Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong
Received November 13, 2002; accepted January 21, 2003
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ABSTRACT |
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Key Words: zebrafish embryos; cadmium; somite patterning; muscle cell type; axon growth; myogenic regulatory factors.
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INTRODUCTION |
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The most prominent feature of teleost trunk development is the process of segmentation. The fish trunk is composed of reiterated blocks of muscles, cartilage, bone, and nerves. These serially homologous blocks are derived from mesodermal segments called somites, which are formed during embryonic development. The zebrafish has provided a wealth of information on the molecular and genetic basis of somitogenesis (reviewed in Holley and Nüsslein-Volhard, 2000). The transparency of the embryos enables easy observation of cells while they differentiate and migrate. The availability of genetic mutants, on the other hand, enables identification of genes involved in various aspects of somitogenesis. We now have an overview of somite development in zebrafish, even though some of the key developmental regulatory factors are yet to be identified (reviewed in Stickney, 2000
). The patterning of the mesoderm occurs during the gastrulation period, from 6 h post fertilization (hpf) to 10.5 hpf. During this period of development, the notochord, which is derived from the axial mesoderm, is formed in the midline, and is an important signaling center that plays pivotal roles in future patterning of the somites and neural differentiation. The first morphologically distinct somite pair appears at approximately 10.5 hpf. During the period of segmentation from 10.5 to 24 hpf, a total of about 30 pairs of somites are formed, and they bracket the notochord. With the formation of somites, the trunk begins to lift off the yolk sac and the tails extends. At 24 hpf, when segmentation completes, the somites have further developed into the myotome and the sclerotome. The myotomes will develop into skeletal muscles in the adult fish. Precursors to adult slow and fast muscles can be identified at 24 hpf. The embryonic fast muscle fibers are the major constituents of the myotome and they are found in the deep portion surrounding the notochord. Embryonic slow muscle fibers, on the other hand, form a superficial monolayer on the surface of the myotome. The myotomes are separated into muscle blocks by myosepta in the anterior/posterior boundaries and by horizontal myosepta dorsoventrally.
Each muscle block is connected to specific motor neurons situated in their central nervous system by axon outgrowth (reviewed in Eisen, 1994). The primary motoneurons consist of three types, namely the caudal primary (CaP), middle (MiP), and rostral (RoP). Their axons pioneer the nerve path connecting the segmental motor nerve to the muscle blocks. Each type of primary motoneuron has a stereotypic axonal trajectory connecting them to their specific axial muscle targets in the somatic hemisegment, precisely. The secondary motoneurons develop later, and their axons extend along the path of the primary motoneurons. These processes of axon growth by which different motoneurons send their axons to reach respective muscle targets along an initially shared common axonal path, and then along divergent cell-specific pathways, are highly regulated events.
In our previous study, we reported that reduced myotome formation was observed in cadmium-exposed embryos with altered axial curvatures (Cheng et al., 2000). We have also reported normal expression of the gene, sonic hedgehog (shh), which is known to act upstream of muscle-specific genes. Our previous work suggests that cadmium might interfere with events subsequent to the inductive signals of shh and before the formation of myotomes, during somitogenesis. In the current study, we investigated the landmark events in somitogenesis in order to find out when and where cadmium might exert its toxicity in this important process of development. We first investigated whether the critical period of exposure occurred preceding or simultaneously with the morphological formation of somites. We then investigated whether the commitment of somitic cells to myogenic lineage and then to specific muscle cell types was affected. The commitment to myogenic lineage was investigated by the expression of two myogenic regulatory factors, myoD and myogenin, which regulate muscle cell differentiation (Weinberg et al., 1996
). myoD expression marks the specification of myogenic precursor cells and the transcripts are expressed in the middle of each chevron-shaped somite at 24 h post fertilization (hpf). Myogenin expression occurs later than myoD during development, and the transcripts are expressed in a subset of myoD-expressing myogenic cells. The differentiation to fast muscle and slow muscle was investigated by staining for the fast skeletal muscle myosin heavy chain (myhz) and the slow myosin-binding protein C (smbpc) (Xu et al., 2000
). We also examined the process of motoneuron innervation in the myotomes in order to find out whether this last step of somitogenesis was affected. We investigated the formation of primary motor neurons and the projection of their axons with the antibodies Islet-1 (Isl-1) and Znp-1, respectively (Inoue et al., 1994
, Trevarrow et al., 1990
). The antibody Isl-1 is specific for two types of primary motor neurons, the MiP and the RoP, while the antibody Znp-1 recognizes primary motor neurons and their axon outgrowths. We then investigated the projection of axons by the secondary motor neurons using the antibody Zn-5, which recognizes DM-GRASP, a cell adhesion molecule found on secondary motor neurons (Fashena and Westerfield, 1999
). Finally, we used the antibody Znt-1, which labeled the notochord in zebrafish embryos, to investigate whether cadmium affected the formation of the notochord, the central organ in providing inductive signals for somite patterning (Trevarrow et al., 1990
).
