Microbead Implantation in the Zebrafish Embryo.

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

Dissection and downstream analysis of zebra finch embryos at early stages of development.

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology, behavior, and memory and learning. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access.

An avian model for the reversal of neurobehavioral teratogenicity with neural stem cells.

A fast and simple model which uses lower animals on the evolutionary scale is beneficial for developing procedures for the reversal of neurobehavioral teratogenicity with neural stem cells. Here, we established a procedure for the derivation of chick neural stem cells, establishing embryonic day (E) 10 as optimal for progression to neuronal phenotypes. Cells were obtained from the embryonic cerebral hemispheres and incubated for 5-7 days in enriched medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) according to a procedure originally developed for mice. A small percentage of the cells survived, proliferated and formed nestin-positive neurospheres. After removal of the growth factors to allow differentiation (5 days), 74% of the cells differentiated into all major lineages of the nervous system, including neurons (Beta III tubulin-positive, 54% of the total number of differentiated cells), astrocytes (GFAP-positive, 26%), and oligodendrocytes (O4-positive, 20%). These findings demonstrate that the cells were indeed neural stem cells. Next, the cells were transplanted in two allograft chick models; (1) direct cerebral transplantation to 24-h-old chicks, followed by post-transplantation cell tracking at 24 h, 6 days and 14 days, and (2) intravenous transplantation to chick embryos on E13, followed by cell tracking on E19. With both methods, transplanted cells were found in the brain. The chick embryo provides a convenient, precisely-timed and unlimited supply of neural progenitors for therapy by transplantation, as well as constituting a fast and simple model in which to evaluate the ability of neural stem cell transplantation to repair neural damage, steps that are critical for progress toward therapeutic applications.

Transplantation of Xenopus laevis ears reveals the ability to form afferent and efferent connections with the spinal cord.

Previous comparative and developmental studies have suggested that the cholinergic inner ear efferent system derives from developmentally redirected facial branchial motor neurons that innervate the vertebrate ear hair cells instead of striated muscle fibers. Transplantation of Xenopus laevis ears into the path of spinal motor neuron axons could show whether spinal motor neurons could reroute to innervate the hair cells as efferent fibers. Such transplantations could also reveal whether ear development could occur in a novel location including afferent and efferent connections with the spinal cord. Ears from stage 24-26 embryos were transplanted from the head to the trunk and allowed to mature to stage 46. Of 109 transplanted ears, 73 developed with otoconia. The presence of hair cells was confirmed by specific markers and by general histology of the ear, including TEM. Injections of dyes ventral to the spinal cord revealed motor innervation of hair cells. This was confirmed by immunohistochemistry and by electron microscopy structural analysis, suggesting that some motor neurons rerouted to innervate the ear. Also, injection of dyes into the spinal cord labeled vestibular ganglion cells in transplanted ears indicating that these ganglion cells connected to the spinal cord. These nerves ran together with spinal nerves innervating the muscles, suggesting that fasciculation with existing fibers is necessary. Furthermore, ear removal had little effect on development of cranial and lateral line nerves. These results indicate that the ear can develop normally, in terms of histology, in a new location, complete with efferent and afferent innervations to and from the spinal cord.

Live dissection of Drosophila embryos: streamlined methods for screening mutant collections by antibody staining.

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Laser surgery of zebrafish (Danio rerio) embryos using femtosecond laser pulses: optimal parameters for exogenous material delivery, and the laser's effect on short- and long-term development.

