Molecular Vision 2024; 30:123-136 <http://www.molvis.org/molvis/v30/123>
Received 16 May 2022 | Accepted 17 March 2024 | Published 19 March 2024

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The generation and characterization of a transgenic zebrafish line with lens-specific Cre expression

Xuyan Peng,1 Xiaolin Jia,1 Guohui Shang,2 Mengjiao Xue,1 Mingjun Jiang,1 Dandan Chen,1 Fengyan Zhang,1 Yanzhong Hu1,3,4

The first two authors contributed equally to this work.

1The Laboratory of Ophthalmology and Vision Science, Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, Zheng Zhou, China; 2Department of Medical Genetics and Cell Biology, School of Basic Medical Sciences, Zhengzhou University, Zheng Zhou, China; 3Joint National Laboratory for Antibody Drug Engineering, Henan University School of Basic Medical Sciences. Kaifeng, China; 4Kaifeng Key Lab for Cataract and Myopia, Institute of Eye Disease, Kaifeng Central Hospital, Kaifeng, China

Correspondence to: Yanzhong Hu, Department of Cell Biology and Genetics, Henan University School of Basic Medical Sciences, Jin-Ming Road, Kaifeng, China, 475014; Phone: 86-18503781944; email: hyz@henu.edu.cn

Abstract

Purpose: Danio rerio zebrafish constitute a popular model for studying lens development and congenital cataracts. However, the specific deletion of a gene with a Cre/LoxP system in the zebrafish lens is unavailable because of the lack of a lens-Cre-transgenic zebrafish. This study aimed to generate a transgenic zebrafish line in which Cre recombinase was specifically expressed in the lens.

Methods: The pTol2 cryaa:Cre-polyA-cryaa:EGFP (enhanced green fluorescent protein) plasmid was constructed and co-injected with Tol2-transposase into one-to-two-cell-stage wild-type (WT) zebrafish embryos. Whole-mount in situ hybridization (ISH), tissue section, hematoxylin and eosin staining, a Western blot, a split-lamp observation, and a grid transmission assay were used to analyze the Cre expression, lens structure, and lens transparency of the transgenic zebrafish.

Results: In this study, we generated a transgenic zebrafish line, zTg(cryaa:Cre-cryaa:EGFP), in which Cre recombinase and EGFP were driven by the lens-specific cryaa promoter. zTg(cryaa:Cre-cryaa:EGFP) began to express Cre and EGFP specifically in the lens at the 22 hpf stage, and this ectopic Cre could efficiently and specifically delete the red fluorescent protein (RFP) signal from the lens when zTg(cryaa:Cre-cryaa:EGFP) embryos were injected with the loxP-flanked RFP plasmid. The overexpression of Cre and EGFP did not impair zebrafish development or lens transparency. Accordingly, this zTg(cryaa:Cre-cryaa:EGFP) zebrafish line is a useful tool for gene editing, specifically with zebrafish lenses.

Conclusions: We established a zTg(cryaa:Cre-cryaa:EGFP) zebrafish line that can specifically express an active Cre recombinase in lens tissues. This transgenic zebrafish line can be used as a tool to specifically manipulate a gene in zebrafish lenses.

Introduction

The ocular lens is an avascular sensory organ without any innervation, and it is responsible for transmitting and focusing light on the retina. It comprises an anterior epithelium and abundant fibers that are derived from the ectoderm visual epithelium [1]. Genetic variation, which affects the development and transparency of the crystalline lens, is a common pathological cause of congenital cataracts [2,3]. Approximately 22.3% of congenital cataracts are caused by genetic variation [4]. Moreover, there are nearly 200 loci and more than 100 genes whose mutations are associated with hereditary congenital cataracts [5]. Some of them have been suggested to be cataractous genes in mouse or zebrafish models using gene editing technology, such as Pax6a [6], Foxe3 [7], Hsf4 [8], and αA-crystallin [9,10]. Cre-mediated conditional knockout technology is a popular tool used to delete gene expression from a specific tissue in mouse models, and this tool has been used to manipulate and determine gene function in lens development and among patients with congenital cataracts [11]. However, the deletion of a specific gene in the zebrafish lens could not be achieved due to the lack of lens-specific Cre-expressing transgenic zebrafish.

