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Keywords:

  • anterior segment;
  • cornea;
  • annular ligament;
  • gsnl1;
  • transgenic zebrafish

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The anterior segment of the eye includes such structures as the cornea, lens, iris, and ciliary body and is essential for many visual and physiological functions of the eye. The zebrafish gelsolin-like 1 (gsnl1) gene encodes an actin regulatory protein and is expressed in the anterior segment of the eye. We report the transgenic analyses of the gsnl1 promoter and enhancer that are required for expression in the anterior segment of the eye. A 6.4-kb genomic fragment upstream from the translation initiation site (ATG) was capable of driving green fluorescent protein (GFP) expression in transient transgenic embryos and stable transgenic adult fish, which mimics the endogenous gsnl1 expression. The GFP expression was localized in the corneal epithelium (CE) and the annular ligament (AL) at the iridocorneal angle. A unique enhancer for each of these two tissues was identified at 3.7-kb upstream from the ATG. The 60-bp AL and 25-bp CE enhancers were separated by 100-bp and functioned independently from each other. Deletion analysis indicated that the proximal promoter was located 1.6-kb upstream from the ATG. Stable GFP transgenic lines were established for future studies of genetic regulation in the anterior segment of the fish eye. Developmental Dynamics 236:1929–1938, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The anterior segment of the vertebrate eye consists of the cornea, lens, iris, ciliary body, and trabecular meshwork (reviewed by Idrees et al.,2006; Gould et al.,2004). The major roles of this subset of the ocular system are (1) physical protection of the posterior portion of the eye such as retina, (2) support of the corneal curvature to keep the refraction angle of the incoming light focused on the retina by means of the lens, and (3) regulation of the intraocular pressure (IOP) by maintaining a dynamic balance of the aqueous humor inside the eye, which is produced by the ciliary body and is drained out primarily at the iridocorneal angle through the trabecular meshwork and Schlemm's canal. Three types of cells developmentally contribute to the heterogeneous tissues of the anterior segment. The surface ectoderm overlaying the optic cup during embryogenesis forms the lens and the corneal epithelium (CE). The neural ectoderm gives rise to the epithelia of the iris and ciliary body. The cranial mesenchyme derived from the neural crest differentiates to form the corneal stroma and endothelium, ciliary stroma and muscles, iris stroma, and trabecular meshwork (Gould et al.,2004; Gage et al.,2005; Idrees et al.,2006). Additionally, these tissues are often affected in human anterior segment dysgeneses and other ocular diseases, including glaucoma, iris hypoplasia, Peters' anomaly, and corneal dystrophy (reviewed by Idrees et al.,2006). Previously, we reported that the zebrafish cornea has three distinct cellular layers (epithelium, stroma, and endothelium) and two extracellular layers (Descemet's and Bowman's) as seen in the human cornea (Zhao et al.,2006). Histological and ultrastructural similarities suggest that the fish and human cornea share common developmental and functional features. Furthermore, the anterior segment organization in zebrafish is also conserved and shares many tissue-specific and structural features with its human counterpart (Soules and Link,2005). These observations demonstrate that the zebrafish is a powerful model organism for investigating genetics, cell and developmental biology, and diseases of the anterior segment of the eye because of its small size, relatively short generation time, rapid external development, large clutch size, transparency of embryos, easy maintenance, and amenability to genetic manipulation.

The gene expression profile of the zebrafish anterior segment excluding the lens by random sequencing of cDNA clones showed that the gsnl1 gene (previously called gelsolin, GenBank AF175294) was the most abundantly expressed transcript (Vihtelic et al.,2005), suggesting it is a prime candidate to study gene regulatory mechanisms and to clone tissue-specific enhancers in the anterior segment of the eye. The gsnl1 gene comprises 17 exons and encodes a 720 amino acid protein. The gsnl1 protein consists of six gelsolin homologous domains or gelsolin repeats, which function in binding with monomeric or filamentous actin (dos Remedios et al.,2002). Zebrafish gsnl1 belongs to the large gelsolin superfamily and shows 58% identity to the two human isoforms of gelsolin (GSN) and human Adseverin (ADSV) at the amino acid (a.a.) level. There are two additional GSN superfamily members in the zebrafish genome, gelsolin (gsn) and gelsolin-like 2 (gsnl2). Homology of the zebrafish gsn to the human GSN (both isoforms) and ADSV are 71% and 57% identity in a.a., respectively, whereas gsnl2 shows less homology to GSN and ADSV (63% and 55% identity, respectively). These comparisons suggest that the zebrafish gsnl1 gene is a diverse homologue of the human GSN gene.

