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Keywords:

  • histone deacetylase;
  • cartilage;
  • fin bud;
  • heart;
  • morpholino;
  • insertional mutant;
  • zebrafish

Abstract

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

Histone deacetylases interact with nucleosomes to facilitate the formation of transcriptionally repressed chromatin. In the present study, we show that histone deacetylase 1 (hdac-1) is expressed throughout embryonic development of the zebrafish. The expression of hdac-1 is ubiquitous in early embryos (2–16 hr postfertilization), but at later stages (36 and 48 hr postfertilization), it is primarily restricted to the branchial arches, fin bud mesenchyme, and hindbrain. We report the phenotypes of hdac-1 homozygous mutant embryos and embryos injected with an hdac-1 antisense morpholino. These embryos possess a complex phenotype affecting several embryonic structures. We observed developmental abnormalities in the heart and neural epithelial structures, including the retina and the loss of craniofacial cartilage and pectoral fins. Developmental Dynamics 231:647–654, 2004. © 2004 Wiley-Liss, Inc.


INTRODUCTION

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

DNA methylation and histone modifications are fundamental epigenetic mechanisms by which eukaryotic gene expression is regulated and maintained. From a transcriptional and chromatin perspective, the most important histone modifications are acetylation and deacetylation, which are accomplished by histone acetyl transferases (HAT) and histone deacetylases (HDAC) (Jones et al., 1998). Transcriptional inactivation is thought to be initiated first by the methylation of DNA, binding of methyl cytosine binding proteins, and the subsequent recruitment of HDAC-1–containing protein complexes (Jones et al., 1998; Nan et al., 1998). In general, there is a correlation between histone acetylation and gene activation and deacetylation and transcriptional repression (for reviews, see Pazin and Kadonaga, 1997; Kuo and Allis, 1998; Thiagalingam et al., 2003).

One of the histone deacetylases, HDAC-1, is important in the embryonic development of several organisms and is generally believed to have a ubiquitous expression pattern (de Ruijter et al., 2003). Absence of HDAC-1 was found to be detrimental in all organisms where HDAC-1 function has been investigated. Mutations in the Drosophila hdac-1 homologue rpd3 are lethal, display a paired-rule segmentation phenotype, and are suppressors of position effect variegation (Mannervik and Levine, 1999; Mottus et al., 2000). Trichostatin A (TSA) inhibits many histone deacetylases in addition to HDAC-1, and treatment of fruit fly larvae with TSA causes lethality and delayed development (Pile et al., 2001). Starfish and sea urchin embryos treated with TSA also undergo growth arrest during the early gastrulation period (Ikegami et al., 1993; Nemer, 1998). Treatment of frog embryos with TSA leads to embryonic lethality and defects in the head and tail region (Almouzni et al., 1994). In addition, TSA blocks thyroid hormone (T3) -induced metamorphosis in Xenopus tadpoles (Sachs et al., 2001a, b). HDAC-1 is also thought to be important for bovine development, because hdac-1 mRNA is found throughout the development of the preimplantation embryo, from immature oocytes to the blastocyst stage (McGraw et al., 2003). Finally, homozygous hdac-1 knockout mice show embryonic lethality before E10.5 and possess severe proliferation defects, growth retardation, and abnormal head and allantois development (Lagger et al., 2002). Thus, proper HDAC-1 function is required for normal embryonic development of both invertebrates and vertebrates.

We have reported previously the characterization of the zebrafish DNA methyltransferase gene dnmt1, including its expression during embryonic development and developmental abnormalities resulting from DNA hypomethylation (Martin et al., 1999). These data indicate that epigenetic gene regulation functions during zebrafish embryonic development. In the present study, we used a combination of northern blot and in situ hybridization to analyze hdac-1 expression during zebrafish development. We report the complex phenotype of zebrafish hdac-1 knockdowns and homozygous hi1618 insertional mutants (Golling et al., 2002).

