Experimental study of early olfactory neuron differentiation and nerve formation using quail-chick chimeras

For the formation of a functional olfactory system, the key processes are neuronal differentiation, including the expression of one or the other olfactory receptors, the correct formation of the nerve and organization of periphero-central connections. These processes take place during embryonic development starting from early stages. Consequently, avian embryos afford an attractive model to study these mechanisms. Taking advantage of species-specific equipment of olfactory receptors genes in different bird species, interspecific avian chimeras were set up by grafting early chick olfactory placodes in same stage quail embryos. Their analysis was performed using different complementary approaches. In situ hybridisation using probes to different chick olfactory receptor (COR) genes indicated that the choice of expression of an olfactory receptor by a neuron is independent of the environment of the olfactory placode and of interactions with the central nervous system. Futhermore, a chick olfactory receptor gene subgroup (COR3 ), absent in the host genome, was expressed by neurons from the graft. The question was then raised of the consequences of such heterospecific differentiation on axonal projections and fiber convergence. The DiI labeling of olfactory fibres in chimeras revealed anomalies in the formation of the nerve from the chick graft. In agreement with the hypothesis of olfactory receptor (OR) involvement in axonal guidance and periphero-central synapse organisation, the presence of migrating cells and axonal fibres from the graft, expressing foreign ORs and having different interactions with the host environment than the host fibres and migrating cells, might explain these anomalies.     

The early development of cranial sensory ganglia and the potentialities of their component cells studied in quail-chick chimeras

The quail-chick marker system has been used to study the early developmental stages of the ganglia located along cranial nerves VII, IX, and X. The streams of neural crest cells arising from the rhombencephalic-vagal neural crest were followed from the onset of their migration up to the localization of crest cells in the trunk and root ganglia of these nerves. It was shown that two different populations of crest cells are segregated early as a result of morphogenetic movements in the hypobranchial region. The dorsal population gives rise to the root ganglia of nerves IX and X located close to the encephalic vesicles, where the crest cells differentiate both into neurons and into glia. In contrast, the ventral stream of neural crest cells contributes together with cells from epibranchial placodes to the trunk ganglia (geniculate, petrous, and nodose ganglia) of cranial nerves VII, IX, and X. The successive steps of the invasion of the placodal anlage by crest cells can be followed owing to the selective labeling of the neural crest cells. It appears that the latter give rise to the satellite cells of the geniculate, petrous, and nodose ganglia while the large sensory neurons originate from the placodes. The nodose ganglion has been the subject of further studies aimed to investigate whether neuronal potentialities can be elicited in the neural crest-derived cells that it contains. The ability to label selectively either the neurons or the glia by the quail nuclear marker made this investigation possible in the particular case of the nodose ganglion whose neurons and satellite cells have a different embryonic origin. By the technique already described (N. M. Le Douarin, M. A. Teillet, C. Ziller, and J. Smith, 1978, Proc. Nat. Acad. Sci. USA75, 2030–2034) of back-transplantation into the neural crest migration pathway of a younger host, it was shown that the presumptive glial cells of the nodose ganglion are able to remigrate when transplanted into a 2-day chick host and to differentiate into autonomic structures (sympathetic ganglion cells, adrenomedullary cells, and enteric ganglia). It is proposed as a working hypothesis that neuronal potentialities contained in the neural crest cells which invade the placodal primordium of the nodose ganglion are repressed through cell-cell interactions occurring between placodal and crest cells.     

The developmental fate of the cephalic mesoderm in quail-chick chimeras.

The developmental fate of the cephalic paraxial and prechordal mesoderm at the late neurula stage (3-somite) in the avian embryo has been investigated by using the isotopic, isochronic substitution technique between quail and chick embryos. The territories involved in the operation were especially tiny and the size of the transplants was of about 150 by 50 to 60 microns. At that stage, the neural crest cells have not yet started migrating and the fate of mesodermal cells exclusively was under scrutiny. The prechordal mesoderm was found to give rise to the following ocular muscles: musculus rectus ventralis and medialis and musculus oblicus ventralis. The paraxial mesoderm was separated in two longitudinal bands: one median, lying upon the cephalic vesicles (median paraxial mesoderm--MPM); one lateral, lying upon the foregut (lateral paraxial mesoderm--LPM). The former yields the three other ocular muscles, contributes to mesencephalic meninges and has essentially skeletogenic potencies. It contributes to the corpus sphenoid bone, the orbitosphenoid bone and the otic capsules; the rest of the facial skeleton is of neural crest origin. At 3-somite stage, MPM is represented by a few cells only. The LPM is more abundant at that stage and has essentially myogenic potencies with also some contribution to connective tissue. However, most of the connective cells associated with the facial and hypobranchial muscles are of neural crest origin. The more important result of this work was to show that the cephalic mesoderm does not form dermis. This function is taken over by neural crest cells, which form both the skeleton and dermis of the face. If one draws a parallel between the so-called "somitomeres" of the head and the trunk somites, it appears that skeletogenic potencies are reduced in the former, which in contrast have kept their myogenic capacities, whilst the formation of skeleton and dermis has been essentially taken over by the neural crest in the course of evolution of the vertebrate head.     

