Developmental Transcriptional Networks Are Required to Maintain Neuronal Subtype Identity in the Mature Nervous System

Summary

During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), we test whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, we find that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply “locked-in,” but must be actively maintained by persistent developmental transcription factor networks. We also examined the cross-regulatory relationships between all transcription factors that persisted in adult Tv1/Tv4 neurons. We show that certain critical cross-regulatory relationships that had existed between these transcription factors during development were no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, our results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain.

Introduction

Tremendous progress has been made in delineating the transcriptional mechanisms that diversify neuronal subtype identities during development. Spatiotemporally-patterned transcription factor cascades act within increasingly diversified progenitor populations to specify postmitotic neuron subtype fate. Within those postmitotic neurons, subtype-specific sets of transcription factors act combinatorially to differentiate subtype identity by initiating expression of the genes that define subtype form and function [1], [2], [3]. These so-called terminal differentiation genes include subtype-specific neuropeptides, neurotransmitter enzymes and ion channels [4]. Developmental transcriptional cascades are progressive and typically nonlinear; many transcription factors act at multiple levels, they exhibit extensive cross-regulation, and their expression undergoes considerable refinement in developing postmitotic neurons [1], [5], [6], [7]. Here, we apply the term ‘subtype transcription network’ to refer to the transcription factors that direct subtype specification and differentiation, their cross-regulatory relationships (or configuration), and the manner in which they direct the expression of subtype-specific sets of terminal differentiation genes.

After subtype-specific gene expression profiles are established by differentiation, continued neuronal function throughout life depends upon their maintenance of subtype gene expression profiles. However, we currently have only a rudimentary understanding of the mechanisms of long-term, subtype-specific gene maintenance. Two extreme models would posit that subtype identity is either actively maintained by persistent subtype transcription network activity or passively maintained by for example stabilized chromatin structure, independent of a subtype transcription network. Here, we test the active model in two Drosophila neuronal subtypes to address the following largely unanswered questions: Do developmental subtype transcription networks persist in adult neurons or are they dispensed with? Are they required to maintain the expression of subtype-specific sets of terminal differentiation genes? If they are required, does maintenance of terminal differentiation genes require the same complex combinatorial codes of transcription factors as for their initiation, or a simplified code involving fewer transcription factors? Finally, do persisting developmental transcription factors retain the same cross-regulatory relationships that regulated their expression during development?

We model these questions in Drosophila Tv1 and Tv4 neurons. There are six clusters of Tv neurons in the Drosophila ventral nerve cord, each comprising four distinct subtypes (Tv1–Tv4). Tv1 and Tv4 express subtype-specific terminal differentiation genes, the neuropeptides Nplp1 (Tv1) and FMRFa (Tv4) and the neuropeptide amidase PHM (peptidylglycine alpha-hydroxylating monooxygenase; in Tv1/Tv4). Using these genes as markers for subtype-specific differentiation, previous work had revealed the elaborate subtype transcription networks that direct Tv1/4 subtype specification and differentiation [6], [8], [9], [10], [11], [12], [13]. The expression of FMRFa [14], Nplp1 and PHM (herein) are stably maintained in Tv1/4 neurons throughout Drosophila life. Thus, our detailed understanding of their subtype-specific initiation in the embryo provides an ideal background to investigate how such terminal differentiation genes, and hence subtype identity, are maintained in the adult. We previously established that persistent retrograde BMP signaling is required to initiate and maintain FMRFa in Tv4 neurons [14]. Here, we examined the adult function of Tv1 and Tv4 subtype transcription networks in the maintenance of Nplp1, FMRFa and PHM. We further examined whether the cross-regulatory interactions observed between the Tv1/Tv4 network transcription factors during development were maintained in adults. We found that each subtype transcription network is largely retained in adult Tv neurons and is required to actively maintain subtype-specific gene expression. Thus, the combinatorial transcription codes for subtype-specific gene expression are not ‘simplified’ or dispensed with for maintenance. Further, we find that certain critical developmental cross-regulatory interactions between transcription factors are no longer utilized in adults for transcription factor maintenance. Thus, we observe a post-developmental switch to a distinct maintenance configuration between individual transcription factors. Collectively, these data provide novel insight relevant to understanding how long-lived cell types maintain their subtype identity.

