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Bipartie axiation follows incomplete epiboly in zebra fish embryos treated with chemical teratogens
Abstract
Medial clefts in the axis of the trunk region are malformations known from many chordates and are mostly referred to as rachischisis anterior. In teleosts, rachischisis was previously ascribed either to secondary rifting of a single uniform axial rudiment, or to the establishment of two (half) axes and body halves physically separate from the very beginning. In order to decide between these conflicting interpretations, we treated zebrafish embryos during blastodisc stages and epiboly with several chemical teratogens causing rachischisis anterior. Treatment with ethanol, Colcemid, hydroxyurea, or cycloheximide was found to delay the proliferation and movements of the deep cells more strongly than the timing of cell differentiation, so that the deep cells embark on organogenesis before having reached their destinations in the uniform germ shield. Treatment with alpha-amanitin, on the other hand, seems primarily to affect the periblast and enveloping layer; the incomplete epiboly observed in these layers appears to restrain deep cell epiboly physically and thus to cause rachischisis. In both instances, the split condition of the embryo's trunk region is clearly due to the ectopic formation of physically separate body halves right from the beginning, a mode we call bipartite axiation. We also describe secondary anomalies specific for individual teratogens, and briefly discuss the possible origins of rachischisis anterior among other chordates including man.
... Previous studies in zebrafish showed that embryos treated with ethanol could cause eye abnormalities (cyclopia), cardiac malformations, skeletal dysmorphogenesis and developmental delays, etc. It was also reported that ethanol can affect axis develop-ment (Baumann and Sander, 1984;Laale, 1971). However, the mechanism responsible for the effects of ethanol exposure on axis development in zebrafish remains unclear. ...
... As discussed above, it seemed impossible to generate split axes by simply affecting those signaling pathways regulating cell movement; then what is the real cause? It was hypothesized that the appropriate ratio of the epiboly speed versus differentiation speed may play a role in maintaining posterior axis uniqueness (Baumann and Sander, 1984). Indeed, there are several lines of evidence suggesting a correlation between split axes phenotype and delayed epiboly in zebrafish. ...
... Indeed, there are several lines of evidence suggesting a correlation between split axes phenotype and delayed epiboly in zebrafish. Treatment during blastula stage with ethanol, colcemid (microtubule assembly inhibitor), hydroxyurea (DNA synthesis inhibitor), cycloheximide (protein synthesis inhibitor), ␣-amanitin (RNA polymerase II inhibitor) or UV irradiation can cause split axes phenotype, and at the same time, all these treatment can cause significant epiboly delay (Baumann and Sander, 1984;Strahle and Jesuthasan, 1993). Knockdown of E-cadherin in zebrafish also delays epiboly, and generate embryos with bifurcated axis (Babb and Marrs, 2004). ...
To explore the toxic effects of ethanol on axis formation during embryogenesis, zebrafish embryos at different developmental stages were treated with 3% ethanol for 3h. The effects of ethanol exposure appeared to be stage-dependent. The dome stage embryo was most sensible to form posterior split axes upon ethanol exposure. Morphological and histological observations and whole-mount in situ hybridization results showed that ethanol exposure at this stage caused a general gastrulation delay, and induced double notochords, double neural tubes and two sets of somites in the posterior trunk. Mechanistically, no ectopic organizer was found by examining the expression patterns of dorsoventral markers including goosecoid, chordin and eve1 at the onset of gastrulation. However, radial intercalation, epiboly and convergence extension were inhibited by ethanol exposure as revealed by cell labeling, phenotypic observation and the expression patterns of axial or paraxial markers. Further investigation showed that the cell aggregation might be affected by ethanol exposure, as indicated by the much more scattered expression pattern of chordin, eve1 and wnt11 at the early gastrula stage, and the discontinuous gsc positive cells during migration. These results imply that ethanol might affect cell movement before and during gastrulation and as a consequence, induces a split axes phenotype.
... Among the solvents examined and with the range of concentrations used, ethanol seemed to ac t quite early during inception stages of embryogenesis and to induce developmental defects. The possible mechanisms leading to the gradual death or failure to hatch or both within the 96-h exposure period have been described in detail in previous studies (Blader and Strahle, 1998;Baumann and Sander, 1984;Laale, 1971). Our present results support the earlier study by Baumann and Sander (1984) on the nature of the effects of ethanol in zebrafish embryos. ...
... The possible mechanisms leading to the gradual death or failure to hatch or both within the 96-h exposure period have been described in detail in previous studies (Blader and Strahle, 1998;Baumann and Sander, 1984;Laale, 1971). Our present results support the earlier study by Baumann and Sander (1984) on the nature of the effects of ethanol in zebrafish embryos. A concentration of 1.0% ethanol induced some developmental defects (e.g., weak pigmentation and edema). ...
... Exposure of zebrafish embryos to even higher concentrations (2.2-3.2%) than those used in the present study showed bipartite axiation characterized by the formation of two separate axial strands when the epibolic movements of the blastodisk are delayed (Maiwald, 1997;Baumann and Sander, 1984). Furthermore, partial duplication of the notochord, spinal cord, and rhoembencephalon has been frequent I y implicated to ethanol exposure (Baumann and Sander, 1984;Laale, 1971). ...
The present study examines the effects of ethanol (ETOH), dimethyl sulfoxide (DMSO), and acetone on zebrafish embryos and the implications of the observed results on the use of these solvents to zebrafish early life stage tests. The embryos were exposed to different concentrations (0.0, 0.0001, 0.001, 0.01, 0.1, 0.05, 1, 1.5, and 2.0% v/v) of the respective solvents by diluting reagent-grade solvent with reconstituted water [DIN 38415-6-Suborganismische Testverfahren (Gruppe T) Teil 6: Giftigkeit gegenüber Fischen. Deutsches Institute für Normung e.V]. The following endpoints were investigated (mortality, hatching rate, abnormalities, heart rate, and hsp 70 induction). No effect on survival was recorded for both acetone and DMSO even up to the highest concentration. On the other hand, embryos exposed to 1.5% and 2.0% ethanol showed a significant reduction in survival rate. No developmental defects occurred with any of the solvents at the 0.1% concentration. However, starting with 1.0%, weak to very pronounced abnormalities (weak pigmentation, edema, crooked bodies, eye defect, tail defect, reduced heartbeat, and abnormal hatching) were observed depending on the solvent type and the concentration used. Ethanol has been shown to be the most embryotoxic solvent while DMSO and acetone have comparably lesser effects. Heat shock protein 70 was induced by all solvents but at different concentration ranges. DMSO has been shown to be the most potent inducer of stress proteins. Based on the study, the chemicals tested here may be used as carrier solvents in the zebrafish embryo assay at levels below 1.5, 1.5, and 1% v/v for acetone, DMSO, and ethanol, respectively. For stress protein analysis of the exposed embryos, however, the solvent levels should be below 0.1%, 0.01%, and 1.5%, respectively. Additional and separate investigations utilizing other biomarkers should be carried out to further validate the suitability of using these solvents in a typical zebrafish embryo assay.
... Animal models are essential to investigate the pattern of biological mechanisms and functions. For long times, the zebrafish D. rerio (Cypriniformes, Cyprinidae) and the Japanese medaka O. latipes (Beloniformes, Adrianichthyidae) have been successfully established as model for many biological and biomedical studies (Baumann & Sander, 1984;Bilotta, Barnett, Hancock, & Saszik, 2004;Sakamotoa, Kozakaa, Takahashib, Kawauchib, & Andoa, 2001). Beside these well-known fish models, species of killifishes have recently been proposed as additional models for various biological and biomedical aspects (Kim, Gil Nam, & Valenzano, 2015;Wendler et al., 2015;Zeinali & Motamedi, 2017). ...
The histomicroscopy and normal anatomy of the major body organ systems were investigated in the adult killifish, Aphanius hormuzensis using histological examination, X‐ray imaging, double staining, light microscopy and scanning electron microscopy (SEM). Based on the histomicroscopic observations, the kidney, liver and swim bladder in the studied species were comparable to other fish models. The anterior portion of the kidney is bulbous, while the posterior portion is narrow and elongated; the liver has a single lobe and the swim bladder is a single‐chambered organ with no connection to the digestive tract (physoclistous). X‐ray imaging and double staining examination showed 12 abdominal and 15 caudal vertebrae and a single hypural plate in the caudal skeleton. According to light microscopy, the scales were rounded to pentagonal in shape with three types of radii (primary, secondary and tertiary), and the urohyal bone was elongated. SEM microscopy showed a single row of tricuspid teeth on the upper and lower jaw, respectively, each tooth has two lateral cusps that are shorter than the middle one. The number of teeth was 17–18 in the upper jaw and 19–20 in the lower jaw. The saccular otoliths were rounded‐trapezoid in shape with a moderately incised and V‐shaped excisura. The members of killifishes are an important group for biologists because of their evolutionary properties, regeneration capacity and usefulness as biological control and also for the ecotoxicological assessment of environmental pollution. The outcomes of this study may provide a useful basis for future research on the genus Aphanius.
... In our previous study, lower doses of alcohol exposure (i.e., 1% [167mM] and 1.5% [251 mM]) for 46 hours during embryogenesis resulted in some defects of octavolateral organs, albeit embryos treated in 2% (334 mM) alcohol had increased defects at 3dpf (Zamora and Lu, 2013). Doses above 2.5% of alcohol exposure are known to result in severe overall developmental defects such as yolk sac and pericardial edema, axial malformations, and cyclopia (Reimers et al., 2004;Baumann and Sander, 1984;Blader and Str€ ahle, 1998). In this study, zebrafish were treated in a 2% alcohol concentration for 2, 3, and 5 hours during early embryonic development. ...
Fetal alcohol exposure can cause Fetal Alcohol Spectrum Disorders (FASD), completely preventable developmental disabilities characterized by permanent birth defects. However, specific gestational timing when developing organs are most sensitive to alcohol exposure is unclear. In this study, we examined the temporal effects of embryonic alcohol exposure on octavolateral organs in zebrafish (Danio rerio), including inner ears and lateral line neuromasts that function in hearing, balance, and hydrodynamic detection, respectively. To determine an alcohol-sensitive period in the first 24 hours post fertilization (hpf), Et(krt4:EGFP)(sqet4) zebrafish that express green fluorescent protein in sensory hair cells were treated in 2% alcohol for 2, 3, and 5-hours. Octavolateral organs of control and alcohol-exposed larvae were examined at 3, 5, and 7 days post fertilization (dpf). Using confocal and light microscopy, we found that alcohol-exposed larvae had significantly smaller otic vesicles and saccular otoliths than control larvae at 3 dpf. Only alcohol-exposed larvae from 12-17 hpf had smaller otic vesicles at 5 dpf, smaller saccular otoliths at 7 dpf and fewer saccular hair cells, neuromasts and hair cells per neuromast at 3 dpf. In addition, auditory function was assessed by microphonic potential recordings from inner ear hair cells in response to 200-Hz stimulation. Hearing sensitivity was reduced for alcohol-exposed larvae from 7-12 and 12-17 hpf. Our results show that 12-17 hpf is an alcohol-sensitive time window when morphology and function of zebrafish octavolateral organs are most vulnerable to alcohol exposure. This study implies that embryonic alcohol exposure timing during early development can influence severity of hearing deficits. © 2017 Wiley Periodicals, Inc.
... Carvan et al., (2004) also reported that EtOH exposure alters the neurobehavioral and skeletal morphogenesis. Another study also documented that it produces severe body malformations (Baumann and Sander, 1984). Daft et al., (1986) had reported that gestational acute EtOH exposure produced abnormal heart and great vessel development in mice. ...
