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First published online 18 March 2009
doi: 10.1242/dev.034967

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Foxg1 promotes olfactory neurogenesis by antagonizing Gdf11

Shimako Kawauchi^1,*, Joon Kim^1,*,, Rosaysela Santos¹, Hsiao-Huei Wu^1,, Arthur D. Lander² and Anne L. Calof^1,

¹ Department of Anatomy and Neurobiology and the Center for Complex Biological Systems, University of California, Irvine, CA 92697, USA.
² Department of Developmental and Cell Biology and the Center for Complex Biological Systems, University of California, Irvine, CA 92697, USA.

View larger version (71K): [in this window] [in a new window]   Fig. 1. Failure of primary neurogenesis in Foxg1-/- OE. (A) Sections of olfactory epithelium (OE) from wild-type and Foxg1-/- mouse embryos at E11, showing the decrease in the numbers of cells expressing stage-specific neuronal markers. D, dorsal; V, ventral. (B) Apoptotic cells visualized by TUNEL labeling in E11 OE from wild-type and Foxg1-/- OE. For comparison, numbers were normalized to an area of 15,000 µm2, the average area of each section of Foxg1-/- OE at this age. Mean values ±s.d. of TUNEL+ cells per 15,000 µm2 OE are: wild type, 7.51±4.24; Foxg1-/-, 6.11±0.095. Data, which showed no significant difference (Student's t-test) (Glantz, 2005), were collected from two animals of each genotype. (C) Fgf8 and Foxg1 expression at E11. Fgf8 is expressed at the rim of the olfactory pit (OP) in wild type, and the pattern is unchanged in Foxg1-/- OE (arrowheads). The Foxg1 expression domain (detected by ISH to Cre), located in the central neurogenic zone of the OE, is reduced in Foxg1-/- OE. Scale bars: 100 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 2. Expression of Foxg1 and Gdf11 in developing mouse OE. (A,B) Coronal sections through heads of E10.5 and E11.5 wild-type mice. Gdf11 expression is detected both in the OE and in a subset of cells in the mesenchyme, possibly migrating pioneer neurons (arrowheads). FB, forebrain; OE, olfactory epithelium. (C-E) Horizontal sections showing the OE in one-half of the nasal region (septum is at bottom) at E12.5, E14.5 and E17.5 in wild-type mice. The expression domains of Foxg1 and Gdf11 overlap, except in regions such as the anterior OE, which has ceased planar expansion at these ages. Insets show high magnification views of the OE at posterior regions of co-expression and anterior regions where co-expression has ceased (anterior is right; posterior, left). NC, nasal cavity; BL, basal lamina. Scale bars: 200 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Cell proliferation and p21Cip1 expression in mutant and wild-type OE. (A) BrdU pulse-fix experiments were performed as described in the Materials and methods, with pregnant dams injected at gestational day 11 or 12. Images show representative anti-BrdU immunostaining results. Graph shows quantification (mean±s.e.m.) of total number of BrdU+ cells per OE section at each age in Foxg1-/- embryos (blue) and wild-type littermates (pink). Histogram shows average OE area per section for mutants versus wild types. The number of BrdU+ cells is significantly lower in Foxg1-/- mutants at each age, as is the average area of OE per section (P<0.01, Student's t-test). Data were collected from two animals of each genotype at each age. (B) Coronal sections of an E10.5 Foxg1-/- embryo and a wild-type littermate, processed for ISH with a p21Cip1 probe. Dorsal is up; ventral, down. OE, olfactory epithelium. Scale bar: 100 µm. (C) Horizontal sections of an E13.5 Gdf11-/- embryo and a wild-type littermate, processed as in B. nc, nasal cavity; A, anterior; P, posterior. Scale bar: 100 µm. (D, top) Immunofluorescence staining of migratory OE neuronal cells in explant cultures (after 14 hours in vitro), grown with or without Gdf11, then processed for anti-p21Cip1 immunoreactivity. (Bottom) Quantification of results from a typical culture experiment. Percentages of p21Cip1+ cells were: 2.5% of 587 counted cells in control cultures; 14.3% of 1065 counted cells in GDF11-treated cultures. P<0.05, Student's t-test.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Rescue of the Foxg1-/- OE phenotype by loss of Gdf11. (A) Cartoon of normal frontonasal structures in mice at P0, shown as a mid-sagittal section. Red square indicates the region photographed for ISH images shown in B-D. G, serous gland; I, right upper incisor; OB, olfactory bulb; OE, olfactory epithelium; T, turbinate bone; NC, nasal cavity. (B) ISH for Ngn1 to show OE neuronal cells in wild-type animals at P0. (C) Olfactory turbinate structures and Ngn1-expressing OE are not observed in Foxg1-/- animals. (D) Foxg1-/-;Gdf11-/- mice show recovery of turbinate structures and OE expressing Ngn1. Note that there is no OB present in either Foxg1-/- or Foxg1-/-;Gdf11-/-; note also that the telencephalon is drastically reduced in Foxg1-/- mice and is not rescued by loss of Gdf11 (see Results). Scale bar: 500 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 5. Significant rescue of the Foxg1-/- OE phenotype occurs by E13.5. Horizontal sections through heads of E13.5 wild-type and Gdf11-/- embryos, hybridized with probes for indicated OE neuronal lineage markers. (A,B) Identical patterns of expression are observed in wild-type and Gdf11-/- OE at this age. OE, olfactory epithelium; nc, nasal cavity; R, retina; Sep, septum; T, turbinate. (C) Foxg1-/- embryos lack OE, a nasal cavity, and all OE neuronal lineage markers by E13.5. III, ventral portion of third ventricle; OC, optic chiasm. (D) Substantial rescue of the OE and nasal cavity structures, as well as cells expressing all OE neuronal lineage markers, is observed in Foxg1-/-;Gdf11-/- embryos. Scale bar: 200 µm.

