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Dual Recombining-out System for Spatiotemporal Gene Expression in C. elegans

Summary Specific recording, labeling, and spatiotemporal manipulating neurons are essential for neuroscience research. In this study, we developed a tripartite spatiotemporal gene induction system in C. elegans, which is based on the knockout of two transcriptional terminators (stops in short) by two different recombinases FLP and CRE. The recombinase sites (loxP and FRT) flanked stops after a ubiquitous promoter terminate transcription of target genes. FLP and CRE, induced by two promoters of overlapping expression, remove the stops (subsequent FLP/CRE-out). The system provides an "AND" gate strategy for specific gene expression in single types of cell(s). Combined with an inducible promoter or element, the system can control the spatiotemporal expression of genes in defined cell types, especially in cells or tissues lacking a specific promoter. This tripartite FLP/CRE-out gene expression system is a simple, labor- and cost-saving toolbox for cell type-specific and inducible gene expression in C. elegans.


INTRODUCTION
Genetic methods are widely used to study functions and neurosignal integration of neural circuits in the nervous system (Dymecki and Kim, 2007;Luo et al., 2008Luo et al., , 2018. Spatiotemporally specific (i.e., cell typespecific and inducible) expression of genes is required to identify neuron type, measure the activity of genetically defined neurons, analyze function(s) of the tested gene(s) in neurons, construct neuronal circuit diagrams, and dissect the process of signal integration in neuronal circuits. The genes expressed include the tested genes of living organisms and those encoding indicators, markers, and proteins used in optogenetic and chemogenetic manipulation of neurons. Owing to the conservation of genes in the animal genera, experimentally tractable animal models are generally used to study the relationships among genes, proteins, and neural circuits that underlie behaviors. Caenorhabditis elegans (C. elegans) is a prevalent animal model for studying conserved molecular and neural bases of the nervous system. The limited specific promoters of single/paired neuron(s) hinder the pace and veracity of neuroscience studies, not only in C. elegans but also in other species (Dymecki and Kim, 2007;Luo et al., 2008Luo et al., , 2018. For spatial and/or temporal gene expression in C. elegans, several strategies have been developed. Bipartite interaction systems of spatial gene expression provide cell type-specific gene expression, which employ two promoters of intersectional expression patterns to drive two interactional parts of a molecule (Chelur and Chalfie, 2007;Zhang et al., 2004). A recently developed strategy of native and tissue-specific fluorescence (NATF) combined with genome editing and split-GFP allows cell-specific fluorescence labeling of native proteins in C. elegans (He et al., 2019). Another method used cell-specific rescue of heat shock factor-1 (hsf-1) mutants to regulate spatial and temporal transgene expression in C. elegans (Bacaj and Shaham, 2007).
The bipartite FLP-out or CRE-out systems induce temporal or spatiotemporal gene expression in C. elegans. It is based on the knockout of a transcriptional terminator (stop) by recombinase FLP (flipase) or CRE (cyclization recombination) derived from the yeast Saccharomyces cerevisiae and bacteriophage P1, respectively (Buchholz et al., 1998;Dymecki, 1996;Golic and Lindquist, 1989;Hoess et al., 1984;Raymond and Soriano, 2007;Sauer and Henderson, 1988). FLP-out and CRE-out are the processes of recombinase-catalyzed intramolecular excision of spacer DNA that lies between tandemly oriented FRT (FLP

Construction and Work Principle of the Tripartite FLP/CRE-out Gene Expression System
Recombining-out transcriptional terminator with recombinase enables spatial, temporal, or spatiotemporal gene expressions (Davis et al., 2008;Guo et al., 2015;Hubbard, 2014;Lopez-Cruz et al., 2019;Macosko et al., 2009;Schmitt et al., 2012;Voutev and Hubbard, 2008;Wang et al., 2017). However, these bipartite systems have challenges providing spatial and temporal gene expression in the neuron(s) or tissue lacking a specific promoter. Therefore, to induce spatiotemporal gene expression in C. elegans, we developed a tripartite gene induction system ( Figure 1A). The system is composed of a core vector for the expression of a tested gene and two recombinase-encoding vectors. The core vector contains eft-3 (strong pan-expression) or hsp-16.48 promoter (heat shock inducible), two different tandem recombinase-sitesflanked transcriptional terminators (stops), and a gene of interest. For an easy change of promoters and genes, we used the Three-Fragment Multisite Gateway method (Magnani et al., 2006) to construct these three vectors (Figures S1A-S1C), using the Invitrogen system from Thermo Fisher Scientific (Thermo Fisher Scientific, MA, USA). To construct the core vector, we used the following sequences: eft-3 or hsp-16.48 promoter with loxP-flanked stop1 and FRT-flanked stop2 as slot1 (promoter::d-stops, in short); the gene of interest as slot2; and unc-54 3 0 UTR as slot3. The loxP and FRT are recombination sites of CRE and FLP. The transcriptional terminators stop1 and stop2, which block the transcription of the target gene, consist of unc-54 3 0 UTR and let-858 3 0 UTR. Recombining-out two stops by recombinase CRE and FLP driven by two distinct promoters with overlapping expression in a type of cell(s), enabled gene transcription in the defined cell(s). CRE and FLP used in the FLP/CRE-out system derived from a nucleolus-transport-enhanced Cre (nCre) of yeast S. cerevisiae (Hoess et al., 1984;Sauer and Henderson, 1988) and mouse codon-optimized FLP (FLPo) (Raymond and Soriano, 2007). We made further modifications to improve their expression and referred to as CRE and FLP. This system provides an "AND" gate strategy for intersectional targeting of a single cell type, i.e., controls gene expression in a type of cell(s) that lacks a specific promoter ( Figure 1A).

