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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 E11.5 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.
The olfactory epithelium (OE) is one of a few exclusive regions in the adult nervous system where lifelong neurogenesis occurs (Graziadei and Graziadei, 1979; Schwob, 2002). Neurogenesis and maintenance of the sense of smell is dependent upon the biological responses of stem and/or progenitor cells. A common problem in stem cell biology however, is the limited availability of markers capable of identifying precursor cells whose ontogeny, in vivo niche and lineage potential can be examined (Weissman et al., 2001).
The postnatal OE has a laminar structure with globose and horizontal basal cell (GBCs and HBCs, respectively) progenitors in the basal region, olfactory receptor neurons (ORNs) in the midsection and sustentacular cells at the apex. In the underlying lamina propria olfactory ensheathing glia wrap around ORN axon bundles as they target the olfactory bulb in the central nervous system (CNS) (Getchell et al., 1984; Farbman, 1992). Bowman’s glands in the lamina propria produce mucous delivered through ducts that extend through the OE to the outer surface (Getchell et al., 1984; Farbman, 1992). In contrast, the embryonic OE lacks laminar structure and is comprised mostly of proliferating progenitors. Several cell types, like HBCs and sustentacular cells, do not emerge until late embryonic or early postnatal development, with ORN numbers gradually increasing as embryonic development proceeds. However, the spatiotemporal contributions of embryonic olfactory progenitors to the postnatal OE are largely unknown.
Although limited, studies using transgenic mice to genetically fate map embryonic progenitor descendants have helped to formulate our current understanding of embryonic OE lineage contributions, which have uncovered either neuron-restricted or glia-restricted embryonic progenitors. Labeled ORNs are detected throughout the OE of FoxG1-cre/reporter mice (Duggan et al., 2008), but regionally restricted to the dorsal-medial OE in Nestin-cre/reporter mice (Murdoch and Roskams, 2008), while BLBP-cre/reporter mice label only olfactory ensheathing glia (Murdoch and Roskams, 2007). Markers of embryonic precursors with the capacity to produce postnatal neurons together with glia, or additional non-neuronal cells like sustentacular cells, have not been identified.
In numerous tissues during embryonic development, mammalian Pax genes, transcription factors of the paired domain family, contribute to the regulation of cell proliferation, lineage specification, differentiation, migration and survival (Lang et al., 2007; Blake et al., 2008). Pax genes also play a role in the development of the OE (Davis and Reed, 1996; LaMantia et al., 2000). For example, although Pax7 mutants have no obvious olfactory abnormality (Mansouri et al., 1996), Pax7 is expressed at early embryonic stages (Jostes et al., 1991; Stoykova and Gruss, 1994) in regions associated with Sox2+ putative OE stem cells (LaMantia et al., 2000; Beites et al., 2005; Kawauchi et al., 2005; Chen et al., 2009), but whose lineage contributions are unknown.
Here we investigate the expression of Pax7 prior to and during OE ontogeny and use Cre-loxP technology to lineage trace Pax7 progeny to investigate the contributions made by Pax7-expressing embryonic progenitors. Our results reveal novel spatiotemporal patterning of the postnatal OE and identify for the first time, embryonic precursors expressing Pax7 that generate multiple nervous system and chemosensory lineages, including CNS, vomeronasal and olfactory neurons, olfactory glia and non-neuronal cells.
Adult and postnatal mice were sacrificed in a CO2 chamber, perfused with cold PBS and 4% paraformaldehyde (PFA) in PBS and post-fixed in 4% PFA at 4°C (Murdoch and Roskams, 2008). Embryos were immersion-fixed in 4% PFA overnight. The day of vaginal plug was defined as E0.5. Tissues were cryoprotected in sucrose, embedded in Tissue-Tek medium (OCT; Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen. 12 μm sections were stored at −20°C for subsequent analysis.
