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Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: Cunming Duan, Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University Avenue, Ann Arbor, Michigan 48109. E-mail: cduan{at}umich.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Among the six known IGFBPs in mammals, IGFBP-2 is one of the most abundantly expressed, particularly during fetal development (6, 7). Whereas in vitro studies suggest that IGFBP-2 is primarily inhibitory to IGF actions (1, 2), a clear understanding of its functions in vivo remains elusive. All known vertebrate homologs of IGFBP-2 contain a putative heparin-binding motif (PKKXRP), and mammalian IGFBP-2 proteins have been shown to interact directly with extracellular matrix proteins (1, 2). These properties suggest that IGFBP-2 may function in a paracrine fashion to coordinate the precise delivery of IGFs to the surface of cells expressing IGF receptors (3, 4).
Quite surprisingly, homologous deletion of the IGFBP-2 gene in mice resulted in few phenotypic effects: knockout progeny were both viable and fertile and had prenatal and postnatal body weights indistinguishable from those of their wild-type litter mates (8). Tissue-specific effects of IGFBP-2 gene deletion (reduced spleen, enlarged liver) were subsequently observed, but only in adult mice (8), leaving in question the functional roles of IGFBP-2 during fetal development.
Two explanations have been offered to account for the lack of developmental and growth defects in IGFBP-2 knockout mice. First, compensatory adjustments in the expression of other IGFBPs may have minimized deleterious effects of gene deletion; for example, serum levels of IGFBP-1, -3, and -4 were significantly increased in IGFBP-2 knockout mice (8). Second, factors delivered through the placental circulation may have compensated for the absence of IGFBP-2. These issues exemplify some of the difficulties inherent to the study of early development in placental vertebrates.
During the last decade, the zebrafish has emerged as an excellent alternative model for the study of early vertebrate development (9). Embryos of this species develop externally, providing the opportunity to study gene and protein function in the absence of maternal compensation. The optical clarity of zebrafish embryos also permits real-time visualization of tissue and organ formation, a property that has been enhanced by the development of transgenic lines expressing reporter genes (e.g. green fluorescent protein, GFP) under the control of tissue-specific promoters. This species is also amenable to genetic loss-of-function approaches, including targeted gene knockdown by morpholino-modified antisense oligonucleotides (MOs), which specifically suppress the translation of target gene mRNAs (10).
The IGF signaling system (ligands, receptors, and binding proteins) is highly conserved among vertebrates, including zebrafish and other teleosts (11, 12, 13, 14, 15). We previously reported the zebrafish IGFBP-2 gene to encode a protein that has high affinity for both IGF-I and IGF-II (but not insulin) and that inhibits IGF-induced cell growth in vitro (14). The objectives of the present study were to examine the expression pattern of IGFBP-2 mRNA during zebrafish embryonic development and to determine the physiological requirements for IGFBP-2 during early development, using a loss-of-function approach.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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], confirming that IGFBP-2 mRNA is provided as a maternal transcript and is expressed by the zygotic genome throughout the cleavage and gastrulation stages of embryonic development.
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, 18h). By 30 hpf, a strong hybridization signal was detected in the peripheral lens (Fig. 1B, 30h): examination of cryosectioned embryos revealed localized expression of IGFBP-2 mRNA in cuboidal epithelial cells on the lateral surface of the lens (Fig. 1B, 30h; inset).
From about 32 hpf onwards, IGFBP-2 mRNA expression became detectable in cranial boundary regions. The location of IGFBP-2 mRNA expression resembled known vascular pathways (16), although double-labeling experiments (using transgenic zebrafish expressing GFP in vascular endothelial cells) suggested that IGFBP-2 mRNA-expressing cells were adjacent to vascular endothelial cells (data not shown). At the present time, the precise identity of the cells expressing IGFBP-2 remains unknown. Expression in these regions persisted throughout the hatching period (Fig. 1B, 56h-a and -b) until approximately 80 hpf; by approximately 60 hpf, however, expression also became detectable in the newly formed liver (Fig. 1B, 72h). By 96 hpf and beyond, IGFBP-2 mRNA expression was detected most abundantly in the liver (Fig. 1, 96h).
