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. Author manuscript; available in PMC: 2026 Jul 7.
Published in final edited form as: Development. 2026 Jul 14;153(16):dev.205402. doi: 10.1242/dev.205402

Zebrafish knock-in lines enabling live visualization of extracellular matrix dynamics during development and regeneration

Jingwen Shen 1,2, Ranjay Jayadev 3, Pierre Gillotay 1,2, Jianhong Ou 1,2, Ashley Rich 4, Madison L Zimmerman 1,2, Kazunori Ando 1,2, Stefano Di Talia 4, David R Sherwood 3, Kenneth D Poss 1,2,5
PMCID: PMC13334507  NIHMSID: NIHMS2187675  PMID: 42240014

Abstract

Extracellular matrix (ECM) plays fundamental roles in animal development, regeneration, and disease. The difficulty of tagging endogenous matrix proteins in vertebrates has limited the understanding of ECM composition and dynamics in complex tissues. To visualize vertebrate ECM components, we tagged zebrafish Laminin, gamma 1 (Lamc1), Collagen, type I, alpha 2 (Col1a2), and Transforming growth factor, beta-induced (Tgfbi) using C-terminus in-fusion genome editing. Analysis of these knock-in lines revealed distinct expression of each protein in various tissues during development and regeneration. Fluorescence recovery after photobleaching analysis further indicated that Lamc1 is stable in fin fold matrix but more dynamic in myoseptal matrix of developing zebrafish, while Col1a2 and Tgfbi are stable matrix components in myosepta. Strikingly, we found that Col1a2-mScarlet protein accumulates at the amputation plane during tailfin regeneration, where it remains concentrated for several days and distant from the regeneration blastema. This “foundation” region also displays a distinct transcriptome suggesting active and dedicated events at the base of the regenerating appendage. Our resource enables live capture of ECM dynamics that can identify new events in developing and regenerating zebrafish.

Keywords: zebrafish, extracellular matrix, fusion protein knock-in, protein dynamics, tissue regeneration

Summary statement

Extracellular matrix resources for zebrafish development and tissue regeneration.

INTRODUCTION

Extracellular matrix (ECM) is composed of structural proteins, modification enzymes and signaling molecules, and is vital for development, organ formation and tissue regeneration. During early development, ECM components and ECM-bound growth factors provide cues for cell proliferation, differentiation, and assembly into complex structures. ECM also exerts key biophysical roles in different stages of tissue regeneration to enable healing and clearance of debris, tissue growth and remodeling.

In vertebrates, direct ECM visualization in models like zebrafish has relied on ectopic overexpression of fluorescent protein-tagged ECM genes using a related regulatory sequence (Morris et al., 2018; Sánchez-Iranzo et al., 2018; Sharma et al., 2019; Hino et al., 2024). CRISPR-Cas9 based knock-in techniques facilitate tagging of the endogenous genomic locus, and studies in C. elegans have used in-frame fusion with fluorescent proteins to visualize 58 basement membrane (BM) proteins (Naegeli et al., 2017; Jayadev et al., 2019; Fan et al., 2020; Keeley et al., 2020; Jayadev et al., 2022; Srinivasan et al., 2025), a specialized form of matrix organized into thin and dense sheets of ECM surrounding most tissues (Feitosa et al., 2011). Studies using these endogenous knock-in strains have revealed surprising insights into different molecules that stably associate with the BM scaffolding versus dynamic BM components that rapidly mobilize on and off BM or diffuse within the scaffolding (Keeley et al., 2020).

Collagens are the main ECM structural proteins, and 28 different collagen types exist in zebrafish. Mutations in type I collagen genocopy and phenocopy human type I collagenopathies, such as osteogenesis imperfecta (Gistelinck et al., 2018). Non-collagenous structural ECM components include the crucial glycoproteins like laminin, which anchors BM to cell surface receptors and exist in heterotrimers with different combination of α, β, γ subunits (Feitosa et al., 2011). Many matrisome-associated secreted factors reside in ECM and regulate diverse cellular events (Skonier et al., 1992; Kim and Ingham, 2009; Fico and Santamaria-Martínez, 2020). For instance, Transforming growth factor, beta-induced (Tgfbi) is a secreted RGD-domain containing protein that binds collagens and also interacts with integrins; it has been described to influence cell adhesion, proliferation, differentiation, and death. Accumulation of insoluble TGFBI has been associated with autosomal, dominant corneal dystrophies in humans (Corona and Blobe, 2021). Here, we tagged three ECM components in zebrafish by in-fusion knock-in -- laminin Lamc1, collagen Col1a2, and Tgfbi – and used live imaging to visualize localization, dynamics and turnover during development and tissue regeneration.

