although this analysis focused on the ap thoracic cluster of neurons, both sqz and nab are co-expressed in many cells in the ventral nerve cord and others expressed either sqz or nab. But no other functions have been identified for sqz and it is not known how the expression of sqz is controlled. It has been reported that the expression of nab in the ventral nerve cord depends on the gene castor (Clements, 2003). Thus, the results presented in this study reveal greater complexity in the mechanisms of neuronal fate specification. The combined expression of genes, whose expression is individually activated by different mechanisms, is required to determine specific neuronal fates (Félix, 2007).

Sqz and Rn share two regions of strong homology: the zinc finger and a stretch of 32 amino acids in the C-terminal domain. By contrast, only rn has a long N-terminal domain. The results indicate that the C-terminal domain mediates the interaction with Nab. By GST pull-down assays, it was shown that Nab binds to the full-length Rn protein but not to the RnΔ894 version, and clones of cells misexpressing rnΔ894 activate wg expression in the nab expression domain. The similarity between sqz misexpression and rn loss-of-function phenotypes in leg and wing suggests that Sqz acts like a dominant-negative form of Rn in the rn domain: both proteins would bind to the same target sites but have opposite effects, and the results indicate that this role of Sqz would not require interaction with Nab. It is possible that the long N-terminal region of Rn is involved in interaction with other partners specifically required for Rn function (Félix, 2007).

Thus, these results indicate that Nab has a dual role as co-repressor of Rn and co-activator of Sqz. Previous studies in vertebrates also suggest that Nab is involved in both repression and activation of transcription. Co-repressors are proteins that bridge the interaction of the repressor with its target. Two main co-repressors have been identified in Drosophila: Groucho and CtBP. CtBP binds to a specific sequence motif (P-DLS-K) that has been found in the sequence of three repressors present in the early embryo: Snail, Knirps and Krüppel. All three are zinc-finger transcription factors, and genetic evidence suggests that they all require CtBP to repress their targets. Neither Rn nor Sqz have a CtBP-binding motif but one has been in Nab (P-DLS--K). Although the functional significance of this motif remains to be confirmed, itis suggested that Nab is acting as a bridge between Rn and CtBP (Félix, 2007).

Targets of Activity

In the wing wingless is expressed in a complex and dynamic pattern that is controlled by several different mechanisms. These involve the Hedgehog and Notch pathways and the nuclear proteins Pannier and U-shaped. The mechanisms that drive wingless expression in the wing hinge have been analyzed. Evidence is presented that wingless is initially activated by a secreted signal that requires the genes vestigial, rotund and nubbin. Later in development, wingless expression in the wing hinge is maintained by a different mechanism, which involves an autoregulatory loop and requires the genes homothorax and rotund. The role of wingless in patterning the wing hinge is discussed (Rodriguez, 2002).

The adult wing is formed by a continuous monolayer of epidermal cells that folds to form the dorsal and ventral surfaces of the wing pouch. The two surfaces contact at the margin of the wing and extend proximally through the wing hinge to the dorsal notum and the ventral pleura. In the presumptive wing region of the wing disc, wg is expressed in a narrow stripe of cells that runs all along the wing margin and in two rings that surround the wing pouch. The phenotypes and wg expression have been examined in several mutants in which the wing hinge is deleted (Rodriguez, 2002).

The effects of removing wg expression in the inner ring (IR) can be observed in spade (spd) mutants. spd mutations are a type of wg allele that specifically removes wg expression from the IR, with no effects on other expression domains. In spdfg wings, the hinge region is deleted, and the wing pouch appears directly joined to more proximal cells. In these wings, both wg-expressing cells and surrounding cells are missing. It has been shown that this phenotype is not caused by cell death but is a consequence of underproliferation in this region, suggesting that one of the functions of Wg in the IR is to promote local cell proliferation (Rodriguez, 2002).

The rotund (rn) gene is a member of the Krüppel family of zinc-finger encoding genes. Among other phenotypes, rn mutations delete the wing hinge and remove wg expression from the IR. nubbin encodes a member of the POU family of transcription factors. In strong nub mutations wings are vestigial, but phenotypic analysis of weaker alleles shows that the wing hinge is deleted and the expression of wg in the IR is missing. The hinge phenotype of the triple mutant spdfg nub2; rnDelta2-2 was examined, and it is similar to the phenotype of each of them, suggesting that the main cause of the phenotype is the lack of wg expression in the IR (Rodriguez, 2002).

vestigial encodes a nuclear protein with no homology with other identified families of nuclear proteins. Based on its interaction with scalloped (sd) it has been suggested that the function of Vg is to mediate transcriptional activation by Sd. vg expression in the wing is regulated by two separate enhancers: the boundary enhancer (BE) and the quadrant enhancer (QE). The BE is activated by the Notch signaling pathway and drives vg expression at the dorsal/ventral boundary in middle/late second instar larval stage. The QE is activated by the combined action of Wg and Dpp, and drives vg expression in the rest of the wing pouch from early third instar larval stage (Rodriguez, 2002).

The expression patterns of vg, rn and nub were examined. In mature wing discs vg, rn and nub are expressed in three concentric domains, the Vg domain being the smallest one. At this stage the wing hinge is lined with several anterior/posterior folds. The boundary of vg expression coincides with the distal-most fold of the disc. The Rn domain is slightly broader and its boundary coincides with a second fold in the disc. The Nub domain contains the Rn domain and coincides with the third fold in the disc. The IR domain corresponds to the proximal-most area of the Rn domain (Rodriguez, 2002).

The expression of these genes was examined in early larval development. In middle/late second instar larvae the expression domains of vg, rn and nub in the presumptive wing pouch are slightly broader than the vg domain. The rest of the cells of the disc, those that do not express nub, express the gene teashirt (tsh). wg is expressed only in a stripe of cells that corresponds to the presumptive wing margin. In early third instar larvae, wg starts to be expressed in the IR. This expression domain corresponds to cells that express rn and nub but do not express vg. wg expression in the IR promotes the growth of the hinge and, in third instar larvae, gives rise to the expression patterns described above for vg, rn and nub. At this stage, the cells that express the wg IR enhancer are located at the limit of the domain 3 (Rn + Nub), and are several cells away from the boundary of vg expression (Rodriguez, 2002).

The results indicate that Vg is required to activate the expression of rn and nub genes in the wing disc. This activation is restricted to the cells that will take wing fate and takes place in the cell that express vg, and also in the surrounding cells, suggesting that a Vg-dependent short-range signal activates rn and nub expression. At this time, the expression of nub and tsh in the wing disc are complementary and cover the whole disc (Rodriguez, 2002).

The expression of these genes in a domain broader than the Vg domain creates a ring of cells that express rn and nub but not vg. Evidence is presented indicating that a signal from vg-expressing cells activates the wg IR enhancer in adjacent rn/nub-expressing cells. Unlike the activation of rn and nub, the activation of wg expression by the IR enhancer is repressed in cells that also express vg. So, the IR enhancer is activated only in cells that surround the Vg domain. During the development of the disc, the position of the IR moves several cells away from the Vg domain. This implies either that the Vg-dependent signaling is able to activate the IR over a long range, or that a different, Vg-independent, mechanism maintains the IR (Rodriguez, 2002).

When artificial Vg/Rn-Nub interfaces are generated experimentally, the IR enhancer is activated in rn-nub-expressing cells that abut the Vg domain. This ectopic IR is around four cells wide, indicating the active range of the signal that activates wg expression. The results indicate that at distances greater than this, a Vg-independent mechanism maintains wg expression in the IR (Rodriguez, 2002).

The rn clonal analysis indicates that Rn is required for wg expression. One interesting observation is that when the IR moves away from the Vg domain, wg-expressing cells are always maintained at the limit of rn expression. rn is activated by a Vg-dependent signal. This implies that the activity range of the signal and the lifetime of the Rn protein together limit the domain of rn expression. So one explanation for why the IR is always maintained in the limit of rn expression is that, as a consequence of cell proliferation, cells drop out of the range of the Vg-dependent signaling. Thus, cells simultaneously lose the expression of both rn and wg. The result of the rn lineage-tracing experiment supports this prediction. Taken together, these results suggest that an autoregulatory loop involving Hth and Rn maintains wg expression. Although hth expression depends on Wg, rn expression depends on Vg, so wg expression in the IR is not maintained by lineage. wg autoregulation has been reported in embryo development, and a negative mechanism of 'self-refinement' has been suggested in wing margin specification. However, in neither of these cases has a role been reported for Hth or Rn (Rodriguez, 2002).