Gelsolin was originally discovered based on its calcium-dependent control of cytoplasmic actin gel–sol transformation (Yin and Stossel,1979). It regulates actin organization by severing filaments, capping filament ends, and nucleating actin assembly in several cellular processes, including cell motility, phagocytosis, and apoptosis (McGough et al.,2003; reviewed by Silacci et al.,2004). It has been recently reported to function as a transcriptional coactivator to regulate gene expression (reviewed by Archer et al.,2005).

Targeted disruption of the gelsolin gene in mice showed that the null mice had normal embryonic development and longevity (Witke et al.,1995). However, these mutant mice had impaired physiological responses in at least two systems. One of them was a prolonged bleeding time, most likely due to diminished severing of actin filaments in platelets and a corresponding reduction in the platelet shape change response. Second, the mutant mice responded more slowly than wild-type mice to a strong inflammatory stimulus, related to a reduced cell migration rate of leukocytes. These observations suggest that gelsolin is required for rapid motile responses such as hemostasis, inflammation, and wound healing (Witke et al.,1995). Mutations in the human GSN gene are responsible for familial amyloidosis V (Finnish type), which results from deposition of the mutant GSN protein (Maury et al.,1990; Ghiso et al.,1990; Levy et al.,1990; Hiltunen et al.,1991), creating unique features of systemic amyloidosis including lattice-like corneal dystrophy and cranial neuropathy.

The zebrafish gsnl1 gene was reportedly expressed during early development in the forebrain and hindbrain by 22 hr postfertilization and in the nose and eye by the hatching stage (Kanungo et al.,2003,2004). It was also expressed in the adult CE (Xu et al.,2000). The gene-specific morpholino oligo against gsnl1 inhibited dorsal structure formation, but overexpression of gsnl1 led to dorsalized development, including axis duplication (Kanungo et al.,2003). These studies demonstrated that gsnl1 plays an important role for dorsal development and head structure formation during early embryogenesis. Xu et al. (2000) proposed a possible role of gsnl1 in the transparency of the adult cornea by preventing polymerization of actin filament, analogous to the crystallin proteins in the lens. However, the function of gsnl1 in the anterior segment during later development and in the adult is not well characterized yet.

The purpose of our study is to isolate functional gene regulatory elements from the zebrafish gsnl1 gene to investigate gene regulation mechanisms in the anterior segment of the eye and to generate transgenic zebrafish models of human diseases of the anterior segment. In this article, we show that gsnl1 is strongly expressed in the anterior segment at the iridocorneal connective tissue, the annular ligament (AL), as well as in the CE during later development stages. A 6.4-kb 5′ flanking region can drive the GPF expression that faithfully mimics the pattern of the endogenous gsnl1 expression in transient transgenic embryos and in stable transgenic lines. We further analyzed the tissue-specific enhancer activity in the upstream region of the zebrafish gsnl1 gene and identified CE-specific and AL-specific enhancers that function independently from each other.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

gsnl1 Expression in the Anterior Segment of the Eye

Zebrafish embryos at stages from 1 to 5 days postfertilization (dpf) were collected and used for whole-mount in situ hybridization using antisense gsnl1 RNA probes. On 1 dpf embryos, the hybridization signals were detected in the lens, the presumptive CE, and the nose region (Fig. 1A,D), confirming previous observations (Kanungo et al.,2003,2004). Although a similar expression pattern of gsnl1 in the lens and nose was detectable, the signal intensity in these tissues was greatly reduced by 3 dpf. At this stage, gsnl1 began to be expressed in the peripheral region of the eye with a unique patchy pattern (Fig. 1B,E). A section through the eye showed that the signal was localized in the connective tissue at the angle between the cornea and iris (data not shown; also see Fig. 4C,E,G), a structure called the annular ligament (AL, Soules and Link,2005). A weak but detectable gsnl1 expression appeared in the central region of the ocular surface ectoderm (the CE; Fig. 1B,E). As embryos developed (5 dpf), gsnl1 was no longer expressed in the nose. Instead, it was strongly expressed in the AL (Fig. 1C,F) and remained to be expressed in the CE of the eye.