BACKGROUND: Femtosecond (fs) laser pulses have recently received wide interest as an alternative tool for manipulating living biological systems. In various model organisms the excision of cellular components and the intracellular delivery of foreign exogenous materials have been reported. However, the effect of the applied fs laser pulses on cell viability and development has yet to be determined. Using the zebrafish (Danio rerio) as our animal model system, we address both the short- and long-term developmental changes following laser surgery on zebrafish embryonic cells. RESULTS: An exogenous fluorescent probe, fluorescein isothiocyanate (FITC), was successfully introduced into blastomere cells and found to diffuse throughout all developing cells. Using the reported manipulation tool, we addressed whether the applied fs laser pulses induced any short- or long-term developmental effects in embryos reared to 2 and 7 days post-fertilization (dpf). Using light microscopy and scanning electron microscopy we compared key developmental features of laser-manipulated and control samples, including the olfactory pit, dorsal, ventral and pectoral fins, notochord, pectoral fin buds, otic capsule, otic vesicle, neuromast patterning, and kinocilia of the olfactory pit rim and cristae of the lateral wall of the ear. CONCLUSION: In our study, no significant differences in hatching rates and developmental morphologies were observed in laser-manipulated samples relative to controls. This tool represents an effective non-destructive technique for potential medical and biological applications.

Heart formation and left-right asymmetry in separated right and left embryos of a newt.

During vertebrate cardiac development, the heart tube formed by fusion of right and left presumptive cardiac mesoderms (PCMs) undergoes looping toward the right, resulting in an asymmetrical heart. Here, we examined the right and left PCMs with regard to heart-tube looping using right- and left-half newt embryos (Cynops pyrrhogaster ). In the half embryos, the rightward (normal) loop of the heart tube was formed from the left PCM, irrespective of the timing of its separation, while the leftward (reversed) loop of the heart tube was formed from the right PCM, separated by stage 18. In addition, the direction of the leftward loop was inverted to the rightward direction in right-half embryos bisected after stage 18. Incision or resection of the embryonic caudal region implicated interactions between the right and left sides of this region as crucial for inverting the direction of the heart-tube loop from leftward to rightward in the right-half embryos. In situ hybridization of CyNodal (Cynops nodal-related gene) suggested that the inversion of heart looping in the right-half embryos has no association with the CyNodal expression pattern. Based on these findings, we propose a mechanism for the rightward looping underlying normal amphibian cardiac development.

Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development.

BACKGROUND: Electroporation is a technique for the introduction of nucleic acids and other macromolecules into cells. In chick embryos it has been a particularly powerful technique for the spatial and temporal control of gene expression in developmental studies. Electroporation methods have also been reported for Xenopus, zebrafish, and mouse. RESULTS: We present a new protocol for zebrafish brain electroporation. Using a simple set-up with fixed spaced electrodes and microinjection equipment, it is possible to electroporate 50 to 100 embryos in 1 hour with no lethality and consistently high levels of transgene expression in numerous cells. Transfected cells in the zebrafish brain are amenable to in vivo time lapse imaging. Explants containing transfected neurons can be cultured for in vitro analysis. We also present a simple enzymatic method to isolate whole brains from fixed zebrafish for immunocytochemistry. CONCLUSION: Building on previously described methods, we have optimized several parameters to allow for highly efficient unilateral or bilateral transgenesis of a large number of cells in the zebrafish brain. This method is simple and provides consistently high levels of transgenesis for large numbers of embryos.

Neptune is involved in posterior axis and tail formation in Xenopus embryogenesis.

In order to elucidate the molecular mechanisms underlying the posterior axis and tail formation in embryogenesis, the function of Neptune, a zinc-finger transcription factor, in Xenopus laevis embryos was investigated. Injection of neptune mRNA into the animal pole area of embryos resulted in the formation of an additional tail structure that included a neural tube and muscle tissue. This activity required FGF signaling since coinjection of a dominant-negative FGF receptor RNA (XFD) completely blocked the formation of a tail structure. A loss-of-function experiment using a fusion construct of neptune and Drosophila engrailed (en-neptune) RNA showed that endogenous Neptune is necessary for formation of the posterior trunk and tail. Furthermore, activity of Neptune was necessary for the endogenous expression of brachyury and fgf-8 at the late gastrula stage. These findings demonstrate a novel function of Neptune in the process of anterior-posterior axis formation through the FGF and brachyury signaling cascades. An experiment using a combination explant with ventral and dorsal marginal tissues showed that cooperation of these two distinct tissues is important for the tail formation and that expression of Neptune in prospective ventral cells may be involved in the activation of the process of tail formation.

