Olfactory ensheathing cells abutting the embryonic olfactory bulb express Frzb, whose deletion disrupts olfactory axon targeting

Abstract

We and others previously showed that in mouse embryos lacking the transcription factor Sox10, olfactory ensheathing cell (OEC) differentiation is disrupted, resulting in defective olfactory axon targeting and fewer gonadotropin-releasing hormone (GnRH) neurons entering the embryonic forebrain. The underlying mechanisms are unclear. Here, we report that OECs in the olfactory nerve layer express Frzb-encoding a secreted Wnt inhibitor with roles in axon targeting and basement membrane breakdown-from embryonic day (E)12.5, when GnRH neurons first enter the forebrain, until E16.5, the latest stage examined. The highest levels of Frzb expression are seen in OECs in the inner olfactory nerve layer, abutting the embryonic olfactory bulb. We find that Sox10 is required for Frzb expression in OECs, suggesting that loss of Frzb could explain the olfactory axon targeting and/or GnRH neuron migration defects seen in Sox10-null mice. At E16.5, Frzb-null embryos show significant reductions in both the volume of the olfactory nerve layer expressing the maturation marker Omp and the number of Omp-positive olfactory receptor neurons in the olfactory epithelium. As Omp upregulation correlates with synapse formation, this suggests that Frzb deletion indeed disrupts olfactory axon targeting. In contrast, GnRH neuron entry into the forebrain is not significantly affected. Hence, loss of Frzb may contribute to the olfactory axon targeting phenotype, but not the GnRH neuron phenotype, of Sox10-null mice. Overall, our results suggest that Frzb secreted from OECs in the olfactory nerve layer is important for olfactory axon targeting.

Keywords: GnRH neurons; OECs; Omp; Sox10; olfactory receptor neurons.

Figure 1

Frzb is expressed by OECs in the embryonic mouse olfactory nerve layer. In situ hybridization for Frzb on parasagittal sections through the developing mouse olfactory system. (a–c³) At E11.5, Frzb expression is seen in the maxillary and mandibular prominences, and in frontonasal mesenchyme caudal to the olfactory epithelium, but not in OECs (identified by nuclear immunoreactivity for the transcription factor Sox10) associated with Tubb3‐positive olfactory nerve fascicles (arrowheads highlight examples). (d–h²) At E13.5, strong Frzb expression can be seen in OECs in the olfactory nerve layer adjacent to the olfactory bulb (white arrowheads, f¹–g²), but in fewer OECs on the olfactory nerve more peripherally (yellow arrowheads, g–g²; white arrowheads, h–h²). Yellow arrowheads in panels h–h² highlight examples of Frzb‐negative peripheral OECs. FB, forebrain; MD, mandibular prominence; MX, maxillary prominence; OB, olfactory bulb; OE, olfactory epithelium; ON, olfactory nerve; ONL, olfactory nerve layer. Scale bar: 100 μm [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

At E16.5, Frzb is expressed most strongly by OECs in the inner olfactory nerve layer. In situ hybridization for Frzb on parasagittal sections through the mouse olfactory system at E16.5. (a–b²) Frzb expression is seen in OECs (identified by nuclear Sox10 immunoreactivity) throughout the olfactory nerve layer, but more strongly in the inner region, adjacent to the olfactory bulb (white arrowheads) than in the outer region (yellow arrowheads). (c–c²) Few OECs associated with peripheral olfactory nerve fascicles on the same section seem to express Frzb (white arrowhead highlights an example): most are Frzb‐negative (yellow arrowheads highlight examples). (d–e²) In situ hybridization for the inner ONL‐specific OEC marker Npy on the adjacent serial section to the section shown in panels a–c². iONL, inner olfactory nerve layer; OB, olfactory bulb; OE, olfactory epithelium; ON, olfactory nerve, ONL, olfactory nerve layer; oONL, outer olfactory nerve layer. Scale bar: 100 μm [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Frzb expression in the olfactory nerve layer depends on Sox10. Coronal sections through the developing olfactory system at E13.5 in Sox10 ^lacZ mouse embryos, in which lacZ replaces one or both alleles of Sox10 (Britsch et al., 2001). (a–d²) In a wild‐type embryo, Frzb expression can be seen in OECs (identified by immunoreactivity for nuclear Sox10) in the Tubb3‐positive olfactory nerve layer (arrowheads, d–d²). (e–h²) In a Sox10 ^lacZ/lacZ (i.e., Sox10‐null) litter‐mate, Frzb expression is lacking in the olfactory nerve layer, where β‐galactosidase‐positive neural crest‐derived cells persist in the absence of Sox10 (Barraud et al., 2013) (arrowheads, h–h²). (i–k) In a more caudal section of the wild‐type embryo in panels a–d² (from a different slide in the same round of in situ hybridization), Frzb expression is seen in mesenchyme in the maxillary and mandibular prominences. (l–n) In a more caudal section of the Sox10 ^lacZ/lacZ (i.e., Sox10‐null) litter‐mate in panels e–h² (from a different slide in the same round of in situ hybridization), Frzb expression is seen in mesenchyme in the maxillary and mandibular prominences. FB, forebrain; MD, mandibular prominence; MX, maxillary prominence; OB, olfactory bulb; OE, olfactory epithelium; ON, olfactory nerve. Scale bar: 100 μm [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Frzb deletion seems to affect development of the olfactory nerve layer. Coronal sections through the mouse olfactory system at E16.5, immunostained for the maturation marker Omp, the outer ONL marker peripherin (Prph), and the general axonal marker Tubb3. (a–c²) In a heterozygous Frzb ^+/− embryo, Omp immunoreactivity labels the olfactory nerve, the full width of the ONL, and many olfactory receptor neurons in the olfactory epithelium, while peripherin immunoreactivity labels the olfactory nerve and the outer ONL. Some peripherin‐positive, Omp‐negative axons are also seen deep to the ONL, in the external plexiform layer (arrowheads, c–c²). (d–f²) In a Frzb‐null embryo, the Omp‐immunoreactive ONL seems to be thinner, and peripherin immunoreactivity is not detectable in the external plexiform layer. EPL, external plexiform layer; OB, olfactory bulb; OE, olfactory epithelium; ON, olfactory nerve; ONL, olfactory nerve layer. Scale bar: 50 μm (a,b,d,e) and 25 μm (c,f) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

