- Email: [email protected]
A novel smoothelin-like, actin-binding protein required for choroidal fissure closure in zebrafish
BBRC Biochemical and Biophysical Research Communications 313 (2004) 1092–1100 www.elsevier.com/locate/ybbrc
A novel smoothelin-like, actin-binding protein required for choroidal fissure closure in zebrafish Ryo Kurita,a Yoko Tabata,a Hiroshi Sagara,b Ken-ichi Arai,a and Sumiko Watanabea,* a
b
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Department of Fine Morphology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Received 27 November 2003
Abstract A gene expressed in the choroidal fissure of the zebrafish eye was isolated. This gene, designated #61, contained significant homology with the previously reported actin-binding protein smoothelin. During zebrafish embryogenesis, #61 expression was first detected in the lateral mesoderm of the mid-trunk region, and then strong expression was observed in the choroid fissure of the eye and in a part of the brain at 30 hpf. Abrogation of #61 activity by an antisense morpholino oligonucleotide resulted in the failure of closure of the choroid fissure at 30 hpf. In addition, hemorrhage was observed at the caudal side of the eye. Detailed analysis indicated that leakage of blood may have arisen from the hyaloid vessels and the primordial midbrain channels. On the other hand, retinal differentiation and optic nerve formation seemed normal. Taken together, our data suggest that gene #61 may play a role in the formation of hyaloid vessels and subsequent choroid fissure closure. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Actin-binding protein; Eye; Choroidal fissure; Hemorrhage; Zebrafish
The development of the vertebrate eye is initiated by lateral evagination of the neuroepithelium of the ventral diencephalon to form the optic vesicle [1]. The optic vesicle makes contact and interacts with the surface ectoderm, from which the lens placode originates, and this interaction leads to the formation of the two-layered optic cup. The outer layer of the optic cup consists of a single layer of epithelial cells, the retinal pigmented epithelium (RPE); and the inner layer develops into the neural retina. The cells of the proximal region of the optic vesicle give rise to the optic stalks. These stalks have a pipe-like structure connecting the optic cup and the diencephalon. At the early stage of eye development, neither optic stalk nor optic cup closes completely and thus they have a gap called the choroid fissure. At this fissure, mesenchymal cells form the hyaloid vessels. Vascular mesenchyme and the hyaloid artery are incorporated into the longitudinal groove of the optic fissure that extends into the optic * Corresponding author. Fax : +81-3-5449-5474. E-mail address: [email protected] (S. Watanabe).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.12.046
stalk and they gain access to the lentoretinal space. Then the growing edges of the choroidal or optic fissure meet and fuse; and thus the hyaloid vessels and associated mesenchyme, situated in the center of the optic stalk, form the future central retinal artery and vein. The fusion or closure of the optic fissure commences at the mid-portion of the optic stalk and continues both proximally and distally. The zebrafish is an excellent model vertebrate to analyze eye development for many practical reasons as well as because of the high degree of genetic and structural organization of its eye that it shares with other vertebrates, including mammals [2]. Indeed, ocular structure and developmental mechanisms between vertebrates have been remarkably conserved during evolution [3]. The zebrafish retina contains the same major categories of cells as mammalian retinas [2], and the involvement of common genes in eye development of zebrafish and other vertebrates is well documented [3,4]. Recently, a panel of zebrafish mutants having abnormalities in their eyes was generated, thus strengthening the value of this organism for genetic studies [5,6].
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
To isolate novel genes that play a role in eye development, we performed in situ screening of an adult zebrafish retinal library in the EST database. We found a novel actin-binding protein that was expressed strongly in the choroid fissure of the eye. Abrogation of the function of this gene resulted in the failure of closure of the choroid fissure and severe hemorrhage around the eye.
Materials and methods Cloning and DNA construction of #61. Total RNAs were prepared from the head and body parts of zebrafish (Danio rerio) embryos (24 or 48 hpf) and reverse transcribed by use of Superscript II (Invitrogen, Carlsbad, CA). The cDNAs were then subjected to PCR amplification using specific primers designed based on the sequences of randomly selected EST clones of adult zebrafish retinal genes in GenBank (NCBI). Full-length #61 (AB110459) was isolated by 50 and 30 RACE using a Gibco RACE kit and subcloned into the pCS2+ vector by insertion of the DraI (blunted)–EcoRI fragment of #61 into pCS2+ at blunted ClaI and EcoRI sites. The EGFP cDNA which was used as a control was subcloned into pCS2+ by using BamHI and EcoRI sites. For co-injection of #61-MO and #61-RNA, a mutant of the #61 gene (#61-mut), which encodes amino acids identical with those of wild-type #61 but contains mutations at the 50 end noncoding region that prevent it from being targeted by the #61-MO, was constructed by PCR mutagenesis. For co-immunoprecipitation analysis, a Myc epitope-tagged #61 was constructed as follows: blunted BamHI–DraIII and DraIII–XbaI fragments of #61 were prepared from #61-mut-pCS2+ and ligated into pTRE-Myc (Clontech, Palo Alto, CA) at the blunted SfiI and XbaI sites. Then, Myc-#61 cDNA was subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) at the EcoRI site. Immunoprecipitation and Western blot analysis. Immunoprecipitation was done as described [7]. Briefly, COS7 cells were transfected with 1 lg of the Myc-#61 or a control plasmid (pcDNA3.1) by lipofection using FuGENE 6 (Roche Diagnostics GmbH, Mannheim, Germany). Two days later, the cells were harvested and the Myc-tagged protein was immunoprecipitated with a rabbit polyclonal anti-Myc antibody (Santa Cruz Biotechnology, Santa Cruz, California). Precipitates were separated by electrophoresis through a polyacrylamide gel containing 0.1% SDS (SDS–PAGE) and electrotransferred onto Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA). Then, Western blotting was carried out by using mouse monoclonal anti-Myc (Clontech, Palo Alto, CA) and mouse monoclonal anti-actin (Sigma, St. Louis, MO). The blots were visualized with horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody (Amersham, Buckinghamshire, England; 1000-fold dilution) and the Lumi-Light Western Blotting system (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s instructions. Whole-mount in situ hybridization. Probes for pax2.1, scl, gata1, myoD, sonic hedgehog (shh), and fli-1 genes were cloned by PCR amplification using the primers designed according to their EST clone sequences. PCR products were subcloned into the pGEM T-easy vector (Promega, Madison, WI). Digoxigenin (DIG)-labeled antisense RNA probes were synthesized by using a DIG RNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany). Whole-mount in situ hybridization was performed as described previously [8]. Photographs were taken with a Zeiss AxioCam (Carl Zeiss, Jena, Germany) attached to a fluorescence dissection microscope, Leica MZFL III (Leica, Wien, Austria). Microinjection of morpholino (MO) or RNA into zebrafish embryos. MO antisense oligo nucleotides (GENE-TOOLS, LLC, Philomath, OR) were designed against 25 bases including the AUG of #61. The sequence was 50 -GCCTGCATCCATCCTGAGTCCAGGT-30 (start codon is
1093
underlined). The MO was diluted to 0.5 mM with 1 Danieau buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4 , 0.6 mM Ca(NO3 )2 , and 5.0 mM Hepes, pH 7.6) and approximately 0.5–3 nl was injected into the yolk of 1- or 2-cell stage embryos. Standard control MO available from the GENE-TOOLS was used as an injection control, which had no effect on embryonic development under our experimental conditions. Capped sense RNAs were synthesized by using a mMessage mMachine in vitro transcription kit (Ambion, Austin, Texas) according to the manufacturer’s instructions. The synthesized RNAs were diluted to an appropriate concentration with RNase-free water and injected into 1- or 2-cell stage embryos. The amounts of injected RNAs were 50 pg to 1 ng/ embryo for #61 gene and 200 pg/embryo for #61-mut gene. The same amounts of EGFP RNA were used as controls for each experiment. Light-, electron-microscopy, and immunohistochemical staining were performed as described previously [8]. Radiation hybrid mapping. Radiation hybrid mapping (RH mapping) of the #61 gene was kindly performed by the RH mapping services of the children’s hospital zebrafish genome project initiative. (http://zfrhmaps.tch.harvard.edu/ZonRHmapper). Microangiography and hemoglobin staining. Microangiography was done as described [9]. Briefly, fluorescein isothiocyanate (FITC)-Dextran with a molecular mass of 2000 kDa (Sigma, St. Louis, MO) in 1 Danieau buffer at 2 mg/ml was injected into the sinus venosa/cardinal vein of anesthetized embryos at various stages. Photographs were taken during observation under the Leica MZFL III dissection microscope, which was equipped with the standard FITC filter set. For hemoglobin staining, embryos were fixed for 30 min at room temperature in 2.5% glutaraldehyde, 2% formaldehyde, and 0.1 M sodium phosphate (pH 7.4). The fixed embryos were washed three times with PBS and pre-incubated for 30 min at room temperature in the dark in DAB staining solution (0.05% 3,30 -diaminobenzidine tetrahydrochloride, 50 mM Tris–Cl, pH 8.0). The embryos were then incubated in H2 O2 at the final concentration of 0.3% for 5–20 min, washed with PBS, and re-fixed as described above.
