Abstract
Retinoic acid (RA) signalling is essential for vertebrate development, but the spatiotemporal dynamics of its regulation remain largely uncharacterized in specific cellular contexts. Here, we introduce a novel gene trap transgenic reporter line for the zebrafish cyp26c1 gene, a key regulator of RA metabolism. Through a screen of a gal4-based gene and enhancer trap collection, we isolated the tg(gSAIGFF104A) line. In this line, a gal4 gene trap cassette is inserted into the third intron of the cyp26c1 gene, allowing it to faithfully drive reporter expression in a pattern that mirrors the endogenous cyp26c1 expression in zebrafish embryos. Using this reporter, we characterized the expression of cyp26c1 with cellular resolution, revealing a highly dynamic and localized expression in the developing telencephalon, diencephalon, hindbrain, otic vesicles, pharyngeal arches, and other ectodermal derivatives, as well as in subdomains of the retina. Furthermore, we demonstrate that this line responds to both exogenous and genetic manipulations of RA signalling, particularly within the retina. Our reporter provides a valuable resource for investigating the intricate biology of RA signalling in zebrafish development and disease, offering a tool for tracing and manipulating cyp26c1-expressing cells.
1 Introduction
Retinoic acid (RA), a bioactive derivative of vitamin A, is an essential signaling molecule that orchestrates a myriad of developmental processes in vertebrates, including cell proliferation, differentiation, patterning and organogenesis (; ; Rhinn and Dollé, 2012; ; ). The functions of RA are mediated by nuclear retinoic acid receptors (RARs), which form heterodimers with retinoid-X receptors (RXRs) to regulate the transcription of target genes by binding to retinoic acid response elements (RAREs) in their promoters (; ; ). Precise regulation of RA levels is critical for proper embryonic morphogenesis, as its dysregulation acts as a potent teratogen, leading to severe congenital abnormalities (; ; ; ; ; Smith, 2001; Wilson et al., 1953; Zile, 2001). This regulation is achieved through a delicate balance between RA synthesis, primarily by retinaldehyde dehydrogenase (Raldh) enzymes, and RA degradation, mediated by the cytochrome P450 family 26 (Cyp26) enzymes (; Sakai, 2001).
The Cyp26 family plays a central role in establishing and shaping controlled domains of RA activity and responsiveness during vertebrate embryogenesis, (; ; Sakai, 2001). To date, three Cyp26 enzymes (Cyp26a1, Cyp26b1, and Cyp26c1) have been identified in human, mice, chick, Xenopus, and zebrafish (; ; ; ; White et al., 1996). These enzymes are, in turn, responsive to RA, creating a feedback loop in which RA regulates its own availability by controlling Cyp26 expression (Ross and Zolfaghari, 2011). While the roles of Cyp26a1 and Cyp26b1 in establishing and maintaining RA gradients are well-established (), the specific physiological function of Cyp26c1 is not well understood. Importantly, cyp26c1 exhibits a highly localized and dynamic expression pattern, suggesting a specialized role in fine-tuning RA signalling at specific spatiotemporal boundaries during development (; Uehara et al., 2007). This precise expression profile suggests that cyp26c1-expressing cells may delineate a sharp RA response, especially within critical territories of the developing central nervous system, such as the forebrain, hindbrain, and retina (; ). A deeper understanding of the expression and regulation of these molecularly defined cells is important for dissecting the regulatory logic of RA signalling throughout organogenesis.
Despite significant progress, a major challenge in RA research has been the limited availability of tools to visualize and manipulate its dynamic signalling within specific cellular contexts of a developing embryo (Theodosiou et al., 2010; ; ). Current reporter lines, often based on RAREs, lack cell-type specificity and can show variable expression patterns due to their genomic integration sites (; ; Reynolds et al., 1991; ; ; Roberts et al., 2014; Wilson et al., 1990; ). Furthermore, these reporters are primarily designed to respond to the presence of RA, but they do not necessarily reflect the precise, endogenous boundaries of RA activity. This highlights a need for new, complementary genetic tools that provide a more accurate and dynamic representation of RA-controlled gene expression, particularly those that faithfully replicate the intricate spatiotemporal patterns of less studied regulatory genes like cyp26c1. Such tools are essential for studying how RA signalling defines morphological boundaries in vivo during development.