The objective of the present study is to evaluate the effect of ethanol on developing zebra fish heart. Male and female fishes are allowed to breed normally and the fertilized eggs were collected and it was exposed to 3 % of ethanol (EtOH) for one hour once in 24 hours for 96 hours. The embryos were subjected to various analyses related to heart development like external morphology, heart morphology, heart rate, heart looping formation, heart length and ventricular stand still. The data showed significant alteration in the length of whole embryo or larva, heart length and heart rate of EtOH exposed embryo when compared to control. The looping also altered like string-like or straight tube appearance, with the ventricle located distinctly anterior to the atrium. Further, the ventricle appeared smaller than normal, the atrium is elongated, and both chambers having a narrow width. The incidence of ventricular standstill and valvular regurgitation is occurring in EtOH treated embryo when compared to control. Taken together, the steady decrease in heart size coupled with the severe defects in ventricular function, failure of cardiac looping formation, heart rate, ventricular standstill and retrograde blood flow would be expected to have a substantial impact on the ability of zebra fish to circulate blood. From the present study it is concluded that EtOH exposure during development results in structural and functional impairment in heart that mimic malformations that occur in patients with fetal alcohol syndrome.
... In addition, ethanol exposure alters gene expression in the ventral aspects of the foreand mid-brain, inhibits acetylcholinesterase activity, and reduces nucleoside triphosphate diphosphohydrolases (NDTPases) 1 and 2 mRNA levels in brain103104105. Furthermore, ethanol induces heat shock proteins [106,107], produces developmental abnormalities of the notochord and spinal cord, and malformations of the body trunk [108]. The effects of ethanol found to be strain-dependent in zebrafish and characterized by modulated aggression and other social behavior [92,93,109110111112113. ...
Contents 1. Introduction 78 2. Fetal Alcohol Spectrum Disorder 79 3. Probable Mechanisms of FASD 80 3.1. Oxidative stress and reactive oxygen species 80 3.2. Interference with retinoid metabolism 81 3.3. Cholesterol deficiency and sonic hedgehog signaling 81 3.4. Interference with neural cell adhesion molecules 82 3.5. Interference in insulin signaling 82 3.6. Disrupting epigenetic mechanisms 82 3.7. Altering placental metabolism 83 4. Use of Japanese Medaka in FASD Research 83 4.1. Ethanol is toxic to medaka morphogenesis and increasing amounts of waterborne ethanol are required to induce FASD phenotypes in medaka 86 4.2. Changes in skeletal structure 89 4.3. Changes in cardiovascular structure 95 4.4. Biochemical parameters 99 5. Future Directions 114 Acknowledgments 117 References 117
... 46,47 In addition to a higher mortality rate at higher concentrations (e.g., 2.5% and 3%), severe developmental defects were present, such as cyclopia and axial malformations. 18,48,49 Lower doses of alcohol (1% and 1.5%) caused many fewer defects in the octavolateral organs than 2% alcohol. Therefore, 2% alcohol solution was chosen to investigate alcohol-induced morphological defects in the inner ear and lateral line in this study. ...
Abstract Prenatal alcohol exposure is known to have many profound detrimental effects on human fetal development (fetal alcohol spectrum disorders), which may manifest as lifelong disabilities. However, how alcohol affects the auditory/vestibular system is still largely unknown. This is the first study to investigate morphological effects of alcohol on the developing octavolateral system (the inner ear and lateral line) using the zebrafish, Danio rerio. Zebrafish embryos of 2 hours post fertilization (hpf) were treated in 2% alcohol for 48 hours and screened at 72 hpf for morphological defects of the inner ear and lateral line. Octavolateral organs from both alcohol-treated and control zebrafish were examined using light, confocal, and scanning electron microscopy. We observed several otolith phenotypes for alcohol-treated zebrafish including zero, one, two abnormal, two normal, and multiple otoliths. Results of this study show that alcohol treatment during early development impairs the inner ear (smaller ear, abnormal otoliths, and fewer sensory hair cells) and the lateral line (smaller neuromasts, fewer neuromasts and hair cells per neuromast, and shorter kinocilia of hair cells). Early embryonic alcohol exposure may also result in defects in hearing, balance, and hydrodynamic function of zebrafish.
... Zebrafish embryos, treated briefly with ethanol at early gastrula stages, developed cyclopia due in part to defects in the migration of the prechordal plate [36]. Exposure to ethanol earlier in development led to incomplete epiboly [37] , whereas the acute treatment of larvae led to a dosedependent locomotor response, with intermediate doses resulting in hyperactivity, and high doses causing hypoactivity and sedation [38]. For these reasons, the use of ethanol as a solvent in behavioral assays is not recommended. ...
Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, drug discovery, and toxicology, and more recently also for natural product discovery. Experiments involving the pharmacological evaluation of small molecules or natural product extracts in zebrafish bioassays require the effective delivery of these compounds to embryos and larvae. While most samples to be screened are first solubilized in dimethyl sulfoxide (DMSO), which is then diluted in the embryo medium, often this method is not sufficient to prevent the immediate or eventual precipitation of the sample. Certain compounds and extracts are also not highly soluble in DMSO. In such instances the use of carriers and/or other solvents might offer an alternative means to achieve the required sample concentration. Towards this end, we determined the maximum tolerated concentration (MTC) of several commonly used solvents and carriers in zebrafish embryos and larvae at various developmental stages. Solvents evaluated for this study included acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG-400), propylene glycol, and solketal, and carriers included albumin (BSA) and cyclodextrin (2-hydroxypropyl-beta-cyclodextrin, or HPBCD). This study resulted in the identification of polyethylene glycol (PEG400), propylene glycol, and methanol as solvents that were relatively well-tolerated over a range of developmental stages. In addition, our results showed that acetone was well-tolerated by embryos but not by larvae, and 1% cyclodextrin (HPBCD) was well-tolerated by both embryos and larvae, indicating the utility of this carrier for compound screening in zebrafish. However, given the relatively small differences (2-3 fold) between concentrations that are apparently safe and those that are clearly toxic, further studies - e.g. omics analyses -should be carried out to determine which cellular processes and signalling pathways are affected by any solvents and carriers that are used for small-molecule screens in zebrafish.
... Ethanol, one of the most widely used drugs in our culture today, has long been employed as a teratogen to study normal development. A study by Baumann and Saunder (1984) also found that ethanol, as well as various other chemicals, caused duplication of the embryonic axis. Ethanol-induced eye defects (Laale 1971) are similar to those generated by exposing embryos to low temperatures (Majima and Ingalls 1966) or overexpressing the secreted cell–cell signalling factor wnt4 in embryos (Ungar et al. 1995 ) or those resulting from the absence of a floor plate (Hatta et al. 1991). ...
The zebrafish has long been the favorite organism in many scientific disciplines. Although its attributes as a model were expounded for many years and thus were no secret, the zebrafish sat in the wings while other more popular vertebrates such as chick, amphibians, and mouse were examined at length. We cannot say there was a resurgence in popularity, but more an explosion of research utilizing the zebrafish beginning in the late 1970s when investigators at the University of Oregon began using it as their model in neuroscience. Prior to this reawakening, the zebrafish was one of the significant organisms in the study of teratology and toxicology, development, and, to some extent, behavior. Recently, however, the field of zebrafish genetics has gained immense popularity and success, in part owing to the fact that zebrafish are diploid and are amenable to genetic manipulations. Here we present an overview of the multidisciplinary research that has laid some of the foundation of our present understanding of the biochemical, cell biological, and molecular genetic events accompanying zebrafish development.
... This finding suggests for an impaired degradation of maternally deposited transcripts in TBP depleted embryos. (Baumann & Sander, 1984;Kane et al, 1996) and causes a block of polymerase II transcription (Kedinger et al, 1970). As a consequence, maternal mRNAs, which are de- ...
The differential expression of protein coding genes in specific cell types and during development requires the interaction of transcription factors with regulatory sequences in the proximal promoter to generate diverse expression patterns. In this thesis I address the differential regulatory function of TBP and the core promoter architecture facilitating this differential response in the complexity of the vertebrate organism by exploiting the experimental advantages of the zebrafish embryo model system. The work presented here demonstrates that only a proportion of genes require TBP-function in early zebrafish development and that TBP has a specific role in the clearance of maternal RNAs that includes the miR-430 pathway. These results indicate that TBP plays a major role in the transition from a transcriptionally inactive state to a transcriptionally active phase of the zebrafish embryo and has distinct functions in regulating gene expression during development. Furthermore, the bioinformatic characterisation of promoters regulated by TBP, as well as the functional analysis of the notail promoter, indicate that the TATA box, the core promoter motif TBP binds to, is not the defining feature of TBP-dependent transcription initiation mechanisms.
... The axial and paraxial tissues of ¢shes can extend as explants, in isolation from the embryo (see Laale 1982), and they can push into the yolk in teratogenized embryos (see Bauman & Sanders 1984 ...
The cells of many embryonic tissues actively narrow in one dimension (convergence) and lengthen in the perpendicular dimension (extension). Convergence and extension are ubiquitous and important tissue movements in metazoan morphogenesis. In vertebrates, the dorsal axial and paraxial mesodermal tissues, the notochordal and somitic mesoderm, converge and extend. In amphibians as well as a number of other organisms where these movements appear, they occur by mediolateral cell intercalation, the rearrangement of cells along the mediolateral axis to produce an array that is narrower in this axis and longer in the anteroposterior axis. In amphibians, mesodermal cell intercalation is driven by bipolar, mediolaterally directed protrusive activity, which appears to exert traction on adjacent cells and pulls the cells between one another. In addition, the notochordal-somitic boundary functions in convergence and extension by 'capturing' notochordal cells as they contact the boundary, thus elongating the boundary. The prospective neural tissue also actively converges and extends parallel with the mesoderm. In contrast to the mesoderm, cell intercalation in the neural plate normally occurs by monopolar protrusive activity directed medially, towards the midline notoplate-floor-plate region. In contrast, the notoplate-floor-plate region appears to converge and extend by adhering to and being towed by or perhaps migrating on the underlying notochord. Converging and extending mesoderm stiffens by a factor of three or four and exerts up to 0.6 microN force. Therefore, active, force-producing convergent extension, the mechanism of cell intercalation, requires a mechanism to actively pull cells between one another while maintaining a tissue stiffness sufficient to push with a substantial force. Based on the evidence thus far, a cell-cell traction model of intercalation is described. The essential elements of such a morphogenic machine appear to be (i) bipolar, mediolaterally orientated or monopolar, medially directed protrusive activity; (ii) this protrusive activity results in mediolaterally orientated or medially directed traction of cells on one another; (iii) tractive protrusions are confined to the ends of the cells; (iv) a mechanically stable cell cortex over the bulk of the cell body which serves as a movable substratum for the orientated or directed cell traction. The implications of this model for cell adhesion, regulation of cell motility and cell polarity, and cell and tissue biomechanics are discussed.
... In addition to the eye phenotypes, up to 24% of injected embryos had a partially duplicated axis with shortened body following microinjection of rx1 or rx2 RNA (Table 1; Fig. 2K). This phenotype is very similar to bipartite axiation in embryos treated with chemical teratogens (Baumann and Sander, 1984). The axis duplication was usually restricted to the region caudal to the midbrain-hindbrain boundary, excluding the tail, although we also observed embryos with axis duplication in the forebrain, and very rarely (Ӷ1%) embryos had a second rostral axis and three (Fig. 2L) or four eyes (data not shown). ...