Results Isolation of a novel smoothelin-like, actin-binding protein To isolate novel genes specifically expressed in the retina, we selected 75 unknown clones randomly from the adult retina EST database of GenBank. Then RTPCR to examine the expression pattern of each gene in head or body part of 24 and 28 hpf zebrafish embryos was performed. Fifteen clones, which had head-specific expression, were used for subsequent whole-mount in situ analysis of 24-hpf zebrafish embryos. Among them, #61 was unique because of its specific expression in the choroid fissure of the eye at 24 hpf. The full-length transcript of #61 was about 2 kb and it contained a putative 1.5-kb ORF. The predicted 493-amino acid sequence contained a leucine zipper motif in its N-terminal region and an actin-binding domain in its C-terminal one (Fig. 1A). A homology search of amino acids in the C-terminal region revealed a 60% homology with mouse and human proteins of the smoothelin family, which comprises smooth muscle-specific actin-binding proteins. No significant homology at its N-terminal region was found with other genes by database analysis. Radiation hybrid mapping showed that #61 was located in LG15 in the zebrafish genome, not corresponding to
1094
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
Fig. 1. Isolation of novel smoothelin-like, actin-binding protein. (A) Deduced full-length amino acid sequence of zebrafish #61 (Z. #61 ) and human and mouse smoothelin A proteins (H. smth. A and M. smtn. A, respectively). Shadowed boxes indicate conserved amino acids. The actin-binding domain is boxed with broken lines and asterisks indicate the leucine-zipper motif found in the #61 sequence. (B) Co-immunoprecipitation of #61 and actin. COS7 cells were transfected with either the Myc-#61 or a control plasmid. Immunoprecipitation using anti-Myc antibody was performed, followed by Western blotting using anti-Myc (lanes 1 and 2) or anti-actin (lanes 3 and 4) antibodies. Arrows indicate bands correspond to Myc-#61 (56 kDa) and actin (42 kDa). (C–I) Whole-mount in situ hybridization of #61 in zebrafish embryos of various stages. (C) 12 hpf, dorsal view with anterior to the left. #61 was initially observed at the lateral mesoderm of the mid-trunk region (arrow head). (D,E) 15 hpf (D) and 18 hpf (E) embryos showing the lateral view with anterior to the left. Strong signals were detected in the mid-anterior lateral mesoderm (arrows). (F,G) Lateral views, with anterior to the left, of 24-hpf (F) and 30-hpf (G) embryos. The expression of #61 was restricted to the choroid fissure (arrowheads) and a part of the lateral mesoderm and brain (arrows). (H) A ventral view of a 30 hpf embryo with anterior at the top. #61 was detected in some parts of the brain (arrow) and in the choroid fissure of the eye (arrowheads). Scale bars: 100 lm. (I) Sagittal section of eye at 30 hpf. Boxed region indicates the choroids fissure.
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
the chromosomal regions of human (22q12.2) and mouse (11 A2-3) smoothelins. Thus, #61 may not be a smoothelin orthologue in zebrafish. Since #61 contained a putative actin-binding region, we examined whether the #61 product bound actin or not by co-immunoprecipitation analysis. The #61 gene tagged with the Mycepitope at its N-terminus (Myc-#61) was expressed in COS7 cells, and immunoprecipitation using anti-Myc antibody was done, followed by Western blotting using anti-Myc or anti-actin antibodies. As shown in Fig. 1B, anti-Myc antibody co-precipitated the actin (42 kDa) together with the Myc-tagged #61 protein (56 kDa). No signal was detected with the sample prepared from cells transfected with the control vector. Although we cannot exclude the possibility of indirect binding between #61 and actin, it is most feasible that the protein encoded by the #61 gene contains a functional actin-binding domain. We next examined the spatio-temporal expression pattern of #61 during zebrafish embryogenesis by whole-mount in situ analysis. The #61 transcript was
1095
first detected in the lateral mesoderm of the mid-trunk at 12 hpf; and within several hours, its expression spread to both rostral and caudal regions until 18 hpf (Figs. 1C– E). Then the expression became restricted to the midanterior lateral mesoderm at 24 hpf (Fig. 1F). By 30 hpf strong expression was observed in the choroid fissure of the retina and a part of the brain (Figs. 1G–H). Sagittally sectioned samples of a 30-hpf whole-mount in situ embryo clearly showed the expression of #61 at margin of the choroidal fissure (Fig. 1I). Ablation of #61 activity by morpholino antisense oligonucleotide To analyze the role of the #61 gene in zebrafish development, we adopted the knock-down approach using a morpholino antisense oligonucleotide (MO). A specific MO was synthesized based on the sequence of the isolated #61 clone and injected into 1- or 2-cell stage embryos. In the #61-MO-injected embryos, developmental abnormality was first detected between 16 and 18 hpf in
Fig. 2. Effects of abrogation of #61 function by #61 specific morpholino oligo. (A–D) Views of 25-hpf zebrafish embryos injected with control (A) or #61 (B,C) MO. Mild (B, type 1) and severe (C, type 2) phenotypes are shown. Asterisks indicate a curly and thickened tail and yolk sac extension. The arrow in “C” indicates failure of closure of the choroidal fissure in the eye. A view of the head part of a #61-MO-injected embryo is also shown with arrowhead pointing to evidence of bleedings at the eye (D). All panels show the lateral view with anterior to the left. Scale bar: 100 lm. (E–H) Structural observation of choroid fissure in control (E,G) and the #61 (F,H) MO-injected embryos. Sagittal sections of the eyes with anterior to the left and dorsal to the top at 30 hpf (E,F) and 54 hpf (G,H). Arrowheads indicate dislocated cells in the inner plexiform layer. CF, choroid fissure. Scale bars: 50 lm. (I–L) Expression of marker genes of the optic stalk and optic nerve in embryos injected with control (I,K) or #61 (J,L) MO. Whole-mount in situ analysis of pax2.1 expression at 22 hpf (I,J) with lateral view, dorsal at the top. Arrowheads show the expression at the optic stalk. Transverse sections of 48 hpf embryos showing retinal ganglion cells and their axons stained with zn-5 antibody (K,L). Asterisks indicate the optic nerves. Dorsal at the top. Scale bars: 100 lm (I,J) and 50 lm (K,L).