Zebrafish (Danio rerio) serve as an ideal model organism for developmental studies due to their transparent embryos, rapid development, and genetic tractability (). The RA signalling pathways are highly conserved in zebrafish, making them a powerful system for investigating the expression of RA-related genes and the role of RA in vertebrate development and disease (). To generate novel transgenic reporter lines two main approaches have been employed: promoter bashing of known RA-regulated genes and forward genetic strategies using enhancer/gene trap screens (; ; ; ; Skarnes et al., 2004). Enhancer/gene trap screens, in particular, offer a powerful approach. They involve the random integration of a gal4 or a fluorescent protein-coding construct into the genome using the Tol2 transposable element, resulting in expression driven by regulatory elements active at that locus (; Vásquez-Ramírez et al., 2025). These “trapping” transgenes often faithfully recapitulate the endogenous expression patterns of the targeted genes, providing valuable resources for studying gene function and addressing the limitations of reverse genetic approaches.
In this study, we report the characterization of a novel gene trap zebrafish line, tg(gSAIGFF104A). This line harbours a gal4 gene trap cassette inserted into the third intron of the endogenous cyp26c1 gene, enabling it to accurately drive reporter expression that mirrors the endogenous cyp26c1 pattern. This line enables high resolution imaging and the manipulation of molecularly defined cells in telencephalon, diencephalon, hindbrain, and the retina, offering insights into the development of the central nervous system and other ectodermal derivatives with high spatial and temporal resolution. The tg(gSAIGFF104A) line represents an advancement in the zebrafish toolkit available for exploring the intricate biology of RA signalling and its implications for neural development.
2 Materials and methods
2.1 Zebrafish husbandry
The zebrafish strains used in this study were: AB*, tg(gSAIGFF104A) (this study), tg(5xUAS:EGFP) (), tg(UAS:RFP)yu9Tg (), tg(neuroD:tagRFP)w69Tg (), tg(rx3:Gal4-VP16) (Weiss et al., 2012) and tg(atoh7:RFP)cu2Tg (Zolessi et al., 2006). Embryos were raised at 28.5 °C in E3 medium and staged by hours or days post fertilization (hpf and dpf, respectively) according to Kimmel et al. (1995). For all experiments, we used stable, germline transgenic lines, and embryos harbouring a single insertion of the transgenes, as assessed by inheritance patterns. To reduce embryo pigmentation for imaging, phenylthiourea (PTU) (0.004%; Sigma) was added to the E3 medium ().
2.2 Screening of transgenic fish
The tg(gSAIGFF104A) line is a gene trap line expressing GFF, a modified version of the Gal4 transcriptional activator consisting of the Gal4 DNA-binding domain fused to a tandem repeat of the VP16 transactivation domain (Takeuchi et al., 2015). This line was generated using the gSAIGFF gene trap construct, which incorporates a gata6 splice acceptor, the GFF sequence, and the SV40 polyadenylation signal (polyA) to ensure proper termination of the trapped transcript. The integration of this construct was achieved through Tol2 transposon-mediated transgenesis, following established protocols involving the co-injection of the construct with transposase mRNA (; ; Takeuchi et al., 2015).
Our initial screening was performed by analysing fluorescent images of gene trap and enhancer trap lines available in the zTrap database (https://ztrap.nig.ac.jp/ztrap/) () that drive expression in developing eyes. From this dataset, carrier lines were selected for adult crossing. Dechorionated embryos from these crosses were screened and observed daily from 24 to 72 hpf using widefield fluorescence microscopy with an MZ16FA (Leica) dissecting microscope available at the National Institute of Genetics (NIG) in Mishima, Japan. Images were captured using a CCD camera (DFC300 FX, Leica). From these lines, tg(gSAIGFF104A) was chosen for further analysis due to its conspicuous transgene expression in a band of cells along the nasal-temporal axis of the retina.
The tg(gSAIGFF104A) line was maintained as a stable germline with a single gal4 integration within the cyp26c1 locus. For all analyses in this study, embryos from the F5 and F6 generations were used. Inheritance stability and expression fidelity were routinely verified during the selection of new carriers for colony maintenance. Experimental embryos were obtained from double-hemizygous crosses (for gal4 and UAS-reporter), yielding the expected Mendelian dihybrid phenotypic ratio of 9/16 fluorescent individuals, while outcrosses to wild-type fish yielded the expected 25% fluorescence ratio. The reported expression patterns remained highly consistent across multiple generations, with no detectable mosaicism or variegation.
