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Microglia are the resident macrophage population of the central nervous system (CNS). Adequate microglia function is crucial for the homeostasis of the CNS in health and disease, as they represent the first line of defence against pathogens, contributing to immune responses, but are also involved in tissue repair and remodeling. It is therefore crucial to better understand microglia origin and homeostasis. Much controversy remains regarding the nature of microglial progenitors, as the exact contribution and persistence of embryonic and post-natal hematopoietic progenitors to the adult microglial pool in the steady state remained unclear. In this study, we show that post-natal hematopoietic progenitors do not significantly contribute to microglia homeostasis in the adult brain in mice. In vivo lineage tracing studies established that adult microglia derives from primitive hematopoietic progenitors that arise before embryonic day 8. These results identify microglia as an ontogenically distinct population in the mononuclear phagocyte system and have implications for the use of embryonically-derived microglial progenitors for the treatment of various brain disorders.

In vertebrate embryos, hematopoiesis takes place in two successive waves of hematopoietic precursors (HP). While the forerunners of the first wave, generated in the yolk sac (YS), present maintenance capacity and reduced differentiation, those from the latter have all the characteristics of hematopoietic stem cells (HSC). They are able to differentiate into all lineages of blood and help ensure long-term hematopoietic reconstitution of irradiated mice lethal. The transcription factor GATA-3 is expressed in the embryo during the stages of determination and generation of HSCs. It's never expressed to equivalent levels in the YS. GATA-3 could play an important role in the generation of HSCs and the acquisition of various properties of hematopoietic precursors of the two sites. We first developed an experimental approach to perform functional analysis of genes with different expression between the two sites generated hematopoietic precursors of the embryo.The in situ electroporation technique is most effective to disrupt gene expression at the time the choice of precursors and retroviral transduction permit HP target at the time of their generation. The analysis of ectopic expression of GATA-3 in the HP YS using retroviral infection show that GATA-3 is able to amplify and maintain immatures HP (article in progress). Tests carried out in parallel by performing the forced expression of GATA-5 (naturally absent from SV), or GATA-1 overexpression, we have concluded to the specificity of these effects. Dramatically, GATA-3 is also capable of amplifying a population responding to a phenotype of type mesodermal / pre-hematopoietic. In conclusion, these results show that GATA-3 could be involved in phenomena related to the maintenance of the quota of HSCs.; Dans l'embryon de vertébrés l'hématopoïèse se met en place à partir de deux vagues successives de précurseurs hématopoïétiques (PH). Alors que les précurseurs de la première vague, générée dans le Sac Vitellin (SV), présentent des capacités de maintenance et de différenciation réduites, ceux qui sont issus de la seconde présentent toutes les caractéristiques des cellules souches hématopoïétiques (CSH). Elles sont en effet capables de se différencier dans tous les lignages sanguins et permettent d'assurer, à long terme, la reconstitution hématopoïétique de souris létalement irradiées.Le facteur de transcription GATA-3 est exprimé dans l'embryon au cours des phases de détermination et de génération des CSH. Il n'est jamais exprimé aux stades équivalents dans le SV. GATA-3 pourrait jouer un rôle important dans la génération des CSH et dans l'acquisition des propriétés différentes des précurseurs hématopoïétiques des deux sites.Nous avons développé une démarche expérimentale permettant d'effectuer l'analyse fonctionnelle de gènes présentant une expression différente entre les deux sites de génération des précurseurs hématopoïétiques de l'embryon. Nous avons sélectionné l'électroporation in situ pour perturber l'expression génique au moment de la détermination de ces précurseurs, et la transduction rétrovirale pour cibler les PH au moment de leur génération.Nous avons réalisé l'expression ectopique de GATA-3 dans les PH du SV en utilisant l'infection rétrovirale. Nos résultats montrent que GATA-3 est capable d'amplifier et de maintenir des PH immatures. Des tests effectués en parallèle en effectuant l'expression forcée de GATA-5 (naturellement absent du SV), ou la surexpression GATA-1, nous ont permis de conclure à la spécificité de ces effets. De façon remarquable, GATA-3 est également capable d'amplifier une population répondant à un phénotype de type mésodermique/pré-hématopoïétique.En conclusion, nos résultats montrent que GATA-3 pourrait être impliqué dans les phénomènes liés au maintien du contingent de CSH.

