InteractiveFly: GeneBrief

nab: Biological Overview | References

BIOLOGICAL OVERVIEW

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. Targeted deletion mutations in the nab gene have been created, and the identification of additional, EMS-induced dnab mutations by genetic complementation analysis has been described. Null alleles in nab cause larval locomotion defects and early larval lethality (L1-L2). A putative hypomorphic allele in nab instead causes early adult lethality due to severe locomotion defects. In the nab -/- CNS, axon outgrowth/guidance and glial development appear normal; however, a subset of eve+ neurons forms in reduced numbers. In addition, mosaic analysis in the eye reveals that nab -/- clones are either very small or absent. Similarly, Nab overexpression in the eye causes eyes to be very small with few ommatidia. These dramatic eye-specific phenotypes should prove useful for enhancer/suppressor screens to identify nab-interacting genes (Clements, 2003; full text of article).

During imaginal disc development, nab is strongly expressed in the wing presumptive domain under the control of vg, and nab is required in proximodistal axis development to control the expression of wg in the wing hinge. 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. This study shows nab is required in proximodistal axis development to control the expression of wg (Félix, 2007).

nab is also a component of the combinatorial code that determines the number of neurons that express the gene apterous (ap) in embryonic neural development, and nab specifies the Tv neuronal fate in the ap thoracic cluster of neurons. 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).

Expression of Nab

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).

Vestigial controls nab expression in the wing

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 vg^83b27r 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).

nab delimits the expression of wg in the wing inner ring

The nab^SH143 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 nab^SH143 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 Rn zinc-finger transcription factor is a potential partner of Nab in wing development

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).

The Sqz zinc-finger transcription factor is a potential partner of Nab in neuronal fate specification

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 nab^SH143 larvae, additional ap-expressing neurons were found in the ap cluster. In nab^SH143 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).

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 (Rn^Δ894). The ability of Nab-GST to retain the [³⁵S]Rn^Δ894 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 rn^Δ894 fragment was cloned into the pUAST vector and clones of cells misexpressing UASrn^Δ894 were generated (Act>Gal4>UASrn^Δ894). 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 nab^SH143 homozygous clones were generated in a sqz^lacZ/+ 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 Rn^Δ894 version, and clones of cells misexpressing rn^Δ894 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).

The co-regulator dNAB interacts with Brinker to eliminate cells with reduced Dpp signaling

The proper development of tissues requires morphogen activity that dictates the appropriate growth and differentiation of each cell according to its position within a developing field. Elimination of underperforming cells that are less efficient in receiving/transducing the morphogenetic signal is thought to provide a general fail-safe mechanism to avoid developmental misspecification. In the developing Drosophila wing, the morphogen Dpp provides cells with growth and survival cues. Much of the regulation of transcriptional output by Dpp is mediated through repression of the transcriptional repressor Brinker (Brk), and thus through the activation of target genes. Mutant cells impaired for Dpp reception or transduction are lost from the wing epithelium. At the molecular level, reduced Dpp signaling results in Brk upregulation that triggers apoptosis through activation of the JNK pathway. This study shows that the transcriptional co-regulator dNAB is a Dpp target in the developing wing that interacts with Brk to eliminate cells with reduced Dpp signaling through the JNK pathway. Both dNAB and Brk are required for cell elimination induced by differential dMyc expression, a process that depends on reduced Dpp transduction in outcompeted cells. A novel mechanism is proposed whereby the morphogen Dpp regulates the responsiveness to its own survival signal by inversely controlling the expression of a repressor, Brk, and its co-repressor, dNAB (Ziv, 2009).

NAB proteins comprise a family of transcriptional co-regulators implicated in various developmental processes in different organisms. Drosophila NAB was found to be required for determining specific neuronal fates in the embryonic CNS and for wing hinge patterning. This study shows that dNAB induces cell elimination through induction of the JNK pathway, which in turn triggers Caspase-3-mediated apoptosis. dNAB acts as a co-repressor that physically interacts with Brk to induce apoptotic cell elimination. This conclusion is based on several lines of evidence. First, dNAB-induced apoptosis is completely nullified by removal of Brk. Second, epistatic analysis placed dNAB in the Dpp signaling pathway downstream of the receptor complex and of brk transcriptional repression and upstream of Brk. Third, dNAB physically associates with Brk through its NCD2 domain in vitro. Fourth, dNAB enhances the killing activity of Brk in the presumptive wing blade region and is required for elimination of Dad-overexpressing cells, a process that is completely dependent upon Brk function. Finally, ectopic expression of dNAB represses the expression of Dpp/Brk target genes (Ziv, 2009).

Competitive interactions occur between cells differing in their levels of dMyc, such that cells expressing more dMyc both outgrow neighboring cells and induce their death. This competitive behavior correlates with, and can be modulated by, the activation of the Dpp survival signaling pathway, showing that dMyc-induced cell competition relies on Dpp signaling. The fact that dNAB, similar to Brk, is crucial for dMyc-induced cell competition strongly supports a role for dNAB as an effector of cell elimination of underperforming cells with reduced Dpp signaling (Ziv, 2009).

Elimination of underperforming cells takes place only during early larval stages. Clones generated later, during the third instar larval stage, persist to adulthood. Consistently, using double staining of wing discs with antibodies directed against Brk and dNAB, it was found that the two do not overlap in the second instar larval stage [60 hours after egg laying (AEL)] and only slightly overlap during the third instar (80 hours AEL). These findings suggest that the Brk-dNAB complex is active in cell elimination only during early development. This might indicate that either another factor required for complex activity is present only during early development, or that a factor is present during later stages that inhibits the complex. Alternatively, intensive growth/proliferation might be required for the execution of the killing activity of the complex (Ziv, 2009).

The morphogen Dpp acts through a well-characterized transduction pathway to simultaneously regulate growth, survival and patterning. To a large extent, Dpp signaling acts through negative regulation of brk expression. This implies that a complete answer to how the Dpp signal directs different cellular and developmental processes requires an understanding of how Brk executes its transcriptional repression functions. The finding that dNAB is a Brk co-repressor is in accordance with recent results showing that overexpression of Brk forms that cannot bind either Gro or CtBP results in repression of sal, omb and vg, and that Brk contains additional co-repressor-binding domains. On contrast to Gro, a known co-repressor of Brk, the function of dNAB is not required for Dpp-dependent patterning. However, Gro does not play a similar role to that of dNAB in promoting JNK-mediated cell killing. These findings imply that the choice of Brk co-repressor determines the specificity of target gene repression, thereby modulating different Dpp outputs. Mechanistically, this could be achieved in a number of ways: for example, dNAB or Gro association might alter the DNA-binding specificity of Brk, or the promoters of Brk target genes might be differentially responsive to dNAB and Gro. In addition, the fact that Gro is ubiquitously expressed throughout the developing wing, and that Dpp induces dNAB expression in the center of the wing disc while restricting Brk expression to lateral regions, provide another means for differentially modulating Dpp outputs (Ziv, 2009).

