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. 2010 Jul 14;30(28):9523-32.
doi: 10.1523/JNEUROSCI.0867-10.2010.

Embryonic Pax7-expressing progenitors contribute multiple cell types to the postnatal olfactory epithelium

Barbara Murdoch  1 , Casey DelConte, Martín I García-Castro
Affiliations

Embryonic Pax7-expressing progenitors contribute multiple cell types to the postnatal olfactory epithelium

Barbara Murdoch et al. J Neurosci. .

Abstract

Prolonged neurogenesis driven by stem/progenitor cells is a hallmark of the olfactory epithelium (OE), beginning at the placodal stages in the embryo and continuing throughout adult life. Despite the progress made to identify and study the regulation of adult OE progenitors, our knowledge of embryonic OE precursors and their cellular contributions to the adult OE has been stalled by the lack of markers able to distinguish individual candidate progenitors. Here we identify embryonic OE Pax7+ progenitors, detected at embryonic day 10.5 (E10.5) in the olfactory pit with an antigen profile and location previously assigned to presumptive OE stem cells. Using Cre-loxP technology (Pax7-cre/ROSA YFP mice), we expose a wide range of derivatives, including CNS and olfactory neurons, non-neuronal cells, and olfactory ensheathing glia, all made from embryonic Pax7+ cells. Importantly, the expression of Pax7 in the embryonic OE is downregulated from E15.5, such that after birth, no Pax7+ cells are found in the OE, and thus the progenitor population here identified is restricted to embryonic stages. Our results provide the first evidence for a population of Pax7-expressing embryonic progenitors that contribute to multiple OE lineages and demonstrate novel insights into the unique spatiotemporal patterning of the postnatal OE.