View larger version (105K): [in this window] [in a new window]   Fig. 6. Rescue of OE neurogenesis in Foxg1-/- mutants is dependent on Gdf11 gene dosage. ISH for OE neuronal lineage markers (Mash1, Ngn1 and Ncam) and sustentacular cells (Otx2), performed on horizontal sections through the OE of E16.5 wild-type and mutant littermates. Insets show high magnification views of septal OE. (A,B) OE and cell types within it are similar in wild-type and Gdf11-/- mice at this age, except that Ngn1- and Ncam-expressing cell layers (and hence OE overall) are thicker, as reported previously (Wu et al., 2003). (C) No OE structure (apart from a truncated septum), nor any cell type-specific markers, are evident in sections from Foxg1-/- mice at the same dorsoventral level. (D) Loss of one Gdf11 allele (Foxg1-/-;Gdf11+/-) rescues all cell types in the OE, and the OE appears to be of normal thickness, although planar expanse of the OE and morphogenesis of nasal cavity are clearly deficient in the compound mutant. (E) Rescue is more pronounced in Foxg1-/-;Gdf11-/- double mutants, particularly in terms of OE planar expanse and nasal cavity morphogenesis. For all panels, posterior is left, anterior is right. Sus, sustentacular cells; BL, basal lamina. Scale bar: 400 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 7. Downregulation of Fst expression in Foxg1-/- nasal mucosa is rescued by loss of Gdf11. (A) ISH for Fst performed on coronal sections through heads of wild-type and Foxg1-/- mice at E10.5 and E12.5, and on horizontal sections at E16.5. At E16.5, when Fst is expressed in both the OE and stroma of nasal mucosa in wild types (A, top right panel), Fst expression is undetectable in Foxg1-/- embryos in rare instances when remnants of nasal mucosa are observed (A, bottom right panel). NE, nasal epithelium; OE, olfactory epithelium; BL, basal lamina; nc, nasal cavity; Str, stroma; fb, forebrain. Scale bars: 100 µm in E10.5 and E12.5; 50 µm in E16.5. (B) Fst expression is restored in the OE and underlying stroma of Foxg1-/-;Gdf11+/- and Foxg1-/-;Gdf11-/-. Scale bar: 50 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 8. Analysis of Gdf11 mRNA expression in the OE of embryos of different genotypes. (A) Gdf11 does not regulate its own transcription. Gdf11 and Gapdh transcript levels at E16.5 and E11.5 were quantified by Q-RT-PCR, and dCT and ddCT values with errors were calculated as described in the Materials and methods. For presentation purposes, data are normalized to wild-type values. Statistics [Dunnett's test for multiple comparisons against a single control (DT) (Glantz, 2005)] were performed using mean dCT values and corresponding errors (s.e.m.), which were: E16.5, wild type=5.32±0.0233; Gdf11+/-=6.89±0.0523, Gdf11-/-=13.53±0.4933; E11.5, wild type=5.22±0.200, Gdf11+/-=6.24±0.009, Gdf11-/-=12.22±0.142. (B) Gdf11 is expressed in Foxg1-/- OE at E12.0. ISH for Gdf11 was performed on coronal cryosections of wild type and Foxg1-/-. Scale bar: 50 µm. (C-E) Relative Gdf11 and Sox2 expression values in OE from E11.5 embryos of different genotypes. Gdf11, Sox2, and Gapdh transcript levels were quantified by Q-RT-PCR as described in the Materials and methods. In C and D, Gdf11:Gapdh and Sox2:Gapdh expression levels are normalized to wild-type values for presentation purposes. Statistics (DT) were performed using mean dCT values and corresponding errors (s.e.m.), which were as follows: Gdf11:Gapdh, wild type=5.22±0.200, n=3; Gdf11+/-=6.24±0.009, n=2; Gdf11-/-=12.22±0.142, n=3; Foxg1-/-=6.07±0.088, n=3; Foxg1-/-;Gdf11+/-=6.39±0.422, n=3. Sox2:Gapdh: wild type=6.81±0.217, n=3; Gdf11+/-=6.30±0.116, n=3; Gdf11-/-=6.42±0.230, n=3; Foxg1-/-=9.09±0.083, n=3; Foxg1-/-;Gdf11+/-=8.85±0.176, n=3. (E) Gdf11 expression plotted as the ratio of the mean Gdf11 transcript level to the mean Sox2 level. Values that differ significantly from wild type (P<0.05, DT) are denoted by asterisks.