RESULTS AND DISCUSSION

A C-terminal knock-in strategy to establish an ECM toolkit

To tag endogenous ECM components in zebrafish, we introduced an eGFP or mScarlet fluorescent protein in frame at the C-terminus, as described (Levic et al., 2021). We designed sgRNAs to cleave a sequence in an intron, as well as a double-stranded DNA donor with part of the last intron and last exon followed by a flexible linker and sequences coding for a fluorescent protein (Fig. 1A, see Methods). Through coinjection of these components with NLS-Cas9, we tagged 3 different important ECM components: the structural ECM protein Lamc1, the fibrous ECM component Col1a2, and the secreted signal protein Tgfbi (Parsons et al., 2002; Kim and Ingham, 2009; Page et al., 2013; Chen et al., 2015; Sánchez-Iranzo et al., 2018; Subramanian et al., 2018; Sharma et al., 2019; Rajan et al., 2020; Metikala et al., 2021; Xia et al., 2022). These ECM proteins were chosen for tagging based on distinct structures and functions and increased expression levels in different adult tissue regeneration contexts (Fig. S1A–D).

Figure 1. C terminal knock-in strategy for ECM knock-ins.

Figure 1.

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(A) C-terminal tagging strategy. gRNAs target sites in last introns for lamc1 and col1a2 genomic loci. The tgfbi gRNA target site is in the fifth intron. Partial intron and last exons were bridged by a flexible linker (GGSGGTGGSGGT) to fluorophore. Red hexagon, stop codon. Pink dashed box, DNA donors for different target genes.

(B) Images of lamc1eGFP/+, col1a2mScarlet/+, and tgfbimScarlet/+ larvae at 5 dpf.

(C) Images of lamc1eGFP/+, col1a2mScarlet/+ and tgfbimScarlet/+ larvae from lateral, dorsal and ventral views at 5 dpf. Scale bars: 500 μm (B, C).

We assessed fluorescence signals from tagged proteins in stable knock-in larvae for correspondence with mRNA localization. lamc1 mRNA is present in transverse myoseptum, notochord, eye structures, pectoral fin bud, cardiac ventricle, and other structures (Fig. S1E) (Parsons et al., 2002). We detected Lamc1-eGFP protein in lamc1egfp/+ fish in an overlapping manner, expressed widely and enriched at 5 days post fertilization (dpf) in otic vesicles, pharyngeal arches, heart, pectoral appendages, myosepta, and notochord (Fig. 1B, C). While commercial antibodies we obtained did not effectively stain Col1a2 or Tgfbi in zebrafish larvae, an antibody against laminin showed immunoreactivity matching most or all of the pattern of Lamc1-eGFP (Fig. S1F). col1a2 transcripts are expressed in muscle, pectoral fin, branchial arches and cleithrum in zebrafish embryos and larvae, and transcriptional reporter lines have indicated similar expression domains (Fig. S1E) (Sharma et al., 2019; Rajan et al., 2020). In col1a2mScarlet/+ animals, Col1a2-mScarlet was detected in cartilage of the lower jaw, cleithrum, and pectoral appendages, and was enriched in joints and myosepta at 5 dpf. Col1a2-mScarlet formed punctate protein signals throughout the trunk, likely indicating microdomains or small nascent fibrils (Fig. 1B, C). tgfbi mRNAs are localized in splanchnocranium, vertebrae precursors, and pharyngeal arches in early stage zebrafish (Fig. S1E) (Kim and Ingham, 2009). Tgfbi-mScarlet was prominent throughout tgfbimScarlet/+ somites and in pectoral fin base and myotomes at 5 dpf (Fig. 1B, C). Thus, C-terminal knock-ins labeled ECM components, with tagged protein localization being consistent with mRNA localization.

lamc1egfp/+, col1a2mScarlet/+, and tgfbimScarlet/+ (all heterozygous) animals were indistinguishable at 3–4 dpf from wild-type siblings (Fig. S2A, B, D, E, G, H). We observed that homozygous lamc1egfp animals survived normally but were shorter than heterozygotes and wild-type siblings at 4 dpf, often with bent notochords reminiscent of lamc1 (sleepy) mutant larvae (Fig. S2A–C) (Parsons et al., 2002), and did not survive to the adult stage. col1a2mScarlet homozygotes appeared normal at 3 dpf but with reduced (Fig. S2D–F), and they did not survive to adulthood. Finally, tgfbimScarlet homozygotes were slightly shorter than heterozygotes and wild-type siblings at 3 dpf but could survive as adults (Fig. S2G–I). Thus, fluorescence tagging has demonstrative effects on gene product function in two of the three knock-in lines.

Larval ECM protein dynamism differs between fin fold and myosepta

ECM proteins are generally thought to be a stable fabric of tissue architecture, locked in place over time (Humphrey et al., 2014; Soans and Norden, 2021). In human arteries, fibrillar collagen was estimated to perdure for 22 days, and elastin can last for decades (Arribas et al., 2006). In adult mouse small intestine, pulse labeling of epithelial BM components like laminin revealed turnover rates of weeks (Trier et al., 1990). Fluorescence recovery after photobleaching (FRAP) is a standard approach to understand the dynamics or kinetics (diffusion and on-off rates) of proteins like ECM at physiological protein concentrations. FRAP measurements showed 2500 kDa aggrecan has a more rapid on-off association with the ECM compared to the 870 kDa hyaluronan in FITC-dextran solutes, due to the more compact and branched structure of aggregan (Hardingham and Gribbon, 2000). Further, broadly endogenous mNeonGreen labeling of matrix proteins in C. elegans revealed static and dynamic ECM networks during morphogenesis and organ development (Keeley et al., 2020). These studies demonstrate that ECM components can vary in dynamics and that mobilities of a protein can be tissue- and stage dependent.

