Segregation of the embryonic vascular and hemopoietic systems.

The origin of endothelial cells and their subsequent assembly into the primary vascular system have been mostly analyzed in the avian embryo. Following the discovery of specific growth factors and their cognate receptors, the molecular mechanisms underlying these processes have been unraveled in both birds and mammals. In particular, experimental studies of the angiogenic vascular endothelial growth factor (VEGF) and its receptors, carried out in both vertebrate classes, have provided significant insight into the developmental biology of endothelial cells. The VEGF receptor VEGFR2 is the earliest marker known to be expressed by endothelial precursor cells of avian and mouse embryos. Based on the localization of VEGFR2+ cells in the avian embryo and on clonal culture experiments, two types of endothelial precursor cells can be distinguished from gastrulation stages onward: posterior mesodermal VEGFR2+ hemangioblasts, which have the capacity to differentiate into endothelial and hemopoietic cells, and anterior VEGFR2+ angioblasts, which can only give rise to endothelial cells.     

Segmentation and compartition in the early avian hindbrain

For the comparative embryologists of the early 20th century, the segment-like bulges that appear transiently during the early stages of vertebrate hindbrain development were both the object of fascination and the subject of vigorous dispute. Conflicting views were held as to the significance of these 'rhombomeres' to brain development and their more general relevance to head evolution. Whether rhombomeres are inconsequential bumps in the embryonic brain or true segments-iterative or metameric units-has only recently been resolved. A number of studies using more modern techniques (such as immunohistochemistry, in situ hybridisation, axonal tracing, single cell labelling, heterotopic and orthotopic grafting, and the manipulation of gene expression by electroporation) have shown that the hindbrain has a truly metameric cellular organisation. The avian embryo has played a particularly prominent role in such studies by virtue of its large size and accessibility, its amenability to microsurgery, and its well-described anatomy. Furthermore, electrophysiological studies, also on avian embryos, have shown that segmentation of the parent neuroepithelium into rhombomeres plays a crucial part in establishing the functional organization of the hindbrain. Segmentation suggests the early allocation of defined sets of precursor cells and is therefore presumed to allow a specific identity for each successive segment to emerge from a common ground plan. This short review will focus on the contribution the avian embryo has made to our understanding of this fly-like region of the vertebrate brain, in respect of its morphology and neuronal architecture, the cellular and molecular mechanisms involved in establishing and maintaining the segments, and the molecular controls of segmental identity.     

Of birds and mice: hematopoietic stem cell development

For many years it has been assumed that the ontogeny of the mammalian hematopoietic system involves sequential transfers of hematopoietic stem cells (HSCs) generated in the yolk sac blood islands, to successive hematopoietic organs as these become active in the embryo (fetal liver, thymus, spleen and eventually bone marrow). Very little was known about early events related to hematopoiesis that could take place during the 4.5 day gap separating the appearance of the yolk sac blood islands and the stage of a fully active fetal liver. Experiments performed in birds documented that the yolk sac only produce erythro-myeloid precursors that become extinct after the emergence of a second wave of intra-embryonic HSCs from the region neighbouring the dorsal aorta. The experimental approaches undertaken over the last ten years in the murine model, which are reviewed here, led to the conclusion that the rules governing avian hematopoietic development basically apply to higher vertebrates.     

Embryonic origins of spleen asymmetry

The spleen is a vertebrate organ that has both hematopoietic and immunologic function. The embryonic origins of the spleen are obscure, with most studies describing the earliest rudiment of the spleen as a condensation of mesodermal mesenchyme on the left side of the dorsal mesogastrium. The development of spleen handedness has not been described previously, presumably because of the difficulty in assaying spleen position in the embryo and the lack of early, organ-specific molecular markers. Here we show that expression of the homeobox gene Nkx2-5 serves as a marker for spleen precursor tissue. Pre-splenic tissue is initially located in symmetric domains on both sides of the embryo but, during subsequent development, only the left side goes on to form the mature spleen. Therefore, the final location of the spleen on the left side of the body axis appears to result from preferential development of the spleen precursor cells on the left side of the embryo. Our studies indicate that the spleen and heart become asymmetric via different cellular mechanisms. Nkx2-5 may function locally as part of the laterality cascade, downstream of nodal and Pitx2, or it may direct asymmetric morphogenesis after laterality has been determined.     