The proximal and distal limits of the IR would be defined respectively by the limit of rn expression and by the limit of the expression of the proposed repressor. In summary, these results suggest that at least four different target genes are independently activated by one or more signals that emanate from vg-expressing cells: rn and nub are activated in second instar larvae; wg is activated in early third instar larvae (this activation requires the function of Rn and Nub and is repressed by Vg); and finally the repressor, which would be activated in middle third instar larvae (Rodriguez, 2002).

SoxF is part of a novel negative-feedback loop in the wingless pathway that controls proliferation in the Drosophila wing disc

Wnt molecules act as mitogenic signals during the development of multiple organs, and the aberrant activity of their pathway is often associated with cancer. Therefore, the production of Wnts and the activity of their signaling pathway must be tightly regulated. This study has investigated the mechanisms of this regulation in the Drosophila hinge, a domain within the wing imaginal disc that depends on the fly Wnt1 ortholog wingless (wg) for its proliferation. The results uncover a new feedback loop in the wg pathway in which the spatially restricted activation of the Sox gene SoxF (Sox15) by wg represses its own transcription, thus ensuring tight regulation of growth control. rotund, a wing proximodistal patterning gene, excludes SoxF from a thin rim of cells. These cells are thus allowed to express wg and act as the source of mitogenic signal. This novel mode of action of a Sox gene on the Wnt pathway -- through transcriptional repression of a Wnt gene -- might be relevant to human disease, as loss of human SoxF genes has been implicated in colon carcinoma (Dichtel-Danjoy, 2009).

One of the long-standing questions in biology is how organ growth is coordinated with tissue patterning. Research during recent decades has shown that a limited set of signals and signaling pathways control this coordination. Some of these signals are mitogenic, and their production at specific sites, called signaling centers, links spatial information to cell proliferation within developing organs. Normal organ growth not only needs mitogens, but also mechanisms to control their production, transport, reception and/or transduction to ensure that proliferation is limited in space and time. Alterations in these control mechanisms often lead to disease (Dichtel-Danjoy, 2009).

The Wnt/β-catenin signaling pathway promotes cell proliferation during normal development and disease. Wnts are lipid-modified glycosylated signaling molecules that can reach distant cells. Binding of Wnts to the receptor complex [composed of a Frizzled family receptor and an Arrow (LRP) co-receptor] results in the stabilization of the transcriptional co-factor β-catenin [armadillo (arm) in Drosophila]. Thereby, β-catenin/Arm accumulates in the nucleus, where it associates with Tcf/LEF DNA-binding transcription factors to regulate the expression of Wnt target genes. Research in a number of model organisms has demonstrated that the Wnt/β-catenin pathway controls cell proliferation in a variety of tissues, including the nervous system and the progenitors of the intestine and hematopoietic systems in mammals, and during imaginal disc development in Drosophila. It is also known that most colorectal tumors, and a number of other tumor types, are caused by aberrant Wnt/β-catenin signaling, which underlines the necessity of tight regulation of this pathway (Dichtel-Danjoy, 2009).

The range and intensity of the signaling elicited by Wnt molecules have been shown to be regulated by many different mechanisms, including negative-feedback loops. These have been particularly well studied for the main Drosophila Wnt gene, wingless (wg). wg is required in the imaginal discs for the growth and patterning of the adult body structures. wg signaling results in the downregulation of its two receptors, Dfz-2 (fz2 - FlyBase) and fz and in the upregulation of Dfz-3 (fz3 - FlyBase), a non-productive low-affinity receptor, and of the extracellular Wg inhibitor Notum (wingful). Intracellularly, high levels of wg/Wnt signaling induce the expression of two inhibitors of the pathway: naked cuticle and nemo. All these feedback loops result in an attenuation of the signal at the sites of maximal wg production and are generally implicated in all processes in which wg is required (Dichtel-Danjoy, 2009).

The Drosophila wing disc gives rise to the wing blade, the notum (body wall) and the hinge, which joins the wing blade to the body wall and articulates its movements. wg is expressed in two concentric rings in the hinge domain and has been shown to be required for the proliferation of hinge cells. Moreover, wg overexpression is sufficient to drive hinge overgrowths without causing major repatterning. Therefore, the precise regulation of the wg pathway is crucial to control the growth of the hinge. The mitogenic effect of wg on hinge cells contrasts with its effect on the neighboring wing pouch cells which, upon similar wg overexpression, are mostly driven into sensory organ differentiation. One prediction from these results is that the hinge-specific proliferative function of wg needs dedicated control mechanisms to ensure normal hinge size and shape. To identify these mechanisms, genes were sought that are differentially expressed in the hinge territory for a role in wg-mediated proliferation. SoxF (Sox15) belongs to the family of sequence-specific HMG Sox transcription factors and has been shown to be expressed in the prospective hinge of third larval stage (L3) wing discs). The functions of Sox genes have been extensively studied in mammals, in which they play essential roles during development. In addition, misregulation of Sox genes is often associated with cancer (Dichtel-Danjoy, 2009).

Only two of the eight Sox family genes present in the Drosophila genome have been studied in detail: Dichaete (D) and SoxNeuro (SoxN). They belong to the SoxB group and have prominent roles in embryonic segmentation and nervous system development. In addition, it has recently been shown that both genes negatively regulate the activity of the wg/Wnt pathway during cell fate specification in the embryonic epidermis (Dichtel-Danjoy, 2009 and references therein).

This paper reports that SoxF, which is the sole member of this Sox group in Drosophila, is also required to restrain wg signaling, but using a novel mechanism: the transcriptional repression of wg. In the absence of SoxF, wg transcription spreads through the hinge causing its overproliferation. SoxF is itself under the control of the canonical wg/Wnt pathway such that wg and SoxF regulate each other's transcription through a feedback loop. Moreover, the expression of rotund (rn), which is part of the proximodistal patterning mechanism of the wing disc, allows the exclusion of SoxF from a thin rim of cells, allowing them to express wg. Thereby, this rim becomes a spatially well-defined mitogen-producing center necessary to ensure normal hinge growth. This novel mode of action of a Sox gene on the Wnt pathway -- the transcriptional repression of a Wnt gene -- might be relevant to human disease, as loss of human SoxF genes has been implicated in colon carcinoma (Dichtel-Danjoy, 2009).

In order to determine the role played by SoxF during hinge development, a SoxF allele, Sox15KG09145 (now renamed SoxFKG09145) was characterized. The SoxFKG09145 allele carries an insertion of the P[SUPor-P] transposon in an intronic region of the gene, which also harbors the CG30071 transcript. Most homozygous SoxFKG09145 flies die as pharate adults, and escapers are weak with held-out wings. This latter phenotype is indicative of hinge defects. In fact, these flies show abnormal proximal hinge structures: the sclerites, the alula and the costa are affected. Although the insertion does not affect SoxF coding sequence, it was observed by RT-PCR and in situ hybridization that SoxF expression is completely lost in the wing disc of mutant L3 larvae. Sice this P-element carries insulator sequences, it was also checked by RT-PCR that expression of CG30071 and of the 5' neighboring gene, RpS23, was not affected by the insertion, which was indeed the case. This study has also generated new alleles by imprecise excision of the P transposon from the original allele. In addition to full revertants, more than ten mutant lines were isolated in which different lengths of intron sequences were deleted, without affecting the coding region, and which showed a range of phenotypic severity. These results suggest that this intronic region carries crucial elements for the regulation of SoxF expression. Some alleles were isolated that disrupt the coding sequence. Among them, SoxF26 is specific to the SoxF gene and deletes the first exon and part of the first large intron, and is therefore likely to be a null allele. This allele has the same phenotype as the initial insertion. In addition, the phenotype and escaper rates of individuals carrying SoxFKG09145 over a deficiency uncovering the SoxF locus, Df(2R)Exel7130, are the same as for homozygous SoxFKG09145 flies. Therefore, SoxFKG09145 behaves as a genetic null allele. It has been reported that SoxF is expressed in the embryonic Peripheral nervous system (PNS). Adult escapers of the molecular null allele SoxF26 exhibit, in addition to their abnormally folded wings, are also weak and die shortly after eclosion. Other hinge mutants, such as wg spd-fg, are much healthier. Therefore, it is possible that the larval lethality and weakness of adult escapers is due to abnormal PNS development (Dichtel-Danjoy, 2009).