Sections were immersed in PBS, permeabilized in 0.1% Triton-X-100/PBS and blocked with 4% normal serum prior to primary antibody incubation. Secondary antibodies (1:200) used were of specific isotypes conjugated to biotin (Vector labs), Alexa 568 or Alexa 488 (Invitrogen Molecular Probes). Prior to blocking, Sus4 and transcription factor detection was enhanced by a 15–60 second incubation of the sections with 0.12% trypsin/EDTA (Gibco) (longer duration for older ages), followed by washing in PBS. Primary antibodies used: mouse anti-rat βIII tubulin (neuron-specific tubulin-TUJ1, 1:500) Covance; mouse anti-Mash1 (1:100) BD Biosciences, Pharmingen; mouse anti -Pax7, -Pax6 Developmental Studies Hybridoma Bank (developed by Kawakami, A and Ordahl, CP), rabbit anti-GFP (1:400, to detect YFP), Millipore; Sox2 (1:300) R & D Systems. Note: all YFP panels show anti-GFP immunofluorescence that was confirmed using non-fluorescent VIP immunohistochemistry with anti-GFP antibodies (Supplementary Fig S2), with the exception of the YFP-containing panels in Fig 2B and Supplementary Fig S2C, which show endogenous YFP fluorescence. Gift antibodies: mouse anti-rat Sus4 (1:100) from Dr. J. Schwob, (Tufts University, Boston, MA), goat polyclonal olfactory marker protein (OMP, 1:5000) from C. Greer (Yale University, New Haven, CT). Nuclei were stained with 0.5 ug/ml diaminopyridine imidazole (DAPI) and sections coverslipped in Vectashield (Vector Laboratories, Burlingame, CA) for fluorescent antigens or 50% glycerol for VIP. Images were visualized with either a Nikon dissecting scope or Nikon Eclipse 80i microscope using a SPOT camera (SPOT Diagnostic Instruments Inc.) with SPOT software (v4.5). Confocal images were collected on a Zeiss LSM 510 microscope with ZEN software (v5) and processed using Image J (v1.43r). All images were compiled using Adobe Photoshop 7.0.
Total RNA was isolated from E11.5 mouse embryos using RNAzol B Reagent (Tel-Test, Friendswood, TX). Total RNA was reverse transcribed with the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Real time PCR was performed in triplicate using the iQ SYBR Green Super Mix in a BioRad iQ5 (Hercules, CA). Each reaction contained cDNA template, 1x iQ SYBR Green Super Mix and 300 μM primers. Each plate was run in triplicate. Conditions for amplification were: 3 min at 95°C, followed by 40 cycles of 10 s at 95°C, 30 s at 55°C, and 30 s at 72°C. Melting curve analysis was performed from 55°C to 95°C, with 1°C/second transitions. The primers used were: Pax7 F 5′-GCTACCAGTACAGCCAGTATG-3′, Pax7 R 5′-GTCACTAAGCATGGGTAGATG-3′ (McKinnell et al., 2008), β-actinF 5′-AAGTGTGACGTTGACATCCG-3 ′, and β-actinR 5′-GATCCACATCTGCTGGAAGG-3′. Cycle threshold values and amplification curves were obtained using iQ5 Optical System Software (v 2.0; Bio-Rad). All data were normalized to β-actin. Relative Pax7 expression was calibrated against wildtype controls where ΔCt1 = wildtype Ct − Pax7 cre or Pax7 null Ct; ΔCt2 = wildtype Ct − β-actin Ct; relative Pax7 expression was calculated as 2 (ΔCt1−ΔCt2).
All experimental procedures were performed in accordance with the Yale Animal Resources Center and Institutional Animal Care and Use Committee policies. Pax7-cre mice (C57Bl/6) obtained from Mario Capecchi (Keller et al., 2004) were crossed with C57Bl/6 female Gt(ROSA)26Sortm(EYFP)Cos mice (gift from D. Krause) expressing enhanced yellow fluorescent protein from the ROSA26 locus (termed ROSA YFP throughout the paper) (Srinivas et al., 2001). Gtrosa26tm1Sor (expressing β-galactosidase protein from the ROSA26 locus) (Soriano, 1999), produced a similar reporter expression pattern after Pax7-cre mediated excision. Pax7-cre/ROSA YFP double transgenic mice, where Cre recombinase expression is under the control of the endogenous Pax7 regulatory elements, express YFP without disrupting Pax7 function (Keller et al., 2004). Pax7 LacZ mutant mice were obtained from A. Mansouri and have been previously reported (Mansouri et al., 1996). Mice were genotyped by PCR for Pax7, Cre (Keller et al., 2004), and YFP. Pax7 primers located within Pax7 exon 10 5′-GCTCTGGATACACCTGAGTCT-3′; 5′-TCGGCCTTCTTCTAGGTTCTGCTC-3′ (ck118 and ck256 respectively; 465 bp product) were combined with an IRES-Cre primer 5′-GGATAGTGAAACAGGGGCAA-3′ (ck172; 340 bp product); ROSA YFP primers: Rosa1 5′-AAAGTCGCTCTGAGTTGTTAT-3′ and Rosa3 5′-GGAGCGGGAGAAATGGATATG-3′ (wildtype ROSA locus, 650bp product); Rosa3 and YRF 5′-CGACCACTACCAGCAGAACA-3′ (ROSA26 YFP, 850 bp product). PCR parameters: 35 cycles of 95°C 30 seconds, 58°C 30 seconds, 72°C 60 seconds. Efficiency of Cre-lox recombination was verified by the specific co-localization of Pax7 with YFP proteins in Pax7-cre/ROSA YFP embryos and the similar reporter expression patterns detected in Pax7-LacZ (Relaix et al., 2004), Pax7-Zs-green (Bosnakovski et al., 2008) and Pax7-cre/Gtrosa26tm1Sor mice (Keller et al., 2004).