Based on these findings, a molecular model is proposed to explain how the morphogen Dpp regulates the cellular response to its own survival signal in the developing wing by inversely controlling the expression of two key factors, Brk and dNAB. In the center of the wing disc, Dpp represses brk and induces dnab expression, so that in situations in which Dpp signaling activity is abnormally reduced, the resulting local increase in the levels of Brk, which complexes with dNAB, activates the apoptotic pathway. Thus, the Dpp signal sensitizes cells in the center of the wing disc to the apoptotic effect associated with reduced Dpp signaling by maintaining dNAB expression. In lateral regions of the wing disc, where Brk expression is normally higher, apoptotic cell elimination is attenuated, at least in part owing to a lack of dNAB. Thus, by invoking dNAB as a Dpp effector molecule that sensitizes cells to the levels of Brk, it can be at least in part explained why cells in the center of the wing disc, near the Dpp source, are more susceptible to cell elimination induced by reduced Dpp signaling, and why high levels of Brk in the periphery do not necessarily bring about apoptosis (Ziv, 2009).

Given that dNAB appears to play no role in Dpp-mediated patterning, it is proposed that dNAB functions in the wing to prevent developmental errors and discontinuities along the Dpp signaling gradient. This mechanism might be a general feature of morphogen gradients that functions to avoid the accumulation of detrimental developmental mistakes that would otherwise lead to embryonic malformation, and is potentially important in cancer, where tumor cells overexpressing oncogenes such as Myc may act as super-competitors. Thus, the molecular principles underlying such developmental fail-safe mechanisms are clearly of biomedical interest (Ziv, 2009).

Neuroblast entry into quiescence is regulated intrinsically by the combined action of spatial Hox proteins and temporal identity factors.

Neural stem cell quiescence is an important feature in invertebrate and mammalian central nervous system development, yet little is known about the mechanisms regulating entry into quiescence, maintenance of cell fate during quiescence, and exit from quiescence. Drosophila neural stem cells (neuroblasts or NBs) provide an excellent model system for investigating these issues. Drosophila NBs enter quiescence at the end of embryogenesis and resume proliferation during larval stages; however, no single neuroblast lineage has been traced from embryo into larval stages. This study establishes a model NB lineage, NB3-3, which allows reproducibly observation of lineage development from NB formation in the embryo, through quiescence, to the resumption of proliferation in larval stages. Using this new model lineage, a continuous sequence of temporal changes is shown in the NB, defined by known and novel temporal identity factors, running from embryonic through larval stages; quiescence suspends but does not alter the order of neuroblast temporal gene expression. NB entry into quiescence is regulated intrinsically by two independent controls: spatial control by the Hox proteins Antp and Abd-A, and temporal control by previously identified temporal transcription factors and the transcription co-factor Nab (Tsuji, 2008).

This study has revealed for the first time the temporal changes in a Drosophila NB lineage from embryonic NB formation, through entry into quiescence, to resumption of proliferation in larval stages. Using a model NB system with which the complete lineage formation can be reproducibly traced at the resolution of individual cell divisions, it was shown that despite considerable differences in extracellular environment the temporal changes (as defined by the switching of transcription factor/co-factor expression) proceeded continuously in each NB throughout the embryonic and larval stages. Moreover, mutual regulation was found between quiescence and the series of the temporal transcription factors/co-factor; the temporal transcription factors/co-factor endogenously control the timing of triggering NB quiescence, which in turn suspends the switching of late temporal transcription factor expression (Tsuji, 2008).

In the Antp mutant and following ectopic expression of Abd-A there was a lack of NB quiescence, and consequently what appeared to be a precocious generation of larval neurons during embryogenesis was observed. This strongly supports the notion that temporal changes in NBs actually continue in sequence before and after quiescence, i.e., through embryogenesis and larval stages, and in the absence of quiescence the changes occur precociously. In addition, this indicates that spatial and temporal factors control NB quiescence through independent routes (Tsuji, 2008).

Antp mutants did not exhibit NB3-3T quiescence in all thoracic T1-T3 segments. In Antp mutants, epidermis in T2 and T3 segments transform into that in the T1 segment, and some thoracic NB lineages retain thoracic-specific features. These facts indicate that the inhibition of NB3-3T quiescence by Antp mutation is not just a consequence of global transformation of thoracic-to-abdominal segments but rather results from specific effects on individual NBs. NB-specific misexpression of Abd-A also repressed Antp and inhibited NB3-3T quiescence. This also provides evidence that the effect is specific to NBs. Furthermore, because the effect could be observed even when Abd-A was induced after several divisions of the NB, thoracic NBs probably maintain plasticity of their identities during lineage formation (Tsuji, 2008).

It was shown that the temporal transcription factors/co-factor Pdm, Cas, Sqz and Nab play a role in triggering NB quiescence intrinsically in NBs. All of these factors also controlled temporal specification within late lineages of embryonic NBs in both thoracic and abdominal segments. This was confirmed by further examining the relationships of the temporal factors. For example, the loss of Pdm function in NB3-3T resulted in precocious transcription factor switching and precocious quiescence. Conversely, in cas mutant embryos, in which Pdm expression was de-repressed, quiescence was inhibited and expression of late-stage-specific temporal factors disappeared. Similar to Pdm upregulation, loss of nab function resulted in loss of both transcription factor switching and quiescence (Tsuji, 2008).

Although Nab and Sqz can form a complex, nab and sqz mutants displayed very different phenotypes. Both mutants showed de-repression of Kr expression; however, sqz mutants showed no other abnormality in transcription factor switching, whereas nab mutants showed the above-mentioned defects in transcription factor switching and timing of quiescence. These mutant phenotypes revealed that regulation of late temporal events is distributed into multiple pathways. Pdm probably coordinately regulates all factors involved in the timing of NB quiescence, because the loss of Pdm alone is sufficient to cause precocious entry into quiescence (Tsuji, 2008).