Amphibian in vitro heart induction: a simple and reliable model for the study of vertebrate cardiac development.

Amphibian embryos are an excellent model system for analyzing the mechanisms of vertebrate cardiogenesis. Studies of heart development in Xenopus have, for example, revealed that the inductive interaction of the heart primordia with the adjacent underlying endoderm and dorsal lip starts at the early stages of gastrulation. However, the molecular basis of those early inductive events and the genes expressed during the early phases of heart differentiation remain largely unknown. Amphibian blastula embryos contain pluripotent cells in their ectodermal region, called the "animal cap," which fortunately can be exploited for understanding a variety of organogenesis processes. Despite an enormous potential for analysis, the use of this system in cardiogenesis research has languished due to a lack of information concerning appropriate culture methods. Herein we report conditions for generating an in vitro heart induction system and present evidence from two types of in vivo transplantations, that the cultured heart rudiment can develop and function in the adult organism. It is expected that the fundamental principles established in this model system will provide a versatile research platform for a variety of organ engineering projects, including modifying in vitro organ growth with exogenous components (e.g. various growth factors) and developing methods for preparing tissue for transplantation.

Left-right positioning of the adult rudiment in sea urchin larvae is directed by the right side.

Indirect-developing sea urchins eventually form an adult rudiment on the left side through differential left-right development in the late larval stages. Components of the adult rudiment, such as the hydropore canal, the hydrocoel and the primary vestibule, all develop on the left side alone, and are the initial morphological traits that exhibit left-right differences. Although it has previously been shown that partial embryos dissected in cleavage stages correctly determine the normal left-right placement of the adult rudiment, the timing and the mechanism that determine left-right polarity during normal development remain unknown. In order to determine these, we have carried out a series of regional operations in two indirect-developing sea urchin species. We excised all or a part of tissue on the left or right side of the embryos during the early gastrula stage and the two-armed pluteus stage, and examined the left-right position of the adult rudiment, and of its components. Excisions of tissues on the left side of the embryos, regardless of stage, resulted in formation of a left adult rudiment, as in normal development. By contrast, excisions on the right side of the embryos resulted in three different types of impairment in the left-right placement of the adult rudiment in a stage-dependent manner. Generally, when the adult rudiment was definitively formed only on the right side of the larvae, no trace of basic development of the components of the adult rudiment was found on the left side, indicating that a right adult rudiment results from reversal of the initial left-right polarity but not from a later inhibitory effect on the development of an adult rudiment. Thus, we suggest that determination of the left-right placement of the adult rudiment depends on a process, which is directed by the right side, of polarity establishment during the gastrula and the prism stages; however, but commitment of the cell fate to initiate formation of the adult rudiment occurs later than the two-armed pluteus stage.

Specification of pharyngeal endoderm is dependent on early signals from axial mesoderm.

The development of taste buds is an autonomous property of the pharyngeal endoderm, and this inherent capacity is acquired by the time gastrulation is complete. These results are surprising, given the general view that taste bud development is nerve dependent, and occurs at the end of embryogenesis. The pharyngeal endoderm sits at the dorsal lip of the blastopore at the onset of gastrulation, and because this taste bud-bearing endoderm is specified to make taste buds by the end of gastrulation, signals that this tissue encounters during gastrulation might be responsible for its specification. To test this idea, tissue contacts during gastrulation were manipulated systematically in axolotl embryos, and the subsequent ability of the pharyngeal endoderm to generate taste buds was assessed. Disruption of both putative planar and vertical signals from neurectoderm failed to prevent the differentiation of taste buds in endoderm. However, manipulations of contact between presumptive pharyngeal endoderm and axial mesoderm during gastrulation indicate that signals from axial mesoderm (the notochord and prechordal mesoderm) specify the pharyngeal endoderm, conferring upon the endoderm the ability to autonomously differentiate taste buds. These findings further emphasize that despite the late differentiation of taste buds, the tissue-intrinsic mechanisms that generate these chemoreceptive organs are set in motion very early in embryonic development.