For each time point, at least three Pax7-cre/reporter positive embryos, pups, or mice were analyzed. Sections from the rostral, middle and caudal OE for coronal sections, or lateral to medial for sagittal sections, were sampled and tested for YFP+ cells from each mouse. To determine the rostral-caudal YFP expression pattern, every 10th section was sampled from adult (60 days or older) Pax7-cre/ROSA YFP mice.
For YFP+ cell quantitation, at least 3 sections per animal, from the rostral, middle and caudal OE, or 72 um flanking the Pax7-expressing region in the E11.5 OE, were assayed to adequately sample the OE, from each of 3 animals per developmental stage. Total YFP+ cells per section, were divided by the length of OE measured, using ImageJ (1.43r). Average total cells per mm, assessed by DAPI nuclear stain, were calculated after measuring 100 um regions of OE from each turbinate visible on the sections, or all of the visible OE for E11.5.
Pax7 protein expression can be detected in mouse embryos at E7.5 to E8.5 in the lateral region of the cephalic neural folds, the caudal neural plate neuroepithelium and in the cephalic mesenchyme (Supplementary Fig S1), in agreement with previous in situ expression beginning at E8 (Jostes et al., 1991). Pax7 is more widely expressed in the E9.5 developing hindbrain, forebrain and frontonasal mesenchyme (Supplementary Fig S1), and reported in the nose at stages E10 to E14.5 (Jostes et al., 1991; Lang et al., 2003). A detailed study showed that E10.5 embryos express Pax7 in the lateral frontonasal mesenchyme and lateral margin of the nasal pit (LaMantia et al., 2000), which was confirmed here (see Fig 2). To better understand the role of Pax7 progenitors during olfactory development, we used immunohistochemistry to determine the changes in Pax7 expression from embryonic to adult development. By E11.5, the mesenchyme of the lateral nasal process robustly expressed Pax7, which in the OE showed a ventral to dorsal-medial gradient (highest to lowest) (Fig 1A). Pax7 was not readily detected in the E15.5 OE, but was found in cells closely juxtaposed to the epithelial-mesenchymal border (Fig 1B). Similarly, the postnatal day 5 lamina propria, but not the OE, contained Pax7+ cells (Fig 1C). Neither the adult OE nor the lamina propria contained cells with detectable Pax7 expression (3 mice sampled, Fig 1D). Thus, the cellular location of Pax7+ cells and dynamic expression levels during development suggest that Pax7 precursors may contribute to multiple cell types in the mature OE.
To test the lineage contributions of Pax7-expressing cells in the OE we crossed Pax7-cre transgenic mice, where an IRES-Cre cassette was inserted into exon 10 of Pax7 (Fig 2A) (Keller et al., 2004), with Gt(ROSA)26Sortm(EYFP)Cos reporter mice (hereafter termed ROSA YFP) (Srinivas et al., 2001). In Pax7-cre/ROSA YFP double transgenic mice, Cre recombinase expression is under the control of the endogenous Pax7 regulatory elements without disrupting Pax7 function. Pax7 dependent Cre expression mediates an excision event that removes a floxed stop cassette, thus allowing continued YFP expression from the ROSA locus in Pax7-expressing cells and their progeny even after Pax7 expression ceases. In wholemount embryos, we detected endogenous YFP expression in the developing midbrain, hindbrain, neural tube, somites, dorsal root ganglia and frontonasal region (Fig 2B) similar to endogenous Pax7 expression. The YFP reporter expression pattern seen in Pax7-cre/ROSA YFP embryos is consistent with that reported previously using Pax7-LacZ (Relaix et al., 2004), Pax7-ZsGreen (Bosnakovski et al., 2008) and Pax7-cre/ROSA LacZ mice (Keller et al., 2004), where LacZ and ZsGreen identify Pax7-expressing cells and their immediate or lifelong progeny.