Nab and Sqz were shown to work for NB quiescence in NBs. The Nab/Sqz-mediated repression of Kr may be controlled in NBs due to changes in NB temporal identity, or in both NBs and their neurons. Nab might inhibit transcription by recruiting the nucleosome remodeling and deacetylase chromatin remodeling complex as does mammalian Nab (Srinivasan, 2006). Mammalian Nab acts with EGR-1, EGR-2 to determine the fate of cells in hematopoiesis (Laslo, 2006; Svaren, 1996), but whether it can act with the mammalian homolog of LIN-29/Sqz has not been reported. Loss of lin-29, a C. elegans homolog of sqz, causes a heterochronic phenotype in which adulthood is not reached and molting is repeated (Ambros, 1984; Rougvie, 1995). C. elegans has a nab homolog gene, mab-10, that acts with lin-29 in a heterochronic genetic cascade (Tsuji, 2008).

It is unclear what molecular mechanisms enable NBs to suspend the switching of transcription factor expression and maintain temporal identity during quiescence. It is known that the mechanisms for switching expression of early temporal transcription factors can be either cell division dependent or independent. Irrespective of the mechanism used, a NB can 'memorize' its temporal state before quiescence and resume the intrinsic temporal changes once cell cycle progression is reactivated. Embryonic stem cells may maintain multipotency during a slow proliferation state by staying in S phase. When quiescent NBs re-entered the cell cycle, their initial progeny incorporated BrdU fed since hatching, indicating that quiescent NBs stay either prior to S phase or early in S phase. It will be important to identify the point in the cell cycle at which NB enters quiescence (Tsuji, 2008).

Another well-established mechanism that governs temporal aspects of lineage formation is the heterochronic gene cascade in C. elegans. This cascade contains one each of the hunchback homolog and lin-29 genes and generates five distinct temporal cell identities within a single cell lineage. Drosophila NB lineage formation uses two types of Zn-finger proteins, namely the Hb/Cas class [Cas shares DNA-binding characteristics with Hb and the Kr/LIN-29 class. These pairs are expressed three times in NB lineages in the following order: (1) Hb and Kr-> (2) Cas, Kr and Sqz--> Cas and DmLin-29-->end of lineage. This sequence seems to produce at least ten distinct temporal identities within an NB lineage. The repetitive use of these temporal transcription factors in three distinct phases appears to have made the NB lineage longer and more diverse. Lack of Hb also generates NB lineage variety; the NB3-3 and NB2-1 lineages lack Hb and initiate their lineage with Kr. Although the model NB employed in this study lacks Hb, the sequence and entry into quiescence described in this study are common to many typical NB lineages that start with Hb. Interesting questions from the perspective of evolution are how do the three phases combine to form a single lineage and how has NB quiescence evolved in the middle of the NB lineages (Tsuji, 2008)?

Neural stem cells in the mouse cerebral cortex go through ~11 divisions and some enter quiescence in late embryo. The possibility has to be considered that mammalian neural stem cell and Drosophila NB share a similar intrinsic mechanism that induces quiescence (Tsuji, 2008).

A genetic cascade involving klumpfuss, nab and castor specifies the abdominal leucokinergic neurons in the Drosophila CNS

Identification of the genetic mechanisms underlying the specification of large numbers of different neuronal cell fates from limited numbers of progenitor cells is at the forefront of developmental neurobiology. In Drosophila, the identities of the different neuronal progenitor cells, the neuroblasts, are specified by a combination of spatial cues. These cues are integrated with temporal competence transitions within each neuroblast to give rise to a specific repertoire of cell types within each lineage. However, the nature of this integration is poorly understood. To begin addressing this issue, this study analyzed the specification of a small set of peptidergic cells: the abdominal leucokinergic neurons. The progenitors of these neurons were identified, along with the temporal window in which they are specified, and the influence of the Notch signaling pathway on their specification. The products of the genes klumpfuss, nab and castor were shown to play important roles in their specification via a genetic cascade (Benito-Sipos, 2010).

Recent findings on NB5-6 demonstrate that Cas and Grh act as crucial temporal genes to specify several cell fates at the end of this lineage. The current findings with NB5-5 reveal similar roles for Cas and Grh, and indicate that the ABLKs are specified in a Cas/Grh temporal window. It was observed that cas mutants generate no ABLKs, that cas misexpression leads to clusters of two to four ABLKs per hemisegment, and that Cas is expressed in all the ABLKs. Thus, these data confirm that cas plays a role as a temporal identity gene, which remains compatible with its proposed role as a switching temporal factor (Benito-Sipos, 2010).

The proposed role of grh as a temporal identity gene remains open to question. It has also been reported that it is required to regulate mitotic activity and apoptosis of post-embryonic NBs. However, recent evidence has emerged indicating that Grh also temporally regulates FMRFamide neuropeptide cell fate and can act in a combinatorial manner with dimm and apterous to trigger ectopic FMRFamide expression. Similarly, it was found that Grh is required for correct specification of the ABLKs. Together, these results suggest that Grh also plays an instructive role in ABLK specification. Thus, many neuropeptidergic neurons are generated late in several lineages, and depend upon the late temporal genes cas and grh for their specification (Benito-Sipos, 2010).

NB5-5 does not express the temporal genes hb and Kr, and genetic analysis confirms that these two genes are not required for specification of ABLK fate. It was observed that NB5-5 initially expresses Pdm at the time of delamination in late stage 11. Pdm is downregulated at early stage 12, when Cas is activated, and there is a brief period in which both proteins can be detected. The lack of molecular markers does not permit determination of whether the Pdm/Cas coexpression stage generates a GMC (Benito-Sipos, 2010).

It is of interest to note that the phenotype observed misexpressing cas with NB-specific drivers was very mildcompared with that obtained using a pan-neuronal driver. NB5-5 expresses cas soon after delamination and generates six to nine neurons. This suggests that NB5-5 probably has a broad Cas temporal window. Thus, the phenotype obtained upon misexpressing cas with elav-Gal4 indicates that Cas might have a later requirement in postmitotic cells that generates subtemporal windows. Consistent with this interpretation, cas misexpression rescues the grh phenotype of loss of ABLKs, which also suggests that Grh, in addition to being required as a temporal factor, would be indirectly required to activate cas expression in postmitotic cells (Benito-Sipos, 2010).