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Figure 1. Expression of gsnl1 in zebrafish embryos detected by whole-mount in situ hybridization. A–C: Lateral view. D–F: Dorsal view. A,D: On 1 day postfertilization (dpf) embryos, gsnl1 was expressed in the lens, future corneal epithelium, and at the nose. B,E: At 3 dpf. C,F: At 5 dpf. At later stages, a strong signal in the annular ligament and a weak signal in patches in the central cornea were detected in the eye. The staining in the lens and nose was not detected at 5 dpf. Al, annular ligament; ce, corneal epithelium; l, lens; n, nose. Scale bar = 200 μm.

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Figure 4. The green fluorescent protein (GFP) expression in 3-month-old stable transgenic fish of gsnl1. A,B: Merged images of whole-mount GFP fluorescence and brightfield microscopy. A: Lateral view. The white lines indicate the positions of sections C–H. B: Dorsal view. The GFP expression was detected in the anterior segment of the eye. C–H: Laser-scanning confocal microscopic images of the transverse sections at the iridocorneal angle. Green, GFP. Blue, nuclear staining by 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI). C,D: Dorsal region. E,F: Ventral region. G,H: Temporal region. The GFP signal was detected in the annular ligament, a specific connective tissue at the iridocorneal angle. Boxes in C,E,G indicate the positions of the high magnification images of D,F,H, respectively. al, annular ligament; cor, cornea; irs, iris; len, lens; ret, retina. Scale bar = 1 mm in A; 50 μm in C,D.

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Regulatory Region of the gsnl1 Gene

To identify cis-regulatory elements that control the anterior segment-specific gene expression, bioinformatic analysis and a database search were performed to determine the genomic structure of gsnl1. Comparison of the sequences between the zebrafish genomic DNA (NW_633561) and a cDNA clone (AF175294), which had the longest 5′ untranslated region among all expressed sequence tags (ESTs) in GenBank, suggested that there was a noncoding exon located 1.5-kb upstream of the translation start site. Therefore, this noncoding exon was considered to be the first exon of the gsnl1 gene. A 6.4-kb genomic region upstream of the initiation ATG was polymerase chain reaction (PCR) amplified and cloned into a Tol2 transposon-based green fluorescent protein (GFP) expression vector. The first exon and first intron were included in this 6.4-kb DNA fragment. To examine whether this upstream region has promoter activity and contains tissue-specific regulatory elements, the plasmid and the in vitro synthesized capped RNA of Tol2 transposase were co-injected into embryos at the one-cell stage.

On embryos of 1 dpf, the GFP signal was detected at the nose region (Fig. 2A), which was in agreement with the in situ hybridization data (Fig. 1). However, expression in the eye was not observed (Fig. 2A). A low level of nonspecific and spotted GFP signals was also detected throughout the body, which is commonly observed in the transient transgenic assay using the Tol2 transposon system (Kawakami et al.,2004). By 3 dpf, the GFP expression was found in both CE and AL of the eye in patches (Fig. 2B). This pattern was identical to the mRNA expression of the endogenous gsnl1 (Fig. 1B). Similar to the in situ hybridization pattern, a reduction of GFP expression in the nose was observed on 3 dpf. The GFP signals were localized in the anterior segment of the eye (Fig. 2C). A strong GFP signal was observed in the AL of the eye by 5 dpf (Fig. 2D). The fluorescent signal at the nasal side was weaker than that at the temporal side of the eye. There was not a significant difference along the dorsoventral axis. The GFP expression in the CE was in a patched pattern (Fig. 2D). These results indicated that the 6.4-kb fragment contained a basal promoter and tissue-specific enhancer(s) required for the anterior segment expression in the eye.