1096
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
the mid-posterior region, especially on the ventral side of the trunk and tail. At 25 hpf, the effects of the MO injection could be classified as type-1 (mild) and type-2 (severe) phenotypes (Figs. 2B and C). Type-1 was characterized by curly, thickened tails and yolk sac extensions, while their head parts seemed to be normal. In the type-2 embryos, failure of closure of choroidal fissure (Fig. 2C, arrow) was observed in addition to the type-1 phenotype. The tail and mid-trunk defects were more severe than those of type-1 morphants. Notably 40% of the total embryos had hemorrhages in the caudal part of their eyes after 30 hpf (Fig. 2D, arrow-head). Hemorrhage was observed in both the type-1 or type-2 embryos and continued to be observed until at least 2.5 dpf. The phenotypic incidence of embryos injected with 8–12 ng of MO was 27% for type 1 and 50% for type 2 (Table 1). Control-MO injection did not affect their embryonic development (Fig. 2A). We next examined whether co-expressed #61 rescued the embryos from the #61-MO effect or not. We prepared #61 mutant containing mis-matched mutations to prevent the inhibition by #61-MO. Over-expression of #61 by capped RNA injection resulted in no morphological defect (data not shown). Synthesized RNA (250 pg) of #61-mutor EGFP as a control was injected together with 6 ng #61-MO into zebrafish embryos (Table 1). The injection of #61-RNA significantly, but not completely, decreased the abnormality observed. MO effects in the #61-mutRNA co-injected embryos were less severe than those in the controls. Retinal differentiation and optic nerve formation in the #61 knock-down embryos We then prepared plastic thin sections from the #61 morphants (type 2) at 30 and 54 hpf and examined the fine structure of their eyes (Figs. 2E–H). In the #61-MOinjected embryo eyes at 30 hpf, failure of the choroid fissure to close was observed (Fig. 2F), whereas the control MO-injected embryos showed only a narrow gap in the choroid fissure (Fig. 2E). But in the later stage, the choroid fissure started to close around at 48 hpf (data not shown), and by 54 hpf, no gap was observed in #61MO-injected embryos (Fig. 2H) or in the control
embryos (Fig. 2G). Layer formation in neural retina occurred indistinguishably in control- and MO-injected embryos at 54 hpf (Figs. 2G and H). Notably, however, cells abnormally located in the inner plexiform layer were observed in the latter (Fig. 2H, arrowheads). We next examined optic stalk and optic nerve formation by using molecular markers. The pax2.1 gene is a member of the paired-box gene family expressed in the optic stalk at early stages during eye development [10,11]. Whole-mount in situ analysis of pax2.1 in the #61-MO-injected embryos revealed that pax2.1 was expressed in optic stalks, midbrain/hindbrain boundaries, and pronephric ducts between 16 and 25 hpf almost just in control embryos. Although the expression of pax2.1 in the optic stalk in the control embryos extended vertically (Fig. 2I), pax2.1 expression in this region of MO-injected embryos was round shaped (Fig. 2J), suggesting failure of extension of the optic nerve. Immunostaining of 54-hpf embryos with antibody zn-5 (The University of Oregon monoclonal facility), which recognizes neurolin of ganglion cell processes, showed no differences in the ganglion cell layer and optic nerve between the eye of control- and #61-MO-injected embryos (Figs. 2K and L), suggesting that optic nerve formation occurred properly in the #61MO-injected embryos at this stage. Taken together, these results indicate that the #61 gene may play an important role in the closure of the choroid fissure, but that it is not necessary for the later eye development including retinal cell differentiation and optic stalk/nerve formation. Blood and vessel development in #61-MO-injected embryos Next we analyzed hemorrhage and vessel formation caused by the absence of #61 in more detail. Hemorrhage around the eye was clearly shown by whole-mount DAB staining (Figs. 3A and D). More detailed analysis of sectioned plastic-embedded samples showed that blood cells remained within the hyaloid vessel in the control-MO-injected embryos (Figs. 3B and C). In contrast, red blood cells outside of the hyaloid vessel were observed with #61-MO-injected embryos (Figs. 3E
Table 1 Summary of MO injection data MO (8–12 ng)
Phenotype Total
Normal
Type 1
Type 2
Others
Lethal
Control MO #61
44 113
37 (84%) 10 (9%)
0 (0%) 30 (27%)
0 (0%) 56 (50%)
0 (0%) 3 (3%)
7 (16%) 14 (12%)
#61-MO (6 ng)+
Total
Normal
Types 1 + 2
Lethal
41 42
13 (32%) 26 (62%)
28 (68%) 12 (29%)
EGFP RNA (250 ng) #61-mut RNA (250 ng)
0 (0%) 4 (9%)
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
1097
Fig. 3. Hemorrhage and blood vessel structures of #61-MO-injected embryos. (A,D) Hemoglobin staining using DAB of 32-hpf zebrafish embryos injected with control (A) or #61 (D) MO. Arrow in “D” shows evidence of hemorrhage. (B,C,E,F) Transverse sections of the eye stained with toluidine blue. (C,F) are magnified views of boxed areas in “B” and “E,” respectively. Scale bars: 100 lm (right panels) and 25 lm (middle and left panels). (G–L) Microangiography in the #61-MO-injected embryos (G,H,J,K) in lateral view with anterior to the left. FITC-dextran was injected at 36 hpf (G,J) or 48 hpf (H,K) into the control (G,H) or #61 (J,K) MO-injected embryos, and vascular network formation of anterior parts (G,J) and caudal parts (H,K) was analyzed by fluorescence dissection microscopy. The white arrow shows a fluorescent lump caused by bleeding (J). Scale bars: 100 lm. (I,L) Light microscopic view of plastic thin transverse-sections of the mid-trunk region derived from 30 hpf zebrafish embryos injected with control (I) or #61 (L) MO (dorsal at the top). NT, neural tube; DA, dorsal aorta; and AV, axial vein.
and F). Since we speculated that failure of blood vessel formation caused hemorrhage, we further examined the vascular network formation by visualizing the network with FITC-Dextran injected into the sinus venosus/cardinal vein (Figs. 3G, H, J, and K). There was a strong FITC signal at the posterior region to the eye where hemorrhage had been observed in the #61-MO-injected embryo (Fig. 3J, arrow). The pattern of the vascular network at other parts showed no significant difference between the control and the #61-MO embryos (Figs. 3H and K). Fluorescence intensity in the #61-MO embryos was weaker than that in the control embryos, which we attribute to the loss of total blood volume due to hemorrhage.
We then prepared transverse sections of the midtrunk region at 30 hpf and examined the development of the dorsal aorta and the axial vein (Figs. 3I and L). As in the control, the dorsal aorta and the axial vein appeared to be well formed and located between the notochord and the gut in the #61-MO embryos (Fig. 3I). We further examined in more detail the structure of blood vessels by electron microscopic analysis of the primordial midbrain channels, but found no evidence of abnormality of the endothelial cells of #61-MO-injected embryos (data not shown). We next examined the expression of an early vascular endothelial cell marker gene, fli-1. Fli-1 encodes an ETSdomain transcription factor and is expressed in the lateral
1098
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
Fig. 4. Whole-mount in situ analysis of vascular and hematopoietic markers in #61 knock-down embryos. Expression of early vascular marker fli-1 (A–D), gata1 (E–H), and scl (I–L) in control and #61-MO-injected embryos of the indicated stages is shown. The arrow in “H” points to ectopic expression of gata1 and the arrowhead in “L” indicates abrogation of expression of scl. Scale bars: 100 lm.
mesoderm [12]. In the #61-MO-injected embryos, the expression pattern of fli-1 appeared to be normal at 20 and 22 hpf (Figs. 4A–D). Since #61 was expressed at the lateral mesoderm of the mid-trunk region, we next examined the expression of the gata1, a hematopoietic tissue marker gene expressed in the lateral mesoderm during early development. At 24 hpf, gata1 was expressed in intermediate cell mass (ICM), which is known as a region for primitive hematopoiesis in zebrafish (Figs. 4E and G). At 18 (Figs. 4E and F) and 22 (data not shown) hpf, the expression pattern of gata1 was similar between the the #61-MO- or control-MO-injected embryos. However, at 25 hpf, expression of gata1 remained in the region anterior to the ICM in the #61-MO-injected embryos (Fig. 4H, arrow), whereas that in the control showed restricted expression in the ICM (Fig. 4G). In contrast, expression of scl, which is another hematopoietic marker [13], was missing from the region anterior to the ICM in #61-MO-injected embryos at 22 hpf (Fig. 4L). The expression pattern of mesoderm markers such as shh, pax2.1, and myoD in #61-MO-treated embryos was indistinguishable from that of control embryos (data not shown).
