Silencer-delimited transgenesis: NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner
© Xie et al; licensee BioMed Central Ltd. 2012
Received: 6 July 2012
Accepted: 30 November 2012
Published: 30 November 2012
We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations.
We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing.
Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.
Keywordszebrafish transgenesis enhancer trap NRSE/RE1 NRSF/REST Gal4/UAS neuron
Accurate characterization of neurons and neural circuits requires that neuronal subtypes be unambiguously identified  and independently manipulated . Typically, neurons are classified based on morphological (e.g., neurite targeting patterns), molecular (e.g., transmitter expression) and/or physiological properties. Cellular subtypes are defined by both activation and repression of specific gene sets. Repression plays a major role in regulating the expression of neural-specific genes. For instance, the transcriptional repressor neuron-restrictive silencing factor (NRSF)/repressor element 1 silencing transcription factor (REST) serves to silence neural gene expression in non-neural tissues [3, 4]. Transgenic techniques provide a means of targeting discrete neuronal subpopulations, affording investigations of the function of specific neuronal cell types and neural subcircuits [5, 6]. However, transgenic labeling of neuronal subpopulations in vertebrates has proven to be complicated, as cis-regulatory elements (e.g., promoters, enhancers) that delineate specific neuronal subtypes are sometimes difficult to define. Equally problematic is that many regulatory elements characterized as being cell-type specific within the nervous system actually label cells in other tissues. Optimally, exclusive transgene expression in neuronal cell subtypes is necessary to assign direct causal effects during assays involving gain- or loss-of-function manipulations.
Gene and/or enhancer trap screens, whereby endogenous cis-regulatory elements are co-opted to regulate transgene expression, eliminate the need to identify cell-specific promoters by allowing visual selection of expression patterns of interest. Transposons, such as Tol2, have been used extensively for gene/enhancer trapping in zebrafish (Danio rerio) and many of the resultant transgenic lines show expression in the nervous system. However, despite the general success of this approach, so-called basal or background expression in heart, skeletal muscle, etc., can compromise the usefulness of these resources . This has been particularly problematic for enhancer traps employing the Gal4/upstream activating sequence (UAS) bipartite expression amplification system [8–10]. The Gal4/UAS system is based on the yeast transcription factor Gal4, which binds to 17 to 23 bp UAS  to drive expression of effector genes/transgenes. A bipartite transgenic system, where Gal4 'drivers' and UAS 'effectors' are derived separately was adapted to the Drosophila system  as a tissue/cell-specific manipulation platform. Rapid expansion of a set of Gal4 driver lines (specifying where and when transgenes are expressed) and UAS effector/reporter transgenic lines (specifying how much and what transgenes are expressed) soon followed. Driver and effector/reporter lines can be brought together in any combination; the bipartite nature of the Gal4/UAS system thus provides a versatile platform for studies of cell and molecular function that has been employed to great effect within the Drosophila community . The source of background expression in zebrafish Gal4/UAS-based transgenic lines has not been resolved. It could be a byproduct of cryptic enhancer elements , promiscuous expression as a result of position effects, or otherwise undetectable gene expression that is revealed by the enhanced transcriptional activity of the Gal4/UAS system. The latter possibility is in keeping with interpretations put forth by Fujimoto et al. , concerning unexpected expression patterns seen in Gal4-VP16 lines (three of three Tg(optb.A:Gal4-VP16) lines show previously uncharacterized expression within eye muscles and retinal cells). Regardless of the underlying mechanism, Gal4/UAS lines in which background expression is reduced or eliminated would provide improved resources for functional studies of the nervous system.
Intersectional and subtractive methods, whereby transgenes are restricted to cells expressing two or more patterning genes, have been developed to promote cell-specific expression . Alternatively, transcriptional repression or silencing could be used to delimit transgene expression to specific cell or tissue types. Interestingly, transcriptional repression plays a prominent role in establishing neuronal specific expression patterns . Accordingly, we were interested in determining whether inserting neuronal silencer binding sites into Gal4 driver and/or UAS effector transgenes would serve to restrict transgene expression to the nervous system in vivo. This approach could potentially solve Tol2-associated background expression issues with regard to studies of the nervous system in zebrafish. Moreover, synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate useful expression patterns not otherwise attainable by standard promoter/enhancer characterizations. Here, we have explored the use of the neuron-restrictive silencer element (NRSE) to delimit transgene expression exclusively to neuronal cells.