The Notch pathway is involved in many cell fate decisions in neural development. This study has shown that the ABLK and its sibling are equivalent cells committed to die, and that activation of the Notch pathway in the ABLK prevents its death. A similar situation has been described for specification of the anterior and posterior Corner Cells (CaCC/pCC) neurons in the grasshopper NB1-1 lineage, in which the siblings start as equivalent cells and interaction between them leads to different fates. By contrast, activation of Notch in the NB7-3 lineage drives PCD). Here, activation of the Notch pathway, or misexpression of p35 in the sibling cell, is sufficient to generate two ABLK neurons. A systematic analysis of the lineage of apoptotic cells in embryos in which apoptosis is prevented has shown that the lineage of abdominal NB5-5 contains twice the normal number of cells, but that they have wild-type-like axonal projections. It is concluded that in this lineage, Notch does not play an instructive role in specifying ABLK neuronal fate, but influences a fate decision by regulating the competence to respond to a program of cell death (Benito-Sipos, 2010).

A set of mutants were identified that produce an altered number of ABLKs. In most cases the effect is very mild. Among the mutants with the strongest phenotypes were jumu, nab and klu. The jumu phenotype was expected because it has been shown that Jumu is required in the NB4-2 lineage for normal segregation of Numb in the asymmetric cell divisions. Consistent with this interpretation, the fact that the phenotype of jumu in the NB5-5 lineage is similar to that seen in spdo explains its phenotype and indicates that in jumu embryos Notch is off in both siblings (Benito-Sipos, 2010).

nab and klu embryos display a strong reduction in the number of ABLKs, suggesting that both genes have direct roles in ABLK specification. Interestingly, Cas activates the expression of both genes via repression of Pdm. The lack of availability of markers for identifying ABLKs in earlier stages did not permit establishing whether Nab and Klu are required in the NB or in postmitotic cells (Benito-Sipos, 2010).

Misexpression of cas in nab embryos showed ectopic ABLKs, suggesting that Cas acts either parallel to, or downstream of, Nab. The lack of molecular markers specific to the NB5-5 lineage does not allow determination of whether all ectopic ABLKs are generated by the NB5-5 or by other lineages. Nevertheless, several results suggest that, most probably, all of them are produced by NB5-5. First, it has been observed that neurons that belong to one lineage form a coherent cluster. Second, all of them express gsb, which labels rows five and six NB, and do not express lbe, an NB5-6-specific marker. Third, ABLKs are the unique cells expressing Lk in the ventral ganglion. However, downregulation of cas was not observed in nab mutants, and the same has been reported in the better characterized lineages of NB3-3 and NB5-6; together, these results indicate that the molecular relationship between Cas and Nab requires a more complex interpretation than a linear genetic cascade (Benito-Sipos, 2010).

klu encodes a zinc-finger protein but does not appear to interact directly with Nab, and no evidence was found that nab and klu regulate each other. Surprisingly, nab misexpression rescues the phenotype of a lack of ABLKs observed in klu. By contrast, it was found that cas misexpression produces more ABLKs in grh, nab or klu than in wild-type background As proposed above, these results suggest that Cas plays a role in postmitotic cells that is crucial for ABLK specification (Benito-Sipos, 2010).

The sqz phenotype is epistatic over the nab phenotype. Thus, although nab embryos have no ABLKs, sqz and nab sqz show a normal pattern of ABLKs. It has been shown by pull-down assay that Nab physically interacts with Sqz, and in vertebrates the Nab homologs act as transcriptional co-factors. Since both genes, sqz and nab, are expressed in the ABLKs, it is proposed that the function of Sqz in NB5-5 lineage is to repress the ABLK fate. In normal development, as both genes are expressed in the ABLKs, Nab binds to Sqz and blocks its repressor activity; in nab embryos Sqz represses the ABLK fate, but in nab sqz the pattern is wild-type because there is no repression by Sqz. This intimate interplay between Sqz and Nab is also found in the NB 5-6 linage, in which sqz is first required to activate cell fate determinants, and then acts with nab to suppress the same determinants (Benito-Sipos, 2010).

The findings reported in this study extend understanding of the mechanisms of ABLK specification. However, more precise analysis of the genes and the mechanisms involved in specification of the different cell fates in the NB5-5 lineage will require additional molecular markers. This would permit identification of the different neurons generated from this NB and the genes required to specify their various fates (Benito-Sipos, 2010).

Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression

During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. This study identified two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers (Ulvklo, 2012).

The NB5-6T lineage utilizes two distinct mechanisms to control daughter cell proliferation. In the early part of the lineage, pros limits daughter cell (GMC) proliferation, whereas in the late part canonical Notch signaling in the neuroblast further restricts daughter cell proliferation, resulting in a switch to the generation of neurons directly. The switch in daughter cell proliferation is integrated with temporal lineage progression and enables the specification of different Ap neuron subtypes and the control of their numbers (Ulvklo, 2012).

The data on Notch activation in the NB5-6T lineage, using both antibodies and reporters, indicate progressive activation in the neuroblast: weak at St10-11 and more robust from St12 onward. Thus, Notch activity coincides with the proliferation mode switch. How is this gradual activation of Notch in the neuroblast controlled? NB5-6T undergoes the typical progression of the temporal gene cascade, with Cas expression preceding strong Notch activation. Thus, one possible scenario is that the late temporal gene cas activates the Notch pathway. However, analysis of the E(spl)m8- EGFP reporter shows that this Notch target is still activated at the proper stage in cas mutants. Although this does not rule out the possibility that other, unknown, temporal factors might regulate Notch signaling, it rules out one obvious player, cas. Alternatively, as Notch signaling is off when neuroblasts are formed (a prerequisite for neuroblast selection), Notch activation in the neuroblast at later stages might simply reflect a gradual reactivation of the pathway. Although such a reactivation might at a first glance appear too imprecise, it is possible that the specificity of this particular Notch output -- proliferation control -- might be combinatorially achieved by the intersection of Notch signaling with other, more tightly controlled, temporal changes (Ulvklo, 2012).