This study describes a novel negative-feedback mechanism in the wg pathway that is required to restrain the expression of wg itself, and which is essential to control organ growth. During Drosophila development, the wg pathway often leads to the activation of genes that attenuate its signaling pathway. This is the case, for example, for Notum and Dfz-3, which are expressed in the wing disc in response to peak levels of signaling to reduce ligand availability for the Wg receptors, and for nemo, which acts intracellularly to block the signal transduction pathway. In all cases described, these negative-feedback components act in all domains of wg expression and none regulates wg expression at the transcriptional level. However, in the case investigated in this study, the putative transcription factor SoxF is activated non-autonomously by wg in a hinge-specific manner. SoxF in turn represses wg transcription driven by the wg spd-fg enhancer, thus restricting the production of wg to the thin inner ring (IR) domain. Interestingly, the SoxF phenotype is similar to those of dominant Dichaete (D) mutations. D is a SoxB gene not normally expressed in the wing disc. However, flies carrying dominant D mutations show reduced hinge structures. This phenotype is caused by ectopic D expression in the prospective hinge region of the disc. One of the salient features of D discs is the repression of the wg IR, which is reminiscent of the wg repression by SoxF described in this study. Therefore, and taking into account the similarity between Sox proteins in their HMG DNA-binding domain, the ectopic D might be mimicking the repression of wg that is normally exerted by SoxF (Dichtel-Danjoy, 2009).

The tight regulation of the growth of the hinge depends critically on the wg-induced activation of SoxF in the growing territory. Nevertheless, this activation is 'polarized' along the PD axis, taking place only in cells adjacent and proximal to the IR. It is proposed that this directionality in SoxF activation results from the mechanisms that pattern the wing disc along its PD axis. It has been suggested that wg is activated non-autonomously by a signal produced by the vg-expressing wing pouch cells, but excluded from them. This would generate a circular domain of wg expression surrounding the wing pouch. However, in the absence of SoxF, the domain of wg is abnormally broad and causes hinge overgrowth. This ectopic wg expression does not seem to result from a misregulation of hinge-specific genes: the expression of nub, tsh, hth and rn and their relative positioning in the hinge are unaffected in SoxF mutant discs. Therefore, it seems that in the absence of SoxF, hinge cells cannot respond to the wg activating signals with enough precision to give rise to a thin ring of wg expression. The results show that this precision is achieved through a double repression mechanism. First, wg activates its own transcriptional repressor, SoxF. This would lead to the extinction of wg expression if it were not for rn, which acts as a repressor of SoxF. Second, rn, by repressing SoxF, permits wg transcription. The result is that wg expression becomes restricted to a narrow circular stripe at the edge of the rn domain that provides a highly localized source of Wg. This signal activates, simultaneously and in the same cells, proliferation and the upregulation of SoxF, which restricts the production of the signal. Therefore, SoxF joins SoxN and SoxD (Sox102F - FlyBase)as the third Drosophila Sox known to antagonize the wg pathway. The vertebrate Sox proteins Sox9, XSox3 and XSox17 have also been shown to downregulate the Wnt/β-catenin pathway. Therefore, this antagonism seems evolutionarily conserved (Dichtel-Danjoy, 2009).

The relationship between SoxF genes, the wg/Wnt pathway and the control of tissue proliferation seems to extend to disease. The SoxF Sox17 is normally expressed in the gut epithelium where it downregulates Wnt signaling via degradation of β-catenin and TCF. In colon carcinomas, the expression of the SoxB gene Sox17 is often reduced, and this is associated with tissue overproliferation. Moreover, inactivation of the SoxE gene Sox9 leads to increased cell proliferation and hyperplasia in the mouse intestine. The authors concluded that Sox9 is essential for the fine-tuning of the transcriptional activity of the Wnt pathway. Interestingly, the expression of Sox9 is regulated by the Wnt pathway itself. These results in Drosophila point to the possibility that the transcriptional regulation of Wnt expression by Sox genes might be a common feature of this proliferation-associated feedback loop (Dichtel-Danjoy, 2009).


DEVELOPMENTAL BIOLOGY

Expression of rotund and roughened eye (roe) (both products of the rotund locus but represented by different transcripts) is detected in developing imaginal discs, as well as in the embryonic and larval CNS. The current analysis focused on the expression in the imaginal discs. Expression of rn commences during the early third larval instar in the leg, wing, haltere and antennal part of the eye-antennal imaginal disc. Expression of rn is observed as a ring in the leg and antenna discs and in the presumptive wing pouch and capitellum of wing and haltere discs respectively. In late third instar, expression of rn in the leg disc is no longer evident, but is maintained in the other discs. The expression of lacZ in both rn89 and in rnGAL4#5/UAS-lacZ larvae was examined to determine rn expression. In both genotypes, expression of lacZ is in agreement with the rn in situ hybridization, except for the persistence of tarsal expression, but in neither line is expression detected in the eye disc. Expression of roe commences in the third instar and is confined to the eye part of the eye-antennal imaginal disc in a band of 4-6 cells at the morphogenetic furrow. No evidence was found of roe expression in other imaginal discs (St Pierre, 2002).

The expression of rn and roe is in agreement with the observed phenotypes. For instance, rn mutants have defects in wings and halteres, and correspondingly rn is expressed in the appropriate presumptive regions in wing and haltere imaginal discs. In the leg, rn mutants display fusion of all 5 leg tarsi into one fused tarsal-like segment. In agreement with this, rn is expressed in a sub-distal ring that represents the presumptive tarsus, as revealed by the persistent tarsal expression of rn-driven lacZ in late third instar discs. Similarly, roe specifically affects the eye, and mutants have rough eyes and reduced numbers of photoreceptors. Accordingly, expression of roe is detected in the eye part of the eye-antennal imaginal disc but not in other imaginal discs. The mutually exclusive patterns of expression of rn and roe raised the issue of whether they may in fact negatively regulate each other. To determine this, the expression was examined of roe in rn mutant imaginal discs and conversely the expression of rn in roe mutant imaginal discs. These studies revealed no apparent changes in the expression of rn and roe when compared to wild type, indicating that there is no cross-regulation between rn and roe (St Pierre, 2002).

Rescue of rn and roe phenotypes

Owing to the complexity of the rn locus it was important to verify the authenticity of the rn and roe cDNAs by rescue experiments. For rn rescue, focus was placed on the leg phenotype and the rnGAL4#5 line (which shows strong leg phenotypes over rn20) was used. By providing rn function with UAS-rn, rescue of the rnGAL4#5/rn20 leg phenotypes was observed, often to a level indistinguishable from the wild-type leg. No dominant effect in the leg of UAS-rn in a heterozygous background was observed (St Pierre, 2002).

The structure of the rn genomic region and the differential expression in imaginal discs explains why rn and roe can be genetically separated and affect different tissues. However, the rn and roe gene products are also different, and the first ZF is truncated in the Roe protein, intriguing given that the first finger of Krüppel-type ZF proteins has been shown to be involved in DNA-binding. Rn and Roe further differ in the N-terminal regions where they contain stretches of glutamine/serine (Roe) or alanine (Rn), often found in transcriptional activator and repressor domains respectively. This raised the possibility that these two proteins may have different activities and may not be interchangeable. To address this issue roe was misexpressed in the leg disc and attempts were made to rescue rn with roe. When roe is misexpressed in the developing leg disc using rnGAL4#5, a negative effect with reduced number of tarsi was observed, similar to rn mutants. Furthermore, in a rn mutant background (rnGAL4#5/rn20) no evidence of rescue by UAS-roe was observed (St Pierre, 2002).

It was also important to attempt rescue of roe mutants using the GAL4/UAS system. The roe rescue was complicated by the fact that no GAL4 insertion in the roe gene was available. This is especially relevant given the dynamic pattern of roe expression in the eye disc, with transient expression in a band of approx. 4-6 cells at the morphogenetic furrow. No GAL4 line was identified that would express precisely in the roe pattern, and instead attempts were made to rescue roe using GAL4 drivers that would drive in photoreceptors. To this end, several eye disc GAL4 driver lines were tested for ectopic effects. Not surprisingly, strong pan-eye drivers such as GMR-GAL4 led to dramatic phenotypes with loss of pigment and bristle cells. A novel sevenless-GAL4 (sev-GAL4) line that expresses GAL4 in the photoreceptors, cone and mystery cells showed little if any sign of rough eye morphology when crossed to UAS-roe. Using sev-GAL4 crossed to UAS-roe in a roe null mutant background (rn16/rn20) partial rescue of the eye phenotypes was observed with increased eye size and reduced roughness. To quantify the roe rescue, the number of adult R1-7 photoreceptors was counted in wild-type, mutant and rescued flies. These results confirm previous studies (Ma, 1996) and show that roe mutants have a reduced number of photoreceptors compared to wild type. In line with the apparent morphological rescue significantly increased numbers of photoreceptors were found in rescued flies when compared to mutants. Given that it was not possible to used a GAL4 driver line that perfectly matched the dynamic expression of roe in eye discs, it is believed that this partial rescue supports the proposed identity of the roe gene. As in the rn rescue experiments, it was important to address whether rn is interchangeable with roe and could provide rescue activity in the eye. First the activity of UAS-rn in the eye was tested by misexpressing it using GMR-GAL4 and sev-GAL4. This led to severe rough eye phenotypes with GMR-GAL4 and little if any sign of rough eye morphology with sev-GAL4. In a roe null mutant background (rn16/rn20) no evidence was found of rescue by adding UAS-rn (St Pierre, 2002).