Evaluation of regulatory mechanisms underlying neural specificity of the synaptic protein stathmin-like 2 gene (STMN2, also known as SCG10) and a voltage-dependent sodium channel revealed that active repression of expression in non-neuronal cells played a central role [17–19]. An NRSE  - also known as restriction element 1 (RE1 ) - was identified as being necessary and sufficient for repression of STMN2 and the type II sodium channel. Subsequently, the Krüppel zinc finger protein NRSF/REST was found to bind NRSE/RE1 sites and repress non-neuronal expression of multiple neural-specific genes [3, 4]. Upon binding to NRSE/RE1 sites, NRSF/REST acts in concert with a corepressor complex of chromatin remodeling proteins including REST corepressor 1 , Sin3A  and histone/lysine deacetylases [23, 24]. NRSF/REST has been reported to repress target gene expression in both embryonic and neural stem cells , whereas corepressors appear to play a role in plasticity of expression in mature neurons . Although NRSE-containing genes can be expressed outside the nervous system - e.g., pancreatic islet cells  - and NRSE sites are thought to act as neuronal enhancers in certain contexts , the predominant effect of NRSE-mediated gene regulation is to repress the expression of neural genes in non-neuronal cell types. This suggests that integrating NRSE sites into the regulatory elements of transgene constructs might serve to promote specificity by delimiting expression to the nervous system. This approach has shown promise when tested in the context of defined regulatory elements in cell culture , viral vectors [30, 31] and transgenic assays in mammalian systems . We were interested in whether NRSE sites would be effective for delimiting transgene expression to the nervous system within the context of enhancer trap screens in zebrafish, and thereby provide improved resources for functional studies of the nervous system.
To test this idea, we integrated a pair of consensus NRSE sites  into several bipartite transgenic expression system constructs and created corresponding transgenic zebrafish lines. The NRSE site used, TTCAGCACCACGGACAGCGCC, is a canonical NRSE site that is highly conserved across species, and is composed of two non-palindromic half-sites separated by a non-conserved 2 bp spacer (underlined). We placed a tandem set of NRSE sites in upstream regulatory regions of several transgenic constructs and compared resulting expression patterns to non-NRSE parental plasmids. In all, this strategy was applied within the context of enhancer trap constructs , defined enhancer constructs , defined promoter constructs  and UAS-based reporter constructs .
The data indicate that transgene expression was strongly biased to the nervous system when NRSE sequences were included in enhancer trap and defined enhancer constructs, thereby effective in delimiting the expression of the driver element of a bipartite expression system (e.g., Gal4-VP16). However, expression biases were not evident when NRSE sequences were added to UAS-based reporter transgenes. Nevertheless, due to the bipartite nature of such systems, delimiting the expression of Gal4-VP16 drivers sufficed to restrict UAS reporters to the nervous system as well - because drivers are required to activate expression of reporters. Morpholino knockdown (this study) and zinc finger nuclease mediated gene disruptions  verified that NRSF/REST is required for NRSE-based transgene silencing. Thus, NRSE-delimited transgenesis may help to overcome difficulties in defining cell-specific expression in the nervous system. Accordingly, we are conducting a large-scale enhancer trap screen coupling NRSE sites with bipartite expression systems to facilitate functional manipulations of trapped neuronal cell subtypes. Several other tissue/cell-specific silencer elements have been reported. Examples include a cartilage-specific element , a cardiac muscle-specific element  and a cell-specific pancreatic silencer element . Thus, transgenes that create novel associations between tissue-specific silencers and more diffusely expressed activator elements may be a useful strategy for labeling and manipulating discrete cell types that are otherwise difficult to demarcate. The existence of tissue-specific silencers in plants  suggests such elements exist throughout most multicellular life forms. In summary, silencer-delimited transgenesis may be a broadly applicable strategy that enhances the capacity to exclusively target specific cell types.