RESULTS AND DISCUSSION

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

As a first step to determine whether histone deacetylase 1 might contribute to epigenetic gene repression during zebrafish embryonic development, we conducted gene expression analysis by using northern blot and whole-mount in situ hybridization. Northern blot analysis was conducted on total RNA collected from nine different embryonic stages: 1, 4, 6, 8, 10, 12, 24, 48, and 72 hours postfertilization (hpf). hdac-1 mRNA was found to be present at all the embryonic stages tested. Relatively stronger expression was observed in stages after gastrulation (8 hpf; Fig. 1a). To determine whether hdac-1 shows any tissue specific expression in zebrafish embryos, we conducted whole-mount in situ hybridization using a zebrafish hdac-1 anti-sense riboprobe. We observed a ubiquitous expression pattern between 1 and 16 hpf (data not shown). However, hdac-1 transcripts show a restricted distribution at later stages of development. In 36 to 48 hpf embryos, we observed specific hdac-1 expression in the pectoral fin bud mesenchyme, anterior and posterior arch structures (mandibular, pharyngeal, and branchial arch primordia), the hind brain (Fig. 1b–d), along with general low-level expression throughout the embryo particularly the central nervous system. Expression of hdac-1 occurs throughout the fin bud mesenchyme but expression was not observed in the apical epidermis (Fig. 1b). Expression of hdac-1 in the brain, branchial arches, and limb buds was also observed in E9.5 mouse embryos (Lagger et al., 2002), suggesting that HDAC-1 may serve a similar function in both fish and mammals.

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Figure 1. hdac-1 is expressed throughout the embryonic development of the zebrafish and shows tissues specific expression in the pectoral fin buds, mid/hind brain, and anterior/posterior arches. a: A Northern blot of total RNA collected from 1 hour postfertilization (hpf), 4 hpf (blastula stages), 6 hpf, 8 hpf (epiboly stages), 10 hpf (tail bud stage), 12 hpf (somitogenesis), 24 hpf (prim-5 stage), 48 hpf (long pec stage), and 72 hpf (protruding mouth stage) zebrafish embryos. The blots were hybridized with a zebrafish hdac-1 cDNA probe; the blots were stripped and hybridized with a zebrafish β-actin cDNA probe. The gel was stained with ethidium bromide (EtBr) before transfer. In situ hybridization of 48 hpf zebrafish embryos using an hdac-1 antisense riboprobe reveals tissue-specific expression. b: Lateral view of a 48 hpf limb bud showing expression of hdac-1 throughout the fin bud mesenchyme (anterior on the left). ae, apical epidermis; fm, fin bud mesenchyme. c: Lateral view of a 36 hpf embryo. d: Dorsal view of 36 hpf embryo. ba, branchial arches; fb, fin bud; h, hindbrain.

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To further address the role of HDAC-1 during zebrafish development, we created hdac-1 knockdowns or morphants by injecting one-cell stage embryos with an antisense morpholino oligonucleotide (hdac1-MO) designed to target the translational start site of the hdac-1 mRNA. We injected 0.3, 0.6, 1.2, 3.0, 6.0, and 12.0 ng of morpholino. This strategy resulted in 19.4% (n = 378), 35.3% (n = 270), 64.7% (n = 269), 70.2% (n = 266), 79.2% (n = 222), and 69.4%(n = 277) of injected embryos displaying an abnormal phenotype at 48 hpf, respectively. Embryos injected with a nonspecific control morpholino did not display any abnormal phenotypes (n = 249). The mortality of embryos between the time of injection and 24 hpf was not significantly different between control and hdac1-MO–injected groups. We did observe slightly higher mortality at the highest injection concentration at 48 hpf, and we believe this is why there is a reduction in the number of phenotypic embryos in this group. The phenotype of the morphant embryos is highly similar, at all stages analyzed, to homozygous insertional mutants (hi1618) that were produced by Golling et al. (2002). Genetic analysis of the hi1618 mutants indicated that the insertion found in these fish is located within 3 cM of the hdac-1 gene. The similarity between the hdac-1 morphants and the hi1618 mutants supports the assertion by Golling et al. (2002) that the hi1618 mutation is in fact disrupting the hdac-1 gene. Further analysis by our laboratory has indicated that the hi1618 mutation displays variable expressivity that appears to be due to genetic background effects. We have analyzed the offspring from two different types of crosses and two generations of hi1618 mutants. Various F4 and F5 matings produced offspring where greater than 75% of the homozygotes displayed a severe phenotype similar to the hdac1-MO–injected embryos. Homozygous mutant offspring derived from mating of two outcross lines with Tuebingin (Tu) and AB (AB) backgrounds, respectively, displayed phenotypes ranging from mild (avr. 60%) to severe (avr. 40%). Despite the variations in phenotype in both cases, we were able to score the 25% of homozygote recessive offspring that are predicted to result from a cross of heterozygotes. These differences in expressivity are likely due to genetic factors and perhaps stochastic events. In this report, we focused our analysis on hdac1-MO–injected embryos and hi1618 mutant embryos displaying the severe phenotype.