Sections from the frontonasal region of E11.5 Pax7-cre/ROSA YFP mice revealed Pax7+ YFP negative cells and Pax7+YFP+ cells, which likely represent cells that only recently initiated Pax7 expression and display a delay between excision and detectable levels of reporter, respectively. Similar delays in Cre-mediated reporter expression have been reported before (Joyner and Zervas, 2006; Murdoch and Roskams, 2008). Additionally, Pax7 negative YFP+ cells, the progeny of Pax7-expressing precursors, which had excised and ceased to express Pax7 were also detected (Fig 2C). At E10.5, most to all Pax7-expressing cells in the frontonasal mesenchyme and developing OE co-express YFP (Fig 2D–G), illustrating the faithful expression of the transgene together with Pax7. Multiple controls ensured the fidelity of our YFP detection in Pax7/reporter transgenic mice (littermate controls without either Pax7-cre or ROSA-YFP alleles, non-fluorescent immunohistochemistry methods, and secondary antibody-only controls (Supplementary Fig S2)). Together this evidence validates the use of Pax7-cre/ROSA YFP transgenic reporter mice to map Pax7 progeny in olfactory lineages.
To determine the contribution of embryonic Pax7+ progenitors to the postnatal OE, we tested for YFP+ reporter cells in postnatal day 5 tissue. YFP+ Pax7 progeny are detected in punctate regions of multiple laminae throughout the postnatal day 5 OE and the vomeronasal organ, a specialized chemosensory organ (Fig 2H–J). In the OE, each postnatal day 5 animal (n=3 for this and all subsequent quantitation) averaged 710 YFP+cells, comprising 87.9 ± 21.2 YFP+ cells/mm out of 1307 ± 16.6 total cells/mm (6.7%, p<0.0001 vs adult, Table 1). The head region also included YFP+ cells in the septal cartilage, head mesenchyme and nasal glands (Fig 2H,I). These results show that embryonic Pax7 precursors contribute to multiple cell lineages that include chemosensory cells.
Using cell type-specific antigens we identified the cell types expressing YFP in the postnatal day 5 vomeronasal organ. Subpopulations of TUJ1+ (β III neuron-specific tubulin) and olfactory marker protein (OMP) positive vomeronasal receptor neurons expressed YFP in their cell bodies, dendrites and axons (Fig 3A–I). YFP+ vomeronasal neurons were seen throughout the apical-basal axis indicative of both V1R and V2R receptor neurons, respectively (Dulac, 2000).
Within the OE and the underlying lamina propria multiple cells expressed YFP (Fig 4A,B), whose location and morphology (summarized in Fig 4C) was combined with candidate antigens in an effort to provide specific identity. The Sus4 antigen identifies sustentacular cells and Bowman’s duct cells in the OE and Bowman’s glands in the lamina propria (Goldstein and Schwob, 1996). With their cell bodies in the apical OE and processes spanning the height of the OE, postnatal day 5 sustentacular cells represent 251 ± 2.9 Sus4+ cells/mm, of which 4.9 ± 0.7 cells/mm (1.9%) are YFP+ (Fig 4D–F; Table 2). Sus4+ cells found in Bowman’s glands did not appear to express YFP (29 glands, 3 animals), but 10.4 ± 1.9% of the 46 ducts examined contained YFP+ duct cells (Fig 4D–F). Coexpression of YFP with cell type-specific antigens, including Sus4, was confirmed by confocal microscopy (Fig S3).