NRSE-delimited enhancer traps
To test whether associating NRSE sites with a minimal promoter would serve to restrict transgene expression to the nervous system, Gal4-VP16-based enhancer trap constructs were assembled with and without NRSE sites. Plasmids were composed of a mouse cfos minimal promoter  upstream of an optimized Gal4-VP16 transcriptional activator, termed KalTA4 , and assembled within the miniTol2 cassette . Initially, transient transgenesis assays were used to compare control plasmids (CK, cfos:KalTA4; see construct diagrams in Additional file 1) and test plasmids having a tandem repeat of two NRSE sites inserted 18 bp upstream of the minimal promoter (NRCK, 2xNRSE-cfos:KalTA4). CK or NRCK transgenes were injected into fertilized eggs from an established UAS effector-reporter line, (14xNTR-Ch, Tg(14xUAS-E1b:nfsB-mCherry)c264 ) and resulting mCherry expression patterns monitored daily until 6 to 7 days post-fertilization (dpf). These preliminary results suggested that the NRSE-containing plasmid, NRCK, was preferentially expressed in neural tissues (data not shown). Following these studies, we created multicistronic 'self-reporting' enhancer trap constructs linking the Gal4-VP16 driver and UAS reporter directly within a single transgene (i.e., in cis). In the NRSE version, a loxP flanked 5xUAS:YFP reporter was placed downstream of NRCK (NRCK-5xMY, 2xNRSE-cfos:KalTA4, loxP-5xUAS-E1b:gap43-EFYP-loxP). The control version consisted of the CK driver element upstream of tandem 5xUAS:nfsB effector and 14xUAS:YFP reporter components (CK-5xN-14xY, cfos:KalTA4, 5xUAS:Eco.nfsB, 14xUAS:gap43-YFP), as previously reported .
Initial evaluations of transgene expression patterns were promising. However, we noted that mCherry patterns often seemed unstable, that is, expression domains would differ slightly from one generation to the next. It was subsequently reported that the Tg(14xUAS-E1b:nfsB-mCherry)c264 line is susceptible to methylation-based silencing [48, 49]. To avoid NRSE-independent silencing issues, which could obviously compromise our expression pattern analyses, we created a new 5xUAS reporter line expressing membrane-tagged yellow fluorescent protein (YFP; 5xMY-HMY, Tg(loxP-5xUAS-E1b:gap43-YFP-loxP, he1a:gap43-YFP)gmc930, see Additional file 1). Importantly, the 5xMY-HMY (gmc930) line has shown no evidence of silencing over three generations, possibly due to our inclusion of 'barrier' insulator sequences  or the reduction in the number of repetitive UAS elements . Barrier insulators are thought to function by maintaining histones of chromatin surrounding the site of transgene integrations in a hyperacetylated state, thereby blocking encroachment of heterochromatin . Either way, the 5xMY-HMY line provided an improved resource for defining expression patterns of CK and NRCK enhancer trap lines. All subsequent expression analyses were performed by crossing KalTA4 enhancer trap lines to 5xMY-HMY and/or by creating driver lines directly in 5xMY-HMY fertilized eggs.
Phenotype characterization of NRSE versus non-NRSE enhancer trap lines.
- NRSE Tg
+ NRSE Tg
To quantify the effect of inserting NRSE sites into enhancer trap transgenes, KalTA4 driver lines were classified between 6 and 9 dpf as displaying one of three phenotypes: neural-restricted, mixed and non-neural. The results show NRSE-containing driver lines (including self-reporting NRCK-5xMY lines) exhibit a clear bias toward neural-restricted expression compared with controls (Figure 1S, Table 1). Of 62 NRSE driver lines evaluated, 66% (41 lines) were classified as having neural-restricted expression. Conversely, of 69 characterized control lines, only 10% (seven lines) showed a neural-restricted pattern. Lines showing mixed expression patterns (see Figure 1B-F) made up 68% of CK controls (47 lines) and 29% of NRSE drivers (18 lines). Non-neural lines made up 22% of controls and only 5% of NRSE lines (Table 1). These data suggest that NRSE sites can have dramatic effects on transgene expression patterns within the context of endogenous enhancer elements trapped by a cfos minimal promoter. Due to the bipartite nature of the Gal4/UAS system, NRSE-delimited expression of an optimized Gal4-VP16 transcriptional activator translated to neural-restricted expression of a UAS:YFP reporter whether Gal4-VP16 and UAS transgenes were in trans (Figure 1G-L) or cis (Figure 1M-R) orientations.