Somatic transgenesis in the avian model system

The chick embryo is a versatile model system, in which classical embryology can be combined with modern molecular approaches. In the last two decades, several efficient methods have been developed to introduce exogenous genes into the chick embryo. These techniques allow alteration of gene expression levels in a spatially and temporally restricted manner, thereby circumventing embryonic lethality and/or eliminating secondary effects in other tissues. Here, we present the current status of avian somatic transgenic techniques, focusing on electroporation and retrovirus-mediated gene transfer. Electroporation allows quick and efficient gain-of-function studies based on transient misexpression of genes. Retroviral vectors, which are capable of integrating exogenous genes into the host chromosome, permit analysis of long-term effects of gene misexpression. The variety of methods available for somatic transgenesis, along with the recent completion of the chicken genome, are transforming the chick embryo into one of the most attractive model systems to examine function of genes that are important for embryonic development.     

Gallera method of chick embryo culture in vitro supports better growth compared with original New method

An avian embryo is a valuable model system for vertebrate embryology. Easy availability, accessibility to various developmental stages and amenability of organ fields makes the chick embryo one of the favored model systems. Seminal discoveries regarding organogenesis and vertebrate morphogenesis have been made using chick embryos cultured in vitro . Dennis A.T. New revolutionized chick embryo culture methodology with his development of a single glass ring explantation technique. Many modifications and/or embellishments were introduced after the New era of embryo culture. A double glass ring method for chick embryo culture introduced by Gallera and Nicolet is compared with the original New method and the EASY method in this study. In addition, a video of culture methods is presented as a valuable tool in learning about and/or teaching techniques of chick embryo culture.     

Evolutionary patterns of MHC class II B in owls and their implications for the understanding of avian MHC evolution

Owing to its special mode of evolution and central role in the adaptive immune system, the major histocompatibility complex (MHC) has become the focus of diverse disciplines such as immunology, evolutionary ecology, and molecular evolution. MHC evolution has been studied extensively in diverse vertebrate lineages over the last few decades, and it has been suggested that birds differ from the established mammalian norm. Mammalian MHC genes evolve independently, and duplication history (i.e., orthology) can usually be traced back within lineages. In birds, this has been observed in only 3 pairs of closely related species. Here we report strong evidence for the persistence of orthology of MHC genes throughout an entire avian order. Phylogenetic reconstructions of MHC class II B genes in 14 species of owls trace back orthology over tens of thousands of years in exon 3. Moreover, exon 2 sequences from several species show closer relationships than sequences within species, resembling transspecies evolution typically observed in mammals. Thus, although previous studies suggested that long-term evolutionary dynamics of the avian MHC was characterized by high rates of concerted evolution, resulting in rapid masking of orthology, our results question the generality of this conclusion. The owl MHC thus opens new perspectives for a more comprehensive understanding of avian MHC evolution.     

Bird Tracks at the Hot Springs Mammoth Site,South Dakota,USA

The Hot Springs Mammoth Site, South Dakota, USA, has been excavated for over three decades, during which time numerous body fossils have been recorded. The site is particularly well known for the skeletal remains of mammalian megafauna. Bedding plane surfaces were studied that displayed the first record of small vertebrate (avian) and invertebrate traces. While large vertebrate tracks, often observed in cross-section, are well known at the site, the new traces form a hitherto unstudied assemblage.

The presence of distinct didactyl and tridactyl avian tracks from the site are described here for the first time. The small (～20 mm long) tracks and associated invertebrate traces suggest relatively high moisture content in the substrate on surfaces that experienced aerial or subaerial exposure. This is consistent with the interpretation that the upper layers of the site represent the latter stages of a sinkhole setting with a pond undergoing cyclical drying out.     

