Creating Avian Forebrain Chimeras to assess Facial Development.

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the strengths of this model system is that the effect of, and interaction among, tissues can be directly assessed in chimeric embryos. We have previously shown that signals from the forebrain contribute to facial morphogenesis by regulating the shape of the expression domain of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ). In this article, the method of generating the forebrain chimeras and provide illustrations of the outcomes of these experiments is described.

Hemomucin, an O-glycosylated protein on embryos of the wasp Macrocentrus cingulum that protects it against encapsulation by hemocytes of the host Ostrinia furnacalis.

It is unclear how endoparasites passively evade their host's immune reactions in most parasite-host systems. Hemomucin from the parasitoid wasp Macrocentrus cingulum (McHEM) is a 97-kDa transmembrane protein containing 51 potential O-glycosylation sites that can be specifically recognized by Arachis hypogaea lectin. Mchem mRNA is highly expressed in M. cingulum eggs, morulae and secondary embryos, and McHEM protein is mainly located on the extraembryonic membrane of embryos. When secondary embryos of M. cingulum were transplanted into naïve larvae of their host, Ostrinia furnacalis, the embryos proliferated to generate dozens of embryos. However, more than 90% of these embryos were encapsulated by host hemocytes after blocking with anti-McHEM serum. Similarly, following knockdown of Mchem expression using double-stranded RNA encoding Mchem (dshem), many more embryos were encapsulated by host hemocytes after transplantation compared to controls (p < 0.01). Furthermore, approximately 70% of the embryos were encapsulated by host hemocytes following digestion with O-glycosidase, which specifically digests ß-gal (1→3) linkages between GalNAc and Ser/Thr of proteins. Western blotting results showed that O-glycosidase digested McHEM into a smaller product. These results indicate that McHEM may protect embryos from being encapsulated by their host and that the McHEM sugar chains play an important role.

Following the fate of neural progenitors by homotopic/homochronic grafts in Xenopus embryos.

The neural plate consists of neuroepithelial cells that serve as progenitors for the mature central nervous system. The neural plate is a highly regionalized structure, harboring neural progenitors with different programs of differentiation, due to signaling or intrinsic differences in their differentiation potential. In the frog neural plate, neural progenitors located in the deep or superficial layer differ in their ability to contribute to early (primary) neurogenesis but intercalate during neurulation. In order to understand the origins and mechanisms of this progenitor heterogeneity, it is necessary to be able to follow directly the fate of different progenitors. Here, we describe a fate mapping method, which is based on homotopic and homochronic grafts of labeled tissue to unlabeled, or differentially labeled, hosts. This method can be combined with immunohistochemical analysis with cell type specific markers, thus allowing one to determine the contribution that each early progenitor type makes to the differentiated nervous system. Such labeling can also be used to examine the morphogenetic movements that take place during neurulation.

Interordinal chimera formation between medaka and zebrafish for analyzing stem cell differentiation.

Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos.

Embryological manipulations in zebrafish.

Due to the powerful combination of genetic and embryological techniques, the teleost fish Danio rerio has emerged in the last decade as an important model organism for the study of embryonic development. It is relatively easy to inject material such as mRNA or synthetic oligonucleotides to reduce or increase the expression of a gene product. Changes in gene expression can be analyzed at the level of mRNA, by whole-mount in situ hybridization, or at the level of protein, by immunofluorescence. It is also possible to quantitatively analyze protein levels by Western and immunoprecipitation. Cell behavior can be analyzed in detail by cell transplantation and by fate mapping. Because a large number of mutations have been identified in recent years, these methods can be applied in a variety of contexts to provide a deep understanding of gene function that is often more difficult to achieve in other vertebrate model systems.

Mef2cb regulates late myocardial cell addition from a second heart field-like population of progenitors in zebrafish.