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Figures

Figure 1.
Figure 1.
Pax7 expression in the mouse OE during ontogeny. A, Nonfluorescent immunohistochemistry detected Pax7 expression in the E11.5 OE that is restricted to the most rostral region (arrowhead), with robust Pax7 signal intensity in the underlying mesenchymal cells of the lateral nasal process (LNP) (arrow; low magnification in inset). B, Pax7 expression was not detected in the E15.5 OE but was found in a few cells at the interface of the OE and underlying mesenchyme (arrows). C, D, These Pax7+ cells persist in the lamina propria (LP) of the P5 OE (C; arrows) but not the adult OE (D; arrowhead). The dashed line indicates the basal lamina. Scale bars: A, 50 μm; B–D, 100 μm.
Figure 2.
Figure 2.
Fate-mapping Pax7+ progenitors in the OE of Pax7-cre/ROSA YFP transgenic mice. A, The Pax7-cre construct has an encephalomyocarditis internal ribosomal entry site with the Cre coding sequence (IRES-Cre; gray box) inserted into the 3′ untranslated region following the exon 10 (black box) stop codon of the Pax7 locus (represented by the black line) (Keller et al., 2004). Pax7-driven Cre expression occurs without disrupting normal Pax7 function. The arrowhead indicates a remaining flippase recognition target site. B, In E11.5 Pax7-cre/ROSA YFP whole-mount embryos, endogenous YFP expression (green) is detected in the midbrain, the hindbrain, the entire anteroposterior axis of the neural tube, the dorsal root ganglia (arrows), and the frontonasal region (arrowhead). C, Pax7 (red) and YFP can be detected in mesenchymal cells of the lateral nasal process (LNP; arrows) and the developing OE (arrowhead; boxed region magnified in inset) of transverse E11.5 sections. D–G, Pax7, together with YFP expression, is detected in the frontonasal mesenchyme (FNM) and olfactory placode/pit (OP) at E10.5 (arrowhead), the first developmental stage in which we recognized olfactory tissue. The boxed region in D is magnified in E–G. H–J, Immunofluorescence on P5 Pax7-cre/ROSA YFP mice show YFP+ cells in the OE (arrowhead) and vomeronasal organ (VNO; arrow) of coronal sections. DAPI nuclear stain is blue in C–E and H. Individual signals are from Pax7 (G), YFP (B, F, I), and DAPI (J); Double asterisks indicate dorsal recess. Sep, Septum; LP, lamina propria; FB, forebrain. The dashed line indicates the basal lamina. Scale bars: B, 1 mm; C, E–G (in C, E), 100 μm; D, 200 μm; H–J (in H), 500 μm.
Figure 3.
Figure 3.
The Pax7 lineage contributes to vomeronasal sensory neurons. A–I, The P5 vomeronasal organ (VNO) contains a subpopulation of YFP+ (green) vomeronasal receptor neurons that can be TUJ1+ (red; D–F, arrows), TUJ1 negative (D–F, arrowheads), or OMP+ (red; G–I, arrows). The boxed region in A is magnified in D–F. Individual signals are from YFP (B, E, H), TUJ1 (C, F), or OMP (I); DAPI nuclear stain is blue in A. Scale bars: A–C (in A), D–I (in D),100 μm.
Figure 4.
Figure 4.
Subpopulations of sustentacular cells and ORNs arise via a Pax7 lineage. A, B, Coronal sections from P5 Pax7-cre/ROSA YFP mice show YFP+ (green) cells in the OE, throughout the lamina propria (LP) and in cartilage of the septum (Sep). DAPI is blue in A. C, Diagram showing the laminar and cellular organization of the postnatal OE that includes from apex to basal lamina, sustentacular cells, ORNs, GBCs, and HBCs; olfactory ensheathing cells, Bowman's glands, and ORN axon bundles are beneath the basal lamina in the lamina propria. D–F, YFP+ cells are detected in the apical OE in a subpopulation of Sus4+ (red) sustentacular cells (Sus; apical arrow) and below the apex in Sus4-negative cells (arrowhead). In the LP, Sus4+ cells of Bowman's gland (BG; arrow) do not appear to express YFP, even though YFP+ cells are detected throughout. Cells of Bowman's ducts that emerge into the OE (asterisk), can express YFP. G–I, A subpopulation of ORNs (TUJ1+; red) express YFP (arrows). Double asterisks indicate dorsal recess. Scale bars: A, B (in A), D–I (in D), μm. Ax, axon bundles.
Figure 5.
Figure 5.
The Pax7 lineage includes olfactory ensheathing cells. A–D, In the lamina propria (LP), TUJ1+ (red) ORN axon bundles (Ax) are surrounded by YFP+ olfactory ensheathing cells that arise via a Pax7 lineage (arrows). E, F, YFP+ (maroon, VIP immunohistochemistry) olfactory ensheathing cells continue to ensheath ORN axons into the nerve fiber layer (NFL) of the olfactory bulb (OB; arrow). The boxed region in E is magnified in F. In addition to olfactory ensheathing cells, YFP+ cells detected in the LP also include presumptive mesenchymal cells, connective tissues, blood vessels, and/or fibroblasts. Individual signals from YFP (B, D–F) and TUJ1 (C). Sep, Septum. The dashed line indicates the basal lamina. Scale bars: A–C (in C), 100 μm; D, 50 μm; E, 500 μm; F, 100 μm; D, 50 μm; E, 500 μm.
Figure 6.
Figure 6.
The Pax7 lineage includes Pax6, Sox2, Mash1, and ICAM-1 postnatal olfactory progenitors. A–C, E–G, I–K, Coronal sections of P5 Pax7cre/ROSA YFP mice shows subpopulations of Pax6-positive (A–C), Sox2-positive (E–G), and Mash1-positive (I–K) progenitors (all red) that are YFP+ (green). B-D) YFP+ cells are seen in a subpopulation of Pax6+ sustentacular cells in the apical layer (arrow) and in Pax6+ progenitors in the basal layers (arrowhead and inset). YFP+ cells in the lamina propria (LP) are closely juxtaposed to Pax6+ YFP-negative cells (asterisk). F–H, In the apical OE, YFP+ cells can be detected in a subpopulation of Sox2+ cells (arrow), but less frequently in basal progenitors (arrowhead). Sox2+ cells of Bowman's glands (BG) in the LP are closely juxtaposed by YFP+ cells (F; asterisk). J–L, YFP is more commonly detected in Mash1+ progenitors (arrowhead) in the middle ORN layer than in the apical or basal layers (arrows). M–O, Most ICAM-1+ HBCs do not express YFP (arrowhead), but rare ICAM-1+ YFP+ HBCs can be detected in regions where most other cells do not express YFP (inset magnifies boxed region). Boxes indicate regions highlighted in panels showing individual signals from Pax6 (C), Sox2 (G), Mash1 (K), ICAM-1 (O), and YFP (D, H, L, N). The dashed line indicates the basal lamina. Scale bars: A, E, I (in A) and B–D, F–H, J–O (in B), 100 μm; M–O, insets (in N), 50 μm.
Figure 7.
Figure 7.
Subpopulations of embryonic olfactory progenitors arise via Pax7 lineage. A, E, I, M, YFP (green) is detected as early as E11.5 on sagittal sections from Pax7-cre/ROSA YFP embryos in the frontonasal process (FNP), developing OE, and a few TUJ1+ neurons and Mash1+ neuronal progenitors in the forebrain (FB; asterisks). DAPI nuclear stain is in blue. B–D, YFP is detected in a subpopulation of TUJ1+ (red) ORNs (arrowhead) in the OE and developing olfactory nerve (ON; arrow). E–H, I–L, YFP can also be detected in subpopulations of Mash1+ (red; E–H) and Sox2+ (red; I–L) progenitors throughout the epithelium (arrowheads). YFP expression is detected in a cytokinetic Sox2+ progenitor (asterisk). M–P, Cells expressing Pax6 (red) are mostly devoid of YFP expression with a distinct boundary formed between the two (N–P, arrow). Inset, Ventral expression of YFP beyond that shown in N. Boxes indicate regions highlighted in panels showing individual signals from TUJ1 (C), Mash1 (G), Sox2 (K), Pax6 (O), and YFP (D, H, L, P). The dashed line indicates the basal lamina. Scale bars: A, E, I, M, (in A), all others (in B), 100 μm.
Figure 8.
Figure 8.
Potential lineages of embryonic Pax7+ olfactory progenitors. Non-neuronal cells, neurons, and olfactory ensheathing glia, which no longer express Pax7 (D), arise via embryonic Pax7+ progenitors. A–C, Potential Pax7 olfactory lineages include a multipotent Pax7+ stem cell (A) or more restricted Pax7+ precursor (B), each with the capacity to clonally produce multiple cell types, or independent lineages through cell type-restricted precursors (C), dependent on the cellular potency of the precursor that first expresses Pax7.
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