Mediolateral somitic origin of ribs and dermis determined by quail-chick chimeras

Somites are transient mesodermal structures giving rise to all skeletal muscles of the body, the axial skeleton and the dermis of the back. Somites arise from successive segmentation of the presomitic mesoderm (PSM). They appear first as epithelial spheres that rapidly differentiate into a ventral mesenchyme, the sclerotome, and a dorsal epithelial dermomyotome. The sclerotome gives rise to vertebrae and ribs while the dermomyotome is the source of all skeletal muscles and the dorsal dermis. Quail-chick fate mapping and diI-labeling experiments have demonstrated that the epithelial somite can be further subdivided into a medial and a lateral moiety. These two subdomains are derived from different regions of the primitive streak and give rise to different sets of muscles. The lateral somitic cells migrate to form the musculature of the limbs and body wall, known as the hypaxial muscles, while the medial somite gives rise to the vertebrae and the associated epaxial muscles. The respective contribution of the medial and lateral somitic compartments to the other somitic derivatives, namely the dermis and the ribs has not been addressed and therefore remains unknown. We have created quail-chick chimeras of either the medial or lateral part of the PSM to examine the origin of the dorsal dermis and the ribs. We demonstrate that the whole dorsal dermis and the proximal ribs exclusively originates from the medial somitic compartment, whereas the distal ribs derive from the lateral compartment.     

Cellular dynamics and molecular control of the development of organizer-derived cells in quail-chick chimeras

Malformations affecting the nervous system in humans are numerous and various in etiology. Many are due to genetic deficiencies or mechanical accidents occurring at early stages of development. It is thus of interest to reproduce such human malformations in animal models. The avian embryo is particularly suitable for researching the role of morphogenetic movements and genetic signaling during early neurogenesis. The last ten years of research with Nicole Le Douarin in the Nogent Institut have brought answers to questions formulated by Etienne Wolff at the beginning of his career, by showing that Hensen's node, the avian organizer, is at the source of all the midline cells of the embryo and ensures cell survival, growth and differentiation of neural and mesodermal tissues.     

Temporospatial study of the migration and distribution of cardiac neural crest in quail-chick chimeras.

It has been demonstrated that the septation of the outflow tract of the heart is formed by the cardiac neural crest. Ablation of this region of the neural crest prior to its migration from the neural fold results in anomalies of the outflow and inflow tracts of the heart and the aortic arch arteries. The objective of this study was to examine the migration and distribution of these neural crest cells from the pharyngeal arches into the outflow region of the heart during avian embryonic development. Chimeras were constructed in which each region of the premigratory cardiac neural crest from quail embryos was implanted into the corresponding area in chick embryos. The transplantations were done unilaterally on each side and bilaterally. The quail-chick chimeras were sacrificed between Hamburger-Hamilton stages 18 and 25, and the pharyngeal region and outflow tract were examined in serial paraffin sections to determine the distribution pattern of quail cells at each stage. The neural crest cells derived from the presumptive arch 3 and 4 regions of the neuraxis occupied mainly pharyngeal arches 3 and 4 respectively, although minor populations could be seen in pharyngeal arches 2 and 6. The neural crest cells migrating from the presumptive arch 6 region were seen mainly in pharyngeal arch 6, but they also populated pharyngeal arches 3 and 4. Clusters of quail neural crest cells were found in the distal outflow tract at stage 23.     