Two populations of cells, termed the first and second heart field, drive heart growth during chick and mouse development. The zebrafish has become a powerful model for vertebrate heart development, partly due to the evolutionary conservation of developmental pathways in this process. Here we provide evidence that the zebrafish possesses a conserved homolog to the murine second heart field. We developed a photoconversion assay to observe and quantify the dynamic late addition of myocardial cells to the zebrafish arterial pole. We define an extra-cardiac region immediately posterior to the arterial pole, which we term the late ventricular region. The late ventricular region has cardiogenic properties, expressing myocardial markers such as vmhc and nkx2.5, but does not express a full complement of differentiated cardiomyocyte markers, lacking myl7 expression. We show that mef2cb, a zebrafish homolog of the mouse second heart field marker Mef2c, is expressed in the late ventricular region, and is necessary for late myocardial addition to the arterial pole. FGF signaling after heart cone formation is necessary for mef2cb expression, the establishment of the late ventricular region, and late myocardial addition to the arterial pole. Our study demonstrates that zebrafish heart growth shows more similarities to murine heart growth than previously thought. Further, as congenital heart disease is often associated with defects in second heart field development, the embryological and genetic advantages of the zebrafish model can be applied to study the vertebrate second heart field.

Tissue determination using the animal cap transplant (ACT) assay in Xenopus laevis.

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye(1,2). From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap (3), we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo (4). While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Creating frog heart as an organ: in vitro-induced heart functions as a circulatory organ in vivo.

Cardiomyocytes have been induced from various pluripotent cells, such as embryonic stem cells and myeloid stem cells; however, the generation of cardiac tissues beyond two-dimensional cell-sheets has not been reported. Creating higher order, three-dimensional structures that are unique to heart is the long-awaited next step in realizing cardiac regenerative medicine. We have previously shown that cardiomyocytes can be induced in vitro from undifferentiated cells (animal caps) excised from Xenopus embryos. Cardiomyocytes were induced by first dissociating the animal caps and then reaggregating them following treatment with activin. Here, we describe an interesting method for creating a complete ectopic heart in vivo, involving the introduction of in vitro-created tissue during early embryogenesis. Thus, animal cap reaggregates were transplanted into the abdomen of late-neurula-stage embryos, resulting in two-chambered hearts being formed. The dual-heart larvae matured into adult animals with transplanted hearts intact. Involvement of transplanted hearts in systemic circulation was demonstrated. Moreover, the ectopic hearts possessed higher order structures such as atrium and ventricle, and were morphologically, histologically, and electrophysiologically identical to original hearts. This system should facilitate the study of heart organogenesis and may promote a shift from tissue to organ engineering for clinical applications.

Tissue targeted embryonic chimeras: zebrafish gastrula cell transplantation.

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered. Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions. Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes. Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras. We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another. We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures. In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo. These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Tissue micromanipulation in zebrafish embryos.

Although a common approach in large vertebrate embryos such as chick or frog, manipulation at the tissue level is only rarely applied to zebrafish embryos. Despite its relatively small size, the zebrafish embryo can be readily used for micromanipulations such as tissue and organ primordium transplantation, explantation, and microbead implantation, to study inductive tissue interactions and tissue autonomy of pleiotropic, mutant phenotypes or to isolate tissue for organotypic and primary cell culture or RNA isolation. Since this requires special handling techniques, tools, and tricks, which are rarely published and thus difficult to apply without hands-on demonstration, this article provides detailed instructions and protocols on tissue micromanipulation. The goal is to introduce a broader scientific audience to these surgical techniques, which can be applied to a wide range of questions and used as a starting point for many downstream applications in the genetically tractable zebrafish embryo.

Chapter 4. Using the zebrafish to study vessel formation.

Danio rerio, commonly referred to as the zebrafish, is a powerful animal model for studying the formation of the vasculature. Zebrafish offer unique opportunities for in vivo analysis of blood and lymphatic vessels formation because of their accessibility to large-scale genetic and experimental analysis as well as the small size, optical clarity, and external development of zebrafish embryos and larvae. A wide variety of established techniques are available to study vessel formation in the zebrafish, from early endothelial cell differentiation to adult vessel patterning. In this chapter, we review methods used to functionally manipulate and visualize the vasculature in the zebrafish and illustrate how these methods have helped further understanding of the genetic components regulating formation and patterning of developing vessels.