To assess ECM dynamics during development, we performed confocal FRAP with all three knock-in lines generated. We first analyzed fluorescence recovery in the fin fold over a 10 min period, imaging every 10 s. Lamc1-eGFP is present in whole fin fold in a universally distributed pattern, whereas Col1a2-mScarlet is more punctate at 5 dpf. Tgfbi-mScarlet was not detected in fin fold at 5 dpf (Fig. 2A). In FRAP experiments, Lamc1-eGFP displayed little or no recovery during the first 10 minutes after bleaching. Longer imaging times revealed less than 10% Lamc1-eGFP recovered after 4 h, with an average recovery half-life in fin fold of 11.3 ± 9.4 h (Fig. 2B–D). The average recovery half-life of Col1a2-mScarlet in the fin fold was 4.1 ± 3.0 h (Fig. 2B). These results are consistent with previous findings that collagen and laminin are stable BM components (Keeley et al., 2020).

Figure 2. ECM component dynamism during embryogenesis.

Figure 2.

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(A) Representative confocal images of Lamc1-eGFP, Col1a2-mScarlet, and Tgfbi-mScarlet in 5 dpf larvae before FRAP analysis.

(B) Average recovery half-life for Lamc1-eGFP, Col1a2-mScarlet and Tgfbi-mScarlet in fin fold and myoseptum at 5 dpf. Tgfbi-mScarlet was not detectable in fin fold.

(C and E) In vivo FRAP analysis of Lamc1-eGFP in fin fold and myoseptum at 5 dpf. Bleached area is highlighted in the dashed box. Time: hour: minute. (n = 6 and n = 4 animals).

(D and F) Line graph indicating quantified relative fluorescence recovery over a time course in fin fold and myoseptum at 5 dpf.

(G, H) FRAP analysis of Col1a2-mScarlet (n = 8) and Tgfbi-mScarlet (n = 7) in myoseptum at 5 dpf. Dashed box, bleached area. Time: hour: minute.

(I) Schematic for experiments with lamb1apd110; lamc1eGFP/+ animals. Embryos were maintained at permissive temperature (28.5 °C) until 5 dpf, then shifted to restrictive temperature (33°C) before analysis.

(J) Representative images of 6 dpf lamb1apd110/+ and lamb1apd110 larvae in the lamc1eGFP/+ background.

(K) Quantification of body length for 6 dpf lamb1apd110/+ (n = 11) and lamb1apd110 (n = 9) larvae in the lamc1eGFP/+ background. Student’s t-test.

(L) FRAP analysis of 6 dpf lamb1apd110/+ and lamb1apd110 larvae in the lamc1eGFP/+ background, assessing myoseptum. n = 5 animals.

(M) Relative fluorescence of 6 dpf lamb1apd110/+ (n = 11) and lamb1apd110 (n = 9) myosepta in the lamc1eGFP/+ background. Student’s t-test.

Scale bars: 50 μm (A), 100 μm (C, E, G, H), 500 μm (J).

Myosepta are specialized structures in somites that act as attachment points for muscle fibers, first visible at 1 dpf and continuing to mature throughout the lifespan (Keenan and Currie, 2019). We found that Lamc1, Col1a2 and Tgfbi fusion proteins are all enriched in myosepta, and thus suitable for FRAP experiments. FRAP experiments with lamc1eGFP/+ myosepta revealed 20% recovery of signal just 10 min after photobleaching at 5 dpf, and its recovery half-life of 0.4 ± 0.2 h was much faster than in fin fold (Fig. 2B, E, F). This faster recovery time is likely due to differences in structure and growth dynamics between the two tissues, with myosepta organized into connective tissue sheets surrounding growing and functioning trunk musculature, versus fin fold, which is a contiguous structure that likely experiences a lower level of stretch and growth at that stage. In contrast, Col1a2-mScarlet and Tgfbi-mScarlet at 5 dpf showed no evidence of signal recovery at 10 min post-bleaching (Fig. 2G, H). These results indicate that Lamc1 is a dynamic matrix protein in myosepta and that Col1a2 and Tgfbi are more stable.

