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.

Developmental changes of keratin expression in chick embryo olfactory epithelium in relation to cellular differentiation and neurogenesis in vivo and in vitro.

Olfactory embryogenesis was studied using an anti-chick keratin antibody on chick embryo sections as well as in vitro. Olfactory placodes form at embryonic day 3 (ED3) in the anterior facial ectoderm and invaginate to form the nasal pits. At ED5, the epidermal ectoderm and respiratory epithelium show the same dense cytokeratin immunoreaction. In contrast, absence of keratin expression in the basal part of olfactory epithelial primordium, in the deeper nasal pit area, coincides with one of the critical first steps of olfactory neurogenesis. However, beginning with periphero-central olfactory synaptogenesis at ED8, a new basal cell population starts to express keratin in the olfactory epithelium. Keratin positive cells appear to correspond, by their epithelial localisation and morphology, to sustentacular and basal cells. This interpretation was confirmed in vitro with ED14 chick primary olfactory cultures where TrKA immunoreactivity was used as a marker of horizontal basal cells (HBCs). After ED15, late keratin expression was detected in forming Bowman's glands. The density of keratin expressing basal cells was measured between ED10 and ED20, and appeared highest in the median part of the olfactory epithelium, the area of most active olfactory neurogenesis and neuronal maturation. Thus, keratin expression corresponds to a specialisation of horizontal basal cells as active neuronal stem cells.

Analysis of the functional maturation of olfactory neurons in chicks before and after birth.

There has been indirect evidence that the olfactory system of mammals could be functional shortly before birth. Taking advantage of the accessibility of bird embryos, we studied the functional maturation of the olfactory mucosa during embryonic development in birds. Using the combination of electrophysiological EOG recordings and immunohistochemical studies, it was possible to directly demonstrate for the first time that the olfactory system is functional during embryogenesis from embryonic day (ED) 13 and that the beginning of olfactory function coincides with the first localization of the calcium dependent calmodulin kinase II (CaMKIIalpha) in the dendrites of the olfactory receptor neurons. CaMKII and olfactory receptor genes are expressed much earlier in olfactory neurons, both involved in the sensory transduction, but the pattern of expression of CaMKIIalpha changes during the ontogenesis. The increase of EOG amplitude between ED13 and ED15 also coincides with the increase of the number of neurons presenting the dendritic localization of CaMKIIalpha. These results suggest that the enzyme CaMKII might play a role in the functional maturation of the olfactory mucosa.

Combination of 3' and 5' IgH regulatory elements mimics the B-specific endogenous expression pattern of IgH genes from pro-B cells to mature B cells in a transgenic mouse model.

To ensure the B cell differentiation stage specificity of the intronic Emu element and of the locus control region (LCR) that lies downstream of the IgH chain locus, we generated transgenic mice harboring a V(H) promoter-GFP reporter gene linked to the 3'LCR region and the Emu element. By flow cytometry, GFP(+) lymphocytes were observed amongst pro-B cells (B220(+)CD43(+)CD117(+)) and at all stages of differentiation up to mature B cells (B220(+)IgM(+)IgD(+)). Expression was strictly confined to cells committed to the B lymphocyte lineage as judged by the lack of GFP(+)Thy1,2(+) cells (T lymphocytes) and GFP(+)B220(-)CD117(+)CD43(+) cells (uncommitted lymphohematopoietic progenitors). Therefore, the Emu-GFP-3'LCR transgene is not expressed by hematopoietic stem cells, begins its expression in pro-B cells and is specifically active at all stages of B cell maturation. The combination of 3' and 5' IgH regulatory elements thus appears as a potentially useful cassette in transgenes that require a stringent and early B lineage-specific expression.