DEV02143 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Co-expression of Fgf8 and definitive olfactory neuroepithelial markers. Double-label in situ hybridization for Fgf8 (orange) and Dlx5, Pax6 or Sox2 (blue) performed on cryosections of E10.5 wild-type embryos. In addition to Sox2 (compare with Fig. 1), a subpopulation of Fgf8-expressing cells also express Pax6 and Dlx5; expression of these two genes is known to mark definitive OE at this stage of development (Acampora et al., 1999; Long et al., 2003; Park et al., 2004). Zones where expression overlaps are indicated with brackets. Scale bar: 200 mm. In situ hybridization was performed as described in the Materials and methods. Additional probes used were 0.9 kb mouse Dlx5 cDNA (Stuhmer et al., 2002) and mouse Pax-6 (295-595 bp of Genbank Accession Number, MN 013627).

• Supplemental Figure 2 -

  Fig. S2. Fgf8 mRNA is expressed in E14.5 OE. (A) In situ hybridization for Fgf8 (ORF probe) in cryosections of E14.5 OE. Arrows indicate cells in basal cell compartment that express Fgf8. NC, nasal cavity; BL, basal lamina; Str, stroma. The cartoon depicts the established OE structure, evident from about E14 onwards, and shows the relative location and numbers of neural stem cells (gold), Mash1-expressing committed neuronal progenitors (dark blue) and Ngn1-expressing INPs (light blue) in the basal cell (BC) layer. ORN, olfactory receptor neurons (green); Sup, supporting (sustentacular) cells (pale green). Scale bar: 50 mm.  (B) RT-PCR for Fgf8 splice variants. Total RNA was isolated from purified E14.5 mouse OE and primary cultures of OE stromal cells (no passage). PCR primers to detect Fgf8 alternative splicing variants have been reported previously (Crossley and Martin, 1995). Lane 1, size markers; lane 2, cDNA from purified E14.5 OE (149 bp=form a; 184 bp=form b; 271 bp=form f); lane 3, OE, no-RT control; lane 4, cDNA from cultured E14.5 OE stroma; lane 5, stroma, no-RT control; lane 6, Fgf8b plasmid control; lane 7, Fgf8a plasmid control.

• Supplemental Figure 3 -

  Fig. S3. Recombinant FGF8 promotes stem cell development and INP proliferation in vitro. OE explant assays were used to demonstrate that FGF8b stimulates neurogenesis in OE cultures, and acts on both INPs and putative neuronal stem cells. (A) Effects on OE neural stem cells were determined by growing explants for 4 days and labeling cells in S phase with BrdU during the final 24 hours in vitro. Only in the presence of an agent that promotes stem cell survival and/or proliferation will a small fraction of explants (normally 5-10%) continue to produce proliferating, late-stage neuronal progenitors at this time in culture (DeHamer et al., 1994; Shou et al., 2000). OE explants were cultured as described previously (DeHamer et al., 1994), with (gray bars) or without (white bars) recombinant FGF8b (50 nM) for 96 hours, with BrdU added for the final 24 hours. Anti-BrdU immunostaining of cultured OE explants was performed as described (DeHamer et al., 1994), except that cultures were fixed for 30 minutes at room temperature in 3.7% formaldehyde in PBS with 5% sucrose, then permeabilized with 0.2% Triton-X 100 in PBS for 30 minutes and treated with 2 M HCl for 1 hour at 37°C before antibody processing and detection. Explant area and numbers of BrdU-incorporating cells surrounding each explant were counted and data normalized for comparison as described (DeHamer et al., 1994). y-axis is the percentage of total explants, in each condition, exhibiting the indicated number of BrdU-immunopositive cells. Recombinant FGF8b is able to stimulate stem cell activity (determined as explants with more than 35 BrdU-labeled progenitors surrounding them; see above) in 10.5% of OE explants; this is similar to that observed with FGF2 treatment (8.5% of explants) (DeHamer et al., 1994). By contrast, no explants grown in control conditions (no FGF8b added) show high stem cell activity. Thus, this action of FGF8b is consistent with the idea that it supports survival and/or proliferation of OE neuronal stem cells (DeHamer et al., 1994; Shou et al., 2000). (B) To test the ability of FGF8b to promote proliferation of INPs, OE explants were cultured in the presence (black bar) or absence of recombinant FGF8b (R&D Systems; 50 nM, replenished daily) for 48 hours. Proliferating cells were labeled with ³H-thymidine for the final 24 hours, then cultures were processed and analyzed as described previously (DeHamer et al., 1994). Mean numbers of ³H-thymidine-positive cells (± s.e.m.) surrounding each explant are indicated. Under control conditions (e.g. without added FGF), INP proliferation is known to largely cease by 24 hours in culture (Calof and Chikaraishi, 1989); however, in cultures treated with FGF2, a significant fraction of INPs continues to undergo division (DeHamer et al., 1994). In the present study, FGF8b treatment produced a threefold increase in the proliferative labeling index of cultured INPs, compared with untreated controls.  In parallel experiments, cultures treated with 10 ng/ml FGF2 showed a similar fold-increase in proliferation [54.042±10.343 (s.d.) in FGF2 versus 16.00±2.223 (s.d.) for untreated cultures; data not shown].

• Supplemental Figure 4 -

  Fig. S4. OE development occurs in the absence of olfactory bulbs. OE development and olfactory bulb (OB) structure were compared in in situ hybridization of sagittal cryosections through the heads of Fgf8 hypomorphs (Fgf8^neo/neo), which lack most OB tissue (Garel et al., 2003; Meyers et al., 1998), and age-matched wild-type animals, at P0. Overall neuronal cell development in brain and OE was assessed by in situ hybridization with a probe to Ncam1. The results show that the normal complement of Ncam1-expressing ORNs is present in the OE of Fgf8 hypomorphs, even though these animals lack an obvious OB. Probes for OB granule cells (Gad67) and neuronal progenitors of the rostral migratory stream (Mash1) were used to examine OB structure more closely, as described previously (Murray and Calof, 1999). Despite a profound loss of both OB granule cells and Mash1-expressing progenitors in the ventricular zone (VZ), the OE of Fgf8 hypomorphs is essentially normal. Scale bars: 200 mm. NC, nasal cavity; OB, olfactory bulb; OE, olfactory epithelium; Tel, telencephalon.

  References

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