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MATERIALS AND METHODS |
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Zebrafish maintenance and embryo treatment.
Zebrafish were maintained and embryos were collected as previously described (Cheng et al., 2000). Embryos were exposed to 100 µM of cadmium chloride in embryo media at different developmental stages categorized in three groups: (1) exposure to cadmium during the gastrulation period (410.5 hpf); (2) exposure to cadmium during the segmentation period (10.524 hpf); (3) exposure to cadmium during both gastrulation and segmentation periods (424 hpf). The untreated controls were embryos maintained in culture medium only. Thirty replicates, consisting of 20 embryos in a 60-mm diameter Petri dish, were set up for each treatment group (i.e., n = 30 x 20 = 600). At 24 hpf, embryos were dechorionated by using a pair of fine forceps under a dissecting stereomicroscope. Embryos were examined for abnormalities in body flexure according to previous reported descriptions and method (Cheng et al, 2000
).
In situ hybridization.
Embryos at 24 hpf were dechorionated and fixed in buffers with 4% paraformaldehyde (PFA) at 4°C overnight. Whole-mount in situ hybridization was carried out as described by Westerfield (1994) with modifications (Cheng et al., 2000
). Briefly, antisense RNA was synthesized by linearizing the plasmid and transcribing with T7 polymerase and Digoxigenin-11-UTP (Roche, Basel, Switzerland). Embryos were transferred to methanol and stored at -20°C to increase permeability. They were then washed and digested with 10 µg/ml proteinase K in PBS (phosphate-buffered saline) with 0.1% Tween 20 before incubating with the antisense probes at 55°C overnight. Following hybridization, probes were removed with high-stringency washes, 2 x SSC and 0.2 x SSC each twice for 30 min, at 62°C. Embryos were subsequently incubated with preabsorbed sheep anti-digoxigenin-alkaline phosphatase Fab fragments (Roche, Basel, Switzerland) for 2 h at room temperature on a nutator. After washing, 5-bromo-4-chloroindolyl phosphate was added as substrate and nitro blue tetrazolium as coupler (Roche, Basel, Switzerland). We constructed plasmids containing cDNA probes for myoD (AF318503) and myogenin (AF202639) from the published nucleotide sequences in GenBank. We obtained plasmids containing cDNA probes for smbpc and myhz from Dr. Z. Gong (Xu et al, 2000
).
Immunohistochemistry.
Embryos were collected at the end of treatment (at 24 hpf), washed three times, and transferred to fresh culture medium without cadmium for further incubation. At 30 or 48 hpf, embryos were dechorionated and fixed in buffers with 4% pfa at 4°C overnight.
Whole-mount immunostaining with antibodies was performed according to the method described (Westerfield, 1994) and (Cheng et al., 2000
). Embryos were frozen at -20°C in acetone to permeabilize the tissues before treating with 2% goat serum in PBS/BSA/dimethyl sulfoxide (DMSO) buffer for 30 min to block non-specific binding sites. Specifically, the PBS/BSA/DMSO buffer was PBS (0.8% NaCl, 0.02% KCl, 0.02M PO4 [pH7.3]) with 1% BSA and 1% DMSO. Embryos were then incubated in primary antibodies at 4°C overnight. After thorough washing, embryos were incubated for 30 min at room temperature in biotinylated goat antimouse secondary antibody that was pre-blocked with zebrafish embryo powder (DAKO, Carpenteria, CA). Horseradish peroxidaseStreptavidin was added before incubation of embryos with chromogenic substrate (diaminobenzidine and hydrogen peroxide). Staining reaction was stopped by PBT (PBS, 0.1% Tween 20).
Culture supernatants against the following marker genes were obtained from the Development Studies Hybridoma Bank (University of Iowa, IA): 39.4D5 for Isl-1 (1:200; Inoue et al., 1994), Znt-1 (1:100; Trevarrow et al., 1990
), and Znp-1 (1:200; Trevarrow et al., 1990
); and from the Oregon Monoclonal Antibody Centre: Zn-5 (1:100; Fashena and Westerfield, 1999
).
