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Differential and overlapping expression patterns of X-dll3 and Pax-6 genes suggest distinct roles in olfactory system development of the African clawed frog Xenopus laevis

Marie-Dominique Franco^*, Michael P. Pape, Jennifer J. Swiergiel and Gail D. Burd

University of Arizona, Department of Molecular and Cellular Biology, Life Sciences South Building 444, PO Box 210106, University of Arizona, Tucson, AZ 85721, USA
^* Present address: Grinnell College, Department of Biology, Robert N. Noyce Science Center, PO Box 805, Grinnell, IA 50112-0806, USA

View larger version (20K): [in a new window]   Fig. 1. Schematic diagrams of the olfactory epithelium during embryonic, larval and metamorphic development in Xenopus laevis. (A) Front views of whole-mount embryos at stages 14, 20 and 32. nr, neural ridge; np, neural plate; pOP, tissue fated to become placodes; op, olfactory placode; sp, sense plate. The asterisks indicate the location of nr and op. (B) Stages 20 and 31 are views from horizontal sections. Stages 50–66 are schematic diagrams of the nasal capsule viewed as whole mounts in the horizontal plane with anterior at the top. By stage 20, superficial ectodermal cells (SE) (precursors of the supporting cells) and neuronal ectodermal cells (NE) (precursors of the olfactory receptor neurons and basal cells) have formed. ‘Water sensing’ and ‘air sensing’ refer to the presumed function of the olfactory receptor neurons. MC, middle cavity; PC, principal cavity; VNO, vomeronasal organ; b, basal cells; n, olfactory receptor neurons; s, supporting cells. Diagrams are not to scale.

View larger version (101K): [in a new window]   Fig. 2. Embryonic expression of X-dll3 and Pax-6 by whole-mount in situ hybridization. (A) X-dll3 expression at stage (St) 15 showing stained anterior neural ridge and future cement gland. (B) X-dll3 expression at stage 18 in the presumptive olfactory placodes and cement gland. (C) X-dll3 expression at stage 25 showing stained olfactory placodes, sense plate and cement gland. (D) X-dll3 expression at stage 37 showing stained olfactory placodes, branchial arches and forebrain. By stage 37, X-dll3 expression begins to disappear in the cement gland. (E) Pax-6 expression at stage 15 showing stained anterior neural ectoderm, hindbrain anlage and spinal cord. (F) Pax-6 expression at stage 21 showing staining in an area of the presumptive olfactory placodes and darker staining in the eye, forebrain, hindbrain and spinal cord. (G) Pax-6 expression at stage 25 showing staining in the eye, forebrain and hindbrain. Note that the olfactory placodes are only very lightly stained at this stage. (H) Pax-6 expression at stage 39 showing stained olfactory placodes, olfactory bulb, forebrain and eyes. (J–M) Paraffin sections of whole-mount stained embryos. (J) X-dll3 expression at stage 26 in all layers of the olfactory placodes and in the non-neuronal and neuronal ectodermal cells of the sense plate. The asterisks indicate the regions of the anterior neural tube that will give rise to the olfactory bulb. (K) Pax-6 expression at stage 29 in the olfactory placodes and in the olfactory bulb, forebrain and eyes. (L) A stage 35 animal showing X-dll3 expression in all layers of the olfactory placodes and olfactory bulb. At this stage, expression has disappeared from the sense plate and a darker stain is visible in the vomeronasal organ of the olfactory placode on the right side. (M) A stage 37 embryo showing Pax-6 expression in the olfactory placodes and olfactory bulb. Staining in the olfactory placodes is light, and staining in the olfactory bulb is primarily located in differentiating mitral cells. Whole-mount photographs are dorsal/anterior views. Sections are horizontal with anterior facing down. ba, branchial arches; cg, cement gland; e, eye; f, forebrain; h, hindbrain; m, midbrain; ne, neuroectoderm; ob, olfactory bulb; OP, olfactory placode; pOP, tissue fated to become olfactory placodes; sc, spinal cord; sp, sense plate; v, ventricle. Scale bars, 150µm.