2.3 PCR and sequencing
Genomic DNA (gDNA) from 72 hpf tg(gSAIGFF104A; UAS:GFP) GFP-positive larvae was isolated using the HotShot method (). Briefly, larvae were incubated in 15 µL of alkaline lysis buffer (1X final concentration; NaOH 1.25 M, EDTA 0.01 M, stock 50X) at 95 °C for 30 min. Afterward, 15 µL of neutralization solution (1X final concentration; Tris HCl 2 M, stock 50X) was added. To confirm the insertion of the transgenic line, PCR was performed using gDNA from transgenic larvae with a primer in one of the arms of the tol2 transposon (T2_For: 5′-ACTCAAGTAAAGTAAAAAAATCCCCAAAAA-3′) and a primer in the third intron of the cyp26c1 gene (cyp26c1_Rev: 5′-TGCAGCCAAAATCAGAGAAGTTAC-3′), generating a 132 bp product. The PCR protocol is as follows: Initial denaturation at 95 °C for 30 s; Denaturation at 95 °C for 30 s; Annealing at 55 °C for 30 s; Extension at 68 °C for 30 s; 34 cycles from Denaturation to Extension; Final extension at 68 °C for 5 min, and hold at 12 °C. 10X Standard Taq Reaction Buffer (NEB B9004S, New England Biolabs), Taq DNA polymerase (NEB M0267S, New England Biolabs), Forward and reverse primers (10 µM), dNTPs 10 mM mix (dATP, dCTP, dGTP, dTTP; #2840655; Cytiva), template DNA, nuclease free water and DMSO (BM-0660; Winkler) were used for the PCR.
The presence and size of the amplicon were verified by running the PCR product on a 2% agarose gel in 1X TAE buffer. The PCR product was then purified using the GFX PCR DNA and Gel Band Purification Kit (#28903470; Cytiva, United Kingdom) and sequenced using the T2_For primer (Macrogen). The genomic sequence surrounding the insertions was analysed by BLAST search against GRCz11 reference genome in Ensembl.
2.4 Microinjection and CRISPR/Cas9
To generate targeted mutations in the gdf6a gene, a CRISPR/Cas injection mix was prepared using the AltR S.p. Cas9 Nuclease V3 (Integrated DNA Technologies, IDT: 1081058) and two guide RNAs (gRNAs) designed using CHOPCHOP (). The gRNAs were synthesized using a cloning-free method according to . For each gRNA, a 60-base forward oligonucleotide was synthesized, containing a T7 viral promoter for in vitro transcription (5′-TAATACGACTCACTATAGG-3′), a 20-base spacer region specific to the target site, and an overlap region complementary to the constant oligonucleotide common to all gRNAs. The reverse primer was common for all the gRNAs ().
The sequences of the forward oligonucleotides were as follows:
Guide RNA 1:
5′-TAATACGACTCACTATAGGCAACACGGTAAACTCCAGGTTTTAGAGCTAGAAATAGCAAG-3′
Guide RNA 2:
5′-TAATACGACTCACTATAGGTGCCAGGAGCGCGTTTGAGTTTTAGAGCTAGAAATAGCAAG-3′
The final concentration of the microinjected solution was 1000 pg/nL of Cas9 and 250 picograms of each gRNA, according to . The injected volume was 4 nanoliters. Injections were performed at the one-cell stage using a Microinjection Pump (World Precision Instruments, SYS-PV820). Crosses were made between the AB* and tg(gSAIGFF104A; UAS:GFP) lines.
2.5 Retinoic acid treatment
Stock solutions of all-trans retinoic acid (at-RA, Sigma) were prepared in dimethyl sulfoxide (DMSO; Sigma) and stored at −20 °C until use. Prior to treatment, embryos were manually dechorionated to ensure proper exposure to the compound. RA was supplied in E3 media at a final concentration of 100 μM, with 2% DMSO as a vehicle control. Embryos were treated with RA from 28 hpf until 52 hpf, a critical window for transgenic expression, and processed for imaging.
2.6 Sample processing for imaging
Embryos at 24, 48, 52, and 72-hpf were anesthetized with 0.4% tricaine (MS-222, Sigma) and transferred to 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for overnight fixation at 4 °C. Whole embryos or dissected brains and eyes were counterstained with TO-PRO-3 (Invitrogen T3605) and reserved for further processing and imaging modalities. Cleared, RA-treated, and HCR samples were embedded in 1% low-melting-point agarose (LMP agarose, Sigma) in PBS, and mounted for confocal and light sheet imaging. Larval brain dissection was performed according to Turner et al. (2014).