The avian embryo as a model to study the development of the neural crest: a long and still ongoing story

The aim of this review is to evoke briefly the progress that has been made in our knowledge about the contribution of the neural crest to the vertebrate body since it was discovered by Wilhelm His in 1868. Although first studied essentially in amphibian embryos, a large amount of what is known on this very special structure was gained by experimental work carried out on the avian embryo. The making of chimeras between quail and chick has permitted not only to analyse the normal course of neural crest cell migration and differentiation but also to reveal some of the cellular interactions that regulate these events. Looking to the future, we can foresee that the novel methods, which now allow to manipulate gene activities in definite groups of cells and at elected times in the developing embryo, will make the avian model even more instrumental than ever to approach the developmental problems raised by neural crest cell differentiation.     

Bone morphogenetic proteins in vertebrate hematopoietic development

During embryonic development, the hematopoietic system is the first to generate terminally differentiated, functional cell types. The urgent necessity for the early formation of blood and blood vessels during embryogenesis means that the induction, expansion, and maturation of these systems must be rapidly and precisely controlled. Bone morphogenic proteins (BMPs) have been implicated in hematopoietic development in the vertebrate embryo and stimulate the proliferation and/or differentiation of human cord blood hematopoietic stem cells (HSC) and embryonic stem cells in vitro. Here we review the mechanisms of action and potential roles of these soluble signaling molecules in vertebrate hematopoiesis.     

Cancer immunotherapy: the past,the present and the future

One of the most controversial issues in immunology for over a century has been whether an effective immune response can be elicited against malignant tumours. Whether the immunology community has believed cancer immunotherapy is feasible or impossible has been largely determined by the prevailing immunological paradigms at that time. In fact, during the last 110 years it is possible to trace at least five dramatic fluctuations in attitude towards cancer immunotherapy. It now appears, however, that overwhelming evidence is available to support the view that both the innate and adaptive immune responses can recognize and eliminate tumours. On the other hand, it remains to be seen if these immune responses can be harnessed to control cancer as, at the time of diagnosis, many tumours have already been immunoselected to be highly resistant to immune elimination. Based on these observations it is argued that immunotherapy approaches, other than the generation of tumour-specific cytotoxic T lymphocytes, must be explored. Alternative strategies include recruiting tumouricidal myeloid cells into tumours, generating antiangiogenic immune responses and directing innate immunity to hypoxia-induced ligands on tumour cells.     

Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens

Conventional assays for quantifying the virulence of microbial pathogens and mutants have traditionally relied upon the use of a range of mammalian species. A number of workers have demonstrated that insects can be used for evaluating microbial pathogenicity and provide results comparable to those that can be obtained with mammals since one component of the vertebrate immune system, the innate immune response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity.     

The Claude Bernard lecture, 1987. Embryonic chimeras: a tool for studying the development of the nervous and immune systems

Chimeras have been constructed in the avian embryo following the observation of the particular structure of the interphase nucleus in the Japanese quail (Coturnix coturnix japonica). In all embryonic and adult cell types of this species a large amount of heterochromatin is associated with the nucleolus, making quail cells readily distinguishable from those of the chick where the constitutive heterochromatin is evenly dispersed in the nucleus. These structural differences have been used to devise a cell-marking technique through which cell migrations and cell interactions during embryogenesis can be followed in the embryo in ovo by grafting quail cells into chick embryos or vice versa. This method was applied to the ontogeny of the neural crest and of the immune system. Recently quail-chick chimeras have been allowed to hatch and the immunological status of the embryonic grafts after birth scrutinized. Xenogeneic tissue grafts made in the embryo are rejected after birth with a more or less prolonged delay according to the nature of the graft. However, rejection can be prevented and a permanent state of tolerance induced for the embryonic tissue grafts by isotopically implanting the thymic epithelium from the same quail donor.     