Antisense MOs Specifically and Efficiently Knock Down IGFBP-2 in Vivo
Microinjection of plasmid DNA (50–100 pg/embryo) encoding an IGFBP-2-GFP fusion protein (BP2:GFP) into zebrafish embryos yielded pronounced mosaic GFP expression, clearly visible at 24 hpf by fluorescence microscopy (Fig. 2A). Coinjection of BP2:GFP plasmid DNA (50 pg) with 2–3 ng of either antisense IGFBP-2 targeting MO (MO1, MO2; Table 1) completely abolished BP2:GFP expression (Fig. 2, B and C), whereas coinjection with a control MO (CTRL; Table 1) failed to abolish GFP expression. Likewise, coinjection of either MO1 or MO2 with an expression plasmid encoding zebrafish IGFBP-3:GFP (Li, Y., and C. Duan, unpublished data) produced embryos with pronounced mosaic GFP expression (Fig. 2C). Collectively, these data confirm that antisense MOs targeted against IGFBP-2 specifically and efficiently suppress the translation of IGFBP-2 mRNA into functional protein but do not suppress the translation of a closely related nontarget gene. Whereas a preferable method would have been to directly measure the quantities of endogenous IGFBP-2 protein, we were unable to achieve this due to the lack of cross-reactivity between a commercially available (bovine) anti-IGFBP-2 antibody and zebrafish IGFBP-2. Similarly, ligand blotting approaches were insufficiently sensitive to detect IGFBP-2 protein levels during early stages of embryonic development in zebrafish.
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, A and B), whereas embryos injected with MO1 or MO2 (morphant embryos) exhibited delayed embryonic development (Fig. 3, C and D). Somite number (Fig. 3E) was used as a quantitative measure of developmental rate during the segmentation period (20). At 24 hpf, control-injected embryos had 29.3 ± 0.17 somites (n = 26) (30 somites at 24 hpf in wild-type embryos); embryos injected with 2.5 ng of MO1 had an average of 29.8 ± 0.45 somites (P = 0.09; n = 13), whereas embryos injected with 5 ng of MO1 had an average of 25.7 ± 0.34 somites (P < 0.0001; n =26). Embryos injected with 5 ng of MO1 were therefore developmentally equivalent to wild-type and control-injected embryos at 22 hpf (20). As shown in Fig. 3F, injection of MO1 also significantly reduced the mean length (ear-tail) from 2.39 ± 0.04 mm (n =19) of the control embryos to 2.69 ± 0.03 mm (n = 14) of the morphant embryos (P < 0.0001).
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) were significantly reduced compared with control-injected embryos (n = 5; P = 0.0016).
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, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Embryos injected with MO1 or MO2 (2.5 ng/embryo) exhibited a visible reduction in the density of circulating blood cells (supplemental Fig. 2). The reduction in circulating blood cells in morphant embryos was confirmed by reduced o-dianisidine staining for hemoglobin, when compared with equivalent stage (48 hpf) control-injected embryos (Fig. 5, A and B). In a representative experiment, severely affected morphant embryos (11/18, 61%) exhibited a complete absence of o-dianisidine staining, whereas more mildly affected morphant embryos (5/18, 27%) exhibited visibly reduced o-dianisidine staining (Fig. 5, A and B). Beyond 30 hpf, the development of cerebral and/or pericardial edema became evident in a majority (91%) of morphant embryos (Fig. 5, C and D), whereas no equivalent phenotype was observed in control-injected embryos.
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). Because the zebrafish heart rate gradually increases during the first few days of embryonic development (21), we sought to determine whether the lower heart rate in morphant embryos could be attributed to delayed development. Heart rates were thus measured again on the same individuals at 32 hpf: the mean heart rate of control-injected embryos at 32 hpf had increased by 15.2% to 110 ± 2.7 beats/min (n = 20); by contrast, the mean heart rate of morphant embryos at 32 hpf had decreased by 13.7% (65.7 ± 2.5 beats/min; n = 20) from the mean value at 28 hpf, indicating progressive cardiac dysfunction in IGFBP-2 morphant embryos.
Knockdown of IGFBP-2 Affects Hematopoietic Transcription Factor Expression Patterns
To determine whether the reductions in blood cell density in morphant embryos were the result of changes in the blood cell differentiation, we performed in situ hybridizations to determine the expression patterns of selected hematopoietic transcription factors. At 18 hpf, we detected robust expression of gata1, a marker for erythroid differentiation, in the intermediate cell mass region of both control-injected (Fig. 6A; n = 8) and morphant (Fig. 6B; n = 8) embryos, in patterns identical with that seen in wild-type embryos (22). At 25 hpf, control-injected embryos continued to exhibit strong expression of gata1 (Fig. 6C), from the intermediate cell mass extending anteriorly along the central axis, whereas gata1 expression in morphant embryos was greatly reduced or abolished at 25 hpf (Fig. 6D). Similar results were observed with the lymphoid marker stem cell leukemia (scl): expression was robust in control-injected embryos at 25 hpf (Fig. 6E) but was reduced or abolished in equivalent-stage morphant embryos (Fig. 6F). These effects were specific to blood markers because expression of selected neuronal transcription factors (e.g. Pax2.1; Fig. 6, G and H) was not affected in 25 hpf morphant embryos.