Results

Genetic analysis has defined cascades of transcription factors that specify Tv1 and Tv4 neuron fates and then differentiates their subtype-specific terminal differentiation gene expression profiles (Figure 1A, 1B) [6], [8], [9], [10], [11], [12]. Tv1–4 neurons are born sequentially from the NB5-6T neuroblast lineage within an expression window of the ‘temporal’ transcription factors castor (cas) and grainy head (grh) [6], [15]. These specify Tv subtype generation together with collier (col), squeeze (sqz) and nab [6], [9], [12], [16]. Within postmitotic Tv1 neurons, ap, eya, dimmed (dimm) and col then differentiate Tv1 identity in part by initiating Nplp1 expression [9]. In Tv4 neurons ap, eya, dimm, dachshund (dac), sqz and grh act combinatorially with target-derived BMP signaling to differentiate Tv4 identity, in part by initiating FMRFa expression [6], [10], [11], [12], [17], [18], [19]. Also, in both Tv1 and Tv4, dimm acts independently of other regulators to induce expression of the neuropeptide amidase, PHM [10], [20]. We refer to these two transcription factor cascades as the Tv1 and Tv4 ‘subtype transcription networks’ (outlined in Figure 1A, 1B). Genetic analysis has placed these transcription factors into two partially overlapping categories; those that are necessary for directing the specification, or generation, of Tv subtypes around the time of postmitotic neuron birth, and those that thereafter direct differentiation of Tv subtypes by initiating subtype-specific terminal differentiation gene expression. Tv1 neurons are not specified (or generated) in cas or col mutants, whereas eya, ap, col and dimm all act combinatorially thereafter to initiate Nplp1 expression [6], [9]. Tv4 neurons are not specified in cas, col or grh mutants. In sqz and nab mutants, Tv4 neurons are not specified in a segment-specific manner [6], [12]. Thereafter, eya, ap, sqz, dimm, dac, grh and BMP signaling all appear to combinatorially initiate FMRFa expression.

Subtype transcription network retention in adult Tv1 and Tv4 neurons

We previously reported that adult Tv4 neurons maintain FMRFa, ap and eya expression, as well as retrograde BMP-signaling. We also demonstrated that FMRFa maintenance in adult Tv4 neurons requires persistent retrograde BMP-signaling [14]. Here, we determined the adult expression of the other transcriptional regulators implicated in Tv1 and Tv4 development (Figure 1C–1J). We found that adult Tv4 neurons retained ap, eya, dimm, dac and sqz (Figure 1C–1E, 1J), but no longer expressed grh, cas or nab (Figure 1G–1I). Additionally, adult Tv1 neurons retained expression of Nplp1, as well as ap, eya, dimm and col (Figure 1C, 1D, 1F). Previously, cas expression was shown to be lost in all Tv neurons prior to neuropeptide initiation [9], and here we find that it does not become re-expressed in adult Tv1 or Tv4 neurons (Figure 1H). These data are summarized (Figure 1K, 1L). Further analysis found that the adult complement of transcription factors was established by the start of the L1 larva stage (Figure S1), shortly after Tv1/4 terminal differentiation.

Which transcription factors persist in adult neurons is intriguing. All those previously implicated in the postmitotic differentiation of Tv1 and/or Tv4 neurons persist. In contrast, transcription factors that act within the neuroblast and newborn postmitotic neuron to specify the fate of Tv1 (cas) or Tv4 (cas, col, grh, nab) neurons are not retained in the adult. The exceptions to this are col and sqz. Both are implicated in Tv subtype specification, but it is notable that both transcription factors have also been implicated, by loss and gain of function genetics, as part of the combinatorial transcription factor codes that initiate Nplp1 or FMRFa expression [6], [9], [12]. Thus, we find that only regulators implicated in postmitotic subtype differentiation are maintained in adult neurons.

Conditional dsRNAi knockdown in adult Tv neurons

To test the function of each transcription factor in maintaining terminal differentiation gene expression in Tv1 and Tv4 neuronal subtypes, we used ap^GAL4 (except where noted) to express UAS-dsRNAi transgenes (abbreviated to dsRNAi) targeted to each transcription factor. We also overexpressed UAS-Dicer2 in all experiments to enhance dsRNAi efficacy [21]. To selectively induce dsRNAi in adults, we utilized the TARGET system wherein a temperature-sensitive GAL4-repressor GAL80 (GAL80^TS) controls the activity of GAL4 [22]. Flies were raised at 18°C to allow functional GAL80^TS to repress GAL4 activity throughout development. Then at adult day 1 (A1), flies were switched to 29°C and kept at that temperature for the remainder of each experiment. At this temperature, GAL80^TS becomes dysfunctional and thus GAL4 is allowed to induce dsRNAi expression (Figure 2A) [14].

eya, ap, and sqz are required for FMRFa maintenance in adults

We induced eya^dsRNAi expression at adult day 1 (A1) and quantified FMRFa transcript levels by fluorescent in situ hybridization, relative to the mean of controls. In adults, eya^dsRNAi dramatically reduced FMRFa transcript to 19.7±4.4% of control (p<0.0001) by adult day A15 (Figure 2B–2D). We observed a similar downregulation in immunoreactivity to the mature amidated FMRFa peptide (Table S1). To confirm dsRNAi specificity, we tested for enhancement of the FMRFa phenotype when expressing eya^dsRNAi in an eya heterozygous background. Indeed, we found that this further reduced FMRFa transcript to 4.6±0.5% of control by A15 (p<0.0001 to eya^dsRNAi alone or eya heterozygosity alone) (Figure 2D). Eya immunoreactivity was eliminated in all cases (Table S4). Previous studies demonstrated that FMRFa is severely downregulated in eya mutants by late embryogenesis [11]. Our data now show that FMRFa maintains this critical dependence on eya in adults.

