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Keywords:

  • Hes5;
  • Cash1;
  • Ngn1;
  • Gap43;
  • HuC/D;
  • Lhx2;
  • neurogenesis;
  • olfactory;
  • chick

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Neurogenesis in the olfactory epithelium begins in early embryos and proceeds throughout life. A comparison of neurogenic marker expression at different developmental stages and at different axes of the olfactory epithelium has not been reported in a coordinated way. In this study, we have in detail compared the temporal and spatial expression patterns of the precursor markers Hes5, Cash1, Ngn1, and the neuronal markers Gap43, HuC/D, Lhx2 in the developing olfactory placode and epithelium in chick embryos from HH10 to HH34. We show that Hes5 starts to be expressed in cells of the prospective olfactory placode at HH10, earlier then previously reported. During olfactory pit stages, the expression of Hes5, Cash1, Ngn1, Gap43, HuC/D, and Lhx2 varies throughout the anterior-posterior and superior-inferior axis of the olfactory epithelium. By HH34, expression of the precursor and neuronal markers show the first signs of apical-basal stratification of the epithelium. Developmental Dynamics 238:1617–1625, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Neurogenesis of olfactory sensory neurons in the olfactory epithelium proceeds throughout life and is often used as a paradigm to study neurogenesis in general because the simple organization of the olfactory epithelium makes it easily accessible.

The olfactory epithelium arises from the olfactory placode (Croucher and Tickle,1989; Kramer et al.,2000). As early as the late gastrula stage, progenitor cells in the anterior neural plate border region, in between the prospective neuroectoderm and epidermis, are specified as olfactory and lens placodal cells (Sjodal et al.,2007). By neural fold stages, Hamburger and Hamilton stage (HH) 8 in chick (Hamburger and Hamilton,1992) olfactory and lens placodal progenitor cells are spatially separated (Bhattacharyya et al.,2004; Sjodal et al.,2007). By HH14, the olfactory placode becomes visible as an ectodermal thickening on the side of the head, which subsequently invaginates to form a bowl-like structure called the olfactory pit at HH17. At later stages, the olfactory epithelium extends further inside the head mesenchyme and distinct olfactory turbinates are formed. The mature olfactory epithelium contains stem-like cells, neuronal precursor cells at different maturity stages, olfactory sensory neurons, and also glial-like supporting cells.

The basic helix-loop-helix (bHLH) repressor gene Hes5, the bHLH transcriptional regulators chick achaete-scute1 (Cash1) and Neurogenin1 (Ngn1), the neuronal markers growth-associated-protein 43 (Gap43) and HuC/D, and the LIM-homeodomain transcription factor Lhx2, define cells at different stages in the olfactory sensory neuronal lineage, and will be referred to as neurogenic markers throughout this study. In the olfactory epithelium, neurogenesis occurs in an ordered fashion; stem-like progenitor cells (Hes5-positive) undergo asymmetric cell division to generate transient amplifying progenitor cells (Cash1-positive), which in their turn generate the immediate neuronal precursor cells (Ngn1-positive) that divide further and finally differentiate into olfactory sensory neurons (Kawauchi et al.,2004; Beites et al.,2005). Olfactory sensory neurons can be characterized by the expression of Gap43 (Baizer et al.,1990), members of the Hu class of RNA-binding proteins that mark post-mitotic neurons [HuC/D (Fornaro et al.,2003)] and Lhx2 (Kolterud et al.,2004).

Genetic studies in mice have addressed the role of some of these transcription factors in olfactory neurogenesis. Mice mutant for Mash1 (the mouse homologue of chicken Cash1) fail to generate olfactory progenitors and thereby also olfactory sensory neurons, among other neurogenic defects (Cau et al.,2002; Murray et al.,2003). In Ngn1 null mutant mice, differentiation of neural progenitors in the olfactory epithelium is blocked (Cau et al.,2002), whereas Lhx2-deficient mice display a relatively normal organization of the olfactory epithelium, but show a perturbed generation of olfactory sensory neurons (Hirota and Mombaerts,2004; Kolterud et al.,2004). Consequently, the expression of several neurogenic markers has been separately described in the olfactory pit in mouse (Cau et al.,1997,2000,2002; Hirota and Mombaerts,2004; Kolterud et al.,2004). However, a comparison of neurogenic expression patterns between species has not been reported, and the expression of neurogenic markers has not been examined in the chick olfactory epithelium. Thus, whether a conserved pattern of neurogenesis in the olfactory epithelium exists among vertebrates still remains to be determined. Moreover, a collective temporal and spatial analysis of these markers in any species has not been examined. Such analyses would define anterior-posterior and superior-inferior positions of precursors, immature and mature neurons at various time points in the olfactory epithelium.