Discussion In this study, we cloned a novel smoothelin-like actinbinding protein that had a unique expression pattern at the choroid fissure during early eye development in the zebrafish. Several actin-binding proteins, such as fascin [14], dystrophin, and b-thymosin [15], were reported to play roles in retinal development, but none of them are expressed at the choroid fissure. The #61 protein had about 60% homology with human and mouse smoothelin at the C-terminal region containing the actin-bind-
ing domain. Smoothelin is a cytoskeletal protein [16] and expressed as two different isoforms, a vascularspecific large isoform and a visceral-specific small isoform [17,18], due to alternative transcriptional start sites [19]. During early embryogenesis in the chick, smoothelin is expressed in the developing somites, heart, and descending aorta [20]. However, expression of it in the choroid fissure had never been reported. The human Smoothelin gene is co-localized with DRG1 at chromosome 22q12.2 and zebrafish drg1 is located on LG10. The results of radiation hybrid mapping showed #61 to be located on LG15, suggesting that the protein encoded by #61 is not a smoothelin counterpart in zebrafish. We searched for a mammalian homologue of #61 in the database and found a smoothelin-like gene at human chromosome 17p13.3, as a predicted protein by an annotation project (RIKEN). Human chromosome 17 is known to have high synteny with zebrafish LG15. #61 showed a total 30% homology with this putative human smoothelin-like gene, which is slightly higher than that with human smoothelin (26%). In the zebrafish EST database, at least two additional genes containing a #61-like actin-binding domain in their C-terminal regions were found as smoothelin-like genes. We isolated full-length clones of these genes and confirmed that #61 and these genes were different. These two genes also showed only low homologies to smoothelin except for the actin-binding domain in their C-terminal region, suggesting the presence of a new family of smoothelin-like genes. In the #61-MO-injected embryos, failure of closure of the choroid fissure in the eye was observed at early stages (25–30 hpf), but the fissure properly closed at 54 hpf. We could not conclude whether the delay in proper closure was caused by a decrease in the effect of the MO or indicated that #61 plays a role only in
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
defining the timing of choroid fissure closure. Since suppression of pax2.1 was only transient, the latter possibility may be more feasible. In zebrafish, vascular development is initiated at the mid and posterior trunk and then extends to the head region containing the eyes and brain [21]. The spatiotemporal pattern of #61 expression corresponds closely to that of vascular development, indicating that #61 has some functions in vascular development. In #61-MO embryos, signs of hemorrhage were observed in the eye and brain, but early vascular formation and the construction of vascular network seemed normal. We tried to clarify the reason for the hemorrhage by observation of the fine structure of vessels by electron microscopy, but we found no clear morphological defect in the vascular endothelial cells. In general, actin-binding proteins regulate the arrangement of filamentous actin [22] and also play important roles as mediators between actin fibers and membrane-associated protein, such as adhesion molecules [23]. In blood vessels, vascular endothelial cells tightly connect each other by junctions made up of various adhesion molecules. It is tempting to speculate that, in #61 knock-down embryos, the location and arrangement of those membrane proteins were perturbed by loss of #61 function, resulting in hemorrhage at local vessels. In #61-MO embryos, hemorrhage was observed only in the brain but not in other parts of the body. Similarly, in most cases of zebrafish mutants with hematopoietic defects, hemorrhage was exclusively observed in the brain [24]. Although we could not conclude by morphological examination whether the #61 plays a role in the development of the local vessels in the brain, irregular expression pattern of gata1 and scl in certain stages in #61-MO-injected embryos implies that #61 has a role in hematopoiesis as well as in angiogenesis. The study of knock-out mice for fli-1, tie-1, and lklf (lung Kruppel-like factor) genes has revealed that the early vasculogenesis and angiogenesis occurred normally but that the vascular structure was fragile, which results in hemorrhage [25–27]. Further analysis of the role of #61 in the regulation of these genes may give important information regarding hematopoietic and angiogenetic processes.
Acknowledgments We thank A. Muto for discussions, C. Kajiwara for technical assistance, and Y. Matsumura for secretarial help. This work was supported by RIKEN Center of Developmental Biology, Kobe.
References [1] B.M. Carlson, The sense organs, in: R. Farrell (Ed.), Human Embryology and Developmental Biology, Mosby, St. Louis, 1994, pp. 252–275.
1099
[2] J. Malicki, Development of the retina, in: Methods in Cell Biology, vol. 59, Academic Press, San Diego, 1999, pp. 273–299. [3] G. Oliver, P. Gruss, Current views on eye development, Trends Neurosci. 20 (1997) 415–421. [4] D. Jean, K. Ewan, P. Gruss, Molecular regulators involved in vertebrate eye development, Mech. Dev. 76 (1998) 3–18. [5] J.M. Fadool, S.E. Brockerhoff, G.A. Hyatt, J.E. Dowling, Mutations affecting eye morphology in the developing zebrafish (Danio rerio), Dev. Genet. 20 (1997) 288–295. [6] J. Malicki, S.C. Neuhauss, A.F. Schier, L. Solnica-Krezel, D.L. Stemple, D.Y. Stainier, S. Abdelilah, F. Zwartkruis, Z. Rangini, W. Driever, Mutations affecting development of the zebrafish retina, Development 123 (1996) 263–273. [7] S. Watanabe, T. Itoh, K. Arai, JAK2 is essential for activating of c-fos and c-myc promoters and cell proliferation through the human granulocyte–macrophage colony-stimulating factor receptor in BA/F3 cells, J. Biol. Chem. 271 (1996) 12681–12686. [8] R. Kurita, H. Sagara, Y. Aoki, B.A. Link, K. Arai, S. Watanabe, Suppression of lens growth by aA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish, Dev. Biol. 255 (2003) 113–127. [9] A. Nasevicius, J. Larson, S.C. Ekker, Distinct requirements for zebrafish angiogenesis revealed by a VEGF-A morphant, Yeast 17 (2000) 294–301. [10] S. Krauss, T. Johansen, V. Korzh, A. Fjose, Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis, Development 113 (1991) 1193–1206. [11] A.W. Puschel, M. Westerfield, G.R. Dressler, Comparative analysis of Pax-2 protein distributions during neurulation in mice and zebrafish, Mech. Dev. 38 (1992) 197–208. [12] L.A. Brown, A.R. Rodaway, T.F. Schilling, T. Jowett, P.W. Ingham, R.K. Patient, A.D. Sharrocks, Insights into early vasculogenesis revealed by expression of the ETS-domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos, Mech. Dev. 90 (2000) 237–252. [13] M. Gering, A.R. Rodaway, B. Gottgens, R.K. Patient, A.R. Green, The SCL gene specifies haemangioblast development from early mesoderm, EMBO J. 17 (1998) 4029–4045. [14] Y. Saishin, R. Ishikawa, S. Ugawa, W. Guo, T. Ueda, H. Morimura, K. Kohama, H. Shimizu, Y. Tano, S. Shimada, Retinal fascin: functional nature, subcellular distribution, and chromosomal localization, Invest. Ophthalmol. Vis. Sci. 41 (2000) 2087–2095. [15] L.W. Roth, P. Bormann, A. Bonnet, E. Reinhard, beta-Thymosin is required for axonal tract formation in developing zebrafish brain, Development 126 (1999) 1365–1374. [16] F.T. van der Loop, G. Schaart, E.D. Timmer, F.C. Ramaekers, G.J. van Eys, Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells, J. Cell Biol. 134 (1996) 401–411. [17] J. Kramer, A.M. Aguirre-Arteta, C. Thiel, C.M. Gross, R. Dietz, M.C. Cardoso, H. Leonhardt, A novel isoform of the smooth muscle cell differentiation marker smoothelin, J. Mol. Med. 77 (1999) 294–298. [18] G.J. van Eys, M.C. Voller, E.D. Timmer, X.H. Wehrens, J.V. Small, J.A. Schalken, F.C. Ramaekers, F.T. van der Loop, Smoothelin expression characteristics: development of a smooth muscle cell in vitro system and identification of a vascular variant, Cell Struct. Funct. 22 (1997) 65–72. [19] J. Kramer, C. Quensel, J. Meding, M.C. Cardoso, H. Leonhardt, Identification and characterization of novel smoothelin isoforms in vascular smooth muscle, J. Vasc. Res. 38 (2001) 120–132. [20] M.C. Deruiter, S.S. Rensen, G.P. Coolen, B.P. Hierck, M. Bergwerff, W.M. Debie, A.C. Gittenberger-De Groot, G.J. Van Eys, Smoothelin expression during chicken embryogenesis: detection of an embryonic isoform, Dev. Dyn. 221 (2001) 460–463. [21] D. Yelon, B.M. Weinstein, M.C. Fishman, Cardiovascular system, results and problems in cell, Differentiation 40 (2002).
1100
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
[22] D.A. Schafer, J.A. Cooper, Control of actin assembly at filament ends, Annu. Rev. Cell Dev. Biol. 11 (1995) 497–518. [23] C. Thompson, C.H. Lin, P. Forscher, An Aplysia cell adhesion molecule associated with site-directed actin filament assembly in neuronal growth cones, J. Cell Sci. 109 (Part 12) (1996) 2843– 2854. [24] D.Y. Stainier, B. Fouquet, J.N. Chen, K.S. Warren, B.M. Weinstein, S.E. Meiler, M.A. Mohideen, S.C. Neuhauss, L. Solnica-Krezel, A.F. Schier, F. Zwartkruis, D.L. Stemple, J. Malicki, W. Driever, M.C. Fishman, Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo, Development 123 (1996) 285–292.
[25] C.T. Kuo, M.L. Veselits, K.P. Barton, M.M. Lu, C. Clendenin, J.M. Leiden, The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis, Genes Dev. 11 (1997) 2996–3006. [26] H.R. Rodewald, T.N. Sato, Tie1, a receptor tyrosine kinase essential for vascular endothelial cell integrity, is not critical for the development of hematopoietic cells, Oncogene 12 (1996) 397–404. [27] D.D. Spyropoulos, P.N. Pharr, K.R. Lavenburg, P. Jackers, T.S. Papas, M. Ogawa, D.K. Watson, Hemorrhage, impaired hematopoiesis, and lethality in mouse embryos carrying a targeted disruption of the Fli1 transcription factor, Mol. Cell. Biol. 20 (2000) 5643–5652.
