|Molecular Vision 2006;
Received 6 September 2005 | Accepted 4 September 2006 | Published 26 October 2006
Egr1 gene knockdown affects embryonic ocular development in zebrafish
Chang-Hao Yang,2 Wei-Yu
Chen,3 Chiu-Ju Huang,4 Hsing-Yen Huang,4 Muh-Shy
Chen,2 Huai-Jen Tsai4
1Department of Ophthalmology, Far Eastern Memorial Hospital, 2Department of Ophthalmology, National Taiwan University Hospital, 3Department of Pathology, Taipei Medical University, 4Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
Correspondence to: Huai-Jen Tsai, PhD, Institute of Molecular and Cellular Biology, National Taiwan University, No.1, Sec. 4, Roosevelt Road, Taipei, Taiwan 106; Phone: +886-2-23641705; FAX: +886-2-23638483, email: firstname.lastname@example.org
Purpose: To identify the changes in zebrafish embryonic ocular development after early growth response factor 1 (Egr1) gene knockdown by Egr1-specific translation inhibitor, morpholino oligonucleotides (MO).
Methods: Two kinds of Egr1-MO were microinjected separately with various dosages into one to four celled zebrafish embryos to find an optimal dose generating an acceptable mortality rate and high frequency of specific phenotype. Chordin-MO served as the positive control; a 5 mismatch MO of Egr1-MO1 and a nonspecific MO served as negative controls. We graded the Egr1 morphants according to their gross abnormalities, and measured their ocular dimensions accordingly. Western blot analysis and synthetic Egr1 mRNA rescue experiments confirmed whether the deformities were caused by Egr1 gene knockdown. Histological examination and three kinds of immunohistochemical staining were applied to identify glutamate receptor one expression in retinal ganglion cells and amacrine cells, to recognize acetylated α-tubulin expression which indicated axonogenesis, and to label photoreceptor cells with zpr-1 antibody.
Results: After microinjection of 8 ng Egr1-MO1 or 2 ng Egr1-MO2, 81.8% and 97.3% of larvae at 72 h postfertilization had specific defects, respectively. The gross phenotype included string-like heart, flat head, and deformed tail. The more severely deformed larvae had smaller eyes and pupils. Co-injection of 8 ng Egr1-MO1 and supplementary 12 pg synthetic Egr1 mRNA reduced the gross abnormality rate from 84.4% to 29.7%, and decreased the severity of deformities. Egr1 protein appeared in the wildtype and rescued morphants, but was lacking in the Egr1 morphants with specific deformities. Lenses of Egr1 morphants were smaller and had some residual nucleated lens fiber cells. Morphants' retinal cells arranged disorderly and compactly with thin plexiform layers. Immunohistochemical studies showed that morphants had a markedly decreased number of mature retinal ganglion cells, amacrine cells, and photoreceptor cells. Retinal axonogenesis was prominently reduced in morphants.
Conclusions: The Egr1 gene plays an important role in zebrafish embryonic oculogenesis. Ocular structures including lens and retina were primitive and lacked appropriate differentiation. Such arrested retinal and lenticular development in Egr1 morphants resulted in microphthalmos.
Organogenesis results from a succession of complex processes including induction, patterning, specification, morphogenesis, and differentiation . Transcription factors including Pax6, Optx2, and Crx play important roles at different developmental stages of oculogenesis and are ultimately important in cell fate decisions [2,3]. Mutations of these factors induce ocular developmental anomalies in humans, including aniridia, anophthalmia, and Leber congenital amaurosis .
Most important genes are evolutionally conservative, although functions of some genes may not be identical among species. Research in zebrafish (Danio rerio) is feasible for functional genomics and reverse genetics [3,4] because the fish develops rapidly, reaching maturity in three months. Furthermore, the fish can be induced to spawn by photoperiod, and has fecund, big, transparent, extra-uterine eggs that are suitable for studying embryonic development and gene modulation [1,3,5,6]. The development of the zebrafish brain, nervous, and visual systems is similar to that of other vertebrates . The remarkable evolutionary conservation among vertebrate eyes provides the basis for using zebrafish as a model system for detection and analysis of genetic defects related to human eye disorders [1,7], such as retinitis pigmentosa, cone dystrophy, and macular degeneration [3,8]. Zebrafish retina and lens show much the same morphology as other vertebrates including humans . Because the zebrafish retina is simple and develops fast, in addition to its accessibility to genetic analysis, it has become an excellent model system for studying retinal neurogenesis in vertebrates . The appearance of retinal ganglion cells between 27 and 28 h postfertilization (hpf) is the initial event in zebrafish retinal neurogenesis . After 10 h, interneurons emerge and form the inner nuclear layer. At about 43 hpf, photoreceptor cells appear in the outer nuclear layer. Zebrafish retina becomes functional between 60 and 80 hpf [3,10,11]. The emmetropization of eye and lens is achieved at 72 hpf . By then most embryos have hatched .