We next tested the role of apterous (ap). As ap^GAL4 is a strong hypomorphic ap allele [23], we used dac^GAL4 to express ap^dsRNAi in wildtype and heterozygous ap backgrounds. We observed a significant downregulation of FMRFa transcript when expressing ap^dsRNAi in ap heterozygotes, falling to 66.8±4.2% of control (p<0.0001 from control or ap heterozygote alone, and p<0.005 from ap^dsRNAi alone). No loss of FMRFa was observed in either ap heterozygotes or ap^dsRNAi alone (Figure 2H–2J). Similar results were obtained for downregulation of the mature FMRFa amidated peptide (Table S1). We also examined FMRFa expression using the strong ap^GAL4 driver to overexpress ap^dsRNAi, and observed a significant reduction of mature amidated FMRFa peptide to 40.0±2.2% of control by A20 (w¹¹¹⁸ control n = 40, ap^dsRNAi n = 38; p<0.0001). Finally, we also confirmed that an ap^dsRNAi targeting different ap sequences also significantly downregulated FMRFa (Table S2). The downregulation of FMRFa that we observed in adults is comparable to that reported for embryonic ap null mutants [12], [17]. Thus, we conclude that ap maintains a persistent role in FMRFa regulation. We were unable to determine the extent of Ap knockdown by either ap^dsRNAi transgene due to a lack of suitable Ap-specific antibodies. Therefore, we tested ap^dsRNAi efficacy by examining another ap phenotype. In ap mutants, the wings fail to form [24]. We found that expression of ap^dsRNAi in the developing wing, using ap^GAL4, could precisely phenocopy this ap phenotype (Figure S2). Thus, we conclude that ap^dsRNAi is specific and highly effective. However, as we could not directly quantify Ap downregulation in Tv neurons, we cannot formally discount the possibility that FMRFa would be further downregulated if Ap were entirely eliminated.

To test the role of sqz in adult Tv4 neurons, we expressed sqz^dsRNAi at A1 and observed a partial downregulation of FMRFa expression in sqz heterozygotes to 53.0±4.8% of control (p<0.0001 from control, sqz heterozygote or sqz^dsRNAi alone) (Figure 2E–2G). Similar results were obtained for immunoreactivity to the mature amidated FMRFa peptide (Table S1). Previous reports established that FMRFa is partially downregulated in embryonic sqz mutants [12]. Thus, our data indicate that sqz maintains its partial requirement for FMRFa expression. Due to ubiquitous but weak Sqz expression in the thoracic nerve cord, we were not able to adequately quantitate Sqz downregulation in Tv neurons. Thus, we do not discount the possibility that Sqz may not have been entirely eliminated, and therefore we may be underestimating its effect on FMRFa expression. Taken together, our data demonstrate that eya, ap and sqz are required to maintain wildtype FMRFa levels in the adult.

dimm maintains FMRFa peptide processing

We induced dimm^dsRNAi at A1 and found that immunoreactivity to the mature amidated FMRFa peptide was rapidly and profoundly reduced by dimm^dsRNAi to 24.0±3.2% of control by A10 (p<0.0001), and this was enhanced to 9.8±1.6% of control in dimm heterozygotes (p<0.0001 to control and dimm heterozygotes, p<0.001 to dimm^dsRNAi alone) (Figure 3A–3C, Figure S3). Immunoreactivity to Dimm demonstrated that it had been eliminated (Table S4). In contrast, FMRFa transcript in adults was downregulated in dimm heterozygotes to 67.1±2.9% of control at A20 (p<0.0001) (Figure 3D). Similar effects were observed using a dimm^dsRNAi that targets different dimm sequences (Table S2). It is notable that downregulation of the transcript was only observed after 20 days of dimm^dsRNAi induction but the peptide was profoundly reduced after only 10 days of induction. In late Stage 17 embryonic dimm mutants, immunoreactivity to the mature amidated FMRFa peptide was profoundly reduced, but the extent to which FMRFa transcript was affected had not been quantified [10], [18]. Here, we find that FMRFa transcript was only modestly downregulated in late Stage 17 embryonic dimm mutants to 71.6±3.9% of controls (wild type control n = 54, dimm mutant n = 34 (p<0.0001)). Thus, we conclude that dimm retains its role in the initiation and maintenance of both FMRFa transcript and mature peptide. Why is the mature peptide more responsive to dimm^dsRNAi than is the transcript? We postulated that this was due to dimm's regulation of proprotein convertases and peptide amidases in secretory neurons, both of which are required to process the FMRFa prepropeptide into amidated neuropeptides [10], [20], [25]. We tested this in adults by examining expression of peptidylglycine α-hydroxylating monooxygenase after dimm^dsRNAi induction (PHM). Confirming our hypothesis, dimm^dsRNAi entirely eliminated PHM immunoreactivity in Tv4 neurons (Figure 3E, 3F). Thus, the maintenance of neuropeptide-processing enzyme expression and biosynthesis of the amidated FMRFa peptide is highly dependent upon persistent dimm function in adult Tv neurons.