The FLP/CRE-out Gene Expression System Is Efficient to Label and Identify a Single Type of Neurons
Spatiotemporally manipulating and recording of neuronal activity is vital for studies of neuronal circuits and circuital neurosignal integration. The FLP/CRE-out system should be a convenient tool for the studies. Neuron-type-specific promoters or promoter pairs are essential for the FLP/CRE-out system. In C. elegans, many neurons, especially sensory neurons, have been identified to possess specific promoters or promoter pairs. However, numerous neurons, especially interneurons, still lack known specific promoters or promoter pairs. Identifying promoter pairs for these neurons will facilitate research in these cells. For easy identification of promoter pairs, we constructed a chromosomally integrated C. elegans transgene ZXW617 strain using a standard UV/trimethylpsoralen (UV/TMP) integration method (Yandell et al., 1994). Its genotype is hkdIs617[eft-3p::d-stops::TagRFP-T::unc-54 3 0 UTR::lin-44p::GFP]. Co-injection of ll OPEN ACCESS 2 iScience 23, 101567, October 23, 2020 iScience Article FLP and CRE plasmids with a promoter pair of a single neuron-type intersectional expression into the transgenic worms should enable single neuron-specific fluorescent labeling. As expected, the hkdIs617 [sra-6p::CRE::sl2::TagBFP; srb-6p::FLP::sl2::GFP] transgene displayed ASHs-specific TagRFP-T labeling ( Figure 1B). The expression patterns of sra-6p and srb-6p were ASHs/ASIs and ASHs/ADLs with an intersection of ASH neurons. To identify whether each of the two stops is sufficient to prevent the expression of the downstream reporter gene, the strains of hkdEx2000[sra-6p::CRE::sl2::TagBFP]; hkdIs617 and hkdEx2001 [srb-6p::FLP::sl2::GFP]; hkdIs617 were generated, respectively. As shown in Figures S2A and S2B, no red fluorescent protein was detected; however, blue and green channels showed the normal expression of CRE and FLP, respectively. This result illustrates that each of the two stops was able to prevent the expression of the downstream reporter gene.
Next, we used the integrated strain ZXW617 to identify various promoter pairs for intersectional targeting of single-neuron type. We successfully identified eight promoter pairs with a single neuron-type intersection. Shown in Figures 2A-2G and Table S1, the following promoter pairs enabled TagRFP ). An FLP-out approach, which used single-copy transgenic strains that stably express an optimized version of FLP in specific tissues or by heat induction, reached 100% recombination efficiencies in several cell types such as muscles, intestine, and serotonin-producing neurons (Munoz-Jimenez et al., 2017). Compared with this system, the FLP/CRE-out system offers efficiencies of 100% in muscle cells and 53%-86% in neurons. The differential efficiencies in  iScience Article neurons may be caused by the varied expression of the recombinases. The ZXW617 strain may be an array of TagRFP-T. In the array, both local excision and excision between different integrated plasmids should be possible. Thus, the array could be reduced to just a single plasmid over time, since every recombination event leaves behind a LoxP or FRT site that can recombine again. After some observations, we observed no obvious variability of TagRFP-T fluorescence in different worms, no gradual dimming of the fluorescence over time, and even brighter fluorescence in older animals.
Since the core plasmid used a strong pan-expression eft-3 promoter to drive target gene expression, the FLP/CRE-out system should be able to control specific gene expression in cells/tissues other than neurons. We thus set out to induce TagRFP-T expression in pharyngeal muscle and body wall musculature. As shown in Figures 2H and 2I, two types of muscle cells showed specific TagRFP-T labeling. In summary, a tripartite cell-specific gene expression system based on recombining-out two different stops was developed. We also have identified eight promoter pairs with intersectional targeting of a single-type neuron(s).
One primary goal of our study is to develop a toolbox for spatiotemporal manipulation of neurons. TeTx, a specific synaptobrevin protease that blocks vesicle fusion with the plasma membrane and thus iScience Article neurotransmitter release (Schiavo et al., 1992), has been successfully used to inhibit chemical synaptic transmission of tested neurons in C. elegans (Guo et al., 2015;Liu et al., 2019;Macosko et al., 2009;Wang et al., 2016). Continual inhibition of neurotransmission by TeTx, beginning from embryonic periods, may interfere with the development of the nervous system. Therefore, the inducible expression of the toxin might be an appropriate solution.