Zebrafish retinal homeobox genes rx1 and rx2 are expressed exclusively in the optic primordia and then in cone photoreceptors of the differentiated neural retina. In this study, we show that the rx expression domain is coextensive with the region identified as the retinal field in published fate maps of the neural plate in zebrafish embryos. Analysis of the spatiotemporal relationships between retinal and forebrain precursors suggests that lateral movement of retinal precursors is responsible for evagination of the optic primordia. Overexpression of either rx1 or rx2 results in the loss of forebrain tissue and the ectopic formation of retinal tissue. We asked whether the deletion of forebrain and expansion of retinal tissue could be explained by the death of telencephalic precursors and enhanced proliferation of retinal precursors, and we found that it could not. Instead, our data are consistent with a change in cell fate of forebrain precursors associated with reduced expression of telencephalic markers (emx1 and BF-1) and ectopic expression of retinal markers (rx1/2/3, pax6, six6, and vsx2) at the neural keel stage. The rx homeodomain alone is sufficient to induce ectopic retinal tissue, although weakly so, and this observation, together with results from deletion constructs, suggests that interactions with unidentified transcriptional regulators are important for rx1 and rx2 function during early eye development. We conclude that regulated expression of zebrafish rx1 and rx2 helps to define the region of the forebrain fated to give rise to retinal tissue and may be involved in the cellular migrations that lead to splitting of the retinal field and formation of the optic primordia.
... These genes function in the Wnt planar polarity pathway (Myers et al., 2002b) and the results, combined with our findings with ntl, might be revealing that Wnt signaling drives extension that is closely coupled to convergence, but that ntldisrupts this relationship leaving other, parallel, mechanisms able to extend the axis. Furthermore, in epiboly mutants (Kane et al., 1996) or in embryos treated with a teratogen (Bauman and Sanders, 1984), both convergence and extension of the notochord can occur in the absence of epiboly, the opposite of the findings with the Wnt signaling mutants. Hence, it is likely that MIB normally functions redundantly with other cellular mechanisms to produce extension of the dorsal mesoderm. ...
Promptly after the notochord domain is specified in the vertebrate dorsal mesoderm, it undergoes dramatic morphogenesis. Beginning during gastrulation, convergence and extension movements change a squat cellular array into a narrow, elongated one that defines the primary axis of the embryo. Convergence and extension might be coupled by a highly organized cellular intermixing known as mediolateral intercalation behavior (MIB). To learn whether MIB drives early morphogenesis of the zebrafish notochord, we made 4D recordings and quantitatively analyzed both local cellular interactions and global changes in the shape of the dorsal mesodermal field. We show that MIB appears to mediate convergence and can account for extension throughout the dorsal mesoderm. Comparing the notochord and adjacent somitic mesoderm reveals that extension can be regulated separately from convergence. Moreover, mutational analysis shows that extension does not require convergence. Hence, a cellular machine separate from MIB that can drive dorsal mesodermal extension exists in the zebrafish gastrula. The likely redundant control of morphogenesis may provide for plasticity at this critical stage of early development.
... This suggests that the notochord actively extends but is not stiff enough to extend straight without buckling in an environment that does not provide appropriate external forces. After treating zebrafish embryos with a teratogen that blocks epiboly of the EVL/YSL, Bauman and Sanders (1984) observed kinked and bent notochords, and extension of the notochord beyond the boundaries of the halted EVL/YSL into the large yolk cell. This again suggests that the teleost notochord is an active extender but is prone to bucking when unassisted. ...
Although it is rarely considered so in modern developmental biology, morphogenesis is fundamentally a biomechanical process, and this is especially true of one of the first major morphogenic transformations in development, gastrulation. Cells bring about changes in embryonic form by generating patterned forces and by differentiating the tissue mechanical properties that harness these forces in specific ways. Therefore, biomechanics lies at the core of connecting the genetic and molecular basis of cell activities to the macroscopic tissue deformations that shape the embryo. Here we discuss what is known of the biomechanics of gastrulation, primarily in amphibians but also comparing similar morphogenic processes in teleost fish and amniotes, and selected events in several species invertebrates. Our goal is to review what is known and identify problems for further research.
... Finally, the practical advantages of this model allows for saturation mutagenesis screens and knockdown approaches that can be used to identify genes involved in toxic responses. Previous studies in zebrafish demonstrated that ethanol leads to craniofacial abnormalities, cardiac and structural malformations, and developmental delays [2,18]. Zebrafish embryos exposed to 1.5% v/v ethanol during development had impaired visual function [4]. ...
Ethanol is a well-established developmental toxicant; however, the mechanism(s) of this toxicity remains unclear. Zebrafish are becoming an important model system for the evaluation of chemical and drug toxicity. In this study, zebrafish embryos were utilized to compare the developmental toxicity resulting from either ethanol or acetaldehyde exposure. Embryos were exposed to waterborne ethanol concentrations for various lengths of time but encompassed the earliest stages of embryogenesis. The waterborne ethanol concentration that causes 50% mortality (LC(50)) following a 45-h ethanol exposure was approximately 340 mM (1.98% v/v). A number of reproducible endpoints resulted from ethanol exposure and included pericardial edema, yolk sac edema, axial malformations, otolith defects, delayed development, and axial blistering. When the exposure period was reduced, similar signs of toxicity were produced at nearly identical ethanol concentrations. To estimate the embryonic dose following a given waterborne ethanol concentration, a kinetic alcohol dehydrogenase (ADH) assay was adapted. The average embryonic ethanol dose was calculated to be a fraction of the waterborne concentration. Embryos exposed to waterborne acetaldehyde resulted in similar, but not identical, endpoints as those induced by ethanol. Embryos were however, almost three orders of magnitude more sensitive to acetaldehyde than to ethanol. Ethanol and acetaldehyde both negatively impact embryonic development; however, ethanol is more teratogenic based on teratogenic indices (TIs). These results demonstrate that the zebrafish model will provide an opportunity to further evaluate the mechanism of action of ethanol on vertebrate development.
... Exposure of zebrafish embryos to alcohol causes cyclopia and craniofacial abnormalities and alters gene expression in the ventral aspects of the fore-and midbrain [8,7]. Alcohol exposure of zebrafish embryos also induces stress proteins [40,42], developmental abnormalities of the notochord and spinal cord [41], and malformation of the body trunk [5]. In this study, we describe the dosedependent effects of developmental ethanol exposure on learning and memory, cell death in the CNS, craniofacial and neurocranial skeleton morphogenesis, and behavior, providing quantitative endpoints for the analysis of mutants and morphants. ...
Exposure to ethanol during development can lead to a constellation of congenital anomalies, resulting in prenatal and postnatal failure to thrive, central nervous system (CNS) deficits, and a number of patterning defects that lead to defects in the cardiovascular system, facial structures, and limbs. The cellular, biochemical, and molecular mechanisms by which ethanol exerts its developmental toxicity and the genes that influence sensitivity to developmental ethanol exposure have yet to be discovered, despite being one of the more common nongenetic causes of birth defects. The zebrafish undergoes much the same patterning and morphogenesis as other vertebrate embryos do--including humans--that are distinct and cannot be studied in invertebrates. Developmental processes in zebrafish are affected by ethanol exposure in a dose-dependent manner, resulting in learning and memory deficits, cell death in the CNS, skeletal dysmorphogenesis, and alterations in startle reflex responses. Interestingly, significant ethanol effects on learning and behavioral endpoints occurred at concentrations well below those that induced cell death in the CNS. This work provides the foundation for identifying genes and pathways involved in developmental alcohol toxicity in vertebrates, leading to a more complete mechanistic understanding of fetal alcohol disorders in humans.
... In addition, ethanol exposure has been shown to alter gene expression in the ventral aspects of the fore-and mid-brain (Blader and Strahle, 1998). Furthermore, in the zebrafish embryo, ethanol induces heat shock proteins Lele et al., 1997), produces developmental abnormalities of the notochord and spinal cord, and malformation of the body trunk (Baumann and Sander, 1984). The effects of ethanol were found to be strain-dependent in zebrafish (Dlugos and Rabin, 2002). ...
Animal models are necessary to investigate the mechanism of alcohol-induced birth defects. We have used Japanese medaka (Oryzias latipes) as a non-mammalian model to elucidate the molecular mechanism(s) of ethanol teratogenesis.
Medaka eggs, within 1 hr post-fertilization (hpf) were exposed to waterborne ethanol (0-1000 mM) in hatching solution for 48 hr. Embryo development was observed daily until 10 days post-fertilization (dpf). The concentration of embryonic ethanol was determined enzymatically. Cartilage and bones were stained by Alcian blue and calcein, respectively and skeletal and cardiovascular defects were assessed microscopically. Genetic gender of the embryos was determined by PCR. Levels of two isoenzymes of alcohol dehydrogenase (Adh) mRNAs were determined by semi-quantitative and real-time RT-PCR.
The concentration of ethanol required to cause 50% mortality (LC50) in 10 dpf embryos was 568 mM, however, the embryo absorbed only 15-20% of the waterborne ethanol at all ethanol concentrations. The length of the lower jaw and calcification in tail fin cartilaginous structures were reduced by ethanol exposure. Active blood circulation was exhibited at 50+ hpf in embryos treated with 0-100 mM ethanol; active circulation was delayed and blood clots developed in embryos treated with 200-400 mM ethanol. The deleterious effects of ethanol were not gender-specific. Moreover, ethanol treatment was unable to alter the constitutive expression of either Adh5 or Adh8 mRNA in the medaka embryo.
Preliminary results suggested that embryogenesis in medaka was significantly affected by ethanol exposure. Phenotypic features normally associated with ethanol exposure were similar to that observed in mammalian models of fetal alcohol syndrome. The results further indicated that medaka embryogenesis might be used as an alternative non-mammalian model for investigating specific alterations in gene expression as a means to understand the molecular mechanism(s) of ethanol-induced birth defects.
... Since zebrafish development has been well characterized, results from zebrafish can be compared to mammalian results. For instance, previous studies in zebrafish demonstrated that ethanol leads to craniofacial abnormalities, cardiac and structural malformations, and developmental delays similar to results observed in mammals [4,10,25,26,28,36]. The role that ethanol biotransformation may play in these ethanol-dependent endpoints in zebrafish remains unknown. ...
Ethanol is a well-established developmental toxicant; however, the molecular and cellular mechanism(s) of toxicity remains unclear. It has been suggested that ethanol metabolism leads to oxidative stress resulting in an increase in cell death. Alcohol developmental toxicity has not been well studied in zebrafish; however, zebrafish represent an excellent vertebrate model for investigating and understanding normal and aberrant development. To evaluate ethanol metabolism dependent toxicity, chemical inhibitors of the ethanol metabolizing enzymes were utilized. Embryos co-exposed to ethanol and a combination of ethanol metabolism inhibitors led to a significant increase in the occurrence of pericardial edema. Further, in the presence of the inhibitor mixture there was an increase in developmental malformations at lower ethanol concentrations. Cell death has been implicated as a potential explanation for ethanol-dependent toxicity. Using cell death assays, ethanol significantly increased embryonic cell death. To determine if oxidative stress underlies cardiovascular dysfunction, embryos were co-exposed to ethanol and several antioxidants. The antioxidants, glutathione and lipoic acid, partially attenuated the incidence of pericardial edema. The effectiveness of the antioxidants to protect the embryos from ethanol-induced cell death was also evaluated. The antioxidants provided no protection against cell death. Thus, ethanol-mediated pericardial edema and cell death appear to be mechanistically distinct.
... Although zebrafish have been widely used for studies of development, their use in the study of the effects of ethanol has been limited. In addition to several reports documenting the teratogenic effects of ethanol on developing embryos (Baumann and Sander, 1984;Blader and Strahle, 1998;Laale, 1971), two recent reports demonstrate that ethanol modifies multiple behaviors of adult zebrafish in a strain-dependent manner (Dlugos and Rabin, 2003;Gerlai et al., 2000). However, it is cumbersome to use adult zebrafish for genetic screens aimed at identifying molecules mediating the biological effects of ethanol. ...