Evolution of the lumbo-sacral neural crest in the avian embryo: Origin and differentiation of the ganglionated nerve of Remak studied in interspecific quail-chick chimaerae

Summary Isotopic and isochronic transplantation of fragments of quail neural tube into chick demonstrates that neural and glial cells of the entire ganglion of Remak (RG) arise from the lumbo-sacral level of the neural crest.The ganglioblasts first accumulate in the mesorectum (stage 24 of Hamburger and Hamilton, in the chick and I8 of Zacchei in the quail) and subsequently migrate cranially.Histochemical studies have been carried out on the rectal and cloacal parts of the quail RG at various stages of development. Cholinesterase activity can be detected as soon as the primordium is in place and the intensity of the reaction increases rapidly. During morphogenesis of the cloacal region the RG and the pelvic plexus become intimately associated. Catecholamine-containing cells are found first in the pelvic plexus, then in the cloacal part of the RG. Fluorescent cells are often grouped close to blood vessels and associated with non-fluorescent ganglia. Cranial to the level of the bursa of Fabricius, the RG is composed only of non-fluorescent neurons whatever the developmental stage considered (up to 1 day after hatching).The developmental capabilities of the RG of the 5-day quail have been tested by transplanting various parts of the hind-gut with the dorsal mesentery onto the chorio-allantoic membrane. Catecholamine-containing cells develop only in grafts including the cloacal region.By using quail-chick chimaerae in which the RG belongs to the quail while mesentery and gut are of chick origin, it was possible to show that neurons which develop in the graft (i.e. in the absence of preganglionic innervation), send nerve fibres into the gut wall. Moreover some neuroblasts located in the primordium of the RG migrate into the gut wall and give rise to some enteric ganglion cells. The contribution of the lumbo-sacral neural crest to the enteric ganglia, by this route, is discussed.List of Abbreviations in Text FIF formol-induced fluorescence - H & H Hamburger and Hamilton - Z Zacchei - CAM chorio-allantoic membrane - SIF small intensely fluorescent (cells)     

A study of the development of the hemopoietic system using quail-chick chimeras obtained by blastoderm recombination

The anterior part of the area pellucida from quail blastoderms extending to the 10th or the 17th somite level was substituted for the corresponding region of chick blastoderms in ovo. Reciprocal grafts were also carried out. In external appearance the resulting chimeras had a composite body, one species contributing the head and neck or the head, neck, and wing regions while the other species provided the remainder. The chimeras were always grafted on a chick yolk sac. The cellular composition of hemopoietic organs according to species was analyzed by means of the quail-chick nuclear difference, in 39 viable chimeras at 11–13 days of incubation. The thymus composition depended on the level of the boundary between the two species. In chimeras in which the quail contributed head and neck, the thymic epithelial stroma was made of quail cells while the lymphoid population was of chick origin. In contrast, when the quail contribution also extended to the wings, both thymic stroma and lymphoid cells were of quail origin. In reciprocal combinations, in which head and neck were of chicken origin on a quail body, a different result was obtained: no lymphoid cells were present in the thymus which was reduced to its epithelial component and this was of chick origin. On the other hand, if the chick contribution extended to the wings, as in the reciprocal combination, all thymus components were of chick origin. The spleen and the bursa of Fabricius in most instances did not differ in their cellular composition from the surrounding tissues; however in some chimeras a minor admixture of cells of the other species was also found. Overall these results suggest that hemopoietic stem cells destined to colonize intraembryonic organs arise in territories derived from the whole area pellucida excluding the prospective head-neck region. Furthermore, each hemopoietic organ rudiment appears to be colonized by precursors derived from adjacent territories.     

Proliferation and differentiation of androgenetic cells in fetal mouse chimeras

The properties of androgenetic cells and their ability to proliferate and differentiate were examined in post-midgestation chimeras. In several tissues, namely the brain, cardiac muscle, skeletal muscle and intestinal epithelium, the rate of proliferation of androgenetic cells was higher than that of normal cells in day 13 embryos. This higher rate of proliferation was however less pronounced by day 17–18 of development. It is possible that IGF2, a major growth factor regulating fetal growth, could play a role in the increased proliferation of androgenetic cells. Igf2 is also an imprinted gene that is expressed only when inherited paternally. Indeed, in the smooth muscle, cartilage and intestinal epithelium, patches of androgenetic (ag) cells exhibited higher levels of IGF2 mRNA than neighbouring wild-type cells. Surprisingly, we also detected expression of Igf2 in ag cells of ectodermal origin; this gene is not normally expressed in this lineage. This expression was observed in the brain, epidermis and in the epithelium of the tongue. We attempted to confirm the identity and differentiation status of ag cells with the help of cell-type specific antibodies and lectins. Evidence for only one of the cell types analysed, i.e. the goblet cells of the gut, suggests a delay or aberrant differentiation of ag cells.     