dac is necessary for FMRFa maintenance

Previous studies found that FMRFa was only modestly downregulated in dac mutants during development [11]. In confirmation, we found here that in L1 larvae, FMRFa immunofluorescence per Tv4 neuron was 68.3±8.5% of control (Figure 3J; P<0.02). We tested dac function in adults and found that dac^dsRNAi dramatically downregulated FMRFa immunoreactivity in adults to 14.8±2.5% of controls, as early as A10 (p<0.0001) (Figure 3G–3I). Correspondingly, FMRFa transcript was reduced to 24.9±4.2% of controls (p<0.0001 to controls), and this was enhanced in dac heterozygotes to 6.5±0.8% (p<0.001 to dac^dsRNAi alone) (Figure S3). Notably, by A15, FMRFa peptide and transcript were entirely eliminated (not shown). In all cases, we found that Dac immunoreactivity was eliminated (Table S4). Moreover, similar effects were observed using a dac^dsRNAi that targets different dac sequences (Figure S2). Thus, dac appears to be unique amongst the Tv4 subtype transcription network factors in that it assumes an increasingly essential role in maintenance compared to developmental initiation.

Post-developmental changes to Tv4 subtype transcription network configuration

The Tv4 subtype transcription network acts through hierarchical and feedforward transcription factor activity, which we refer to here as the network's configuration (summarized in Figure 1A, 1B) [6], [10]. Initiation of grh, dac, sqz and col requires transient cas activity [6]. Expression of ap and eya requires transient col expression [9]. The induction of dimm then requires eya, ap and grh [6], [9], [10]. BMP signaling is dependent upon eya [11]. Finally, ap, eya, dimm, dac, sqz, grh and BMP signaling are all required for FMRFa initiation [6], [11], [12], [17], [18]. This cascade represents a progressive and dynamic set of interactions during Tv4 neuron specification and differentiation. However, for long-term maintenance of subtype gene expression, the subtype transcription network presumably resolves into a stable configuration. As grh, cas and col are lost by early L1 (Figure S1), network configuration must change as the remaining transcription factors become independent of those that initiated their expression. However, we wished to ask whether the developmental cross-regulatory interactions between the persisting transcription factors are retained in the adult to help stabilize the network post-developmentally. Thus, we examined the configuration (cross-regulatory interactions) of all Tv4 subtype transcription network factors.

BMP signaling in embryonic Tv4 neurons is dramatically reduced in eya mutants [11]. We expressed eya^dsRNAi in adults until A15 and found that nuclear pMad, an indicator of BMP activity [12], was significantly downregulated to 47.9%±2.9 of control (Figure 4A, 4B, 4D). As BMP signaling is required for FMRFa expression in embryos and adults, we asked whether eya-dependence of FMRFa in adults is due to reduced BMP signaling. To do this, we simultaneously expressed eya^dsRNAi and restored BMP signaling, using constitutively-activated type I BMP-receptors, thickveins and saxophone. Even though nuclear pMad was robustly activated in all Tv neurons, eya^dsRNAi-induced FMRFa downregulation was not rescued (Figure 4C, 4E). Thus, in adults, eya independently maintains both BMP signaling and FMRFa expression.

In the embryo, initiation of dimm expression in Tv4 is absolutely dependent upon eya and grh [6], [9] and partially dependent upon ap [10]. As grh is not expressed in adult Tv4 neurons, dimm maintenance must become independent of grh. However, as eya and ap are retained, we tested their role in dimm maintenance. We expressed ap^dsRNAi in adults until A20 using ap^GAL4 (a strong hypomorphic allele) and found that Dimm immunoreactivity was significantly downregulated to 58.5%±6.6 of control (Figure 4I–4K). In contrast, we found that Dimm expression in adult Tv4 neurons was entirely unaffected by eya^dsRNAi (Figure 4F–4H). These data indicate that Dimm becomes independent of eya in adult Tv4 neurons, even though eya expression persists in adults and eya is absolutely required for the initiation of dimm expression [9]. We conclude that maintenance of dimm remains dependent on ap but becomes independent of eya and grh post-developmentally.

We also examined all other potential cross-regulatory relationships within the Tv4 subtype transcription network (Table S3), but found no instances of a transcription factor requiring the presence of another for its maintenance.

Maintenance of subtype transcription network output, but not configuration, in mature Tv1 neurons

Is the maintained role found for the Tv4 subtype transcription network common to other subtype transcription networks? To determine this, we examined the output and configuration of the adult Tv1 subtype transcription network. In the neuroblast lineage that gives rise to the Tv1 neuron, cas induces col. Upon birth of the postmitotic Tv1 neuron col initiates eya and ap expression. Initiation of dimm is then absolutely dependent on each of col, ap and eya. Then all four regulators are required for Nplp1 initiation [6], [9].