Over 70% of annotated human genes have at least one obvious zebrafish ortholog [12]. Therefore, zebrafish became a time- and cost-efficient model to study human disease at the end of the last century because of their multiple advantages compared to established vertebrate genetic models, such as mice models [13]. Many human-disease-associated genes, including cataracts, are expressed and modeled in zebrafish [14]. For example, the conventional deletion of pax6 in zebrafish impairs the development of the brain, retina, and lens, which clinically imitates PAX6-mutant-associated Aniridia diseases [15]. Conventional gene knockout via CRISPR-Cas9 or TALENs and gain-of-function via creating transgenic lines are widely used to manipulate genes in zebrafish lenses [8,16]. The limitation of conventional gene knockout is that some gene mutations lead to embryonic lethality or affect multiple organs, affecting the definition of a gene’s precise function during lens development.

As in the human lens, α-crystallin is the dominant crystallin in zebrafish lenses. The α-crystallin content of the zebrafish lens has been reported with a proportion of 22% in the total lens protein [17]. α-crystallin contains two isoforms, αA-crystallin and αB-crystallin [18]. They act as chaperone proteins to regulate crystallin folding in the lens and protect lens proteostasis from divergent stresses [19]. Their chaperone activity and stability in zebrafish have been examined and compared with those in mammals [20]. Missense mutations in both the cryaa and cryab genes cause congenital cataracts and other diseases [18]. αA-crystallin is predominantly expressed in lens tissues, while αB-crystallin is expressed in other tissues (e.g., the brain, muscle, lung, liver, and heart) in addition to the lens. Therefore, the promoter of cryaa has been used to specifically initiate the expression of extrinsic proteins in lenses within mouse models [21]. Transgenic mice with αA-crystallin-promoter-driven Cre can specifically express Cre recombinase in the lens, and these Cre-Tg mice have been widely used to conditionally delete genes from the mouse lens [11]. Zebrafish have one cryaa gene and two duplicated cryaba and cryabb genes [22]. The expression of cryaa occurs uniquely in the lens for both larval [23] and adult zebrafish [24]. By contrast, the cryaba and cryabb genes are almost entirely expressed in non-lens tissues at the larval stage. During this stage, cryaba is expressed predominantly in non-lens tissues; for example, cryaba’s mRNA is mainly detected in the tissues of the lateral line, the hindbrain, muscle progenitors, and olfactory system. Conversely, cryabb’s mRNA is detected in hair cells and otic capsule cells, according to single-cell transcriptomic analysis [23]. As zebrafish mature, their expression of cryaba is restricted to the lens [25], while their cryabb is expressed in multiple tissues, including the lens, muscles, and the brain [22]. Additionally, cataract-linked mutants of αA-crystallin and αB-crystallin have caused cataracts in zebrafish [26]. Transgenic zebrafish with cryaa-promoter-driving green fluorescent protein (GFP) can specifically express GFP in their lenses [27]. These results suggest that the cryaa promoter is an ideal driver of extrinsic protein expression in zebrafish lenses.

The Cre-loxP recombination system is a powerful genetic tool for conditionally achieving gene deletion, and this system has been widely used in mice [28]. In this system, two recombinant mice are needed: loxP-carrying mice, in which loxP sequences are introduced to a targeting gene, and tissue-specific Cre-expressing transgenic mice. Cre recombinase recognizes and cleaves the loxP-flanked sequences to delete the target gene [29]. Tg:cryaa-Cre mice have been successfully used to conditionally delete the gene of Rac1 from the mouse lens [30]. In zebrafish, Cre-mediated gene deletion has been successfully used to inactivate the supv3l1 and shha genes in hepatocytes [31] and the epicardium [32], respectively. This use suggests that the Cre-loxP system could efficiently work in zebrafish. Furthermore, loxP-flanked transgenic zebrafish have been successfully created via homologous recombination-mediated gene editing [33] or the UFlip alleles generated by the CRISPR-Cas9 targeted integration strategy, which provide robust conditional inactivation and rescue [34]. Several Cre-Tg-zebrafish lines have been generated using Tol2-mediated transgenesis [35]. However, transgenic zebrafish with lens-specific Cre expression are still unavailable.