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, A and B), but examination of secondary vascular structures revealed vascular patterning defects in regions associated with IGFBP-2 mRNA expression. Specifically, vessels medial to the lens were clearly distinguishable in control-injected embryos at 30 hpf (Fig. 6C), whereas corresponding vessels were generally absent or disorganized in equivalent-stage morphant embryos (Fig. 6D). Similarly, angiogenic sprouting of secondary vessels between the primordial hindbrain channel (PHBC) and the basilar artery was clearly visible in control-injected embryos by 36 hpf (Fig. 6E); by contrast, there was a visible reduction in secondary vessel development in equivalent-stage IGFBP-2 morphant embryos (Fig. 6F).
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). The mean number of secondary vessels in control-injected embryos (5.1 ± 0.3; n =12) did not differ significantly from the mean number of secondary vessels in wild-type embryos (5.0 ± 0.4; n = 8; P > 0.05); however, the mean number of secondary vessels sprouting from the PHBC in morphant embryos (1.8 ± 0.3; n =18) was significantly lower than the mean values of both control-injected and wild-type embryos (P < 0.0001). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The detection of IGFBP-2 mRNA in gastrula embryos indicates that IGFBP-2 mRNA is initially endowed as a maternal transcript. mRNA transcripts encoding IGF-I, IGF-II, and type-1 IGF receptors were previously reported in unfertilized zebrafish eggs (12), suggesting that the IGF signaling system as a whole is present and may be functional during the initial stages of embryonic development. These findings are of interest, in light of considerable debate regarding the functional requirements for IGF signaling in embryonic development in mammals (8, 25). Later in the segmentation period (10–24 hpf), IGFBP-2 begins to exhibit spatially distinct patterns of expression, although these differ somewhat from the patterns reported in other vertebrate embryos. For example in the chick embryo, IGFBP-2 mRNA expression was initially localized to the primitive streak region and was later expressed in anterior lateral plate mesoderm and in regions adjacent to developing somites (26). In the ovine fetus, IGFBP-2 mRNA was initially widely expressed, before becoming restricted to liver, kidney and choroid plexus during later fetal development (27). Interestingly, the lens vesicle consistently exhibited detectable IGFBP-2 mRNA expression in the embryonic mouse (embryonic d 9.5-13.5), which is consistent with our observations in early (18–32 hpf) zebrafish embryos. However, the distribution of detectable IGFBP-2 expression in the fetal mouse increased throughout development (28), whereas in zebrafish it retained high tissue specificity, becoming specifically localized to brain boundary regions in close proximity to vascular pathways. Finally, near the completion of embryonic development, the zebrafish liver becomes the primary source of IGFBP-2, which is consistent with an endocrine role for IGFBP-2 during juvenile and adult development (13, 14, 27, 29).
Using MOs, we next examined the phenotypic consequences of targeted IGFBP-2 gene suppression. Knockdown of IGFBP-2 in zebrafish embryos resulted in delayed development, as indicated by the reduced rate of somitogenesis during the segmentation stage, and reduced linear body growth during later stages. These data suggest that IGFBP-2 serves a growth-promoting function in early development, which is seemingly contradictory to the conventional view of IGFBP-2 function. Data from a large number of studies suggest that IGFBP-2 functions primarily to inhibit IGF-mediated cell growth, presumably by sequestering IGFs and preventing their interaction with IGF-I receptors (2, 4). However, most of these data are derived from in vitro model systems, which cannot reproduce the complexity of molecular interactions in vivo. These in vitro data are also inconsistent with the high levels of IGFBP-2 observed during fetal development, a period of distinctly rapid somatic growth and differentiation. Importantly, there are precedents for our data suggesting that IGFBP-2 plays a growth-promoting function in vivo: for example, targeted knockout of IGFBP-4, which has consistently been shown to inhibit IGF-induced cell growth in vitro, similarly resulted in reduced growth in mice (30). Likewise, targeted deletion of IGFBP-2 in mice resulted in increase liver size but reduced spleen size (8).