Origin and progress of nuclear transfer in nonmammalian animals.

This chapter traces the origin and progress of nuclear transfer that later became the paradigm for cloning animals. Classic studies in cytology, embryology, or genetics spanning more than five centuries that led to nuclear transfers in unicellular animals and to those in oocytes of insects, fish and amphibians are reviewed. The impetus for the development of successful nuclear transfers in amphibian oocytes in 1952 was to determine whether or not differentiated somatic cell nuclei are developmentally equivalent to zygote nuclei. Experiments in amphibians demonstrated several important results: (1) specialized somatic cell nuclei are extensively multipotent; (2) fertile adult amphibians can be cloned from embryonic and larval nuclei; (3) serial cloning expands the number of clones; (4) transplanting nuclei into oocyte cytoplasm induces reprogramming of their gene function; and (5) amphibian cloning became the model for cloning mammals. Subsequent studies in mice, a more technically favorable species, revealed that specialized cell nuclei are equivalent to zygote nuclei.

Chimeric honeybees (Apis mellifera) produced by transplantation of embryonic cells into pre-gastrula stage embryos and detection of chimerism by use of microsatellite markers.

The production of chimeras, by use of cell transplantation, has proved to be highly valuable in studies of development by providing insights into cell fate, differentiation, and developmental potential. So far, chimeric honeybees have been created by nuclear transfer technologies. We have developed protocols to produce chimeric honeybees by use of cell transplantation. Embryonic cells were transplanted between pre-gastrula stage embryos (32-34 hr after oviposition) and hatched larvae were reared in vitro for 4 days. Chimeric individuals were detected by use of microsatellite analysis and a conservative estimation approach. 4.8% of embryos, posteriorly injected with embryonic cells, developed into chimeric honeybee larvae. By injection of cells pre-stained with fluorescent cell tracer dye, we studied the integration of transplanted cells in the developing embryos. Number of injected cells varied from 0 to 50 and cells remained and multiplied mainly in the area of injection.

Generation of live fry from intraperitoneally transplanted primordial germ cells in rainbow trout.

Germ cell transplantation has tremendous applications in transgenic animal production, assisted reproductive technology, and germline stem cell research. Here, we report for the first time the production of individuals from intraperitoneally transplanted primordial germ cells (PGCs) in animals. To trace the behavior of exogenous PGCs in recipients, PGCs visualized by a green fluorescent protein gene were used as donors. The PGCs prepared from the genital ridges of hatching embryos were transplanted into recipients at various developmental stages. The PGCs injected into the peritoneal cavities of hatching embryos had the ability to migrate toward, and to colonize, the genital ridges of recipient embryos. Furthermore, donor-derived PGCs proliferated and differentiated into mature eggs and sperm in the allogenic gonads; the resulting gametes produced live fry, showing the donor-derived phenotype, through fertilization. Combined with in vitro culture, genetic modification, and cryopreservation of PGCs, this technique provides new approaches for fish bioengineering.

Long-term culture of decapsulated gastropod embryos: a transplantation study.

Encapsulated embryos of the pond snail Helisoma trivolvis have been useful for examining neural development and neural circuit function during development. However, their full potential in developmental studies is limited by the lack of an effective method for long-term culture of decapsulated embryos. In the present study, decapsulated early embryos were either cultivated ex ovo in various media under different environmental conditions or transplanted into host egg capsules. Although diluted capsular fluid, 30% M199, and albumen-gland-conditioned medium were partially effective in promoting embryonic growth for a short time, none of the media promoted normal embryonic development in long-term tests. In contrast, after previously decapsulated and experimentally manipulated embryos were transplanted into host capsules, their growth and development were similar to their intact siblings. In combination with laser ablation, this transplantation technique was used to demonstrate the role played by a pair of serotonergic neurons in regulating an embryonic rotational behavior. These results suggest that embryonic transplantation is an extremely effective technique for achieving long-term growth and development of previously decapsulated embryos and therefore can be instrumental in investigating cell lineage, function, and development in encapsulated embryos.