Larval zebrafish are used extensively for developmental genetic studies due to their salient features, such as small size, external development, optical transparency, and accessibility in large numbers. However, their use for the study of drug and alcohol abuse has not been explored. Here we investigated the response of larval zebrafish to acute treatment of alcohol. Our analyses showed that like adults, the larval zebrafish exhibited a dose-dependent locomotor response to ethanol: intermediate doses led to hyperactivity, whereas high doses have a neurodepressive effect resulting in hypoactivity and sedation. Alcohol also induced morphological changes of melanocytes, providing a visible cellular measure of the biological effects of alcohol in vivo. In addition, alcohol induced thigmotaxis behavior (preference for the edge of a compartment). In the behaviors we analyzed, genetic background influenced the locomotor responses to alcohol. The present study demonstrates that larval zebrafish exert a response to the acute treatment of alcohol, which is genetically modifiable. Therefore, the larval zebrafish represent a tractable vertebrate model system for a large-scale genetic analysis of the biological effects of alcohol.
... Studies are presented below according to species and year of publication. Baumann and Sander (1984), supported by the German Research Council, examined the effects of hydroxyurea exposure on developmental toxicity in zebrafish. Other compounds were examined but the effects will not be discussed here. ...
The National Toxicology Program (NTP) and the National Institute of Environmental Health Sciences (NIEHS) established the NTP Center for the Evaluation of Risks to Human Reproduction (CERHR) in June 1998. The purpose of CERHR is to provide timely, unbiased, scientifically sound evaluations of human and experimental evidence for adverse effects on reproduction and development caused by agents to which humans may be exposed.
Hydroxyurea was selected for evaluation by a CERHR expert panel because of (1) its increasing use in the treatment of sickle cell disease in children and adults, (2) knowledge that it inhibits DNA synthesis and is cytotoxic, and (3) published evidence of its reproductive and developmental toxicity in rodents. Hydroxyurea is FDA-approved for reducing the frequency of painful crises and the need for blood transfusions in adults with sickle cell anemia who experience recurrent moderate-to severe crises. Hydroxyurea is used in the treatment of
cancer, sickle cell disease, and thalassemia. It is the only treatment for sickle cell disease aside from blood transfusion used in children. Hydroxyurea may be used in the treatment of children and adults with sickle cell disease for an extended period of time or for repeated cycles of therapy. Treatment with hydroxyurea may be associated with cytotoxic and myelosuppressive effects, and hydroxyurea is mutagenic.
To obtain information about hydroxyurea for the CERHR evaluation, the PubMed (Medline) and Toxline databases were searched through January 10, 2007 using ‘‘hydroxyurea’’ and its CAS RN (127–07–1). References were also identified from databases such as REPROTOX, HSDB, IRIS, and DART and from the bibliographies of reports being reviewed. This evaluation results from the efforts of a 13-member panel of government and non-government scientists that culminated in a public expert panel meeting held January 24–26, 2007. This report is a product of the hydroxyurea expert panel and is intended to (1) interpret the strength of scientific evidence that hydroxyurea is a reproductive or developmental toxicant based on data from in vitro, animal, or human studies, (2) assess the extent of human exposures, especially for patients receiving hydroxyurea in the treatment of sickle cell disease and other health disorders, (3) provide objective and scientifically thorough assessments of the scientific evidence that adverse reproductive/developmental health effects may be associated with such exposures, and (4) identify knowledge gaps to help establish research and testing priorities to reduce uncertainties and increase confidence in future evaluations. This report has been reviewed by members of the hydroxyurea expert panel and by CERHR staff scientists. Copies have been provided to the CERHR Core Committee, which is made up of representatives of NTP-participating government agencies.
The relationship between cell density distributions and differentiation is of interest to workers in many different areas of developmental biology. Traditionally, such studies have required tedious counting of individual cells through a microscope eyepiece grid or on photographs. We have developed a rapid, reliable, and inexpensive microcomputer based system for counting cells in histological sections. The system employs an Apple He computer, Digisector video board (Micro Works, Del Mar, CA), Super Scan program (Magna Soft, La Canada, CA), Gibson Light Pen System (Gibson Laboratories, Laguna Hills, CA), and Telestar video camera (Bell and Howell). We have applied this to identify specific patterns of cell density in feathers. Our system is significantly different from other image analysis systems available because its low price (about $2500) makes it accessible to average laboratories, as opposed to the expensive image analysis systems which are available commercially or in a few special research laboratories. Normal feather germs, serial sectioned at 6 μ, and stained using the cold hydrolysis Feulgen technique were photographed at 400× using Kodak Tech Pan film and printed on Kodabromide paper (no. 5). One inch squares of the 4×6 inch print were exposed to the video camera. The correlation coefficient for the number of black pixels counted by the computer and the number of cells counted by hand from the photograph was >·98. Dramatic local increases in mesenchymal cell density in the area of the germ which protrudes distally were observed in the saggital sections of all the specimens examined. (Supported by NIH Training Grant 5T32AM07376.)
Highlighting latest advances in genetics and biochemistry, the completely revised Third Edition reviews the field from basic science, clinical, epidemiological, and regulatory perspectives. Contributions from top opinion leaders in the field bring together developments in molecular embryology and cell biology as they apply to problems in developmental toxicology. It covers testing of pharmaceutical and environmental agents and interpretation of developmental toxicology data, highlighting mathematical and statistical techniques, as well as the effects of toxic exposure on the functional development of various organs. The relationship between maternal and developmental toxicology is examined, in addition to current techniques for studying chemical disposition, metabolism, and placental transfer. Close attention is given to the regulatory aspects of testing and risk assessment. Pre and postconceptional clinical care and genetic factors in clinical developmental toxicology are also discussed.
Teleost gastrulation has been studied for a long time, albeit with rather controversial results (Oppenheimer, 1936; Pasteeis, 1936; Hisaoka and Battle, 1958; Hisaoka and Firlit, 1960; Hamano, 1964; Ballard, 1973 a,b,c; Sander et al., 1984). The separation of mesoderm and endoderm and the cell movements involved in the further development of these layers in teleosts have been described in salmonids, but conflicting conclusions were drawn (Pasteels, 1936; Ballard, 1973b). In the zebrafish, the cells of the future inner germ layers derive from cells involuting along the blastodisc rim (Kimmel, 1989), starting from about mid-epiboly. Their initial movements are incompletely known, but while Hamano (1960) described a strong convergence movement in the germ ring towards the future body axis, Sander et al. (1984) observed only cell movements perpendicular to the blastodisc rim, i.e. towards the animal pole rather than the future body axis (*). The latter authors at first drew the conclusion that these cells move directly underneath the enveloping layer which covers the blastodisc surface (Sander et al., 1984). A later study using radial instead of tangential focal planes (K. Sander and H. Vollmar, pers.comm.) and my SEM-studies showed however that these cells move between the yolk surface and the epiblast as has been described for the rosy barb (Wood and Timmermanns, 1988). In the present report I reconstruct from SEM-preparations the movements of these cells from their segregation at the germ disc margin until the time when the notochord anlage becomes visible.
Alcohol is recognized as a classic teratogen capable of inducing a wide range of developmental abnormalities. Alcohol abuse during pregnancy produces permanent brain damage in the fetus and is associated with the development of a life-long behavioral, social, and cognitive disorder now known as fetal alcohol spectrum disorder (FASD). The most clinically recognizable form of FASD is fetal alcohol syndrome (FAS) which is characterized by a set of characteristics including facial dysmorphogenesis, mental dysfunction, growth retardation, and cardiovascular and limb defects. Due to ethical constraints, human studies of FASD are very limited; however, our current understanding of FASD is mainly based on studies on several model vertebrate and invertebrate organisms. The fish embryo, especially zebrafish (. Danio rerio) and Japanese medaka (. Oryzias latipes), have proven utility for studying ethanol's damaging effects during morphogenesis. These two fish are long-established models for research in developmental biology and have been used to explore ethanol's effect on neurogenesis, cardiogenesis, intracellular signaling, neurobehavioral aspects, and apoptosis. Zebrafish are the center of attraction as a fish model of FASD; however, we have demonstrated that medaka embryos exposed to ethanol during development have several phenotypic features in craniofacial, cardiovascular structures and many biochemical parameters which are comparable to FASD phenotypes observed in human. In this chapter, we reviewed our findings and propose that medaka embryogenesis, like zebrafish, may be a very useful model for investigating the molecular endpoints of FASD.
1.1. Changes in tolerance to low temperature during embryonic development were investigated in red sea bream, olive flounder and multicolorfin rainbowfish.2.2. The tolerance decreased at cleavage stages, early gastrula stage, the stage of embryo appearance and the stage of blastopore closure in each species.3.3. Exposure to low temperature at cleavage stages appears to obstruct mitotic division.4.4. Cooling at the early gastrula stage caused delay in epiboly of periblast.5.5. The embryos of eggs that died as a result of cooling at the stage of embryo appearance were torn physically at the margin between the germ ring or embryo shield and the other part of embryo.6.6. In morphological observations, no abnormality could be found in the embryos exposed to low temperature at the stage of blastopore closure.
“Entwickelungsmechanik, like every new approach in science, will have to acquire its deserved status step by step. Nonetheless, posterity might well be amazed on learning that the now ruling descriptive school ignored the secure findings of Entwickelungsmechanik for a long time, until descriptive research had led to the same views, and especially that this school placed more confidence in the latter kind of findings than in the former. This will bear permanent testimony to the insufficient appreciation that those researchers had for the value of experiments.” This was Wilhelm Roux’s verdict at the end of a discussion of gastrulation and the fate map in frogs (Roux 1892, p. 441). In the preceding paragraph he had summarized his conclusions on these topics, and at the same time paved the way for the verbal showdown just quoted: “The conclusion, based on three different types of experiment, that gastrulation in the frog occurs by way of bilateral epiboly and concrescence on the lower side of the egg and that the outer face of the blastoporal lip is the site of the neural folds, is therefore so secure that its certainty could neither be increased by the consent of descriptive researchers, which is per se pleasing, nor could it be diminished by their opposition; the facts thus established must rather be viewed as the firm foundations of our knowledge of developmental processes, meaning at the same time that all views clearly incompatible with these facts can for sure be called mistaken” (Roux 1892, p. 440/41).
Objectives:
The aim of this study was to determine the effects of 96 hour exposure to selected solvents on the embryonic stages of zebrafish (Danio rerio). We investigated mortality and various types of changes which appeared (oedema, tail and eye defects, weak pigmentation, and deformation of the body). Based on the results, values of NOEC and LOEC for embryos of D. rerio were determined.
Methods:
Embryonal toxicity tests were conducted according to OECD guideline 212. Ethanol and methanol were tested at concentrations of 0.1, 0.5, 1, 1.5, 2%; acetone at concentrations of 0.1, 0.5, 1, 1.25, 1.5%; and dimethylsulfoxide at concentrations of 1, 1.5, 2, 2.5, 3%.
Results:
The LOEC values of ethanol and methanol were detected in the 1% concentration. Statistically significant changes (oedema) were reported in 1% ethanol, and oedema, weak pigmentation and deformation of the body were observed in 1% methanol. After exposure to acetone, the most common occurrence of oedema was in the 0.5% concentration (LOEC = 0.5%). The solvent dimethylsulfoxide caused oedema and body deformation at the 2% concentration (LOEC = 2%).
Conclusions:
The NOEC concentrations of the individual solvents were as follows: ethanol and methanol, 0.5%; acetone, 0.1%; and dimethylsulfoxide, 1.5%. These concentrations of individual solvents were higher than the maximum recommended concentration for toxicity tests on fish. For this reason, it can be assumed that the concentration of solvent allowed by the norm does not affect the procedure or results of such tests.