In this study, we generated the transgenic zebrafish line zTg(cryaa:Cre-cryaa:EGFP), in which Cre and EGFP cDNA were subcloned downstream of the cryaa promoter. The zTg(cryaa:Cre-cryaa:EGFP) zebrafish exclusively expressed Cre and EGFP in the lens but not in their other tissues, and their expression patterns completely recapitulates the endogenous cryaa expression pattern during lens development. This ectopic Cre can specifically and efficiently delete the red fluorescent protein (RFP) signal from the lens when zTg(cryaa:Cre-cryaa:EGFP) embryos are injected with plasmids containing loxP-flanked RFP. These results suggest that zTg(cryaa:Cre-cryaa:EGFP) is a powerful tool for the manipulation of a specific gene in zebrafish lenses.

Methods

Zebrafish maintenance

Danio rerio zebrafish and transgenic zTg(cryaa:Cre-cryaa:EGFP) zebrafish were maintained at 26–28.5 °C in a circulating water system with a light–dark cycle of 14:10 h according to the zebrafish protocol [36]. The fish were fed three times daily with newly hatched brine shrimp. Embryos were obtained via natural spawning and kept in egg water at 28.5 °C. The developmental stages were determined using days post-fertilization (dpf) or months post-fertilization (mpf) [37]. The use of all the animals in this project followed the guidelines on the use of animals by the Association for Research in Vision and Ophthalmology (Baltimore, MD). The procedures used in our study were approved by the ethics committee of The First Affiliated Hospital at Zhengzhou University, Zheng Zhou, China.

Plasmids

pTol cryaa:Cre-polyA-cryaa:EGFP plasmid:-- A 1,028 bp DNA fragment of the cryaa promoter (−1 to −1,028 bp) upstream of the ATG (the protein translation initiation site) site was amplified via PCR using the wild type (WT) zebrafish genome as a template [27]. The fragment of the cryaa promoter was subcloned into the pGEMT-easy vector (A1360, Promega) and used as a template. Fragments of the cryaa promoter, Cre recombinase cDNA, and SV40-ployA were amplified via primers, respectively, and then subcloned into the PstI site of the pTol2-MCS vector (CZP10, China Zebrafish Resource Center) via homologous recombination using the ClonExpress MultiS One Step Cloning Kit (C113–02, Vazyme Biotech Co. Ltd., Nanjing, China), generating pTol cryaa:Cre-polyA plasmids. To generate the pTol cryaa:Cre-polyA-cryaa:EGFP plasmid, a fragment of the cryaa promoter, EGFP/cDNA, and SV40 ploy A was amplified with the primers and subcloned into the XmaI site of pTol cryaa:Cre-polyA via homologous recombination.

The plasmids pcryaa:RFP-cmlc:EGFP and pcryaa:loxP-RFP-loxP-cmlc:EGFP:-- DNA fragments containing the cryaa promoter, RFP cDNA, and the cmlc promoter (specifically controlling the expression of the cardiac myosin light chain (CMLC) protein in the heart tissue)-EGFP cDNA, or containing the cryaa promoter- loxP -RFP cDNA- loxP- cmlc promoter-EGFP cDNA, were synthesized in vitro and subcloned into the pUC57 vector (Dongxuan Jiyin Jiangsu Technology Co., Ltd., Jiangsu, China). The primer sequences are listed in Table 1. All recombinant plasmids were verified via DNA sequencing.

Whole-mount in situ hybridization and cryosectioning

For the whole-mount in situ hybridization of embryos older than 24 hpf, at 12 hpf, the embryos were incubated in egg water containing 0.003% 1-phenyl-2-thiourea (PTU, Sigma) to prevent pigmentation. To make sense and antisense probes for cryaa, egfp, and cre, the cDNA fragments of cryaa, egfp, and cre were amplified using the primers with T7-RNA-polymerase-recognizing sequences. The sense and antisense probes for cryaa, egfp, and cre were transcribed in vitro using T7 RNA polymerases, and they were labeled with digoxigenin-UTP, following the protocol provided with the TranscriptAid T7 High Yield Transcription kit (catalog no. K0441, Thermo Scientific), with the modification of the replacement of the nucleoside triphosphates (NTPs) mix with nucleic acid labeling (catalog no. 11277073910, Roche, offered by Sigma-Aldrich, Darmstadt, Germany). Whole-mount in situ hybridization was performed following previous reports [38]. The sense probes were used as negative controls. The signals were tested with the anti-digoxigenin antibody conjugated to alkaline phosphatase (Anti-Digoxigenin-AP Fab fragments, catalog no. 11,093,274,910, Roche). Then, they were developed in the NBT/BCIP solution (catalog no. 11,681,451,001, Roche) and photographed under a DM4/Leica light microscope (M205 FA, Leica, Germany). To test the exact distribution of ISH signaling in the lens, after ISH, the embryos were cryosectioned at an 8 µm thickness, and the ISH signals were photographed under a Leica DM4 light microscope. The primer sequences are listed in Table 1.