To determine if the lamc1eGFP allele could help dissect dynamic properties of Lamc1 in development, we crossed temperature-sensitive mutations in laminin beta 1a (lamb1a) into this background (lamb1apd110) (Chen et al., 2015). Laminins forms heterotrimers containing one alpha, one beta and one gamma chain (Timpl et al., 1979; Yurchenco and Cheng, 1993). The pd110 mutation alters a conserved lysine that in the N-terminal domain that mediates interactions with other Laminin members, which at the restrictive temperature results in mislocalized Laminin in the basal epidermal layer of regenerating fins (Chen et al., 2015). To permit developmental progression and analyze Lamc1-eGFP dynamics in myosepta, we retained animals at permissive temperature for 5 days before shifting to the restrictive temperature of 33°C for 1 day, after which FRAP was performed (Fig. 2I). lamb1apd110; lamc1eGFP/+ animals shifted to 33°C had shorter trunks than lamb1apd110/+; lamc1eGFP/+ clutchmates (Fig. 2J, K). Photobleaching of lamb1apd110; lamc1eGFP/+ embryos revealed that Lamc1-eGFP in lamb1apd110 mutants has a ~20% lower recovery rate compared to lamb1apd110/+ clutchmates (Fig. 2L). We noted that visualized Lamc1-eGFP protein amounts were not grossly changed in lamb1apd110 mutants (Fig 2M). These results indicate that the lamb1apd110 mutation reduces the on-off rate at which laminin populates myoseptal BM, which might ultimately influence mechanical properties and resilience and/or have effects on tissue patterning.

Visualization of ECM components across different adult organs

ECM components such as collagens and laminins have been investigated by many groups for their roles in contexts of tissue regeneration in zebrafish (Wang et al., 2013; Chen et al., 2015; Jessen, 2015; Mokalled et al., 2016; Mukherjee et al., 2021; Tsata et al., 2021; Xia et al., 2022). We examined several different tissues of lamc1eGFP/+, col1a2mScarlet/+, and tgfbimScarlet/+ zebrafish for protein localization. Lamc1-eGFP was evident in the extracellular space within the compact muscle layer of adult cardiac ventricles, as compared to inner trabecular cardiomyocytes (Fig. 3A, Fig. S3A). Upon resection of the ventricular apex, which stimulates vigorous regeneration (Poss et al., 2002), we noted increased expression in regenerating compact muscle. Col1a2-mScarlet expression was not detected in uninjured ventricles, but punctate signals were apparent at 14 dpa in the injury site adjacent to fibronectin deposits (Fig. 3B, Fig. S3B), resolving by 30 dpa. In uninjured spinal cord, all three knock-in products were undetectable. However, Lamc1-eGFP was induced in cells of the central canal at both anterior and posterior stumps after a paralyzing complete transection injury at 7 dpi, which typically regenerates within 6 weeks (Becker et al., 1998; Mokalled et al., 2016) (Fig. 3C). In scales, Lamc1-eGFP was present in vasculature that runs through the bony canals as well as that penetrating the overlying skin (Fig. S3C). Col1a2-mScarlet was evident as patterned fibrils as well as puncta throughout scales (Fig. 3D and Fig. S3C). In zebrafish kidney, which has filtering and hematopoietic functions, Lamc1-eGFP localized to renal tubules and glomeruli, whereas Col1a2-mScarlet and Tgfbi-mScarlet were in proximal tubules (Fig. 3E and Fig. S3D). Thus, the resources we present here enable investigation of ECM components in a variety of adult organs in uninjured or regenerating contexts.

Figure 3. Localization and regenerative responses of different ECM components in adult zebrafish.

Figure 3.

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(A) Confocal images of uninjured and 14 dpa lamc1eGFP/+ ventricles. Dashed lines indicate approximate injury site; bracket indicates compact muscle layer. n = 13 (uninjured) and 18 (14 dpa).

(B) Confocal images of uninjured and 14 dpa col1a2mScarlet/+ ventricles. White box, area of Col1a2-mScarlet accumulation. n = 10 (uninjured) and 14 (14 dpa).

(C) Lamc1-eGFP localization in uninjured and regenerating spinal cord (arrowheads; 7 dpi). n = 5 (uninjured) and 11 (7 dpi).

(D) Confocal imaging of scales in live col1a2mScarlet/+; fgf20aEGFP adults, indicating Col1a2-mScarlet localization in anteroposteriorly-oriented fibrils. n = 2.

(E) Confocal images of sections of adult lamc1eGFP/+; tcf21:DsRed2 (left), col1a2mScarlet/+ (middle), and tgfbimScarlet/+ (right) kidneys. Phalloidin (green) labels apical surface of tubules, and DAPI labels nuclei in blue. White arrowhead, glomerulus. n = 3.

(F) Representative images of Lamc1-eGFP, Col1a2-mScarlet and Tgfbi-mScarlet in adult caudal fins. White arrowheads, joints. Yellow arrowhead, intraray vasculature.

Scale bars:100 μm (A-D), 50 μm (E), 1 mm (F).

Long-lasting accumulation of Col1a2 at the foundation of regenerating fins

Zebrafish fins comprise several connected bony fin rays, each with repetitive segments connected by collagenous joints. Lamc1-eGFP in fins mainly localized to fin rays but was also found in blood vessels (Fig. 3F). Col1a2-mScarlet also localized to fin rays, especially at joints, while Tgfbi-mScarlet localization was similar to Col1a2-mScarlet, but present at lower levels (Fig. 3F).

