Olfactory ensheathing glia are required for embryonic olfactory axon targeting and the migration of gonadotropin-releasing hormone neurons

Summary Kallmann's syndrome is caused by the failure of olfactory axons and gonadotropin-releasing hormone (GnRH) neurons to enter the embryonic forebrain, resulting in anosmia and sterility. Sox10 mutations have been associated with Kallmann's syndrome phenotypes, but their effect on olfactory system development is unknown. We recently showed that Sox10 is expressed by neural crest-derived olfactory ensheathing cells (OECs). Here, we demonstrate that in homozygous Sox10lacZ/lacZ mouse embryos, OEC differentiation is disrupted; olfactory axons accumulate in the ventromedial olfactory nerve layer and fewer olfactory receptor neurons express the maturation marker OMP (most likely owing to the failure of axonal targeting). Furthermore, GnRH neurons clump together in the periphery and a smaller proportion enters the forebrain. Our data suggest that human Sox10 mutations cause Kallmann's syndrome by disrupting the differentiation of OECs, which promote embryonic olfactory axon targeting and hence olfactory receptor neuron maturation, and GnRH neuron migration to the forebrain.


Introduction
The anosmia and sterility of Kallmann's syndrome arise when olfactory axons and gonadotropin-releasing hormone (GnRH) neurons, which are needed for pituitary gonadotropin release, fail to enter the embryonic forebrain (Cadman et al., 2007;Cariboni et al., 2007;Hardelin and Dodé, 2008). GnRH neurons migrate from the embryonic olfactory epithelium along olfactory and/or vomeronasal nerves into the forebrain (Cariboni et al., 2007;Wray, 2010;Wierman et al., 2011). Recently, spontaneous mutations in the transcription factor gene Sox10 were associated with Kallmann's syndrome phenotypes: anosmia, hypogonadism and cryptorchidism (Bondurand et al., 2007;Barnett et al., 2009). Sox10 is expressed by migrating neural crest cells and required for the specification and differentiation of neural crest-derived Schwann cells and satellite glia (Herbarth et al., 1998;Southard-Smith et al., 1998;Britsch et al., 2001;Paratore et al., 2002;Finzsch et al., 2010). We recently showed that olfactory ensheathing cells (OECs), which ensheath olfactory axons from the epithelium to their targets in the olfactory bulb (Ekberg et al., 2012), are neural crest-derived and express Sox10 (Barraud et al., 2010). Sox10 expression was subsequently reported in mouse OECs from E10.5 (Forni et al., 2011), when olfactory axons and migratory neurons first emerge from the olfactory epithelium (Valverde et al., 1992;Miller et al., 2010). Here, we test the hypothesis arising from the association of Sox10 mutations with Kallmann's syndrome, namely that Sox10 is required for OEC differentiation and that OECs are required for the entry of olfactory axons and GnRH neurons into the embryonic forebrain.

Embryo collection and sectioning
Sox10 lacZ mutant mice (Britsch et al., 2001) and wild-type litter-mates of C3HeB/FeJ background were obtained from heterozygous crosses. Embryos were immersionfixed overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4˚C. Genotypes were determined from tail biopsies as described (Britsch et al., 2001). Embryos were embedded for wax or cryosectioning and sectioned at 5-6 mm (or at 30 mm, for some E16.5 embryos).

OECs and olfactory development 751
from the snout and part of the forebrain using Trizol (Invitrogen), and single-strand cDNA generated using Invitrogen's Superscript III First-Strand Synthesis System kit. GnRH1 was amplified by PCR (forward primer: CTCAACCTACCAACGGAAGC; reverse primer: GGGCCAGTGCATCTACATCT). The 344 bp product was cloned into pDrive (Qiagen) using the Qiagen PCR Cloning Kit and sequenced (Biochemistry Department DNA Sequencing Facility, Cambridge, UK). Digoxigenin-labelled antisense riboprobes were generated (Henrique et al., 1995) and in situ hybridization performed on sections as described (Xu et al., 2008).