View larger version (100K): [in this window] [in a new window]   Fig. 9. Absence of Gdf11 does not rescue defects in Foxg1-/- cerebral cortex. (A) Hematoxylin and Eosin staining of horizontal sections through the brains of wild type, Foxg1-/-, and Foxg1-/-;Gdf11-/- double mutants at E13.5. The cortex is severely reduced in Foxg1-/- embryos; absence of Gdf11 does not rescue this phenotype. Scale bar: 200 µm. (B) Expression of Foxg1 and Gdf11 in coronal sections through developing mouse brain. Foxg1 is abundantly expressed in the telencephalon (except for the cortical hem) at E11.5 (expression boundary indicated by arrowheads). Gdf11 expression is apparent in the ventral telencephalon and OE at E11.5, but by E12.5 is restricted to ventral midline of the telencephalon and the nascent hippocampus (arrows). At E15.5, no Gdf11 expression is apparent in the rostral telencephalon, whereas Foxg1 levels are high, especially in dorsal areas. Scale bars: 400 µm. C, cortex; CB, cerebral cortex; CH, cortical hem; CP, cortical plate; Di, diencephalon; H, hippocampus; LV, lateral ventricle; OE, olfactory epithelium; S, striatum; POA, preoptic area; SVZ, subventricular zone.

View larger version (15K): [in this window] [in a new window]   Fig. 10. Schematic model of Foxg1-Gdf11 interactions controlling OE neurogenesis. (A) In wild-type OE, Foxg1 and Gdf11 are both produced by OE neuronal cells, but Foxg1 pro-neurogenic activity antagonizes both the anti-neurogenic activity of Gdf11 and the production of Gdf11 by OE neuronal cells. Fst is also expressed by OE neuronal cells, and Fst action antagonizes Gdf11 activity. This default network of gene activities controls the normal steady-state level of neurogenesis in the OE. (B) In Foxg1-/- OE, Foxg1 activity is absent, Fst expression is downregulated, and Gdf11 expression is upregulated, resulting in hypersensitivity of the frontonasal region and OE to the action of Gdf11. Both OE neurogenesis and planar expansion of the OE fail. (C) In the Foxg1-/-;Gdf11-/- double mutant, Fst expression is restored and histogenesis (neurogenesis) within the OE is rescued, as the anti-neurogenic activity of Gdf11 is now removed and any similar anti-neurogenic factors are antagonized by Fst. (D) Foxg1 activity strongly inhibits both Gdf11 activity and expression, which would allow the OE to undergo planar expansion in sites where Foxg1 is highly expressed in wild-type OE (e.g. posterior recess of the nasal cavity). Once expansive growth is finished, Foxg1 expression is downregulated (e.g. in the anterior septum), and OE neurogenesis returns to its default state.

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