Neural stem cell separation from the embryonic avian olfactory epithelium by sedimentation field-flow fractionation.

The aim of the present study was to isolate neural stem cells from a complex tissue: the avian olfactory epithelium; by using sedimentation field flow fractionation (SdFFF). By using "Hyperlayer" elution mode, fraction collection and cell characterization methods, results shows that SdFFF could be a useful cell sorter to isolate an enriched, viable and sterile immature neural cell fraction from which the reconstitution of a complete epithelium was possible. In culture, SdFFF eluted cells first led to a "pseudoplacodal" epithelioid cell type from which derived "floating cells". These cells were then able to generate neurosphere-like structures which were composed of cell having many features of immature cells: undifferentiated, self-renewable and multipotentiality. Such a population might be used as a model to improve our understanding of the mechanisms of olfactory neoneurogenesis.

Differential responses of olfactory neurons to axotomy at embryonic and postnatal stages.

In the nervous system, apoptosis is a major process during embryonic and postnatal stages. In chick, experimental cell death can be obtained by axotomy. We have compared the responses of olfactory neurons to axotomy at embryonic stage E17 and postnatal stages.Forty-three chicken embryos and 32 young chickens less than 3 weeks old were used. We combined optic microscopy, electronic microscopy, terminal (TdT)-mediated dUTP-nick end labeling (TUNEL) method and gel electrophoresis of genomic DNA to analyze cell death. Cells in synthetic phase were labeled by bromodeoxyuridine injected i.p. and detected by immunohistochemistry. Apoptotic index and 5-bromo-2'-deoxyuridine (BrdU) labeling index were calculated for each stage. The Wilcoxon test was used for statistical analysis. A P value <0.05 was considered significant. Within 3 days following axotomy in E17 chicken embryos, there was no significant increase of apoptosis on the lesion side analyzed 3, 6, 12, 18, 24, 48 or 56 h later. A slight increase of bromodeoxyuridine incorporation appeared at 3 h, was weakly significant at 24 h (P=0.045) and the return to basal rate took place at 48 h. In postnatal stages, an apoptotic wave appeared 12 h after axotomy, reached a maximum at 24 h, and then decreased between 48 h and 72 h. A significant elevation of bromodeoxyuridine incorporation occurred on 48 h with a 24 h delay after the peak of apoptotic death. This differential response to axotomy in embryos and postnatal chickens might be due to a less complete maturation and higher plasticity of embryonic olfactory neurons corresponding to different requirement for survival and differentiation factors. Thus the embryonic or immature neurons would depend more on local epithelial environment and mature or postnatal neurons would require target-derived survival factors and die from apoptosis after their shortage resulting from axotomy.

Insulin-like growth factor I induced survival of axotomized olfactory neurons in the chick.

Insulin-like growth factor 1 (IGF1) receptor is expressed in avian olfactory neurons and IGF1 in the bulb. To explore the function of IGF1 in olfactory system in the chick, we infused IGF1 at the lesion site 0 and 12 h after olfactory axotomy. The animals were killed 1-3 days later. TdT mediated dUTP nick end labeling method and bromodeoxyuridine incorporation allowed the evaluation of programmed cell death and mitotic activity respectively in the olfactory epithelia of IGF1 treated or untreated lesioned animals and controls. IGF1 treatment suppressed the apoptotic wave, stimulated mitosis which peaked within 24 h (instead of 48 h), to return promptly to normal, and transiently maintained the number of calmodulin related kinase II expressing neurons at normal levels. It reveals a long lasting effect of IGF1 on the survival of lesioned olfactory neurons and transient effects on maintenance of differentiation and mitosis stimulation.

[The avian peripheral olfactory system: model for study of apoptosis and cellular regeneration]. / Le système olfactif périphérique aviaire: modèle d'étude de l'apoptose et de la régénération cellulaire.

UNLABELLED: A reliable model, usable in vitro and in vivo, is necessary for analysis of processes engaged during cell death, regeneration and differentiation. The peripheral olfactory system is an attractive model for studying these processes through its dynamic neurogenesis that occurs continually throughout the lifetime. STUDY AIM: The aim of this study was the analysis of these processes on an animal model. MATERIAL AND METHODS: We performed axotomy of the nerve olfactory on young animals and chicken embryos E17. Then we infused IGF-I (insulin-like growth factor-I) in the lesioned site. Death, regeneration and differentiation of cells were studied by immunocytology. RESULTS: After hatching, the section of the olfactory nerve induced a rapid neuronal apoptosis at the 24th hour followed by a wave of mitosis 24 hours later. In prenatal stages, the response to the axotomy was rather similar to a dedifferentiation. In postnatal stages, the IGF-I infusion at the lesioned site had a triple function: survival of mature neurons, maintenance of differentiation and stimulation of mitosis. The neoneurogenesis, which occurred from neuronal stem cells would depend on the maturation and environment of the olfactory neurons protected from apoptosis by IGF-I. CONCLUSION: The avian olfactory epithelium is a good model for analysis of cell death, regeneration and differentiation. The capacity of these neuronal stem cells to dedifferentiate makes then more primitive than the pluripotent cells, closer to totipotent embryonic stem cells.