Developing nervous tissue induces formation of blood-brain barrier characteristics in invading endothelial cells: A study using quail-chick transplantation chimeras

Brain capillaries have structural and functional characteristics that constitute a regulatory interface, or “barrier,” between the blood and the brain. We have investigated the role of the neural tissue environment in the differentiation of the endothelial barrier, by transplanting embryonic brain fragments to the coelomic cavity, where they were vascularized by nonneural vessels, and fragments of embryonic mesoderm to the brain, where they were vascularized by neural vessels. A major problem in this approach is that when embryonic tissues are transplanted to an ectopic site, their own blood vessels survive and form a part of the new vascular system. This has made the results of previous experiments difficult to interpret. We overcame this problem by transplanting fragments of tissue that had not yet been vascularized from very young quail embryos to host chick embryos. These grafts did not contain vascular channels that could form part of a new vascular system. Furthermore, the distinctive quail nuclear morphology allowed us to demonstrate that the grafted tissue was, in fact, vascularized by the host vessels. Abdominal vessels vascularizing grafted neural tissue formed structural, functional, and histochemical features of the blood-brain barrier. In contrast, brain vessels vascularizing grafted mesodermal tissue were devoid of barrier characteristics. These results indicate that endothelial blood-brain barrier characteristics develop in response to some aspect of the neural environment.     

Mapping of the early neural primordium in quail-chick chimeras : I. Developmental relationships between placodes, facial ectoderm, and prosencephalon

Defined fragments of the anterolateral neural ridge and of the associated region of the neural plate of presomitic to three-somite stage quail embryos were grafted isotopically and isochronically into chick hosts. This resulted in the development of apparently normal brain and facial structures to which the contribution of the grafted tissue could be observed by means of the quail nuclear marker. It was shown that the anterolateral neural ridge contains the progenitor cells of the adenohypophyseal and olfactory placodes and also of the superficial ectoderm lining the nasal cavity and conchae and the superficial ectoderm of the beak. When the appropriate region of the neural ridge was involved in the quail-chick substitution, the egg tooth was made up of graft-derived cells. Grafting of the neural plate area adjacent to the "ridge" territory containing the placodal ectoderm revealed that the presumptive region of the hypothalamus is in contiguity with that of the adenohypophyseal placode. The same observation was made for the olfactory placode and the floor of the telencephalon from which the olfactive bulb later develops.     

Vasculogenesis and angiogenesis in the mouse embryo studied using quail/mouse chimeras

Using quail/chick chimeras, we have previously shown that different embryonic territories are vascularized through two distinct mecanisms, angiogenesis and vasculogenesis. Angiogenesis occurs in tissues of somatopleural origin, vasculogenesis occurs in territories of splanchnopleural origin. The aim of this work was to establish if these modes of vascularization were conserved in the mammalian embryo. Since in vivo manipulations with mammalian embryos are difficult to perform, we used a quail/mouse chimera approach. Mouse limb buds of somatopleural origin, and visceral organ rudiments of splanchnopleural origin, were grafted into the coelomic cavity of 2.5 day-old quail embryos. After four to seven days, the hosts were killed and the origin of the endothelial cells in the mouse tissues was determined by double staining with the quail endothelial and hematopoietic cell-specific marker, QH1 and mouse-specific VEGFR2 and VEGFR3 probes. Our findings show that the great majority of vessels which developed in the mouse limbs was QH1+, indicating that these tissues were vascularized by angiogenesis. Conversely, visceral organs were vascularized through the vasculogenesis process by mouse endothelial cells which differentiated in situ. These results demonstrate for the first time that in the mouse embryo, as previously shown in avian species, the tissues from somatopleural origin are vascularized by angiogenesis, while rudiments of a splanchnopleural origin are vascularized by vasculogenesis, both at vascular and lymphatic levels.     

Fate map of the avian anterior forebrain at the four-somite stage, based on the analysis of quail-chick chimeras.