In adult Tv1 neurons, col, eya, ap and dimm were maintained (Figure 1). Induction of col^dsRNAi [9] at A1 reduced Nplp1 to 10.7±1.7% of control by A15 (Figure 5A–5C). We also found that induction of ap^dsRNAi, eya^dsRNAi or dimm^dsRNAi significantly reduced Nplp1 expression levels to 22.7±2.7%, 45.3±4.2% and 19.0±2.4% of control, respectively (all p<0.0001 to control) (Figure 5G, 5J, 5O). We verified that Col, Eya and Dimm immunoreactivity in Tv1 were eliminated by their respective dsRNAi (Table S4). In addition, we found that dimm^dsRNAi also eliminated PHM expression in Tv1 neurons (Figure 5N). Thus, the Tv1 subtype transcription network is required to maintain the expression of Tv1-specific terminal differentiation gene expression.

Next, we examined the configuration of the adult Tv1 subtype transcription network. Intriguingly, even through col is essential for eya, ap and dimm expression in the embryo, col^dsRNAi had no effect on dimm, ap, or eya expression in Tv1 (Figure 5D–5F). In contrast, expression of either ap^dsRNAi or eya^dsRNAi led to a significant downregulation of Dimm levels in Tv1 to 52.4±5.1% and 42.9±3.7% (Figure 5H, 5K). These data are highly intriguing. The loss of col-dependence of ap, eya and dimm on col was unexpected, notably because Nplp1 retains its col-dependence. Moreover, it is intriguing that dimm retains its eya-dependence in adult Tv1 neurons, but not in adult Tv4 neurons. Thus, the cross-regulatory interactions between persistent transcription factors can be significantly altered after the process of differentiation.

Genetic studies in the embryo had established that col, ap, eya and dimm act non-redundantly to initiate Nplp1 expression during development [6]. We tested whether these transcription factors also act non-redundantly in the adult. As col^dsRNAi dramatically downregulated Nplp1 but did not affect ap, eya or dimm, we conclude that col acts non-redundantly in this case. However, dimm is dependent on both ap and eya in Tv1. Therefore, to test for redundancy between these transcription factors, we restored dimm (UAS-dimm) in either ap^dsRNAi or eya^dsRNAi backgrounds. UAS-dimm expression was found to only partially rescue Nplp1 expression in an ap^dsRNAi background, from 22.7±2.7% to 57.0±5.4% (p<0.0001 compared to ap^dsRNAi and also w¹¹¹⁸ control) (Figure 5I). However, UAS-dimm expression failed to rescue Nplp1 expression in an eya^dsRNAi background (Figure 5L). Thus, as during development, all regulators are required combinatorially for normal Nplp1 expression in adult Tv1 neurons.

Adult neurons respond predictively to ectopic reconstitution of subtype transcription network activity

During development, late-acting subtype transcription networks can override earlier-acting transcriptional codes to dominantly activate ectopic expression of their target genes and/or subtype identity [6]. For example, in embryos, overexpression of col in all Tv neurons ectopically activated Nplp1 in Tv4, presumably reconstituting the col/ap/eya/dimm Tv1 subtype transcription network (Baumgardt et al., 2007). Interestingly, this did not disrupt native FMRFa expression in Tv4 neurons, nor its known subtype transcription network profile. Here, we verify these data in embryos (Figure 6A, 6C). Additionally, we demonstrate here that the reciprocal subtype transcription network reconstitution, dac and BMP activation in all Tv neurons, is sufficient to initiate FMRFa expression in embryonic Tv1 neurons. Interestingly, we found that this also occurred without a concomitant disruption of Nplp1 expression in Tv1 (Figure 6A, 6B).

We tested whether Tv1 and Tv4 subtype transcription networks retained this capacity in adult neurons. This would prove that subtype transcription networks are capable of inducing expression of their pertinent target gene in a mature cell that never had expressed that gene. We found that activation of BMP signaling and dac in adult Tv1 neurons for 5 days robustly activated ectopic FMRFa expression in 100% of Tv1 neurons (Figure 6D, 6E), without affecting Nplp1 in Tv1. We next ectopically expressed col in adult Tv neurons, but this failed to induce ectopic Nplp1 expression (n = 21 Tv4 neurons) (data not shown). However, co-expression of col and dimm in adult Tv neurons was sufficient to trigger ectopic Nplp1 expression in 100% of Tv4 neurons (n = 42) (Figure 6D, 6F). These data show that subtype transcription networks are sufficient to initiate pertinent target gene expression, even in adult neurons that had never expressed the gene.

Discussion

Our data provide novel insight supporting the view of Blau and Baltimore [26] that cellular differentiation is a persistent process that requires active maintenance, rather than being passively ‘locked-in’ or unalterable. We make two primary findings regarding the long-term maintenance of neuronal identity. First, we find that all known developmental transcription factors acting in postmitotic Tv1 and Tv4 neurons to initiate the expression of subtype terminal differentiation genes are then persistently required to maintain their expression. Second, we found that key developmental cross-regulatory relationships that initiated the expression of certain transcription factors were no longer required for their maintained expression in adults. Notably, we found this to be the case even between transcription factors whose expression persists in adults.