Paraffin section and H&E staining

The zebrafish were anesthetized and fixed in 4% paraformaldehyde for 24 h at 4 °C and then washed in 50% ethanol. The fixed fish were dehydrated and embedded in paraffin, sectioned (to a thickness of 10 µm), and stained with hematoxylin and eosin.

RNA isolation and quantitative real-time PCR

The total RNA was isolated from the embryos at various developmental stages using the TRIzol Reagent (Life Technology, USA), following the manufacturer’s instructions for the kit. Fifty WT embryos at each stage (0, 3, 6, 12, 24, 36, 48, 60, 72, and 96 hpf) were collected and pooled together for RNA extraction. An equal amount of RNA was retrotranscripted to the first strand of cDNA using the First Strand cDNA Synthesis Kit (AE341, TransGen Biotech, Beijing, China). Quantitative real-time PCR was performed using the TransStart Green qPCR SuperMix (AQ101, TransGen Biotech, Beijing, China) on the QuantStudio 3 Real-Time PCR System (Applied Biosystems by Thermo Fisher Scientific). According to the manufacturer’s instruction, the following program was engaged: 30 s at 94 °C (initial denaturation), 5 s at 94 °C, 15 s at 60 °C, 10 s at 72 °C, and 40 cycles. actb2 was used as the internal reference gene [39]. The experiments were repeated independently three times. The primer sequences are presented in Table 1.

Western blot

Proteins were extracted from the lens, lens-excluded eye tissues, and eyes-excluded body tissues of zTg (transgenic zebrafish) and WT zebrafish at 15 dpf using protein extraction buffer (Beyotime). The antibodies used in this study were anti-Cre recombinase (15036S, Cell Signaling Technology, Danvers, United States) and anti-Gapdh (10,494-1-AP, Proteintech, Wuhan, China) at a dilution of 1:1,000. The extracted proteins were separated using 10% SDS–PAGE (sodium dodecyl sulfate-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in 5% dry milk/phosphate buffer solution and Tween-20 (PBST), the membranes were incubated in a solution with appropriate primary antibodies. Next, they were incubated with a secondary antibody conjugated with horseradish peroxidase. The signal was developed in the ECL buffer and detected using a charge coupled device (CCD)-camera-based imager (Amersham Imager 680, Amersham, CA; GE).

Statistical analysis

The Statistical Product and Service Solutions (SPSS) software 24.0 version was used for data analysis. The unpaired Student’s t-test was used for statistical analysis of the data on the zebrafish’s standard length and eye area, and one-way analysis of variance (ANOVA) was used for the data analysis of quantitative real-time PCR. The results were expressed as means ± standard deviations (SDs), and p < 0.05 was considered statistically significant.

Results

The generation of zTg(cryaa:Cre-cryaa:EGFP) zebrafish

To generate a transgenic zebrafish with Cre overexpressing specifically in the lens, about 1.5 nL of a solution containing the plasmid of pTol cryaa:Cre-polyA-cryaa:EGFP (80 ng/ul) and Tol2-transposase mRNA (80 ng/ul) were microinjected into one-to-two-cell-stage embryos (Figure 1A). Tol2-transposase is responsible for catalyzing the recombination of the DNA fragment of cryaa:cre-polyA-cryaa:EGFP in the plasmid of pTol cryaa:Cre-polyA-cryaa:EGFP into the zebrafish genome. Cryaa is a lens-specific protein [27]. We used the promoter of the cryaa gene to drive Cre and GFP expression in this plasmid. The specific expression of GFP in zebrafish lenses was used as a selective marker for transgenic zebrafish. After injection, the embryonic zebrafish showed GFP-positive signals at 3 dpf under a fluorescence microscope, and these zebrafish were defined as F0 founders (Figure 1B). Adult GFP-positive F0 founders were bred with WT zebrafish to generate heterozygous F1 offspring (Figure 1B). The F1 GFP-positive zebrafish were selected using a fluorescence microscope and considered zTg(cryaa:Cre-cryaa:EGFP) individuals for further use.

