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 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).
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 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).
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).
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).
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).
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date revised: 5 October 2007
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