NRSE-delimited expression of a defined enhancer
NRSE sites fail to restrict UAS transgene expression patterns
Temporal aspects of NRSE-delimited transgenesis
Changes in REST expression are correlated with delayed transgene repression
NRSE-mediated transgene repression is dependent on NRSF/REST expression
Adaptation of the Gal4/UAS system to the zebrafish system  was initially met with great enthusiasm due to the versatile and powerful nature of bipartite transgene expression systems. However, this effort soon became fraught with problematic issues, such as low expressivity, Gal4-VP16 toxicity and, more recently, methylation-based transgene silencing of UAS reporter lines. Solutions to these problems have been developed [43, 49, 63, 64], as well as strategies for Gal4-VP16-based lineage tracing . However, a general lack of cell-specific expression of many of the Gal4-VP16 driver lines generated to date presents an obstacle to the widespread deployment of such resources. Efforts to address this issue have included the development of a miniTol2 cassette , characterizations of alternative minimal promoters [10, 43, 64, 65], the use of gene traps as opposed enhancer traps , and modulating Gal4 expression through interactions with Gal80 [14, 66]. We reasoned that an alternative strategy would be to leverage tissue-specific silencer elements to delimit transgene expression to tissue types of interest by eliminating expression in non-targeted regions. More specifically, we were interested in a strategy that would enhance the neuronal specificity of transgene expression, thus facilitating exciting new functional assay platforms such as optogenetics .
It remains unclear whether the background patterns evident in Gal4-VP16 lines are predominantly an artifact (e.g., cryptic enhancers), promiscuous position effects, Gal4-VP16 based amplification of previously undetectable gene/enhancer activity , or a combination of these. Interestingly, the latter possibility is in keeping with stochastic resonance analyses suggesting many genes are active at low levels as part of natural circadian oscillations . In addition, Fujimoto et al., favored this explanation regarding unexpected but consistent expression patterns seen in multiple Tg(optb.A:Gal4-VP16) lines . Combined with data presented here, this would suggest that NRSE sites are capable of delimiting endogenous enhancers, not simply reducing an artificial byproduct of the technique. Unfortunately, due to inherent ambiguities of identifying distinct regulatory elements acting on gene/enhancer traps [69, 70], the transgenic lines generated here can provide only limited insight to this question. Nevertheless, the data show that regardless of the mechanism, NRSE sites serve to bias enhancer traps toward neuronal specific expression patterns, thus providing useful new resources for neurobiological research.
The addition of NRSE sites to UAS reporter lines was not sufficient to restrict reporter expression to neural tissues (Figures 3 and 7, Table 1). This is unfortunate, as NRSE-delimited UAS lines would have been useful to restrict the expression of existing Gal4-VP16 lines. How is it that NRSE sites can function to alter the expression of Gal4-based enhancer traps but not UAS reporter lines? The answer to this may be related to the fact that NRSE sites only served to bias expression; they were not 100% effective. This is in keeping with our increasing understanding of the complex multifactorial nature of gene regulation. Transcriptional activity cannot be adequately described in binary on/off terms; rather each element impinging on gene expression provides relative 'rheostat'-type modulations that are summed for final effect. Accordingly, our data suggest that, the majority of the time, REST/NRSE interactions are adequate to dampen non-neural expression when integrated with endogenous regulatory elements. Thus, in the context of an enhancer trap screen, REST/NRSE interactions are sufficient to reduce Gal4-VP16 expression to non-effective levels in non-neural populations. However, when non-neural expression of Gal4-VP16 is unchecked - i.e., when NRSE sites are solely associated with UAS reporters - the artificially enhanced strength of Gal4-VP16 transcriptional activators is the dominant element in the equation and the silencing activity of REST/NRSE is rendered ineffective.