There are more than 100 different biophenols reported in olive samples. This chapter covers the chemistry, pharmacodynamics, pharmacokinetics, posology, adverse effects, and potential drug interactions of olives and major olive biophenols (OBP). Major biophenols detected in olive samples include hydroxytyrosol, tyrosol and their secoiridoid derivatives (oleuropein, oleuropein aglycone, and elenolic acid dialdehydes), verbascoside, lignans, and flavonoids. By far the majority of reports on the chemistry of OBP pertain to their ability to function as antioxidants, but other bioactivities include binding to lipids, proteins, carbohydrates, and nucleic acids. The majority of pharmacological
studies have focused on just four compounds hydroxytyrosol, tyrosol, oleuropein, and verbascoside. Reported pharmacological properties include antioxidant, anti-inflammatory, cardiovascular, immunomodulatory, gastrointestinal, respiratory, autonomic, central nervous system, antimicrobial, anticancer and chemopreventive. While OBP are generally regarded as safe, further studies on potential adverse reactions may be required to demonstrate the safety of supplements with elevated levels of compounds.
This chapter explains the utility of zebrafish as a model for toxicological research. Zebrafish (Danio rerio) have long been the genetic model of choice for vertebrate developmental biologists, as it provides several advantages for investigating organ and tissue development. Zebrafish have become a powerful model organism for investigating the molecular and cellular mechanisms by which environmental chemicals disrupt normal developmental processes. The utility of zebrafish as a laboratory model organism also makes it an excellent system for studying processes in juvenile and adult organisms, including reproductive development, carcinogenesis, aging, and the influence of environmental chemicals on these processes. The advent of modern genetic tools and genome sequencing projects has elevated zebrafish as a suitable model to effectively study human disease and pathophysiology, ushering in a new era of comparative biology and medicine. The chapter reviews the mechanistic toxicology and advantages of the zebrafish model system.
Early embryonic development is dependent on proteins translated from messenger ribonucleic acid (mRNA) molecules of maternal origin and the embryonic genome. The present study examined the importance of maternal mRNA during early embryogenesis in the rainbow trout (Oncorhynchus mykiss), and developmentally, when the embryonic genome becomes transcriptionally active. To address the significance of maternal mRNA, eggs were exposed to the translational inhibitor cycloheximide (CHX) for 12h post-fertilization. Concentrations of CHX beginning at 1M significantly delayed the initial stages of cleavage. Exposed embryos had only reached the 4-cell stage, while a significant proportion of the eggs treated with 0.1M CHX or no CHX (i.e., control) had attained the 8-cell stage. Treatment of fertilized eggs with the transcriptional inhibitor actinomycin D (AMD) was utilized to demonstrate when synthesis of embryonic mRNA begins. Rainbow trout eggs exposed to 16M AMD were unaffected after 12h, but by 9days post-fertilization had not developed the embryonic keel characteristic of the controls. To determine when transcriptional inhibition could first be demonstrated AMD exposed embryos were dissected free of the egg, between days 2 and 6 of incubation, and individual blastomeres prepared for diameter measurements. After 3days of exposure to 16M AMD embryonic blastomeres were significantly larger than the control group (P
Carp embryos were dechorionated and their early development was studied in the presence or absence of a-amanitin. Cleavage and the formation of the enveloping layer and yolk syncytial layer were not influenced by the drug. However, a-amanitin largely blocked epiboly which started 6 h after fertilization in controls. Involution of deep cells, taking place during gastrulation movements, appeared to be blocked to a lesser degree. This might reflect differences in the degree to which maternal transcripts influence these developmental steps.
The eggs of African mouth-brooders are of unusual size and shape. Studying their development may help to more clearly understand epiboly, gastrulation, and the relation between enveloping layer (periderm) and epidermis. When epiboly has progressed over just one fifth of the yolk mass, the germ ring and embryonic shield are already well established. Behind the germ ring very few deep cells are present at this early stage of epiboly, except in the embryonic shield. When the blastodisc covers the animal half of the yolk mass, the future body is already well established with notochord, somites and developing neural keel. Apart from these structures, no deep cells can be detected between enveloping layer and yolk surface; not even a germ ring remains behind the advancing edge of the enveloping layer. Epiboly over the greater part of the yolk is achieved only by the enveloping layer and the yolk syncytial layer. As the margin of the enveloping layer begins to reduce its circumference when closing around the vegetal pole, groups of cells in the advancing edge become spindle-shaped, with a single cell in between of each of these groups broadening along the edge. The enveloping layer (called periderm after epiboly) remains intact until after hatching, when, together with the underlying ectoderm, it forms the double-layered skin of the larval fish. Thereafter, cells deriving from the subperipheral ectoderm gradually replace the decaying periderm cells to form the final epidermis. Thus, in the cichlids studied, the enveloping layer alone forms the yolk sac to begin with, and it covers the larval body until some days after hatching.
The present study compares two possible alternative methods to replace the acute fish test. Fertilized eggs of zebrafish, Brachydanio rerio were used to investigate the acute toxicity of chemicals. Different toxicological endpoints such as coagulation of the eggs, development of gastrulation, number of somites, development of organs, circulation, heartbeat, otolithanlage and pigmentation have been examined during the embryonic development of zebrafish within the first 48 h. Differences to the normal development were measured by these endpoints. Chemicals have been examined in a preliminary approach with respect to their cytotoxicity on the RTG-2 cells (Rainbow Trout Gonad). The vitality of cells was measured by Neutral Red-and MTT assay.Urea, sodium chloride, 2-nitroanisole, 3,4-dichloroaniline, 4-nitrophenol, phenol, 2,4-dinitrophenol, dinitro-ortho-cresole, carbaryl and malathion were used as compounds with different modes of actions. The results of the cytotoxicity tests with Neutral Red uptake (NR50) and MTT (MTT50) have been compared with data of zebrafish, Brachydanio rerio. We could show that the embryo test in most cases is more sensitive than the acute toxicity test with adult zebrafish and in all cases is more sensitive than the RTG-2 cell test. Therefore, considering the wide range of possibilities the embryo of zebrafish offers, we think that the embryo test is a very good candidate to replace the acute fish test. Whether the cytotoxicity test with RTG-2 cells is also an alternative requires further, more detailed studies.
SP600125 (anthrapyrazolone) is a synthetic polyaromatic chemical that inhibits c-Jun N-terminal kinase (JNK) signaling by interfering with phosphorylation of c-Jun. To determine the pharmacological impact of SP600125 on zebrafish development, we incubated embryos in various concentrations of SP600125 from 18 h postfertilization (hpf) to 48 hpf. Embryos treated with 1.25 µm appeared with occasional pericardium edema. Treatment with 12.5 µm resulted in complete mortality by 120 hpf, preventing an assessment of physiological defects. Embryos treated with 5 µm exhibited slowed overall growth, a delay in hatching and numerous morphological defects such as pericardium edema, yolk sac edema, swim bladder deflation, bent vertebrae and eye and jaw malformations. Whole-mount immunohistochemical studies using an anti-acetylated β-tubulin antibody confirmed developmental defects in the nervous system. Within the retina, fish treated with 1.25 µm showed a mild reduction of immunoreactivity. Immunoreactivity in the retina was further reduced in fish treated with 5 µm of SP600125. In these fish, eyes and olfactory organs were half the size compared with other groups. Multiple lenses were observed in 67% of these fish. A second experiment with a shorter exposure period of SP600125 (6 h) presented significantly fewer morphological defects. The treatment led to a delay in hatching, and increased incidences of swim bladder deflation and pericardium edema with increasing concentrations. In summary, SP600125 caused developmental abnormalities during zebrafish organogenesis starting at 1.25 µm and the defects were exacerbated with increasing concentrations. Our study suggests that SP600125 at 1.25 µm and beyond has devastating consequences for zebrafish development. Copyright © 2011 John Wiley & Sons, Ltd.
The early development of the zebrafish Brachydanio rerio is being investigated as a possible test system for prescreening drugs suspected to be hazardous to humans. Experiments have been carried out to evaluate the teratogenic effect of the human antiepileptic drug valproic acid (VPA) and several chemically related substances. The experiments show the highly synchronous development of zebrafish embryos, even of batches laid by different parents. Effects due to drug treatment can be easily and accurately determined. VPA causes retardation and cessation of development. It generates malformations such as oedema, brain deformities, a shortened and bent tail, and bipartite axiation of the posterior trunk. The effects of the various substances tested were estimated and compared by measuring retardation of development. The related substances display similar effects, but differ in their effectiveness as they do in other test systems: 2-en-valproic acid and 4-en-valproic acid display weaker effects than VPA and propylhexanoic acid. Valpromide, methylhexanoic acid, pentenoic acid and diethylacetic acid display a weak effect or no effect at all. The effects in zebrafish appear to be more similar to those observed in hydroids and in mammalian whole embryo culture systems than to the effects in mammals in vivo. Differences may be best explained by differences in uptake and degradation of the drugs, and by the influence of the maternal organism in mammals.
The response of zebrafish (Brachydanio rerio) embryos, cultured in reconstituted water, to continuous treatment with N,N-dimethylformamide (DMF) or its degradation products until hatching, was investigated. The embryos were distinctly susceptible to DMF, N-(hydroxymethyl)-N-methylformamide (DMF-OH), N-methylformamide (NMF), N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), and formamide (FA). Concentrations of 18 (DMF-OH), 121 (DMF), 163 (AMCC), 186 (NMF) and 203 (FA) mmol/litre produced 50% embryo lethality (LC(50)). Only treatment with DMF, NMF or FA induced malformations, involving curvature of the spine and tail and reduction of heart size and action with decreased blood circulation.
The early development of the zebrafish Brachydanio rerio is shown to be suitable as a test system for prescreening drugs suspected to be hazardous to humans. The teratogenic effects of all-trans-retinoic acid (tretinoin) and eight chemically related substances, such as 3,4-didehydro-retinol (vitamin A(2)), 4-oxo-retinoic acid and several cis isomers, are analysed. Zebrafish development offers several advantages as a test system: large amounts of eggs can be collected throughout the year. Because the embryo is translucent, organ development can be monitored in vivo. After 1 day, eyes, heart and blood circulation is visible already, and the development is synchronous up to day 3 after fertilization. The effects of the retinoids can be scored easily, and the substances can be ranked and be compared by their effectiveness. Further, a new scoring method is presented which also allows comparison of the relative strength of the teratogenic effects of the substances on the various organs enabling investigation of the structure-activity relationship. Concentrations of 10(-19)-10(-6)M all-trans-retinoic acid generate malformations such as oedema, brain deformities (anophthalmy, microcephaly and acephaly), duplication of the otic placodes and otoliths, and a shortened and bent tail. The related substances display similar effects but they differ in their effectiveness. With respect to the chemical structure, the order of potency is found to be: acid > aldehyde > alcohol; all-trans 9-cis > 13-cis; tretinoin = 3,4-didehydro > 4-oxo. The same order of potency is reported for mammals. Because of this similarity early zebrafish development appears to be a proper model system for predicting teratogenic effects of drugs in mammals, especially of chemicals and their metabolites, which are able to cross the placental barrier and reach the mammalian embryo in utero.