To better understand the topological organization of the primordia within the anterior forebrain, we made a fate map of the rostral neural plate in the chick. Homotopic grafts at the four-somite stage were allowed to survive for up to 9 days to enable an analysis of definitive brain structures. In some cases, the topography of the grafted neuroepithelia was compared with gene expression patterns. The midpoint of the anterior neural ridge maps upon the anterior commissure in the closed neural tube, continuing concentrically into the preoptic area and optic field. Non-neural epithelium just in front of this median ridge gives rise to the adenohypophysis. Areas for the presumptive pallial commissure, septum, and prosencephalic choroidal tissue lie progressively more posteriorly along the ridge, peripheral to the telencephalic entopeduncular and striatopallidal primordia (the subpallium), and the pallium (olfactory bulb, dorsal ventricular ridge, and cortical domains). Subpallial structures lie topologically anterior to the pallial formations, and both are concentric to the septum. Within the pallium, the major cortical domains (Wulst and caudolateral, parahippocampal, and hippocampal cortices) appear posterior to the dorsal ventricular ridge. The amygdaloid region appears concentrically across both the subpallial and pallial regions. This fate map shows that the arrangement of the prospective primordia in the neural plate is basically a flattened representation of topological relationships present in the mature brain, though marked phenomena of differential growth and selective tangential migration of some cell populations complicate the histogenetic constitution of the mature telencephalon.     

脑的可塑性变化和功能重组

脑的可塑性和功能重组是近十多年来颇受关注的课题,导致人们对脑功能的认识更深化了一步。人们已逐渐认识到,神经系统折结构和机能完整靠外周伟入和效应器官的正常机能活动维持。用建立条件反射条件反射的训练可以使脑功能动态性调节得到加强和皮层功能定位域扩大,而一旦失去这种功能联系,各级中枢的替代性功能重组即不可避免地发生。     

Plasticity and rigidity in adaptor protein-2-mediated internalization of the TCR:CD3 complex

Many cell surface proteins are internalized via dileucine- or tyrosine-based motifs within their cytoplasmic domains by the heterotetrameric adaptor protein complex, AP-2. In this study we have examined how AP-2 mediates internalization of large cell surface receptors, such as the eight-chain TCR:CD3 complex. Although most receptors have a single signal that drives internalization, the TCR complex has two (D/E)xxxL(L/I) motifs and 20 Yxx? motifs. Using 293T cells, we show that AP-2 is completely dependent on both signals to mediate TCR internalization, because deletion of either completely blocks this process. Significant plasticity and redundancy were observed in the use of the Yxx? motifs, with a clear hierarchy in their use (CD3delta > CD3gamma >or= CD3zeta > CD3epsilon). Remarkably, a single, membrane-distal Yxx? motif in CD3delta could mediate approximately 75% of receptor internalization, whereas its removal only reduced internalization by approximately 20%. In contrast, significant rigidity was observed in use of the (D/E)xxxL(L/I) motif in CD3gamma. This was due to an absolute requirement for the position of this signal in the context of the TCR complex and for a highly conserved lysine residue, K128, which is not present in CD3delta. These contrasting requirements suggest a general principle by which AP-2 may mediate the internalization of large, multichain complexes.     

Plasticity of DNA methylation in mouse T cell activation and differentiation

Background

Circulating CD4⁺ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4⁺ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells.

Results

Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status.

Conclusion

We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.     

Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras.

Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.     

Temperature-reversible eruptions of vesicles in model membranes studied by NMR.

Deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) and freeze-fracture electron microscopy were used to study spontaneous vesiculation in model membranes composed of POPC:POPS with or without cholesterol. The NMR spectra indicated the presence of a central isotropic line, the intensity of which is reversibly and linearly dependent upon temperature in the L alpha phase, with no hysteresis when cycling between higher and lower temperatures. Freeze-fracture microscopy showed small, apparently connected vesicles that were only present when the samples were frozen (for freeze-fracture) from an initial temperature of 40-60 degrees C, and absent when the samples are frozen from an initial temperature of 20 degrees C. Analysis of motional narrowing was consistent with the isotropic lines being due to lateral diffusion in (and tumbling of) small vesicles (diameters approximately 50 nm). These results were interpreted in terms of current theories of shape fluctuations in large unilamellar vesicles which predict that small daughter vesicles may spontaneously "erupt" from larger parent vesicles in order to expel the excess area created by thermal expansion of the bilayer surface at constant volume. Assuming that all the increased area due to increasing temperature is associated with the isotropic lines, the NMR results allowed a novel estimate of the coefficient of area expansion alpha A in multilamellar vesicles (MLVs) which is in good agreement with micromechanical measurements upon giant unilamellar vesicles of similar composition. Experiments performed on unilamellar vesicles, which had been placed upon glass beads, confirmed that alpha A determined in this way is unchanged compared with the MLV case. Addition of the highly positively charged (extrinsic) myelin basic protein (MBP) to a POPC:POPS system showed that membrane eruptions of the type described here occur in response to the presence of this protein.