2.7 Tissue clearing
Embryos, dissected brains and eyes were cleared using CUBIC (Susaki et al., 2014; ), with modifications. CUBIC-1.1 solution consists of 10% (v/v) N, N, N′, N′-Tetrakis(2-Hydroxypropyl) ethylenediamine (Sigma-Aldrich, 122262), 5% (v/v) Triton X-100 and 5% (m/v) urea (Sigma-Aldrich, U5128). CUBIC-2 solution consists of 10% (m/v) 2,2′,2″-Nitrilotriethanol, Tris(2-hydroxyethyl) amine (Sigma-Aldrich, 90279), 50% (m/v) sucrose (Sigma-Aldrich, S0389) and 25% (m/v) urea. Tissues were cleared in CUBIC-1.1 solution at 37 °C until transparency was reached and imaged using confocal microscopy or transferred and equilibrated in CUBIC-2 solution for refractive index (RI) matching at 1.45 for light sheet microscopy.
2.8 Hybridization Chain Reaction (HCR)
72-hpf embryos were fixed in 4% PFA overnight at 4 °C and dehydrated in a methanol gradient for permeabilization and long-term storage at −20 °C if required. Embryos were gradually rehydrated to PBSTr (PBS with 0.05% Triton X-100), permeabilized by Proteinase K treatment (10 ng/mL in 0.05% PBSTr) and post-fixed in 4% PFA for 20 min at room temperature. HCR protocol was performed as described by the manufacturer (Molecular Instruments). Custom HCR 3.0 probes for cyp26c1 mRNA (Supplementary Table S1) were designed with a published script () using the coding sequence of cyp26c1 and aligned against the transcriptome to exclude off-target detection.
2.9 Confocal imaging
Imaging was performed on a Zeiss LSM 710 confocal microscope using the HeNe633, HeNe543 and 458/488/514 multi-line Argon lasers. 16-bit images were acquired at a pinhole size of 1 airy unit and employing the Plan-Apochromat 10x/0.3 M27 and EC Plan-Neofluar 20x/0.50 M27 objectives. Samples were imaged in 1% LMP agarose within custom-made glass-bottom chambers. For cleared samples, the imaging chamber was filled with CUBIC-1.1 solution for refraction index matching at 1.38.
2.10 Light sheet imaging
Imaging of fixed and live samples was performed in a Zeiss Light Sheet 7 microscope with 405/488/561/638 nm laser lines, using glass capillaries for mounting. For fixed material, cleared samples were RI matched at 1.45 in CUBIC-2 solution. Images were acquired with a Clr Plan-Neofluar 20x detection objective (1.0 Corr, nd = 1.45) and two LSFM 10×/0.2 illumination objectives. Image acquisition utilized dual-side illumination with pivot scan, a bit depth of 16-bit and a light sheet thickness of 2 µm.
For live imaging, embryos were maintained in a chamber with E3 medium heated to 28.5 °C. tg (gSAIGFF104A; UAS:GFP) embryos were anaesthetized with 0.04% tricaine and mounted in 1% low-melting-point agarose (Sigma) in E3 medium with PTU. Time-lapse z-stacks were collected from a lateral view at 3-min intervals for 12 h, beginning at 54 hpf. Images were acquired by dual-side illumination with pivot scan, a bit depth of 16-bit, and a light sheet thickness of 2 µm.
2.11 Image analysis
Fiji (Schindelin et al., 2012) was employed for general image processing. Particle tracking analysis on maximal intensity projection (MIP) time lapse image sequences was performed using the Mastodon plugin, which is optimized for cell tracking in large datasets and derived from the TrackMate plugin (Tinevez et al., 2017). Dual-side fusion, color-coded projection, and light sheet processing were conducted using Zen Blue 3.4 (Zeiss). Imaris 9 (Oxford Instruments) was utilized for three-dimensional (3D) rendering and surface segmentation.
3 Results
3.1 The tg(gSAIGFF104A) transgene drives GFP expression in the developing nervous system and other ectoderm derived tissues
To identify transgenic lines that drive gene expression in developing zebrafish eyes, we initially screened incrosses of 71 lines from a pool of 255 candidates. These lines were preselected based on their preliminary expression patterns in the developing brain and eye after being crossed with a tg(UAS:GFP) responsive fish. From this screening, we isolated a novel transgenic line, tg(gSAIGFF104A), which exhibits a striking and highly specific expression pattern in the retina. This line displayed a distinct band of GFP-positive cells along the nasal-temporal axis of the developing retina at 2 dpf (Figure 1A). By 3 dpf, this well-defined band of GFP-positive cells was readily detectable, showing a broader distribution within the nasal retina (Figure 1B). At both 2 and 3 dpf, a sparse number of cells were also labelled in the lens. The highly specific retinal expression pattern reflects targeted activation of the reporter transgene within a retinal subdomain, underscoring its value as a tool for investigating retinal development and the signalling pathways that define specific cell populations.