Our findings indicate that new NRSE -delimited Gal4-VP16 driver lines will need to be derived to take advantage of the neural expression bias provided by this approach. Accordingly, we have begun a large-scale NRSE-delimited enhancer trap screen to create new Gal4-VP16 lines useful for dissecting neural circuit functions. To date, 62 NRSE-delimited KalTA4-expressing lines have been created. In a related screen, we are creating a series of NRSE-delimited LexA-based driver lines (manuscript in preparation). The use of two bipartite transgene expression systems (e.g., Gal4/UAS and LexA/LexA Operon) would allow two neuronal subpopulations to be independently manipulated. Optimally, complementary platforms of this nature could be used to differentially modulate pre- and post-synaptic elements of discrete subcircuits - a possibility that improved trans-synaptic transporters would facilitate. In addition, the use of an inducible LexA-based transactivation system in transgenic zebrafish  provides an additional level of temporal control over transgene expression; a strategy that increases the versatility of such systems even further.
The mechanism behind NRSE-delimited transgene expression is likely due to a repressive action of NRSF/REST in non-neural tissues [3, 4]. Our data are consistent with this; a possible explanation for the reduction in non-neural phenotypes we see with NRSE lines (Figure 1S) is that, at some frequency, lines that would have expressed solely in non-neural cells are rendered silent by NRSE. However, context-dependent transcriptional regulatory functions have been proposed for NRSF/REST that span the gamut of embryogenesis: from embryonic stem cells , to neural progenitors , to differentiating neurons [28, 73]. In neural lineages, NRSF/REST is thought to repress gene expression in progenitors until differentiation commences. In agreement with these models, REST expression levels decrease over time in neural lineages [26, 58, 59]. Conversely, NRSF/REST is thought to repress neural gene expression in non-neural cells, in keeping with NRSF/REST expression becoming progressively restricted to non-neural tissues as development proceeds. Thus, REST may play several roles during development, including repression of neuronal genes in the developing nervous system and later non-neuronal cells, and regulation of the terminal differentiation of neurons (reviewed by [74, 75]). Several of our observations are in keeping with the view that REST action is more complex than simply repressing expression in non-neural cells; however, our data are not consistent with REST playing a major role in regulating neurogenesis .
REST is expressed nearly ubiquitously during early zebrafish development, including within the nervous system. Yet, early reporter expression (e.g., 1 to 2 dpf) is observed in the majority of NRSE-containing Gal4-VP16 driver lines (Additional file 4). This observation conflicts somewhat with reports suggesting that REST acts to repress neural differentiation programs in stem cells  and/or neuronal progenitors , which predicts that NRSE-delimited transgene expression would be limited to late neural differentiation stages. However, because NRSF/REST translation is tightly regulated [26, 58, 59], evaluations of REST protein expression levels and/or subcellular localization are necessary to better determine the degree to which REST expression patterns correlate to function. In addition, almost half of the NRSE-containing driver lines established to date (44%) show delayed non-neural repression of transgene expression (Figure 4). The timing of the delayed repression phenomenon is associated with a nearly two-fold increase in REST expression (Figure 5); this suggests that REST-mediated silencing requires a threshold of expression. This possibility is supported by data showing a two-fold increase in REST expression that coincides with downregulation of NRSE-containing neuronal genes in differentiating oligodendrocytes , and a corresponding increase in the number of REST-occupied target genes in these cells as they mature . Additionally, our results are consistent with data from Kok et al., showing that early neural patterning is largely unaltered in rest mutants .
It is important to note that lines displaying the delayed repression phenotype will be less useful for experiments concerning early neural development. However, their applicability to tests in late stage larvae (6 dpf) and beyond - for instance, to assay behavioral consequences of altering neuronal activity - remains viable.
To further test whether REST was required for NRSE-delimited expression patterns, we knocked down REST expression in transgenic NRCK zebrafish embryos and larvae using a previously characterized morpholino. The data showed clear evidence that when REST function is disrupted, NRSE-mediated neural expression biases are lost, with spatially expanded and temporally extended strong skeletal muscle expression seen in rest MO-injected NRSE-containing Gal4-VP16 driver lines (Figure 6). In addition, investigations of a rest mutant line (rest sbu29 ), generated by zinc finger nuclease targeting [78, 79], showed similar results with four different NRCK transgenic lines (gmc606, 607, 632 and 641) . Interestingly, evidence of expanded neural expression in rest sbu29/sbu99 mutants suggests REST can repress expression in the nervous system as well. This is in keeping with studies suggesting that REST may act to repress gene expression in neuronal subsets  and that REST expression is detected in neurons of certain brain regions . More recently, the possibility of REST acting as a transcriptional activator has been attributed to the expression of a dominant-negative splice variant, REST4, that disrupts REST-mediated gene repression . Future analyses could determine whether the presence of REST4 alters the expression of NRSE-containing transgenes.