EFFECTS OF MUTATION

bric à brac (bab) is required and expressed in a distinct proximal-distal domain of the limbs; the central region of the tarsus of the leg and the basal cylinder of the antenna. The domain of bab activity in limbs is apparently identical to the domain defined by the phenotype and expression pattern of rotund. In addition, this leg domain is characterized by the gene deadpan, which is expressed in a distal circumferential stripe in each of the segments TS1 to TS4. bab and rotund appear to act rather late in limb development, in contrast to genes that control the whole proximal-distal axis and appear to be required from embryogenesis onward, such as Distal-less and wingless. The subdivision of the tarsal primordium is a late event in the pattern formation of the leg and is also an evolutionarily recent step. Primitive insects only have one tarsal segment and the number of tarsal segments differs widely among more advanced insects. Taken together, this indicates that the bab/rotund domain is a distinct field for pattern formation during leg and antenna development (Godt, 1993).

Previous studies suggested that rn and roe act late during development of their respective tissues, perhaps during terminal differentiation (Godt, 1993; Renfranz, 1989). To further explore the function of rn and roe during leg and eye development, the expression of genes that play key roles during development of these tissues was examined. The leg disc was studied and genes whose expression abuts or overlaps that of rn were examined. Dachshund (Dac), a nuclear factor required for normal leg development, is expressed at early stages of leg development in a ring pattern that abuts the early rn-expressing ring. Bric a brac (Bab), a BTB-domain containing transcription factor, has been suggested to be active late in limb development and is expressed in a similar pattern to rn in the leg (Godt, 1993). Furthermore, bab mutants show similar (though not identical) phenotypes as rn mutants in the tarsal segments of the leg (Godt, 1993). Interestingly, neither Dac nor Bab appears to be regulated by rn as revealed by staining of third instar leg discs. These results suggest that rn might act in parallel to, or downstream of, dac and bab to specify tarsal segment identity. Ser, a ligand for the Notch (N) receptor, is expressed in presumptive joint areas in larvae and pupa leg discs and controls the development of the leg joints. In wild-type mid-third instar leg discs, Ser is expressed in the first tarsal fold, which coincides with the rn-expressing ring. In rn, Ser is down-regulated in the tarsal ring but not outside it. In pupal leg discs, Ser expression, normally present in four stripes within the presumptive tarsal area, is present in fewer and less defined stripes in rn (St Pierre, 2002).

The roe rough eye phenotype is reflected in reduced numbers of photoreceptors present in adult ommatidia. To determine whether roe mutants show early patterning defects in the eye-antennal disc, expression of Dac, which plays an early role in the eye disc and is expressed in a broad domain spanning both sides of the morphogenetic furrow (MF), was analyzed. Since dac mutants have a more severe eye phenotype than roe it was anticipated that Dac would not be regulated by roe, and as expected no change was observed in the pattern of Dac staining in roe when compared to wild type. Next, third instar eye-antennal discs were analyzed with antibodies to Elav and to Bride of Sevenless (Boss), a marker of R8 photoreceptors. In wild-type eye discs, Elav and Boss are expressed in a stereotyped pattern immediately posterior to the MF. In roe mutants, expression of Elav and Boss reveals abnormal photoreceptor differentiation with apparent gaps in the expression of both markers posterior to the MF. Elav expression also indicates that photoreceptor clusters frequently have fewer photoreceptors than normal. Expression of Elav and Boss further reveals an apparent failure of the MF to progress in a straight line from dorsal to ventral. The MF appears to progress more slowly in some areas, creating a wave-like appearance of developing photoreceptor clusters near the MF. These results indicate that roe function is centered around the MF, a notion that fits well with the strong but transient roe expression seen at the MF. Markers expressed at the MF were analyzed, and since roe has been shown to interact genetically with the NSpl mutation (Brand, 1990), expression of Delta (Dl), a N ligand, and Scabrous (Sca), a secreted glycoprotein implicated in N signaling, were analyzed. In wild type, Dl and Sca are expressed in clusters of cells at the MF, and expression is maintained posterior to the MF in subsets of cells. In roe mutants, the punctate expression of Dl and Sca is lost at the MF and replaced by a diffuse band of expression. Posterior to the MF, expression is punctate but appears disorganized (St Pierre, 2002).

Basolateral junctions utilize warts signaling to control epithelial-mesenchymal transition and proliferation crucial for migration and invasion of Drosophila ovarian epithelial cells

Fasciclin2 (Fas2) and Discs large (Dlg) localize to the basolateral junction (BLJ) of Drosophila follicle epithelial cells and inhibit their proliferation and invasion. To identify a BLJ signaling pathway a genome-wide screen was performed for mutants that enhance dlg tumorigenesis. Two genes were identified that encode known BLJ scaffolding proteins, lethal giant larvae (lgl) and scribble (scrib), and several not previously associated with BLJ function, including warts (wts) and roughened eye (roe/rotund), which encode a serine-threonine kinase and a transcription factor, respectively. Like scrib, wts and roe also enhance Fas2 and lgl tumorigenesis. Further, scrib, wts, and roe block border cell migration, and cause noninvasive tumors that resemble dlg partial loss of function, suggesting that the BLJ utilizes Wts signaling to repress EMT and proliferation, but not motility. Apicolateral junction proteins Fat (Ft), Expanded (Ex), and Merlin (Mer) either are not involved in these processes, or have highly spatio-temporally restricted roles, diminishing their significance as upstream inputs to Wts in follicle cells. This is further indicated in that Wts targets, CyclinE and DIAP1, are elevated in Fas2, dlg, lgl, wts, and roe cells, but not Fat, ex, or mer cells. Thus, the BLJ appears to regulate epithelial polarity and dynamics not only as a localized scaffold, but also by communicating signals to the nucleus. Wts may be regulated by distinct junction inputs depending on developmental context (Zhao, 2008).

The purpose of this work was to gain greater insight into how the BLJ suppresses epithelial tumorigenesis and invasion by identifying and understanding the function of new genes important for BLJ function. To do so, a genomewide screen was completed for enhancers of dlg, which encodes a scaffolding protein that is a crucial organizer of the BLJ and is a potent repressor of follicle epithelial cell tumorigenesis and invasion. Deficiencies that cumulatively span ∼80% of the autosomes, or 64% of the Drosophila genome were systematically screened. A relatively small number of enhancers, ∼1 per 1000 genes screened, were detected indicating that the screen selected for loci specifically required for dlg function. Thus, the novel dlg enhancer genes that were identified, wts, roe, ebi, as well as at least two genes yet to be identified, are likely to be key collaborators with dlg in suppressing epithelial invasion. The specificity of the interactions between dlg and these enhancers is further indicated in that more than one allele of each gene showed an interaction, in several dlg backgrounds, and the strengths of enhancement were similar to deficiencies defining each locus. wts, roe, and ebi also enhanced Fas2 and lgl, indicating that they are not just important for dlg function, but for the function of the BLJ as a whole. In addition, overexpression of all enhancers except ebi suppressed dlg and Fas2 tumorigenesis, further confirming that the identified genes function in a BLJ network (Zhao, 2008).