In the middle OE layers, the cell bodies of TUJ1+ immature ORNs express YFP, as do their axons, dendrites and dendritic knobs (Fig 4G–I). YFP+ mature OMP+ ORNs were detected in postnatal day 5 (data not shown) and adult animals (Fig S4). In postnatal day 5 mice, of the 944 ± 16.8 total ORNs/mm, 80.6 ± 6.2 (8.5%) express YFP (p<2×10−5 vs adult, Table 2). Approximately 3% of basal cells (3.7 ± 0.7 YFP+ cells/mm from a total of 112 ± 1.1 basal cells/mm) express YFP (p<0.02 vs adult, Table 2).
The adult OE averaged 755 YFP+ cells (Table 1) with 16.2 ± 2.1 YFP+ cells/mm and 1796 ± 43.0 total cells/mm (0.9%, Table 1). YFP was expressed in approximately 1% of total sustentacular cells and ORNs, and in 0.004% of basal cells (Table 2). Within the YFP population of both adult and P5 animals, ORNs, sustentacular cells and basal cells, had the highest to lowest distribution of YFP+ cells, respectively (Table 3).
A greater than 2 fold increase of YFP+ cells was consistently detected unilaterally compared to contralaterally (but not on the same side in all animals; n=3/time point), in the postnatal day 5 (121.8 ± 34.5 vs 54.2 ± 16.2; p<0.05) and adult OE (16.9 ± 0.2 vs 8.2 ± 2.0; p<0.05), indicating a bilateral asymmetry in Pax7-derived progeny.
To test if the insertion of the Cre recombinase gene into the Pax7 locus could modify Pax7 expression and potentially account for the non-uniform pattern of YFP+ cells in the OE, we compared the level of Pax7 expression in transgenic embryos homozygous for Pax7-cre to non-transgenic wildtype embryos (3 embryos/genotype). Quantitative RT-PCR showed no significant difference in the relative expression of Pax7 in wildtype compared to Pax7-cre homozygous embryos, but significant differences in both compared to Pax7 null controls (p<0.001, Supplementary Fig S5). Likewise, neither the OE (85.5 ± 1.8% vs 87.3 ± 1.5%) nor the frontonasal mesenchyme (98.7 ± 0.7% vs 98.1 ± 1.0%) revealed any significant differences (p>0.05) in the percentage of Pax7+ cells in Pax7-cre homozygotes compared to wildtype controls, respectively (Supplementary Fig S5). These analyses show that Pax7 expression is stable upon Cre insertion and suggest that Pax7 instability cannot account for the unique expression patterns detected in olfactory YFP+ cells. Our results demonstrate that Pax7 embryonic progenitors contribute diverse cell types in unique patterns to the perinatal and adult OE and vomeronasal organ.
The postnatal lamina propria contains multiple cell types including olfactory ensheathing glia, mesenchymal cells, connective tissues, blood vessels and fibroblasts. Of particular interest due to their possible use in therapies of spinal cord injury (reviewed in (Raisman and Li, 2007; Richter and Roskams, 2008; Kocsis et al., 2009), are the olfactory ensheathing glia. Olfactory ensheathing glia are a special type of glial cell that supports the growth of olfactory axons from the peripheral OE to their central nervous system (CNS) target, the olfactory bulb, where they form the nerve fiber layer. Identified by their morphology and location, immediately adjacent to, and surrounding TUJ1+ axon bundles in the lamina propria, YFP+ OECs were found in postnatal day 5 and adult Pax7cre/ROSA YFP mice (Fig 5A–D; Supplementary Fig S3), where 94.5 ± 0.5% of adult OECs are YFP+ (1163 OECs counted). A strong YFP signal is also evident in the nerve fiber layer surrounding the olfactory bulb in the CNS (Fig 5E,F). These results demonstrate that Pax7-derivatives contribute to various cell types in the lamina propria, which include olfactory ensheathing glia found both in the peripheral and central nervous systems.
To more precisely identify cells arising via a Pax7 lineage and further elucidate their cellular diversity, we assessed the expression of cell type-specific transcription factors and cell surface molecules in postnatal day 5 Pax7cre/ROSA YFP mice together with YFP. The Pax6 transcription factor is required for OE development and is expressed postnatally in basal cells, sustentacular cells and Bowman’s gland/duct cells (Davis and Reed, 1996). Sox2, an Sry-related HMG (high mobility group) box transcription factor, is expressed in apical and basal cells in the embryonic (Kawauchi et al., 2004; Beites et al., 2005), and postnatal OE. In postnatal day 5 Pax7cre/ROSA YFP animals, YFP together with either Sox2 or Pax6 OE expression are detected in a subpopulation of sustentacular and basal cells (Fig 6A–H). In the lamina propria, YFP+ cells closely juxtapose Sox2+ cells of Bowman’s glands (Fig 6F).
