By HH18, the olfactory placode begins to invaginate and by HH22 (∼E12.25 in mouse) the olfactory pit has formed in a bowl-like shape (Fig. 4). The rim of the olfactory pit is termed the inferior region, while the invaginated part of the epithelium is called the superior region (Fig. 4). In mouse, E10.5–E12 are the earliest stages in which separate neurogenic markers in the olfactory epithelium have been studied (Cau et al.,1997,2000,2002). However, these studies have not analyzed whether the expression of various neurogenic markers differs at different anterior-posterior levels of the olfactory epithelium. To analyze this issue in detail, we collectively examined the expression pattern of Hes5, Cash1, Ngn1, Gap43, HuC/D, and Lhx2 at HH22. Similar to earlier stages of development, Hes5 is expressed in a majority of cells in the olfactory pit, and excluded from the inferior regions of the pit (Fig. 4B1–6). At HH22, the expression domain of Hes5 in the olfactory pit appears to be patchier (Fig. 4B2–6), in line with a role for Hes-genes in Notch-mediated lateral inhibition in the neurogenic area of the olfactory pit (Cau et al.,2000). At HH22, Cash1 expression is excluded from the most anterior region of the olfactory pit (Fig. 4C1). In the medial part of the pit, Cash1 expression is located in the inferior region of the olfactory epithelium (Fig. 4C2,3), whereas in the posterior part of the olfactory pit Cash1-positive cells are detected in the superior region of the epithelium (Fig. 4C4–6). At HH22, Ngn1, Gap43, and HuC/D are expressed in scattered patterns throughout the anterior-posterior axis of the olfactory pit, with the majority of expressing cells in the superior region at anterior to medial levels (Fig. 4D2–4, E2–4, F2–4). In the posterior part of the olfactory pit, the numbers of Ngn1-positive cells are higher (Fig. 4D5,6), compared to Gap43- and HuC/D-positive neurons (Fig. 4E5,6, F5,6). Ngn1 expression is detected in both the apical and basal part of the olfactory epithelium evenly distributed along the anterior-posterior axis (Fig. 4D1–6). In contrast, Gap43- and HuC/D-positive cells are located in the basal part of the epithelium adjacent to the mesenchyme and the strongest Gap43 and HuC/D expression is detected in the medial-superior region of the olfactory pit (Fig. 4E2–4, F2–4). Furthermore, at HH22, Lhx2 is expressed in a subset of HuC/D-positive cells in the medial part of the olfactory pit in the most superior region of the epithelium (Fig. 4G3,4, H). The more restricted Gap43, HuC/D, and Lhx2 expression domains in the medial-superior part of the olfactory pit, compared to the broader Cash1 and Ngn1 expression, clearly resembles the fact that Cash1 and Ngn1 are expressed by neuronal precursor cells, while Gap43, HuC/D, and Lhx2 are expressed by olfactory sensory neurons. At this stage, there is little obvious divergence in the expression patterns of Hes5, Cash1, Ngn1, Gap43, and Lhx2 in the middle of the anterior-posterior axis of the olfactory pit in chick compared to published mouse expression (Cau et al.,1997,2000,2002; Hirota and Mombaerts,2004; Kolterud et al.,2004). However, our results show that neurogenesis in the olfactory region proceeds along distinct axes in the olfactory pit, with the most differentiated neurons being located at medial-superior positions. In addition, at this stage Ngn1, Gap43, HuC/D, and Lhx2 expression are also detected in migratory neurons that have emerged from the olfactory epithelium (Fig. 4D1,2, E1,2, F1,2, G1–3). Previous studies in chick have shown that, around HH22, a small fraction of the migratory cells are GnRH (gonadotropin-releasing hormone) neurons that will migrate via the olfactory nerve to the hypothalamus (Mulrenin et al.,1999).
Figure 4. Expression of Hes5, Cash1, Ngn1, and Gap43 detected by in situ hybridization (B–E) and HuC/D and Lhx2 detected by immunohistochemistry (F–H) in the olfactory pit of HH22 chick embryos. Schematic chick embryo to the left indicates the transversal plane of sections and the sectioned area, and schematic illustration of the invaginated olfactory epithelium indicates the nomenclature describing the spatial location of cells within the bowl-like olfactory pit. A: The boxed area in the DAPI-stained section indicates the invaginated olfactory pit close to the telencephalon. Sections are represented from anterior at the top to posterior at the bottom. B:Hes5 is expressed in the majority of cells in the olfactory pit except in the medial to posterior inferior regions (arrowheads in B3–5). C:Cash1 is expressed in cells in the medial-inferior and posterior-superior regions of the olfactory pit (C2–6). D–F:Ngn1 (D1–6), Gap43 (E1–5), and HuC/D (F1–5) are expressed in scattered patterns throughout the anterior-posterior axis of the olfactory pit, with the majority of expressing cells in the medial-superior region. Ngn1 (D1,2), Gap43 (E1,2), and HuC/D (F1,2) are also expressed in migratory cells (arrowheads in D2, E2, F2). G, H: Lhx2 is expressed in a subset of HuC/D-positive cells in the medial-superior part of the olfactory pit (G3,4, yellow cells in H), and in cells of the migratory mass (G1-3, arrowhead in G2). The boxed area in G3 indicates the selection used for the blow-up in H. Cells expressing Hes5, Cash1, Gap43, HuC/D, and Lhx2 can also be detected in the telencephalon. A-Me-P, anterior-medial-posterior axis; In, inferior; M, mesenchyme; Opit, olfactory pit; S, superior; T, telencephalon. Scale bars = 100 μm.
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By HH25, the expression patterns of Hes5, Cash1, Ngn1, Gap43, HuC/D, and Lhx2 are similar to the expression at HH22, but the numbers of neurons have increased (data not shown), which is in agreement with progressive neurogenesis in the olfactory epithelium.