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Figure 2. Transient transgenic expression of the gsnl1 6.4-kb reporter in zebrafish embryos. A–D: Green fluorescent protein (GFP) expression. E–H: Brightfield images. A,E: On 1 days postfertilization (dpf), GFP was detected at the nose like the endogenous gsnl1 expression, but a specific signal was not detected in the lens or the future cornea. B,C,F,G: Lateral view (B,C) and dorsal view (C,G) of 3-dpf embryos. D,H: The 5-dpf embryos. At later stages (B–D,F–H), the annular ligament and a subpopulation of the corneal epithelium were GFP-positive. The transgenic expression between 3–5 days was localized in the anterior segment of the eye, which was consistent with the endogenous gsnl1 expression pattern shown in Figure 1. al, annular ligament; ce, corneal epithelium; n, nose. Scale bar = 200 μm.

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Six stable transgenic lines were established from this 6.4-kb fragment plasmid. All positive F1 embryos showed essentially the same GFP expression pattern as the transient transgenic embryos (Fig. 3). The GFP expression in the nose was at the nasal pit and in a subpopulation of the head mesenchyme cells at the most anterior part (Fig. 3A,B,D,E,G). Two of these six transgenic lines had additional ubiquitous expression at a lower level, which may be due to an influence of the integration site in the zebrafish chromosome. It is interesting that the GFP expression in the central cornea was not uniform in the 5 dpf F1 embryos (Fig. 3C). This pattern of expression was identical to that observed in the F0 embryos (Fig. 2B). Adult fish of the stable transgenic lines contained GFP-positive cells in the AL (Fig. 4C,E,G). The cells in this tissue were characterized by a large intracellular vesicle with GFP signals in inclusions (Fig. 4D,F,H). No GFP signal was detected in the cornea of the 3-month-old transgenic fish in all three stable lines, although GFP signals were found in the cornea of 20 dpf transgenic fish (data not shown).

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Figure 3. Green fluorescent protein (GFP) expression of the gsnl1 6.4-kb reporter in stable transgenic embryos. A–F: Lateral view of GFP (A–C) and brightfield images (D–F). G–L: Dorsal view of GFP (G–I) and bright field images (J–L). A,D,G,J: The 1 dpf embryos showed GFP expression in the nose region, which was primarily localized at the nasal pit and in a subpopulation of the head mesenchyme cells at the most anterior part. B,E,H,K: On 3 dpf embryos, the strong GFP signal was detected in the nose and the weak signal was observed on the future cornea in a mosaic pattern. C,F,I,L: The GFP signal was detected in the annular ligament of the eye and in the corneal epithelium in patches on 5-dpf embryos. Scale bar = 200 μm.

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Promoter and Enhancer Analysis

Several deletion vectors derived from the 6.4-kb expression plasmid were constructed to identify cis-regulatory elements necessary for the anterior segment of the eye. In these vectors, the 5′ portion of the 6.4-kb fragment was deleted at appropriate restriction enzyme sites (Fig. 5). The number of GFP-positive embryos was scored to evaluate the promoter and enhancer activity of each construct. The 4.3-kb construct (RV) showed a similar pattern of GFP expression to the 6.4-kb plasmid at a high percentage (> 70%; Fig. 5). However, a significant reduction in the number of positive embryos with GFP in the eye and nose was found with the 3.5-kb (Sac) and the 2.4-kb (H3) constructs (Fig. 5). These results suggested that the 0.8-kb region between EcoRV and the second SacI sites contained a regulatory element for the gsnl1 expression in the eye and nose. A 3′ deletion construct was used to examine whether the first intron is essential for the expression in the eye and nose. The intron-less construct (Dint) showed 95% of GFP-positive embryos, which was similar to the RV construct that contained the same DNA fragment plus the intron (Fig. 5), indicating that the first intron was not required for the transgenic expression of the GFP reporter.

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Figure 5. Transient transgenic analysis of the gsnl1 5′ upstream region. The chart shows the percentage of positive embryos with GFP in the eyes observed on 5 dpf. The 0.8-kb region between the EcoRV and the second SacI sites, located 4.3–3.7 kb upstream from the translation start site (ATG), was essential for the reporter expression. The intron was dispensable for the tissue-specific expression. RV, EcoRV; S, SacI; H3, HindIII; Bg, BglII.