Early embryonic development appears to proceed normally in HDAC-1–deficient embryos. At 12 hpf, nt-1 expression shows a thickened anterior notochord (data not shown). At approximately 24 hpf, a slightly curved and shortened tail is observed compared with wild-type embryos. Often a small kink is observed in the anterior notochord at the point of flexion during embryo movement. At later stages the abnormalities include reduced pigmentation, shortened body axis, and a curved tail (Fig. 2a). The head is smaller, and larvae have a shortened and round snout compared with controls (Figs. 2, 4). After 48 hpf, moderate developmental delay is observed relative to wild-type or uninjected control embryos. Hatching is delayed in HDAC-1–deficient embryos, and often it must be assisted. The choroid fissure fails to undergo complete closure, and as a result, the larvae possess cleft eyes (Fig. 2a inset). Although the HDAC-1–deficient phenotype is not compatible with a long lifespan, we have raised some morphant and mutant embryos until 14 days postfertilization (dpf). The mortality rate (stopping of heart) of these embryos is relatively uniform between 48 hpf and 10 days dpf with 50% of embryos dying by 8 dpf.

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Figure 2. External phenotype of 72 hours postfertilization (hpf), hi1618 mutant zebrafish embryos: a,b: hi1618 mutant embryos develop a small head, curved tail, edema, blood pools, cleft eye (a and inset), and an abnormal heart (arrow in b).

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Figure 4. Alcian blue staining of craniofacial cartilage in 8 days zebrafish embryos. a: Control. b: Homozygous mutant (hi1618). c:hdac1-MO–injected embryo. The cartilaginous structures Meckel's cartilage (mc), palatoquadrate (pq), ceratohyals (ch), hyosymplectic (hs), gill arches (g), ethmoid plate (eth), and pectoral fins (pf) are completely absent in hi1618 homozygous mutants and morphants.

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The heart begins to beat normally at approximately 24 hr, but at 72 hpf, the heart rate is approximately half the rate (60–80 beats per min) that is counted in wild-type embryos. The heart does not appear to undergo looping and, therefore, forms a stretched tube (Fig. 2b). In 48 hpf and later, hdac-1 mutant embryos, there is reduced blood flow in the heart but circulation is absent in the head and tail regions. Blood pools are observed after 48 hpf in the vasculature at various locations, including the head, ventral tail, and above the yolk and heart (Fig. 2a,b). Hemorrhages do not appear to account for formation of these blood pools. An edema begins to become prominent around the heart by 48 hpf and gradually enlarges over the short lifespan of the HDAC-1–deficient embryo. In extreme cases, this edema can enlarge to a size several times that of the actual larvae. After 120 hpf, we begin to observe necrosis in the tail. The absence of blood flow is the likely cause of this necrosis and developmental delay in later stages.