Cell-type specific lineages are often defined by multifactorial 'codes' of overlapping subsets of transcription factors . Thus, identifying individual promoter/enhancer elements providing cell-type exclusive expression patterns can be challenging. A potential strategy suggested by the data presented here is to create artificial associations between tissue-specific silencer elements and regulatory elements that target cell types of interest, albeit not exclusively. By eliminating expression in non-targeted tissues, the silencer element serves to produce the desired expression pattern; e.g., a cell-specific expression domain that might not otherwise be attainable. As an example of this approach, we are creating neuronally restricted Gal4-VP16 driver transgenic lines. These resources should facilitate the functional dissection of neural circuits in zebrafish - e.g., optogenetic  and/or toxin-mediated inhibition/activation of neuronal activity , and inducible cell ablations [46, 83–85] - by restricting manipulations to targeted neuronal cell subpopulations, thus facilitating delineations of causal relationships.
These studies validate the use of tissue-specific silencer elements to promote enhanced transgene expression specificity. NRSE sites served to bias the expression of trapped and defined DNA regulatory elements to the nervous system, providing a means of targeting neuronal cell subtypes by silencing expression in non-neural tissues. Transgene silencing effects were dependent on the expression of REST, in keeping with a well-characterized role of this NRSE-binding transcriptional repressor in maintaining neural-specific gene expression. Using the strategy, promoter/enhancer elements that would otherwise be too broadly expressed can be harnessed for functional assays. This approach also affords a solution to non-specific background expression issues that can compromise large-scale enhancer trap screens, as has been the case in the zebrafish field. NRSE-delimited transgenes can provide useful new tools for functional studies of the nervous system. Inclusion of bipartite expression systems, such as Gal4/UAS, ensures that a multitude of functional assays can be performed with NRSE-delimited transgenic resources. For instance, integrating new toolsets for manipulating neuronal activity or targeted cellular ablation systems into bipartite effectors will provide a versatile platform for the genetic dissection of neural circuit function. More broadly, similar genetic mechanisms may be used to reinforce expression specificity in other tissues. Thus, creating synthetic associations between endogenous regulatory sequences and tissue-specific silencer elements could provide a means of targeting unique cellular subsets for which cell-specific regulatory elements are lacking.
This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. An animal use protocol was approved by the Institutional Animal Care and Use Committee (Approval Identification Number: BR10-12-391) of Georgia Health Sciences University, which has an Animal Welfare Assurance on file in the Office of Laboratory Animal Welfare (Assurance Number: A3307-01). Using approved anesthetics, all efforts were made to minimize discomfort and suffering during experimental procedures.
Zebrafish husbandry, transgenes and transgenic lines
Zebrafish were maintained using established temperature (28.5°C) and light cycle conditions (14 hours on, 10 hours off). Embryos and larvae were cultured in standard growth media supplemented with paramecia and Sera micron flake (Sera; Heinsberg, Germany) starting at 5 dpf. Previously described transgenic zebrafish strains used in this study included Tg(elavl3:EGFP)knu3  and Tg(14xUAS-E1b:nfsB-mCherry)c264 .
