The identification of sequences that control transcription in metazoans is a major goal of genome analysis. In a previous study, it was demonstrated that searching for clusters of predicted transcription factor binding sites could discover active regulatory sequences, and identified 37 regions of the Drosophila melanogaster genome with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. This study reports the results of in vivo functional analysis of 27 remaining clusters. Transgenic flies were generated carrying each cluster attached to a basal promoter and reporter gene, and assayed embryos for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three drive expression in patterns unrelated to those of neighboring genes; the remaining 18 do not appear to have enhancer activity. The Drosophila pseudoobscura genome was used to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. Conservation of binding-site clustering was incorporated into a new genome-wide enhancer screen, and several hundred new regulatory sequences were predicted, including 85 adjacent to genes with embryonic patterns. It is cocluded that measuring conservation of sequence features closely linked to function -- such as binding-site clusterin -- makes better use of comparative sequence data than commonly used methods that examine only sequence identity (Berman, 2004).
Individual neurons express only one or a few of the many identified neurotransmitters and neuropeptides, but the molecular mechanisms controlling their selection are poorly understood. In the Drosophila ventral nerve cord (VNC), the six Tv neurons express the neuropeptide gene FMRFamide (FMRFa). Each Tv neuron resides within a neuronal cell group specified by the LIM-homeodomain (LIM-HD) gene apterous (ap). The zinc-finger gene squeeze acts in Tv cells to promote their unique axon pathfinding to a peripheral target. There, the BMP ligand Glass bottom boat activates the Wishful thinking receptor, initiating a retrograde BMP signal in the Tv neuron. This signal acts together with apterous and squeeze to activate FMRFamide expression. Reconstituting this 'code,' by combined BMP activation and apterous/squeeze misexpression, triggers ectopic FMRFamide expression in peptidergic neurons. Thus, an intrinsic transcription factor code integrates with an extrinsic retrograde signal to select a specific neuropeptide identity within peptidergic cells (Allan, 2003).
FMRFa is specifically expressed in the six Tv neuroendocrine neurons located bilaterally in the three thoracic (T1-3) segments of the embryonic and larval VNC. apterous is expressed in three interneurons per VNC hemisegment, as well as in a lateral cluster of four neurons (the ap-cluster) in each of the T1-3 hemisegments. One of the four ap-cluster cells is the FMRFa-expressing Tv neuron. All ap interneurons in the VNC, except for the Tv, join a common ipsilateral axon tract termed the ap-fascicle. The Tv axon instead projects to the midline and exits the VNC dorsally to innervate the dorsal neurohemal organ (DNH). The DNH is a club-like neuroendocrine structure formed by two glial cells protruding from the midline of each thoracic segment. Anteriorly, two additional FMRFa-expressing cells are found, denoted SE2 cells. The SE2 cells do not express, nor depend upon, any regulators described in this study for their FMRFa expression. ap is important for the expression of FMRFa in the Tv neurons, but since most ap neurons do not express FMRFa, other regulators are likely needed for FMRFa regulation (Allan, 2003).
To determine whether sqz regulates FMRFa expression, immunoreactivity for the FMRFa peptide was compared in wild-type and sqz mutant L1 larvae. In wild-type, FMRFa immunoreactivity is robust (98%) in all six Tv neurons. In sqz mutants (sqzlacZ/sqzDf), FMRFa staining was found to be reduced in all Tv neurons and was detected in 75% of cells. The T1 segment was most affected, with FMRFa expressed in 40% of T1 Tv neurons. To verify that the observed effects reflected regulation of the FMRFa gene, antibodies recognizing the C-terminal of the FMRFa precursor peptide (proFMRF) were used, as well as an FMRFa-lacZ reporter that faithfully reports FMRFa expression in Tv neurons. An equivalent effect on proFMRF (75%) and FMRFa-lacZ (77%) was found in sqz mutants (sqzlacZ/sqzDf and sqzGAL4/sqzDf, respectively) when compared to wild-type. Again, segment T1 is most affected with FMRFa-lacZ expressed in only 50% of T1 Tv neurons. These results show that sqz in part regulates the expression of the FMRFa gene in Tv cells (Allan, 2003).
To determine whether sqz regulates axon pathfinding of the Tv neuron, apGAL4 was used to drive the expression of a membrane-targeted reporter (UAS-EGFPF). In sqz mutants, a frequent failure of the Tv axon to innervate the DNH was observed, instead, it apparently joins the ap-fascicle. This phenotype is most pronounced within the most anterior thoracic segment (T1). In wild-type embryos, the DNH was innervated in 100% of thoracic segments, whereas sqz mutants (apGAL4/+;sqzie/sqzDf,UAS-EGFPF) show axonal innervation in 69% of T1 segment DNHs. Failure of innervation did not result from the absence of the DNH itself, since its profile was evident in affected segments. These results show that sqz is important for proper pathfinding of Tv axons and that the Tv axon often fails to diverge from the ap-fascicle in sqz mutants, apparently reverting to an 'ap-only' phenotype (Allan, 2003).