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An additional series of 5′ deletions between the EcoRV and the second SacI sites was generated to further define the regulatory element(s) within the 0.8-kb region (Fig. 6). All reporter constructs that had the 3.9-kb fragment (BsrD, RV, Dra, and Stu) displayed the eye- and nose-specific GFP expression in over 65% embryos (Fig. 6). The two shorter constructs (SacA and SacB) showed only a background level of expression in the eye (Fig. 6). A significant GFP expression in the nose region was detected in the SacA construct, but not in the SacB (detail shown in the later section). These results indicated that the 200-bp fragment between the BsrDI and the first SacI sites was necessary for expressing GFP in the fish eye as a tissue-specific enhancer.

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Figure 6. Transgenic enhancer analysis of the gsnl1 upstream region for the anterior segment-specific expression. A deletion series of the regulatory region between the EcoRV and the second SacI sites was examined. The 200-bp region between the BsrDI and the first SacI sites was necessary for the transgenic expression in the anterior segment of the eye. RV, EcoRV; Dr, DraIII; St, StuI; Bs, BsrDI; S, SacI; Nd, NdeI.

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The enhancer region identified above was located 2.1-kb distant from the first exon. To examine if this 2.1-kb region included basal proximal promoter activity, four GFP reporters with internal deletion were constructed (Fig. 7). All four of these reporters contained the EcoRV–NdeI fragment that contained the enhancer region. Transient transgenic analysis showed that all reporters containing DNA fragments that were longer than that in the BbvC construct expressed GFP in the eye and nose in over 94% embryos. On the other hand, the 76-bp 5′ flanking region (BbvC) was not sufficient for the GFP expression in both eye and nose (3%). These results suggested that the EcoRV–NdeI region did not include a promoter activity. Instead, it functioned as an enhancer. The results revealed that the 310-bp region that is 5′ upstream of the first exon in the Msc construct had basal (or minimal) promoter activity for the expression in both eye and nose and that our predicted first exon was actually expressed in vivo.

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Figure 7. Transgenic promoter analysis of the gsnl1 gene. An internal deletion series between the NdeI site and the predicted first exon was examined. The 310-bp fragment (MscI), 5′ upstream of the first exon was essential for the expression in both eye and nose. RV, EcoRV; Nd, NdeI; H3, HindIII; Bg, BglII.

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Tissue-Specific Enhancers

It is possible that the cis-regulatory elements for the tissue-specific expression in the nose and the eye could be separated. To test this possibility, several deletion reporter plasmids were generated (Fig. 8) and used for transient transgenic experiments. The results of the GFP expression in the eye were consistent with the previous observations that the 200-bp upstream region of the first SacI site was sufficient for the anterior segment-specific expression and that the SacI–NdeI fragment was dispensable (Fig. 8). On the other hand, the reporters lacking the internal region (D1 and D2) showed a significant reduction in the number of embryos positive for GFP in the nose. The GFP expression of the D1 and D2 constructs in the eye was still at a comparable level to the other reporters (Fig. 8). Therefore, these results, combined with the SacB data in Figure 6, indicated that the enhancer for the nose was localized between two SacI sites and could be separated from the anterior segment-specific enhancer.

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Figure 8. Transgenic enhancer analysis of the gsnl1 upstream region for the tissue-specific expression in the eye and nose. Five deletion constructs lacking either 5′ or the internal region between the StuI and NdeI site, located 4.0–3.2 kb upstream from the transcription start site, were examined by transient transgenic assay. The 200-bp fragment between the BsrDI and the first SacI (see also Fig. 6) was necessary for the green fluorescent protein (GFP) expression in the central cornea and/or the annular ligament. The 480-bp fragment between the first SacI and NdeI sites was necessary for the nasal expression. These results indicate that the gsnl1 expression in the eye and nose is regulated by different enhancers. St, StuI; Bs, BsrDI; S, SacI; Nd, NdeI.