zTg(cryaa:Cre-cryaa:EGFP) specifically expresses Cre protein in embryonic lenses at the 22 hpf stage

Before we determined when Cre was expressed in zebrafish lenses, we analyzed cryaa transcripts during the zebrafish’s development in qRT-PCR and in situ hybridization (ISH) assays. The qPCR results indicated that the expression of cryaa mRNA was regulated during zebrafish development. The cryaa mRNA was expressed in the 0 hpf embryos at a relatively high level (with the relative value of 1±0.24), followed by a decrease to the lowest level (with the relative value of 0.07±0.03) at the 12 hpf stage. After this, the cryaa mRNA expression again increased at the 24 hpf stage, and thereafter, the expression level increased gradually with embryo growth (Figure 2A). Accordingly, we postulated that the tested cryaa mRNA in the embryos from 0 to 24 hpf originated from maternity and that this maternal cryaa mRNA decreased with embryo development. The zygotic cryaa started to express its own mRNA in the embryos after 12 hpf. The ISH results indicated that the relative expression level of cryaa mRNA was consistent with the qPCR results (Figure 2B–D). As the results indicated, the maternal cryaa mRNA was expressed in whole embryo tissues during the early embryo stages (3 hpf to 24 hpf; Figure 2B–D); the cryaa mRNA was detected in the eye at 18 hpf and solely expressed in lens tissues at the 48 and 72 hpf embryo stages (Figure 3A). The expression level and pattern of the cryaa mRNA showed no difference between the WT and zTg(cryaa:Cre-cryaa:EGFP) zebrafish, suggesting that the overexpression of Cre or EGFP did not impair cryaa mRNA expression in the lens. Next, we performed an ISH assay to determine the expression of cre and egfp in zTg(cryaa:Cre-cryaa:EGFP). The results showed that zTg(cryaa:Cre-cryaa:EGFP began to express Cre and EGFP was weak in other position of the embryos (such as the trunk part) at 16 hpf (Figure 2C). After this, both Cre and EGFP were entirely expressed in lens tissues in a similar pattern (Figure 2D and Figure 3A). Like cryaa, cre and egfp were expressed in lens cortical fiber tissue (Figure 3B), and this finding was consistent with previous reports that cryaa is expressed almost exclusively in 2 and 5 dpf lens fiber cells [23]. No expression of Cre or EGFP was detectable in the WT zebrafish (Figure 2D and Figure 3A). These results indicated that Cre is predominantly expressed in the lens fibers and that the EGFP signal could represent the expression pattern of Cre during lens development. The EGFP fluorescence analysis results indicated that EGFP was constitutively expressed in the zTg(cryaa:Cre-cryaa:EGFP) lenses at various stages, including the larval stage (20 dpf and 1 mpf) and the adult stage (3 mpf, 6 mpf, 9 mpf, and 12 mpf; Figure 3C). Furthermore, we performed immunoblots to test the expression of Cre protein in zTg(cryaa:Cre-cryaa:EGFP) lenses. The 15 dpf WT and zTg(cryaa:Cre-cryaa:EGFP zebrafish were dissected into lenses, lens-excluded eye tissue, and eyeless zebrafish bodies. The results showed that Cre was expressed only in the lens tissues and not in the other eye tissues or body tissues of the zTg(cryaa:Cre-cryaa:EGFP) individuals (Figure 3D). No expression of Cre protein was detected in WT zebrafish (Figure 3D). Accordingly, we proposed that two sets of cryaa mRNA occurred in early embryo development that originated from maternal transportation or new zygotic synthesis. The zygotic embryo started to express cre at 16 hpf, which implies that the zygotic embryo starts to express its own cryaa at 16 hpf. The expression patterns of cryaa, egfp, and cre at each stage were summarized in Table 2.

