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FIGURE 1
S100Z expression in the main olfactory system of larval Xenopus laevis. (a) Immunohistochemical staining for S100Z revealed cells with neuron-like morphology, bipolar shape with dendritic knobs, throughout the whole MOE of the peripheral olfactory organ. The axon-like processes of S100Z-positive cells extended into the ON. (b) Axons of S100Z-positive cells projected into glomeruli within the glomerular layer of the OB in premetamorphic tadpoles. No S100Z-positive cell bodies were visible in the different layers of the OB. Structures of the olfactory system and glomerular clusters are outlined with a dotted white line. A, anterior; IC, intermediate cluster; L, lateral; LC, lateral cluster; M, medial; MOE, main olfactory epithelium; OB, olfactory bulb; ON, olfactory nerve; P, posterior.
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FIGURE 2
S100Z expression exclusively in ORNs. (a) Schematic overview of the peripheral olfactory organ of premetamorphic X. laevis. The major cell types of the main olfactory epithelium are highlighted. (b) Stem cell marker TP63 (magenta) was localized in cell nuclei in basal cell layers, highlighting basal stem cells. S100Z-positive cells (yellow) occurred in more intermediate layers of the olfactory epithelium. No co-localization of S100Z and TP63 was found. (c) Supporting cells expressing cytokeratin type II (Cytok II, magenta) did not express S100Z (yellow). (d) Membrane-localized NCAM1 (magenta) is a marker for developing neurons. A subset of S100Z-expressing cells (yellow) was clearly surrounded by NCAM1 immunoreactivity (white dotted circle) and dendrites showed co-localization. (e) Microtubules were labeled with antibodies against beta tubulin (TUBB, magenta). TUBB was found at cellular boundaries and apical appendages, presumably cilia (arrow). S100Z signal only clearly occurred in soma, dendrite, and knob. (f) Individual S100Z-positive neurons show no co-localization of S100Z and TUBB in their dendritic appendages. TUBB expression is expected in cilia, whereby S100Z was located in shorter protrusions from the dendritic knob, potentially indicating microvilli (white dotted circle). MOE, main olfactory epithelium; NC, nasal cavityl; OB, olfactory bulb; ON, olfactory nerve; ORN, olfactory receptor neuron.
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FIGURE 3
Laterally shifted distribution of S100Z-positive cells. (a) Maximum projection of a larval X. laevis MOE with an overlay of all S100Z-positive cells. The color gradient from blue (lateral) to red (medial) indicates the angular position within the MOE (zero equals the middle of the MOE). Crosses mark the average coordinates of the manually drawn apical and basal MOE boundaries. Angles were determined between each cell position and the vector that defined the middle of the MOE (dashed line). (b) The bimodal distribution of S100Z-positive cells (blue) was shifted toward a lateral position (n = 11 larvae). The general ORN population was labeled via a biocytin backfill and showed a unimodal distribution centered around the middle of the MOE (gray, n = 4 larvae). (c) The mean number of S100Z-positive cells in the lateral MOE was significantly higher than in the medial MOE (n = 11 larvae). KruskalWallis test. **, p < .01. (d) The number of S100Z-positive cells in the lateral MOE was always higher than in the medial MOE in all animals investigated (n = 11 larvae). (e) The relative position within the MOE on the apical-basal axis was not different between the lateral and medial S100Z-positive populations, a tendency toward a more basal position on the lateral side notwithstanding. (f) The medial S100Z-positive population featured a correlation between the position on the ventral-dorsal and basal-apical axis. Cells deeper in the medial MOE were shifted to apical positions. MOE, main olfactory epithelium.