BLJ pathway components in the nucleus and their putative relationship to Notch: ebi encodes an F-box protein with WD repeats that promotes protein degradation of specific targets. The failure of ebi overexpression to suppress Fas2 or dlg, and the relatively mild ebi phenotypes (midoogenesis small-nucleus and epithelial-organization defects, but no defects in germinal vesicle localization), suggest that ebi may function in only one of the three branches of BLJ signaling or in a parallel pathway to the BLJ. In the eye, ebi is important for promoting differentiation and inhibiting proliferation, which appear to be separable functions. Thus ebi could enhance Fas2 and dlg tumorigenesis by functioning within the proliferation-repressing branch of the BLJ, or the importance of ebi for differentiation suggests that it could function in the EMT branch of the BLJ or both. In contrast, ebi promotes protein degradation in response to Notch (N) and Drosophila EGF receptor (EgfR) signals, suggesting that it may act in a parallel pathway. Both Ebi and its mammalian homolog, TBL1, function in a corepressor complex through association with nuclear hormone transcriptional corepressor SMRTER/SMRT (Zhao, 2008).

Interestingly, although most N appears to be localized on the apical surface of follicle cells, some N is also localized in BLJs. Thus, it is possible that N localized to the BLJ may signal directly to Ebi. Consistent with this possibility, it was found that all of the genes in the BLJ network share some midoogenesis defects with N, including the small nucleus phenotype, epithelial stratification defects, and mislocalization of the germinal vesicle. The epithelial defects are also reminiscent of N-pathway mutants brainiac and egghead, which are required in the germ line for regulating N that is localized on the apical surface of the follicle cells abutting the germ line. Thus one possibility is that N signaling activity is regulated by its localization to apical vs. basolateral junctions in response to several signaling pathways acting during midoogenesis (Zhao, 2008).

The other modest dlg enhancer that was identified, roe, encodes a Krüppel-family zinc-finger protein that appears to be a transcription factor. Roe is also implicated in Notch signaling and thus may function with Ebi in N-dependent processes as proposed above. However, in contrast to ebi, roe loss caused follicle cell tumors, suggesting that roe may function more directly in a BLJ pathway than ebi. Consistent with a direct role for Roe in BLJ signaling, it was found that roe overexpression suppressed Fas2 and dlg tumorigenesis. Further, as for Fas2, dlg, and wts, roe represses CycE and DIAP1 expression (Zhao, 2008).

Warts was of special interested because of the many similarities observed in the quality and strength of wts and scrib phenotypes, suggesting that they are components in a BLJ signaling pathway, rather than a parallel pathway that cross talks with BLJ signaling. wts encodes a serine/threonine kinase that is an ortholog of human tumor suppressors Lats1 and Lats2, both of which have been linked to highly aggressive breast cancers. The prevailing model for Wts signaling in Drosophila is based on signaling in eye and wing tissue. Wts appears to relay signals from apicolateral junction proteins Ft, Ex, and Mer in wing and eye tissues. However, the results from almost every assay, including early tumor formation, border cell migration, BrdU, PH3, CycE, and DIAP1 expression, indicated little functional overlap between Ft, ex, mer, or mer; ex and wts, thus diminishing the importance of apicolateral Ft-Ex-Mer for Wts activation in follicle cells. The exceptions were that during midoogenesis, Mer is required for border cell migration and Ex is required for the endocycle switch, while both are required for maintenance of epithelial integrity and positioning of the germinal vesicle. However, the involvement of Ex and Mer in these processes are fundamentally distinct from how they act in Wts-dependent processes in other tissues. (1) Ft is not involved; (2) no indication was observed of Ex-Mer synergism; (3) ex, mer, and mer; ex phenotypes are relatively mild when compared to wts. It is concluded that the model for Wts activation in which apicolateral junction proteins Ft, Ex, and Mer play the predominant role cannot be universally applicable in all cell types. Rather, the relative importance of Ex and Mer for Wts regulation appears to depend on developmental context (Zhao, 2008).

Consistent with this proposal, strong functional interdependence and phenotypic similarities were found between Fas2, dlg, lgl, scrib, and wts, thus indicating that the BLJ, not the apicolateral junction, plays the predominant role in Wts regulation during oogenesis. Although genetic evidence alone cannot completely rule out that Wts may act in a parallel pathway to the BLJ and impinge on a set of downstream targets that overlap with those targeted by the BLJ, the following observations favor a model in which the BLJ is more directly involved in Wts regulation (it is noted that these are not mutually exclusive alternatives): (1) over 50 tumor suppressor genes have been identified in Drosophila, but lgl, scrib, and wts were the only strong dlg enhancers identified in this genomewide screen; (2) wts showed strong genetic interactions with Fas2, dlg, and lgl, similar to or stronger than scrib, which encodes a known BLJ protein; (3) wts has early tumor phenotypes similar to dlg partial loss of function and to scrib; (4) wts has the same border cell migration phenotype as scrib; (5) wts has similar small nucleus, epithelial stratification, and germinal vesicle defects as Fas2, dlg, lgl, and scrib; (6) like lgl and scrib, wts overexpression suppressed Fas2 and dlg tumorigenesis; (7) Fas2, dlg, and wts have similar proliferation defects, and (8) Fas2, dlg, and wts similarly repress CycE and DIAP1 expression, which is especially crucial, because CycE and DIAP1 are downstream targets of Wts signaling, and ex and mer had no impact on their expression, contrary to results in other tissues. Thus, the data strongly indicate that the BLJ signals through Wts, and may impinge on Roe in the nucleus, thus suggesting the first BLJ signaling pathway in animal cells. This implies that the BLJ not only acts as a localized scaffold, but also signals to the nucleus to control gene expression, both of which cooperate to regulate epithelial polarity and dynamics (Zhao, 2008).

How can these results in follicle cells, which suggest that Wts acts predominantly downstream of the BLJ, be reconciled with findings in eye tissue, which indicate that Wts acts downstream of the apicolateral junction? Interestingly, the genetic data in the eye suggest that Ft, Ex, and Mer cannot account for all of the signals that activate Wts, because wts overgrowth and tissue disorganization phenotypes are more severe than ft or mer; ex. On the basis of these findings in follicle cells, it is possible that Wts activation in the eye requires additional input from the BLJ. This possibility may have been overlooked thus far because dlg does not appear to have an overgrowth phenotype in the eye. dlg may be essential for additional functions in the eye that are epistatic to its tumor suppressor function, thus preventing loss of cells from the epithelium that could mask an overgrowth phenotype. Consistent with this, when activated Rasv12 is combined with dlg loss, dramatic tumors develop that are larger and more invasive than those produced by Rasv12 alone (Zhao, 2008).

In contrast, Dlg may have a diminished role in Wts signaling in the eye, much as the evidence indicates a diminished role for Ex and Mer in Wts signaling in the ovary. According to this model, Wts receives predominant input from distinct lateral junctions depending on tissue context. One distinction is that ovarian follicle cells are derived from a mesodermal lineage, while the eye and wing tissues are from ectodermal lineages. Further, many genes that disrupt apical-basal polarity and epithelial morphology have only subtle phenotypes in the eye by comparison to the ovary or embryo. Finally, the follicular epithelium requires input from junctions on all three follicle cell surfaces, lateral, apical, and basal, whereas most epithelia require only two, lateral and apical or basal. Thus, ovarian and imaginal tissues are likely to organize signaling pathways acting downstream of epithelial junctions in similar, yet fundamentally different ways to meet the unique organizational requirements of their cell-tissue morphologies. Some or all of these differences may contribute to the suggested specificity observed in Wts signaling downstream of BLJs in follicle cells. In general, these findings raise the possibility for future investigation that depending on the cell-tissue morphologies of a given organ, one lateral junction may play a predominant organizational role, and Wts signaling may act as a universal signaling adapter for mediating contact inhibition from that junction (Zhao, 2008).

An especially interesting aspect of Mer and Ex function that was uncovered in follicle cells is that it appears to be restricted to predominantly postmitotic, differentiated cells, in contrast to the role of Mer and Ex in other tissues. Further, given the absence of an involvement of Ft and lack of Mer-Ex synergism it is concluded that if Mer and Ex would be involved in Wts activation in follicle cells, they would have to function via a fundamentally distinct mechanism than in other tissues. It is proposed that during early oogenesis, the BLJ alone may provide the predominant input to Wts. Then, during midoogenesis, Ex and Mer may become involved in novel interactions with Dlg or other components of the BLJ to activate Wts in spatiotemporally distinct populations of differentiating cells to help achieve their unique developmental functions (Zhao, 2008).