DISCUSSION
In the present study, we develop a tripartite FLP/CRE-out gene induction method/system for spatiotemporal control of transgene expression in single/single type of neuron(s) and other cells or tissues in C. elegans. In addition, the system provides an "AND" gate strategy for the spatial expression of a gene. The advantages of the FLP/CRE-out method are as follows.
(1) Ability to spatiotemporally control gene expression in the neuron(s) or other cells that lack a specific promoter. Especially this system is useful for optogenetic and chemogenetic manipulation of single types of neuron(s).
(2) Simple and cost saving. Changes of promoter pairs, which direct CRE and FLP expression, are needed only for expressing a target gene in varied types of neurons and other cells. Meanwhile, the change of a target gene in the core vector is needed to control the spatiotemporal expression of different genes in defined cell types. Replacing promoters and genes in the FLP/CRE-out system is easy by the use of the MultiSite gateway method. All plasmids and worms used in this study are available to the scientific community. The integrated worm strain ZXW617 of genotype hkdIs617[eft-3p::d-stops:: TagRFP-T::unc-54 3 0 UTR::lin-44p::GFP] is a useful tool to identify new promoter pairs for spatial control of gene expression in a single type of neuron(s). Using this worm strain, we identified eight promoter pairs for controlling spatial gene expression in the neuron(s) lacking a known specific promoter or promoter pair. Our work lays an important foundation for studies in these neurons. Importantly, 6,000 promoters in the C. elegans promoterome (Dupuy et al., 2004) are available for constructing recombinase vectors by the three fragment gateway method. Moreover, single-copy transgenic strains that stably express an optimized version of FLP in specific tissues or by heat induction (Munoz-Jimenez et al., 2017) are useful for the FLP/CRE-out system.
(3) The strong pan-expression eft-3p and hsp-16.48p promoters in the core gene expression plasmid of the FLP/CRE-out system lay a foundation for the use of the system in cells and tissues other than neurons in C. elegans. (4) In some bipartite systems, the effector transgene is placed downstream of a tissue-specific promoter; therefore, it can only be expressed as long as the promoter is active. Compared with these bipartite induction systems, the use of eft-3 promoter in the FLP/CRE-out system offers the possibility of prolonged and continuous expression of the effector transgene.
Multiple methods have been developed for controlling spatial or spatiotemporal gene expression in C. elegans. Bipartite systems, such as FLP-out and CRE-out systems, are not able to induce spatial and temporal gene expression in a single type of neuron(s) and other cells lacking a specific promoter ( . To temporally and spatially control gene expression and protein activity/localization are essential in modern biology and even modern medicine. Given the advantages of the FLP/CRE-out-mediated gene control system, this system expands the genetic toolbox for C. elegans.

Limitations of the Study
All of the transgenic strains except ZXW617 were not integrated. The ZXW617 strain was generated by a standard ultraviolet/trimethylpsoralen (UV/TMP) integration method, not by single-copy insertion. The strain may be an array of TagRFP-T.

Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Zheng-Xing Wu (ibbwuzx@mail.hust.edu.cn).

Materials Availability
All data generated or analyzed in this study are included in this published article (and its Supplemental Information files). The strains and plasmids will be deposited at CGC (https://cgc.umn.edu/) and Addgene (https://www.addgene.org/), respectively.

Data and Code Availability
The published article includes all data generated or analyzed during this study.

METHODS
All methods can be found in the accompanying Transparent Methods supplemental file.

SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.101567. The system works on the base of recombining-out two transcriptional terminators by recombinases FLP and CRE, and subsequent FLP/CRE-out.

Animals
The C. elegans worms of various genotypes were cultured on nematode growth medium (NGM) plates at 20 °C using E. coli OP50 bacteria as food according to standard methods (Brenner, 1974), unless otherwise indicated. All transgenic worms were generated by microinjection with standard techniques (Mello et al., 1991). Plasmids were injected at concentrations of 50 ng µl -1 . All strains used in this study are listed in Table S2.