Anosmin-1 is a regionally restricted component of basement membranes and interstitial matrices during organogenesis: implications for the developmental anomalies of X chromosome-linked Kallmann syndrome.

Kallmann syndrome is a developmental disease characterized by gonadotropin-releasing hormone (GnRH) deficiency and olfactory bulb hypoplasia. The gene underlying the X chromosome-linked form, KAL-1, has been identified for several years, yet the pathogenesis of the disease is not understood. By immunohistofluorescence and immunoelectron microscopy, we establish that the KAL-1 encoded protein, anosmin-1, is a transient and regionally restricted component of extracellular matrices during organogenesis in man. Anosmin-1 was detected in the basement membranes and/or interstitial matrices of various structures including bronchial tubes, mesonephric tubules and duct, branches of the ureteric bud, muscular walls of the digestive tract and larger blood vessels, precartilaginous models of skeletal pieces, muscle tendons, head mesenchymes, inner ear, and forebrain subregions. Our results suggest that this protein acts as a local, rather than a long-range, cue during organogenesis. In the olfactory system, anosmin-1 was detected from week 5 onward. The protein was restricted to the olfactory bulb presumptive region and later, to the primitive olfactory bulbs. We therefore suggest that the genetic defect underlying X-linked Kallmann syndrome disrupts the terminal navigation of the early olfactory axons or directly affects the initial steps of olfactory bulb differentiation. The mechanism of the GnRH deficiency is also discussed, relying on the evidence that anosmin-1 is present in the medial walls of the primitive cerebral hemispheres, along the rostro-caudal migratory pathway of the GnRH-synthesizing neurons, at 6 weeks. Finally, the present results strongly suggest that the renal aplasia observed in about one third of the affected individuals results from primary failure of the collecting duct system.

Avian olfactory receptors: differentiation of olfactory neurons under normal and experimental conditions.

The bird olfactory system has a simple structure and affords an attractive developmental model for the study of olfactory morphogenesis and differentiation. We have cloned and characterized several chick olfactory receptor (COR) genes belonging to the superfamily of seven-transmembrane domain proteins. In situ hybridization analysis of their spatiotemporal patterns of expression during development reveals several important characteristics. COR expression starts early in placodal cells (Embryonic Day 5, E5). Changes in their expression pattern then correlate with the onset of synaptogenesis (E8). The adult pattern, achieved before hatching, shows that cells expressing a particular COR are not regionalized within the epithelium. By double-label in situ hybridization, we clearly demonstrate that a single cell does not coexpress different COR genes (or subsets of CORs) at any stage of development. Following bulbar deafferentation, COR expression ceases more rapidly than expected from previous axotomy experiments. Concomitantly, a reactivation of the Cash-1 gene, which is involved in early neuronal specification, could be an early sign of olfactory neuronal regeneration. Modulation of COR and Cash-1 expression points to a simultaneous process of neuronal degeneration and regeneration in the olfactory epithelium after axotomy. COR expression is restricted to the olfactory epithelium except during early stages (before synaptogenesis). At that time, cells distributed along the olfactory nerve, from the placode to the anterior telencephalon, also express CORs. This cell population is different from the luteinizing hormone releasing hormone neurons migrating from the placode. Our results show that the olfactory neurons or neuroblasts choose to express one COR before establishing functional connections with the bulb. Later on, bulboepithelial connections seem important not only for olfactory neuron survival but also for stimulation of COR expression. In addition, beyond their implication in functional odor detection, CORs could be involved, at early stages, in processes of olfactory morphogenesis, including the establishment of a bulbar chemotopy.

Early expression of the KAL gene during embryonic development of the chick.

The human KAL gene is responsible for the X chromosome-linked Kallmann syndrome, which consists of the association of hypogonadotropic hypogonadism and anosmia. The human and chicken KAL genes have been isolated. Using in situ hybridization, we studied KAL gene expression during development of the chick. We have previously reported that, from embryonic day 8, the expression is almost restricted to definite neuronal populations in the central nervous system, most of which still express the gene after hatching. Here we report that the KAL gene is also expressed during early embryonic development (days 2-8) in various endodermal, mesodermal, and neurectodermal derivatives. In most endodermal and mesodermal derivatives, the expression is transient and precedes cell differentiation. In contrast, the expression in the nervous system concerns postmitotic central neuroblastic populations, most of which still express the gene after differentiation. In accordance with such a spatio-temporal pattern of expression, we suggest that the KAL gene is involved both in morphogenetic events and in neuronal late differentiation. In addition, the absence of detectable expression of the KAL gene either in the embryonic olfactory epithelium or in the surrounding nasal mesenchyme reinforces the hypothesis that Kallmann's syndrome results from a central olfactory target cell defect.