Epicardial outgrowth inhibition leads to compensatory mesothelial outflow tract collar and abnormal cardiac septation and coronary formation.

In the present study, we investigated the modulatory role of the epicardium in myocardial and coronary development. Epicardial cell tracing experiments have shown that epicardium-derived cells are the source of interstitial myocardial fibroblasts, cushion mesenchyme, and smooth muscle cells. Epicardial outgrowth inhibition studies show abnormalities of the compact myocardial layer, myocardialization of cushion tissue, looping, septation, and coronary vascular formation. Lack of epicardial spreading is partly compensated by mesothelial outgrowth over the conotruncal region. Heterospecific epicardial transplant is able to partially rescue the myocardial development, as well as septation and coronary formation.

A role for GATA5 in Xenopus endoderm specification.

The endoderm gives rise to the gut and tissues that develop as outgrowths of the gut tube, including the lungs, liver and pancreas. Here we show that GATA5, a zinc-finger transcription factor, is expressed in the yolk-rich vegetal cells of Xenopus embryos from the early gastrula stage onwards, when these cells become committed to form endoderm. At mid-gastrula stages, GATA5 is restricted to the sub-blastoporal endoderm and is the first molecular marker for this subset of endodermal cells so far identified. We show that GATA4 and GATA5 are potent inducers of endodermal marker genes in animal cap assays, while other GATA factors induce these genes only weakly, if at all. When injected into the dorsal marginal zone, GATA5 respecifies prospective mesoderm towards an endodermal fate, thereby disrupting the convergence and extension movements normally undergone by the dorsal mesoderm. The resulting phenotype is very similar to those seen after injection of dominant negative versions of the FGF-receptor or the T-box transcription factor, Xbra and can be rescued by eFGF. The ability of GATA5 to respecify ectodermal and mesodermal cells towards endoderm suggests an important role for GATA5 in the formation of this germlayer. In animal cap assays, GATA5 is induced by concentrations of activin above those known to induce dorsal mesoderm and heart, in an FGF-independent manner. These data indicate that the emerging view for endodermal induction in general, namely that it is specified by high levels of TGF-beta in the absence of FGF signalling, is specifically true for sub-blastoporal endoderm.

Cytochalasin B inhibits morphogenetic movement and muscle differentiation of activin-treated ectoderm in Xenopus.

Xenopus ectodermal explants (animal caps) begin to elongate after treatment with the mesoderm inducing factor activin A. This phenomenon mimics the convergent extension of dorsal mesoderm during gastrulation. To analyze the relationship between elongation movement and muscle differentiation, animal caps were treated with colchicine, taxol, cytochalasin B and hydroxyurea (HUA)/aphidicolin following activin treatment. Cytochalasin B disrupted the organization of actin filaments and inhibited the elongation of the activin-treated explants. Muscle differentiation was also inhibited in these explants at the histologic and molecular levels. Colchicine and taxol, which are known to affect microtubule organization, had little effect on elongation of the activin-treated exp ants. Co-treatment with HUA and aphidicolin caused serious damage on the explants and they did not undergo elongation. These results suggest that actin filaments play an important role in the elongation movement that leads to muscle differentiation of activin-treated explants.

Induction of cardiac myogenesis in avian pregastrula epiblast: the role of the hypoblast and activin.