How do wts, scrib, and roe promote motility? It is proposed that Scrib, Wts, and Roe are all crucially involved in EMT. In EMT, cells (1) loose apical-basal polarity and become mesenchymal-like, and (2) adopt a polarity conducive to movement. scrib, wts, and roe cells clearly lose epithelial polarity and become mesenchymal-like as indicated by their rounded morphology and lateralized phenotype. However, scrib, wts, and roe tumors do not invade, and scrib, wts, and roe border cells do not move, suggesting that the second aspect of EMT, adoption of a polarity conducive to movement, is defective. Consistent with this, mammalian Scrib is required for migration and epithelial wound healing of cultured human breast epithelial cells, and is also required in vivo for wound healing in mice. Human Scrib directs migration by organizing several polarities crucial for migration, including the orientation of the microtubule and Golgi networks and the localization of Cdc42 and Rac1 to the cell's leading edge. Thus Scrib has a conserved function in directed cell migration by organizing a polarity conducive to movement. In mammalian PC12 cells Scrib is in complex with Rac1. Fly Rac1 is essential for border cell migration and invasion of Fas2 and dlg tumors, suggesting that an essential role of Scrib in Rac1 function may be of crucial importance for movement. The apparent conserved role of BLJ proteins in organizing EMT, and both promoting and repressing movement, reemphasizes the suggestion that BLJ proteins do more than merely maintain apical-basal polarity, but rather repress a cellular transformation from epithelial polarity to a mesenchymal, lateralized signature conducive to movement (Zhao, 2008).

How is the function of scrib, wts, and roe in promoting border cell movement consistent with the requirement of Fas2, dlg, and lgl in repressing border cell movement? Further, how do scrib and wts act as enhancers of dlg tumor invasion even though scrib and wts tumors are noninvasive? For border cell movement, Fas2 and dlg mutations not only accelerate movement, but also delay border cell delamination. The delay in border cell delamination suggests that the BLJ normally promotes motility, but this promoting function can be bypassed when the repression of motility branch of the BLJ pathway is simultaneously lost. Cumulative data indicate that scrib, wts, and roe act predominantly within the EMT and proliferation branches of the BLJ pathway, and not the repression of motility branch. It is suggested that without simultaneous loss of the repression of motility branch of the BLJ pathway, scrib and wts border cells cannot bypass the essential requirement for the second step of EMT, thus border cell motility is blocked (Zhao, 2008).

This interpretation is also consistent with the seemingly paradoxical function of scrib and wts as enhancers of dlg tumor invasion, even though Scrib and Wts promote rather than repress border cell movement. The noninvasive scrib and wts tumor phenotypes indicate that they are crucial for repressing the first step of EMT, loss of epithelial polarity and adoption of a lateralized, mesenchymal-like phenotype. It has been suggested that scrib and wts enhance dlg invasive tumorigenesis by increasing the rate at which dlg mutant follicle cells undergo EMT and further facilitate invasion by depressing proliferation control and increasing the number of follicle cells available for movement. Thus, even though scrib and wts are required to promote movement, it is suggested that in dlg; scrib/+ or dlg; wts/+ tumors this requirement can be bypassed because the branch of the BLJ pathway that represses motility is simultaneously disrupted (Zhao, 2008).

The noninvasive tumor phenotypes of scrib and wts are very similar to the phenotypes of dlg mutants that specifically disrupt Dlg SH3 and GuK domains. Thus Scrib and Wts may act specifically downstream of the Dlg SH3 and GuK domains. Consistent with this, Scrib appears to associate with the Dlg GuK domain in neuronal synapses via the linker protein GuK-holder. Further, whereas Fas2, dlg, and lgl cause faster border cell migration, border cell migration is very similar to wild type in the dlg SH3/GuK-specific mutants, suggesting that Dlg SH3/GuK predominantly represses the first step of EMT and proliferation but not motility. On the basis of this specificity, it is suggest that one reason that lgl may be a stronger dlg enhancer than scrib and wts is that lgl represses motility in addition to EMT and proliferation. For example, the de novo tumor formation observed when one copy of lgl, scrib, or wts is removed in dlghf/dlgsw ovaries suggests that a threshold level of BLJ activity essential for maintenance of polarity has been lost. However, the lgl interaction may be much stronger than scrib and wts because lgl additionally represses motility (Zhao, 2008).

Increased expression of CycE and DIAP1, known Wts targets, was observed in Fas2, dlg, lgl, scrib, wts, and roe cells. Thus the importance of CycE for proliferation control, and DIAP1 for control of EMT and motility, suggests that part of the mechanism by which Fas2-Dlg represses tumorigenesis is through activating Wts signaling. DIAP1 is in a complex with Rac1 and Profilin and enables border cell motility apparently by promoting actin turnover. Further, in the embryo, DIAP1 loss leads to Dlg cleavage and cellular rounding and dispersal. Too much DIAP1 also appears to be deleterious to movement, because targeted overexpression of DIAP1 specifically in border cells slows their migration (data not shown). Thus maintaining the proper balance of DIAP1 is critical for directed movement, and it may be part of the mechanism by which Scrib and Wts influence border cell movement, suggesting that interaction with Dlg and Rac1 may be another level at which Scrib regulates EMT and movement, consistent with the possibility that it functions downstream of Scrib and Wts in follicle cells to repress both EMT and proliferation (Zhao, 2008).

In contrast to the strong enhancement of dlg by scrib, Fas2 was only weakly enhanced by scrib. Given the complexity of coordinating EMT, proliferation, and motility within an epithelial field, perhaps the simplest model is that multiple Dlg complexes reside within the BLJ, each with a distinct set of ligands that control one or more morphogenetic activities (Zhao, 2008).

Another interesting difference in the enhancement of dlg and Fas2 by lgl, scrib, wts, and roe was that they all enhanced both dlg tumorigenesis and invasion, but only enhanced Fas2 tumorigenesis, without invasion. An important difference between these experiments may be that in Fas2null follicle cells, Dlg is missing Fas2 as a ligand, whereas in dlghf/dlgsw, dlghf/dlgip20, and dlghf/dlglv55 follicle cells, Fas2 is localized at sites of contact between follicle cells in both the native epithelium and in streams of invading cells, suggesting that Fas2 continues to act as a Dlg ligand in these cells. This is probably an important difference because Fas2-Dlg binding is expected to control the conformation of Dlg. Dlg conformations in turn may specify Dlg intra- and intermolecular interactions that determine the relative balance of EMT, proliferation, and invasion factors that associate with the BLJ scaffold. For example, in neuronal cells intramolecular interactions between Dlg SH3 and GuK domains regulate the strength of intermolecular binding of GuK-holder, which binds Scrib. The SH3-GuK intramolecular interaction is further modulated by intramolecular interactions with PDZ3, which are regulated by intermolecular interactions with neurolignin, a transmembrane ligand for PDZ3 (Zhao, 2008).

On the basis of this molecular model, it is proposed that in the absence of Fas2, Dlg has a distinct conformation that tilts the balance toward EMT and proliferation over invasion, when Lgl, Scrib, Wts, or Roe are reduced. This study has shown that lgl, scrib, wts, and roe are expected to act predominantly downstream of Dlg SH3 and GuK domains to repress EMT and proliferation. Thus, removal of one copy of lgl, scrib, wts, or roe in Fas2 cells may tip the ratio of factors controlling EMT, motility, and proliferation toward derepression of EMT and proliferation, masking the Fas2 requirement for invasion. One possibility is that lgl, scrib, wts, or roe are especially important for expression of a protein in the apicolateral junction, such as Par-3/Bazooka, which is essential for dlg invasion. Consistent with this, Ex upregulation is seen in both dlg and wts clones. Further, lgl enhancement at the lglts permissive temperature showed essentially the opposite trend from Fas2. Rather than enhance tumorigenesis over invasion, removal of one copy of Fas2, dlg, scrib, wts, or roe in lgl egg chambers favored invasion. Thus, it is suggested that tumor invasiveness associated with particular combinations of mutated BLJ proteins may be masked or unmasked on the basis of the balance of activities that are disrupted, rather than disruption of particular activities per se (Zhao, 2008).

In summary, this study has identified the first signaling pathway that acts downstream of the BLJ that specifically controls EMT and proliferation, and important clues have been gained as to how this signaling may be organized. Like the Drosophila follicular epithelium, the human ovarian surface epithelium, which is thought to be the site of origin of most ovarian cancers, is derived from a mesodermal lineage. The data suggest that the BLJ plays an especially crucial role in the follicle cells compared to ectodermal lineages in repressing epithelial invasion and that the follicular epithelium appears to organize signaling from epithelial junctions in distinct ways compared to other epithelia. Given the conservation in the lineage of the fly and human epithelia, and the sensitivity of this screen for detecting molecules important for invasive carcinogenesis, it is proposed that the fly egg chamber may serve as a prototype for identifying early molecular events that are crucial for invasion of human ovarian cancer and possibly other malignancies that remain undetected before they start to invade (Zhao, 2008).


