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First published online 2 November 2005
doi: 10.1242/dev.02143

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Fgf8 expression defines a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the mouse

Shimako Kawauchi¹, Jianyong Shou^1,*, Rosaysela Santos¹, Jean M. Hébert², Susan K. McConnell³, Ivor Mason⁴ and Anne L. Calof^1,

¹ Department of Anatomy and Neurobiology, and Developmental Biology Center, University of California, Irvine, CA 92697-1275, USA
² Department of Neuroscience and Molecular Genetics, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA
³ Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
⁴ MRC Centre for Developmental Neurobiology, New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK

View larger version (97K): [in a new window]   Fig. 1. Expression of Fgf8 and neuronal cell markers in developing OE. (A) Five successive images show in situ hybridization for Fgf8 (full-length ORF probe) and OE neuronal lineage markers in invaginating nasal pit (NP) at E10.5. In whole-mount in situ hybridization, Fgf8 is detected in commissural plate and olfactory placode (white asterisk), branchial arches, mid-hindbrain junction, and limb and tail buds. Scale bar: 1 mm. In serial sections, locations of neuronal lineage markers within the OE are shown: arrowheads indicate Mash1-expressing cells, arrow indicates Ncam1-expressing neurons. Scale bar: 200 µm. (B) Double label in situ hybridization for Fgf8 (full-length ORF probe, orange) and Sox2 (blue) demonstrates overlap of the two markers in a small rim of surface ectoderm and adjacent invaginating neuroepithelium (brackets). Scale bar: 50 µm. (C) In situ hybridization for unprocessed, intronic RNA (Fgf8int probe) expression and processed Fgf8 mRNA (Fgf8ex2,3 probe) expression. Red and blue arrowheads indicate the basolateral extent of intronic RNA versus processed mRNA expression, respectively. The domain of mRNA expression subsumes that of intronic expression. Scale bars: 50 µm. LNP, lateral nasal process; MNP, medial nasal process. (D) Model of peripheral-to-central process of neuronal differentiation in developing OE and origin of Sox2-expressing neural stem cells from Fgf8-expressing ectoderm.

View larger version (28K): [in a new window]   Fig. 2. Localization of Foxg1-driven Cre expression and strategy for generating Fgf8 conditional knockouts. (A) X-gal staining of R26RlacZ/+;Foxg1Cre/+ embryos at E9.5, E10.5 and E12.5. Top panels show whole-mount images and bottom panels show corresponding sections from embryos processed as whole mounts. At E9.5, staining is detected in forebrain (FB) and olfactory placode (arrows). At E10.5, staining is observed at rim of invaginating nasal pit (NP; arrows). OC, optic cup. At E12.5, prominent staining is observed in OE. Scale bars: 500 µm in the top panels; 200 µm in the bottom panels. (B) Fgf8flox/flox,Foxg1+/+ females were crossed with Fgf8d2,3/+;Foxg1Cre/+ males and embryos genotyped with three different primer sets to detect different alleles: Fgf8-flox and wild-type (F1 and F2), Fgf8-d2,3 (F1 and F3) and Cre (C1 and C2). One-quarter of offspring had the Fgf8d2,3/flox;Foxg1Cre/+ (mutant) genotype.

View larger version (84K): [in a new window]   Fig. 3. Deficits in anterior neural structures and FGF-mediated signaling in mutant embryos. (A) Pictures of mutant animals and control littermates from E9.5 to birth. White arrowheads indicate forebrain-midbrain boundary. Black asterisks indicate nasal pits in E10.5 mutants. P, pinna. Scale bars: 1 mm. (B) Whole-mount in situ hybridization with Fgf8 exon2,3 probe in E10.5 embryos. White arrows indicate nasal pits. CP, commissural plate. Insets show forelimbs of embryos hybridized with Fgf8 ex2,3 probe. Scale bar: 0.5 mm. (C) RT-PCR for Fgf8 exons 2 and 3 using cDNA from E10.5 forebrain/frontonasal tissue of control and mutant animals. Lane 1, 100 bp marker; lane 2, control tissue with Fgf8 ex2,3 primers; lane 3, control tissue and no RT; lane 4, mutant tissue with Fgf8 ex2,3 primers; lane 5, mutant tissue with no RT; lane 6, Fgf8 exon2,3 plasmid control; lane 7, control cDNA and Hprt primers; lane 8, mutant cDNA with Hprt primers. (D) Pyst1 expression is reduced in OE and underlying mesenchyme (arrows in insets). R, retina; FB, forebrain. No change in Shh expression is apparent in mutants. MB, midbrain. Scale bar: 200 µm.