The ectopic expression of Cre and EGFP does not affect zebrafish development or lens transparency

To determine whether ectopic Cre and EGFP impair zebrafish development, including lens development, we compared the development of WT zebrafish to zTg(cryaa:Cre-cryaa:EGFP) zebrafish. No differences in standard length from the snout to the origin of the tail fin or the eye area were observed between the WT and zTg(cryaa:Cre-cryaa:EGFP) zebrafish (Figure 4A,B). Histology analysis showed that ectopic Cre and GFP in the zTg(cryaa:Cre-cryaa:EGFP) lenses did not impair the lens structure compared to that of WT lenses (Figure 4C). We further analyzed the transparency of 2 mpf and 9 mpf WT and zTg(cryaa:Cre-cryaa:EGFP) lenses via split-lamp microscopy and grid transmission images. The results revealed no cataract phenotype in the zTg(cryaa:Cre-cryaa:EGFP) zebrafish (Figure 4D), though EGFP signals were observed. The transparency of the zTg(cryaa:Cre-cryaa:EGFP) lens was similar to that of the WT lens (Figure 4D). These results indicate that lens development is not affected in this Cre-transgenic zebrafish.

zTg(cryaa:Cre-cryaa:EGFP) expresses an active Cre recombinase in the lens

To test Cre activity in the zTg(cryaa:Cre-cryaa:EGFP) lens, the plasmids pcryaa:RFP-cmlc:EGFP or pcryaa:Flox-RFP-Flox-cmlc:EGFP were constructed and microinjected into one-cell-stage embryos of zTg(cryaa:Cre-cryaa:EGFP) and WT zebrafish, respectively. Within these two constructs, the cmlc (cardiac myosin light chain) promoter was used to drive EGFP expression specifically in heart tissue, and it was used to indicate the successful administration of pcryaa:RFP-cmlc:EGFP and pcryaa:Flox-RFP-Flox-cmlc:EGFP into the embryos. The cDNAs of RPF and Flox-RFP-Flox were cloned downstream of the cryaa promoter, which specifically expresses fluorescent RFP in lens tissue. To determine Cre activity, 3 dpf embryos with GFP-positive hearts were collected to analyze RFP’s expression in the lens. As the results in Figure 5 indicate, the plasmid pcryaa:RFP-cmlc:EGFP constitutively expressed RPF in the lens of WT zebrafish and zTg(cryaa:Cre-cryaa:EGFP) zebrafish (Figure 5A). In contrast, the pcryaa:Flox-RFP-Flox-cmlc:EGFP plasmid, which expresses RFP and GFP in WT zebrafish lenses and hearts, respectively (Figure 5B’s upper panel) did not express RFP in the zTg(cryaa:Cre/cryaa:EGFP) zebrafish lenses (Figure 5B’s lower panel). These results suggested that Cre exhibited recombinase activity toward Flox-RFP-Flox when pcryaa:Flox-RFP-Flox-cmlc:EGFP was injected into the zTg(cryaa:Cre-cryaa:EGFP) embryos. To determine Cre efficiency, we quantitated the RFP-positive lenses versus the GFP-positive hearts in the zTg(cryaa:Cre-cryaa:EGFP):pcryaa:Flox-RFP-Flox-cmlc:EGFP zebrafish. Two of the 126 heart-EGFP positive zTg(cryaa:Cre-cryaa:EGFP): Flox-RFP-Flox-cmlc:EGFP embryos exhibited lens-RFP-positivity; therefore, 98.4% of Cre-Tg-zebrafish exhibit Cre recombinase activity in the lens (Table 2). These results suggest that this novel transgenic line, zTg(cryaa:Cre-cryaa:EGFP), expresses active Cre in the lens. Therefore, it could be used as a tool to specifically target gene deletion in the zebrafish lens.

Discussion

We successfully generated a transgenic zebrafish, zTg(cryaa:Cre-cryaa:EGFP), which specifically expresses a functional Cre recombinase in the lens. In this Tg-cre zebrafish, the expression of Cre and EGFP is driven by the cryaa promoter, and the expression patterns of EGFP and Cre recombinase faithfully recapitulate the endogenous expression pattern of cryaa in the lens (Figure 2 and Figure 3A and 3B). The expression pattern of EGFP was the same as that of Cre in lens tissues at 16 hpf, 2 dpf, 3 dpf, 1 mpf, 3 mpf, 6 mpf, 9 mpf, and 12 mpf for the zTg(cryaa:Cre-cryaa:EGFP) zebrafish (Figure 2 and Figure 3). Interestingly, the whole-mount ISH results showed that cre and egfp were weakly detected in the whole bodies of Tg-zebrafish at 16 hpf, and with the embryo’s growth, cre and egfp were only visualized in lens tissues. The weak expression of cryaa-promoter-controlled Cre in the tissues outside the lens during early embryo development might have caused tissue specificity problems when it was used to edit gene expression specifically in the lens. To avoid this problem and increase lens specificity, the promoters of cryaa were tested at different lengths in our laboratory. Taken together, our results demonstrate that zTg(cryaa:Cre-cryaa:EGFP) can constitutively express Cre recombinase in lens tissue (Figure 3) and that EGFP is a primary selective marker for transgenic zebrafish.