The reverse genetic approach is faster and discloses a more precise structure-function relationship than forward genetics . Morpholino oligonucleotides (MO) are designed to bind a target mRNA, resulting in translation blockage by preventing 40S subunit of ribosomes from binding, and ultimately suppress target gene expression. Alternatively, it can be designed to bind the junction of intron and exon of pre-mRNA, and causes the splicing machinery to effectively skip an entire exon [3,13,14]. Currently MO is the most widely used approach for studying the loss-of-function of zebrafish genes [13,15,16].
Early growth response factor 1 (Egr1) gene (also known as NGFI-A, zif268, Krox24, tis8, cef5, and ZENK) is an immediate early transcriptional regulator gene . This factor encodes a highly conserved C2-H2 zinc finger DNA-binding protein, which is necessary for vertebrate development; this then leads to proliferation and differentiation [18-20]. However, the functions of Egr1 on oculogenesis are still unclear. Postnatally induced myopia by a diffuser or a minus lens in chicks was associated with elongated eyeballs and decreased ZENK expression in retinal glucagon-containing amacrine cells [21,22]. By contrast, the Egr1 mRNA and protein, especially in ganglion cells and bipolar cells, were strongly regulated by the transitions between light and darkness in form-deprived myopic mice, whereas image contrast exerted only a minor influence on Egr1 expression . Since the mechanisms of eye growth control are different among species, studies of gene expression patterns between species during emmetropization and myopic progression are valuable . By using NCBI protein-protein BLAST, the zebrafish Egr1 protein (NCBI accession number P26632) shares 55%, 56%, and 62% identities of amino acid sequence with human Egr1 protein (P18146), mouse Egr1 protein (NP_031939), and chicken ZENK protein (NP_989467), respectively. However, they share at least 95% identity in the three zinc finger C2-H2 domains that bind to the major groove of B-DNA . In the zebrafish embryo, Egr1 mRNA is initially detected in the presomitic mesoderm between 11 and 20 hpf [19,26]. It is expressed in hypothalamus, mesencephalon, tegmentum, pharynx, otic vesicle, and heart between 24 and 48 hpf . In retina, it is observed at 40 hpf and disappears at 48 hpf .
In this study, we microinjected Egr1-MO targeting at Egr1-mRNA into zebrafish embryos and observed their changes in appearance and ocular histology after loss of Egr1 gene function. Our findings should be helpful to further research in ocular development.
Animals and embryos
Adult AB-strain zebrafish were bred with a controlled light cycle of 14 h of light and 10 h of darkness at 28.5 °C [26,27]. After the lights were turned on, we removed the existing embryos, collected the newly laid eggs within 2 to 3 h, and then sorted them into petri dishes according to the developmental stages defined by Kimmel et al. . The one to four celled embryos were used for microinjection . All procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Microinjection of morpholino oligonucleotides
Microinjection is an effective delivery system for MO in zebrafish . In this study, two different sequences of Egr1-MO were produced by Gene Tools (Philomath, OR) referring to the genomic DNA sequence reported by Drummond et al. (GenBank accession number U12895) . The Egr1-MO1 is composed of 5'-GCA GCC ATC TCT CTG GAG TGT GCT C, which is the antisense of the last 17 nucleotides in the 5'-untranslated region (5'-UTR) and the leading 8 nucleotides of coding region. The Egr1-MO2 is composed of 5'-GAG AGA ATT GTG CTG GAG AAA TCA G, which is complementary to the 5'-UTR sequence of Erg1 located at the upstream 24 to 48 nucleotides from the start code (ATG). Since their effects were presumed to be dose-dependent, various dosages of Egr1-MO, mixed with Phenol Red, were microinjected with Nanoject II (Drummond, Broomall, PA) into one to four celled zebrafish embryos to find an optimal dose generating a low mortality rate and a high frequency of specific phenotype . We also microinjected 4.5 ng of chordin-MO per embryo as a positive control . Theoretically, at least 75% of chordin morphants at 72 hpf should have severe defects including expanded blood island, u-shaped somites, reduced head size, and an abnormal tail fin with multiple folds . A 5 base mismatch MO (5'-GCA CCC ATG TCT GTG GAC TGT CCT C) of Egr1-MO1 and a nonspecific MO (5'-CCT CTT ACC TCA GTT ACA ATT TAT A) served as negative controls [27,28]. These morphants and wildtypes were raised with a similar density of embryos in incubators at 28.5 °C.