FIGURE 1
In addition to the expression in the retina, significant tg(gSAIGFF104A)-driven expression was observed in various other embryonic regions. Specifically, we detected strong GFP fluorescence in the telencephalon, hindbrain, pharyngeal arches, and anterior lateral line (Figures 1C–F). At 24 hpf, GFP expression in the hindbrain closely aligns with developmentally regulated compartmentalization events (Figures 1C,D), serving as a reliable marker for delineating tissue boundaries crucial for proper morphogenesis. At this stage, expression was also prominent in part of the otic vesicle (Figure 1D). As development progressed, expression in the hindbrain, otic vesicle and pharyngeal arches remained prominent, with additional labelling detected in the boundary between telencephalon and diencephalon, and the anterior lateral line (Figures 1E,F), highlighting the progressive activation of expression in both central and peripheral nervous domains.
The observed GFP expression pattern in the developing brain partially overlaps with the neuronal marker neuroD, as demonstrated by outcrossing the tg(gSAIGFF104A; UAS:GFP) line with the tg(neuroD:tagRFP) reporter line (Figures 1C,F). These results are consistent with the idea that RA activity intersects with regions of early neuronal differentiation. Overall, our data demonstrates that the tg(gSAIGFF104A; UAS:GFP) transgenic line marks relevant nervous system subdomains, sensory organs, and other ectoderm-derived tissues throughout early development. This precise and dynamic expression pattern makes it a valuable tool for delineating tissue boundaries crucial for morphogenesis.
3.2 The expression of the tg(gSAIGFF104A) transgenic line is driven by the cyp26c1 locus
Given the transgenic pattern observed, we aimed to identify the gene responsible for driving this expression. According to the zTRAP database, the tg(gSAIGFF104A) construct was inserted within the third intron of the cyp26c1 gene, which encodes a 7-exon retinoid 4-hydroxylase responsible for degrading RA, thereby restricting its signalling and controlling its concentration levels (Figure 2A) (Zhong et al., 2018). To validate the transposon insertion, we performed a genotyping PCR using primers located in the Tol2 sequence of the transgene and the adjacent genomic region of cyp26c1 (Figure 2B). The amplification and sequencing of a 132 bp product confirmed that the tg(gSAIGFF104A) transgene is correctly inserted 326 bp downstream of the start of the third intron of cyp26c1, thereby creating a gene trap (Figures 2C,D).
FIGURE 2
The insertion of the tg(gSAIGFF104A) transgene within the third intron of the cyp26c1 gene supports the idea that the gal4-encoding sequence is actively transcribed and expressed alongside cyp26c1. Consistently, we compared the retinal GFP expression pattern with the spatial distribution of cyp26c1 mRNA using HCR. The two expression patterns showed close correspondence (Figures 3A–C), reinforcing the reliability of the tg(gSAIGFF104A) line as a driver that recapitulates the expression of cyp26c1.
FIGURE 3
3.3 The tg(gSAIGFF104A) transgene drives gene expression in both neuronal and non-neuronal cell types during retinal development
At 72 hpf, the zebrafish retina is laminated. At this stage, the tg(gSAIGFF104A) line drives gene expression in a striking and spatially refined pattern within the medial region of the developing retina (Figure 1). Using high-magnification captures of the retina we found UAS-driven GFP expression across the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) (Figure 4A). Optical sections of tg(gSAIGFF104A; UAS:GFP) along the dorsal ventral axis of the retina reveal trails of cells emerging from the ciliary marginal zone (CMZ), a reservoir of multipotent retinal progenitors in fish (Figure 4B) (; ).
FIGURE 4
To confirm expression in different retinal cell types, we conducted co-labelling experiments by outcrossing our driver line into established transgenic markers for progenitors and differentiated cells in the retina. At 48 hpf, GFP expression was found in cells that also expressed the neuronal marker tg(neuroD:tagRFP), as well as in cells expressing tg(atoh7:mRFP) that define committed progenitors and subsets of amacrine and bipolars cells, as well as retinal ganglion cells, respectively (Figures 4C,D) (Zolessi et al., 2006; ). Notably, tg(gSAIGFF104A; UAS:GFP) line selectively labels a distinct subset of Müller glia, the last lineage to be born in the embryonic zebrafish retina (), which are prominently marked by GFP (Figures 4E,F). This widespread expression pattern in the retina highlights the utility of the tg(gSAIGFF104A) line to mark both neuronal and non-neuronal cell types as the tissue matures. Together, our co-labelling results and detailed morphological description validate tg(gSAIGFF104A) as a robust driver line that faithfully labels retinal cells, from progenitor to differentiated neurons and glia, during embryonic and larval retinal development.