To evaluate whether the absence of blood circulation is due to abnormal development of the peripheral vascular system, we injected Tg(fli1:EGFP) (Y1) transgenic embryos, a fish line that expresses eGFP in the vascular endothelium, with 6 ng of the hdac1-MO (Fig. 3). The tail vasculature of morphant embryos appeared normal compared with control embryos, and we observed little or no delay in the formation of the vascular system. We did observe, however, an overall decrease in fluorescence particularly in the head and cardiac region of 72 and 96 hpf morphant embryos compared with control embryos. Therefore, the absence of peripheral blood flow must be the result of either a defect or weakness in the heart or a defect in the vessels connecting the heart to the dorsal aorta and carotids. A small number of mutant embryos possessed a milder heart phenotype (partially looped) and exhibited blood flow in the large tail vessels (dorsal aorta, caudal artery, caudal vein, and posterior cardinal vein). In these embryos, blood flow is still not observed in the smaller vessels of the tail (dorsal longitudinal anastomotic vessel and intersegmental vessels). Microscopic observations of the flow of blood cells through the heart of mutants and morphants revealed that blood cells were regurgitated after a beat of the heart. Taken together, these data suggest that the absence of blood flow is due to defects and weakness in the heart. Class I and class II HDACs have been implicated in the regulation of cardiac specific genes and the regulation of cardiac hypertrophy (Hamamori and Scheider, 2003). HDAC-2, a class I HDAC, interacts with Hop (homeodomain-only protein) and serum response protein to block the transcriptional program that opposes hypertrophy. The involvement of other class I HDACs, including HDAC-1, has not been excluded, but knockdown of Hop by injection of an antisense Hop morpholino into zebrafish embryos results in severe heart defects. These embryos display a phenotype, including pericardial edema, reduced heart rate and impaired blood circulation, which is remarkably similar to HDAC-1–deficient embryos (Chen et al., 2002). These data suggest that HDAC-1 may also interact with Hop to facilitate normal heart development or be involved in cardiac gene regulation.

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Figure 3. a: Enhanced green fluorescent protein (eGFP) expression in Tg(fli1-eGFP) (Y1) transgenic embryos injected with 6 ng of a hdac1-MO: 24 hours postfertilization (hpf) hdac-1 morphant embryo. b,c: The head of a 72 hpf control embryo (b) and a 72 hpf hdac-1 morphant embryo (c), which were imaged concurrently using identical exposures, gain, and offset settings of the camera sensor.

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Two prominent phenotypes of hi1618 mutants and hdac-1 morphants are the absence of craniofacial cartilage structures and a reduction in pectoral fin development (Fig. 4). Alcian blue staining showed an apparent absence of all the mandibular, pharyngeal, and branchial arch-derived cartilages that would be normally present in a wild-type embryo. To confirm the absence of craniofacial cartilage elements, we conducted histological sections of 5-day hi1618 mutant embryos (Fig. 5). In severely affected embryos, we were unable to identify any structures that resemble or exhibit characteristics of normally differentiated cartilages. In mildly affected embryos, we often observed the presence of the trabeculae and parachordal cartilages. This finding might suggest that there is a greater requirement for HDAC-1 in some cartilage elements. In addition to confirming the absence of craniofacial cartilage, histological analyses of HDAC-1–deficient embryos revealed abnormalities in the organization of the retina (Fig. 6).

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Figure 5. a,b: Histological sections of the posterior buccal cavity region of 5-day-old wild-type (a) and hi1618 mutant (b) larvae. In hi1618 homozygous mutant larvae, the cartilaginous structures such as the hyosymplectic (hs), ceratohyal (ch), and the ceratobranchial (cb) are absent. The parachordal cartilage (pc) is the only cartilage element that is sometimes observed in HDAC-1–deficient larvae.

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Figure 6. Patterning of the retina in HDAC-1–deficient embryos. a–c: Histological sections of the eye of 5-day-old wild-type (a), hi1618 mutants (b), and hdac1-MO–injected embryos (c). pe, pigmented epithelium; pcl, photoreceptor layer; opl, outer plexiform layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer.

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The lens of hi1618 mutants and hdac-1 morphants is often reduced and/or misshapen. The HDAC-1–deficient embryos displayed a neuronal patterning defect in the retina similar to that observed in the Group I eye mutants such as oko meduzy (ome), glass onion (glo), and nagie oko (nok; Malicki et al., 1996). The eyes of hi1618 mutants and hdac1-MO–injected embryos do not form the typical laminar pattern of nuclear and plexiform layers of the retina. Instead, the retinal neurons appear disorganized and the pigmented epithelium is often seen broken or patchy. Of interest, differences between mutants and morphants are observed with respect to the pigmented epithelium. Mutants appear to have reduced pigment compared with morphants. In addition to the eye phenotype of the previously mentioned Group I mutants, these mutants and the HDAC-1–deficient embryos also show abnormalities in brain shape, a curved body axis, and reduced or absent circulation (Schier et al., 1996). This finding could indicate that the mutations in these zebrafish lines affect a common biochemical or signaling pathway.