View larger version (161K): [in a new window]   Fig. 3. Expression of X-dll3 in the olfactory epithelium and forebrain during metamorphosis. (A) The principal cavity (PC) and vomeronasal organ (VNO) of a stage 57 tadpole hybridized with the sense X-dll3 riboprobe as a negative control. (B–L) Sections through the nasal cavities and forebrain hybridized with the antisense X-dll3 riboprobe. (B,E) The PC at stages 51 and 54, respectively, showing X-dll3 expression in the neuronal and basal cell layers. (C,F,I,L) Sections through the forebrain at stages 51, 54, 59 and 64 respectively. X-dll3 expression is seen around the lateral and tectal ventricles, in the granular cell layer and in the ventrothalamus. (D) At stage 54, X-dll3 expression occurs throughout the VNO and middle cavity (MC). (G) At stage 59, X-dll3 expression is present throughout the VNO and in the neuronal and basal cell layers of the MC. (H) The PC at stage 59 with X-dll3 expression in deep neuronal and basal cell layers. (J) The MC and PC at stage 64 showing X-dll3 expression in the basal cell/deep neuronal layer of the MC. (K) The PC and VNO at stage 64 showing X-dll3 expression in a few basal cells or deep olfactory receptor neurons and throughout the VNO. The inset at the upper right is an enlargement of the basal cell layer showing arrows pointed to individually stained cells. Nose sections are transverse sections of the olfactory cavity with ventral facing up and lateral to the left except for B and E, in which dorsal is upwards. Dense black material delimiting regions of the olfactory epithelium in the nasal cavities is composed of pigment granules in A, B, D, E, G, H, J and K. Brain sections are horizontal, with anterior facing the left. b, basal cell layer; g, glomerular cell layer; gr, granular cell layer; lV, lateral ventricle; m, mitral cell layer; n, neuronal cell layer; OB, olfactory bulb; on, olfactory nerve; s, supporting cell layer; tV, tectal ventricle; VT, ventral thalamus. Scale bars, 200µm.

View larger version (107K): [in a new window]   Fig. 4. Immunocytochemistry showing metamorphic and adult expression of Distal-less protein in the olfactory epithelium. Sections in A, C and E are stained with the blue nuclear stain DAPI and sections in B, D and F are stained with anti-Distal-less antibody followed by Cy3-labeled secondary antibody. (A,B) Staining in the olfactory epithelium of a stage 53 tadpole, (C,D) staining in the principal cavity (PC) of an adult, and (E,F) staining in the middle cavity (MC) of an adult. Note that the staining in the PC at stage 53 is scattered throughout the neuronal cell layer and in the basal cell layer, while in the adult PC, most of the staining is concentrated in the deeper neuronal and basal cell layers. In the MC, the staining is distributed throughout the olfactory epithelium at stage 53, but also becomes concentrated in deeper layers in the adult. (G,H) Negative control (no primary antibody) that illustrates areas of background staining at the most apical surface of the olfactory epithelium (arrow) and in the region at and below the basal lamina (arrowhead); the dots define the superficial (top) and basal (bottom) surfaces of the olfactory epithelium. Scale bars, 100µm.

View larger version (89K): [in a new window]   Fig. 5. Expression of Pax-6 in the olfactory epithelium and forebrain during metamorphosis using the antisense Pax-6 riboprobe. (A) Middle cavity (MC) and vomeronasal organ (VNO) at stage 58 showing Pax-6 expression in the supporting cell layer of the VNO and in the neuronal and basal cell layers of the MC. (B,D,F,H) Sections through the principal cavity (PC) at stages 49, 58 61 and 66, respectively, demonstrate that Pax-6 is not expressed in the remodeling olfactory epithelium. (C,G,I) Sections through the forebrain at stages 50, 58 and 65, respectively, showing Pax-6 expression mainly in the granular cell layer of the olfactory bulb, in a few mitral cells and in very few cells around the ventricles. (E) The MC at stage 61 showing Pax-6 expression in the neuronal and basal cell layers. Sections A, B, D, E, F, G are transverse sections of the olfactory cavity with ventral upwards and lateral to the left; B, dorsal upwards. Dense black material delimiting regions of the olfactory epithelium in the nasal cavities is composed of pigment granules in A, D and F. Brain sections are horizontal with anterior facing the left and lateral facing upwards. AB, accessory olfactory bulb; b, basal cell layer; g, glomerular cell layer; gr, granular cell layer; lV, lateral ventricle; ml, mitral cell layer; n, neuronal cell layer; on, olfactory nerve; s, supporting cell layer; VT, ventrothalamus. Scale bars, 150µm.

View larger version (15K): [in a new window]   Fig. 6. Summary of the spatial and temporal expression of X-dll3 and Pax-6 in the developing olfactory epithelium. The presumptive water-sensing epithelia of the larval principal cavity (PC) and the adult middle cavity (MC) are represented in blue and the presumptive air-sensing epithelium of the adult PC is represented in green. The time course is not to scale. b, basal cell layer; n, neuronal cell layer; s, supporting cell layer. Stages are according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1994).

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