Unlike the IGFBP-2 knockout mice, knockdown of IGFBP-2 in zebrafish embryos did not alter the levels of other IGFBPs, at least not detectably at the mRNA level; neither were changes observed in IGF-II mRNA levels. The IGF-I mRNA levels, however, were reduced in morphant embryos. At present, it is unclear whether the reductions in IGF-I mRNA expression are the direct result of reduced IGFBP-2 protein or represent a secondary defect of disrupted development and/or metabolism. It is possible that the reductions in IGF-I mRNA expression could be secondary to the observed cardiovascular dysfunction. It is well established in both mammals and lower vertebrates that IGF-I gene expression is influenced by nutritional status, during both fetal and postnatal stages (31, 32, 33, 34). Because one of the primary functions of the circulatory system during early development is to distribute nutritional resources derived from the yolk, the reduced circulatory function of IGFBP-2 knockdown embryos likely results in reduced distribution of nutritional resources to the growing embryo. Further studies are needed to distinguish between the proximate and ultimate causes of altered IGF-I mRNA expression after IGFBP-2 knockdown.
The development of brain edema, as observed in morphant embryos, is indicative of disruptions to the microvascular interface (choroid plexus) between the general circulation and the cerebral spinal fluid. The choroid plexus is a site of abundant IGFBP-2 expression in mammals (27, 35), and IGFBP-2 protein is found in abundance in rat cerebral spinal fluid (35). In this study, we detected strong expression of IGFBP-2 in brain boundary regions closely associated with cranial vascular tracts. We furthermore demonstrated, in flk1:GFP embryos, that angiogenic sprouting of secondary blood vessels between the primordial hindbrain channel and the basilar artery (16) was visibly disrupted in IGFBP-2 knockdown embryos. Our observations indicating malfunction of the choroid plexus after knockdown of IGFBP-2 are consistent with the reported expression patterns of IGFBP-2 in mammals and suggest that IGFBP-2 may be specifically required for early development of cranial microvascular structures.
The reductions in blood cell density observed in our study were temporally coincident with the onset of primary blood circulation. Before the onset of circulation, we did not detect any differences in hematopoietic transcription factor expression between knockdown and control-injected embryos. These data suggest that IGFBP-2 is probably not required for hematopoietic differentiation but may be involved in the survival of blood cells after the onset of circulation. We also observed a reduction in the heart rate of IGFBP-2 knockdown embryos. For two reasons, we suspect this to be a secondary defect resulting from the failure to develop a functional circulatory system. First, we did not detect IGFBP-2 expression in cardiac tissues during development; second, cardiac function in morphant embryos progressively declined as development proceeded, which is consistent with the progressive angiogenic defects.
Our experiments with flk1:GFP transgenic embryos provide novel in vivo evidence that IGFBP-2 is required for vascular development in zebrafish embryos. Whereas primary vessels (e.g. dorsal aorta, posterior cardinal vein) develop in the IGFBP-2 knockdown embryos, we observed distinct disruptions to angiogenic sprouting of secondary blood vessels. These defects were most evident in the lens and the brain, regions that exhibit high levels of IGFBP-2 mRNA expression during embryogenesis. Collectively, our findings in the zebrafish embryo contribute to an increasing body of evidence, indicating the importance of IGF signaling for vascular development in vertebrates. For example, IGF-I was shown to be necessary for normal vascularization of the human retina (36), whereas overexpression of IGF-I in mice ocular tissues recapitulated vascular defects associated with diabetes-like eye disease (37). In addition, IGF-I was shown to be required to stimulate blood vessel growth and remodeling in the rat brain under various metabolic circumstances (38).
Whereas cultured mammalian vascular endothelial cells have previously been shown to secrete IGFBPs (39), our preliminary colocalization experiments (data not shown) indicated that IGFBP-2 mRNA in the brain boundary regions did not directly colocalize with vascular endothelial cells, suggesting that that cells adjacent to vascular tissues, but not vascular endothelial cells per se, are the primary source of IGFBP-2 in zebrafish embryos. These findings are suggestive of a paracrine function for IGFBP-2 during angiogenic development.