To further characterize the phenotype of HDAC-1–deficient embryos, we conducted in situ hybridization by using probes that are expressed in affected structures. For example, the area of dlx4 expression in the region of the branchial arches in 48 hr hi1618 mutant (Fig. 7) and hdac1-MO–injected (data not shown) embryos is significantly reduced compared with wild-type embryos. The expression pattern of dlx4 also revealed that the size of the otic vesicle is significantly reduced. The distance between the eye and the otic vesicle is larger, and expression of dlx4 in the forebrain is reduced or absent. Abnormalities in the neuronal patterning of hi1618 mutant embryos have been observed (Cunliffe, 2004). By 48 hpf, a very small fin bud slightly raised above the body surface is observed in HDAC-1–deficient embryos (Fig. 7b). The cells of the fin bud expressing dlx4 show a diffuse and weak pattern of expression (Fig. 7c). Normal expression of dlx4, however, was observed in the median fin fold of 48 hpf HDAC-1–deficient embryos (data not shown). Expression of tbx5 in 48 hpf HDAC-1–deficient zebrafish embryos was normal in the eyes (Fig. 8) and was also observed at high levels in the heart (data not shown), although the heart itself was abnormal. The expression of tbx5 in the fin bud region was similar to the expression of dlx4, occurring as a more diffuse patch of cells compared with control embryos. To investigate whether the pattern of expression that we observed might be the result of developmental delay we conducted in situ hybridization on 72 hpf embryos by using a tbx5 probe. These embryos displayed very weak tbx5 expression in the fin bud cells and the development of the limb bud appeared to progress little from that observed in 48 hpf embryos. Thus, although cells in the pectoral fin field express early epidermal and mesenchymal markers, little or no fin bud growth occurs.

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Figure 7. Expression of dlx4 in a 48 hours postfertilization (hpf) hi1618 mutant embryo. a,b: Lateral views of a wild-type zebrafish embryo (a) and an hi1618 HDAC-1 mutant embryo (b). c: A dorsal view an hi1618 HDAC-1 mutant embryo. f, forebrain; ba, branchial arches; ov, otic vesicle; fb, fin bud; ff, fin fold.

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Figure 8. Expression of tbx5 in the fin bud of hi1618 mutant embryos. a: Dorsal view of a 48 hpf wild-type zebrafish embryo. b: A 48 hours postfertilization (hpf) hi1618 mutant embryo. c: A 72 hpf hi1618 mutant embryo.

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The precise function of HDAC-1 in zebrafish heart, fin, and cartilage development is still unclear. Lagger et al. (2002) attributed the phenotype of hdac-1 mutant mouse embryos to a lack of cell proliferation. HDAC-1–deficient embryonic stem cells possess decreased levels of cyclin-dependent kinase activity, whereas hdac-1 homozygous mutant mouse embryo have elevated levels of the cyclin-dependent kinase inhibitors p21 and p27 in the absence of increased apoptosis. The phenotype in hdac-1–deficient zebrafish embryos is much less severe, and the embryos develop to significantly later embryonic stages than that observed in mouse mutants. It would be worthwhile to investigate defects in the extraembryonic tissues of mutant mice to see whether this finding may account for some of these differences. Additionally, zebrafish may possess a gene duplication of the hdac-1 gene leading to less severe effects of mutation through redundancy or subfunctionalization. Absence of a growing fin bud in these zebrafish, however, could be indicative of a lack of proliferation in the fin bud mesenchyme—a tissue in which hdac-1 is highly expressed. The complex phenotype of hi1618 mutants and hdac1-MO–injected embryos is not just a defect in cell proliferation but also involves defects in tissue patterning.