Obwohl fetale Mißbildungen seit langem bekannt sind, wird eine intensive Prüfung von Arzneimitteln und anderen Substanzen erst seit den 1960er Jahren durchgeführt. Dem Tierschutzbericht von 2001 ist zu entnehmen, daß im Jahr 1999 insgesamt etwa 1,6 Millionen Wirbeltiere zu Versuchszwecken benötigt wurden. Als mögliche Alternative zu Untersuchungen mit Säugetieren wurde ein Testmodell mit Embryonen des Zebrabärblings (D. rerio) entwickelt. Ziel der vorliegenden Arbeit ist es zu klären, ob sich mit den Embryonen von Danio rerio ein teratogenes Potential von Substanzen erkennen und quantifizieren läßt. Dazu wurde DarT (?Danio rerio Teratogenicity Assay?) als Teratogenitäts-Screening Test entwickelt. Es können anhand von toxikologischen Endpunkten sowohl die letalen als auch die subletalen Wirkungen von Substanzen bestimmt werden. Darüber hinaus werden anhand von teratogenen Endpunkten speziell Malformationen erfaßt. Der Vergleich der beobachteten Effekte und der daraus berechneten Wirkkonzentrationen gestattet eine Einschätzung des teratogenen Potentials von Substanzen. Die im DarT erzielten Ergebnisse werden mit der bekannten Zuordnungen des ?säugerteratogenen? Potentials verglichen. Für 88 % der getesteten Substanzen gibt DarT die aus säugertoxikologischen Untersuchungen bekannten Einordnungen hinsichtlich des teratogenen Potentials wieder. Für 10 % der Testsubstanzen wurde das teratogene Potential zu hoch, für 2 % zu niedrig eingeschätzt. Mit dem Testsystem ?Danio rerio Teratogenicity Assay ? DarT? ist ein Vergleich von Substanzen hinsichtlich ihres teratogenen und allgemein toxischen Potentials möglich. In einem Modell können Wirkkonzentrationen und Konzentrations-Wirkungs-Beziehung ermittelt und direkt verglichen werden. Mit DarT kann eine große Anzahl von Substanzen zeit- und kostengünstig untersucht werden. Die aus Untersuchungen mit Säugetieren bekannten Zuordnungen der teratogenen Potentiale von Substanzen wird gut wiedergegeben.
Pollutants, by disrupting metabolic processes, can interfere with development, and, at critical periods of development, can act as teratogens. Such interference with normal development can be used as a bioassay. Some screening tests are based on this phenomenon. As teratogens, pollutants are fairly nonspecific. Many different classes may elicit the same developmental responses. Mechanisms of teratogenicity include disruption of mitosis, interference with transcription and translation, metabolic disturbances in energy utilization, and nutritional deficits. These in turn interfere with cell interactions, migration, and growth. In aquatic organisms, environmental conditions can be critical. Interactions of pollutant effects with salinity and with temperature have been reported. Interactions between toxicants have also been studied; both synergism and antagonism have been reported. Most reports of teratogenesis have been qualitative. Quantitation has usually been in the form of percentages of embryos affected, but when severity of effect is indexed, more critical analysis is allowed. When effects of other developmental processes such as growth are analyzed, quantitation is readily achieved. Regeneration is an especially useful model of both differentiation and growth. These two components of regeneration can be separately analyzed. Dose-response relationships are readily apparent. In comparison to mammalian embryos, the use of embryos of many aquatic species for testing toxicants has certain advantages, including lower cost and maintenance and shorter development times. They respond to many of the same teratogens. A special advantage is availability for continual examination during development so that abnormalities can be observed and recorded as they arise.
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Members of the Wnt family have been implicated in a variety of developmental processes including axis formation, patterning of the central nervous system and tissue morphogenesis. Recent studies have shown that a Wnt signalling pathway similar to that involved in the establishment of planar cell polarity in Drosophila regulates convergent extension movements during zebrafish and Xenopus gastrulation. This finding provides a good starting point to dissect the complex cell biology and genetic regulation of vertebrate gastrulation movements.
The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1-2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.
Developmental ethanol exposure from maternal consumption of alcoholic beverages and many other consumer products has been linked to developmental abnormalities in humans and animal models. The sensitivity of an individual to ethanol-induced perturbation of developmental processes is strongly influenced by genetic factors. In this study, we show that there are strain- and dose-dependent differences in sensitivity to developmental ethanol exposure in zebrafish (Danio rerio), suggesting that genetic variation within regulatory factors, influencing critical developmental pathways, is responsible for these differences. Embryos/larvae from genetically distinct strains of zebrafish [Ekkwill (EK), AB, and Tuebingen (TU)] were treated with different concentrations of ethanol. Embryo/larval survival, neurocranial and craniofacial skeletal development, and CNS cell death were analyzed. EK was the most resistant strain to the embryolethal effects of ethanol exposure but had the greatest increase in ethanol-induced cell death. AB survival was affected moderately, as were the neurocranial and craniofacial skeletal structures and ethanol-induced cell death. TU had the lowest survival rate but was the most resistant to alterations in neurocranial and craniofacial skeletal elements. No single strain is the most sensitive or the most resistant to any of the phenotypes examined, suggesting that alcohol influences each of these pathways independently. Further analysis of the molecular and biochemical pathways underlying the strain-dependent differences reported herein could lead to a significant advancement in our mechanistic understanding of the teratogenic effects of ethanol in humans.
Prenatal exposure to alcohol has been shown to produce the overt physical and behavioral symptoms known as fetal alcohol syndrome (FAS) in humans. Also, it is believed that low concentrations and/or short durations of alcohol exposure can produce more subtle effects. The purpose of this study was to investigate the effects of embryonic ethanol exposure on the zebrafish (Danio rerio) in order to determine whether this species is a viable animal model for studying FAS. Fertilized embryos were reared in varying concentrations of ethanol (1.5% and 2.9%) and exposure times (e.g., 0-8, 6-24, 12-24, and 48-72 h postfertilization; hpf); anatomical measures including eye diameter and heart rate were compared across groups. Results found that at the highest concentration of ethanol (2.9%), there were more abnormal physical distortions and significantly higher mortality rates than any other group. Embryos exposed to ethanol for a shorter duration period (0-8 hpf) at a concentration of 1.5% exhibited more subtle effects such as significantly smaller eye diameter and lower heart rate than controls. These results indicate that embryonic alcohol exposure affects external and internal physical development and that the severity of these effects is a function of both the amount of ethanol and the timing of ethanol exposure. Thus, the zebrafish represents a useful model for examining basic questions about the effects of embryonic exposure to ethanol on development.
The effects of acute and chronic ethanol administration on the wild-type (WT), long-fin striped (LFS), and blue long-fin (BLF) strains of zebrafish were investigated. In the LFS strain, acute exposure to 0.25% (v/v) ethanol inhibited the startle reaction and increased both the area occupied by a group of subjects and the average distance between each fish and its nearest neighbor. Similar effects were found in the WT fish although higher concentrations of ethanol were required. No effects on the behavior of the BLF fish were observed with up to 1.0% (v/v) ethanol. Brain alcohol levels were comparable among the three strains precluding a pharmacokinetic explanation for the behavioral results. In LFS zebrafish, behavioral tolerance was observed after 1 week of continual exposure to ethanol. Conversely, chronic ethanol exposure of the WT fish for up to 2 weeks did not result in the development of tolerance, but rather appeared to increase the disruptive action of the drug. The present results suggest the observed strain differences in the effects of ethanol reflect genotypic differences in both the response of the central nervous system (CNS) to ethanol as well as the ability of the CNS to adapt to ethanol exposure. Although preliminary, the present study indicates that the zebrafish is an excellent model system to investigate the genetic determinants involved in regulating the responses to ethanol.
Vitellogenins (Vtgs) are the major yolk proteins in all oviparous animals. Systematic and regulated processing of these during embryogenesis is crucial for embryonic development. In the present study, toxicant-induced disturbance of Vtg degradation processes during Danio rerio (DR) embryogenesis was analysed to establish a sensitive tool for monitoring toxic stress at the molecular level. A 2-DE-based proteomic approach for whole DR embryos was established to study Vtg cleavage products (lipovitellin (Lv) derivatives). Ethanol was chosen as a positive control for a toxicity related change in the proteome of whole zebra fish embryos. Protein extracts from embryos treated with two ethanol concentrations, 0.5 and 2% v/v, showing either no or very strong visible effects, like absent heartbeat and blood circulation, were examined. Significant changes in the Lv pattern were detected for both conditions. The results are interpreted as scope for the use of the high abundant Lv derivatives as sensitive stress indicators in zebra fish embryos reflecting the overall fitness of the intact organisms.
An attempt was undertaken to develop a model system based on artificial cell cycle synchronization by means of reversible mitosis blocking in zebrafish embryos for studying the role of cell cycle synchrony in embryogenesis. Dechorionized and intact embryos at the stages of 512-cell blastula and 75% epiboly were treated with nocodazole and then washed within several times of exposure. When working on dechorionized embryos, we succeeded to obtain complete block mitosis in the presence of low nocodazole concentrations: 0.5-1.0 microg/ml. Block of mitosis was relieved in all experimental series within a certain time after the beginning of washing. This inertia depended on both nocodazole concentration and duration of treatment. The nocodazole elimination was significantly accelerated only after five (or more) changes of washing medium containing DMSO. As a result, the conditions were established for obtaining a parasynchronous cell population in the zebrafish gastrulas with a peak of mitosis up to 17.2%.
It is largely accepted that vertebrates are more susceptible to chemical insult during the early life stage. It is implied that if a chemical such as ethanol is developmentally toxic, it must interfere with, or modulate, critical signaling pathways. The probable molecular explanation for increased embryonic susceptibility is that collectively there is no other period of an animal's lifespan when the full repertoire of molecular signaling is active. Understanding the mechanism by which ethanol exposure disrupts vertebrate embryonic development is enormously challenging; it requires a thorough understanding of the normal molecular program to understand how transient ethanol exposure disrupts signaling and results in detrimental long-lasting effects. During the past several years, investigators have recognized the advantages of the zebrafish model to discover the signaling events that choreograph embryonic development. External development coupled with the numerous molecular and genetic methods make this model a valuable tool to unravel the mechanisms by which ethanol disrupts embryonic development. In this chapter we describe procedures used to evaluate and define the morphological, cellular and molecular responses to ethanol in zebrafish.
Two distinct patterns of chimaerism were found in conceptuses produced by injecting dissociated 4.5-day inner cell mass cells into genetically dissimilar blastocysts. Pattern 1: donor cells were found in the endoderm layer of the visceral yolk sac, but not in the adjacent mesoderm layer of this organ or in the foetus itself. Pattern 2: donor cells were found in the mesoderm layer of the visceral yolk sac and/or foetus, but never in the yolk-sac endoderm as well. Primitive endoderm cells of donor inner cell masses are responsible for the first pattern and primitive ectoderm cells for the second. These results, together with those of previous studies, suggest that the entire foetus, including its endodermal components, is formed from the primitive ectoderm, and that primitive endoderm forms only extra-embryonic endoderm of the conceptus.
Conclusions about the fate maps and morphogenetic movements of vertebrate embryos seem to be unduly influenced by antique homologic theory. Diversities within the phylum, greater than usually admitted, could extend to the behavior of individual moving cells and the factors generating and controlling that movement.
Chalk granules implanted among the cells of the stage 7 blasto-disc are carried along with the cells to final location in the differentiating tissues and organs. The results, more often than not, are inconsistent with predictions suggested by an earlier fate map for Salmo (Pasteels, (36). Granules vertically thrust into a given spot on the blastodisc are usually carried to various levels of the trunk and to a variety of organs, presumably because cells at different depths of one spot and at different distances from the center are moving at different rates. Data from hundreds of such implants are assembled into charts showing the areas from which cells are drawn, to contribute to formation of the endoderm, notochord, five separate zones of the central nervous system, the head mesoderm, anterior and posterior trunk somites, tail somites, lateral plate, and heart. Using evidence of the overlapping and intergradation of these areas, as well as evidence from vital dyes when allowed to penetrate to various depths of the pregastrular blastodisc, a three-dimensional fate map is synthesized for Salmo gairdneri, which is very different from the earlier map, but consistent with the previously reported patterns of morphogenetic movements in this species (Ballard, (73b). In contrast with the gastrulation of amphibia and birds, Salmo germ layers arise without any invagination, and there is at no time an anterior mesoderm-free area. Previous efforts to generalize upon vertebrate gastrulation and its evolution within the phylum seem to have been premature.