Plasmids construction
We constructed plasmids by L-R recombination reactions (Fig S1A-C) using the Multisite Gateway system (Invitrogen™, Thermo Fisher Scientific, MA, USA). Three entry clones, entry clones A, B and C, and a destination vector, were used. We utilized BP recombination reactions to construct the B and C entry clones. The destination vectors used in this study were modified from the pBCN44-R4R3 vector. The primers for PCR products used in the plasmid construction are listed in Table S3.
The destination vector used to construct CRE and FLP plasmids was modified from the pDEST-R4-R3 vector. An unc-54 3' UTR was added into the vector between attR3 and AmpR. In short, as shown in Figure S4B, the pDEST-R4-R3 vector was linearized by the PCR-linearized method. Then, an unc-54 3' UTR was added to the location by the In-fusion method (ClonExpress ® Ⅱ One Step Cloning Kit, Vazyme company, Nanjing, Jiangsu Province, China). The unc-54 3' UTR sequence was amplified from C. elegans DNA genome by PCR.
Construction of entry clones. Entry clones B and C used in the MultiSite Gateway system were constructed by BP reactions. While the most entry "A" clones were constructed by other methods ( Figure S3), except some promoter clones in the C. elegans Promoters Library (Thermo Fisher Scientific, Waltham, MA, USA) were directly used.
Entry clone A. The "A" entry clones contribute to a fragment of slot1 in the expression plasmids.
Three types of "A" entry clones were used to construct different expression plasmids. The vector A used in the construction of the recombinase-encoding plasmid contains a promoter sequence. Most parts of "A" entry clones in this study were constructed by the In-Fusion method ( Figure S3A), using ClonExpress ® Ⅱ One Step Cloning Kit and primers listed in Table S3. Alternatively, some entry clones in the C. elegans Promoters Library were directly utilized. The "A" entry clones used to construct expression plasmids containing sequences of a promoter, two recombinase sites-flanked and ER exportation, ttctgctacgagaacgaggtg. CRE and FLP fragments were amplified by PCR with primers containing attB1, attB2, Kozak, NLS, linker, and ER export: CRE; from pDD104 plasmid (from Addgene); FLP from pPGKFLPpA plasmid (from Addgene). TeTx sequence was amplified from pWD170 plasmid (by courtesy of Dr. Erik M. Jorgensen) by PCR with the primers containing attB1 and attB2 recombination sites. TagRFP-T fragment was amplified by PCR with primers containing attB1 and attB2 recombination sites.
Entry clone C. An entry clone C contributing to sequences of slot 3 in the expression plasmid contains a fragment of unc-54 3' UTR, sl2::GFP or sl2::TagBFP. The corresponding PCR products with attB2R and attB3 sites were amplified by the PCR method. A pDONR-P2R-P3 donor vector was used to construct "C" entry clones by BP reactions ( Figure S3D). The "C" entry clones containing unc-54 3' UTR, sl2::GFP and sl2::TagBFP were used to construct expression plasmids containing a tested gene, CRE, and FLP, respectively.

Confocal fluorescence imaging
All confocal fluorescence imaging were performed by an Andor Revolution XD laser confocal microscope system (Andor Technology plc., Springvale Business Park, Belfast, UK) based on a spinning-disk confocal scanning head CSU-X1 (Yokogawa Electric Corporation, Musashino-shi, Tokyo, Japan). The confocal system was mounted on an Olympus IX-71 inverted microscope (Olympus, Tokyo, Japan) and controlled by Andor IQ 3.2 software. All fluorescent images were imaged by a ×60 objective lens (numerical aperture = 1.45, Olympus) and captured by an Andor iXon EM + DU-897D EMCCD camera.

Calcium imaging
The calcium responses in neuronal soma were measured by detecting changes in the fluorescence intensity of R-GECO1, a sensitive and rapid kinetic calcium indicator of weak photobleaching (Zhao et al., 2011). A home-made microfluidic device was used for calcium imaging as previously described (Chronis et al., 2007;Guo et al., 2015;Liu et al., 2019;Wang et al., 2016). Briefly, a young adult worm was transferred from food to M13 buffer solution (in mM, Tris-HCl 30, NaCl 100 and KCl 10, PH 7.0) to wash bacteria from the body. Then, the animal was loaded into the worm channel of the microfluidic chip with its nose exposed to the buffer or solution of CuSO4 dissolved in M13 buffer (final concentration 10 mM) under laminar flow for each Ca 2+ assay. The solutions were delivered via