Transgenes used to establish new transgenic lines during the course of these studies are diagrammed in Additional file 1. New transgenic lines include four different types of enhancer trap driver lines (two sets, ±NRSE) expressing an optimized Gal4-VP16 fusion, termed KalTA4 ; two motor neuron targeted (i.e., zCREST1 enhancer ) lines based on two multicistronic self-reporting Gal4/UAS plasmids; and two 5xUAS-based YFP reporter lines based on two different plasmids (±NRSE sequences). Two control transgenes that did not contain NRSE sites were used to establish enhancer trap lines, designated CK (Et(cfos:KalTA4)) and CK-5xN-14xY (Et(cfos:KalTA4, 5xUAS-E1b:nfsB, 14xUAS-E1b:tagYFP)). Two NRSE-containing transgenes were used to create new NRSE-delimited enhancer trap lines, designated NRCK (Et(2xNRSE-cfos:KalTA4)) and NRCK-5xMY (Et(2xNRSE-cfos:KalTA4,loxP- 5xUAS-E1b:gap43-YFP-loxP)). CREST1 enhancer containing transgenes were used to make lines designated C1CK-5xY2N (Tg(CREST1- cfos:KalTA4,5xUAS-E1b:gap43-tagYFP-2A-nfsB)) and NR C1CK-5xY2N (Tg(2xNRSE-CREST1- cfos:KalTA4,5xUAS-E1b:gap43-tagYFP-2A-nfsB)). In the CREST1 lines, a 'self-cleaving' viral peptide sequence, derived from porcine teschovirus-1 (P2A ), was used to promote equimolar expression of a bicistronic message  consisting of a YFP reporter and nitroreductase [51, 88] (i.e., YFP-2A-nfsB). A non-NRSE 5xUAS-based reporter transgene was used to make a reporter line designated 5xMY-HMY (Tg(loxP-5xUAS-E1b:gap43-YFP-loxP, he1a:gap43-YFP)gmc830). In addition, a NRSE-containing 5xUAS-based reporter transgene was used to make a reporter line designated NR5xMY-HMY (Tg(loxP-2xNRSE-5xUAS-E1b:gap43-YFP-loxP, he1a:gap43-YFP)gmc835). All transgenes were assembled in the miniTol2 background to facilitate transgenesis efficiency . A set of core cloning vectors were synthesized (GenScript; Piscataway, New Jersey, USA, or BioMatik; Wilmington, Delaware, USA) from which all transgenes were constructed. This was done to optimize codon usage (i.e., 'zebrafish-ize' codons), eliminate GC content where possible, incorporate elements promoting improved transgene stability, and provide unique cloning sites allowing functional subunits to be easily exchanged. UAS reporter transgenes generated by our laboratory were flanked by an AT-rich 'barrier' insulator sequence thought to separate methylated and unmethylated domains near CpG islands . This was done in an effort to circumvent variegated transgene expression resultant to methylation-induced silencing of UAS-based transgenic lines in zebrafish, specifically demonstrated to effect expression of the Tg(14xUAS:nfsb-mCherry)c264 line [48, 49]. Most coding sequences were followed by a rabbit β-globin intron sequence shown to improve viral and transgene expression efficiency  that was previously evaluated for the ability to promote mRNA stability and nuclear export of KalTA4 transgenes . Finally, a transgene 'tracer' strategy was used to facilitate identification of UAS reporter transgene carriers in the absence of Gal4-VP16 expression. A 365 bp promoter of the zebrafish hatching enzyme 1a (he1a), which contained three regions highly conserved between he1a, he1b and he2, was used to drive expression of membrane-tagged YFP (i.e., he1a:gap43-YFP) in the hatching gland, a set of cells located within the yolk sac that are resorbed after hatching. This allows UAS reporter carriers to be identified by a 'temporary tracer' that fades by 4 dpf, thus does not impinge on late larval imaging experiments (see Additional file 5), unlike similar strategies using heart- and lens-specific promoters that are expressed into adulthood. Complete transgene sequences and cloning details are available upon request. Stable lines were established in the roy orbison (roy) pigmentation mutant background with Tol2-based transgenesis methods.
RNA isolation and real-time qRT-PCR analysis
Ten wild-type embryos were collected at the indicated developmental stages and total RNA was isolated using TRIzol (Life Technologies; Grand Island, New York, USA) according to the manufacturer's protocol and treated with DNase (Promega; Madison, Wisconsin, USA) to remove genomic DNA contamination. The first-strand cDNA synthesis was performed using the SuperScript II First-Strand System (Life Technologies; Grand Island, New York, USA). qRT-PCR reactions were carried out as described previously [90, 91]. In brief, cDNA amplification was performed in triplicate using the Bio-Rad iQ SYBR Green Supermix (Bio-Rad; Hercules, California, USA) on a Bio-Rad iCycler (Bio-Rad; Hercules, California, USA). Gene expression levels were normalized to β-actin by 2-ΔΔCT methods. The primers used in this study were as follows: β-actin: forward 5'- CGAGCAGGAGATGGGAACC - 3'; reverse 5'- CAACGGAAACGCTCATTGC - 3'; REST: forward 5'- GAGAGCGCAGAGAGCAACTC - 3'; reverse 5'- GCGCAGATGGTGCACTTGAA - 3'.