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The 200-bp fragment between the BsrDI and the first SacI sites showed enhancer activity for two independent anterior segment tissues, the CE and the AL. The embryonic CE develops from the surface ectoderm overlaying the lens. The origin of the AL cells is not well characterized, but they may derive from the mesenchyme (Idrees et al.,2006). Because CE and AL may have different origins, it is possible that the tissue-specific expression in the CE and AL is controlled by two independent enhancers. To test this hypothesis, one additional deletion vector (D5) in this 200-bp region was constructed (Fig. 9). Embryos injected with this and other deletion constructs showed three patterns of GFP expression in the anterior segment of the eye, that is, (1) both in the CE and AL, (2) predominantly in the CE and weak or no expression in the AL (Fig. 10A), and (3) predominantly in the AL and weak or no expression in the CE (Fig. 10B). The D4 construct showed a significant reduction in the AL-positive embryos (26%), although this level was still higher than that of the D5 (Fig. 9). The actual expression level of the D4 construct in the AL could be lower than 26% because we observed that the number of the GFP-positive cells per D4-injected embryo was less than those per D3-injected embryo (data not shown). These results suggest that the expression in the CE and AL was regulated independently. The enhancer for the AL located upstream of the CE enhancer (Fig. 9). These experiments narrowed down the size of the regulatory region to 60 bp for the AL and 25 bp for the CE enhancers (Fig. 9).

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Figure 9. Transient transgenic analysis of the annular ligament (AL) -specific and corneal epithelium (CE) -specific enhancers. The constructs from Stu to D4 were the same as those in the Figure 8. The AL-enhancer was localized 60-bp downstream of the BsrDI site and the CE-enhancer was 25-bp upstream the first SacI site. St, StuI; Bs, BsrDI; S, SacI; Nd, NdeI.

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Figure 10. Transient transgenic expression of green fluorescent protein (GFP) controlled by the tissue-specific enhancers. A: Typical expression pattern of GFP in the embryo injected with the corneal epithelium (CE) -specific enhancer D4 in Figure 9. B: The embryo injected with the AL-enhancer D2. The GFP fluorescence and the brightfield images were taken at 5 days postfertilization (dpf) and merged. Scale bar = 200 μm.

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Annular Ligament-Specific Enhancer

Possible transcription factor binding sites in each of the two enhancers (CE and AL) were analyzed by using Transcription Element Search System (TESS, http://www.cbil.upenn.edu/cgi-bin/tess/tess/) with the TRANSFAC database. The CE enhancer included a CANNTG motif, known as the E-box, for basic helix–loop–helix (bHLH) transcriptional factors, while the AL enhancer contained multiple candidate sites (Fig. 11). To investigate which of the transcription factors may be essential for the AL enhancer activity, a new plasmid was constructed, which included the minimal promoter and the 0.8-kb enhancer-containing fragment. Five mutation constructs were generated from this plasmid, in which five-nucleotide substitutions were introduced into the 60-bp AL enhancer at different positions (Fig. 11B, M1–M5). Two mutation constructs, M3 and M4, displayed 40% and 42% AL-positive embryos, respectively, which were less than half of the wild-type (95%). The remaining three plasmids (M1, M2, and M5) did not show a significant change in GFP-positive embryos compared with the wild-type enhancer (Fig. 11B). These results suggested that the 15-bp region covered by these two mutation constructs (M3 and M4) might contain essential elements for the AL-specific expression. The mutated nucleotides in M3 and M4 changed the AT-rich feature, which overlapped the consensus of POU3F2 (also known as N-Oct-3, BRN2), suggesting that POU3F2 may be the key regulator of the AL enhancer.

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Figure 11. The enhancer sequences and the transient transgenic point mutation assay in the annular ligament enhancer. A: The DNA sequence of the corneal epithelium-enhancer indicates an E-box motif. B: The sequence of the annular ligament-enhancer and possible transcription factor binding sites are shown at the top. Nucleotide substitutions (shown in lower cases) were introduced to the mutant constructs (M1–M8), and transgenic analysis was performed. The M3 and M4 constructs revealed a reduction of the number of positive embryos with GFP in the annular ligament. This region included an AT-rich sequence.