Several determinants critical for proper FMRFa expression have been identified. These include a general peptidergic cell identity, co-expression of sqz and ap, axon projection out of the VNC, and competence to respond to a retrograde signal by activating the BMP pathway. When these criteria are met, either in the endogenous or ectopic case, FMRFa expression is triggered. Importantly, none of these events are individually exclusive to the Tv cell, but they are uniquely combined in only these 6 out of the 10,000 cells in the VNC. Reconstituting this scenario in other peptidergic neurons can trigger FMRFa expression. These results are in line with the emerging theme of a critical interplay between combinatorial transcription factor codes and signal transduction pathways in regulating gene expression and provide a clear example of how these general mechanisms also apply to the specific regulation of a terminal differentiation gene in the nervous system (Allan, 2003).
Why is ectopic FMRFa expression restricted to peptidergic neurons? Conceivably, cells responding to BMP activation and sqz/ap co-misexpression may arise from precursor cells utilizing a common genetic program, resulting in a chromatin state where the FMRFa gene is accessible to activation. Currently, the lineage from which most neuropeptidergic neurons arise is unknown, and any common theme behind their generation is uncertain. FMRFa expression may also be constrained by the presence of activators common to peptidergic neurons and/or by repressors present in non-peptidergic neurons. Common properties of peptidergic neurons, such as the dense core vesicle secretory machinery and the processing of precursor peptides, may indicate the existence of common regulatory programs for all peptidergic neurons. In support of this notion, recent studies of a novel basic helix-loop-helix transcription factor, dimmed, show that this gene is specifically expressed in most if not all peptidergic neurons. In dimmed mutants, peptidergic and secretory properties of the majority of peptidergic neurons are affected, including the expression of processing enzymes and several neuropeptides, such as FMRFa. This shows that dimmed plays a key role in specifying the peptidergic fate and supports the notion of a common regulatory program for this cell type (Allan, 2003).
Previous studies found that ap is essential for axon pathfinding of the majority of ap-neurons. However, ap does not affect Tv axon pathfinding, suggesting that the role of ap in Tv cells may exclusively be to regulate FMRFa expression. In line with these results, ap mutants do not show any apparent loss of pMad accumulation in the Tv neurons. In contrast, sqz mutants have Tv axon pathfinding phenotypes, and, consequently, a partial loss of pMad staining specifically in Tv neurons. Observations of Tv axons at the midline suggest that in the absence of sqz, the Tv axon likely reverts to an 'ap-only' axonal phenotype and turns to grow along the common ap-fascicle. Given the importance of DNH innervation for FMRFa expression, axon pathfinding defects in sqz mutants likely contribute to the loss of FMRFa in some hemisegments. However, the great difference in the loss of FMRFa expression between sqz (75%) and wit (0%) argues that sqz is not critical for BMP signaling, but rather affects it indirectly by affecting Tv axon pathfinding. Moreover, the sqz axon pathfinding phenotype is only partially penetrant and fails to explain either the reduction of FMRFa expression observed in all hemisegments, or the potency of sqz (acting together with ap) to trigger ectopic expression in Va and Vap peptidergic neurons (cells whose axons already exit the VNC and are pMad-positive). Misexpression of sqz in all ap cells occasionally leads to an additional pMad/FMRFa positive cell in the ap-cluster. In these cases, no ectopic FMRFa expression is detected in any axons extending in the common ap-fascicle, only in axons projecting into the DNH. Therefore, sqz misexpression likely alters the identity of another ap-cluster cell, imposing a Tv-like axonal pathfinding behavior and causing it to ectopically innervate the DNH. Thus, it appears that sqz regulates two critical features of Tv cell identity: differential pathfinding, and FMRFa expression (both directly and indirectly) (Allan, 2003).