To visualize ECM components in live regenerating fins, we amputated caudal fins and monitored regeneration at 4, 7, and 14 dpa. Lamc1-eGFP was present in the distal regions of regenerating rays and remained distally throughout regeneration, gradually decreasing in levels (Fig. 4A, and Fig. S4A, C). In contrast, Col1a2-mScarlet was not detected before ~4 dpa in regenerating tissue (Fig. 4B, Fig. S4B, C). At 7 dpa, Col1a2-mScarlet was present throughout the regenerate though especially enriched at the amputation site, where it was retained at 14 dpa and even as long as 60 dpa (Fig. 4B, C, Fig. S4D). Excess Col1a2-mScarlet at the amputation site was structurally less organized as compared with fibrous sheets in uninjured fins (Fig. 4C). Tgfbi-mScarlet localization was similar to Col1a2 and accumulated at amputation plane during fin regeneration (Fig. S4E, F).

Figure 4. Spatiotemporal deposition of ECM components during fin regeneration indicates a Col1a2-mScarlet-enriched foundation.

Figure 4.

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(A) Regenerating lamc1eGFP/+ tail fins at 4, 7, and 14 dpa. White arrowheads, tip of regenerate. Cyan arrows, amputation plane. n = 5.

(B) Regenerating col1a2mScarlet/+ tail fins at 4, 7, and 14 dpa. n = 4.

(C) Uninjured (left; n = 3) and regenerating col1a2mScarlet/+ fin rays at 2, 4, and 7 dpa (n = 4), shown as multiray overviews (top) and individual rays (bottom). The same individual was imaged longitudinally over time.

(D, E) Regenerating sp7:FUCCI fin rays at 4 and 7 dpa, indicating cycling (Geminin) and non-cycling (zCdt1) osteoblasts. White arrowheads, signals at joints. n = 5.

Scale bars: 500 μm (A, B), 100 μm (C, D).

We were intrigued by the retention of Col1a2-mScarlet at the amputation plane -- the junction between newly regenerating tissue and existing tissue. Amputation of zebrafish fins does not lead to fibrotic scarring and leaves a callous at the amputation plane detectable by bright field microscopy. This suggested a potential functional role during regeneration in securing the base or “foundation” of the regenerate. To assess if osteoblast cycling showed distinct patterns at the amputation site, we assessed sp7:FUCCI fins, which harbor reporters that mark cycling (Geminin+) and resting (Cdt+) phases in osteoblasts and recently were extensively analyzed in a study of fin regeneration (Rich et al., 2025). While we found a concentration of Geminin+ osteoblasts at distal areas of growth and occasionally at areas of future joints, we did not see an association of this signal with Col1a2 accumulation (Fig. 4D). We also noted expansion of vascular bundles and a more complex vascular pattern at the amputation plane compared with pre-amputation vasculature (Fig. S4G).

To identify molecular changes at the amputation site, we analyzed recently generated bulk RNA-seq from samples collected from the amputation site of regenerating fins at 3.5, 7, 14, and 21 dpa and compared these with samples collected from uninjured fins at the same proximodistal region (0 dpa) (Fig. S5A) (Rich et al., 2025). We found ECM organization and cartilage development genes are enriched at the amputation site, including col1a2, collagen, type XI, alpha 2 (col11a2), cellular communication network factor 4b (ccn4b), as well as skeletal development genes such as keratin 93 (krt93) and microfibril associated protein 2 (mfap2) (Fig. S5B, C). We also observed various ECM genes and matrix metalloproteinase genes such as mmp9 and mmp14b enriched at amputation site. We then assessed molecular differences between 7 dpa tissue at the amputation site and the 1 dpa early regenerate, based on published 1 dpa bulk RNA-seq datasets (Kang et al., 2016; Thompson et al., 2020). DNA replication and cell cycle related genes like MCM complex components mcm2, mcm3, mcm4, mcm5 and mcm6, Origin recognition complex subunits such as orc3, orc5, orc6 and cyclin E2 (ccne2), cyclin dependent kinase 2 (cdk2), cell division cycle 45 (cdc45), and DNA polymerase alpha 1 (pola1) were highly enriched both in early and amputation site samples. Notably, collagen genes such as col1a2 and col1a1 and collagen metabolism-related genes such as mmp2, mmp9 and mmp14, which break down and restructure ECM, accumulated predominantly at the amputation site and not in 1 dpa regenerates (Fig. S5D, E). Thus, regenerating fins maintain a region at their base transcriptionally distinct from the more experimentally targeted distal portions and includes long-term presence of collagen, likely providing mechanical properties and structural integrity for actively growing and maturing rays.