Pros and Notch control daughter proliferation in different parts of NB5-6T, and no evidence of cross-regulation between these pathways was found. The limited overproliferation of the lineage when each pathway is separately mutated results not from redundant functions, but rather stems from the biphasic nature of this lineage. Specifically, in pros mutants, Notch signaling is likely to be on in all 'A' type sibling daughter cells, as Numb continues to be asymmetrically distributed between daughter cells. Thus, Notch signaling in 'A' cells may preclude each 'A' cell from dividing even once. This notion is in line with recent studies showing that postmitotic Notch activated cells ('A' cells) within the Drosophila bristle lineages are particularly resilient to overexpression of cell cycle genes. Similarly, in Notch pathway mutants, as Ap cells now divide (in essence becoming GMC-type cells), Pros will still play its normal role in these 'GMCs' and limit their proliferation to a single extra cell division. However, in kuz;pros double mutants, Ap cells are relieved of both types of daughter cell proliferation control and can thus divide for many additional rounds. This notion also applies to early parts of the NB5-6T lineage and probably to the majority of other VNC lineages, as indicated by the extensive overproliferation of the entire NB5-6T lineage, and to the general overproliferation of the VNC. However, based on the findings that neither the Notch pathway nor pros controls neuroblast identity or its progression, it is postulated that these large clones contain a single, normally behaving NB5-6T neuroblast. In fact, the neuroblast is likely to exit the cell cycle and undergo apoptosis on schedule, as neither of these decisions depends upon pros or the Notch pathway. Of interest with respect to cancer biology is that the findings point to a novel mechanism whereby mutation in two tumor suppressors (e.g., Pros and Notch) cooperate to generate extensive overproliferation: not by acting in the same progenitor cell at the same time, but by playing complementary roles controlling daughter cell proliferation (Ulvklo, 2012).

As an effect of alternate daughter cell proliferation patterns, both vertebrates and invertebrates display variability in neural lineage topology. Similarly, progenitors in these systems undergo temporal changes in competence, as evident by changes in the types of neurons and glia generated at different time points. Hence, the temporal-topology interplay described in this study is likely to be extensively used and to be conserved in mammals. As a proof of principle of this novel developmental intersection, single and double mutants were examined for kuz and nab, thereby independently versus combinatorially affecting temporal progression and daughter cell proliferation. Strikingly, these mutants show the predicted combined effect, with the appearance of additional Ap1/Nplp1 neurons beyond those found in each individual mutant (Ulvklo, 2012).

If programmed proliferation switches are conserved, how might such a topology-temporal interplay become utilized in mammals? There are several examples in which different clusters/pools/nuclei of neurons of distinct cell fate are generated from the same progenitor domain in the developing mammalian nervous system. Such pools often contain different numbers of cells, but the underlying mechanisms controlling the precise numbers of each subtype are poorly understood. Based on previous studies in a number of models, at least three different mechanisms can be envisioned. Based on the current study, a novel fourth mechanism is proposed, whereby alteration of daughter cell proliferation is integrated with temporal progression to control subtype cell numbers. These four mechanisms are not mutually exclusive, and given the complexity of the mammalian nervous system it is tempting to speculate that all four mechanisms are utilized during development (Ulvklo, 2012).

Drosophila NAB (dNAB) is an orphan transcriptional co-repressor required for correct CNS and eye development

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. Targeted deletion mutations in the nab gene have been created, and the identification of additional, EMS-induced dnab mutations by genetic complementation analysis has been described. Null alleles in nab cause larval locomotion defects and early larval lethality (L1-L2). A putative hypomorphic allele in nab instead causes early adult lethality due to severe locomotion defects. In the nab -/- CNS, axon outgrowth/guidance and glial development appear normal; however, a subset of eve+ neurons forms in reduced numbers. In addition, mosaic analysis in the eye reveals that nab -/- clones are either very small or absent. Similarly, Nab overexpression in the eye causes eyes to be very small with few ommatidia. These dramatic eye-specific phenotypes should prove useful for enhancer/suppressor screens to identify nab-interacting genes (Clements, 2003; full text of article).

The mammalian NAB proteins have been shown to repress the transcriptional activity of EGR proteins by binding specifically to the R1 domain. A single-point mutation in the R1 domain (I298F) of EGR1 abrogates binding to mNAB1 and mNAB2 (Russo, 1993; Russo, 1995b). The NCD1 domains of both mNAB1 and mNAB2 have been shown to be necessary and sufficient for interaction with the EGR1 R1 domain. Attempts were made, by means of the yeast two hybrid system, to determine whether dNAB, like its mammalian counterparts, can bind and repress a mammalian EGR protein containing an R1 domain. To evaluate whether dNAB interacts directly with the EGR1 R1 domain, dNAB NCD1 was cloned as a fusion protein C terminal to the GAL4 DNA binding domain in pAS-1 (Gal4DBD:dNCD1) and was tested for interaction in yeast strain AH109 with either the rat EGR1 R1 domain (aa 269-304) or R1 domain (I298F) point mutant fused C terminal to the Gal4 activation domain in pBM2462 (Gal4ACT:rR1 or Gal4ACT:rR1pm, respectively). Multiple colonies of doubly transformed yeast were selected on -Leu-Trp drop-out medium, and the colonies were restreaked onto -His-Leu-Trp drop-out medium to test for Gal4-mediated activation of the HIS3 reporter gene in AH109. Yeast expressing both Gal4DBD:dNCD1 and Gal4ACT:rR1 were able to grow on -His-Leu-Trp medium, whereas yeast expressing Gal4DBD:dNCD1 and Gal4ACT:rR1pm were not. Similar results were obtained in multiple repeat experiments. These data indicate that the NCD1 domain of dNAB interacts directly with the rEGR1 R1 domain in yeast but does not interact with the I298F rEGR1 R1 domain point mutant. In this respect, dNAB acts similarly to the mammalian NAB proteins (Clements, 2003).