An in vitro assay has been developed to investigate tissue interactions regulating myocardial cell specification in birds. Explants from the posterior region of stage XI-XIV blastulas were found to form heart muscle at high frequency with a timing that corresponded to onset of cardiac myocyte differentiation in vivo. Isolation and recombination experiments demonstrated that a signal from the hypoblast was required to induce cardiac myogenesis in the epiblast, and regional differences in epiblast responsiveness and hypoblast inductiveness restrict appearance of cardiac myocytes to the posterior region. Explantation studies provided evidence that myocardial cell specification is underway by stage 3, indicating that the hypoblast-derived signal occurs shortly before specification is detected. Recombinations were also performed to compare cardiac-inducing capacities of pregastrula hypoblast and stage 5 anterior lateral endoderm. The hypoblast possessed broad capacity to induce heart muscle cells in pregastrula and mid-gastrula epiblast, and modest ability to induce cardiac myogenesis in stage 4 posterior primitive streak. Stage 5 anterior lateral endoderm, in contrast, showed no ability to induce heart development in epiblast cells but was a potent inducer of cardiac myogenesis in cells from stage 4 posterior primitive streak. These findings suggest that the hypoblast-derived signal likely acts upstream of proposed heart-inducing signals provided by anterior lateral endoderm. Experiments were also performed to investigate whether activin, or an activin-like molecule, is involved in regulating cardiac myogenesis. Follistatin blocked cardiac myogenesis in stage XI-XIV posterior region explants and activin induced cardiac myogenesis in a dose-dependent fashion in posterior epiblast. These findings indicate that activin, or an activin-like molecule, is required for and is sufficient to stimulate cardiac myogenesis in posterior region pregastrula epiblast. Three models are presented to explain these results.

Avian gastrulation and neurulation are not impaired by the removal of the marginal zone at the unincubated blastoderm stage.

In the present study, we removed the whole area marginalis, Rauber's sickle and the peripheral part of the area centralis from unincubated chicken blastoderms (st IV, Vakaet, 1962a). By placing a fragment of a quail Rauber's sickle (functioning as early gastrulation organizer: Callebaut and Van Nueten, 1994) at different places and oriented in different directions on the remaining central part of the area centralis, we observed, after in vitro culture, a normal embryonic development. This indicates that the area marginalis itself is not indispensable for gastrulation and neurulation. Our study also indicates that none of the three elementary tissues (Rauber's sickle, endophyll and upper layer) of the avian unincubated blastoderm present an irreversible functional polarity.

IGF-I and insulin in the acquisition of limb-forming ability by the embryonic lateral plate.

Acquisition of limb-forming ability by discrete regions of the lateral plate of the chick embryo is dependent on a medial-lateral inductive signaling cascade moving sequentially from the area of Hensen's node to the somitic mesoderm, the intermediate mesoderm, and then to the prospective limb-forming regions of the lateral plate. IGF-I and insulin are expressed by medial tissues as they are influencing the prospective limb-forming regions of the lateral plate. Here we report that IGF-I and insulin, but not FGF-2 or FGF-4, induce the formation of limb bud-like structures in vitro from prospective limb regions before they have acquired the ability to form limbs independent of medial tissues, and also induce the formation of limb bud-like structures from the prospective flank. The limb bud-like structures induced by IGF-I and insulin possess a thickened cap of ectoderm along their distal tips that resembles the apical ectodermal ridge (AER) and this thickened distal apical ectoderm expresses the AER-characteristic homeobox-containing gene Msx-2. Like in normal limb buds, a population of highly proliferating cells which express the homeobox-containing gene Msx-1 are localized in the mesoderm directly subjacent to the thickened AER-like structures induced by IGF-I and insulin. However, the limb bud-like structures induced by IGF-I and insulin do not express sonic hedgehog, which encodes a secreted signaling molecule that has been implicated in regulating the anteroposterior patterning of the developing limb bud. IGF-I- and insulin-treated prospective limb explants give rise to rudimentary limbs containing identifiable skeletal elements when grafted into the coelom or to somites of host embryos. Overall, these results suggest that IGF-I and insulin may be endogenous signals produced by medial tissues that are involved in conferring limb-forming ability to the lateral plate and may promote the initial outgrowth of limb buds and possibly induce the AER. However, other signals are necessary to promote the expression of genes such as sonic hedgehog that regulate the patterning of the developing limb.