3.4 Versatility of the tg(gSAIGFF104A) transgenic line
The pattern of labelled cells across all three retinal lineages in tg(gSAIGFF104A; UAS:GFP) is reminiscent of the ARCoS that arise from the CMZ, as described by . To gain deeper insights into the behaviour of the CMZ retinal progenitors labelled by the tg(gSAIGFF104A; UAS:GFP) line, we conducted time-lapse imaging from 54 to 66 hpf, a period when the CMZ is active (; Valdivia et al., 2016). This approach revealed that GFP-labelled cells derived from the CMZ actively migrate and integrate within the central retina, demonstrating the dynamic nature of retinal growth and the contribution of CMZ progenitors to retinal expansion and architectural maintenance (Figure 5; Supplementary Movie S1). Collectively, these findings establish the tg(gSAIGFF104A) driver as a powerful tool for studying the spatial and temporal dynamics of post-embryonic retinal progenitors in vivo.
FIGURE 5
Finally, to illustrate additional versatility of the tg(gSAIGFF104A) line, we outcrossed it with the tg(rx3:Gal4-VP16); tg(UAS:RFP) (Weiss et al., 2012) reporter line. This crossing enabled visualization of tg(gSAIGFF104A)-driven expression using an alternative fluorescent marker. Consistent with the previous observations, RFP expression closely mirrored the GFP pattern confirming the ability of the tg(gSAIGFF104A) line to drive expression of other UAS-based transgenes. Importantly, we detected no evidence of variegation or ectopic expression (Figure 6).
FIGURE 6
3.5 The tg(gSAIGFF104A) transgenic line shows tissue-specific response to retinoic acid signalling
RA signalling plays a critical role in dorsoventral retinal patterning, with components of RA synthesis expressed in a polarized manner in the retina (). Furthermore, RA has been shown to induce the expression of cyp26 enzyme-coding genes, which degrade RA and thus regulate its own levels (; ; Thatcher and Isoherranen, 2009; ). Given the expression of the tg(gSAIGFF104A; UAS:GFP) reporter in the medial region of the developing eye, we hypothesized that modulating RA levels could influence the transgene expression. To test this, we first treated tg(gSAIGFF104A; UAS:GFP) heterozygous embryos with exogenous RA from 28 hpf to 52 hpf, a timing where the cyp26c1 reporter is expressed in the retina. We used heterozygous embryos to control for transgene copy number. Consistent with the known role of RA, acute treatment with a 100 µM RA concentration resulted in craniofacial malformations (Figure 7) (). Notably, together with a small eye phenotype, the reporter expression is downregulated in the retina but increased within the developing lens (Figures 7E,F). This behaviour is consistent with the tissue specific regulation previously described for cyp26c1, which can be either upregulated or downregulated depending on the cellular context (; ; Reijntjes et al., 2005; ; White et al., 2007). These findings indicate that, to some extent, the tg(gSAIGFF104A) line responds to RA, highlighting its utility for studying RA signalling dynamics in aspects of eye development.
FIGURE 7
To further explore this idea, we analyzed the loss-of-function of the gdf6a gene, which is known to counteract ventrally produced RA and arrest the growth of the eye (; Valdivia et al., 2016). We found that somatic mutagenesis of the gdf6a locus using CRISPR/Cas results in smaller eyes, together with a reduction in the transgenic reporter signal, suggesting that increased levels of RA signalling accompany this phenotype (Figures 7D–H).
4 Discussion
In this study, we introduce tg(gSAIGFF104A), a novel zebrafish gene trap transgenic line. The line features a gal4 insertion within the cyp26c1 locus, which encodes a key regulator of RA metabolism. Through its combination with tg(UAS:GFP), this line drives GFP expression within specific subdomains of the developing nervous system, including the retina, as well as in other ectoderm-derived tissues. This versatile tool is particularly well suited for studies aiming to delineate tissue boundaries and track cell behaviour during early embryonic development.
The utility of the tg(gSAIGFF104A) line hinges fundamentally on its structure and fidelity. The line was generated via a Tol2-mediated gene trap insertion localized within the third intron of the cyp26c1 locus. The construct architecture, which integrates both splicing acceptor and donor sites, suggests that gal4 transcription proceeds through an internal fusion event with the endogenous transcript (). While this insertion holds the potential to disrupt gene function, the assessment of a direct mutagenic effect falls outside the scope of this work; we have not detected an obvious morphological phenotype in incrosses of the line (data not shown). Crucially, the observed GFP reporter expression (Figure 1) closely mirrors the endogenous expression domains of cyp26c1 (; ). This robust correspondence validates the transgene as a precise reporter of cyp26c1 transcriptional activity.