A novel smoothelin-like, actin-binding protein required for choroidal fissure closure in zebrafish Ryo Kurita,a Yoko Tabata,a Hiroshi Sagara,b Ken-ichi Arai,a and Sumiko Watanabea,* a
b
Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Department of Fine Morphology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Received 27 November 2003
Abstract A gene expressed in the choroidal fissure of the zebrafish eye was isolated. This gene, designated #61, contained significant homology with the previously reported actin-binding protein smoothelin. During zebrafish embryogenesis, #61 expression was first detected in the lateral mesoderm of the mid-trunk region, and then strong expression was observed in the choroid fissure of the eye and in a part of the brain at 30 hpf. Abrogation of #61 activity by an antisense morpholino oligonucleotide resulted in the failure of closure of the choroid fissure at 30 hpf. In addition, hemorrhage was observed at the caudal side of the eye. Detailed analysis indicated that leakage of blood may have arisen from the hyaloid vessels and the primordial midbrain channels. On the other hand, retinal differentiation and optic nerve formation seemed normal. Taken together, our data suggest that gene #61 may play a role in the formation of hyaloid vessels and subsequent choroid fissure closure. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Actin-binding protein; Eye; Choroidal fissure; Hemorrhage; Zebrafish
The development of the vertebrate eye is initiated by lateral evagination of the neuroepithelium of the ventral diencephalon to form the optic vesicle [1]. The optic vesicle makes contact and interacts with the surface ectoderm, from which the lens placode originates, and this interaction leads to the formation of the two-layered optic cup. The outer layer of the optic cup consists of a single layer of epithelial cells, the retinal pigmented epithelium (RPE); and the inner layer develops into the neural retina. The cells of the proximal region of the optic vesicle give rise to the optic stalks. These stalks have a pipe-like structure connecting the optic cup and the diencephalon. At the early stage of eye development, neither optic stalk nor optic cup closes completely and thus they have a gap called the choroid fissure. At this fissure, mesenchymal cells form the hyaloid vessels. Vascular mesenchyme and the hyaloid artery are incorporated into the longitudinal groove of the optic fissure that extends into the optic * Corresponding author. Fax : +81-3-5449-5474. E-mail address: [email protected] (S. Watanabe).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.12.046
stalk and they gain access to the lentoretinal space. Then the growing edges of the choroidal or optic fissure meet and fuse; and thus the hyaloid vessels and associated mesenchyme, situated in the center of the optic stalk, form the future central retinal artery and vein. The fusion or closure of the optic fissure commences at the mid-portion of the optic stalk and continues both proximally and distally. The zebrafish is an excellent model vertebrate to analyze eye development for many practical reasons as well as because of the high degree of genetic and structural organization of its eye that it shares with other vertebrates, including mammals [2]. Indeed, ocular structure and developmental mechanisms between vertebrates have been remarkably conserved during evolution [3]. The zebrafish retina contains the same major categories of cells as mammalian retinas [2], and the involvement of common genes in eye development of zebrafish and other vertebrates is well documented [3,4]. Recently, a panel of zebrafish mutants having abnormalities in their eyes was generated, thus strengthening the value of this organism for genetic studies [5,6].
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
To isolate novel genes that play a role in eye development, we performed in situ screening of an adult zebrafish retinal library in the EST database. We found a novel actin-binding protein that was expressed strongly in the choroid fissure of the eye. Abrogation of the function of this gene resulted in the failure of closure of the choroid fissure and severe hemorrhage around the eye.
Materials and methods Cloning and DNA construction of #61. Total RNAs were prepared from the head and body parts of zebrafish (Danio rerio) embryos (24 or 48 hpf) and reverse transcribed by use of Superscript II (Invitrogen, Carlsbad, CA). The cDNAs were then subjected to PCR amplification using specific primers designed based on the sequences of randomly selected EST clones of adult zebrafish retinal genes in GenBank (NCBI). Full-length #61 (AB110459) was isolated by 50 and 30 RACE using a Gibco RACE kit and subcloned into the pCS2+ vector by insertion of the DraI (blunted)–EcoRI fragment of #61 into pCS2+ at blunted ClaI and EcoRI sites. The EGFP cDNA which was used as a control was subcloned into pCS2+ by using BamHI and EcoRI sites. For co-injection of #61-MO and #61-RNA, a mutant of the #61 gene (#61-mut), which encodes amino acids identical with those of wild-type #61 but contains mutations at the 50 end noncoding region that prevent it from being targeted by the #61-MO, was constructed by PCR mutagenesis. For co-immunoprecipitation analysis, a Myc epitope-tagged #61 was constructed as follows: blunted BamHI–DraIII and DraIII–XbaI fragments of #61 were prepared from #61-mut-pCS2+ and ligated into pTRE-Myc (Clontech, Palo Alto, CA) at the blunted SfiI and XbaI sites. Then, Myc-#61 cDNA was subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) at the EcoRI site. Immunoprecipitation and Western blot analysis. Immunoprecipitation was done as described [7]. Briefly, COS7 cells were transfected with 1 lg of the Myc-#61 or a control plasmid (pcDNA3.1) by lipofection using FuGENE 6 (Roche Diagnostics GmbH, Mannheim, Germany). Two days later, the cells were harvested and the Myc-tagged protein was immunoprecipitated with a rabbit polyclonal anti-Myc antibody (Santa Cruz Biotechnology, Santa Cruz, California). Precipitates were separated by electrophoresis through a polyacrylamide gel containing 0.1% SDS (SDS–PAGE) and electrotransferred onto Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA). Then, Western blotting was carried out by using mouse monoclonal anti-Myc (Clontech, Palo Alto, CA) and mouse monoclonal anti-actin (Sigma, St. Louis, MO). The blots were visualized with horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody (Amersham, Buckinghamshire, England; 1000-fold dilution) and the Lumi-Light Western Blotting system (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s instructions. Whole-mount in situ hybridization. Probes for pax2.1, scl, gata1, myoD, sonic hedgehog (shh), and fli-1 genes were cloned by PCR amplification using the primers designed according to their EST clone sequences. PCR products were subcloned into the pGEM T-easy vector (Promega, Madison, WI). Digoxigenin (DIG)-labeled antisense RNA probes were synthesized by using a DIG RNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany). Whole-mount in situ hybridization was performed as described previously [8]. Photographs were taken with a Zeiss AxioCam (Carl Zeiss, Jena, Germany) attached to a fluorescence dissection microscope, Leica MZFL III (Leica, Wien, Austria). Microinjection of morpholino (MO) or RNA into zebrafish embryos. MO antisense oligo nucleotides (GENE-TOOLS, LLC, Philomath, OR) were designed against 25 bases including the AUG of #61. The sequence was 50 -GCCTGCATCCATCCTGAGTCCAGGT-30 (start codon is
1093
underlined). The MO was diluted to 0.5 mM with 1 Danieau buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4 , 0.6 mM Ca(NO3 )2 , and 5.0 mM Hepes, pH 7.6) and approximately 0.5–3 nl was injected into the yolk of 1- or 2-cell stage embryos. Standard control MO available from the GENE-TOOLS was used as an injection control, which had no effect on embryonic development under our experimental conditions. Capped sense RNAs were synthesized by using a mMessage mMachine in vitro transcription kit (Ambion, Austin, Texas) according to the manufacturer’s instructions. The synthesized RNAs were diluted to an appropriate concentration with RNase-free water and injected into 1- or 2-cell stage embryos. The amounts of injected RNAs were 50 pg to 1 ng/ embryo for #61 gene and 200 pg/embryo for #61-mut gene. The same amounts of EGFP RNA were used as controls for each experiment. Light-, electron-microscopy, and immunohistochemical staining were performed as described previously [8]. Radiation hybrid mapping. Radiation hybrid mapping (RH mapping) of the #61 gene was kindly performed by the RH mapping services of the children’s hospital zebrafish genome project initiative. (http://zfrhmaps.tch.harvard.edu/ZonRHmapper). Microangiography and hemoglobin staining. Microangiography was done as described [9]. Briefly, fluorescein isothiocyanate (FITC)-Dextran with a molecular mass of 2000 kDa (Sigma, St. Louis, MO) in 1 Danieau buffer at 2 mg/ml was injected into the sinus venosa/cardinal vein of anesthetized embryos at various stages. Photographs were taken during observation under the Leica MZFL III dissection microscope, which was equipped with the standard FITC filter set. For hemoglobin staining, embryos were fixed for 30 min at room temperature in 2.5% glutaraldehyde, 2% formaldehyde, and 0.1 M sodium phosphate (pH 7.4). The fixed embryos were washed three times with PBS and pre-incubated for 30 min at room temperature in the dark in DAB staining solution (0.05% 3,30 -diaminobenzidine tetrahydrochloride, 50 mM Tris–Cl, pH 8.0). The embryos were then incubated in H2 O2 at the final concentration of 0.3% for 5–20 min, washed with PBS, and re-fixed as described above.