Statistical analysis.
All data were presented as the mean ± SEM, which is indicated by bars in the figures. Significant difference between control and different cadmium-exposed groups was determined by one-way ANOVA with post hoc analysis using Dunnetts multiple comparison tests (p < 0.05).
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RESULTS |
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Taken together, it appears that exposure to cadmium caused defects in axon growth but not in the cell fate specification nor in the differentiation of motoneurons.
Cadmium affects notochord development.
The notochord plays a crucial role in somite patterning (reviewed by Bumcrot and McMahon, 1995). In order to find out whether the observed defects in somitogenesis arose from notochord defects, we examined the morphological features of the notochord in cadmium-treated embryos by in situ hybridization with probes against the myogenic tissues flanking the notochord. In untreated embryos at 24 hpf, the notochord appeared as a straight and rod-like stripe along the trunk set against the myotomes in a dorsal view (Figs. 8A
, 8C
, and 8E
). In cadmium-treated embryos with altered curvature, a variety of notochord malformations could be detected. For example, looping of the notochord in the caudal region caused it to end abruptly and fail to extend to the tail tip (Fig. 8B
). Flexure of the notochord was observed in another embryo with disarrayed and interrupted fast muscle fibers (Fig. 8D
). Bulging of the notochord was observed when unilateral absence of fast muscle occurred (Fig. 8F
). Another common malformation was the bent and undulated notochord forming a knob-like structure in the dorsal region of the trunk (Figs. 3F
and 3H
).
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The fate of axial mesodermal cells destined to become notochord was investigated. Where the notochord failed to form in the caudal region, the somitic cells simply committed to the myogenic lineage as they expressed the myoD and myogenin genes instead (Figs. 3D and 3H
). Infringement by differentiated muscle cells in the notochord area could also be found in some cadmium-treated embryos (Fig. 4
). In untreated controls, the fast muscle fibers appeared as bilateral rows flanking the notochord (Figs. 4A
and 4C
). In cadmium-treated embryos, the fast muscle fibers could be found in the midline mesoderm (Figs. 4B
and 4D
). Taken together, it appears that the cell fate decision of axial mesodermal cells to notochord may be affected by cadmium and hence some of these cells take up the default pathway and become muscle cells.
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DISCUSSION |
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We are, however, aware that the observed effects could either be direct actions of cadmium in interfering with various cellular mechanisms, possibly by replacing other divalent ions such as Ca2+ or Mg2+, or indirect actions of cadmium on inductive signaling molecules important in embryonic patterning and cell-fate specification.
Myogenic commitment and muscle type diversification.
Myotomes are the major components of each somite. The process of myogenesis in embryonic development is a multistep event involving the specification, differentiation, and migration of different cell types. We examined two landmark events in this study: namely, the commitment and the diversification of myogenic cells.
We found that the commitment to myogenic lineage by the axial mesodermal cells was not affected (Fig. 3). Axial mesodermal cells commit to the muscle lineage when they express the myogenic regulatory factors (reviewed in Currie and Ingham, 1998
). These transcription factors are specific to skeletal muscle and bind to DNA. These factors regulate the expression of muscle structural genes such as myosins. Our data shows unchanged expression of myoD, which involves early muscle differentiation, and of myogenin, which is expressed more broadly at a later stage (Fig. 3
).
The formation of fast and slow fibers, however, was affected (Figs. 4 and 5
). The bulk of the myotome is composed of fast muscle fibers and the slow muscle forms only a monolayer surrounding the fast muscles in zebrafish. Myosins are hexameric molecules composed of two heavy chains and two light chains. They can be found in both fast and slow muscles. We showed gaps of missing fast muscle fibers in regions of altered curvatures when we labeled the fast skeletal muscle myosin heavy chain with the marker myhz (Fig. 4
). Taken together, the cause of the loss of myosin heavy chain observed in cadmium-treated embryos in our previous study might be attributed mainly to the loss of fast muscle fibers but not to failures in myogenic commitment.
Axon growth.