Loss of the maintenance methyltransferase, xDnmt1, induces apoptosis in Xenopus embryos.

DNA methylation is necessary for normal embryogenesis in animals. Here we show that loss of the maintenance methyltransferase, xDnmt1p, triggers an apoptotic response during Xenopus development, which accounts for the loss of specific cell populations in hypomethylated embryos. Hypomethylation-induced apoptosis is accompanied by a stabilization in xp53 protein levels after the mid-blastula transition. Ectopic expression of HPV-E6, which promotes xp53 degradation, prevents cell death, implying that the apoptotic signal is mediated by xp53. In addition, inhibition of caspase activation by overexpression of Bcl-2 results in the development of cellular masses that resemble embryonic blastomas. Embryonic tissue explant experiments suggest that hypomethylation alters the developmental potential of early embryo cells and that apoptosis is triggered by differentiation. Our results imply that loss of DNA methylation in differentiated somatic cells provides a signal via p53 that activates cell death pathways.

Expression of Frzb-1 during chick development.

We cloned the chick homolog of Xenopus and mouse Frzb-1, a secreted Wnt antagonist and performed in situ hybridizations to determine the pattern of cFrzb-1 expression in the developing chick embryo. At early stages, cFrzb-1 transcripts are located exclusively in the ectodermal layer corresponding to the neural plate. The labelling continues in the neural tube, but is always excluded from the floor plate. cFrzb-1 mRNA is expressed by migrating cephalic and truncal neural crest cells. Later, cFrzb-1 transcripts are found in a subset of neural crest derivatives such as cephalic cartilage, nerves and spinal ganglia. In addition to ectodermal derivatives, cFrzb-1 transcripts were also observed in mesodermal derivatives such as vertebral and limb cartilage, the adrenal cortex, the gonads, and a subpopulation of blood cells.

The specification of the pronephric tubules and duct in Xenopus laevis.

We have examined the timing of specification of the pronephric tubules and duct in Xenopus laevis by explanting the presumptive pronephric rudiments into blastula ectodermal wraps. We have established the time point of specification using the monoclonal antibody markers 3G8 and 4A6 which recognize antigens in pronephric tubule and duct, respectively. We show that, by experimental analysis in explants, kidney tubules are specified by stage 12.5 in the pronephric anlagen whereas pronephric duct is specified later between stages 13 and 14. Furthermore we show that signals involved in tubulogenesis of the pronephric tubules are normally received between stage 12.5 and 13. These experiments unambiguously pinpoint the timing of pronephros specification analyzed by explant experimentation to a developmental stage prior to that demonstrated for urodele amphibia, and provide an essential biological backdrop to a search for the molecular nature of pronephric inducers.

Production of donor-derived offspring by transfer of primordial germ cells in Japanese quail.

We transfused concentrated primordial germ cells (PGCs) of the black strain (D: homozygous for the autosomal incomplete dominant gene, D) of quail into the embryos of the wild-type plumage strain (WP: d+/d+) of quail. The recipient quail were raised until sexual maturity and a progeny test of the putative germline chimeras was performed to examine the donor gamete-derived offspring (D/d+). Thirty-one percent (36/115) of the transfused quail hatched and 21 (13 females and 8 males) of them reached maturity. Five females and 2 males were germline chimeras producing donor gamete-derived offspring. Transmission rates of the donor derived gametes in the chimeric females and males were 1.8-8.3% and 2.6-63.0%, respectively. Germline chimeric and the other putative chimeric males were also test-mated with females from the sex-linked imperfect albino strain (AL: d+/d+, al/W, where al indicates the sex-linked imperfect albino gene on the Z chromosome, and W indicates the W chromosome) for autosexing of W-bearing spermatozoa: No albino offspring were born.