Statistical analysis of olfactory receptor neuron maturation and olfactory epithelium thickness
Confocal images covering an optical depth of 15 mm were captured from 30 mm sections through the olfactory mucosa of E16.5 embryos (two wild-type, two Sox10 lacZ/+ and three Sox10 lacZ/lacZ embryos). Adjacent sections were immunostained for OMP and neuronal bIII tubulin. The region of interest covered a 200 mm length of the nasal septum in the middle portion of the dorsal-ventral span of the olfactory mucosa. Three sections were quantified/ embryo for each marker, with each section being 240 mm apart (480 mm total rostral-caudal distance); the first section was 300 mm from the most rostral portion of the olfactory bulb. All cells expressing OMP or neuronal bIII tubulin within the imaged regions of interest were counted. For each of the three sections quantified/ embryo, the number of OMP-positive and neuronal bIII tubulin-positive cells within the olfactory epithelium on each side of the nasal septum was counted (i.e., 6 measurements/embryo for each marker), and the thickness of the epithelium (from the nasal surface to the basal lamina) measured at three different positions on each side of the septum (i.e., 18 measurements per embryo). The mean/embryo was determined for each measurement, which was converted from pixels to mm and presented as OMP-positive or neuronal bIII tubulin-positive cell count/100 mm of olfactory epithelium, or thickness of olfactory epithelium in mm. GraphPad Prism (GraphPad Software, La Jolla, California, USA) was used to perform oneway ANOVA using Tukey's multiple comparison test (comparing every mean with every other mean) and unpaired 2-tailed t-tests.