Conclusions

Here we established resources to visualize endogenous ECM protein dynamics for zebrafish development and tissue regeneration. We detected localization of selected Lamc1, Col1a2 and Tgfbi ECM proteins in various tissues during development and regeneration, and we measured mobility of ECM proteins in FRAP assays. Col1a2 localization revealed different molecular properties at the amputation site than the distal tip or uninjured regions, highlighting a region of the regenerating appendage that has received little research attention. We propose that persistent Col1a2 enrichment at the amputation site is not part of a scarring process, but rather a mechanism to strengthen the foundation of an actively growing and extending appendage. Future work can expand this toolset to additional ECM proteins, assess regenerative foundations in other models for appendage regeneration, and establish mechanistically how collagens and other proteins contribute to stability at the base.

MATERIALS AND METHODS

Zebrafish

Zebrafish of the Ekkwill strain background were maintained at 28.5 °C with a 14:10 hour light:dark cycle and were used according to protocols approved by Institutional Animal Care and Use Committee (IACUC) guidelines at Duke University or University of Wisconsin--Madison. Adult male and female fish between 3 and 12 months old were used for injury and regeneration experiments. Larvae from 2 to 6 dpf were used for imaging. For larval FRAP experiments, lamb1a mutants were maintained at 28°C until 5 dpf, then shifted to a restrictive temperature of 33°C for 24 hours for further analysis. Strains generated in this study are: lamc1eGFP (allele pd410), col1a2mScarlet (allele pd411), tgbfimScarlet (allele pd412). Other strains used in this study were: TgBAC(tcf21:DsRed2)pd37 (Kikuchi et al., 2011). sp7:FUCCI (Tg(osx:mCherry-zCdt1)pd270; Tg(osx:Venus-hGeminin)pd271) (Cox et al., 2018), fli1:EGFPy1 (Lawson and Weinstein, 2002), fgf20a:EGFP enhancer trap (allele nkhgn21aEt) (Shibata et al., 2016), and lamb1apd110 (Chen et al., 2015). Larvae were anesthetized with 0.4 mg/ml Tricaine (MS-222; Sigma, A5040) during handling. Adult fish were anesthetized in 0.075% 2-phenoxyenthonal (77699, Millipore Sigma) for surgery. Ventricular resection injuries were performed as previously described (Poss et al., 2002). Zebrafish spinal cord injuries were performed as previously described (Mokalled et al., 2016).

Generation of C-terminal PCR donors

We decided to use a C-terminal in-fusion knock-in methods by precise genomic editing for a few key reasons. First, C-terminal tagging works well with most of the BM ECM components, without impairing their function and localization (Keeley et al., 2020). Second, a construct with splice donor and acceptor arms that does not require homologous recombination-based integration is effective with small DNA donor templates. Third, the gRNA is located in intron regions, which is likely to have less impact on the coding sequence.

C-terminal knock-in constructs were gifts from Michel Bagnat’s lab at Duke University. In pUC19 vector backbone, this construct contains a multiple cloning site (MCS), a fluorescent protein (eGFP or mScarlet) coding sequence, stop codon, another MCS, and ubiquitin B (ubb) poly(A) sequence. To modify it to an in-fusion backbone, a flexible linker (aa: GGSGGTGGSGGT) was added between the gene of interest and fluorescent protein(Chen et al., 2013). Gene fragments of partial intron sequence and the last exon from the gene of interest were inserted into the modified pUC19 vector. PCR donors for injection were purified using a GeneJET PCR Purification Kit (Thermo Scientific, K0702). The final products were eluted by nuclease free water and stored at −20°C before injection. Primers for DNA donor: lamc1-forward: 5’-CCTTCCAAGCACTGCATTCAAATTT-3’; lamc1-reverse: 5’-TGGCCGCTCTAGAGACGGCGTGTTA-3’; col1a2-forward: 5’-AATGTACCAAATTTGGTTTAATACC-3’; col1a2-reverse: 5’-TTTGAAACAGACTGGGCCAATGTCC-3’; tgfbi-forward: 5’-GGGGTGAACTCAATTATGCTGAGCA-3’; tgfbi-reverse: 5’-CTGGACACGAGACAATGTTCTGCTG-3’. Primers for genotyping heterozygous and homozygous knock-in animals: lamc1_WT_FW: 5’-TCGACAGGGAAAATCAAGAGAA-3’; lamc1_WT_Rv: 5’- GACATTTGGGTTGTGATCCGT-3’; lamc1_KI_Fw: 5’- AAGGTGGATCTGAGCAAGCT-3’; eGFP_KI_Rv: 5’- TCCTTGAAGTCGATGCCCTT-3’; Col1a2_WT_FW: 5’- AACAGCCGCTTCACTTTCAG-3’; Col1a2_WT_Rv: 5’- TCAGTCCATTGGTCCACTCC-3’; Col1a2_KI_Fw: 5’- CGACATTGCACCTTTGGACA-3’; mScarlet_KI_Rv: 5’- CTTCTTCTGCATTACGGGGC-3’; tgfbi_WT_Fw: 5’- ACCTACACCTTCTTCGCACC-3’; tgfbi_WT_Rv: 5’- CAAATGACTCAGACTCGGGG-3’; tgfbi_KI_Fw: 5’- TGAGAAGTCACAGCAGCAGA-3’.