View larger version (135K): [in a new window]   Fig. 4. Histological analysis of mutant OE. (A) Schematic model of mouse OE development. OP, olfactory placode; NP, nasal pit; FB, forebrain; MNP, medial nasal process; LNP, lateral nasal process; S, nasal septum; NC, nasal cavity. (B) Hematoxylin-eosin staining was performed on 20 µm cryosections through the entire frontonasal region of control and mutant embryos from E10.5-E17.5. Scale bar: 200 µm. CP, cartilage primordium of nasal capsule; H, heart; L, lens; MB, midbrain; NR, neural retina; Tel, telencephalon.

View larger version (85K): [in a new window]   Fig. 5. Cessation of neuronal lineage development in mutant OE. (A) In situ hybridization on serial coronal sections through E10.5 nasal pit. No Fgf8 ex2,3 is detected in mutant. (B) In situ hybridization on serial coronal sections of E12.5 Type A mutant. Arrowhead indicates region of Sox2-expressing epithelium in sections hybridized with other probes. (C) In situ hybridization on horizontal serial sections of E14.5 Type B mutant. Arrow indicates apparent OE remnant. (D) In situ hybridization on horizontal sections of E17.5 Type B mutant. Arrowheads indicate presumptive remnant of OE. NP, nasal pit; MNP, medial nasal process; VG, Vth ganglion; OC, oral cavity; T, tongue; VNO, vomeronasal organ; FB, (presumptive) forebrain; NR, neural retina; S, nasal septum. Scale bars: 200 µm.

View larger version (58K): [in a new window]   Fig. 6. Fgf8 is required for cell survival in the neurogenic domain. (A) In situ hybridization for Fgf8 ex1 probe indicates areas where (nonfunctional) Fgf8 is expressed in mutants (Fgf8 ex2,3 probe signal was not detected in mutants). TUNEL panel shows high number of apoptotic cells in mutants in ectoderm (white asterisk) and OE (white arrowhead; magnified in inset) of invaginating nasal pit (NP). Hoechst panel shows extent of invaginating NP. LNP, lateral nasal process; MNP, medial nasal process. Sox2 expression is reduced in OE of MNP (black arrowhead). Scale bars: 100 µm. (B) High-power micrographs of TUNEL staining in OE of E12.5 Type B mutant and control littermate. Broken white line indicates basal lamina (BL) of OE. AL, apical surface; Str, stroma. Scale bar: 50 µm. (C) Total TUNEL+ cells in identifiable nasal epithelium (NE) were counted, and area of NE measured, in multiple sections at each age indicated. Data for each NP were summed and normalized to 0.1 mm2, the average total NE area in a section at E12.5. Mean±s.d. for data from individual NPs are shown; NPs from a minimum of 2 animals of each genotype at each age were counted. Differences between mutants and controls were statistically significant at E10.5 (P=0.009, Student's t-test) and E12.5 (P=0.003), but not E14.5. (D) High-power micrographs of anti-phosopho-Histone H3 immunostaining at E10.5. Scale bar: 50 µm. (E) Quantification of data illustrated in D. (F) High-power micrographs of anti-BrdU immunostaining at E10.5. Scale bar: 50 µm. (G) Quantification of data illustrated in F. There are no significant differences between datasets in E and G. Data for each NP were summed and normalized to 0.03 mm2, the average total OE area in a section at E10.5. Mean±s.d. for data from individual NPs is shown; NPs from a minimum of two animals of each genotype at each age were counted.

View larger version (21K): [in a new window]   Fig. 7. Role of Fgf8 in olfactory neurogenesis. Cartoon illustrating the relative positions of the Fgf8 expression domain (anterior morphogenetic center) and different neuronal cell types during primary neurogenesis at E10.5 in normal OE (wild type) and a model for the role of Fgf8 in primary olfactory neurogenesis based on the consequences of inactivating Fgf8 in the anterior morphogenetic center using Foxg1-driven Cre (mutant). Fgf8 expression domain, orange; Sox2 expression domain (definitive neuroepithelium), yellow; primordial neural stem cells (co-expressing Sox2 and Fgf8), green; Mash1-expressing committed neuronal progenitors, dark blue; INPs, light blue; Ncam1-expressing neurons, pink. Cells in the Fgf8 expression domain that undergo apoptosis when Fgf8 is inactivated are shown in red, and apoptotic primordial neural stem cells in green with a red jagged border. Vestigial populations of other neuronal cell types are shown in their corresponding colors, but with jagged borders. For discussion, see text.