Early arterial differentiation and patterning in the avian embryo model

Of the many models to study vascular biology the avian embryo remains an informative and powerful model system that has provided important insights into endothelial cell recruitment, assembly and remodeling during development of the circulatory system. This review highlights several discoveries in the avian system that show how arterial patterning is regulated using the model of dorsal aortae development along the embryo midline during gastrulation and neurulation. These discoveries were made possible through spatially and temporally controlled gain-of-function experiments that provided direct evidence that BMP signaling plays a pivotal role in vascular recruitment, patterning and remodeling and that Notch-signaling recruits vascular precursor cells to the dorsal aortae. Importantly, BMP ligands are broadly expressed throughout embryos but BMP signaling activation region is spatially defined by precisely regulated expression of BMP antagonists. These discoveries provide insight into how signaling, both positive and negative, regulate vascular patterning. This review also illustrates similarities of early arterial patterning along the embryonic midline in amniotes both avian and mammalians including human, evolutionarily specialized from non-amniotes such as fish and frog.     

Basal metabolic rate and the evolution of the adaptive immune system

Vertebrates have evolved an adaptive immune system in addition to the ancestral innate immune system. It is often assumed that a trade-off between costs and benefits of defence governs the evolution of immunological defence, but the costs and benefits specific to the adaptive immune system are poorly known. We used genetically engineered mice lacking lymphocytes (i.e. mice without adaptive, but with innate, immunity) as a model of the ancestral state in the evolution of the vertebrate immune system. To investigate if the magnitude of adaptive defence is constrained by the energetic costs of producing lymphocytes etc., we compared the basal metabolic rate of normal and lymphocyte-deficient mice. We found that lymphocyte-deficient mice had a higher basal metabolic rate than normal mice with both innate and adaptive immune defence. This suggests that the evolution of the adaptive immune system has not been constrained by energetic costs. Rather, it should have been favoured by the energy savings associated with a combination of innate and adaptive immune defence.     

Evolution and distribution of class II-related endogenous retroviruses

Endogenous retroviruses (ERVs) are widespread in vertebrate genomes and have been loosely grouped into "classes" on the basis of their phylogenetic relatedness to the established genera of exogenous retroviruses. Four of these genera-the lentiviruses, alpharetroviruses, betaretroviruses, and deltaretroviruses-form a well-supported clade in retroviral phylogenies, and ERVs that group with these genera have been termed class II ERVs. We used PCR amplification and sequencing of retroviral fragments from more than 130 vertebrate taxa to investigate the evolution of the class II retroviruses in detail. We confirm that class II retroviruses are largely confined to mammalian and avian hosts and provide evidence for a major novel group of avian retroviruses, and we identify additional members of both the alpha- and the betaretrovirus genera. Phylogenetic analyses demonstrated that the avian and mammalian viruses form distinct monophyletic groups, implying that interclass transmission has occurred only rarely during the evolution of the class II retroviruses. In contrast to previous reports, the lentiviruses clustered as sister taxa to several endogenous retroviruses derived from rodents and insectivores. This topology was further supported by the shared loss of both the class II PR-Pol frameshift site and the class II retrovirus G-patch domain.     

Knowing left from right: the molecular basis of laterality defects

The apparent symmetry of the vertebrate body conceals profound asymmetries in the development and placement of internal organs. Asymmetric organ development is controlled in part by genes expressed asymmetrically in the early embryo, and alterations in the activities of these genes can result in severe defects during organogenesis. Recently, data from different vertebrates have allowed researchers to put forward a model of genetic interactions that explains how asymmetric patterns of gene expression in the early embryo are translated into spatial patterns of asymmetric organ development. This model helps us to understand the molecular basis of a number of congenital malformations in humans.     

Tumor vaccines and beyond

For the last two decades the immunotherapy of patients with solid and hematopoietic tumors has met with variable success. We have reviewed the field of tumor vaccines to examine what has worked and what has not, why this has been the case, how the anti-tumor responses were examined, and how we can make tumor immunity successful for the majority of individuals rather than for the exceptional patients who currently show successful immune responses against their tumors.