Chick tail buds labeled with tritiated thymidine were transplanted homotopically to unlabeled embryos to determine the specific body levels and structures formed by tail buds. In addition, labeling patterns of posterior structures were studied in normal embryos labeled for progressively longer time periods.
Tail buds contributed labeled cells primarily to tail neural tube, somites, mesenchyme, and caudal arteries, but not to tail notochord, skin ectoderm, hindgut, or tail gut. Labeled somites were often located three to four somite levels anterior to labeled portions of neural tubes. In labeled normal embryos central notochordal cells were labeled only occasionally. This labeling pattern extended posteriorly into a region designated as prospective notochord, which merged posteriorly with the tail bud. The tail bud labeled uniformly and contained about the same density of labeled cells as the segmental plates, indicating that proliferation was not excessive in the former.
Defects in operated embryos occurred primarily in the neural tube, somites, and notochord. The tail portion of the notochord, presumably formed from the prospective notochordal region, was never isolated from the trunk portion. The tail portion of the neural tube rarely seemed isolated from the trunk portion. Large, unpaired midline somites frequently developed. They appeared to form only when a space was available in which paired segmental plates could approach the midline and fuse.
Amphibian (Triturus alpestris) ectoderm was isolated and after 2–16 days in culture examined by electron microscopy. It has been shown that the ectoderm, formerly called “undifferentiated ectoderm”, forms in part ciliated epithelial cells, which cannot be distiguished from the cells of the epidermis, which has developed in situ (except for the alignment of the cells which depends on the mesenchyme underlying the epidermis). The ultrastructure of the cilia is similar to that of cilia of protozoa or sperms of insects or vertebrates. A zone at the periphery of the epidermal cells, free of yolk platelets, mitochondria and pigment granules (embryonic pigment) is observed after 4 days. This area is rich in vacuolous structures and basal bodies of the cilia.
Ectoderm, which was treated with the vegetalizing factor differentiates into mesodermal and endodermal tissues. Cilia, as well as the characteristic peripheral zone, are not formed in the induced ectoderm.
In ectoderm treated with actinomycin D (1 μg/ml for 6 h) the differentiation of the peripheral zone and the cilia is delayed.
Before examination of biological specimens can take place all volatiles must be removed. This presented paper details a method of sample preparation that reduces distortion and maintains 3-dimensional structure. Freeze drying disrupts cell structures as phase boundaries move through the specimen twice, first a solid boundary in the freezing process then a solid-vapor boundary during sublimation. To eliminate this the temperature of the ambient liquid is raised above its critical point. When it reaches the region where two phases cannot exist surface tension vanishes and fluid can escape without disrupting structures. Specimen preparation is given. Place droplet on a formvar-coated screen and fix with osmic acid vapor. Replace water with alcohol and alcohol with amyl acetate. Place specimen in a bomb and flood this with liquid carbon dioxide to replace the amyl acetate. Raise the temperature in the bomb to 35 Centigrade. Previous determinations show this to be above the critical point for carbon dioxide. Stereoscopic views of electron micrographs show three prepared specimens. There remains some minor distortion and flattening against the formvar screen. Outlines a method to retain cell structure in samples destined for the scanning electron microscope.
Aus: Biologisches Zentralblatt. Bd 84. 1964, H. 2 Köln, Math.-naturwiss. F., Diss. v. 26. Febr. 1964 (Nur in beschr. Anz. f. d. Aust.).
Aus: Wilh. Roux' Archiv f. Entwicklungsmechanik d. Organismen. Bd 134, H. 1. Zürich, Med. Hab.-Schr.
Desmosomes (macula adherens) have been associated with the function of adhesion. Their possible role in aggregation and sorting of chick and mouse epithelial cells has been investigated. Treatment of aggregates with 2-5 microgram/ml of actinomycin D which inhibited RNA synthesis also inhibited both desmosome formation and aggregation if administered at the beginning of the aggregation process. In contrast, if the drug was administered at six hours, when the cells had recovered from the process of dissociation, then aggregation over the following six hours appeared normal from observation of living samples. Such aggregates incorporated leucine-3H at roughly 85% of the control level. A quantitative comparison was made of desmosome formation in aggregates treated with actinomycin D for hours 6-12 and those cultured in normal medium. Desmosome formation was inhibited by the drug, although aggregation could proceed. Combinations of chick corneal and mouse skin cells sorted out in the presence of actinomycin D to the same extent as controls. Thus desmosome formation, which normally occurs during aggregation of the epithelial cells studied here, is not coupled with the aggregation or cell sorting process in these cells of stratified epithelia. When cells were treated with cycloheximide (100 muM) both desmosome formation and the progressive rounding up of aggregates was inhibited.
The development of the olfactory organ in the rainbow fish, Nematocentris maccullochi, was studied using scanning and transmission electron microscopy; it was compared with the developmental process in other teleosts, especially in the closely related atherinids and cyprinodonts. The formation of the nares parallels that in atherinids, salmonids, cyprinids and heterosomats, but differs from that found in cyprinodonts. Another ontogenetic feature in which the olfactory organs of the rainbow fish and also of atherinids differ from those of cyprinodonts, is the occurrence of transitory kinociliary cells which disappear during the postlarval period. The divergent evolutionary pathways are discussed with reference to experimental investigations. During development, ciliated and microvillous receptor cell types occur. At the primary larval stage ciliated receptor neurons are exclusively present. At a later stage the microvillous type develops and becomes equal in frequency. Thus, the microvillous receptor represents a separate type of olfactory neuron and is not a progenitor of the ciliated receptor cell.
In cleavage mitoses colchicine (2 x 10(-3) M) does not interrupt the cycle of chromatin and its action is exerted on all the mitotic phases. Cycloheximide (10(-3) M) prevent normal condensation of the chromosomes and breakdown of the nuclear envelope; it brings on various nuclear abnormalities, particularly irregularities in chromosome condensation. At 10(-4) M, cycloheximide allows some mitoses, often abnormal ones, but anaphase is inhibited. The action of colchicine, more efficient upon the spindle, obscures the action of cycloheximide when both substances are associated. In return, cycloheximide imposes its action upon chromosome condensation, so that the structure of the nuclei treated by both substances is determined by cycloheximide.
Surface contour, contact relations, and cortical structure of marginal cells of the enveloping layer (EVL) and of the yolk syncytial layer (YSL) of Fundulus heteroclitus were studied before and during epiboly with transmission and scanning electron microscopy. The contacts of the marginal cells of the EVL with the underlying YSL involve only the most marginal part of each cell and consist of a mixture of tight and close junctions apically and wider appositions more proximally. This junctional complex is very extensive (2.1–2.4 μm) prior to the onset of epiboly, very restricted (0.5–0.8 μm) during early epiboly up to a mid‐gastrula, more extensive again (0.8–1.0 μm) at late midgastrula, and still more extensive (2.0–2.3 μm) at late gastrula toward the end of epiboly. At this time, the margin of each marginal cell is embedded in the YSL. Several lines of evidence suggest that these marginal contacts are stable and therefore that epiboly of the EVL occurs passively, in response to pull exerted by the independently expanding YSL.
As the external YSL (E‐YSL) narrows during the earliest phase of epiboly, its surface becomes more and more convoluted. Since the network of 4–6 nm microfilaments in the cortical cytoplasm of the E‐YSL thickens with increasing convolution of its surface, it seems possible that a contractile force resides in the E‐YSL cortex which simultaneously throws the surface of the E‐YSL into folds, narrows the E‐YSL, and exerts tension on the attached margin of the EVL. Networks of thin microfilaments are also found in the cortex of EVL cells, especially in the leading edge of the marginal cells (as in fibroblasts and epithelial cells in vitro) and in the cortex of the yolk cytoplasmic layer (YCL), where they are presumably responsible for the contractile tension of this layer; 10‐nm microfilaments are also present but have a different distribution. They are arranged in bundles in both the marginal cytoplasm of each marginal cell and in the YSL beneath the marginal contact, running parallel to the contact and circumferentially relative to the whole egg. This arrangement in this location coincides with constriction of the egg in this marginal region and suggests that these thick filaments might provide the contractile force for the constriction, along with the thin filaments with which they are associated. The morphological relationship between the E‐YSL and the YCL during epiboly is also described.
SEM reveals that the surface of the internal YSL (I‐YSL) is covered with long microvilli at the beginning of gastrulation and that they disappear and are replaced by shorter microvilli as epiboly progresses. Estimation of the amount of surface in the long microvilli at the beginning of epiboly indicates that there is enough membrane on the surface of the I‐YSL at this time to account for epibolic expansion up to a late gastrula. Although it is not known how the surface membrane in these microvilli might be redistributed, the presence within them of abundant microfilaments that appear to insert in the plasma membrane suggests that this process might be accomplished by these presumed contractile elements.
Finally, measurements of cell surface expansion and calculations of cell number show that the number of cells in the EVL remains approximately constant during epiboly. Clearly, cell division is not a factor in the epibolic expansion of the enveloping layer. Instead, there is a marked thinning of individual cells.
Exposure to ethanol retards growth and differentiation in cultured rat embryos during organogenesis. The development of untreated embryos is indistinguishable from growth in utero. These data suggest that the hypoplastic features of children born to chronically alcoholic mothers are due, at least in part, to a direct action of ethanol, which causes reduced embryonic cellular proliferation early in gestation.
The origin of periderm and epidermis has been studied in trout embryos from stage 20 (2 days after fertilization at 10° C) to hatching (stage 410).
Between stages 20 and 50, the blastodisc consists of an inner mass of blastomeres covered by a superficial layer of closely packed blastomeres, the peripheral layer. This layer gives rise to both peripheral cells and to cells joining the inner mass of blastomeres.
Between stages 50 and 110, junctions differentiate between peripheral cells. This newly formed superficial epithelium, the enveloping layer, no longer gives rise to inward migrating cells.
From stage 110 on, a basement membrane differentiates beneath a one-cell thick subperipheral layer, which thus becomes the ectodermal basal layer, the prospective epidermal basal layer.
From these and ultrastructural observations, it is concluded that 1) the epidermis apparently originates, at least in part, from the peripheral cells, between stages 20 and 50; 2) the periderm assumes a protective function over the body of the embryo and also a secretory function over the yolk sac (probably producing the hatching enzymes); 3) the periderm, which is a temporary structure, appears to play the role of an embryonic membrane in teleosts.
The reversible inhibition of protein synthesis at the 75–95% level in the early zygote of Fundulus results in a specific series of developmental failures dependent upon the times of inhibitor pulse initiation. The severity of the morphogenetic failure is inversely related to the time of initiation and directly to the length of the pulse. The defects reflect the time dependent serial order of events in morphogenesis. The defects range from failure of cleavage through disorders of blastulation, failure of axiation, anencephaly to microcephaly and are entirely predictable. With the exception of cleavage failure the pattern is identical with that found using pulses of actinomycin D in a similar manner. The agent used was pactamycin, an antibiotic which reversibly inhibits amino acid incorporation into protein by disturbing the assembly of the functional ribosomal complex. The significance of time dependent protein synthesis as an active expression in morphogenesis of similarly time dependent information flow via RNA synthesis is discussed.