Expression of IGF-I and -II mRNA in the brain and craniofacial region of the rat fetus.

Insulin-like growth factors (IGF-I and -II) are present in the brain during development, with high levels of both being also found in the periphery particularly in the embryo. IGFs in the brain are believed to stimulate the proliferation of neuronal and glial precursors and their phenotypic differentiation. Using in situ hybridization, we have investigated the distribution of cells producing IGF-I and -II in the rat fetus during the second half of prenatal development with special emphasis on the peripheral and central nervous system. High levels of IGF-I mRNA were found in the olfactory bulb and in discrete neurons of the cranial sensory ganglia, notably in the trigeminal ganglion, as early as 13 days of gestation, in the pineal primordium of 18 day old fetuses, and in discrete groups of cells in the cochlear epithelium located laterally outside the forming spiral organ, in day 13 to 21 fetuses. High levels of IGF-II mRNA in the brain, besides the choroid plexus and the leptomeninges, were detected in hypothalamus, in the floor of the 3rd ventricle at all stages studied, in the pineal primordium at 18 days and in the pars intermedia of the pituitary or in the Rathke's pouch epithelium from which it is derived, with progressive fading towards the end of the gestation. In the peripheral nervous system the IGF-II mRNA was only found in association with the vascular endothelia of the ganglia. IGF-II mRNA in the nervous system was found in highly vascularized areas, meninges, blood vessels and choroid plexuses. It is thus associated with structures involved in the production of extracellular fluids and/or substrate transport and supply in the nervous tissues. A more specific role in the differentiation or fetal endocrine function should be considered for IGF-II in cells producing melatonin and melanocyte stimulating hormone (MSH) in the pineal and pituitary glands, respectively. The presence of IGF-I mRNA in the nervous system could be associated with fiber outgrowth and synaptogenesis in the cases of olfactory bulb and developing iris. The role of IGF-I in restricted populations of cells of the cochlear epithelium and in the pineal gland is unclear and requires further investigations including a search for IGF-I receptors in possible target cells. In the sensory ganglia, the presence of high levels of IGF-I mRNA eventually corresponds to the production, by post-translational processing, of the amino-terminal tripeptide of IGF-I, which might represent a neurotransmitter for these sensory neurons.

Expression of beta-nerve growth factor receptor mRNA in Sertoli cells downregulated by testosterone.

Nerve growth factor (NGF) is synthesized in male germ cells. The NGF receptor (NGFR) mRNA was found in the Sertoli cells of rat testis. Hypophysectomy increased both NGFR mRNA in testis and the number of NGFR hybridizing cells in seminiferous tubules. This was suppressed by treatment with chorionic gonadotropin or testosterone, but not with follicle-stimulating hormone. The NGFR mRNA also increased after destruction of Leydig cells or blocking of the androgen receptor. This suggests that NGF produced by male germ cells regulates testicular function in an androgen-modulated fashion by mediating an interaction germ and Sertoli cells.

Detection of nerve growth factor and its mRNA by separate and combined immunohistochemistry and in situ hybridization in mouse salivary glands.

Intense labelling of secretory cells in the male mouse submandibular gland was observed after in situ hybridization using mouse nerve growth factor (NGF) cDNA probes. Under the same conditions, sparse less intensely labelled cells were also found in the sublingual gland. Hybridization to a chicken NGF cDNA probe gave weak labelling on the glands in accordance with a weak cross-hybridization between mouse NGF mRNA and chicken NGF cDNA probes, whereas no labelling was seen using pUC9 DNA as a hybridization probe. A combination of in situ hybridization and immunohistochemistry was also carried out on the same sections of submandibular gland. A good correlation was seen between actively synthesizing and intensely immunoreactive cells in the gland. The technique described here allows the detection of individual cells synthesizing relatively low levels of NGF. The combination of in situ hybridization and immunocytochemistry on the same section should be particularly useful in cases where NGF is transported away from its site of synthesis.

Parasympathetic neurons of salivary gland ganglia in the fetal and postnatal rat are substance P-like immunoreactive.

Substance P-like immunoreactivity (SPLI) of neuron cell bodies is described here in the parasympathetic ganglia of salivary glands in rat fetuses. When grafted to the anterior eye chamber of adult rats, outgrowth of SPLI fibers was also observed around fetal and postnatal ganglia. These observations are significant for the understanding of salivation mechanisms. They also imply the importance of substance P or related compounds in parasympathetic peripheral neurons. The graft experiments indicate a substantial morphological plasticity of these SPLI neurons on perturbation.