Guide RNA (gRNA) design

gRNA target sites were identified by CHOPCHOP (http://chopchop.cbu.uib.no)(Montague et al., 2014; Labun et al., 2016; Labun et al., 2019). crRNAs and tracrRNAs were purchased from IDT (https://www.idtdna.com/). gRNAs used in this study were: lamc1-gRNA: 5’- TACTCACTATTATATAGTCGGGG-3’; col1a2-gRNA: 5’-TTCCTTTAGCACACTTAGCATGG-3’; tgfbi-gRNA:5’-GACCAAATATTGCCAATCGATGG-3’. Two gRNAs were designed for each gene. gRNAs were diluted with nuclease-free duplex buffer and stored at −80°C. To test the gRNA efficiency, every gRNA was injected with tracrRNA and NLS-Cas9 (IDT, 10007807) into 100 embryos. 16 injected embryos and 16 uninjected sibling embryos were chosen to prepare genomic DNA, and PCR products from injected and sibling embryos were amplified and annealed. T7EI (T7 Endonuclease I, NEB, M0302) digestion was used to confirm the efficiency of gRNA cutting. Those gRNAs with higher cutting efficiency were used for knock-in establishment.

Whole mount in situ hybridization (WISH)

Antisense RNA probes were synthesized by in vitro transcription from PCR-generated DNA templates containing a T3 RNA polymerase promoter sequence. Briefly, purified DNA templates (100–200 ng) were incubated with T3 RNA polymerase in transcription buffer supplemented with DTT, RNase inhibitor, and a digoxigenin (DIG)-labeled nucleotide mix for 2 h at 37 °C. Following transcription, DNA templates were removed by DNase I treatment, and the labeled RNA probes were purified using spin columns. The resulting DIG-labeled antisense RNA probes were quality-checked and stored at −80 °C until use for in situ hybridization.

Whole-mount in situ hybridization on 3 dpf zebrafish embryos was performed over three days following fixation in 4% paraformaldehyde and storage in 100% methanol at −20 °C. Embryos were rehydrated through a graded methanol/PBS-Tween (0.1%) series, washed, and permeabilized using proteinase K digestion (10 μg/mL, ~30 min for 3 dpf embryos), followed by postfixation in 4% paraformaldehyde to preserve tissue integrity. After additional washes, embryos were prehybridized for 2–5 h at 65°C in hybridization buffer (50% formamide, 5× SSC, 0.1% Tween 20, heparin, and yeast tRNA - Thermo Fisher Scientific # 15401029) to reduce nonspecific binding, and then hybridized overnight at 70 °C with 30–50 ng of digoxigenin-labeled antisense RNA probe. Stringent post-hybridization washes were performed at 70 °C using decreasing concentrations of hybridization buffer/SSC and high-stringency (0.2× SSC) washes to eliminate nonspecific hybridization, followed by gradual equilibration to PBS-Tween (0.1%). Embryos were blocked for 3–4 h in serum-containing buffer (PBS-Tween supplemented with BSA −2mg/ml- and 2% v/v sheep serum) and incubated overnight at 4 °C with alkaline phosphatase–conjugated anti-digoxigenin antibody (1:6000 Roche #11093274910). After extensive washes in PBS-Tween, embryos were equilibrated in alkaline Tris buffer and developed in NBT/BCIP (EMD Millipore # 11681451001) chromogenic solution in the dark until optimal signal intensity was reached. Chromogenic staining reaction was inhibited using repeated PBS-Tween (0.1%) washes at room temperature. Samples were conserved in 50% glycerol at 4 °C until imaged.

Whole mount immunofluorescence

Whole-mount immunofluorescence on zebrafish embryos was carried out over 8 days. Embryos were fixed overnight in 4% paraformaldehyde (PFA), washed in PBS-Tween (0.1%), and dehydrated through a graded methanol/PBS-Tween (0.1%) series before storage at −20 °C for at least 16 h. Samples were subsequently rehydrated through an inverted graded methanol/PBS-Tween (0.1%) series, subjected to cold acetone treatment (−20 °C, 10 min), and permeabilized using proteinase K (20 μg/mL for 20 min, or 30 μg/mL for 30 min for embryos older than 6 dpf), followed by a brief post-fixation in 4% PFA. After extensive washing using PBS-Tween (0.1%), embryos were incubated in blocking solution (PBS-Tween supplemented with 1% BSA, 4% horse serum, 1% DMSO, and 0.8% Triton X-100) for at least 4 h at room temperature. Primary antibodies (1:300–1:500 for laminin, Sigma, L9393) were applied for three nights at 4 °C with agitation, followed by a series of PBS-Tween (0.1%) washes and incubation with secondary antibodies (1:250) for two nights at 4 °C, with optional DAPI staining. After final washes in blocking solution and PBS-Tween, embryos were either processed for imaging or stored in 50 glycerol at 4 °C for long-term preservation until imaging.