The mammalian NAB proteins mediate dose-dependent transcriptional repression by means of the NCD2 domain (Russo, 1995a; Svaren, 1996; Swirnoff, 1998). When rNAB1 and rEGR1 expression constructs are transiently cotransfected into CV1 cells along with an EGR1-responsive luciferase reporter construct, EGR1-induced luciferase activity is repressed in a dose-dependent manner; similar results are observed for hNAB2. To determine whether dNAB can repress transcription mediated by rEGR1, tests were made to see whether dNAB can repress luciferase activity in the same assay. Luciferase activity is reduced in a dose-dependent manner when dNAB is coexpressed with rEGR1. These data suggest that dNAB contains a functional transcriptional repression domain, because it represses transactivation by EGR1 in a dose-dependent manner (Clements, 2003). In Drosophila, two EGR-like zinc finger transcription factors have been identified: Stripe and Klumpfuss. stripe is expressed in the embryonic epidermis, and mutants exhibit a disruption in muscle attachment and myotubule patterning. The Stripe protein contains a zinc finger DNA-binding domain that is highly homologous to the EGR1 DBD; amino acids known to make contact with the EGR consensus binding site are completely conserved in Stripe. However, it does not contain an identifiable R1 domain. In addition, Stripe expression occurs in a pattern that does not overlap dnab expression. That stripe lacks an R1 domain and that it is not expressed in dnab-expressing cells indicates that stripe is unlikely to be a dNAB binding partner in vivo. In contrast, klumpfuss is expressed widely in NBs of the embryonic and larval CNS, as well as in several imaginal disc tissues. Mutants exhibit distal leg defects (tarsal segments 3-5 are fused together, as are the trochanter and femur), loss of bristles at certain positions, and homeotic changes in ganglion mother cell fate within certain neuroblast lineages of the central nervous system. Klumpfuss also contains three EGR-like zinc fingers; amino acids in the mammalian EGR proteins, which are known to make direct contact with the consensus binding site, are completely conserved in Klumpfuss. However, unlike the mammalian EGR proteins, the Klumpfuss protein contains a fourth zinc finger, making it more similar to the Wilm's tumor protein (WT-1) than to the EGR protein family. Klumpfuss also lacks the R1 domain found in several mammalian EGR proteins. Still, Klumpfuss expression in the CNS does overlap dnab expression, indicating that it could be a bona fide binding partner for dNAB in vivo (Clements, 2003).

To determine whether dNAB binds Klumpfuss by means of its NCD1 domain, a klumpfuss cDNA lacking the zinc finger DNA-binding domain was cloned as a fusion protein C-terminal to the GAL4 activation domain in pACT2 (GAL4ACT:kluDZf). The klumpfuss DBD was removed in this case, because expression of EGR-like zinc fingers is known to inhibit the growth of yeast. Interaction between GAL4DBD:dNCD1 and GAL4ACT:kluδZf was tested as above in yeast strain AH109. Yeast expressing both GAL4DBD:dNCD1 and GAL4ACT:kluδZf failed to grow on -His-Leu-Trp medium, indicating that the nutritional reporter HIS3 is not activated. These results strongly suggest that dNAB does not bind Klumpfuss by means of the NCD1 domain, although binding by means of some other portion of the dNAB molecule cannot be formally ruled out. The data are not surprising, perhaps, when one considers that Klumpfuss, like Stripe, lacks an identifiable R1 domain. The lack of other EGR-like proteins in Drosophila strongly suggests that dNAB interacts with other families of transcription factors in both Drosophila and mammals (Clements, 2003).

To determine the wild-type expression pattern of dnab, in situ hybridizations was performed on embryos and third instar larvae to detect dnab RNA. In embryos, dnab transcript appears to be expressed solely in a cluster of midline cells and a subset of neuroblasts (NBs); it is undetectable in the peripheral nervous system or in any other embryonic tissue. Neuroblast-specific expression was confirmed by double staining for dnab RNA and Klumpfuss protein. Of interest, dnab expression is also absent from ganglion mother cells (GMCs) and neurons of the embryonic CNS. Expression begins at stage 11 in midline cells, which are most likely midline mesectodermal cells, and in one NB of every hemisegment (Clements, 2003).

By stage 13, dnab transcripts are expressed in 10-12 of the 30 identified NBs of each hemisegment. By stage 14, dnab transcript levels begin to decrease, and by stage 16, expression is undetectable, except in a few cells at the lateral edge of the CNS (Clements, 2003).

The exclusive expression of dnab in only a subset of NBs of the CNS suggests that it might function in determining the identity of neuronal lineages derived from those NBs after the onset of dnab expression. Alternatively, dnab might play a more general role in the differentiation or functioning of neurons derived from these cells. Although dnab RNA is expressed only in NBs, a function for dNAB protein in ganglion mother cells or neurons cannot be precluded. This phenomenon has been observed for several genes expressed in the Drosophila CNS. The RNA for the zinc finger transcription factor Castor, for instance, is expressed exclusively in NBs, whereas the protein perdures and is expressed in ganglion mother cells and neurons (Clements, 2003).

dnab expression in third instar larvae is observed in both the CNS and in several imaginal discs. In the larval ventral ganglion, dnab expression is limited to a subset of proliferating neuroblasts in the cephalic lobes and the thoracic region; expression is excluded from the abdominal segment of the ventral ganglion, where proliferative NBs are not observed. Whatever its role, dnab likely performs similar functions during both embryonic and larval CNS development (Clements, 2003).

The embryonic and larval CNS expression patterns for dnab are recapitulated by lacZ expression in a dnab enhancer trap line, dnab^e310. Gal staining of third instar larvae containing the dnab^e310 enhancer trap also reveals that dnab is expressed, albeit at lower levels, in several imaginal discs. In the eye disc, expression is observed in a pattern consistent with expression either in or behind the morphogenetic furrow. Expression in the wing imaginal disc occurs in the prospective wing area early in the third larval instar. Expression also occurs in the antennal and haltere discs. These imaginal disc expression patterns match those observed for the dnab transcript by in situ hybridizationand suggest a potential role for dnab in imaginal disc development (Clements, 2003).

Because dnab is expressed in only a subset of the 30 identified neuroblasts in each hemisegment, it was hypothesized that it may promote the formation of specific neuronal subgroups. To examine several unique neuronal lineages in dnab mutant embryos for defects, cells expressing even-skipped and engrailed were examined. In the stage 15/16 CNS, the homeodomain protein Eve is expressed in approximately 16 CNS neurons per abdominal hemisegment. Medially, pCC and fpCC interneurons and the aCC and RP2 motorneurons each express Eve, as do four mediolateral CQ neurons and 8 to 10 Eve lateral (EL) neurons. The homeodomain protein En, in contrast, is expressed in a larger complement of cells: three large ventral midline cells (VUMs), a cluster of dorsal medial (DM) cells, two pair of non-neuronal median support (MS) cells, a bilateral group of 4-6 posterior intermediate (PI) cells, a group of 8-10 posterior lateral (PL) cells, and two pair of more anterior NH cells (Clements, 2003).