A comparative study of fixation techniques was conducted on cleavage stage amphibian embryos. Different combinations of primary fixatives, buffers and postfixation procedures were tested, as well as variations in the duration and temperature of fixation. Differences in preservation resulting from individual fixation mixtures and procedures were examined. A new procedure is described which provided a uniform, excellent preservation of early embryos. The initial fixative consisted of 3% glutaraldehyde, 2% formaldehyde, 1 % acrolein, and 2.5% dimethyl sulfoxide (DMSO), in an 0.1 M cacodylate buffer. Postfixation with buffered 2% osmium tetroxide in sucrose was carried out for an extended period of time, up to 24 hours, ensuring adequate membrane contrast throughout the embryo. Procedures for proper rinsing of fixatives, and for embedding of these embryos are also described. Results are also presented which indicate that the DMSO-trialdehyde combination followed by postosmication has application to other embryonic organisms in addition to amphibians.
Embryos of Brachydanio rerio, stage 10 (Hisaoka and Battle, '58) were exposed for 24 hours to graded concentrations of ethanol from 1 to 3%. Developmental abnormalities included defects of the eye, i.e., approximation, confluence and fusion of the optic cups mid-ventrally; as well as duplications of notochord and spinal cord. Duplication of the spinal cord in the hind brain region has not been reported previously. The duplicatory effects are discussed and a mechanism of duplication by infolding roof plate material is proposed.
Comparative studies of annual fish development reveal a unique developmental pattern in which embryogenesis occurs within a reaggregated mass of previously dispersed cells. The normal development of Austrofundulus myersi Dahl, a representative annual fish, has been divided into 46 stages. Cleavage during Stages 1–11 produces a typical teleost blastula. At Stage 12 (a flat, hollow blastula), the blastomeres segregate into two populations. One population composed of deep blastomeres will disperse as amoeboid cells, while the other, an hemispherical shell of outer blastomeres, flattens to form the enveloping cell layer. The cells of the enveloping cell layer become multinucleate and the layer functions as an extra-embryonic membrane which is shed at hatching. When epiboly commences (Stage 14), the deep blastomeres come together as a consolidated mass and then migrate outward as individual amoeboid cells into the space formed between the expanding enveloping cell layer and periblast. When epiboly is concluded (Stage 19), the deep blastomeres have completely dispersed over the surface of the periblast. The dispersed phase usually lasts for several days. Prior to epiboly and during the dispersed phase, no germ ring, embryonic shield, or axial organization is present. The dispersed cells come together to form a definitive aggregate (Stage 22) by day 4. Embryogenesis within the reaggregated mass of previously, dispersed cells produces a typical teleost embryo. The definitive embryonic axis and solid neural keel appear by Stage 28 (day 10). Growth and organogenesis proceeds and hatching occurs at Stage 44. Development through Stage 44 requires 39–40 days in non-diapausing eggs. Eggs of A. myersi may enter diapause at three distinct stages: (1) dispersed cell phase; (2) long somite embryo; and (3) late pre-hatching.
Fertilized eggs of the zebrafish, Brachydanio rerio, were subjected at 26 °C to three concentrations of chloramphenicol, 0.125, 0.250, and 0.500 mg/ml, at seven developmental stages ranging from cleavage to optic cup formation for periods of 12, 24, and 36 h. Subsequently, they were transferred to aquarial water to complete 48 h of development. Most extreme anomalies were induced by chloramphenicol in eggs initially subjected during cleavage and blastulation and less extreme anomalies in eggs exposed during or after gastrulation. In addition to general retardation of development, anomalies of the nervous, muscular, and vascular systems were induced. A unique type of spina bifida was of frequent occurrence. The teratogenic responses of the zebrafish egg are discussed in relation to the present concept of the mode of action of this antibiotic.
The effects of cyanide on the development of embryos of F. heteroclitus and A. maculatum were studied. Cyanide did not interfere with the development of the blastula but did prevent further embryogenesis. Inhibition of post-blastular development was reversible since upon removal of the inhibitor development proceeded normally. Utilization of oxygen was not required for maintenance of cells of the post-blastular embryo although it was required for further morphogenesis. Oxygen consumption by these embryos was arrested by cyanide and was resumed upon removal of the inhibitor. No accelerated rate of development was observed in those embryos released from cyanide inhibition. No oxygen debt was demonstrated upon removal of cyanide from these embryos. 2,4-Dinitrophenol was shown to have an effect similar to cyanide on the development of Fundulus embryos. Lactic acid accumulated in embryos incubated in cyanide. The extent of this accumulation was greatest during pregastrula development. Calculations demonstrated an increased rate of glycolysis during this period. The level of ATP was unaffected by cyanide during pregastrula development in these embryos and was diminished at subsequent states. The increased glycolytic rate was shown to be capable of sustaining a normal rate of ATP synthesis in the pregastrula.
Fertilized eggs and embryos of Fundulus heteroclitus reared in solutions of Actinomycin D for 24 hr exhibited stikingly different failures of morphogenesis dependent upon the time of initiation of the treatment. While morphogenesis was irreversibly inhibited cellular differentiation occurred. These two areas of development are therefore separable in time. The most severe effects, failure of gastrulation and axiation, occurred when the incubation period began immediately upon fertilization. Failure of cephalogenesis occurred when the incubation period began 1 hr after fertilization. Incubation of later onset (2 hr) led to microcephaly. Incubation beginning at blastulation had no effect on embryogenesis. Preincubation in cyanide until high blastula (stage 9) followed by incubation in Actinomycin D, led to failure of axiation similar in all details to the effect seen with incubation in Actinomycin D which included the first hour after fertilization. The Actinomycin D sensitive period of development therefore is dependent upon aerobic metabolism. A serial order of morphogenetic effects has been demonstrated by these inhibition studies: failure of gastrulation, axiation, cephalogenesis. Incubation of sperm in Actinomycin D prior to fertilization led to a severe mortality but no effect on morphogenesis in the survivors. Incubation of eggs, or eggs and sperm, led to failure of development past the blastula. The implications of these data on the synthesis of morphogenetically meaningful macromolecules is discussed. 27 references, 14 figures.
Fertile Xenopus eggs were irradiated with low doses of ultraviolet irradiation (254 nm) and permitted to develop through neural morphogenesis. They were then examined with the scanning electron microscope. The effects of irradiation on the development of the notochord, neural tube, and somites were observed. Embryos which displayed defects in notochord structure frequently exhibited normal development of both neural tube and somites. Those embryos were examined in detail. The apparent normal character of axial structures in “notochord-defective” embryos prompted a discussion of the role of the notochord in normal axial structure morphogenesis.
This paper is concerned with the influences which cause undifferentiated mesoderm to become converted into somites in the chick embryo. The experiments that are reported were designed to test several theories which already exist in the literature. Each of these theories ascribes an essential, inductive role to one of the following regions:
1. The ‘somite centres’. Spratt (1955) put forward the idea that two ‘somite centres’ exist in the chick blastoderm and that these actively induce the formation of somites. His evidence is based mainly on a series of transection experiments.
2. Hensen's node. This theory is based on the results of experiments in which the node is damaged or extirpated (e.g., Peebles, 1898; Wetzel, 1929; Fraser, 1954). The literature already contains evidence against this theory.
3. The neural tissue. This theory, which has been put forward by various authors (e.g. Grünwald, 1936; Fraser, 1960), is based on the fact that neural tissue and somites are usually closely associated.
1.1. The effects of actinomycin D (an inhibitor of DNA-primed RNA synthesis) and of puromycin (an inhibitor of protein synthesis) have been studied in two different biological systems: amphibian eggs and the unicellular alga Acetabularia mediterranea.2.2. Actinomycin D, even after microinjection, does not stop cleavage in amphibian eggs; it interferes with gastrulation and, especially, nervous system formation.3.3. Puromycin also inhibits nervous system differentiation in amphibian eggs.4.4. Anucleate fragments of Acetabularia, in contrast to nucleate ones, can form caps in the presence of actinomycin D. Later growth of these caps is, however, inhibited.5.5. Puromycin inhibits growth and morphogenesis in both nucleate and anucleate halves of Acetabularia; the effect is reversible in the former, but abnormalities of regeneration are frequent under these conditions.6.6. The results are discussed in the context of modern theories in molecular biology (intervention of messenger RNA's, formation of polysomes, possible role of cytoplasmic DNA, etc.).
Morphogenetic movements in gastrulation of Fundulus heteroclitus occur in a normal fashion in cases in which cytokinesis is prevented. In most cases the rate of occurrence of the process of gastrulation is uninterrupted by absence of cell division. Therefore, it is concluded thai in Fundulus cell division plays little, if any, role in the morphogenetic movements of gastrulation.
Recherches sur les monstruosités du brochet observées dans l'oeuf et sur leur mode de production
- Lereboullet A.
Lereboullet, A. (1863) Recherches sur les monstruositks du brochet observkes dans l'oeuf et sur leur mode de production. Ann. Sci. Natur. (Paris), Skrie 4, 20:177-271.
Develop-ment of the olfactory organ in the rainbow fish Nema-tocentris macullochi (Atheriniformes, Melanotaeni-idae)
- H Breucker
- E Zeiske
- R Melinkat
Breucker, H., E. Zeiske and R. Melinkat (1979) Develop-ment of the olfactory organ in the rainbow fish Nema-tocentris macullochi (Atheriniformes, Melanotaeni-idae). Cell Tissue Res., 20053-68.
Die Organisation der Hemididymi und Anadidymi der Knochenfische und ihre Bedeutung fur die Theorien uber Bildung und Wachstum des Kno-chenfischembryos Ethanol induced notochord and spinal chord duplications in the embryo of the zebrafish (Brachydanio rerio)
- F Kopsch
- H W Laale
Kopsch, F. (1899) Die Organisation der Hemididymi und Anadidymi der Knochenfische und ihre Bedeutung fur die Theorien uber Bildung und Wachstum des Kno-chenfischembryos. Internat. Monatssch. Anat. Phys., Laale, H.W. (1971) Ethanol induced notochord and spinal chord duplications in the embryo of the zebrafish (Brachydanio rerio). J. Exp. Zool., 177:51-64.
Asyntaxie blastoporale chez I'Am-phioxus. Mitt
- R Legros
Legros, R. (1907) Asyntaxie blastoporale chez I'Am-phioxus. Mitt. Zool. Stat. Neapel, 18:440-534.
Urmund und Spina bifida. Arch. Mik rosk
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Hertwig, 0. (1892) Urmund und Spina bifida. Arch. Mik rosk. Anat., 39:353-503.
Differentiation of the trunk mes-oderm in Amblystoma maculatum Terata mesodidyma von Salmo sal-uelinus nebst Bemerkungen uber einige andere a n Fischen beobachtete DoppelmiPbildungen. Sitzungs-ber
- W B Muchmore
Muchmore, W.B. (1951) Differentiation of the trunk mes-oderm in Amblystoma maculatum. J. Exp. Zool., Oellacher, J. (1873) Terata mesodidyma von Salmo sal-uelinus nebst Bemerkungen uber einige andere a n Fischen beobachtete DoppelmiPbildungen. Sitzungs-ber. Akad. Wiss. Wien, Math. Naturwiss. K1, 68:299-325.
Formbildung und Formstörung in der Entwicklung von Wirbeltieren
- Rauber A.
Rauber, A. (1880) Formbildung und Formstorung in der Entwicklung von Wirbeltieren. Morph. Jahrbuch, 6:129-184.
Chemisch ausgelöste Teratogenese beim ZebrabärblingBrachydanio rerio: Die getrennte Anlage der Körperhalften (Spina bifida) und ihre Be-gleitanomalien
- M Baumann
Baumann, M. (1982) Chemisch ausgeloste Teratogenese beim Zebrabarbling Brachydanio rerw Die getrennte Adage der Korperhalften (Spina bifida) und ihre Be-gleitanomalien. Ph.D. Thesis, University of Freiburg, Federal Republic of Germany.
The mechanism of amphib-118:137-186
- A M Schechtmann
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Praparationsmethoden fur Vital-beobachtungen an Mikroorganismen
- G Hertwig
- Jena Fischer
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