Immunohistochemistry was used to detect markers of the vascular, stromal and hematopoietic cell compartments in the human embryo and early fetus, from 3 to 15 weeks of gestation. CD34 expression was consistently observed at the surface of vascular endothelial cells from off earliest stages tested, at the single exception of embryonic liver blood vessels. Yolk sac hematopoiesis was very transient and limited to primitive erythropoiesis. Clusters of erythroblasts, monocytes and granulocytes appeared from 4 to 5 weeks of gestation in the liver rudiment. The early development of the bone marrow was marked by the rapid invasion, at 8 weeks, of long bone cartilaginous rudiments by CD68+ osteoclast precursors, CD34+ endothelial cells and by preosteoblasts, leading to the development of large vascular sinuses between ossifying trabeculae. Endogenous erythro- and granulopoiesis developed from week 11 in primary logettes always organized around an arteriole, in a loose stromal mesenchymal network established between the media of these arterioles and the sinusal endothelium. Round, hematopoietic CD34+ cells were seen occasionally in yolk sac blood vessels. In the liver they were rare and intermingled as single cells in the hepatocyte cords; strikingly, CD34+ hematopoietic cells could seldom be detected in the developing bone marrow. In contrast, compact clusters of non-endothelial, round CD34+ CD45+ hematopoietic cells were detected, during the 5th week of development, in close association with the ventral wall of the dorsal aorta. These cells exhibit phenotypic and functional characteristics of very primitive hematopoietic progenitors. This observation is in striking correlation with the evidence accumulated in animal models that stem cells for the late embryonic and adult hematopoietic systems develop inside the embryo per se, in the vicinity of the dorsal aorta. We thus suggest that these aorta-associated CD34+ cells, that exhibit an anatomic localization similar to that of the intraembryonic stem cells identified in the avian and murine embryo, are the real stem of human hematopoiesis.

In a search for assays that might facilitate identification of pluripotent stem cells with extended potentialities, we analysed the properties of hematopoietic progenitor cells detected in the extraembryonic yolk sac and in the intraembryonic part of human embryos between approximately 28 and 45 days of development. Cells from the yolk sac, the liver rudiment and the remainder of the embryo were plated in semi solid methylcellulose colony-assays supplemented with combinations of cytokines. Large BFU-E-derived colonies as well as granulocytic colonies were detected in every yolk sac sample. Interestingly, progenitor cells were also detected in the intraembryonic part, outside the liver and a subclass of these progenitors were detected that generated large granulomacrophagic colonies capable of generating secondary colonies when replated. These were preferentially located in the embryo. Colony-assays initiated with CD34+ cells sorted from the different tissues confirmed these data. These results first indicate that embryonic progenitors exhibit unique phenotypic features, and second, analysis of the distribution of progenitors between the different tissues may suggest the existence of other sites of hematopoietic production. More detailed analysis of the potentialities of these progenitors should now be assessed in vitro in cocultures assays and in vivo by reconstituting immunodeficient mice.

Chimeras built between a quail embryo and a chick yolk sac have led to the demonstration that, in the avian model, intra-embryonic hemopoietic stem cells (HSC) relay HSC originating from the yolk sac. Converging experimental approaches have localized the emergence of intraembryonic HSC to the region of the aorta. Today's session will be dedicated to reviewing experimental data in mouse and man that clearly indicate that this model of hemopoietic ontogeny is also operating in mammals.