Why do sqz and ap function to activate FMRFa expression within only three neuropeptidergic cell types (the Tv, Va, and Vap cells) which together comprise only 18 out of ~200 peptidergic neurons in the developing Drosophila VNC? Using the specific GAL4 lines, apGAL4, VaGAL4, and VapGAL4 to drive the expression of UAS-EGFPF, it was found that all three neuronal subsets exit the VNC: Tv axons via the DNHs, Va axons via the transverse nerves, and Vap axons via the posterior A8 nerves. This observation is important in light of previous studies of tinman (tin) mutants. In tin mutants, a number of mesodermally derived tissues, including the DNHs, fail to develop. As a result, Tv axons stall at the presumptive midline exit point and, intriguingly, FMRFa expression is strongly reduced. This suggests that the DNHs may be necessary for proper FMRFa expression in Tv cells. These findings have been confirmed; in tin mutants, the DNHs are absent, and proFMRF staining is weak and only detected in 10% of Tv neurons. To address the putative target requirement for FMRFa expression in an alternative way, apGAL4 was used to express molecules that either alter Tv axon pathfinding or interfere with Tv axonal transport. roundabout (robo), a receptor that mediates repulsion from the VNC midline, was tested. In apGAL4/UAS-robo L1 larvae, Tv axons avoid the midline and fail to innervate the DNH. As predicted, this results in a loss (2%) of FMRFa-lacZ expression. Next, dominant-activated rac (UAS-racV12) was tested; it causes Tv axons to stall before reaching the midline and they fail to innervate the DNHs. This results in a complete loss (0%) of FMRFa-lacZ expression. To interfere with axonal transport, apGAL4 was used to express a dominant-negative version of the P150/Glued dynactin motor complex component (UAS-GluedDN), a molecule shown to specifically interfere with retrograde axonal transport. In apGAL4/UAS-GluedDN L1 larvae, a complete loss (0%) of FMRFa-lacZ expression was detected. Similarly, expression of the microtubule binding Tau protein, shown to interfere with axonal transport in Drosophila led to a near complete loss (4%) of FMRFa-lacZ expression (apGAL4/UAS-τ-myc). In both UAS-GluedDN and UAS-τ-myc, normal Tv axon innervation of the DNH was observed in all segments (Allan, 2003).
By co-expressing UAS-EGFPF in all scenarios outlined above, it was found that loss of FMRFa expression was not due to loss of the Tv cell, since the number of cells within the ap-cluster was unaltered in tin, UAS-robo, UAS-racV12, UAS-GluedDN, and UAS-τ-myc. Using α-Glutactin, it was found that the DNH itself is only affected in tin mutants, not in the other genotypes. Together, these results show that innervation of the DNH and retrograde signaling is essential for the expression of FMRFa (Allan, 2003).
What is the identity of the retrograde FMRFa-inducing signal? Recently, a Drosophila BMP type-II receptor, wishful thinking (wit), was implicated in mediating a retrograde signal from muscles to motor neurons, responsible for presynaptic maturation. Signaling by the TGF-β/BMP superfamily occurs via activation of a receptor complex, consisting of two type I and two type II receptors, leading to phosphorylation and nuclear translocation of a receptor Smad protein. In Drosophila, BMP signaling leads to the phosphorylation and nuclear translocation of the Smad protein Mothers against dpp (Mad), which can be monitored using antibodies specific to phosphorylated Mad (pMad) (Allan, 2003).
Using antibodies to pMad, BMP activation in peptidergic neurons was assayed. Nuclear pMad was detected not only in motor neurons, but also in the Tv, Va, and Vap neurons, demonstrating that peptidergic neurons projecting out of the VNC also show evidence of BMP activation. Accumulation of pMad in the Tv neurons commences during stage 17, immediately following DNH innervation. These results led to a test of whether Tv innervation of the DNH would be critical for pMad accumulation and consequently for FMRFa expression. Indeed, it was found that the absence of the DNH (in tin mutants), Tv axon pathfinding alterations (in apGAL4/UAS-robo and apGAL4/UAS-racV12) and interference with Tv axonal transport (in apGAL4/UAS-GluedDN and apGAL4/UAS-τ-myc) are all accompanied by loss of pMad staining specifically in Tv neurons. The ectopic ap-cluster FMRFa-expressing cell induced by sqz misexpression is also pMad positive. Given the role of sqz in Tv axon pathfinding, this is interpreted as resulting from sqz dominantly altering the projection of one other ap-cluster cell, forcing it to innervate the DNH. Thus, in all genotypes examined, Tv axonal projection to the DNH is critical for pMad accumulation (Allan, 2003).
Since Wit is expressed in a restricted pattern in the developing VNC, attempts were made to address whether the Tv neurons express Wit. However, single-cell resolution could not be obtained with the Wit antibody and Wit could not be definitely localized in Tv cells. However, the wit-dependent pMad accumulation in Tv neurons, the apGAL4/UAS-tkvA, UAS-saxA-mediated rescue of wit mutants, and the UAS-gbb-mediated 'rescue' of UAS-robo misexpression, provide genetic evidence supporting the expression of wit in Tv cells. Previous studies have shown that gbb is expressed in developing endoderm and visceral mesoderm, but it has not been detected in the VNC. By in situ hybridization, no apparent expression was detected in the DNH. Given that the DNH only contains two cell bodies, low-level gbb expression may be beyond detection. Moreover, since the anterior midgut is positioned in very close proximity to the DNHs, it is possible that Gbb diffuses from the visceral mesoderm to the DNH (Allan, 2003).