Measurement of ocular dimensions
To decrease the possible individual differences, we compared the ocular dimensions between the wildtype and Egr1 morphants collected from the same batch of embryos and cultured them in a similar density under the same circumstances. After anesthesia with 0.02% volume of 2-phenoxyethanol (Sigma-Aldrich, St. Louis, MO) for 1 min, the 72-hpf larvae were observed with a stereo dissecting microscope (MZFL III, Leica, Wetzlar, Germany) and photographed with a digital camera (FinePix S2pro, Fujifilm, Tokyo, Japan). Parameters were measured with image-processing software (PhotoImpact 6.0, Ulead, Taipei, Taiwan). The severity of the deformity was graded according to the changes in appearance. We measured the ocular and pupillary diameters of the left eye, which represented the gross development of retinal and lenticular structures, respectively . The horizontal and vertical diameters were determined accordingly with the maximal length of line segments parallel to, or right-angled with the assumed line connecting its mouth and otic capsule. These measurements were analyzed with one-way analysis of variance, and the differences between every two groups were verified with Scheffe's posthoc test (SPSS 11.0, SPSS, Chicago, IL). P values less than 0.05 were considered significant.
Rescue experiment of Egr1 mRNA
The total RNA of 48-hpf zebrafish embryos was extracted with Trizol (ProTech, Taipei, Taiwan) and treated with RNase-free DNase (Promega, Madison, WI). The first strand of cDNA was obtained with the reaction of total RNA, oligo(dT)12-18 primers, dNTP, and M-MLV reverse transcriptase (Promega). The coding region of Egr1 DNA was acquired with polymerase chain reaction (PCR) using VioTaq DNA polymerase (Viogene, Taipei, Taiwan), the above product after reverse transcription and the primers of CCA GCA TGC ATC TCT CCT CTC CGA GCA CAC TCC AGA G and TTT CTG ACT CGA GGG AGA GCT TGG AGT TCT GGG GAA G. The expected 1.6-kb PCR product was extracted with a gel extraction kit (Geneaid, Taoyuan, Taiwan) and then ligated to the pGEM-T Easy vector (Promega). The resultant plasmid pGEM-egr1 was obtained from Escherichia coli DH5α after transformation. The Egr1 DNA was separated from pGEM-egr1 by SacII and was transcribed to 5'-capped Egr1 mRNA with 3'-poly(A) tail with a MESSAGE mMACHINETM Kit (Ambion, Austin, TX). Various amounts of the in vitro synthetic Egr1 mRNA, mixed with the selected dose of Egr1-MO, were microinjected into one to four celled embryos. The abnormality rates at 72 hpf were compared to confirm the effect of Egr1 mRNA rescue.
Western blot analysis
To establish the effect of Egr1 gene knockdown by Egr1-MO, we compared the amounts of 55-kD Egr1 protein (NCBI accession number NP_571323) between the Egr1-MO-injected embryos and the wildtype embryos at 24, 48, and 72 hpf. We also evaluated its expression in the mRNA rescued morphants. The loading dose of extracted total proteins was quantified separately by absorbance measurements at 595 nm (DU 640B, Beckman Coulter, Fullerton, CA). One piece of gel after SDS-PAGE served for coomassie blue stain to determine adequate loading. The other part of gel was blotted onto a polyvinylidene difluoride membrane, hybridized with the Egr1 antibody (sc-110, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1000 dilution, washed extensively with Tris buffered saline, incubated with horseradish peroxidase-conjugated secondary antibody (number 12-348, Upstate Biological Inc., Lake Placid, NY) at 1:5000 dilution, and finally developed by using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Pierce, Rockford, IL).