The transgenic line reveals several biologically relevant features. The conspicuous hindbrain labelling at 24 hpf is particularly notable, given its consistency with the established role for RA signalling during hindbrain compartmentalization and rhombomere formation (White et al., 2007; ; ). Additional expression in otic vesicles and pharyngeal arches points to a potential involvement in the development of early ectodermal derivatives (; ; ). The partial overlap with the neuronal marker neuroD in tg(neuroD:tagRFP) embryos further suggests that cyp26c1 may influence specific neuronal populations, underscoring the complexity of RA signalling in the developing nervous system (). These observations indicate the substantial utility of our line in revealing fine morphological and molecular events during neuronal differentiation, particularly when combining the line with other relevant reporters.
A remarkable strength of the tg(gSAIGFF104A) lies on its ability to drive expression in retinal progenitor cells, neurons and Müller glia while confining this expression to a distinct horizontal stripe within the zebrafish retina. Through co-labelling experiments with the transgenic markers tg(neuroD:tagRFP) and tg(atoh7:RFP), 3D reconstruction, and assessment based on retinal layer position, we confirmed that the tg(gSAIGFF104A; UAS:GFP) broadly labels diverse retinal cell types (; ; ). Indeed, high-magnification imaging at 72 hpf revealed GFP expression across all retinal layers, demonstrating the utility of the line for studying the dynamic contributions of retinal progenitors to retinal architecture. Furthermore, time-lapse imaging from 54 to 66 hpf showed that GFP-positive cells originating in the CMZ actively contribute to the central retina, highlighting the critical role of CMZ progenitors in retinal growth and maintenance (). This restricted expression within the CMZ subdomain along the dorsal-ventral axis is a powerful asset, as it allows for precise manipulation of gene expression within the stem cell niche. Combining the precise labelling of tg(gSAIGFF104A) with blastula cell transplantations will constitute a robust tool for single cell lineage tracing and high-resolution analysis.
The tg(gSAIGFF104A) line exhibits a tissue-specific response to RA signalling, a critical regulator of dorsoventral retinal patterning in vertebrates (Wagner et al., 2000; Sen, 2005; ). Our results show that loss-of-function of gdf6a, which normally counteracts ventrally produced RA, resulted in smaller eyes and reduced GFP expression in the retina. This observed phenotype is likely due to an increase in the expression of genes involved in RA synthesis, as gdf6a loss-of-function is known to elevate RA signalling in developing zebrafish eyes (; Valdivia et al., 2016). However, we cannot entirely exclude the possibility that primary patterning defects also contribute to the observed phenotype. Consistent with the established role of RA in modulating cyp26c1 expression, exogenous RA treatment led to classic craniofacial malformations and a downregulation of GFP in the retina, while upregulating it in the lens (Figure 7). Taken together, both genetic manipulation of RA levels and exogenous RA treatment support the idea that increased RA levels can modify cyp26c1 expression or the formation of cells that express it. This strongly underscores the utility of the tg(gSAIGFF104A) line for investigating RA signalling dynamics, particularly the function and regulation of the poorly characterized cyp26c1 enzyme and its role in eye development ().
Another significant advantage of the tg(gSAIGFF104A) line is its combinatorial utility within the Gal4-UAS system, allowing for versatile experimental applications. When crossed with tg(UAS:RFP), the driver line exhibited the same precise expression pattern as observed with tg(UAS:GFP), with no evidence of variegation or ectopic expression (). This demonstrated tunability confirms that the line provides robust, interchangeable expression control over any UAS-driven reporter, serving as a powerful and flexible tool for studying cyp26c1-mediated developmental processes and probing the dynamics of various gene expression cascades.
It is worth noting that another cyp26c1 gene trap line has been previously reported, featuring a gal4 insertion in an exon, which likely disrupts the function of the endogenous gene (). While that line could serve as a loss-of-function allele, its phenotypic consequences have not yet been analyzed. Despite this, the expression pattern of the published line is consistent with our tg(gSAIGFF104A) line, suggesting that both lines capture the endogenous cyp26c1 expression profile ().