Results Isolation of a novel smoothelin-like, actin-binding protein To isolate novel genes specifically expressed in the retina, we selected 75 unknown clones randomly from the adult retina EST database of GenBank. Then RTPCR to examine the expression pattern of each gene in head or body part of 24 and 28 hpf zebrafish embryos was performed. Fifteen clones, which had head-specific expression, were used for subsequent whole-mount in situ analysis of 24-hpf zebrafish embryos. Among them, #61 was unique because of its specific expression in the choroid fissure of the eye at 24 hpf. The full-length transcript of #61 was about 2 kb and it contained a putative 1.5-kb ORF. The predicted 493-amino acid sequence contained a leucine zipper motif in its N-terminal region and an actin-binding domain in its C-terminal one (Fig. 1A). A homology search of amino acids in the C-terminal region revealed a 60% homology with mouse and human proteins of the smoothelin family, which comprises smooth muscle-specific actin-binding proteins. No significant homology at its N-terminal region was found with other genes by database analysis. Radiation hybrid mapping showed that #61 was located in LG15 in the zebrafish genome, not corresponding to
1094
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
Fig. 1. Isolation of novel smoothelin-like, actin-binding protein. (A) Deduced full-length amino acid sequence of zebrafish #61 (Z. #61 ) and human and mouse smoothelin A proteins (H. smth. A and M. smtn. A, respectively). Shadowed boxes indicate conserved amino acids. The actin-binding domain is boxed with broken lines and asterisks indicate the leucine-zipper motif found in the #61 sequence. (B) Co-immunoprecipitation of #61 and actin. COS7 cells were transfected with either the Myc-#61 or a control plasmid. Immunoprecipitation using anti-Myc antibody was performed, followed by Western blotting using anti-Myc (lanes 1 and 2) or anti-actin (lanes 3 and 4) antibodies. Arrows indicate bands correspond to Myc-#61 (56 kDa) and actin (42 kDa). (C–I) Whole-mount in situ hybridization of #61 in zebrafish embryos of various stages. (C) 12 hpf, dorsal view with anterior to the left. #61 was initially observed at the lateral mesoderm of the mid-trunk region (arrow head). (D,E) 15 hpf (D) and 18 hpf (E) embryos showing the lateral view with anterior to the left. Strong signals were detected in the mid-anterior lateral mesoderm (arrows). (F,G) Lateral views, with anterior to the left, of 24-hpf (F) and 30-hpf (G) embryos. The expression of #61 was restricted to the choroid fissure (arrowheads) and a part of the lateral mesoderm and brain (arrows). (H) A ventral view of a 30 hpf embryo with anterior at the top. #61 was detected in some parts of the brain (arrow) and in the choroid fissure of the eye (arrowheads). Scale bars: 100 lm. (I) Sagittal section of eye at 30 hpf. Boxed region indicates the choroids fissure.
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
the chromosomal regions of human (22q12.2) and mouse (11 A2-3) smoothelins. Thus, #61 may not be a smoothelin orthologue in zebrafish. Since #61 contained a putative actin-binding region, we examined whether the #61 product bound actin or not by co-immunoprecipitation analysis. The #61 gene tagged with the Mycepitope at its N-terminus (Myc-#61) was expressed in COS7 cells, and immunoprecipitation using anti-Myc antibody was done, followed by Western blotting using anti-Myc or anti-actin antibodies. As shown in Fig. 1B, anti-Myc antibody co-precipitated the actin (42 kDa) together with the Myc-tagged #61 protein (56 kDa). No signal was detected with the sample prepared from cells transfected with the control vector. Although we cannot exclude the possibility of indirect binding between #61 and actin, it is most feasible that the protein encoded by the #61 gene contains a functional actin-binding domain. We next examined the spatio-temporal expression pattern of #61 during zebrafish embryogenesis by whole-mount in situ analysis. The #61 transcript was
1095
first detected in the lateral mesoderm of the mid-trunk at 12 hpf; and within several hours, its expression spread to both rostral and caudal regions until 18 hpf (Figs. 1C– E). Then the expression became restricted to the midanterior lateral mesoderm at 24 hpf (Fig. 1F). By 30 hpf strong expression was observed in the choroid fissure of the retina and a part of the brain (Figs. 1G–H). Sagittally sectioned samples of a 30-hpf whole-mount in situ embryo clearly showed the expression of #61 at margin of the choroidal fissure (Fig. 1I). Ablation of #61 activity by morpholino antisense oligonucleotide To analyze the role of the #61 gene in zebrafish development, we adopted the knock-down approach using a morpholino antisense oligonucleotide (MO). A specific MO was synthesized based on the sequence of the isolated #61 clone and injected into 1- or 2-cell stage embryos. In the #61-MO-injected embryos, developmental abnormality was first detected between 16 and 18 hpf in
Fig. 2. Effects of abrogation of #61 function by #61 specific morpholino oligo. (A–D) Views of 25-hpf zebrafish embryos injected with control (A) or #61 (B,C) MO. Mild (B, type 1) and severe (C, type 2) phenotypes are shown. Asterisks indicate a curly and thickened tail and yolk sac extension. The arrow in “C” indicates failure of closure of the choroidal fissure in the eye. A view of the head part of a #61-MO-injected embryo is also shown with arrowhead pointing to evidence of bleedings at the eye (D). All panels show the lateral view with anterior to the left. Scale bar: 100 lm. (E–H) Structural observation of choroid fissure in control (E,G) and the #61 (F,H) MO-injected embryos. Sagittal sections of the eyes with anterior to the left and dorsal to the top at 30 hpf (E,F) and 54 hpf (G,H). Arrowheads indicate dislocated cells in the inner plexiform layer. CF, choroid fissure. Scale bars: 50 lm. (I–L) Expression of marker genes of the optic stalk and optic nerve in embryos injected with control (I,K) or #61 (J,L) MO. Whole-mount in situ analysis of pax2.1 expression at 22 hpf (I,J) with lateral view, dorsal at the top. Arrowheads show the expression at the optic stalk. Transverse sections of 48 hpf embryos showing retinal ganglion cells and their axons stained with zn-5 antibody (K,L). Asterisks indicate the optic nerves. Dorsal at the top. Scale bars: 100 lm (I,J) and 50 lm (K,L).