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FIGURE 4 Mature and immature receptor neurons express S100Z exclusively in the MOE and not in the VNO of X. laevis tadpoles. (a) Biocytin-streptavidin-stained ORNs (magenta) and S100Z-positive ORNs (yellow) were present throughout the whole MOE. Some ORNs showed co-localization with both proteins (highlighted with circles). (b) The axonal projection of biocytin-streptavidin labeled ORNs (magenta) was found in all glomerular clusters within the main OB and AOB of X. laevis tadpoles, but axonal projection of S100Z-positive neurons (yellow) was exclusively detected in the LC and IC. (c) Biocytin-streptavidin stained ORNs (magenta) were located in the VNO and MOE of X. laevis tadpoles. S100Z-expressing neurons (yellow) occurred in the MOE. (d) Immature pax6-positive cells (magenta) and S100Z-positive ORNs (yellow) were present throughout the whole MOE. Although many, not all cells were double positive (e.g., white dotted oval). (e) Axonal projections of pax6-positive neurons, indicating an immature status, were detected in the LC and MC within the glomerular layer and different cell types (magenta) within the OB. Some, but not all glomeruli within the LC, showed co-localization for S100Z and pax6. A, anterior; AOB, accessory olfactory bulb; IC, intermediate cluster; L, lateral; LC, lateral cluster; M, medial; MC, medial cluster; MOE, main olfactory epithelium; OB, olfactory bulb; ON, olfactory nerve; ORN, olfactory receptor neuron; P, posterior; SC, small cluster; VNO, vomeronasal organ.
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FIGURE 5
Distinct, but partially overlapping expression patterns of calretinin/S100Z. (a) In premetamorphic X. laevis, ORNs across the whole MOE expressed calretinin (magenta) and S100Z (yellow). Not all but many cells showed co-localization of the two calcium-binding proteins (e.g., white dotted circles). (b) In premetamorphic animals, the axonal projection of calretinin-expressing neurons (magenta) targeted the AOB, LC, and MC. Within the LC, calretinin- and S100Z-positive (yellow) glomeruli were detected and some were double-positive (white dotted ovals). (c) Expression patterns of calretinin (magenta) and S100Z (yellow) in glomeruli of the lateral cluster in four different premetamorphic animals. The expression patterns were very variable between individuals, but some trends for recurring patterns were apparent. The outer lateral glomeruli within the LC had the tendency to be either S100Z- or calretinin-positive (asterisks), whereas in the medial part of the LC, more glomeruli were S100Z-positive. The most caudal glomerulus in the middle of the LC was often double positive for S100Z/calretinin (arrows). (d) Immunohistochemical staining for calretinin (magenta) labeled neurons throughout the whole VNO in premetamorphic animals, but no S100Z expression (yellow) was found in the VNO. A anterior; AOB, accessory olfactory bulb; L, lateral; LC, lateral cluster; M, medial; MC, medial cluster; MOE, main olfactory epithelium; ORN, olfactory receptor neuron; P, posterior; VNO, vomeronasal organ.
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FIGURE 6 S100Z expression restricted to the middle cavity after metamorphosis. (a) In postmetamorphic X. laevis calretinin-positive neurons (magenta) were localized in the middle cavity (MiC) and principal cavity (PC). S100Z expression (yellow) occurred only in ORNs of the MiC. (b) In postmetamorphic animals, S100Z-expressing neurons within the olfactory epithelium of the MiC exclusively projected into the lateral glomerular cluster. No expression of S100Z was found in the PC. (c) Immunohistochemical staining for calretinin (magenta) labeled neurons throughout the whole VNO in postmetamorphic animals. No S100Z expression (yellow) was detected in the VNO. A, anterior; L, lateral; LC, lateral cluster; M, medial; MiC, middle cavity; ON, olfactory nerve; ORN, olfactory receptor neuron; P, posterior; PC, principal cavity; VNO, vomeronasal organ.
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calb2 (calbindin 2m aka calretinin) expression in X. laevis embryo, NF 48/52, assayed via immunohistochemical staining (magenta) showing labeled neurons throughout the whole vomeronasal organ/Jacobsons organ in premetamorphic tadpoles.
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