The volume of the Omp‐immunoreactive olfactory nerve layer is reduced after Frzb deletion. (a–g) Coronal sections moving rostrally to caudally through the olfactory bulbs of a heterozygous Frzb ^+/− mouse embryo, immunostained for Omp, with the Omp‐positive regions outlined. (h) Schematic showing a parasagittal view through an E16.5 mouse head, with dotted lines showing the approximate location of the most rostral and most caudal sections shown in (a–g). (i,j) Low‐power (i) and high‐power (j) views of a hematoxylin and eosin‐stained parasagittal section through an E16.5 mouse head (Plate 38c, image a, from the eHistology Atlas with Kaufman annotations; Graham et al., 2015). The dotted lines in (j) show the approximate locations of the sections shown in (a–g). (k–q) Coronal sections moving rostrally to caudally through the olfactory bulbs of a Frzb‐null mouse embryo, immunostained for Omp, with the Omp‐positive regions outlined. (r) Scatter plot showing the mean volume of one‐tenth of the Omp‐positive ONL at E16.5 in heterozygous Frzb ^+/− embryos (n = 4) and Frzb‐null embryos (n = 5). Error bars show SD. Scale bar: 200 μm [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

Frzb deletion disrupts olfactory receptor neuron maturation, not overall neuron number or the thickness of the olfactory epithelium. (a,b) Example coronal sections through a region of dorsal olfactory epithelium at E16.5, immunostained for the maturation marker Omp and the general neuronal/axonal marker Tubb3 and counter‐stained with DAPI, from a heterozygous Frzb ^+/− mouse embryo (a) and a Frzb‐null embryo (b). All Omp‐positive (mature) neurons (cyan dots) have been counted within the 200 μm region highlighted by the cyan bar. (c) Scatter plot showing that there is a significant difference between the mean number per embryo of Omp‐positive (mature) olfactory receptor neurons per 200 μm of epithelium for heterozygous Frzb ^+/− embryos (n = 4) versus Frzb‐null embryos (n = 4). Error bars show SD. (d,e) The same sections as in (a,b), showing Tubb3 and DAPI only, with all Tubb3‐positive neurons counted (cyan dots) within the 200 μm region highlighted by the cyan bar. (f) Scatter plot showing there is no significant difference between the mean number per embryo of olfactory receptor neurons per 200 μm of epithelium for heterozygous Frzb ^+/− embryos (n = 4) versus Frzb‐null embryos (n = 4). Error bars show SD. (g,h) The same sections as in (a,b), showing example measurements of the thickness of the olfactory epithelium at three points within the 200 μm region highlighted by the cyan bar. (i) Scatter plot showing there is no significant difference between the mean thickness per embryo of the olfactory epithelium for heterozygous Frzb ^+/− embryos (n = 4) versus Frzb‐null embryos (n = 4). Error bars show SD. LP, lamina propria; OE, olfactory epithelium. Scale bar: 50 μm [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7

Frzb deletion does not significantly affect GnRH neuron entry into the forebrain. (a–d) Schematic representations of the distribution of GnRH neurons (black spots) at E16.5 on coronal sections at different rostrocaudal levels of a heterozygous Frzb ^+/− mouse embryo (a,b) and a Frzb‐null mouse embryo (c,d). Each dot represents a single GnRH neuron counted on one slide of a 10‐slide series. (e) Scatter plot showing the mean percentage of GnRH neurons found inside the brain for heterozygous Frzb ^+/− embryos (n = 3) versus Frzb‐null embryos (n = 3). Error bars show SD. FB, forebrain; hypo, hypothalamus; OB, olfactory bulb, OE, olfactory epithelium; VNO, vomeronasal organ [Color figure can be viewed at wileyonlinelibrary.com]