To test if GBC or HBC progenitors could descend from Pax7 precursors, we monitored the expression of the basic helix-loop-helix (bHLH) transcription factor Mash1 (mammalian achaete-scute homolog 1) (Gordon et al., 1995; Cau et al., 1997; Shou et al., 1999) and the intercellular adhesion molecule-one (ICAM-1) (Carter et al., 2004), respectively. Mash1+ cells are detected in the basal layers, in addition to the neuronal and sustentacular cell layers of the postnatal day 5 Pax7cre/ROSA YFP OE (Fig 6I-L), subsets of which co-express YFP (Fig 6J–L). Most ICAM-1+ HBCs do not co-express YFP, but rare ICAM-1+YFP+ cells could be detected in regions devoid of YFP+ ORNs and sustentacular cells (Fig 6M–O). These results show that subsets of multiple postnatal olfactory progenitors descend from Pax7+ precursors.
Expression of YFP was used to test for the temporal contributions of YFP+ cells prior to and during olfactory pit development. Rare YFP+ cells were detected in the cephalic mesenchyme and lateral neural folds of E8.5 Pax7cre-ROSA YFP embryos, whereas more abundant contributions to the hindbrain, forebrain and frontonasal region were first seen by E9.5 that persisted to E10.5 (Supplementary Fig S6). Several cells in the E10.5 frontonasal mesenchyme were Pax7+YFP+ as were cells in the olfactory pit, the only region at this developmental stage known to contribute to the OE proper (Fig 2D–F).
At E11.5, of the 630 ± 36.9 cells/mm, 102.8 ± 9.1 cells/mm express YFP (16.3%, Table 1, p<0.0001 vs adult); 43% of YFP+ cells are immature TUJ1+ neurons (Table 3), suggesting additional contributions from embryonic progenitors. However, only 35% of TUJ1+ ORNs (343/972 counted), throughout the developing E11.5 OE express YFP (Fig 7A–D), some of which likely arise via Mash1+ neuronal progenitors (Fig 7E–H). TUJ1 and Mash1 expression was detected also in the few YFP+ cells found in the forebrain (Fig 7A,E). Abundant YFP+ cells were detected in the lateral nasal process, many of which co-expressed the presumptive embryonic olfactory stem cell marker Sox2 (Kawauchi et al., 2004; Beites et al., 2005) (Fig 7I–L). In general, those progenitors most highly expressing Pax6 did not express YFP, producing a distinct border between YFP-expressing and highly expressing Pax6+ cells (Fig 7M–P). These patterns display a regional restriction, and suggest an early embryonic commitment to the Pax7 lineage in multiple embryonic olfactory progenitors, a small proportion of CNS neurons and neuronal progenitors.
The identity and spatiotemporal location of embryonic OE progenitors, as well as their contributions to the postnatal OE are largely unknown. Here, we identified Pax7-expressing cells in the early embryonic OE, which stop expressing Pax7 around E15.5 (Fig 1). The progeny of embryonic cells expressing Pax7 were genetically fate mapped, leading to the identification of various Pax7-derivatives including olfactory progenitors, non-neuronal cells, CNS, olfactory and vomeronasal neurons and glia (Fig 2–7). These results exemplify the heterogeneity of derivatives generated by embryonic Pax7 precursors.
One limitation of the standard Cre-loxP fate mapping approach is the inability to spatiotemporally label Pax7 progenitors, hence the precise developmental time point from which Pax7 precursors first emerge and their temporal lineage allocations remain unknown. Prior to olfactory placode development (before E10.5), we detected YFP+ cells in the anterior neural folds and frontonasal mesenchyme. In the chick embryo, portions of the anterior neural folds are thought to generate the OE (Couly and Le Douarin, 1985; Bhattacharyya and Bronner-Fraser, 2008) however, the frontonasal mesenchyme has no known cellular contribution to the OE. Coincident expression of Pax7 with the YFP reporter in the E10.5 olfactory placode, which subsequently forms the OE, suggests that labeled cells detected within the OE arose via a Pax7+ embryonic olfactory progenitor. The precise origin of olfactory ensheathing cells however, is currently debated (discussed below).