Results and Discussion
Sox10 expression in the developing olfactory system is restricted to OECs (and, at later stages, Bowman's gland/duct cells) We aimed to understand how Kallmann's syndrome phenotypes could result from Sox10 mutations (Bondurand et al., 2007;Barnett et al., 2009). We used in situ hybridization (ISH) and immunostaining to examine Sox10 expression during mouse olfactory system development from E10.5 to neonatal stages ( Fig. 1A-O 1 ). Our results confirm and extend previous reports (Barraud et al., 2010;Forni et al., 2011) showing that Sox10 expression is restricted to OECs (which are found along the entire length of the olfactory nerve throughout its development), apart from Bowman's gland/duct cells in the olfactory epithelium at later stages (as we previously described for avian embryos; Barraud et al., 2010). Sox10 expression was not seen (by either ISH or immunostaining) in neurons in the olfactory epithelium at any stage examined ( Fig. 1A-O 1 ). Likewise, Sox10 expression was not seen in neurons in the vomeronasal organ epithelium (e.g. Fig. 1C-D 2 ,G-J 1 ), or in the neurons (which include GnRH neurons) migrating along olfactory and/or vomeronasal nerves (e.g. Fig. 1C-J 1 ). At E16.5, non-neuronal Sox10-positive cells were clearly visible within the olfactory epithelium ( Fig. 1M-M 2 ). From E17.5 until at least neonatal stages, these were found in large clusters protruding into the mesenchyme ( Fig. 1N-O 1 ), and as strands projecting across the width of the epithelium (Fig. 1O,O 1 ). As we previously reported for avian embryos [ figure S8 in Barraud et al. (Barraud et al., 2010)], these nonneuronal Sox10-positive cells in the olfactory epithelium can be identified as developing Bowman's gland/duct cells, which start to protrude from the mouse olfactory epithelium at E17.5 (Cuschieri and Bannister, 1975). Overall, therefore, while GnRH neurons are migrating (Cariboni et al., 2007) and olfactory axons reach the olfactory bulb, Sox10 expression is restricted to OECs during mouse olfactory system development.
Sox10 deletion disrupts olfactory axon targeting and olfactory receptor neuron maturation The absence of lamina propria OECs at E16.5 in homozygous Sox10 lacZ/lacZ embryos was associated with defasciculation of olfactory axon bundles and inappropriate migration of axons within the lamina propria ( Fig. 4A-B 1 ). We also noticed an apparent reduction in the number of olfactory receptor neurons (ORNs) expressing the maturation marker olfactory marker protein (OMP) (compare Fig. 4A 1 ,B 1 ; Fig. 4C-E). To investigate this further, we calculated the mean/embryo (6 standard error of the mean, s.e.m.) of OMP-positive cells and neuronal bIII tubulin-positive neurons/100 mm of olfactory epithelium, and the thickness of the olfactory epithelium. One-way analysis of variance (ANOVA) using Tukey's multiple comparison test showed no significant difference for any measurement between wild-type (n52) and heterozygous Sox10 lacZ/+ embryos (n52), so we combined wild-type and heterozygote data (n54) for comparison with homozygotes (n53). We confirmed that the mean/embryo (6 s.e.m.) of OMP-positive cells/100 mm of epithelium ( Fig. 4F)  Immunostaining for the axonal marker NCAM also showed that, relative to wild-type, the ONL in dorsal and lateral regions of the olfactory bulb was much thinner after Sox10 deletion, while the ventromedial ONL was much thicker (Fig. 4I,J). In two homozygous Sox10 lacZ/lacZ embryos, we noticed a ventromedial accumulation of olfactory axons so pronounced that axons from both sides of the nasal cavity merged together ventrally, apparently forming whorls/balls (similar to what is observed in Gli3 Xt extra-toes mutant mice, which lack olfactory bulbs; St John et al., 2003) rather than a uniform ONL as in wild-type mice (Fig. 4K-L 1 ).
These data suggest that the disruption of OEC differentiation arising from Sox10 deletion results in olfactory axons failing to find their targets in the lateral and dorsal regions of the olfactory bulb, leading to axon accumulation in the ventromedial region and a significant reduction in ORN maturation. When combined with the lack of detectable NPY expression in OECs in the ONL of homozygous Sox10 lacZ/lacZ embryos ( Fig. 3E-F 2 ), our results are consistent with the previously proposed hypothesis (based on the timing of onset of NPY expression) that NPY secreted from inner-ONL OECs may be involved in the final stages of olfactory axon outgrowth towards glomerular targets (Ubink and Hökfelt, 2000). The effect on maturation is presumably a consequence of defective axon targeting: the maturation marker OMP is only expressed in ORNs that have already contacted the olfactory bulb (Graziadei et al., 1980).
We conclude that human Sox10 mutations cause Kallmann's syndrome phenotypes (Bondurand et al., 2007;Barnett et al., 2009) by disrupting the differentiation of OECs, which, as shown here, promote olfactory axon targeting, ORN maturation (most likely because of their importance for olfactory axon targeting) and GnRH neuron migration. A neural crest defect in Kallmann's syndrome is supported by its inclusion within CHARGE syndrome (Pinto et al., 2005), an autosomal dominant disorder caused by heterozygous mutations in CHD7, encoding a chromatinremodeling protein that controls neural crest formation (Bajpai et al., 2010), and by the demonstration that anosmin1, loss-offunction mutations in which cause X-linked Kallmann's syndrome (Cadman et al., 2007;Hardelin and Dodé, 2008), promotes cranial neural crest cell formation in an autocrine fashion (Endo et al., 2012). Overall, our results highlight the interplay between neural crest-derived OECs and olfactory placode-derived axons and neurons (Sabado et al., 2012) that seems to be required for both olfaction and fertility.
Note added in proof While our manuscript was in revision, another study was published showing that loss-of-function mutations in SOX10 cause Kallmann's syndrome with deafness and describing the same OEC phenotype in Sox10 mutant mice, thus implicating neural crest-derived OECs in the aetiology of Kallmann's syndrome (Pingault et al., 2013).