Zebrafish motoneurons follow stereotyped pathways and project to appropriate regions of the myotome by integrating various cues (reviewed in Eisen, 1994 and in Beattie, 2000
). The primary motor neurons are born at the end of gastrulation at around 10 hpf and the axonal growth takes place from 18 to 24 hpf. In this study, we showed that exposure to cadmium when these processes were taking place only affected the axonal growth, but not the formation of primary motor neurons (Figs. 6
and 7
). Axons of the secondary motor neurons start to extend at approximately 26 hpf. They enter the common pathway pioneered by the primary motor axons, suggesting that the growth cones of secondary motor neurons might require the axons of the primary motor neurons to facilitate path finding (reviewed in Eisen, 1994
). Ablation of primary motor neurons caused delayed axonal growth of the secondary motor neurons (Pike et al., 1992
). In this study, we observed stalling of the axonal extension of secondary motor neurons in cadmium exposed embryos at 48 hpf, even though the process of axonal extension occurred when the embryos were no longer exposed to cadmium (Figs. 6
and 7
). The stunted growth of secondary axons at 48 hpf could be a consequence of delayed axonal growth caused by abnormal primary axon extension in cadmium-treated embryos. For example, in areas of the trunk where altered axial curvatures were observed, the region of abnormal primary axonal growth coincided with the region of abnormal secondary axonal growth (Figs. 7B
and 7C
).
The growth cone at the tip of an embryonic axon samples its local environment and makes decisions on where to navigate by motile responses. Calcium is recognized as the key second messenger in growth cone extension and in growth cone turning (reviewed in Doherty et al., 2000). Specifically, changes in the intracellular and extracellular Ca2+ concentrations induce different turning responses of the growth cones (Zheng, 2000
). We could only speculate here that the presence of cadmium might cause imbalances of the Ca2+ concentrations, which in turn lead to failures of some motoneuron axons to reach the designated targets in the myotome (Fig. 6
).
Molecular cues, which can be repulsive or attractive and act at short or long ranges, are also crucial in proper axon growth (reviewed in Beattie, 2000). Numerous classes of molecules have been identified and they include cell adhesion molecules, semaphorins, netrins, receptor phosphatases and Eph receptor protein tyrosine kinases. The divalent nature of cadmium ion could enable it to replace Mg2+ in events involving phosphatases and kinases.
On the other hand, zebrafish mutants stumpy, unplugged, and diwanka show defects such as stalled or bifurcated axons (reviewed in Beattie, 2000). The functional roles of these genes have not been fully revealed. Similar defects are also observed in cadmium-treated embryos (Figs. 6E
and 6H
). We cannot postulate whether cadmium interacts with them directly to inhibit their functions or whether these genes utilize calcium as the messenger to mediate their effects on axon growth. Our report, however, provides a tentative link to the toxic effects of cadmium to embryonic axon growth in vivo. Other toxicants, such as ethanol, have been shown to disrupt central neuron migration in vitro (Liesi, 1997
).
Cadmium affects induction of somitogenesis.
Exposure to cadmium during the induction, rather than the formation, of the somites is more critical (Fig. 2). The gastrulation period denotes the formation of the notochord and the notochord precursor cells begin to express signaling molecules that exert patterning influences on the surrounding mesoderm. The notochord is the central organ in the induction of somitogenesis as analyses of zebrafish mutants defective in notochord formation invariably have disrupted mesoderm development, such as formation of U-shaped somite, lack of horizontal myosepta, and see review in Currie and Ingham (1998)
. These mutant studies reveal that a stepwise series of inductive events originating from the notochord act to pattern the somite. We observed malformed notochord with a variety of phenotypes such as looping, bulging, and flexures in cadmium-treated embryos (Figs. 1
, 3
, and 8
).
It is also of interest to note that analyses of the zebrafish mutants reveal that cells fated to become notochord would only do so when muscle differentiation of these cells is suppressed and that the muscle development pathway is the default route for these midline cells (Amacher and Kimmel, 1998; Halpern, et al., 1995
). In our observed data, exposure to cadmium caused some of the notochord cells to become muscle cells (Figs. 3D
, 3H
, 4B
, and 4D
).
Taken together, it appears that cadmium may exert its actions on the notochord during the gastrulation period, skews the developmental pathways of this organ resulting in morphological changes, and consequently, perturbs the inductive activities of this organ. Exposure to cadmium during the segmentation period, on the other hand, might affect other developmental mechanisms involved in formation of embryonic axis. For example, the convergence and extension cell movements during segmentation continue to shape the embryonic body by narrowing and lengthening the body axis (reviewed in Myers et al., 2002). This process involves the noncanonical Wnt pathway which utlizes the intracellular calcium as the second messenger (Slusarski et al., 1997
). The additional effect on trunk malformation when the embryos were exposed during the segmentation period might due to perturbations to the Wnt signaling pathway mediated by the intracellular calcium.
Somitogenesis and motility.