An epidemiological survey on avian chlamydiosis, carried out by serological probing in 8 pigeon breeders representative of New Caledonian livestock, combined with bacteriological research on pigeon organs and droppings was set up in New Caledonia in order to determine the prevalence rate of this infection and to adapt sanitary regulations concerning pigeon imports. All sera collected (230) were analysed by complement fixation test (CFT). The organs were diluted in sucrose solution, then stored frozen (-70 degrees C), until inoculation of the yolk-sac of 3 6-8-day-old embryonated eggs (2 blind passages). Yolk-sac smears stained according to the Gimenez method were made in order to detect intracellular chlamydial organisms. Seventeen sera out of 230 were found to be positive, ie 7.4% of the test sample (confidence interval to 95% = 4.0 to 10.8%). The carrier pigeons were significantly more infected (17.8%) than pigeons of other breeds in New Caledonia. These results resulted in the sanitary authorities easing restrictions on imports of seropositive pigeons by imposing a 45-day compulsory quarantine with daily administration of chlortetracycline at the rate of 150 mg per 1 of drinking water.

Histological sections of a stage 6b human embryo (13-15 days old) were immunohistochemically stained for specific alphafoetoprotein (AFP). Conspicuous AFP+ endodermal cells were observed in the yolk-sac wall using the ABC peroxidase technique, which gave consistent results on dismounted and decolorized old slides.

Iodine is accumulated in the egg yolk during oogenesis. It is transferred during incubation through the walls of the yolk sac (extraembryonic endomesoderm surrounding the yolk) to the extraembryonic, and thence to the embryonic circulation. Iodine exchanges would probably occur between both compartments, the egg yolk and the embryonic blood in which the iodine level increases from day 10 to 20 of incubation. The increase of thyroid gland iodine content parallels that of the plasma iodine level. Our results suggested that the pituitary gland, which acts directly on the developing thyroid gland, might also influence the iodine exchanges across the yolk sac wall, thereby affecting the thyroid by increasing the iodine supply.

With the aid of scanning electron microscopy the normal pattern of both faces of amnion and visceral yolk sac were described. The inner face of the amnion turned towards the amniotic cavity is overlaid by large pavimentous cells bearing numerous microvilli. The opposite face, which delimits the exocoelome, consists of flattened fibroblasts separated by a fundamental connective substance. The two faces of the visceral yolk sac also show very different patterns. The exocoelomic face is covered by pavimentous cells ; these are smaller and bear less microvilli than amniotic cells. The yolk sac endoderm appears built up with prismatic or cubic cells, whose apical part is bulging and possesses a dense covering of microvilli. Amniocentesis effects are then reported. This intervention seems to provoke fetal membrane modifications only after 15 hours. These modifications essentially concern the visceral yolk sac whose exocoelomic cells are covered by more numerous microvilli which may show an intensification of a secretory process.

In the African locust, the secretory dynamics of the A1 and A2 median protocerebral neurosecretory cells (M-NSC) is evaluated by the product content and the gonadotropic action of these cells. The axotomy of the A1 and A2 M-NSC disturbes their secretory dynamics. The axonal regeneration, if it leads to a reconnexion with the corpora cardiaca (CC), restores a normal secretory dynamics of the M-NSC. Without reconnexion to the CC, the nervi corpori cardiaci interni (NCCI) do note regenerate or regenerate to form new nerve endings. In these cases, the secretory dynamics of the M-NSC remains more or less inhibited specially that of the A2 M-NSC which can be completely suppressed. A functional state of the A2 M-NSC could be necessary for the beginning of the vitellogenesis. In the CC, separated from the regenerating M-NSC, the A1 and A2 neurosecretory products are transformed into large globules which became intensively basophil before disappearance.

Morphogenesis like cyto-differentiation is expressed in Mouse teratocarcinomas. The presence in certain tumours, of yolk-sac cavities combining trophoblast, Reichert's membranes and parietal distal endoderm in spite of the absence of included embryoïd structures, suggests that there is a precursor cell common to there extra-embryonic components.

The guinea-pig yolk sac endoderm has been observed through scanning and transmission electron microscopy to correlate ultrastructure and function of the cell surface. Using ferritin, different stages of protein transfer have been traced and a secretory activity of the vitelline endoderm during the last third of pregnancy has been suggested.