Disruption of NRSF/REST expression
To knock down REST production, a previously characterized splice inhibiting morpholino targeted to the intron-exon boundary of zebrafish rest exon 3 (5'-GGCCTTTCACCTGTAAAATACAGAA-3') was used. The control morpholino was the standard provided by GeneTools (5'-CCTCTTACCTCAGTTACAATTTATA-3'; Philomath, Oregon, USA). Morpholinos were diluted to 0.1, 0.25, 0.5, 1 or 2 μM and a 1- to 2-nL volume injected into eggs as previously described . Images from a 0.1-μM injection are shown in Figure 6. Zinc finger nuclease targeting of the rest locus was as previously described .
All single time point and time lapse confocal imaging of transgenic zebrafish larvae was performed as previously described .
Statistical comparisons were performed using an independent sample t-test to compare across treatment conditions, or a repeated measures t-test for time series data; i.e., when data from individuals were compared across time. Where symbols are present in figures, P-values were minimally ≤ 0.05.
a porcine 2A viral peptide sequence
a tandem repeat of a 21 bp consensus NRSE site
Tg(loxP-5xUAS-E1bgap43-YFP-loxP: he1a:gap43-YFP) :transgene or transgenic reporter line (e.g.: allele number gmc930)
transgene sequence composed of five serial repeats of UAS binding sites
14xUAS-E1bnfsB-mCherry)c264 :transgenic line
Tg(CREST1- cfosKalTA4:5xUAS-E1b:gap43-tagYFP-2A-nfsB) :transgene or transgenic line (allele number lmc002)
minimal promoter element from mouse cFos gene
Tg(cfosKalTA4) :transgene or transgenic lines (allele numbers gmc675-gmc699)
Et(2xNRSE-cfosKalTA4: 5xUAS-E1b:nfsB: 14xUAS-E1b:tagYFP:) transgene or enhancer trap transgenic lines (allele numbers gmc700-gmc724)
highly conserved enhancer element from the zebrafish Islet-1 gene
basal promoter from carp beta-actin
yeast transcription activator protein
a bipartite transgene expression amplification system
fusion protein linking the DNA binding domain of Gal4 and transcriptional activation domain of VP16
Gal4-VP16 fusion variant optimized for expression in zebrafish
loxP recombination site
membrane-tagged yellow fluorescent protein
bacterial gene encoding nitroreductase B
Tg(2xNRSE-loxP-5xUAS-E1bgap43-YFP-loxP: he1a:gap43-YFP) :transgene or transgenic reporter line (e.g.: allele number gmc932)
Tg(2xNRSE-CREST1- cfosKalTA4:5xUAS-E1b:gap43-tagYFP-2A-nfsB) :transgene or transgenic line (e.g.: allele number lmc003)
Et(2xNRSE-cfosKalTA4) :transgene or enhancer trap transgenic lines (allele numbers gmc600-gmc674)
Et(2xNRSE-cfosKalTA4: 5xUAS-E1b:gap43-YFP) :transgene or enhancer trap transgenic lines (allele numbers gmc725-gmc774)
neuron-restrictive silencer element
neuron-restrictive silencing factor
quantitative reverse transcriptase polymerase chain reaction
restriction element 1
RE1-silencing transcription factor
stathmin-like 2 gene (aka SCG10)
a member of the hAT family of transposons
upstream activating sequence
viral protein 16, a strong transcriptional activator
yellow fluorescent protein.
The authors wish to thank Drs Bruce Appel and Michael Parsons for providing transgenic lines and Dr Koichi Kawakami for providing Tol2 transposase reagents. Thanks to the Mumm and Luminomics laboratories for helpful discussions during the course of this work. We also owe a huge debt of gratitude to Olga Lositsky for agreeing to share web design codes she developed with MD. Funding was provided by the following: R21 MH083614 (NIMH) and R43 HD047089 (NICHD) to JSM; R44 HD047089 (NICHD) to MTS, NYSTEM C026414 to HIS, funds from Helmholtz Zentrum München to RWK, and by fellowships to MD (Studienstiftung des deutschen Volkes and the German Academic Exchange Service (DAAD)).
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