Lineage tracing of GBCs (Goldstein et al., 1998; Huard et al., 1998; Chen et al., 2004) and HBCs (Leung et al., 2007; Iwai et al., 2008) has shown that the most potent clones generate HBCs, GBCs, ORNs, sustentacular cells, and Bowman’s duct/gland cells. HBCs can also produce olfactory ensheathing glia in vitro (Carter et al., 2004). However, neither GBCs nor HBCs have demonstrated the in vivo production of olfactory ensheathing cells. Thus compared to postnatal basal progenitors, Pax7+ embryonic progenitors share some common cellular descendants, but are unique in their ability to additionally produce olfactory ensheathing glia and CNS neurons. These analyses suggest a possible switch to more restricted progenitors in the OE with aging.
Transgenic mice used to fate map embryonic olfactory progenitors identified either glia-restricted (Murdoch and Roskams, 2007) or neuron-restricted embryonic progenitors (Duggan et al., 2008; Murdoch and Roskams, 2008), but not progenitors that contribute to non-neuronal cells, neurons and glia, such as Pax7 descendants (Figs 2,,44–6). Additionally, Pax7 neuronal and sustentacular cell descendants revealed a unique pattern of YFP+ progeny, not seen in the aforementioned lineage tracing studies, which is found in patches throughout the postnatal OE. This pattern could not be attributed to the instability of Pax7 expression associated with Cre in the Pax7 locus (Supplementary Fig S5).
The mosaic pattern of YFP expression could alternatively be due to incomplete recombination. However, since Pax7 expression is not altered, Cre recombinase expression is also likely unaltered, and thus full recombination should result. Multiple data support this view. Incomplete recombination would lead to random excision events resulting in variable YFP expression patterns. Contrary to this, the YFP expression pattern is consistent between equivalently staged samples and in an independent transgenic reporter line, R26R, expressing the LacZ gene (data not shown). The overall YFP expression pattern seen in Pax7-cre/ROSA YFP embryos is consistent with that reported previously using several transgenic mouse lines (Pax7-LacZ (Relaix et al., 2004), Pax7-ZsGreen (Bosnakovski et al., 2008) and Pax7-cre/ROSA LacZ mice (Keller et al., 2004)). Finally, although the signal intensity of Pax7 protein expression in the olfactory placode and OE is much lower compared to the frontonasal mesenchyme, all Pax7+ cells in the olfactory placode appear to express the YFP reporter (Fig 2). These results do not support an “incomplete recombination” model and suggest that the pattern of YFP expression seen in Pax7-cre/ROSA YFP mice is reflective of complete recombination events that are Pax7-driven. Discrediting these possible technical caveats highlights the surprising nature of the varied cell types, and mosaic distribution of Pax7 derivatives, calling for a mechanistic explanation.
Our study exposes an intriguing difference in the proportion of Pax7-derived cells contributed to the OE versus those contributed to olfactory ensheathing cells. While the first set is composed of a modest and declining contribution, the second seems stable and robust. A possible explanation for such disparate contributions is that the precursors for both OE and olfactory ensheathing cells are the same (Fig 8A–D), and simply one fate is favored, while the others are temporally and quantitatively restricted. Alternatively different Pax7-expressing progenitors could be responsible for OE versus olfactory ensheathing cell derivation (Fig 8), each displaying different regulation of proliferation, survival and differentiation.
The mosaic pattern of Pax7 progeny suggests a considerable contribution from Pax7 negative progenitors to the postnatal OE. Indeed, as the OE expands during embryonic to adult stages, there is a 27 fold decrease (16.3% divided by 0.6%, Table 1) in the percentage of total cells expressing YFP, even though the average number of YFP+ cells does not appreciably increase beyond postnatal day 5 (Table 1). Decreases in the YFP contributions to the OE are seen in all cell types examined (Table 1). The proportionate decline in Pax7-derived OE progeny with aging could be explained by a progressive depletion in Pax7-progenitors. This makes obvious the participation of alternative, yet undescribed Pax7-negative progenitors, which contribute more highly throughout the OE. If these alternative progenitors are restricted to embryonic stages, like Pax7-precursors, they likely possess greater survival and/or proliferative potential in comparison. Another option considers a set of progenitors present during both embryonic and postnatal development that is responsive to cues for embryonic tissue expansion and postnatal maintenance.