Although all En+ cells are present in dnab^e310/dnab^e310 mutant embryos, a subgroup of eve+ neurons develops in reduced numbers. dnab^e310/dnab^e310 and dnab^G26/dnab^G26 embryos are missing 1-4 EL neurons in a subset of abdominal hemisegments per embryo. Because this loss of EL neurons is variably expressed, the loss was quantified by counting the number of abdominal hemisegments containing seven or fewer EL neurons. More abdominal hemisegments contain reduced numbers of EL neurons in dnab^e310/dnab^e310 and dnab^G26/dnab^G26 embryos vs. wild-type (Clements, 2003).

The loss of EL neurons is more pronounced in dnab^A23/dnab^A23 embryos. Here 92.5% of abdominal hemisegments are affected, and the mean number of neurons lost per hemisegment is 3.5. Because dnab^A23 appears by other measures to be a hypomorphic mutation in dnab, the more dramatic loss of EL neurons in dnab^A23/dnab^A23 embryos cannot be currently explained (Clements, 2003).

In mammals, NAB2 overexpression has been shown to preclude axon outgrowth in cultured PC12 cells and to maintain those cells in a proliferative state (Qu, 1998). In Drosophila, dnab is also expressed in a proliferating preneural cell type, and strong loss-of-function mutants exhibit slow, uncoordinated movements as early larvae. Therefore attempts were made to assess whether dnab plays a role in axon outgrowth/guidance during embryonic CNS development. Within the CNS, the longitudinal, anterior commissural, and posterior commissural fascicles can be labeled with mAbBP102, whereas the peripheral motor neuron branches that exit the CNS to innervate muscle groups dorsally along the body wall express the antigen Fasciclin II (FasII). By using these markers, dnab^e310/dnab^e310, dnab^G26/dnab^G26, and dnab^A23/dnab^A23 embryos were examined for defects in axon outgrowth and targeting, but none were found (Clements, 2003).

The human NAB proteins have been indirectly implicated in peripheral glial development; several patients with congenital hypomyelinating neuropathies have been identified who have mutations in EGR2, a known hNAB1/hNAB2 binding partner. Therefore, glial development was examined in dnab mutant Drosophila embryos by staining for RK2, a glia-specific homeodomain protein that is expressed in all CNS glia, with the exception of the midline glia. In dnab^e310/dnab^e310 embryos, glia appeared to develop in normal numbers, and migrated appropriately to form the longitudinal glial array and exit glia (Clements, 2003).

To identify regulators of dnab gene expression, mutants of several NB-expressing transcription factors were screened for loss of dnab RNA expression. Castor is a zinc finger transcription factor expressed in Drosophila NBs and GMCs; castor loss of function causes reductions in CNS axonal density and reductions in the number of neurons expressing the homeodomain protein engrailed. Furthermore, castor has been shown to positively regulate expression of the POU domain transcription factors drifter and acj6 and to negatively regulate expression of the POU domain transcription factors pdm-1 and pdm-2 in the embryonic CNS. dnab expression was analyzed in ^casH23A1/cas^H23A1 and cas^H23A3/cas^H23A3 embryos, which contain deletions removing the entire castor gene; dnab expression is affected in these mutants. dnab expression in midline cells at stage 11 appears normally; however, expression fails to spread to NBs during stages 12/13. In the cephalic lobes, only a few cells express dnab. This finding is in contrast to wild-type, where dnab is robustly expressed in many cephalic NB. These results indicate that dnab is either a direct or indirect target gene of castor. The temporal expression patterns of castor and dnab support these conclusions. Castor expression first appears in NBs at stage 10 and spreads to 9-10 NBs per hemisegment by late stage 11, the stage at which dnab expression is first observed. The castor protein has been shown to bind the consensus DNA sequence (G/C)C(C/T)(C/T)AAAAA(A/T). A genomic region containing the entire dnab transcription unit, as well as 10 kb upstream and 10 kb downstream of dnab was scanned for Castor binding sites. This analysis revealed that no consensus Castor binding sites occur in the putative dnab promoter region or in dnab introns, although at least two closely related sites occur in the putative promoter region. It may be possible that Castor directly regulates dnab expression through these sites, or through sites in a distal enhancer element. Alternatively, dnab expression might be directly regulated by the transcription factors encoded by the Castor target genes drifter, acj6, pdm-1, or pdm-2. In these scenarios, Drifter and Acj6 might normally function as positive regulators of dnab expression, whereas pdm-1 and pdm-2 might function as negative regulators of dnab expression (Clements, 2003).

The Drosophila eye consists of approximately 800 precisely patterned light sensing units, or ommatidia. To initially survey whether the dnab gene is essential for Drosophila ommatidial development, both gain- and loss-of-function experiments were performed for dnab in the developing eye (Clements, 2003).

dnab loss of function in the eye was achieved by using the FLP/FRT system to induce somatic recombination between chromosomes bearing mutations in the dnab gene and wild-type chromosomes bearing a w+ transgene. By inspection of gross eye morphology, it was found that dnab^e310/dnab^e310 (w-/w-) cell clones failed to form altogether, resulting in a rough eye phenotype and completely w+ eyes. In contrast, eyes bearing dnab^G26/dnab^G26 cell clones grossly appeared normal, although the dnab^G26/dnab^G26 clones were moderately reduced in size compared with wild-type. dnab^A23/dnab^A23 eye clones were not analyzed, due to the possible presence of closely linked second site mutations on the dnab^A23 chromosome. Whether dnab -/- cell clones could be detected in the presence of chromosomes bearing the Minute mutation Rps174 along with a w+ transgene was analyzed. Minute mutations are dominantly cell proliferation-defective, so they would give dnab -/- cells a competitive advantage in populating the eye if they are present. By using this method, no dnab^e310/dnab^e310 clones formed, resulting again in a rough eye phenotype and completely w+ eyes. In contrast, dnab^G26/dnab^G26 clones formed and appeared grossly normal, populating greater than 80% of the eye (Clements, 2003).

The absence/reduced size of dnab^e310/dnab^e310 eye clones suggests that there might be defects in cell proliferation or, alternatively, increases in cell death during eye development. That dnab^G26/dnab^G26 eye clones are capable of proliferating to populate a phenotypically normal eye suggests the possibility that the dnab^G26 allele retains some functionality; it may be that even slight amounts of dnab function are sufficient for normal eye development (Clements, 2003).