EVOLUTIONARY HOMOLOGS

The C. elegans gene lin-29 is required for the terminal differentiation of the lateral hypodermal seam cells during the larval-to-adult molt. lin-29 protein accumulates in the nuclei of these cells, consistent with its predicted role as a zinc finger transcription factor. The earliest detectable LIN-29 accumulation in seam cell nuclei is during the last larval stage (L4), following the final seam cell division, which occurs during the L3-to-L4 molt. LIN-29 accumulates in all hypodermal nuclei during the L4 stage. The time of LIN-29 appearance in the hypodermis is controlled by the heterochronic gene pathway: LIN-29 accumulates in the hypodermis abnormally early, during the third larval stage, in loss-of-function lin-14, lin-28 and lin-42 mutants, and fails to accumulate in the hypodermis of lin-4 mutants. LIN-29 also accumulates stage-specifically in the nuclei of a variety of non-hypodermal cells during development. Its accumulation is dependent upon the upstream heterochronic genes in some, but not all, of these non-hypodermal cells (Bettinger, 1996).

C. elegans vulval development culminates during exit from the L4-to-adult molt with the formation of an opening through the adult hypodermis and cuticle that is used for egg laying and mating. Vulva formation requires the heterochronic gene lin-29, which triggers hypodermal cell terminal differentiation during the final molt. lin-29 mutants are unable to lay eggs or mate because no vulval opening forms; instead, a protrusion forms at the site of the vulva. It has been demonstrated, through analysis of genetic mosaics, that lin-29 is absolutely required in a small subset of lateral hypodermal seam cells, adjacent to the vulva, for wild-type vulva formation and egg laying. However, lin-29 function is not strictly limited to the lateral hypodermis: (1) LIN-29 accumulates in many non-hypodermal cells with known roles in vulva formation or egg laying; (2) animals homozygous for one lin-29 allele, ga94, have the vulval defect and cannot lay eggs, despite having a terminally differentiated adult lateral hypodermis; (3) vulval morphogenesis and egg laying requires lin-29 activity within the EMS lineage, a lineage that does not generate hypodermal cells (Bettinger, 1997).

The C. elegans gene lin-29 encodes a zinc-finger transcription factor that is required for hypodermal cell terminal differentiation and proper vulva morphogenesis. lin-29 is also required in males for productive mating. lin-29 males can perform the early mating behaviors including response to hermaphrodite contact and vulva location, but they do not perform the subsequent steps of vulva attachment via spicule insertion and sperm transfer. Consistent with this observation, lin-29 mutant spicules are on average 43% shorter than wild-type spicules while other male mating structures appear unaltered. In lin-29 mutants, spicule development goes awry after the generation of spicule cells, when spicule morphogenesis occurs in wild-type males. LIN-29 accumulates in many cells of the wild-type male tail, including those that form the spicules. It has been demonstrated, through analysis of genetic mosaics, that the formation of wild-type-length spicules requires lin-29(+) in the AB.p lineage, the lineage that gives rise to the spicules and other male copulatory structures. Mosaic analysis also reveals a role for lin-29(+) in the P1 lineage, which mainly produces sex muscles, cells of the somatic gonad, and body wall muscles (Euling, 1999).

The development of a connection between the uterus and the vulva in the nematode C. elegans requires specification of a uterine cell called the 'utse', and its attachment to the vulva and the epidermal seam cells. The uterine pi cells generate the utse and uv1 cells, which also connect the uterus to the vulva. The uterine anchor cell (AC) induces the vulva through LIN-3/epidermal growth factor (EGF) signaling, and the pi cells through LIN-12/Notch signaling. A gene required for seam cell maturation is also required for specification of the utse and for vulval differentiation, and thus helps to coordinate development of the vulval-uterine-seam cell connection. The egl-29 gene is necessary for induction of uterine pi cells; it is allelic to lin-29, which encodes a zinc finger transcription factor that is necessary for the terminal differentiation of epidermal seam cells. In the uterus, lin-29 functions upstream of lin-12 in the induction of pi cells and is necessary to maintain expression in the AC of lag-2, which encodes a ligand for LIN-12. It is concluded that the lin-29 gene controls gene expression in the epidermal seam cells, uterus and vulva, and may help to coordinate the terminal development of these three tissues by regulating the timing of late gene expression during organogenesis (Newman, 2000).

Null mutations in the C. elegans heterochronic gene lin-41 cause precocious expression of adult fates at larval stages. Increased lin-41 activity causes the opposite phenotype, reiteration of larval fates. let-7 mutations cause similar reiterated heterochronic phenotypes that are suppressed by lin-41 mutations, showing that lin-41 is negatively regulated by let-7. lin-41 negatively regulates the timing of LIN-29 adult specification transcription factor expression. lin-41 encodes an RBCC protein, and two elements in the lin-41 3'UTR are complementary to the 21 nucleotide let-7 regulatory RNA. A lin-41::GFP fusion gene is downregulated in the tissues affected by lin-41 at the time that the let-7 regulatory RNA is upregulated. It is suggested that late larval activation of let-7 RNA expression downregulates LIN-41 to relieve inhibition of lin-29 (Slack, 2000).

p130(cas) (Cas) is a docking protein that contains an SH3 domain and multiple tyrosine residues. p130(cas) is located at focal adhesions, is tyrosine phosphorylated in response to integrin stimulation, and is thought to transmit signals, via c-Crk and other proteins, for the remodeling of actin stress fibers and cell movement. In a search for the ligands of the SH3 domain of p130(cas) by far-Western screening, a novel protein named CIZ (for Cas-interacting zinc finger protein) was cloned. CIZ consists of the following: a putative leucine zipper; a serine/threonine-rich region; a proline-rich sequence; five, six, or eight Krüppel-type C(2)H(2) zinc fingers, and the glutamine-alanine repeat. CIZ binds Cas in cells and is located in the nucleus and at focal adhesions. CIZ has been identified as a nucleocytoplasmic shuttling protein, by using the transient interspecies heterokaryon formation assay. In order to search for the targets of CIZ in nucleus, the DNA binding consensus of CIZ has been identified as (G/C)AAAAA(A) by cyclic amplification and selection of targets analysis. The consensus-like sequences are found in several promoters of matrix metalloproteinases (MMPs), which are the enzymes used to degrade the extracellular matrix proteins. CIZ binds to a consensus-like sequence in the MMP-1 (collagenase) promoter. Overexpression of CIZ upregulates the transcriptions from MMP-1, MMP-3 (stromelysin), and MMP-7 (matrilysin) promoters, and this transactivation is enhanced in the presence of Cas. Furthermore, the stable overexpression of CIZ promotes the production of MMP-7 in culture medium. In summary, CIZ, a novel zinc finger protein, binds Cas, is a nucleocytoplasmic shuttling protein, and regulates the expression of MMPs (Nakamoto, 2000).

MAB-10/NAB acts with LIN-29/EGR to regulate terminal differentiation and the transition from larva to adult in C. elegans

In Caenorhabditis elegans, a well-defined pathway of heterochronic genes ensures the proper timing of stage-specific developmental events. During the final larval stage, an upregulation of the let-7 microRNA indirectly activates the terminal differentiation factor and central regulator of the larval-to-adult transition, LIN-29, via the downregulation of the let-7 target genes lin-41 and hbl-1. This study identifies a new heterochronic gene, mab-10, and shows that mab-10 encodes a NAB (NGFI-A-binding protein) transcriptional co-factor. MAB-10 acts with LIN-29 to control the expression of genes required to regulate a subset of differentiation events during the larval-to-adult transition, and the NAB-interaction domain of LIN-29 is conserved in Kruppel-family EGR (early growth response) proteins. A similar interaction between Drosophila NAB and the two Drosophila LIN-29 homologs RN and SQZ was reported recently. In mammals, EGR proteins control the differentiation of multiple cell lineages, and EGR-1 acts with NAB proteins to initiate menarche by regulating the transcription of the luteinizing hormone β subunit. Genome-wide association studies of humans and various studies of mouse recently have implicated the mammalian homologs of the C. elegans heterochronic gene lin-28 in regulating cellular differentiation and the timing of menarche. This work suggests that human homologs of multiple C. elegans heterochronic genes might act in an evolutionarily conserved pathway to promote cellular differentiation and the onset of puberty (Harris, 2011).