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Because A/T bases were thought to be essential for MEF-2 binding, a three-nucleotide substitution was introduced at the middle portion from A/T to C/G (Fig. 11B, M7). Similarly, a mutation construct (M8) was constructed to evaluate whether A/T stings were required for IRF-1 (Fig. 11B, M8). As a control, M6 with a three-nucleotide substitution outside of these consensus binding sites was made. Transient transgenic assays showed that, although the M7 displayed a slight reduction in positive embryos (70%), it was still much higher than the M3 and M4. The control construct M6 did not show a significant change, but the M8 exhibited an intermediate level of positive embryos (55%, Fig. 11B). These results suggested that MEF-2 and IRF-1 might not be required for the AL-enhancer activity.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In this study, we have isolated regulatory elements in the upstream region of the zebrafish gsnl1 gene and analyzed their function for the tissue-specific expression in the anterior segment of the eye. We confirmed previous observations by in situ hybridization that the expression in the ocular surface ectoderm, lens, and nose (Kanungo et al.,2003,2004) was transient and disappeared by 3 dpf. We extended this analysis to later stages of fish development up to 5 dpf. Although the hybridization signals were still observed in the anterior segment between 3 to 5 dpf, the pattern of expression was altered. The gsnl1 mRNA was localized predominantly in the AL and weakly in the CE. A 6.4-kb genomic fragment upstream of the translation start site was sufficient to mimic the endogenous gene expression in the nose and the eye of 3 dpf or older embryos. However, this reporter did not express GFP in the lens and the surface ectoderm on 1 dpf embryos. The regulatory element necessary for the early expression in the lens may be located in the other introns, downstream, or far upstream of the gene.

Of interest, the expression of GFP in the surface ectoderm of the eye during early development was not uniform, even in the stable transgenic embryos (Fig. 3). This finding was consistent with the endogenous gsnl1 expression pattern (Fig. 1). These results suggest that the embryonic corneal epithelium may consist of more than one type of cells. We previously reported that the secondary corneal stroma is not formed by 5 dpf and that there are only two cell layers in the CE at this stage (Zhao et al.,2006). Therefore, the endogenous gsnl1-expressing cells and the transgenic GFP-positive cells by 5 dpf are thought to be in the CE layer. Sections through the eye of embryos derived from transgenic fish and the whole-mount in situ hybridization did confirm their localization in the CE (data not shown).

Strong GFP expression was observed in the AL of the eye in transient transgenic embryos and adult fish of stable transgenic lines. The biological role of the AL is not well understood, but is thought to support the shape of the anterior segment to keep an appropriate angle of the cornea (Soules and Link,2005). The specific expression of gsnl1 in this tissue suggests it may participate in the development and maintenance of the structure of the iridocorneal angle by regulating the actin polymerization/depolymerization, similar to the proposed function in the CE by Xu et al. (2000). Ultrastructural studies by electron microscopy will help determine whether the AL cells have unique features of microfilament-based cytoskeleton. Anatomically, the fish AL occupies an analogical position of the human trabecular meshwork at the iridocorneal angle, which is a major drainage pathway for the aqueous humor outflow. Because the trabecular meshwork plays a crucial role in maintaining a normal level of the IOP in human and is implicated in glaucoma, the fish AL may also be involved in IOP control. Therefore, the gsnl1 AL-specific enhancer could be a useful tool to generate transgenic fish models of human disease related to the anterior segment of the eye, including anterior segment dysgenesis and diseases of the cornea such as corneal dystrophy and of the angle structure such as glaucoma.

We have shown that a 310-bp proximal cis-regulatory element was located 1.6-kb upstream from the ATG, which was 5′ to the predicted noncoding exon. This region itself was not sufficient for tissue-specific GFP expression. However, all GFP reporters lacking this region lost their GFP expression in both eye and nose (Fig. 7). We, therefore, propose this 310-bp region is a basal promoter of the gsnl1 gene, although it does not contain cis-elements that are typical of eukaryotic promoters such as TATA or CAAT box. Using this basal promoter, we found that the Pax6 enhancers derived from the mouse showed a telencephalon-specific and lens-specific expression (unpublished observation). Thus, this basal promoter from the zebrafish gsnl1 gene can be used for studying enhancer activity and enhancer trapping experiments as a minimal promoter in zebrafish.

