Supplementary figure legends

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1 Supplementary figure legends Supplementary Figure 1. Shep1 knockout mice have small olfactory bulbs. A, Photographs from the side of dissected brains from P0.5 Shep1 wild-type (WT), heterozygous (HET), and knockout (KO) mice. B, Nissl staining of sagittal sections does not reveal major abnormalities in the P0.5 forebrain architecture of Shep1 knockout mice compared to wild-type or heterozygous mice, except for a much smaller olfactory bulb. Arrows point to the olfactory bulb. Residual portions of the olfactory epithelium were left attached to the dissected wild-type and heterozygous forebrains through the OSN axons to avoid damaging the olfactory bulb. Scale bars = 1 mm. Supplementary Figure 2. The OSN axons appear to stall outside the pial basement membrane at E16.5. A series of 40 µm thick sagittal sections, taken at approximately 200 µm intervals from wild-type (WT) and Shep1 knockout (KO) mice, were labeled with an anti-olfactory marker protein (OMP) antibody to visualize OSN axons and an anti-laminin antibody to visualize the pial basement membrane. The wild-type axons extend inside the olfactory bulb whereas the knockout axons appear to be stalled on the outside of the basement membrane. Scale bar, 500 µm. Supplementary Figure 3. Apoptosis is not increased in the Shep1 knockout olfactory bulb or olfactory epithelium at E14.5. A, C, Coronal sections of wild-type and Shep1 knockout olfactory bulb and olfactory epithelium were double-labled for cleaved caspase-3 to label apoptotic cells (red) and for βiii-tubulin to label immature neurons (green). Scale bar, 20 µm. B, Quantification of the number of caspase-3 positive cells/mm 2 in sections of the olfactory bulb. The histogram 1

2 shows averages from 2 pairs of wild-type (n = 7 sections) and Shep1 knockout (n = 6 sections) mice ± standard error. p >0.5, Student s t-test. D, Quantification of the number of caspase-3 positive cells/mm length of the olfactory epithelium. The histogram shows averages from 2 pairs of wild-type (n = 6 sections) and Shep1 knockout (n = 8 sections) mice ± standard error. p >0.5, Student s t-test. Supplementary Figure 4. Shep1 mrna is present in the OSNs. At E.13.5 (A), strongest expression of Shep1 transcripts is found in the OE (D) and in the primordium of the OB. At E15.5 (B), Shep1 mrna expression in the olfactory system is strongest in OSNs of the OE (E). Furthermore, individual cells within the OB, suggestive of Mitral cells, are positive for Shep1. At P1 (C), there is still weaker but obvious expression of Shep1 in OSNs (F). Furthermore, the thin band of Mitral cells can be distinguished in the OB (C). BC, basal cells; MCL, mitral cell layer; OB, olfactory bulb; OE, olfactory epithelium; OSN, olfactory sensory neurons; SC, sustentacular cells. Original magnification 5X in A, B, C and 40X in D, E, F. Supplementary Figure 5. Shep1 does not colocalize with the ensheating cell marker S100β. Consecutive coronal sections (10 µm apart) from E14.5 wild-type mice were labeled for Shep1 and GAP43 to label OSN axons, or S100β to label ensheating cells. The panels on the lower right show enlargments of the regions outlined in the squares. Parallel staining of a Shep1 knockout section serves as a control to demonstrate the specificity of the Shep1 staining. Shep1 is localized in OSN axons and not in ensheating cells. Shep1 immunoreactivity is also low in the olfactory epithelium, where OSN cell bodies are located. OB, olfactory bulb; OE, olfactory epithelium. Scale bar = 400 µm. 2

3 Supplementary Figure 6. Shep1 does not colocalize with the ensheating cell marker p75ntr. Consecutive horizontal sections (10 µm apart) from E12.5 wild-type mice were labeled for Shep1 and p75ntr neurotrophin receptor (A-C) or for βiii tubulin (D) to label OSN axons. (E,F) Show enlargments of the areas outlined by squares in (C), illustrating the complementary distribution of Shep1 and p75ntr. Shep1 is localized in OSN axons and not in the ensheating cells. Shep1 immunoreactivity is also not detectable in the olfactory epithelium, where OSN cell bodies are located. The arrows in A and D point to OSN axon fascicles; the asterisk in E marks a large blood vessel. OB, olfactory bulb; OE, olfactory epithelium. Scale bars = 400 µm in D and 25 µm in F. Supplementary Figure 7. Tyrosine phosphorylated Cas is concentrated in E12.5 wild-type OSN axons and greatly reduced in Shep1 knockout axons. Sagittal sections from wild-type and knockout mice were double-labeled for phosphocas Y410 and βiii-tubulin to label immature axons. Tyrosine phosphorylated Cas is concentrated in wild-type OSN axons but essentially undetectable in the olfactory epithelium, where OSN cell bodies are located. The arrows point to OSN axons. OB, olfactory bulb; OE, olfactory epithelium. Scale bar = 100 µm. Supplementary Figure 8. Collagen I is a component of the pial basement membrane at E13.5. E13.5 sagittal section double-labeled with an antibody specific for collagen I and an anti-gap43 antibody. Scale bar = 250 µm. 3

4 Supplementary Figure 9. Small olfactory bulb in adult Shep1 knockout mice. Top panels, Nissl staining of sagittal sections from adult brain does not reveal major abnormalities in the brain architecture of Shep1 knockout mice (KO) compared to wild-type, except for a much smaller olfactory bulb. Bottom panels, DAPI staining (blue) and staining for PSA-NCAM (red) allows visualization of the rostral migratory stream (rms), which appears to be of normal size in Shep1 knockout mice. Scale bar = 1 mm. Supplementary Figure 10. Shep1 knockout mice have fewer gonadotropin releasing hormone (GnRH) positive cells in the preoptic region of the hypothalamus and smaller testes. A, The images shown are representative of coronal serial sections from 3 pairs of P0.5 Shep1 wild-type (WT) and Shep1 knockout (KO) mice that were stained for GnRH (red) and DAPI (white). Bar, 200 µm. B, Examples of testes from the few surviving adult Shep1 knockout (KO) mice in comparison with wild-type (WT) testes. Scale bars = 3 mm. 4

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