To evaluate Cre activity in the zTg(cryaa:Cre-cryaa:EGFP) lens, we constructed and administered two plasmids (Figure 5): pcryaa:RFP-cmlc:EGFP and pcryaa:Flox-RFP-Flox-cmlc:EGFP. A total of 98.4% of the zTg(cryaa:Cre-cryaa:EGFP) zebrafish exhibited Cre activity in the lens (Table 3). These results demonstrate that zTg(cryaa:Cre-cryaa:EGFP) expresses an active Cre recombinase in lens tissues. Cre-transgenic mouse lines are a common tool for studying lens development or cataracts by manipulating specific gene expression in the lens. There are transgenic Le-Cre, MLR10, MLR39, Nes-Cre, P0-P3.9GFPCre, and LR-Cre mice [11]. Among them, MLR39 Cre-transgenic mice use the αA-crystallin promoter to drive Cre expression in lens fiber cells. αA-crystallin promoter sequences in zebrafish are conserved with those in mice [27]. The results presented in Figure 2 and Figure 3 indicate that the cryaa gene promoter could drive Cre expression in lens fibers in the zTg(cryaa:Cre-cryaa:EGFP) zebrafish model. Like transgenic MLR39 mice, our zTg(cryaa:Cre-cryaa:EGFP) zebrafish is an alternative tool to edit gene expression in the lens fibers. By breeding this Tg-zebrafish with the zebrafish carrying the Flox-targeted genes, the researchers can narrow down the genes’ specific regulation toward the lens, rather than another organ—for example, the pax6 [40], six3 [41], or sox1 [42] genes.

Additionally, using this zTg(cryaa:Cre-cryaa:EGFP) zebrafish model, we also ensured the regulation of cryaa mRNA expression during zebrafish embryo development. We found that maternal cryaa mRNA was distributed in early zebrafish embryos (Figure 2). The embryos started to express their own cryaa mRNA at the 16–18 hpf stage, and following the embryos’ growth, this expression occurred predominantly in lens fiber tissue (Figure 2C,D and Figure 3A–C).This expression pattern of cryaa in zebrafish is consistent with previous reports that cryaa mRNA was detectable at 10 somites (14 hpf) as a ubiquitous expression pattern [43] and that cryaa mRNA was detectable with qRT-PCR at 0.5 dpf (12hf) even at a low level [27].

In conclusion, we established a zTg(cryaa:Cre-cryaa:EGFP) zebrafish line with lens-specific expression of an active Cre recombinase. The Cre recombinase in zTg(cryaa:Cre-cryaa:EGFP) could efficiently recognize and cleave loxP-flanked RFP (Figure 5 and Table 2), and the ectopic expression of Cre and EGFP did not influence lens development or transparency (Figure 4). Accordingly, this zTg(cryaa:Cre-cryaa:EGFP) zebrafish is a useful tool for specifically manipulating gene expression in zebrafish lenses when the fish are crossed with another transgenic zebrafish line in which the target’s particular sequences are flanked by the LoxP site.

Acknowledgments

Funding: This work is supported by the National Natural Science Foundation of China (Grand number: 81,970,785, 81,570,825, U1604171 and 31,802,314), the Joint Construction Project of Henan Medical Science and Technology Research Plan (Grand number: SBGJ202102157 and SBGJ202103068), and the Key Science and Technology Program of Henan Province (Grant No. 222,102,310,467). Authors’ contributions: YH, and XP designed the experiments, analyzed the data, and wrote the manuscript. XP and XJ performed the experiments. FZ, GS, MX, MJ and DC co-analyzed and discussed the results. Yanzhong Hu, professor, (hyz@henu.edu.cn) Department of Cell Biology and Genetics, Henan University School of Basic Medical Sciences, Jin-Ming Road, Kaifeng, China, 475014; Tel. 86-18503781944. Fengyan Zhang,Ph.D./ M.D. (Zhangfengyanx@aliyun.com). The Laboratory of Ophthalmology and Vision Science, Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, No.1 Long-Hu-Zhong Huan Road, Zhengzhou, China, 450052.

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