Histological examination and immunohistochemical staining
The 72-hpf larvae derived from Egr1-MO-injected embryos and from the wildtype were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5 μm sections, stained with hematoxylin and eosin. They were examined with a light microscope (BX51, Olympus, Tokyo, Japan) and photographed with a camera (PM-C35DX, Olympus). To identify the differentiation of retinal ganglion cells and amacrine cells, we performed immunohistochemical staining using 3 μg/ml of glutamate receptor 1 antibody (AB-1504, Chemicon, Temecula, CA) at 4 °C overnight [26,30]. In addition, the axonogenesis was identified by acetylated α-tubulin antibody (T6793, Sigma-Aldrich) at 1:400 dilution, 4 °C overnight [31,32]. After deparaffinization, rehydration, and antigen retrieval with water bath in 121 °C citrate buffer for 5 min, the sections were washed with phosphate buffered saline, and incubated with those primary antibodies. The sections then were incubated sequentially with the biotinylated horseradish peroxidase, avidin, and diaminobenzidine following the manufacturer's instructions (Ultratech HRP Kit, Immunotech, Marseilles, France). Finally, the sections were counterstained with hematoxylin and recorded with a camera mounted on a light microscope. On the other hand, to identify the differentiation of photoreceptor cells, we immersed the 72-hpf wildtype larvae and Egr1 morphants in OCT separately, stored them in -20 °C freezer, and cryosectioned them into 6 μm slices. The zpr-1 antibody labeled most cone cells at 1:100 dilution for 2 h at room temperature .
The effect of morpholino oligonucleotide injection on embryonic development
About 82.6% of 72-hpf larvae (n=113) after chordin-MO injection exhibited severe defects, whereas only less than 1% of the nonspecific negative control (n=166) changed in appearance. In a pilot study, we microinjected various amount of Egr1-MO into the one to four celled embryos and found that higher dose of Egr1-MO produced more severely deformed embryos and more deaths. We chose 8 ng of Egr1-MO1 and 2 ng of Egr1-MO2 per embryo as the study concentration because they generated an acceptable mortality rate (<10%) and a high specific abnormality rate (>80%). The most common abnormalities that appeared in the Egr1-MO-injected embryos were enlarged pericardium, small eye, and deformed body axis. According to the severity of gross deformities at 72 hpf, Egr1 morphants were classified into five grades (Figure 1):
Grade 1: No difference between the morphant and wildtype; Grade 2: Enlarged pericardium only; Grade 3: Mildly deformed body axis; Grade 4: Markedly deformed body axis; Grade 5: Agenesis of tail or eyes.
About 81.8% of the 8 ng-Egr1-MO1-injected embryos (n=142) and 97.3% of the 2 ng-Egr1-MO2-injected embryos (n=400) exhibited Grade 3 or more severe deformity, whereas only 2.5% of embryos (n=200) injected with 8 ng of our five nucleotide mismatch control MO showed abnormal appearance. Besides pericardial edema found in Grade-2 morphants, Grade 3 morphants additionally had a string-like heart, bent tail and flat head. Similar deformities appeared but were more severe in Grade 4 morphants.
Egr1-morphants with higher grade states had smaller eyes and pupils (Table 1, Figure 2). These changes were especially prominent in Grade 2 and above. The horizontal ocular diameter was the most sensitive indicator of Egr1 depression because it reduced significantly even when there was no detectable change in appearance of the Grade-1 morphants. It also decreased more significantly than did the vertical ocular diameter. By contrast, the horizontal pupillary diameter changed in proportion to the vertical pupillary diameter.
Expression of Egr1 gene in wildtype, morphants, and Egr1-mRNA-rescued morphants
Coinjecting various amounts of synthetic Egr1 mRNA and 8 ng Egr1-MO1 resulted in different rescue effects (Figure 3). When 12 pg of Egr1 mRNA was supplied, the abnormality rate decreased prominently from 84.4% to 29.7% without altering the survival rate. Among the larvae with abnormalities in appearance, the average grade of deformity in the rescued morphants also decreased markedly from 3.5 to 2.0.