In summary, we have shown that IGFBP-2 mRNA is expressed in temporally and spatially specific patterns during zebrafish embryonic development and that targeted knockdown of IGFBP-2 results in reduced embryo growth and distinct disruptions to embryonic vascular development. The defects in vascular development are most severe in regions associated with high levels of IGFBP-2 mRNA expression, suggesting a paracrine function for IGFBP-2. To our knowledge, this study is the first to provide in vivo evidence for the requirement of an IGFBP in vertebrate vascular development. In vitro, zebrafish IGFBP-2 binds to IGFs and was shown to inhibit IGF-induced cell proliferation (9). Likewise, overexpression of IGFBP-2 in zebrafish embryos inhibited IGF-stimulated somatic growth (40). It is therefore possible that IGFBP-2 affects embryonic vascular development in the zebrafish through its interaction with IGFs. Future studies will focus on the mechanisms of IGFBP-2 action and how IGFBP-2 works together with IGFs and/or other molecules to regulate vascular development.
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MATERIALS AND METHODS |
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Standard chemicals and reagents were purchased from Fisher (Pittsburgh, PA) unless otherwise specified. RNA polymerase enzymes and ribonuclease-free deoxyribonuclease were purchased from Promega (Madison, WI). Restriction endonucleases were purchased from New England BioLabs (Beverly, MA). The Eppendorf MasterTaq Kit (Brinkmann, Westbury, NY) was used for all PCRs. Superscript II reverse transcriptase and oligonucleotide primers (Table 2) were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA). MOs (Table 1) were purchased from Gene Tools, LLC (Philomath, OR).
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To detect IGFBP-2 mRNA expression in pregastrula embryos (0–10 hpf), we used an oligonucleotide primer pair (BP2-F1, BP2-R1; Table 2) designed to amplify a 426-bp fragment of the IGFBP-2 cDNA. A denaturing step (94 C for 5 min) was followed by 35 cycles in the following conditions: denaturation (94 C for 1 min), annealing (58 C for 1 min), and extension (72 C for 1 min), in an Eppendorf Mastercycler gradient thermocycler. To determine whether knockdown of IGFBP-2 alters the expression of other components of the IGF system, total RNA was purified from pools of control and IGFBP-2 morphant embryos (n = 20 embryos for each) at 48 hpf, and reverse-transcribed to cDNA as described above. Target genes were PCR-amplified using specific primers (Table 2) and examined after an appropriate number of cycles, ensuring amplification was terminated during the exponential phase; this was empirically determined for each target gene. PCR products were size-fractionated in 1% agarose gels, stained with ethidium bromide, and measured densitometrically using National Institutes of Health Image software (NIH, Bethesda, MD). The density of PCR products in control-injected and morphant embryos were directly compared in parallel reactions, using odc as an internal reference standard.
Whole Mount in Situ Hybridization
An 830-bp DNA fragment encoding the full-length zebrafish IGFBP-2 and its complete 3'-untranslated region (UTR) was subcloned into the pCS2+ vector (a gift from Dr. V. Prince, University of Chicago, Chicago, IL) using Xho1 and BamH1 restriction sites. Plasmid DNA for zebrafish gata1, scl, and Pax2.1 was provided by Drs. Susan Lyons and Pamela Raymond (University of Michigan). Purified plasmid DNA was linearized by restriction enzyme digestion to generate template for riboprobe synthesis. Digoxigenin-labeled riboprobes were synthesized by in vitro transcription as previously described (11). Whole-mount in situ hybridizations were performed on fixed embryos as previously described (11). Images were captured with a digital camera (Nikon COOLPIX995) mounted to a dissecting stereomicroscope, or with a Nikon DC50NN camera mounted to a Nikon Eclipse E600 microscope equipped with Nomarski optics (Melville, NY).
For cryosectioning analysis, embryos were refixed in paraformaldehyde as described, washed in PBS containing 0.1% Tween 20 (PBS-T), permeated with 15% sucrose in PBS-T, and embedded in OCT medium. OCT-embedded samples were frozen at –20 C and sectioned (10 µm) onto charged microscope slides (SuperFrost+) using a Leica CM3050 S cryostat (Solms, Germany). Embedding medium was removed by washing in room temperature PBS-T, and sections were examined with Nomarski optics.
MOs
Two MOs targeted against zebrafish IGFBP-2 were designed according to criteria provided by the commercial supplier (Gene Tools, LLC.): MO1 targeted nucleotides –45 to –20, and MO2 targeted nucleotides –26 to –2 (Table 1). A control MO (CTRL), composed of the inverted sequence of MO2, was used as a control for nonspecific effects of MO injection. Stock MO solutions [3 mM in Danieau buffer: 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES (pH 7.6)] were diluted to working strength concentrations in Danieau buffer containing 0.25% phenol red.