The tg(gSAIGFF104A) line represents a significant resource that opens exciting avenues for future research. One particularly promising direction is to further investigate the behaviour of a subset of CMZ progenitors and their contribution to the central retina, analogous to the ARCoS described by . This approach could yield insights into the cellular and molecular mechanisms underlying retinal growth. Additionally, it is noteworthy that the expression pattern observed in zebrafish appears to be conserved across vertebrates. In both mouse (Wagner et al., 2000; Sakai et al., 2004; ) and chick (), a conspicuous tripartite retinal compartmentalization has been described: a ventral RA-rich zone, a dorsal RA-poor zone and a horizontal RA-poor strip (Wagner et al., 2000); this central strip is marked by expression of Cyp26c1 and Cyp26a1, where the latter is also detected in the developing fovea of rhesus monkeys () and human, particularly in Müller glia (; ). These findings suggest a conserved role for Cyp enzymes to establish and delineate critical boundaries during retinal patterning across species, potentially influencing photoreceptor/RGC production or topographic map formation. Therefore, the tg(gSAIGFF104A) line may help to unveil previously unrecognized structural or functional features of this retinal region in zebrafish. Finally, the tg(gSAIGFF104A) line holds substantial utility for exploring the role of cyp26c1 in other developmental contexts, such as craniofacial development, neurogenesis, and hindbrain compartmentalization, where RA signalling is known to relevant (; ; ; ). The ability to cross the tg(gSAIGFF104A) line with other UAS-based responsive lines, such as optogenetic tools or biosensors, provides a direct means to enable sophisticated functional studies of cyp26c1-expressing cells in vivo.
In summary, the tg(gSAIGFF104A) transgenic line provides a novel and versatile resource for studying retinal development, RA signalling, and the behaviour of retinal progenitors in vivo. Its ability to faithfully recapitulate cyp26c1 expression, label diverse cell types, and drive expression of other UAS-based constructs makes it a valuable addition to the zebrafish toolkit. Future studies using this line will be instrumental in illuminating the molecular mechanisms underlying retinal progenitor behaviour, deepening our understanding of the role of RA signalling in eye and retinal development, and uncovering broader functions of cyp26c1 in vertebrate embryogenesis and disease.
Statements
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
Ethics statement
The animal study was approved by Bioethics Committee of Universidad Mayor. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
RR-P: Writing – review and editing, Conceptualization, Investigation, Writing – original draft, Methodology, Formal Analysis. DA-A: Investigation, Formal Analysis, Writing – review and editing, Methodology. MV-R: Investigation, Writing – review and editing. JB: Writing – review and editing, Investigation. AV: Writing – review and editing, Investigation. ML: Writing – review and editing, Resources. RBM: Resources, Writing – review and editing. KK: Resources, Writing – review and editing. LEV: Writing – review and editing, Project administration, Funding acquisition, Formal Analysis, Supervision, Writing – original draft, Conceptualization.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was funded by grants from regular FONDECYT 1211508 (to LEV), FONDEQUIP 190087 (to LEV), LISIUM-Chile CZI S-2022008-03 (to LEV), postdoc FONDECYT 3220417 (to RR-P), JSPS KAKENHI JP24K02008 to KK, as well as BBSRC David Phillips Fellowship (BB/S010386/1) to RBM.
Acknowledgments
We thank Pauline Sallaberry for excellent fish care and the members of the Valdivia lab for their valuable and insightful discussions. The authors also gratefully acknowledge “Unidad de Microscopía de la Universidad Mayor” UM2 for their technical support and assistance with this work, as well as the “Unidad de Microscopía” of Facultad de Ciencias at Universidad de Chile for their technical services. We specially thank Jorge Mpodozis for providing resources necessary for some of the imaging experiments and Anibal Vargas for helping with the time lapse analysis.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.
Publisher’s note
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2026.1753548/full#supplementary-material
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Summary
Keywords
CYP26C1, Gal4/UAS, retina, retinoic acid, transgenic, zebrafish
Citation
Reyes-Pinto R, Arancibia-Altamirano D, Vásquez-Ramírez MJ, Benítez JM, Villanueva A, Lahne M, MacDonald RB, Kawakami K and Valdivia LE (2026) A novel cyp26c1-driven reporter line to study boundaries of retinoic acid signalling during zebrafish development. Front. Cell Dev. Biol. 14:1753548. doi: 10.3389/fcell.2026.1753548
Received
24 November 2025
Revised
20 January 2026
Accepted
09 February 2026
Published
25 May 2026
Volume
14 - 2026
Edited by
Natália Martins Feitosa, Federal University of Rio de Janeiro, Brazil
Reviewed by
Surendra Rajpurohit, Augusta University, United States
Carla Lima, Butantan Institute, Brazil
Updates
Copyright
© 2026 Reyes-Pinto, Arancibia-Altamirano, Vásquez-Ramírez, Benítez, Villanueva, Lahne, MacDonald, Kawakami and Valdivia.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Leonardo E. Valdivia, leonardo.valdivia@umayor.cl
† Present address: Rosana Reyes-Pinto, Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France
Disclaimer
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