That HDAC-1 is a transcriptional regulator leads us to hypothesize that HDAC-1 loss of function could potentially alter the normal expression of a large number of genes affecting a large number of developmental processes. HDACs have been implicated in several developmental processes, including homeobox gene repression (Chang et al., 2001), Fgf signaling (Weinstein et al., 1998; Xu et al., 2000), retinoic acid signaling and chondroblast differentiation (Weston et al., 2003), and cardiac hypertrophy (Kook et al., 2003; Hamamori and Schneider, 2003). Microarray analysis currently being initiated in our laboratory will allow us to identify specific genes and developmental processes that should be evaluated as targets for HDAC-1 regulation. This investigation will facilitate a better understanding of the role of epigenetic gene silencing in zebrafish embryonic development.

EXPERIMENTAL PROCEDURES

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

Zebrafish

Zebrafish and embryos were maintained and staged according to Westerfield (1995). Embryos that contain an hdac-1 insertional mutation (hi1618) were generously provided by Adam Amsterdam and Nancy Hopkins (Golling et al., 2002).

Northern Blot

Total RNA was isolated with Trizol reagent (Invitrogen). A purified zebrafish hdac-1 cDNA fragment (2.2 kb) was labeled with 32P following random hexamer labeling procedure. Electrophoresis and membrane hybridizations were carried out according to Sambrook and Russel (2001). The northern blot was subsequently stripped and reprobed with a full-length β-actin cDNA probe to ensure RNA integrity. Equal loading was determined by staining the RNA before transfer with ethidium bromide. Transfer efficiency was determined by visualization of stained RNA within the gel after transfer.

In Situ Hybridization

In situ hybridization was performed using an Intavis In situ Pro automated system and a modified protocol of Akimenko et al. (1994). An antisense digoxigenin-labeled RNA probe was prepared by in vitro transcription using a linearized HDAC-1 cDNA clone (2.2 kb) and SP6 RNA polymerase.

Morpholino Injection

Embryos were injected with a morpholino oligonucleotide targeted to zebrafish HDAC-1 mRNA containing the sequence 5′-TTGTTCCTTGAGAACTCAGCGCCAT-3′ (designed by GENE TOOLS, LLC). A nonspecific positive control oligo with the sequence 5′-CCTCTTACCTCAGTTACAATT TATA 3′ was also injected in some experiments. Morpholino (HDAC-1-MO) was injected into early (1- to 2-cell stage) zebrafish. The concentration of injected morpholino was modified to do concentration studies (12 ng, 6 ng, 3 ng,1.2 ng, 0.6ng, and 0.3 ng). The morpholino was dissolved and injected in 1× Danieau solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca (NO3)4, 5 mM HEPES pH 7.6) with 0.05% sterile phenol red dye. The microinjection was carried out using a Narishige 1M 300 microinjector manipulator and a Nikon SMZ 1500 microscope.

Alcian Blue Staining and Histological Analysis

Eight-day-old zebrafish larvae were fixed overnight in 4% paraformaldehyde and stained in 0.1% solution of Alcian blue dissolved in 80% ethanol and 20% glacial acetic acid for 24 to 72 hr. Embryos were then rinsed in 40% ethanol in phosphate-buffered saline (PBS) and followed by rinses in PBS alone. Stained embryos were cleared and mounted in 80% glycerol. Fixed embryos were embedded in LR White embedding medium, sectioned to 5–8 μm, and photographed by using a Nikon E600 stereomicroscope. Fluorescence images of enhanced green fluorescent protein (eGFP) expressing transgenic embryos were taken by using a Nikon SMZ-1500 fitted with epifluorescence, GFP filters and a Qimaging Retiga 1300 CCD camera.

Acknowledgements

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

We thank Adam Amsterdam and Nancy Hopkins for the generous gifts of the hdac-1 mutant zebrafish (hi1618). We thank Louise Labelle for conducting some of the histological analysis. This work was supported by grants to C.C. Martin by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Ottawa. We thank Marc Ekker and Marie-Andree Akimenko for helpful suggestions with the manuscript. This paper is dedicated in loving memory of our faithful friend Merlin.

REFERENCES

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