PMC

Wnt reporter activated “connecting zone” cells in developing olfactory nerve layer (ONL) during glomerulus formation from E18.5 to P14 of mice. (A–A2) X-gal staining of whole forebrain (A) and 150-µm-thick sections of sagittal (A1) and coronal (A2) olfactory bulb at E18.5. Arrowheads indicate the intensely stained X-gal products on the bulb surface/olfactory nerve layer. Arrows indicate the sharp border between the stained bulb and non-stained forebrain surface. Note that no positive staining was present inside the bulb in the thick sections (A1,A2). (B) Double immunolabeling of βgal (green) and NCAM (red) on the sagittal section of E18.5 heads. Intensely immunolabeled βgal⁺ cells formed a multiple-cell layer (arrowheads) in the connecting zone/developing inner ONL where some NCAM⁺ olfactory axons crossed the connecting zone and entered into the forming glomerular layer (arrows). Asterisks indicate the thick bundles of NCAM⁺ olfactory axons crossed through the cribriform plate (cp). (C,C1) The βgal⁺ cells (green) in the ONL of P3 horizontal bulb sections. Arrow (in C,C1) indicates the same region with different magnifications for the βgal⁺ process as a capsule-like structure surrounding the forming glomerulus immunolabeled by OMP (red) on the bulb side. Arrowheads (in C) indicate the area of significantly expanded βgal⁺ multiple-cell layer in ONL. (D,D1) The thickness and density of the βgal⁺ multiple-cell layer was increased further (arrowheads) in P5 ONL. βgal⁺ processes were still found in the periglomerulus (arrows) on the bulb side. (E,E1) The thickness and density of βgal⁺ cells were significantly declined and restricted to the inner most ONL at P14 (arrowheads) when most glomeruli are formed. The βgal⁺ process mixed with OMP⁺ olfactory axons were found in the olfactory axonal entrance to the glomerulus (arrows). OB, olfactory bulb; Glo, glomerulus (visualized by OMP⁺ olfactory axons); OMP, olfactory marker protein. The cell nuclei were counterstained by DAPI (blue in C–E1).

a, ultrastructural detail of the numerous collagen fibrils (double arrowheads) connecting cells of the connective sheath to the basement membrane of epidermal cells of the hair peg of mouse skin at about 18 days of embryonic life. 12 000×. b, two hair pegs from ventral epidermis in embryonic rat of 18 days, showing a radial papilla (double arrowhead). 450×. c, bent hair bulb (double arrowhead) of a 7–8 day postpartum mouse tail. A group of fibroblasts (arrowhead) are external to the bulb while the IRS (arrow) is partially cornified. 250×. c, immunofluorescence for trichohyalin in IRS and medulla of longitudinally sectioned ventral hairs of adult rat. 350×. d, immunofluorescence for trichohyalin in IRS and medulla of longitudinally sectioned ventral hairs of adult rat. 350×. e, immunofluoresent IRS and medulla cells in cross-sectioned ear rabbit hairs. 400×. E, epidermis; F, fibroblasts; I, IRS; ME, medulla; P, hair peg; TH, trichohyalin immunolabelling.

Localization and lineage identity of the TOPgal-labeled cells in the olfactory bulb anlage. (A) The Wnt reporter TOPgal construct. (B) Wholemount X-gal staining (blue) of E12 heads of TOPgal mice demonstrated the positively stained (blue) island in the presumably olfactory bulb anlage (red arrow). Note that the dominantly stained X-gal products in the rostral telencephalon and facial regions (asterisks) of the adjacent area faithfully reflected the known activation of Wnt signaling in these sites. (C) Double immunolabeling for β-galactosidase (green) and NCAM (red) on sagittal head sections demonstrated that the Wnt reporter activated cells were single-cell layered and tangentially localized in the connecting zone (white arrow indicates the yellow labeled cell layer with the overlapped red and green signals) where olfactory sensory neuron axons attached to the olfactory bulb anlage at E13. Note that no intense βgal immunolabeling was found in other adjacent tissues including the thick olfactory axon bundles that is a part of migratory mass (mm) on the projecting way from olfactory epithelium to the bulb. (D) Double immunolabeling for βgal and NCAM on coronal sections through olfactory bulb and epithelium at E14. The dash-enclosed areas indicate relevant regions of different tissue sections with a higher magnification in panels E–F2 (single optical sections by confocal microscopy). (E–E2) Double immunolabeling of βgal and Nestin in the middle region between two olfactory bulbs at E14. Most intensely immunolabeled βgal⁺ cells are co-labeled with weak Nestin-immunoreactivities in the connecting zone (arrows). (F–F2) βgal⁺/nestin⁺ cells (arrows) in the connecting zone of the migratory mass. ob, olfactory bulb; oe, olfactory epithelium. The cell nuclei were counterstained by DAPI (blue in C–F2).

Double immunolabeling for the TOPgal-labeled “connecting zone” cells with OEC markers on coronal sections of E18.5 olfactory bulb. (A–B2) βgal⁺ cells (green) consist a multiple-cell layer intermingled with NCAM⁺ olfactory axons (red) in the inner ONL (dashed lines in parallel). Some of them were co-immunolabeled with NCAM in both perinuclear cytoplasm (small arrows in B–B2) and processes (large arrows in B–B2). Arrowheads (in B–B2) indicate a βgal⁺/NCAM⁻ cell located in the front line of the connecting zone. (Panels B–B2 were magnified from the dash-lined area indicated in A2). (C–D2) Weakly immunolabeled p75⁺ OECs (green) were also found in the inner ONL that most of them were co-immunolabeled with NCAM in the perinuclear cytoplasm (arrows). Note that p75⁺ OECs were dominantly distributed in outer ONL by surrounding the large olfactory axonal bundles (asterisks) but no overlapping (arrowheads) with NCAM immunolabeling. (Panels D–D2 were magnified from the dash-lined area indicated in C2; Arrowheads in D–D2 indicate an intense p75⁺ cells that was not stained with NCAM in boundary between ONLi and ONLo). (E–E2) A few βgal⁺ (green) and p75⁺ (red) double-immunolabeled OEC-like cells were found in the E18.5 ONLo (arrows). Arrowheads indicate an intensely immunolabeled βgal⁺ cell without p75-immunolabeling in the inner ONL (that divided by a dashed line). (F–F2) Double immunolabeling of βgal and Nestin on a coronal section with left and right bulbs shown at E18.5. Most βgal⁺ cells (green) in the ONLi were co-immunolabeled with Nestin (red). Note that Nestin immunoreactivities were also found in the intermediate zone between two bulbs and in the developing glomerular layer. cp, cribriform plate. Asterisks (in A–C2) indicate a large olfactory axonal bundle crossed the cribriform plate. The cell nuclei were counterstained by DAPI (blue).