Our data showed that muscle cell type differentiation and motor axonal growth were affected in cadmium-treated embryos. We have also examined the physiological consequences in terms of locomotor activities. We touched the heads or tails of 24 hpf and 48 hpf embryos. Untreated controls displayed vigorous rapid coiling at 24 hpf and swam away swiftly at 48 hpf, as described in Saint-Amant and Drapeau (1998). Cadmium-treated embryos with normal trunk curvature displayed much less vigorous coiling at 24 hpf and swam away slowly at 48 hpf. On the other hand, cadmium-treated embryos with altered axial curvatures did not respond to touching at all. In other words, they did not coil nor swim away upon touching. It appears that the combination of losing muscle fibers and abnormal axon growth result in defects in locomotion and might have consequence to the long-term survival of these cadmium-treated embryos.
Signaling molecules.
A number of molecules secreted by the axial structures, in particular the notochord and the neural tube, have been implicated in the control of myogenesis (reviewed in Currie and Ingham, 1998). Many of them belong to the Hedgehog and WNT families. In our previous report, we found that the expression of the signaling molecule shh was not perturbed in the trunks of embryos with altered axial curvature (Cheng et al., 2000
). In this report, we provide evidence that some of developmental processes mediated by the shh molecule are not affected by cadmium while others are.
The formation of primary motoneurons required signaling of shh and its related members (Lewis and Eisen, 2001). We observed unchanged numbers of 2 subsets of primary neurons in cadmium-treated embryos, denoting lack of perturbation of shh-induced processes (Fig. 6B
). On the other hand, signaling from shh and other Hedgehog family members were essential for slow muscle development (Bladgen et al., 1997
; Lewis et al., 1999
). We observed bilateral and unilateral losses of slow muscle fibers in cadmium-treated embryos (Fig. 5B
and 5C
). Therefore, the loss of slow muscle fibers could be attributed partly to ectopic functions of the shh and its related members.
The shh signaling pathway is comprised of many mediators and quite a number of them are known to be involved in specific processes of somitogenesis. The presence of cadmium, although it did not influence shh expression, might perturb the functions of these mediators by various mechanisms. We can only postulate on some of the possibilities here. For example, most of Hedgehog signaling leads to the activation of transcriptional effectors that are zinc finger-containing proteins of the Gli family. The metal binding sites in other zinc finger binding proteins have been shown to be attractive targets for cadmium to bind in competition with Zn2+ (Makowski and Sunderman, 1992).
Tissue body burden.
In this study, we set the exposure concentration at 100 µM cadmium as this concentration was found to induce total adverse effects in just under 40% of the embryos exposed to cadmium from 4.5 to 28 hpf (Cheng et al., 2000). We needed to gather enough embryos with malformations before we could proceed to determine the molecular mechanisms using in situ hybridization and immunohistochemistry. While this concentration of cadmium can hardly be found in nature, our experimental setting is in line with other tissue culture studies on the molecular mechanisms of cadmium toxicity at µM ranges (Alvarez-Barrientos et al., 2001
; Bagchi et al., 2000
; Chao and Yang, 2001
; Dong et al., 2001
; Hader et al., 1996
).
We performed chemical analyses on the embryos to determine the cadmium concentrations. We found that the tissue body burden of cadmium was 6.867 ± 0.332 ppm from 700 embryos exposed to 100 µM cadmium from 4 to 24 hpf. In contrast, the tissue body burden of control embryos was 0.554 ± 0.012 ppm from the same number of embryos. The embryos examined were wet and we did not obtain an estimate of the dry weight from each batch of 700 embryos.
Zebrafish as a model organism of developmental toxicology.
Fish embryos from different species have been used successfully in screening tests for aquatic toxicity assessment. The obvious advantages include the large numbers of embryos that can be obtained for better data analysis and the transparency of the fish embryos for morphological and functioning examinations. This report, however, illustrates additional advantages afforded by studying zebrafish embryos. The availability of marker gene probes and antibodies has enabled us to identify the affected morphological components, such as the muscle fibers, the axons, and the notochords. The wealth of information on the events in somitogenesis obtained from mutant analyses helps us to postulate on the respective molecular cues being affected by cadmium. The trunk malformation is among the most prominent deformities observed in fish embryos. The techniques employed in this study could well be used to study the developmental toxicology of other pollutants. It will be interesting to compare whether other toxicants perturb similar or different events in somitogenesis. With that, we will be able to devise read-out systems on morphological and molecular changes to assess toxicant-specific and -nonspecific effects.
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ACKNOWLEDGMENTS |
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NOTES |
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