We have also shown that the distal cis-regulatory element located between 3.8- and 3.5-kb upstream from the ATG. This region included three independent tissue-specific enhancers for the AL (60 bp), CE (25 bp), and nose (150 bp; Fig. 12). The gsnl1 expression in these three tissues is likely regulated by different transcriptional mechanisms. The CE enhancer contained an E-box motif CANNTG that could be recognized by bHLH transcription factors (Lassar et al.,1989; Blackwell and Weintraub,1990). The point mutation analysis suggested the AT-rich region of the AL enhancer, containing consensuses of several transcription factors, was important for the AL-specific expression (Fig. 11). Further characterization of their expression pattern, biochemical analysis of their direct binding to the AL enhancer, and functional studies such as morpholino knock-down will be required to determine which of these factors are essential for the AL-specific gene regulation in vivo.

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Figure 12. Scheme of the promoter and tissue-specific enhancers of the zebrafish gelsolin-like 1 (gsnl1) gene. The proximal promoter and the distal enhancer locate 1.6- and 3.7-kb upstream from the translation start site (ATG), respectively. The enhancer region includes three independent tissue-specific enhancers for the annular ligament (AL), corneal epithelium (CE), and nose, in the order from the 5′ side.

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In conclusion, the zebrafish gsnl1 gene is predominantly expressed in the two tissues, the CE and AL, in the anterior segment of the eye during early development and the expression in each tissue is controlled by an independent cis-regulatory element. The stable transgenic lines we established in this work are the first transgenic zebrafish displaying tissue-specific GFP expression in the cornea and the AL of the eye. They are very useful resources for future screening of mutant zebrafish with anterior eye defects, which will facilitate studies of human diseases such as anterior segment dysgenesis, corneal dystrophy, and glaucoma.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Animals, Microinjection, and Transgenic Analysis

The zebrafish (Danio rerio) was purchased from a local pet shop and maintained at 27 to 28°C as described in the Zebrafish Book (Westerfield,2000). Super-coiled plasmid and in vitro synthesized capped RNA of Tol2 transposase (Kawakami et al.,2004) were co-injected to one-cell stage embryos with a glass micro-needle using FemtoJet microinjection machine (Eppendorf). The estimated amount of DNA and RNA injected was 100 pg and 50 pg per embryo, respectively. For semiquantitative analysis of promoter and enhancer activity in transient transgenic embryos, the plasmid-injected embryos at 1–5 dpf were anesthetized and the numbers of embryos which had more than one cluster of GFP-positive cells in the target tissue were scored.

In Situ Hybridization

An EST clone of gsnl1, B986 (gift of Drs. Christine and Bernard Thisse), was used as the template for in vitro transcription and the RNA probe was used for whole-mount in situ hybridization. The cDNA was amplified with the M13 forward and reverse primers and the SP6 RNA polymerase was used to generate digoxigenin (DIG) -labeled antisense probe, according to the manufacturer's instructions (DIG RNA synthesis kit, Roche). Whole-mount in situ hybridization was performed on 1, 3, and 5dpf embryos as described previously (Thisse et al.,2004).

DNA Construction

A 6.4-kb genomic DNA fragment upstream of the translation start site was amplified by PCR and cloned to a Tol2 transposon-based vector pTol2000 (Kawakami et al.,2004) with enhanced GFP and SV40 polyA signal in the opposite direction of the Tol2 element. The deletion plasmids were constructed by modifying this 6.4-kb reporter with either restriction enzyme digestion or replacement with a PCR fragment. For point mutation constructs, nucleotide substitutions were introduced by recombinant PCR and the 0.8-kb PCR products between the StuI and NdeI sites were subcloned to the upstream of the minimum promoter at the MscI site. All sequences of PCR primers are available by request.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Drs. Christine and Bernard Thisse for the EST clone B986, Dr. Koichi Kawakami for the Tol2 transposon vector and transposase cDNA, and Dr. Louvenia Carter-Dawson for critical reading of early drafts of this article. X.C.Z. was funded by the Hermann Eye Fund, R.W.Y. was funded by the National Eye Institute, and E.N. was supported by the NIH Predoctoral Fellowship Award for Minority Students.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES