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First published online 29 September 2004
doi: 10.1242/dev.01416

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The Lim homeobox gene Lhx2 is required for olfactory sensory neuron identity

¹ Department of Molecular Biology, Umeå University, Umeå, SE901 87, Sweden
² Umea Centre for Molecular Medicine, Umeå University, Umeå, SE901 87, Sweden

View larger version (103K): [in a new window]   Fig. 1. Unaltered distribution of Lhx2 expression and lack of OR expression in OE of Lhx2 -/- embryos. (A-C) Double-immunohistochemical analysis of Lhx2 and TubIII expression in OE of E15.5 mouse embryo is shown. (A) Nuclear Lhx2 immunoreactivity in the neuronal cell layer (N) and in a fraction of cells in the basal progenitor cell layer (P). No Lhx2-positive cells are present in the sustentacular cell layer (S) and the lamina propria (l.p.). (B) TubIII immunoreactivity in neuronal soma and neuronal processes. (C) Images in A and B combined, showing that Lhx2 and TubIII are co-expressed in OSNs. A limited number of TubIII-negative Lhx2-positive cells are present in the progenitor cell layer (arrows in C). (D-I) In-situ hybridization analyses of serial OE sections from control (left panel) and Lhx2-/- embryos (right panel) with Dig-labeled cRNA probes that hybridize to Lhx2 and mutated Lhx2 transcripts (Lhx2m) (D,E), and OR genes (K21, L45, M50 and A16) (F-I). (J) Autoradiographs of in-situ hybridization analyses with a 35S-labeled cRNA probe specific to the OR gene K20. Scattered cells expressing the different OR genes are present in control mice. The zones in which each the OR gene is expressed is indicated, i.e. dorsomedial (dm), the middle (m) or the ventrolateral (vl) zone. In Lhx2 -/- mice, there are no cells expressing ORs in any epithelial region (representative results are shown).

View larger version (134K): [in a new window]   Fig. 2. No expression of the zone-specific genes Nqo1 and Ncam2 in OE of Lhx2-/- embryos. (A) Autoradiographs of in-situ hybridization analyses of consecutive coronal sections (dorsal up and medial left) of one nasal cavity of control embryos (A-C) and Lhx2-/- embryos (D-F). Sections were hybridized with 35S-labeled cRNA probes specific for Lhx2 and mutated Lhx2 transcripts (Lhx2m) (A,D), Nqo1 (B,E) and Ncam2 (C,F). Lhx2 expression is distributed throughout the OE in both control (A) and Lhx2-/- mice (D). Nqo1 is expressed in OSNs located in the dorsomedial zone in control embryos (arrow in B), whereas no signal is present in the neuronal cell layer of Lhx2-/- embryos (E). Ncam2 is expressed in OSNs located in the middle and the ventrolateral zones in control embryos (arrow in C), whereas no hybridizing signal is present in the neuronal cell layer of Lhx2-/- embryos (F). The border between neurons in the dorsomedial zone and the middle zone is indicated (dotted line in B,C). Nqo1-specific hybridization to cells in lamina propria (arrowheads in B,E) and Ncam2-specific hybridization to cells in the telencephalon (arrowhead in F) are also indicated. (B) High-power magnifications of OE sections hybridized with Dig-labeled cRNA probes specific for Nqo1 (A,C) and Ncam2 (B,D) are shown. Both probes hybridize to OSN in control embryos (A,B), whereas no signal over background is evident in sections of Lhx2-/- embryos (C,D).

View larger version (81K): [in a new window]   Fig. 3. Unaltered neurogenesis and altered neuronal differentiation in Lhx2-/- embryos. In-situ hybridization analyses of OE sections with Dig-labeled cRNA probes specific for Mash1 (A), Ngn1 (B), Neurod1 (C), Golf (D), and Omp (E). Control embryos (left panel) and Lhx2-/- embryos (right panel) show an equal distribution and number of apical and basal progenitor cells expressing Mash1 (A) and basal progenitors expressing Ngn1 (B). The OE in Lhx2-/- embryos contains an increased number of progenitors and/or neurons that express Neurod1 (C), whereas the expression of OSN-enriched and late differentiation markers Golf (D) and Omp (E) is reduced. (F) Immunohistochemical analyses of the dorsomedial zone (dorsal left and lateral up) with a reduced number of Omp-positive cells in OE of Lhx2-/- embryos.

View larger version (68K): [in a new window]   Fig. 4. The neuronal cell layer in OE of Lhx2-/- embryos largely contains newly differentiated neurons that have acquired pan-neuronal traits and form axon bundles. Coronal sections (dorsal up and medial left) of one nasal cavity from control embryos (A,C,E,G,I,K,M,O) and Lhx2-/- embryos (B,D,F,H,J,L,N,P). (A-F) Immunohistochemical analyses for expression of immature neuronal markers is shown. Ncam1 (A,B), Gap43 (c-d), and Stathmin/SCG10 (E,F) are expressed in OE and axon bundles of the lamina propria (arrows) in both control and Lhx2-/- embryos. (G,H) Double phospho-H3 (PH3; in green) immunohistochemistry and Neurod1 (ND; in red) in-situ hybridization analyses showing that the increased expression of Neurod1, primarily apparent in the ventral OE (arrowhead in H), does not correlate with the number and distribution of mitotic cells. (I-L) Analyses of consecutive OE sections, showing that cells immunoreactive using an anti-TubIII antibody (I,J) and cells that express Neurod1 transcripts (K,L) co-localize predominantly in ventrolateral regions of OE (arrowheads in J,L). Expression of TubIII in axon bundles of the lamina propria is indicated (arrows in I,J). (M,N) High magnification confocal images of double TubIII immunohistochemical (in green) and Neurod1 in-situ hybridization (in red) analyses showing an increased number of cells in Lhx2-/- embryos that co-express Neurod1 and TubIII (yellow signal; insert in N). (O,P) High magnification confocal images of double phospho-H3 immunohistochemical (in green) and Neurod1 in-situ hybridization (in red) analyses showing that OE in Lhx2-/- embryos does not contain an increased number of progenitors that co-express phospho-H3 and Neurod1 (in yellow; arrowheads).

View larger version (75K): [in a new window]   Fig. 5. Abnormal region-specific OSN differentiation and normal region-specific gene expression in sustentacular and progenitor cells. In-situ hybridization analyses of serial coronal (dorsal up and medial left) sections of OE from control (A,C,E,G,I,K,M) and Lhx2-/- embryos (B,D,F,H,I,L,N) using Dig-labeled cRNA probes specific for Golf (A,B), Omp (C,D), Nqo1 (E,F), Alk6 (G-J) and Msx1 (K-N). (A-F) Golf- and Omp-positive cells are present in all OE zones in control embryos (A,C) but show a restricted dorsomedial distribution in OE of Lhx2-/- embryos (arrows in B,D). The distribution of Golf- and Omp-positive cells in Lhx2-/- embryos is similar to the distribution of the dorsomedial-specific marker Nqo1 in control embryos (arrows in E). No Nqo1 expression over background is detected in Lhx2-/- embryos (F). (G-N) The gradient of Alk6 expression (G,H; high dorsomedial and low ventrolateral) and the reverse gradient of Msx1 expression (K,L) are present in both control and Lhx2-/- embryos. High-power magnifications of Alk6 expression in the sustentacular cell layer (I,J) and Msx1 expression in the basal progenitor cell layer (M,N) is shown. Thus, cells expressing Alk6 and Msx1 show a similar distribution in OE cell layers in control and Lhx2-/- mice.

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