In this study, all transcription factors implicated in the initiation of subtype-specific neuropeptide expression in Tv1 and Tv4 neurons were found to maintain subtype terminal differentiation gene expression in adults (summarized in Figure 7). In Tv1, col, eya, ap and dimm are required for Nplp1initiation during development (Figure 1A). In this study, knockdown of each transcription factor in adult Tv1 neurons was shown to dramatically downregulate Nplp1. In Tv4 neurons, FMRFa initiation during development requires eya, ap, sqz, dac, dimm and retrograde BMP signaling (Figure 1B). Together with our previous work showing that BMP signaling maintains FMRFa expression in adults [14], we now demonstrate that all six regulatory inputs are required for FMRFa maintenance. Most transcription factors, except for dac, also retained their relative regulatory input for FMRFa and Nplp1 expression. In addition, individual transcription factors also retained their developmental subroutines. For example, as found during development [10], [11], [20], dimm was required in adults to maintain PHM (independently of other regulators) and FMRFa/Nplp1 expression (combinatorially with other regulators).

The few genetic studies that test a persistent role for developmental transcription factors support their role in initiating and maintaining terminal differentiation gene expression. In C. elegans, where just one or two transcription factors initiate most neuronal subtype-specific terminal differentiation genes, they then also appear to maintain their target terminal differentiation genes. In ASE and dopaminergic neurons respectively, CHE-1 and AST-1 initiate and maintain expression of pertinent subtype-specific terminal differentiation genes [27], [28]. In vertebrate neurons, where there is increased complexity in the combinatorial activity of transcription factors in subtype-specific gene expression, certain transcription factors have been demonstrated to be required for maintenance of subtype identity. These are Hand2 that initiates and maintains tyrosine hydroxylase and dopa β-hydroxylase expression in mouse sympathetic neurons [29], Pet-1, Gata3 and Lmx1b for serotonergic marker expression in mouse serotonergic neurons [30], [31], and Nurr1 for dopaminergic marker expression in murine dopaminergic neurons [32].

However, while these studies confirm a role for certain developmental transcription factors in subtype maintenance, it had remained unclear whether the elaborate developmental subtype transcription networks, that mediate neuronal differentiation in Drosophila and vertebrates, are retained in their entirety for maintenance, or whether they become greatly simplified. Our analysis of all known subtype transcription network factors in Tv1 and Tv4 neurons now indicates that the majority of a developmental subtype transcription network is indeed retained and required for maintenance. Why would an entire network of transcription factors be required to maintain subtype-specific gene expression? The combinatorial nature of subtype-specific gene expression entails cooperative transcription factor binding at clustered cognate DNA sequences and/or synergism in their activation of transcription. In such cases, our data would indicate that this is not dispensed with for maintaining terminal differentiation gene expression in mature neurons.

How the transcription factors of the subtype transcription networks are maintained is less well understood. An elegant model has emerged from studies in C. elegans, wherein transcription factors stably auto-maintain their own expression and can then maintain the expression of subtype terminal differentiation genes [33]. The transcription factor CHE-1 is a key transcription factor that initiates and maintains subtype identity in ASE neurons. CHE-1 binds to a cognate DNA sequence motif (the ASE motif) in most terminal differentiation genes expressed in ASE neurons, as well as in its own cis-regulatory region. Notably, a promoter fusion of the che-1 transcription factor failed to express in che-1 mutants, indicative of CHE-1 autoregulation [27], [34]. Similar observations were made for AST-1 [28], and for the cooperatively-acting TTX-3 and CEH-10 transcription factors in AIY neurons [35]. Thus, subtype maintenance in C. elegans is anchored by auto-maintenance of the transcription factors that initiate and maintain terminal differentiation gene expression.

In contrast, all available evidence in Tv1 and Tv4 neurons fails to support such an autoregulatory mechanism. An ap reporter (apC-τ-lacZ) is expressed normally in ap mutants [12], [36], and in this study ap^dsRNAi was not found to alter ap^GAL4 reporter activity (Figure 2I). Moreover, col transcription was unaffected in col mutants that express a non-functional Col protein [9]. This leaves unresolved the question of how the majority of the transcription factors are stably maintained. For transcription factors that are initiated by transiently expressed inputs, a shift to distinct maintenance mechanisms have been invoked and in certain cases shown [35]. In this study, this was found for the loss of cas expression in Tv1 (required for col initiation) and the loss of cas, col and grh in Tv4 (required for eya, ap, dimm, sqz, dac initiation). However, we were surprised to find that the cross-regulatory relationships between persistently-expressed transcription factors were also significantly altered in adults. Notably, eya initiated but did not maintain dimm in Tv4. In Tv1, col initiated but did not maintain eya, ap or dimm. This was particularly unexpected as eya remained critical for FMRFa maintenance and col remained critical for Nplp1 maintenance. Indeed, although we tested for cross-regulatory interactions between all transcription factors in both the Tv1 and Tv4 subtype transcription networks, only Dimm was found to remain dependent upon its developmental input; Eya and Ap in Tv1 as well as Ap in Tv4. However, even in this case, the regulation of Dimm was changed; it no longer required eya in Tv4, and in Tv1 it no longer required col, in spite of the fact that both col and eya are retained in these neurons. We anticipate that such changes in transcription factor cross-regulatory relationships will be found in other Drosophila and vertebrate neurons, which exhibit high complexity in their subtype transcription networks [1], [5]. Indeed, recent evidence has found that in murine serotonergic neurons, the initiation of Pet-1 requires Lmx-1b, but ablation of Lmx-1b in adults did not perturb the maintenance of Pet-1 expression [31].