Silver impregnation of intrinsic sensory innervation of rat iris in toto.

A silver impregnation technique (Linder, 1978) has been applied to whole mounts of the rat iris. The results suggest that only sensory fibres, both myelinated and non-myelinated, are stained. They disappear only after a trigeminal lesion and their distribution is different from that of catecholaminergic intrinsic fibres. Staining of the iris reveals a conspicuous pattern of innervation, characterised by a circular bundle and a thin plexus in the ciliary body, and by prominent bundles of fibres with a loose network of thin smooth fibres in the external part of the dilator plate and with a denser network in the central area. Nerve endings are seen on the dilator plate, in the sphincter as well as in the ciliary body. It is possible by a slight modification of the technique to stain myelinated and non-myelinated fibres separately. It results in a deep staining of the myelin while thin fibres are relatively clear. This method provides clear and reproducible staining of the nerves of the iris. It can be combined with various histochemical and immunocytochemical techniques. This will permit further studies to be made on the development of sensory and central nervous tissues, when grafted to the normal or the selectively denervated iris.

Restrictions of developmental capacities in the dorsal root ganglia during the course of development.

By grafting ganglia from embryonic quails into the neural crest migration pathway of 2-day chick embryos, it was previously demonstrated that all type of ganglia possess more developmental potentialities than those normally expressed in the normal course of development. Namely autonomic neurones with catecholamine and adrenomedullary cells can be obtained from grafted spinal ganglia. The latter also yield sensory neurons to the host dorsal root ganglia (DRG) but only if they are taken from the donor before 8 days of incubation. In the present article we show that the capacity to differentiate sensory neurons in back-transplantation experiments can be correlated with the presence in the donor DRG of cycling neuronal precursors. Once all the neurons have been withdrawn from the cell cycle - an event which occurs first in the mediodorsal and then in the lateroventral area of the ganglion - the DRG cell population gives rise exclusively to autonomic ganglion cells in the host. It is concluded that in the conditions of the back-transplantation experiments, the postmitotic neurons contained in the donor ganglion do not survive. Therefore, the neurons and paraganglion cells which differentiate in the host arise from still undifferentiated precursor cells. This indicates that besides sensory neuron precursors the embryonic DRG cell population also contains precursor cells for the autonomic differentiation pathway.

The neural crest: its relations with APUD and paraneuron concepts.

The neural crest cells give rise to a large variety of derivatives including neural, mesenchymal, APUD and/or paraneuron cell types. A better knowledge of these derivatives was partly achieved through studies using Le Douarin's quail/chick marker system. We review here evidences which were thus provided for a neural crest origin of calcitonin containing cells, carotid body, aortic paraganglia, adrenomedulla, and against a neurectodermal origin of enterogastric and respiratory tract endocrine cells. The role of neural crest cells in Pearse's APUD system is discussed. The results implicate that an explanation for the common properties of these cell types and their pathological and biochemical significance should not be looked for in a common embryological origin but at another level. The place of neural crest and, more generally, neurectoderm derivatives in the paraneuron concept of Fujita is examined. The relevance of the epithelial origin of these cell types to their "receptosecretory" function is stressed. Considering neural crest itself as a unique system is still questioned and discussed here. Its ubiquity and penetration of other systems is pointed out as a widespread phenomenon which is not restricted to APUD and paraneuron systems.

Mesectodermal capabilities of the trunk neural crest of birds.

Orthotopic transplantation experiments have shown that in birds, under normal conditions, mesectodermal capabilities seem restricted to the cephalic neural crest down to the level of the 5th somite. In the present study the mesectodermal capabilities of trunk and lumbar neural crest were investigated at early stages of development by heterotopic, heterospecific transplantation of the neural primordium. The quail-chick nuclear marker system was used to identify the grafted cells. Mesectodermal cells did not arise from the trunk neural crest when this was implanted orthotopically, even though the neural primordium was taken early in development at the level of unsegmented plate mesoderm just anterior to Hensen's node. Mesectodermal derivatives (connective tissues, dermis and muscle but no cartilage or bone) developed from the same trunk neural crest fragments when they were heterotopically grafted at the cephalic level and mixed with host cephalic neural crest cells. These host cephalic neural crest cells emigrated from the contralateral neural primordium when the graft was unilateral or from the fringe area of the operation in cases of bilateral transplantations. As a control, unsegmented paraxial mesoderm was inserted alongside the cephalic neural tube; its cell did not migrate ventrally in the neural crest-derived area and they differentiated in the dorsal region of the host. These results indicate that mesectodermal capabilities, thought reduced, exist in the trunk neural crest at early stages of development but the differentiation of these mesectodermal derivatives is largely dependent upon environmental influences which may be found in early cephalic levels.