Western blot analysis demonstrated that the wildtype had the presumed 55-kD Egr1 protein at 24, 48, and 72 hpf, whereas the Grade-3 Egr1 morphants did not (Figure 4). When supplementary 12 pg Egr1 mRNA was coinjected with 8 ng Egr1-MO1 into one to four celled embryos, the Egr1 protein appeared in the rescued morphants (Figure 4). None of our blotting showed a band like the 75 kDa nuclear Egr1 protein found in mouse retina .
Histological observation and immunohistochemical staining
In histological examination, lenses of the 72-hpf Egr1 morphants were smaller than those of the wildtype and had some residual nucleated lens fiber cells; however, their retinas were more markedly affected (Figure 5). Morphants' retinal cells arranged disorderly and compactly with little extracellular matrix, but there were no apparent vacuoles or pyknotic cells in their retinas. Their inner and outer plexiform layers were thin. The retinal pigmented epithelium (RPE) layer was also thin and irregular. In the morphants of higher-grade deformity, these changes became more prominent. The Grade 4 morphants were even devoid of retinal lamination.
Immunohistochemical study showed that morphants' retina had markedly less glutamate receptor 1 expression, namely decreased cellular differentiation of ganglion cells and amacrine cells (Figure 6A,B). The reduced expression of acetylated α-tubulin in retina represented decreased axonogenesis (Figure 6C,D). The remaining retinal axonongenesis of morphants was sometimes found in the presumed ganglion cell layer or inner nuclear layer. Zpr-1 antibody can label most cone cells in the wildtype, but it failed in the Grade 3 morphants (Figure 7). The decrement of all protein markers in the present study was parallel to the increasing grading and became obvious in the Grade 3 morphants and higher.
MO has been applied successfully in zebrafish genetic studies [3,27]. The increasing number of defined gene sequences and the success of MO technology in zebrafish open the door for modeling nearly any inherited developmental defect [8,16,28,35,36]. Previous researchers have successfully applied this technique to prove the essential role of receptor protein-tyrosine phosphatase α in zebrafish retinal development . In the present study, Egr1-MO effectively inhibited Egr1 gene expression for at least three days and generated phenotypes including string-like heart, small eyes, flat head, and deformed tail. The vertical eye diameter of wildtype in this study is comparable with a former histological study that measured 240 μm at 74 hpf . Small ocular and pupillary dimensions of Egr1 morphants suggest arrested retinal and lenticular development, respectively . Such reduced retinal and lenticular mass generated the picture of microphthalmos. The disarranged retinal cells were primitive proven by the depressed axonogenesis, and decreased cellular differentiation in ganglion cells, amacrine cells, and photoreceptor cells. Most of the Egr1 morphants could not survive longer than 96 h and did not hatch out. Such a fatal "inborn error" prohibited further evaluation [21-23]. It is reasonable that if a vital gene is inhibited immediately after MO injection, embryos cannot survive long or recover from their severe developmental retardation. Although few low-grade hypomorphic larvae survived for more than seven days, their changes in expression were too obscure to be differentiated. In addition, because MO spreads evenly in the embryo [13,28], we cannot confine its effect to a specific tissue only. In this study, the defective phenotypes we observed are a result of Egr1 gene knockdown in the whole body. Nevertheless, we cannot rule out the possible influence from other tissues. Such problems should also exist in knockout animals, although researchers did not find that Egr1 knockout mice had obvious developmental or behavioral defects except female infertility [37,38]. To conquer the problems of limited temporal and unlimited spatial expression, we planned to apply a tetracycline-controlled transcriptional regulation system [39,40]; however, the lack of a cell-type-specific promoter of the presumed target cells like the glucagon-containing amacrine cell prohibited its use . The other method of specific gene knockdown, double-strand RNA interference (RNAi), has been also considered [3,41]. But in zebrafish, RNAi is limited by frequent nonspecific effects [3,36,42,43]. Other shortcomings includes problems of delivery, off-target effects, poor tissue distribution, varying response between genes, and difficulty in tackling neurological targets .