We are pursuing the potential role of autoregulation for the other factors in the Tv1/Tv4 subtype transcription networks. However, we consider there to be three additional, potentially overlapping, models for subtype transcription network maintenance. First, regulators may act increasingly redundantly upon one another. Second, unknown regulators may become increasingly sufficient for transcription factor maintenance. Third, transcription factors may be maintained by dedicated maintenance mechanisms, as has been shown for the role of trithorax group genes in the maintenance of Hox genes and Engrailed [37], [38]. Moreover, chromatin modification is undoubtedly involved and likely required to maintain high-level transcription of Tv transcription factors as well as FMRFa, Nplp1 and PHM. However, the extent to which these are instructive as opposed to permissive has yet to be established [39]. In this light, it is intriguing that MYST-HAT complexes, in addition to the subtype transcription factors Che-1 and Die-1, are required for maintenance of ASE-Left subtype identity in C. elegans [31].

Taken together, our studies have identified two apparent types of maintenance mechanism that are operational in adult neurons. On one hand, there are sets of genes that are maintained by their initiating set of transcription factors. These include the terminal differentiation genes and the transcription factor dimm. On the other, most transcription factors appear to no longer require regulatory input from their initiating transcription factor(s). Further work will be required to better understand whether these differences represent truly distinct modes of gene maintenance or reflect the existence of yet unidentified regulatory inputs onto these transcription factors. One issue to consider here is that the expression of certain terminal differentiation genes in neurons, but perhaps not subtype transcription factors, can be plastic throughout life, with changes commonly occurring in response to a developmental switch or physiological stimulus [40], [41], [42]. Thus, terminal differentiation genes may retain complex transcriptional control in order to remain responsive to change. It is notable, however, that FMRFa, Nplp1 and PHM appear to be stably expressed at high levels in Tv1/4 neurons, and we have not found any conditions that alter their expression throughout life. Thus, we consider these to be stable terminal differentiation genes akin to serotonergic or dopaminergic markers in their respective neurons that define those cells' functional identity and, where tested, are actively maintained by their developmental inputs [30], [32]. Tv1/4 neurons undoubtedly express a battery of terminal differentiation genes, and sets of unknown transcription factors are likely required for their subtype-specific expression. We consider subtype transcription networks to encompass all regulators required for differentiating the expression of all subtype-specific terminal differentiation genes. Further, we view differentiation of subtype identity as the completion of a multitude of distinct gene regulatory events in which each gene is regulated by a subset of the overall subtype transcription network. As highly restricted terminal differentiation genes expressed in Tv1 and Tv4 neurons, we believe that Nplp1, FMRFa and PHM provide a suitable model for the maintenance of overall identity, with the understanding that other unknown terminal differentiation genes expressed in Tv1 and Tv4 may not be perturbed by knockdown of the transcription factors tested herein. In the future, it will be important to incorporate a more comprehensive list of regulators and terminal differentiation genes for each neuronal subtype. However, we believe that the principles uncovered here for FMRFa, Nplp1 and PHM maintenance will hold for other terminal differentiation genes.

Finally, we propose that the active mechanisms utilized for maintenance of subtype differentiation represent an Achilles heel that renders long-lived neurons susceptible to degenerative disorders. Nurr1 ablation in adult mDA neurons reduced dopaminergic markers and promoted cell death [32]. Notably, Nurr1 mutation is associated with Parkinson's disease [43], and its downregulation is observed in Parkinson's disease mDA neurons [44]. Adult mDA are also susceptible to degeneration in foxa2 heterozygotes, another regulator of mDA neuron differentiation that is maintained in adult mDA neurons [45]. Studies in other long-lived cell types draw similar conclusions. Adult conditional knockout of Pdx1 reduced insulin and β-cell mass [46], [47] and, importantly, heterozygosity for Pdx1 leads to a rare monogenic form of non-immune diabetes, MODY4 [48]. Similarly, NeuroD1 haploinsufficiency is linked to MODY6 [49] and adult ablation of NeuroD in β-islet cells results in β-cell dysfunction and diabetes [50]. These data, together with our results here, underscore the need to further explore the transcriptional networks that actively maintain subtype identity, and hence the function, of adult and aging cells.

Materials and Methods

Fly stocks

Flies were maintained on standard cornmeal food and maintained at stable temperatures in environment rooms set at 70% humidity at 18°C, 25°C or 29°C.