How might local expression of Pax7 define a unique progenitor domain? Regional expression and level of transcription factors, as seen in the developing spinal cord (Briscoe et al., 2000; Bel-Vialar et al., 2007), and detected here between Pax7 and Pax6, could form initial progenitor zones responsible for the fine architecture of the OE. The unique transcriptional profiles characteristic of independent progenitors may confer proliferative and/or survival advantages compared to their competitors. Further analysis of the contribution of Pax6 and other putative progenitors, may resolve these seemingly random mosaic patterns into more coherent organizational domains. Importantly, our capacity to identify additional OE progenitors and the expression patterns of their progeny, is currently limited by a lack of markers at earlier stages.
In muscle, Pax7+ satellite cells are stem/progenitor cells that contribute to postnatal muscle growth and regeneration (Charge and Rudnicki, 2004). Like Pax7+ muscle satellite cells (Collins et al., 2005; Kuang et al., 2007), Pax7+ olfactory precursors likely also represent a hierarchical heterogeneous progenitor cell population with mostly committed progenitors, but whose stem cell function remains to be tested.
Neural crest cells are multipotent migratory cells critical for vertebrate development that contribute to multiple tissues including the peripheral nervous system, cardiovascular, craniofacial bone and cartilage (Kirby et al., 1983; Le Douarin et al., 1998; Chai et al., 2000; Jiang et al., 2000; Baker, 2005; Nakamura et al., 2006). In the chick, Pax7 has been identified as an early marker of neural crest cells that is required for neural crest development (Basch et al., 2006). Accordingly, Pax7 is expressed in neural crest cell precursors in the dorsal neural tube (Jostes et al., 1991; Mansouri et al., 1996; Lang et al., 2003; Basch et al., 2006) and Pax7-cre/ROSA YFP mice label neural crest derivatives (Murdoch and Garcia-Castro, in preparation). Interestingly, in dorsal root ganglia, we have detected the Pax7 lineage in neurons but not S100β+ peripheral glia (data not shown), suggesting that subpopulations of peripheral and olfactory sensory neurons share similar transcriptional lineage profiles.
Our data leads one to question whether neural crest cells contribute to some of the OE derivatives here identified, olfactory ensheathing cells in particular. Quail-chick transplants of the anterior neural folds, a region with Pax7 expression (Basch et al., 2006; Otto et al., 2006; Khudyakov and Bronner-Fraser, 2009), contribute to the olfactory placodes (Couly and Le Douarin, 1985; Bhattacharyya and Bronner-Fraser, 2008), from which olfactory ensheathing cells are thought to be derived (Farbman, 1992). Evidence from zebrafish however, suggests that at least some olfactory ensheathing cells, could have a neural crest origin (Whitlock, 2004). Little is known about the lineage of olfactory ensheathing cells in the mouse, beyond that they arise via a BLBP+ precursor (Murdoch and Roskams, 2007). The Pax7 progeny detected in the olfactory mucosa of our Pax7-cre/ROSA YFP mice could arise via Pax7-expressing cells of the olfactory placode, from pre-placodal cells, or from neural crest cells. Our experimental model can’t resolve this issue, but transgenic mice used to fate map neural crest derivatives, like Wnt1-cre/reporters (Chai et al., 2000; Jiang et al., 2000), offer encouraging alternatives.
Here we identify embryonic Pax7+ progenitors, whose postnatal progeny reveal novel spatiotemporal patterning of the OE and contribute to multiple cell lineages including neurons, glia and non-neuronal cells. We propose that Pax7 expression thus identifies the first documented population of embryonic progenitors capable of producing multiple committed olfactory cell types in vivo.
We thank the following colleagues for sharing the reagents that made this project possible – Charles Greer for antibodies; James Schwob, (SUS4 antibody); Mario Capecchi (Pax7-cre mice); Diane Krause (ROSA -YFP and –LacZ mice). Thanks to Dorothy Wierzbicki for Fig 4 artwork; Carson Miller and Elke Stein for technical advice and support. Funding was provided by a Seessel Postdoctoral scholarship (BM) and NIH RO1 DE017914 (MIGC).