The Egr1 gene is multipotent and important for cell proliferation, differentiation, embryonic ocular development, and neuronal plasticity [18-20,45]. As an inducible transcription factor, its expression can be used as a functional assay for neuronal inputs and receptor systems . Basal retinal Egr1 expression is maintained by synaptic plasticity occurring in response to physiological stimuli, and it is higher in the highly visual animals like monkeys [46,47]. In adult monkey retinas, the Egr1 protein is located mainly in cells of the inner nuclear layer and also in the ganglion cells. Amacrine cells are likely to mediate neural plasticity . However, Zhong et al.  showed that at least 90% of Egr1 protein was found in the ON-bipolar cells and the remaining in the GABAergic amacrine cells of infant macaque retinas. These cells were stimulated to produce more Egr1 when retinal images were enriched in focus without form-deprivation . Similarly, Egr1 protein in young mice appears in retinal ganglion cells, amacrine cells, and bipolar cells . However, mouse retinal Egr1 mRNA and protein, especially in ganglion cells and bipolar cells, are strongly regulated by the transitions between light and darkness, whereas differences in retinal illumination either play no role or only a minor one . Rat retinal Egr1 mRNA also increases transiently after the rat is exposed to light from the dark , but enhanced environments dramatically increase the Egr1 expression in retinal ganglion cell layer and inner nuclear layer of young rats . The number of ZENK-expressing bipolar cells and amacrine cells in chick retinas increase after light onset and declined later . The light intensity but not focus-defocus modulates ZENK synthesis in chick retinal ON-bipolar cells . Thus, there exist minor but notable differences in Egr1 response among species, and this evidence suggests a circadian influence on Egr1 expression as well . In rodents, zif268 mRNA is increased in suprachiasmatic nuclei (SCN), the circadian pacemaker, by retinal illumination at night . The SCN clock is reset by daily exposure to light by transmission of photic information from retina via the retinohypothalamic tract, a monosynaptic glutamatergic projection . The photosensitive retinal ganglion cells innervating SCN express melanopsin and might be the primary photoreceptor for this system . However, there are pronounced species differences in such zif268 response as well as melanopsin expression [51,54]. In zebrafish, the melanopsin mRNA is confined to the eye, merely in a subset of retinal horizontal cells (type B) . In contrast, chicken melanopsin is expressed in brain, pineal gland, and all retinal layers . ZENK gene in chick retina is downregulated in postnatal myopic chicks with elongated eyeballs [21,22], but the causal relationship between ZENK gene expression and ocular growth has not been well established. Except ZENK studies, no other research documents the association between Egr1 expression and ocular growth.
Egr1 expression is also upregulated in response to diverse stimuli including basic fibroblast growth factor (bFGF), focal ischemia, hypoxia, physical forces, injury, etc. . It is activated through multiple protein-protein interactions with co-activators such as CREB-binding protein (CBP) and p300 [56,57]. The expression of numerous target genes like platelet-derived growth factor (PDGF) A-chain, PDGF B-chain, transforming growth factor-β1, and tumor necrosis factor is regulated by Egr1 protein . Similarly, microphthalmia-associated transcription factor (Mitf) is also responsive to bFGF, activated by CBP/p300, and associated with cellular differentiation as well as cell division . The mutation of Mitf in mice results in retinal degeneration and microphthalmia . The Mitf/Pax6 interactions are important for RPE development . Our study proves the effect of Egr1 expression on embryonic oculogenesis. Egr1 and Mitf may share some common downstream mechanisms in oculogenesis, although the latter mainly affects RPE and is critical for RPE-neural retina communication . Leung et al. proposed that cyclin-dependent kinase 5 (Cdk5)/p35 activity was essential for eye development and retinal lamination in zebrafish and was likely regulated by Egr1 .
In conclusion, early knockdown of zebrafish embryonic Egr1 gene arrested retinal and lenticular development and then caused microphthalmos. This is quite different from the findings in postnatal chicks and implies Egr1 may have diverse functions at various developmental stages in varied species. MO is a powerful tool to study gene function precisely and quickly. However, since the outcomes result from gene knockdown in all tissues, further research to identify the role of Egr1 in the isolated tissue is favored.
The whole study was supported by the National Science Council Grant 92-2314-B-418-006, Taiwan. We thank the Zebrafish International Resource Center (NIH-NCRR Grant number RR12546) for providing zpr-1 antibody.
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