Why is BMP activation necessary for FMRFa expression? Neither forced axonal exit from the VNC (apGAL4/UAS-Unc5) nor autocrine presentation of the Gbb ligand (apGAL4/UAS-gbb) leads to activated pMad and FMRFa expression in ap cells other than the Tv cell. This indicates that the Tv cell is uniquely predetermined to respond to the Gbb ligand. In fact, even direct activation of the BMP pathway (UAS-saxA, -tkvA;apGAL4/+) in all ap neurons does not trigger ectopic FMRFa expression, showing that the Tv cell is further uniquely capable of responding to BMP activation. The misexpression results show that both of these properties of the Tv cell are specified by sqz/ap co-expression. Given this level of Tv cell predetermination, it begs the question as to why Tv cell FMRFa expression evolved to be dependent upon a retrograde BMP signal. Perhaps dependence upon a retrograde signal provides precise control over the onset of FMRFa expression during embryogenesis. In fact, Tv neurons are born by stage 14 (as evident by ap expression) but do not activate FMRFa expression until late stage 17, upon DNH innervation. Alternatively, the presence of a small number of sqz/ap co-expressing cells in the developing brain that do not express FMRFa may necessitate additional regulatory control over FMRFa expression. Dependence upon a signal transduction pathway also provides several unique means of control and amplification of target gene expression. Finally, the fact that sqz, ap, and BMP activation only act to trigger FMRFa expression within a neuropeptidergic cellular context reveals additional complexity underlying the control of specific neuropeptide expression. Given the large number of diverse cell types in the CNS, what may appear to be an almost excessive complexity of combinatorial coding may in fact be essential for high fidelity of gene expression (Allan, 2003).
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 (Svaren, 1998) or activate (Sevetson, 2000) 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 (Swirnoff, 1998). 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 (Le, 2005). In zebrafish, egr2 controls expression of the Nab gene homologs in the r3 and r5 rhombomeres of the developing hindbrain (Mechta-Grigoriou, 2000). 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 (Mechta-Grigoriou, 2000). 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 (Le, 2005; 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 (Clements, 2003). 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).
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).
Although nab loss-of-function alleles are larval lethal, the rn-null condition is homozygous viable. This suggests that Nab may have at least one other partner in embryonic development. Rn belongs to a conserved subfamily of zinc-finger proteins that include Drosophila Sqz, C. elegans LIN-28 and rat Ciz. Sqz and Rn have two highly homologous domains: the zinc-finger domain (90% identity) and a 32 amino acid C-terminal domain (over 80% identity). sqz mutant alleles are larval lethal and have a motility defect. sqz is first required in embryonic CNS development to define the number of cells that express the LIM-homeodomain gene ap in the ap thoracic cluster of interneurons. Later on, it is also involved in the combinatorial code of transcription factors that specifies the fate of the Tv neuron in the ap cluster. The Tv neuron is distinguished from the rest of the neurons in the cluster by the fact that it contains the neuropeptide FMRFa [FMRFamide-related (Fmrf)]. In sqz mutant embryos, additional ap-expressing neurons are generated and the Tv neuron is not specified as no FMRFa expression is found (Allan, 2003). To determine whether Nab is a co-factor of Sqz, the expression of nab and sqz was examined in stage-17 embryos. It was found that a subset of the CNS neurons that express sqz also expressed nab, whereas other neurons express either sqz or nab. Two or three neurons in the ap cluster of stage-17 embryos express nab, one typically at a relatively high level of expression. By the first instar larval stage only one neuron in the ap cluster expressed nab. By double staining with anti-FMRFa and anti-Nab it was possible to identify this as the Tv neuron. At this stage, sqz was expressed at high levels in the Tv neuron and at low levels in two other neurons of the ap cluster. Next the expression of ap and FMRFa was analyzed in nab mutant larvae. In first instar nabSH143 larvae, additional ap-expressing neurons were found in the ap cluster. In nabSH143 embryos, additional cells expressed the bHLH gene dimmed (dimm), as shown for sqz mutants. The expression of FMRFa was analyzed in the ap clusters of first instar larvae, and FMRFa staining was lost or reduced in all the Tv neurons, mainly in the T1 cluster. It is concluded that lack-of-function alleles of nab and sqz generate the same embryonic phenotypes: the number of ap-expressing cells in the ap thoracic clusters is increased, additional dimm-expressing neurons are detected in the clusters, and Tv neuronal fate is absent. These results strongly suggest that, unlike the situation in imaginal disc development where Nab acts as a co-repressor of Rn, in CNS development Nab is required as a co-activator of Sqz (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, it was asked whether 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).
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