Fluorescence Recovery after Photobleaching (FRAP) Analysis

Zebrafish larvae were anesthetized by 0.01% Tricaine and embedded in 1% low-melt agarose on a standard agarose plate. After low-melt agarose solidified, egg water with 0.01% Tricaine was added to dish. FRAP analysis of Lamc1-eGFP, Col1a2-mScarlet and Tgfbi-mScarlet were performed using a Zeiss 880 confocal microscope, using a 488 nm or 568 nm laser and a 20x water immersion lens. Higher laser power was used for photobleaching with 300 repeated cycles. For each experiment, imaging before bleaching was acquired. Images were collected every 10 second for myosepta, and every 30 minutes for fin fold. For FRAP image acquisition of fin folds at 5 dpf, prior to photobleaching, the total fluorescence intensity of an area of 75 × 150 μm was measured before bleaching for the pre-bleach measurement. For bleaching, 100% laser power at 488 nm was used to bleach the region of interest. Laser bleaching involved 300 iterations at this intensity, and one image was then acquired immediately for time 0 post-bleach. Images for recovery assays were then acquired at 30, 60, 120, and 240 min post-bleaching. For FRAP image acquisition using myosepta, a region 100 × 150 μm was bleached--this region contained two myosepta. Bleaching parameters were the same as for fin fold assays. Images of fluorescence recovery were acquired every 10 s for myosepta, versus every 30 min for fin fold. For quantifications, the net fluorescence intensity was calculated by subtracting the background fluorescence intensity from the raw total fluorescent intensity. The relative fluorescence intensity recovery was quantified by normalizing the total fluorescence intensity during the recovery period against the total fluorescence intensity of the protein of interest prior to photobleaching at different time points. The fluorescence recovery half-time (t1/2) was calculated based on the duration needed for the fluorescence intensity of different ECM proteins to recover 50% of the maximum recoverable fluorescence for each animal. The mean recovery quantification was averaged from 5–7 animals.

Immunohistochemistry

Zebrafish heart, spinal cord, fin or kidney were fixed in 4% paraformaldehyde (PFA) overnight at 4°C. Heart, spinal cord or kidney were suspended in 30% sucrose and frozen, and 10 μm (heart) or 16 μm cryosections (fin, spinal cord and kidney) were used for immunostaining. Fins were embedded into 1% low melting agar before sectioning to retain tissue structure. Tissue slides were treated by cold methanol for 10 minutes before immunostaining. Slides were dried in room temperature on 37°C for 10 minutes and circled with a hydrophobic pen. Slides were rinsed 2 times in PBST (phosphate buffered saline with 0.1% Tween-20) and blocked with NCS-PBT (10% NCS, heat inactivated newborn calf serum, 90% PBST, 1% DMSO and 2% horse serum) for 1 hour. Primary antibody was applied overnight at 4°C. Slides were washed 4 times for 5 minutes in PBST, then secondary antibody was incubated at 4°C overnight. Slides were rinsed 4 times for 5 minutes in PBST, then treated with Vectashield mounting medium (H-1900, Vector) and DAPI (1:1000, D3571, Thermo fisher). Primary antibodies used in this study were: anti-GFP (1:1000, SKU: GFP-1020, Antibodies inc), anti-RFP (1:500, 5f8, Proteintech), anti-Fibronectin (1:300, F3648, Millipore Sigma), anti-GFAP (Glial Fibrillary Acidic Protein) (1:500, G3893, Millipore Sigma). Secondary antibodies Alexa Fluor 488, Alexa Fluor 567, and Alexa Fluor 633 goat anti-rabbit/mouse/rat/chicken (1:250–1:500, Invitrogen) used in this study.

RNA sequencing dataset analysis

RNA-seq reads were trimmed by Trim Galore (v 0.6.7, with cutadapt v 3.5) and counted by salmon (v 1.4.0) by supplying the UCSC danRer11 annotation (Patro et al., 2017). Bioconductor package DESeq2 (v 1.34.0) was used for differential expressions (DE) analysis (Love et al., 2014). Gene set enrichment analysis was performed using GSEA (v 4.4.0).

Datasets

We analyzed the datasets GSE291478 (Rich et al., 2025), GSE76564 (Kang et al., 2016), GSE146960 (Thompson et al., 2020), GSE75894 (Kang et al., 2016), and GSE193503 (Cigliola et al., 2023).

Supplementary Material

Supplementary Materials

Acknowledgments

We thank the Duke Zebrafish Core and Jim Burris and Mackenzie Klemek at Morgridge Institute for Research for zebrafish care; C.-H. Chen and J. Cao for helpful comments on the manuscript. The authors acknowledge funding from NIH (R35 HL150713 and R01 HD105033 to K.D.P; R01 AR076342 to S.D.T. and K.D.P.) and from Pew Charitable Trusts to D.R.S. and K.D.P.

Footnotes

Competing interests statement

Ken Poss is an Academic Editor of Development. Ken Poss was not involved in the editorial assessment of this submission. The authors declare no other competing interests.

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Supplementary Materials

Supplementary Materials
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