To complement the loss of function analysis in the eye, the GAL4/UAS transgenic system was used to overexpress a UAS-dnab transgene. When dnab is expressed under the control of eyeless-GAL4, which permits expression very early in the embryonic eye primordium, eyes are completely absent; occasionally a tiny patch of eye tissue can be seen. A more weakly expressing UAS-dnab transgene permits development of extremely small eyes with few ommatidia. In contrast, when dnab is expressed under the control of GMR-GAL4, which initiates expression in cells at the morphogenetic furrow, eyes are glassy from fused facets and have an irregular surface. Expression of dnab under the control of sevenless-GAL4, which expresses behind the morphogenetic furrow in the R7 photoreceptor neuron as well as several other retinal cells, causes a rough eye phenotype with many dark, necrotic patches (Clements, 2003).

The results from the above survey suggest that dnab has the ability to affect multiple aspects of eye development at several developmental stages. The complete lack of eyes observed when dnab is expressed under the control of eyeless-GAL4 may be due to an inability of precursor cells to proliferate, for instance. The GMR-dnab phenotype mimics the phenotype of the glass gene itself, which causes a complete failure of photoreceptors to form, as well as aberrant projection of retinal axons and complete absence of the larval optic nerve. The sevenless-dnab phenotype, in contrast, is reminiscent of the eye phenotype for Blackpatch mutations, which cause neural degeneration in the eye and optic lobe of adult brains beginning at approximately 60 hr after pupariation. These dramatic initial eye-specific phenotypes obviate the need for more detailed study of dnab function during ommatidial development, and should prove invaluable in identifying dnab-interacting genes through enhancer-suppressor screens (Clements, 2003).

Search PubMed for articles about Drosophila Nab

Ambros, V. and Horvitz, H. R. (1984). Heterochronic mutants of the nematode Caenorhabditis elegans. Science 226: 409-416. PubMed ID: 6494891

Benito-Sipos, J., Estacio-Gomez, A., Moris-Sanz, M., Baumgardt, M., Thor, S. and Diaz-Benjumea, F. J. (2010). A genetic cascade involving klumpfuss, nab and castor specifies the abdominal leucokinergic neurons in the Drosophila CNS. Development 137: 3327-3336. Pubmed: 20823069

Clements, M., Duncan, D. and Milbrandt, J. (2003). Drosophila NAB (dNAB) is an orphan transcriptional co-repressor required for correct CNS and eye development. Dev. Dyn. 226: 67-81. Medline abstract: 12508226

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

Laslo, P., Spooner, C. J., Warmflash, A., Lancki, D. W., Lee, H. J., Sciammas, R., Gantner, B. N., Dinner, A. R. and Singh, H. (2006). Multilineage transcriptional priming and determination of alternate hematopoietic cell fates. Cell 126: 755-766. PubMed ID: 16923394

Le, N., Nagarajan, R., Wang, J. Y., Svaren, J., LaPash, C., Araki, T., Schmidt, R. E. and Milbrandt, J. (2005). Nab proteins are essential for peripheral nervous system myelination. Nat. Neurosci. 8: 932-940. Medline abstract: 16136673

Mechta-Grigoriou, F., Garel, S. and Charnay, P. (2000). Nab proteins mediate a negative feedback loop controlling Krox-20 activity in the developing hindbrain. Development 127: 119-128. Medline abstract: 10654606

Qu, Z., et al. (1998). The transcriptional corepressor NAB2 inhibits NGF-induced differentiation of PC12 cells. J. Cell Biol. 142(4): 1075-82. PubMed ID: 9722618

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. PubMed ID: 7671813

Russo, M. W., Matheny, C. and Milbrandt, J. (1993). Transcriptional activity of the zinc finger protein NGFI-A is influenced by its interaction with a cellular factor. Mol. Cell Biol. 13: 6858-6865. PubMed ID: 8413279

Russo, M. W., Sevetson, B. R. and Milbrandt, J. (1995a). Identification of NAB1, a repressor of NGFI-A and Krox20 mediated transcription. Proc. Natl. Acad. Sci. 92: 6873-6877. PubMed ID: 7624335

Russo, M. W., Sevetson, B. R. and Milbrandt, J. (1995b). Identification of NAB1, a repressor of NGFI-A- and Krox20-mediated transcription. Proc. Natl. Acad. Sci. 92: 6873-6877. PubMed ID: 7624335

Sevetson, B. R., Svaren, J. and Milbrandt, J. (2000). A novel activation function for NAB proteins in EGR-dependent transcription of the luteinizing hormone beta gene. J. Biol. Chem. 275: 9749-9757. Medline abstract: 10734128

Srinivasan, R., Mager, G. M., Ward, R. M., Mayer, J. and Svaren, J. (2006). NAB2 represses transcription by interacting with the CHD4 subunit of the nucleosome remodeling and deacetylase (NuRD) complex. J. Biol. Chem. 281: 15129-15137. PubMed ID: 16574654

Svaren, J., et al. (1996). NAB2, a corepressor of NGFI-A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli. Mol. Cell Biol. 16: 3545-3553. PubMed ID: 8668170

Svaren, J., Sevetson, B. R., Golda, T., Stanton, J. J., Swirnoff, A. H. and Milbrandt, J. (1998). Novel mutants of NAB corepressors enhance activation by Egr transactivators. EMBO J. 17: 6010-6019. Medline abstract: 9774344

Swirnoff, A. H., Apel, E. D., Svaren, J., Sevetson, B. R., Zimonjic, D. B., Popescu, N. C. and Milbrandt, J. (1998). Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain. Mol. Cell. Biol. 18: 512-524. Medline abstract: 9418898

Tsuji, T., Hasegawa, E. and Isshiki, T. (2008). Neuroblast entry into quiescence is regulated intrinsically by the combined action of spatial Hox proteins and temporal identity factors. Development 135(23): 3859-69. PubMed ID: 18948419

Ulvklo, C., et al. (2012). Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression. Development 139(4): 678-89. PubMed ID: 22241838

Ziv, O., et al. (2009). The co-regulator dNAB interacts with Brinker to eliminate cells with reduced Dpp signaling. Development 136(7): 1137-45. PubMed ID: 19270172

date revised: 10 February 2013

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