1096
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
the mid-posterior region, especially on the ventral side of the trunk and tail. At 25 hpf, the effects of the MO injection could be classified as type-1 (mild) and type-2 (severe) phenotypes (Figs. 2B and C). Type-1 was characterized by curly, thickened tails and yolk sac extensions, while their head parts seemed to be normal. In the type-2 embryos, failure of closure of choroidal fissure (Fig. 2C, arrow) was observed in addition to the type-1 phenotype. The tail and mid-trunk defects were more severe than those of type-1 morphants. Notably 40% of the total embryos had hemorrhages in the caudal part of their eyes after 30 hpf (Fig. 2D, arrow-head). Hemorrhage was observed in both the type-1 or type-2 embryos and continued to be observed until at least 2.5 dpf. The phenotypic incidence of embryos injected with 8–12 ng of MO was 27% for type 1 and 50% for type 2 (Table 1). Control-MO injection did not affect their embryonic development (Fig. 2A). We next examined whether co-expressed #61 rescued the embryos from the #61-MO effect or not. We prepared #61 mutant containing mis-matched mutations to prevent the inhibition by #61-MO. Over-expression of #61 by capped RNA injection resulted in no morphological defect (data not shown). Synthesized RNA (250 pg) of #61-mutor EGFP as a control was injected together with 6 ng #61-MO into zebrafish embryos (Table 1). The injection of #61-RNA significantly, but not completely, decreased the abnormality observed. MO effects in the #61-mutRNA co-injected embryos were less severe than those in the controls. Retinal differentiation and optic nerve formation in the #61 knock-down embryos We then prepared plastic thin sections from the #61 morphants (type 2) at 30 and 54 hpf and examined the fine structure of their eyes (Figs. 2E–H). In the #61-MOinjected embryo eyes at 30 hpf, failure of the choroid fissure to close was observed (Fig. 2F), whereas the control MO-injected embryos showed only a narrow gap in the choroid fissure (Fig. 2E). But in the later stage, the choroid fissure started to close around at 48 hpf (data not shown), and by 54 hpf, no gap was observed in #61MO-injected embryos (Fig. 2H) or in the control
embryos (Fig. 2G). Layer formation in neural retina occurred indistinguishably in control- and MO-injected embryos at 54 hpf (Figs. 2G and H). Notably, however, cells abnormally located in the inner plexiform layer were observed in the latter (Fig. 2H, arrowheads). We next examined optic stalk and optic nerve formation by using molecular markers. The pax2.1 gene is a member of the paired-box gene family expressed in the optic stalk at early stages during eye development [10,11]. Whole-mount in situ analysis of pax2.1 in the #61-MO-injected embryos revealed that pax2.1 was expressed in optic stalks, midbrain/hindbrain boundaries, and pronephric ducts between 16 and 25 hpf almost just in control embryos. Although the expression of pax2.1 in the optic stalk in the control embryos extended vertically (Fig. 2I), pax2.1 expression in this region of MO-injected embryos was round shaped (Fig. 2J), suggesting failure of extension of the optic nerve. Immunostaining of 54-hpf embryos with antibody zn-5 (The University of Oregon monoclonal facility), which recognizes neurolin of ganglion cell processes, showed no differences in the ganglion cell layer and optic nerve between the eye of control- and #61-MO-injected embryos (Figs. 2K and L), suggesting that optic nerve formation occurred properly in the #61MO-injected embryos at this stage. Taken together, these results indicate that the #61 gene may play an important role in the closure of the choroid fissure, but that it is not necessary for the later eye development including retinal cell differentiation and optic stalk/nerve formation. Blood and vessel development in #61-MO-injected embryos Next we analyzed hemorrhage and vessel formation caused by the absence of #61 in more detail. Hemorrhage around the eye was clearly shown by whole-mount DAB staining (Figs. 3A and D). More detailed analysis of sectioned plastic-embedded samples showed that blood cells remained within the hyaloid vessel in the control-MO-injected embryos (Figs. 3B and C). In contrast, red blood cells outside of the hyaloid vessel were observed with #61-MO-injected embryos (Figs. 3E
Table 1 Summary of MO injection data MO (8–12 ng)
Phenotype Total
Normal
Type 1
Type 2
Others
Lethal
Control MO #61
44 113
37 (84%) 10 (9%)
0 (0%) 30 (27%)
0 (0%) 56 (50%)
0 (0%) 3 (3%)
7 (16%) 14 (12%)
#61-MO (6 ng)+
Total
Normal
Types 1 + 2
Lethal
41 42
13 (32%) 26 (62%)
28 (68%) 12 (29%)
EGFP RNA (250 ng) #61-mut RNA (250 ng)
0 (0%) 4 (9%)
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
1097
Fig. 3. Hemorrhage and blood vessel structures of #61-MO-injected embryos. (A,D) Hemoglobin staining using DAB of 32-hpf zebrafish embryos injected with control (A) or #61 (D) MO. Arrow in “D” shows evidence of hemorrhage. (B,C,E,F) Transverse sections of the eye stained with toluidine blue. (C,F) are magnified views of boxed areas in “B” and “E,” respectively. Scale bars: 100 lm (right panels) and 25 lm (middle and left panels). (G–L) Microangiography in the #61-MO-injected embryos (G,H,J,K) in lateral view with anterior to the left. FITC-dextran was injected at 36 hpf (G,J) or 48 hpf (H,K) into the control (G,H) or #61 (J,K) MO-injected embryos, and vascular network formation of anterior parts (G,J) and caudal parts (H,K) was analyzed by fluorescence dissection microscopy. The white arrow shows a fluorescent lump caused by bleeding (J). Scale bars: 100 lm. (I,L) Light microscopic view of plastic thin transverse-sections of the mid-trunk region derived from 30 hpf zebrafish embryos injected with control (I) or #61 (L) MO (dorsal at the top). NT, neural tube; DA, dorsal aorta; and AV, axial vein.
and F). Since we speculated that failure of blood vessel formation caused hemorrhage, we further examined the vascular network formation by visualizing the network with FITC-Dextran injected into the sinus venosus/cardinal vein (Figs. 3G, H, J, and K). There was a strong FITC signal at the posterior region to the eye where hemorrhage had been observed in the #61-MO-injected embryo (Fig. 3J, arrow). The pattern of the vascular network at other parts showed no significant difference between the control and the #61-MO embryos (Figs. 3H and K). Fluorescence intensity in the #61-MO embryos was weaker than that in the control embryos, which we attribute to the loss of total blood volume due to hemorrhage.
We then prepared transverse sections of the midtrunk region at 30 hpf and examined the development of the dorsal aorta and the axial vein (Figs. 3I and L). As in the control, the dorsal aorta and the axial vein appeared to be well formed and located between the notochord and the gut in the #61-MO embryos (Fig. 3I). We further examined in more detail the structure of blood vessels by electron microscopic analysis of the primordial midbrain channels, but found no evidence of abnormality of the endothelial cells of #61-MO-injected embryos (data not shown). We next examined the expression of an early vascular endothelial cell marker gene, fli-1. Fli-1 encodes an ETSdomain transcription factor and is expressed in the lateral
1098
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
Fig. 4. Whole-mount in situ analysis of vascular and hematopoietic markers in #61 knock-down embryos. Expression of early vascular marker fli-1 (A–D), gata1 (E–H), and scl (I–L) in control and #61-MO-injected embryos of the indicated stages is shown. The arrow in “H” points to ectopic expression of gata1 and the arrowhead in “L” indicates abrogation of expression of scl. Scale bars: 100 lm.
mesoderm [12]. In the #61-MO-injected embryos, the expression pattern of fli-1 appeared to be normal at 20 and 22 hpf (Figs. 4A–D). Since #61 was expressed at the lateral mesoderm of the mid-trunk region, we next examined the expression of the gata1, a hematopoietic tissue marker gene expressed in the lateral mesoderm during early development. At 24 hpf, gata1 was expressed in intermediate cell mass (ICM), which is known as a region for primitive hematopoiesis in zebrafish (Figs. 4E and G). At 18 (Figs. 4E and F) and 22 (data not shown) hpf, the expression pattern of gata1 was similar between the the #61-MO- or control-MO-injected embryos. However, at 25 hpf, expression of gata1 remained in the region anterior to the ICM in the #61-MO-injected embryos (Fig. 4H, arrow), whereas that in the control showed restricted expression in the ICM (Fig. 4G). In contrast, expression of scl, which is another hematopoietic marker [13], was missing from the region anterior to the ICM in #61-MO-injected embryos at 22 hpf (Fig. 4L). The expression pattern of mesoderm markers such as shh, pax2.1, and myoD in #61-MO-treated embryos was indistinguishable from that of control embryos (data not shown).