This study identified mab-10 as a new heterochronic gene that is required for specific aspects of the larval-to-adult transition, specifically molting cycle exit and seam cell exit from the cell cycle. mab-10 encodes the only C. elegans NAB transcriptional co-factor. NAB proteins are thought to physically interact with Kruppel family EGR transcription factors to regulate their activity (Harris, 2011).

Previous work demonstrated that MAB-10 (then known only as the C. elegans NAB protein R166.1) could interact with mammalian EGR proteins in a yeast two-hybrid assay; no corresponding C. elegans EGR protein was identified. This study has demonstrate that MAB-10 interacts with the terminal differentiation factor LIN-29 through an evolutionarily conserved NAB binding domain (R1 domain) and that MAB-10 is required for a subset of LIN-29-dependent activities. This work identifies LIN-29 as a C. elegans EGR-like protein and demonstrates that the C. elegans heterochronic pathway controls the timing of NAB/EGR-mediated differentiation (Harris, 2011).

Several experiments using mammalian tissue culture suggest that NAB proteins negatively regulate EGR activity by binding EGR proteins at specific target genes and preventing EGR-mediated transcription. However, loss of either EGR2 function or NAB function in mice and humans results in hypomyelination, suggesting that EGR and NAB proteins need not act antagonistically in vivo (Harris, 2011).

In C. elegans, MAB-10 and LIN-29 both act to promote terminal differentiation and the onset of adulthood. Furthermore, mab-10 promotes the formation of precocious adult alae in a lin-41 mutant background, suggesting that MAB-10 does not specifically act to control genes required for exit from the molting cycle and seam cell exit from the cell cycle, but more likely acts as a general enhancer of LIN-29 activity (Harris, 2011).

EGR and NAB proteins have been shown to operate in a negative-feedback loop wherein an EGR protein promotes the expression of its NAB co-factor, which then inhibits EGR activity. mab-10 transcription does not depend on LIN-29, despite a dramatic increase of mab-10 transcription during the L4 stage. Thus, mab-10 is not a transcriptional target of LIN-29 (Harris, 2011).

Whereas mab-10 is not a transcriptional target of LIN-29, MAB-10::GFP localization to seam cell nuclei during the L4 stage required LIN-29, indicating that LIN-29 might promote MAB-10 seam cell nuclear localization via a post-transcriptional mechanism or via direct physical interaction (Harris, 2011).

This work demonstrates that MAB-10 and LIN-29 do not operate in a negative-feedback loop. It is proposed that other components of the heterochronic pathway directly regulate mab-10 transcription to temporally regulate MAB-10/LIN-29 activity and that LIN-29 or some factor downstream of LIN-29 controls MAB-10/LIN-29 activity by promoting the accumulation of MAB-10 in seam cell nuclei (Harris, 2011).

By showing that MAB-10 acts with LIN-29 through an evolutionarily conserved EGR R1 domain, LIN-29 and the Drosophila LIN-29 homologs RN and SQZ are identified as EGR-like molecules. It is proposed that NAB proteins and EGR proteins act together in temporal developmental programs to control terminal differentiation. In Drosophila, the LIN-29 homolog SQZ acts with Drosophila NAB to control neuroblast differentiation. In C. elegans, LIN-29 and MAB-10 act together to control the differentiation of a hypodermal stem cell lineage during the transition from larva to adult by regulating the expression of the nuclear hormone receptors nhr-23 and nhr-25 and the cell cycle regulator cki-1. Recently, a study of C. elegans demonstrated that nhr-25 is itself a heterochronic gene and possibly functions with lin-29 to promote aspects of the larval-to-adult transition, including seam cell exit from the cell cycle. Though the mechanism by which nhr-25 regulates seam cell exit from the cell cycle is not known, it is speculated that LIN-29 and NHR-25 might act together to promote cki-1 expression (Harris, 2011).

EGR proteins were originally identified as immediate-early genes and generally have been regarded as differentiation factors. Like mab-10 and lin-29 mutants, Nab and Egr mutant mice are defective in the terminal differentiation of several cell lineages. For example, in Schwann cells, EGR2 promotes the expression of P27, the homolog of C. elegans CKI-1, and acts with NAB proteins to promote terminal differentiation. Mammalian homologs of other C. elegans heterochronic genes also control differentiation. Similar to the role of LIN-28 in C. elegans, mammalian LIN28 and LIN28B promote stem cell identity and prevent differentiation by repressing the let-7 microRNA gene. As in C. elegans, increasing levels of let-7 drive differentiation, and the mouse homolog of LIN-41, LIN41, has been shown to be a let-7 target acting in stem cell niches to prevent premature differentiation (Harris, 2011).

Mammalian LIN-28 controls the timing of the onset of puberty in mice and possibly humans. Mice lacking EGR1 function, like lin-29 mutants of C. elegans, fail to undergo puberty. EGR1 and NAB proteins act with SF1, the homolog of C. elegans NHR-25, in the gonadotrope lineage of the pituitary gland to regulate the expression of luteinizing hormone and the onset of puberty. The molecular mechanism by which mammalian LIN-28 regulates the onset of puberty is not known. This work raises the possibility that homologs of C. elegans heterochronic genes might act in an evolutionarily conserved pathway that controls the terminal differentiation of cell lineages and the onset of adulthood by regulating the activity of NAB and EGR proteins (Harris, 2011).


REFERENCES

Search PubMed for articles about Drosophila rotund

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Agnel, M., Roder, L., Vola, C. and Griffin-Shea, R. (1992b). A Drosophila rotund transcript expressed during spermatogenesis and imaginal disc morphogenesis encodes a protein which is similar to human Rac GTPase-activating (racGAP) proteins. Mol. Cell Biol. 12: 5111-5122. 1406685

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Bettinger, J. C., Euling, S. and Rougvie, A. E. (1997). The terminal differentiation factor LIN-29 is required for proper vulval morphogenesis and egg laying in C. elegans. Development 124(21): 4333-42. 9334281

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Euling, S., Bettinger, J. C. and Rougvie, A. E. (1999). The LIN-29 transcription factor is required for proper morphogenesis of the C. elegans male tail. Dev. Biol. 206(2): 142-56. 9986728

Felix, J. T., Magarinos, M. and Diaz-Benjumea, F. J. (2007). Nab controls the activity of the zinc-finger transcription factors Squeeze and Rotund in Drosophila development. Development 134(10): 1845-52. Medline abstract: 17428824

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Harris, D. T. and Horvitz, H. R. (2011). MAB-10/NAB acts with LIN-29/EGR to regulate terminal differentiation and the transition from larva to adult in C. elegans. Development 138(18): 4051-62. PubMed Citation: 21862562

Hartl, M., Loschek, L. F., Stephan, D., Siju, K. P., Knappmeyer, C. and Kadow, I. C. (2011). A new Prospero and microRNA-279 pathway restricts CO2 receptor neuron formation. J Neurosci 31: 15660-15673. PubMed ID: 22049409

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Nakamoto, T., Yamagata, T., Sakai, R., Ogawa, S., Honda, H., Ueno, H., Hirano, N., Yazaki, Y. and Hirai, H. (2000). CIZ, a zinc finger protein that interacts with p130(cas) and activates the expression of matrix metalloproteinases. Mol. Cell Biol. 20: 1649-1658. 10669742

Newman, A. P. et al., (2000). The Caenorhabditis elegans heterochronic gene lin-29 coordinates the vulval-uterine-epidermal connections. Curr. Biol. 10(23): 1479-88. 11114514

Raymond, K., et al. (2001).The Rac GTPase-activating protein RotundRacGAP interferes with Drac1 and Dcdc42 signalling in Drosophila melanogaster. J. Biol. Chem. 276(38): 35909-35916. 11468292

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Rougvie, A. E. and Ambros, V. (1995). The heterochronic gene lin-29 encodes a zinc finger protein that controls a terminal differentiation event in Caenorhabditis elegans. Development 121: 2491-2500. 7671813

Slack, F. J., et al. (2000). The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol. Cell 5(4): 659-69. 10882102

St Pierre, S. E., Galindo, M. I., Couso, J. P. and Thor, S. (2002). Control of Drosophila imaginal disc development by rotund and roughened eye: differentially expressed transcripts of the same gene encoding functionally distinct zinc finger proteins. Development 129: 1273-1281. 11874922

Zhao, M., Szafranski, P., Hall, C. A. and Goode, S. (2008). Basolateral junctions utilize warts signaling to control epithelial-mesenchymal transition and proliferation crucial for migration and invasion of Drosophila ovarian epithelial cells. Genetics 178(4): 1947-71. PubMed Citation: 18430928


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