Fly strains

apterous^md544 (referred to as ap^GAL4); ap^P44; sqz^IE; sqz^GAL4; UAS-thickveins activated (UAS-tkv^A); UAS-saxophone activated (UAS-sax^A) [12]; dac^GAL4 [51]; rev4; UAS-dimm [18]; dac³ [52]; ; eya^CliIID [53]; grh^GAL4 [6]. tubP>GAL80^TS (temperature-sensitive GAL80 under the control of the Drosophila tubulin 84B promoter) (McGuire et al., 2003); UAS-nEGFP (nuclear localized EGFP); UAS-dicer2 [21].

dsRNAi lines

Strains used for primary data: UAS-col#24E [9]; UAS-ap^dsRNAi 8376R-2; UAS-dac^dsRNAi 4952R-2; UAS-sqz^dsRNAi 5557R-2 (NIG-FLY); UAS-dimm^dsRNAi GD44470; UAS-eya^dsRNAi GD43911 (VDRC). Strains used secondarily to verify data: UAS-dimm^dsRNAi KK103356; UAS-eya^dsRNAi 108071KK (VDRC); UAS-eya^dsRNAi JF03160; UAS-dac^dsRNAi JF02322 (TRiP).

Spatial and temporal regulation of transgene expression using the TARGET system

Flies for TARGET-mediated transgene induction were generated by crossing utility flies (UAS-dicer2/UAS-dicer2; ap^Gal4; tubP>GAL80^TS, UAS-nEGFP/SM6-TM6,Tb) or (UAS-dicer/UAS-dicer; dac^Gal4; tubP>GAL80^TS, UAS-nEGFP/SM6-TM6,Tb) to UAS-dsRNAi (experimental group) or w¹¹¹⁸ flies (control). Experiments were performed on resulting progeny bearing appropriate genotypes (screened by loss of SM6-TM6, Tb balancer chromosome). All experimental and control flies were raised at 18°C until eclosion (hatching from the pupal case). On adult day 1 (A1), flies were switched to 29°C for the duration of the induction period indicated. Throughout the text, we present dsRNAi data for the induction period at which we observe the maximal phenotype of Nplp1 and FMRFa expression. Further details for each dsRNAi are provided in the text.

Antibodies

Primary antibodies

Sheep anti-digoxygenin (1∶1500; Roche); rabbit anti-GFP (1∶100; A6455 Invitrogen); rabbit anti-FMRFa (1∶1000; T-4757 Peninsula Labs); mouse anti-β-Galactosidase (1∶100; 40-1a); chicken anti-Nplp1 (1∶1000), guinea pig anti-Dimm (1∶1000) and rat anti-Cas (1∶1000) (gifts from S. Thor, Linkoping U, Sweden); guinea pig anti-Collier (1∶1000; gift from Adrian Moore, RIKEN, Japan); rabbit anti-pMad (1∶100; cell signaling); rabbit anti-nab (gift from Fernando Jimenez Diaz-Benjumea, Universidad Autónoma de Madrid, Spain); mouse anti-Eya (1∶100; clone 10H6) and mouse anti-Dac (1∶2; Mab Dac 2–3) (both from Developmental Studies Hybridoma Bank; Iowa U. Iowa).

Secondary antibodies

Donkey anti-sheep Alexa 555 (1∶10; Invitrogen, Carlsbad, USA); donkey anti-mouse Cy5 and donkey anti-rabbit Cy2 (1∶200; Jackson Immunoresearch, West Grove, USA). Antisense DIG-RNA Probe: DIG-Uracil tagged RNA probes were generated using T3 RNA polymerase from clone RH03963 (DGRC: Drosophila Genomic Resource Centre, Indiana, USA) containing a 1584 bp FMRFa cDNA (using the Roche DIG-U-RNA Labelling Kit). Probe synthesis was confirmed using gel electrophoresis.

Multiplex fluorescent in situ hybridization (FISH) and immunohistochemistry

Standard in situ and immunohistochemistry protocols were carried out as described [14]. All tissues compared for fluorescence intensity were processed at the same time using the same aliquots of all solutions under the same conditions. They were then mounted on the same slide and confocal settings were calibrated to control staining levels.

Image and statistical analysis

All images acquired on an Olympus FV1000 confocal microscope. Fluorescent intensity of individual neurons was measured using Image J (US National Institutes of Health). The mean pixel intensity for each neuron was measured from compressed Z-slices, and corrected for background fluorescence. Analysis was performed on every identifiable Tv1 and Tv4 neuron in segments T1 and T3. The resulting value for each Tv neuron was then incorporated as a single datum point towards the mean intensity for each experiment. Each datum point is represented as a percentage of the mean of the w¹¹¹⁸ control for that experiment. Data are presented as Mean ± SEM. Representative images of Tv neurons that were directly compared in figures were processed in an identical way, simultaneously, using Adobe Photoshop CS4. Normally distributed unpaired data were compared using a two-tailed T-test assuming equal variance, to identify significant differences between means. All statistical analysis and graphs data were performed using Prism 5 software. (Graphpad).

Supporting Information

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