Discussion In this study, we cloned a novel smoothelin-like actinbinding protein that had a unique expression pattern at the choroid fissure during early eye development in the zebrafish. Several actin-binding proteins, such as fascin [14], dystrophin, and b-thymosin [15], were reported to play roles in retinal development, but none of them are expressed at the choroid fissure. The #61 protein had about 60% homology with human and mouse smoothelin at the C-terminal region containing the actin-bind-
ing domain. Smoothelin is a cytoskeletal protein [16] and expressed as two different isoforms, a vascularspecific large isoform and a visceral-specific small isoform [17,18], due to alternative transcriptional start sites [19]. During early embryogenesis in the chick, smoothelin is expressed in the developing somites, heart, and descending aorta [20]. However, expression of it in the choroid fissure had never been reported. The human Smoothelin gene is co-localized with DRG1 at chromosome 22q12.2 and zebrafish drg1 is located on LG10. The results of radiation hybrid mapping showed #61 to be located on LG15, suggesting that the protein encoded by #61 is not a smoothelin counterpart in zebrafish. We searched for a mammalian homologue of #61 in the database and found a smoothelin-like gene at human chromosome 17p13.3, as a predicted protein by an annotation project (RIKEN). Human chromosome 17 is known to have high synteny with zebrafish LG15. #61 showed a total 30% homology with this putative human smoothelin-like gene, which is slightly higher than that with human smoothelin (26%). In the zebrafish EST database, at least two additional genes containing a #61-like actin-binding domain in their C-terminal regions were found as smoothelin-like genes. We isolated full-length clones of these genes and confirmed that #61 and these genes were different. These two genes also showed only low homologies to smoothelin except for the actin-binding domain in their C-terminal region, suggesting the presence of a new family of smoothelin-like genes. In the #61-MO-injected embryos, failure of closure of the choroid fissure in the eye was observed at early stages (25–30 hpf), but the fissure properly closed at 54 hpf. We could not conclude whether the delay in proper closure was caused by a decrease in the effect of the MO or indicated that #61 plays a role only in
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
defining the timing of choroid fissure closure. Since suppression of pax2.1 was only transient, the latter possibility may be more feasible. In zebrafish, vascular development is initiated at the mid and posterior trunk and then extends to the head region containing the eyes and brain [21]. The spatiotemporal pattern of #61 expression corresponds closely to that of vascular development, indicating that #61 has some functions in vascular development. In #61-MO embryos, signs of hemorrhage were observed in the eye and brain, but early vascular formation and the construction of vascular network seemed normal. We tried to clarify the reason for the hemorrhage by observation of the fine structure of vessels by electron microscopy, but we found no clear morphological defect in the vascular endothelial cells. In general, actin-binding proteins regulate the arrangement of filamentous actin [22] and also play important roles as mediators between actin fibers and membrane-associated protein, such as adhesion molecules [23]. In blood vessels, vascular endothelial cells tightly connect each other by junctions made up of various adhesion molecules. It is tempting to speculate that, in #61 knock-down embryos, the location and arrangement of those membrane proteins were perturbed by loss of #61 function, resulting in hemorrhage at local vessels. In #61-MO embryos, hemorrhage was observed only in the brain but not in other parts of the body. Similarly, in most cases of zebrafish mutants with hematopoietic defects, hemorrhage was exclusively observed in the brain [24]. Although we could not conclude by morphological examination whether the #61 plays a role in the development of the local vessels in the brain, irregular expression pattern of gata1 and scl in certain stages in #61-MO-injected embryos implies that #61 has a role in hematopoiesis as well as in angiogenesis. The study of knock-out mice for fli-1, tie-1, and lklf (lung Kruppel-like factor) genes has revealed that the early vasculogenesis and angiogenesis occurred normally but that the vascular structure was fragile, which results in hemorrhage [25–27]. Further analysis of the role of #61 in the regulation of these genes may give important information regarding hematopoietic and angiogenetic processes.
Acknowledgments We thank A. Muto for discussions, C. Kajiwara for technical assistance, and Y. Matsumura for secretarial help. This work was supported by RIKEN Center of Developmental Biology, Kobe.
References [1] B.M. Carlson, The sense organs, in: R. Farrell (Ed.), Human Embryology and Developmental Biology, Mosby, St. Louis, 1994, pp. 252–275.
1099
[2] J. Malicki, Development of the retina, in: Methods in Cell Biology, vol. 59, Academic Press, San Diego, 1999, pp. 273–299. [3] G. Oliver, P. Gruss, Current views on eye development, Trends Neurosci. 20 (1997) 415–421. [4] D. Jean, K. Ewan, P. Gruss, Molecular regulators involved in vertebrate eye development, Mech. Dev. 76 (1998) 3–18. [5] J.M. Fadool, S.E. Brockerhoff, G.A. Hyatt, J.E. Dowling, Mutations affecting eye morphology in the developing zebrafish (Danio rerio), Dev. Genet. 20 (1997) 288–295. [6] J. Malicki, S.C. Neuhauss, A.F. Schier, L. Solnica-Krezel, D.L. Stemple, D.Y. Stainier, S. Abdelilah, F. Zwartkruis, Z. Rangini, W. Driever, Mutations affecting development of the zebrafish retina, Development 123 (1996) 263–273. [7] S. Watanabe, T. Itoh, K. Arai, JAK2 is essential for activating of c-fos and c-myc promoters and cell proliferation through the human granulocyte–macrophage colony-stimulating factor receptor in BA/F3 cells, J. Biol. Chem. 271 (1996) 12681–12686. [8] R. Kurita, H. Sagara, Y. Aoki, B.A. Link, K. Arai, S. Watanabe, Suppression of lens growth by aA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish, Dev. Biol. 255 (2003) 113–127. [9] A. Nasevicius, J. Larson, S.C. Ekker, Distinct requirements for zebrafish angiogenesis revealed by a VEGF-A morphant, Yeast 17 (2000) 294–301. [10] S. Krauss, T. Johansen, V. Korzh, A. Fjose, Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis, Development 113 (1991) 1193–1206. [11] A.W. Puschel, M. Westerfield, G.R. Dressler, Comparative analysis of Pax-2 protein distributions during neurulation in mice and zebrafish, Mech. Dev. 38 (1992) 197–208. [12] L.A. Brown, A.R. Rodaway, T.F. Schilling, T. Jowett, P.W. Ingham, R.K. Patient, A.D. Sharrocks, Insights into early vasculogenesis revealed by expression of the ETS-domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos, Mech. Dev. 90 (2000) 237–252. [13] M. Gering, A.R. Rodaway, B. Gottgens, R.K. Patient, A.R. Green, The SCL gene specifies haemangioblast development from early mesoderm, EMBO J. 17 (1998) 4029–4045. [14] Y. Saishin, R. Ishikawa, S. Ugawa, W. Guo, T. Ueda, H. Morimura, K. Kohama, H. Shimizu, Y. Tano, S. Shimada, Retinal fascin: functional nature, subcellular distribution, and chromosomal localization, Invest. Ophthalmol. Vis. Sci. 41 (2000) 2087–2095. [15] L.W. Roth, P. Bormann, A. Bonnet, E. Reinhard, beta-Thymosin is required for axonal tract formation in developing zebrafish brain, Development 126 (1999) 1365–1374. [16] F.T. van der Loop, G. Schaart, E.D. Timmer, F.C. Ramaekers, G.J. van Eys, Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells, J. Cell Biol. 134 (1996) 401–411. [17] J. Kramer, A.M. Aguirre-Arteta, C. Thiel, C.M. Gross, R. Dietz, M.C. Cardoso, H. Leonhardt, A novel isoform of the smooth muscle cell differentiation marker smoothelin, J. Mol. Med. 77 (1999) 294–298. [18] G.J. van Eys, M.C. Voller, E.D. Timmer, X.H. Wehrens, J.V. Small, J.A. Schalken, F.C. Ramaekers, F.T. van der Loop, Smoothelin expression characteristics: development of a smooth muscle cell in vitro system and identification of a vascular variant, Cell Struct. Funct. 22 (1997) 65–72. [19] J. Kramer, C. Quensel, J. Meding, M.C. Cardoso, H. Leonhardt, Identification and characterization of novel smoothelin isoforms in vascular smooth muscle, J. Vasc. Res. 38 (2001) 120–132. [20] M.C. Deruiter, S.S. Rensen, G.P. Coolen, B.P. Hierck, M. Bergwerff, W.M. Debie, A.C. Gittenberger-De Groot, G.J. Van Eys, Smoothelin expression during chicken embryogenesis: detection of an embryonic isoform, Dev. Dyn. 221 (2001) 460–463. [21] D. Yelon, B.M. Weinstein, M.C. Fishman, Cardiovascular system, results and problems in cell, Differentiation 40 (2002).
1100
R. Kurita et al. / Biochemical and Biophysical Research Communications 313 (2004) 1092–1100
[22] D.A. Schafer, J.A. Cooper, Control of actin assembly at filament ends, Annu. Rev. Cell Dev. Biol. 11 (1995) 497–518. [23] C. Thompson, C.H. Lin, P. Forscher, An Aplysia cell adhesion molecule associated with site-directed actin filament assembly in neuronal growth cones, J. Cell Sci. 109 (Part 12) (1996) 2843– 2854. [24] D.Y. Stainier, B. Fouquet, J.N. Chen, K.S. Warren, B.M. Weinstein, S.E. Meiler, M.A. Mohideen, S.C. Neuhauss, L. Solnica-Krezel, A.F. Schier, F. Zwartkruis, D.L. Stemple, J. Malicki, W. Driever, M.C. Fishman, Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo, Development 123 (1996) 285–292.
[25] C.T. Kuo, M.L. Veselits, K.P. Barton, M.M. Lu, C. Clendenin, J.M. Leiden, The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis, Genes Dev. 11 (1997) 2996–3006. [26] H.R. Rodewald, T.N. Sato, Tie1, a receptor tyrosine kinase essential for vascular endothelial cell integrity, is not critical for the development of hematopoietic cells, Oncogene 12 (1996) 397–404. [27] D.D. Spyropoulos, P.N. Pharr, K.R. Lavenburg, P. Jackers, T.S. Papas, M. Ogawa, D.K. Watson, Hemorrhage, impaired hematopoiesis, and lethality in mouse embryos carrying a targeted disruption of the Fli1 transcription factor, Mol. Cell. Biol. 20 (2000) 5643–5652.