Confirmation of MO Specificity
A 984-bp fragment of the zebrafish IGFBP-2 cDNA corresponding to the complete 5'-UTR region (including the MO target regions), the complete coding region, and a portion of the 3'-UTR region was generated by RT-PCR using designed primers (BP2MO-F1, BP2MO-R2; Table 2). The amplified cDNA product was subcloned into the pCR2.1-TOPO vector (Invitrogen Life Technologies). Plasmids with an insert in the sense direction were identified by restriction digestion analysis, and sequence accuracy was confirmed by automated sequencing. A 922-bp fragment of the subcloned insert (containing the MO target regions) was removed by digestion with BamH1 and Xho1, gel-purified (QIAQuick gel extraction kit), and subcloned into the pEGFP-N1 plasmid (CLONTECH, Palo Alto, CA) using Xho1 and BamH1 restriction sites. Expression of this plasmid insert (driven by the cytomegalovirus promoter) was predicted to yield a fusion protein of 513 amino acids (57.4 kDa), consisting of truncated IGFBP-2 (amino acids 1–268) fused to GFP at the C terminus (BP2:GFP).
Microinjection
MO or plasmid DNA solutions were microinjected into embryos at the 1–2 cell stage using drawn glass microcapillary pipettes attached to a micromanipulator. Injection was driven by compressed N2, under the control of a PV830 Pneumatic PicoPump (World Precision Instruments, Sarasota, FL). Microinjection volumes were estimated at 1 nl/embryo, based upon calibrations using known quantities of solution.
In preliminary experiments, control and antisense MOs were injected at 1.2, 2.5, 5.0, and 10 ng per embryo (final MO concentrations 0.12 –1.0 µM, assuming an oocyte volume of 1 µl); a nominal concentration of 2.5 ng MO per embryo yielded consistent, reproducible effects with both antisense MOs, whereas an equivalent amount of control MO (CTRL) yielded embryos indistinguishable from wild-type embryos. A nominal concentration of 2.5 ng MO/embryo was thus chosen for the majority of MO injection experiments.
Growth and Development
To measure embryo growth, embryos were anesthetized in a dilute solution of tricaine methanesulfonate, mounted in low-melting point agarose in a plastic tissue culture plate, and photographed under a dissection scope with a digital camera. The number of somite pairs/embryo was used as an indicator of developmental rate up to 24 hpf. Ear-tail length was measured in 56-hpf embryos to determine linear body growth in the hatching period.
Animated Images
To visualized blood flow in real-time, selected body regions were photomicrographed as sequential tagged-image files with a Nikon DC50NN camera mounted to a Nikon Eclipse E600 microscope. Images were captured using Scion Image software (Beta 4.0.2, Scion Corp., Frederick, MD), and converted to audio video interleave (.avi) format using ImageJ software (NIH).
Hemoglobin Staining
Erythrocytes were visualized by staining live embryos with o-dianisidine (Sigma), as previously described (19). Embryos were then fixed for 1 h in paraformaldehyde, washed in PBS-T, and digitally photographed as described above.
Heart Rate
Microinjected embryos at the desired stages were immobilized in low-melting point agarose, and the number of heart contractions in a 30-sec interval was determined under a dissecting microscope.
Embryonic Vascular Development
To visualize and quantify developing vascular structures, we used a line of transgenic zebrafish (flk1:GFP) with stable integration of a reporter construct (GFP) fused to the promoter region of the vascular endothelial growth factor receptor (flk1; Chan, S. J., and D. Stainier, personal communication). Transgenic embryos were generously provided by Dr. Didier Stainier (University of California at San Francisco), in liaison with Drs. John Kuwada and Qin Li (University of Michigan).
Data Analyses
Data are shown as means ± SEM. Differences in somite number, body length, heart rate, and cranial vessel number between morphant and control-injected embryos were statistically compared using an unpaired Student’s t test, after ensuring homogeneity of variances between groups. For semiquantitative RT-PCR, relative band densities were compared between morphant and control-injected groups using one-way ANOVA. Statistical significance was accepted when P < 0.05.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was funded in part by National Science Foundation (NSF) Grant IBN 0110864 (to C.D.). A.W.W. was supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council (NSERC) of Canada and by NSF Grant IBN 0110864.
First Published Online December 23, 2004
Abbreviations: GFP, Green fluorescent protein; hpf, hours post fertilization; IGFBP, IGF binding protein; MOs, morpholino-modified antisense oligonucleotides; odc, ornithine decarboxylase; PHBC, primordial hindbrain channel; scl, stem cell leukemia; UTR, untranslated region.
Received for publication October 1, 2004. Accepted for publication December 13, 2004.
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