In this study, we have analysed the temporal and spatial expression of Hes5, Cash1, Ngn1, Gap43, HuC/D, and Lhx2 during the early development of the chick olfactory placode and the olfactory epithelium by analysing HH10, before the placode is morphologically apparent, to HH34 when distinct olfactory turbinates are formed. The selected markers define cells at different stages in the olfactory sensory neuronal lineage, which enables us to visualize the progression from stem and progenitor cells to differentiated neurons. When possible, we compare our results with published mouse data, and we have correlated chick and mouse developmental stages based on comparisons of olfactory epithelial/head morphology throughout the study (Kaufman,1998; Bellairs and Osmond,1998).

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Onset of Hes5 Expression in Prospective Olfactory Placodal Cells

Hes-genes are known targets of the Delta-Notch signalling pathway (Jarriault et al.,1995). The role of Hes-genes in olfactory epithelial development is dual; at early stages of development, Hes-genes are implicated as pre-patterning genes defining the domain of neurogenesis within the olfactory placodal region, whereas at later stages, Hes-genes are involved in controlling the number of neural progenitors emerging in this domain by negatively regulating neurogenesis (Cau et al.,2000). Previous studies in mouse have shown that Hes5 is expressed in the olfactory epithelium at E10.5 (∼HH18 in chick) (Cau et al.,2000). In chick, three Hes5-like genes (Hes5-1, Hes5-2, Hes5-3) have been described (Fior and Henrique,2005). Since Hes5-1, Hes5-2, and Hes5-3 show similar expression patterns in the olfactory epithelium (data not shown), in this study the Hes5 expression is represented by Hes5-1, and referred to as Hes5. Our results show that Hes5 can be detected in the anterior-lateral head ectoderm in chick already at HH10 (Fig. 1B1–2). Based on fate maps in chick at HH10, which have shown that progenitor cells of the olfactory placode are located at the most anterior ectoderm (Bhattacharyya et al.,2004), we conclude that the Hes5-positive region corresponds to prospective placodal cells. The early expression domain of Hes5 is uniform, suggesting that in chick Hes5 is involved in pre-patterning of the prospective olfactory placode. Moreover, at this stage no Cash1, Ngn1, GAP43, HuC/D, or Lhx2 expression is detected (data not shown). Thus, at HH10 prospective olfactory placodal cells express Hes5, which appears to be the earliest marker associated with neuronal determination in the chick olfactory placode.

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Figure 1. Expression of Hes5 detected by in situ hybridization (B) in the prospective olfactory placode in a HH10 chick embryo. Schematic chick embryo to the left indicates transversal plane of sections. A: The boxed area in the DAPI-stained section indicates the prospective olfactory placode adjacent to the telencephalon. The arrows outline the prospective olfactory region. B: At HH10 (10 somites), expression of Hes5 is detected in the head ectoderm in the region of the prospective olfactory placode. Hes5 expression can also be detected in the telencephalon. T, telencephalon. Scale bar = 100 μm.

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At HH14, Olfactory Placodal Cells Express Hes5, Cash1, Ngn1, Gap43, and HuC/D

By HH14 (∼22 somites), the olfactory placodal epithelium starts to thicken (Fig. 2A). Previous studies in chick have monitored the first migratory cells derived from the olfactory placode, termed epithelioid cells (Croucher and Tickle,1989; Drapkin and Silverman,1999), around HH14 by analyzing HuC/D-positive cells, detecting early post-mitotic neurons (Fornaro et al.,2001). However, the expression pattern of Hes5, Cash1, Ngn1, Gap43, or Lhx2 have not been examined in either chick or mouse at this stage. Our results show that at HH14, apart from Hes5-expression, expression of Cash1, Ngn1, Gap43, and HuC/D can be detected in a few cells in the olfactory placodal region (Figs. 2B–F). No Lhx2 expression is detected in or around the olfactory placode at this stage (data not shown). Hes5 is expressed in a broad domain spanning almost the entire olfactory placodal region (Fig. 2B1–4). In contrast, Cash1, which marks transient amplifying progenitor cells, can only be detected in a few cells in the anterior part of the olfactory placode (Fig. 2C1–4). Moreover, at this stage the neuronal markers Ngn1, Gap43, and HuC/D are expressed in a few cells in the anterior-medial region of the olfactory placode (Fig. 2D2,3, E2,3, F1–3), but excluded from the most posterior part of the placode (Fig. 2D4, E4, F4). Furthermore, at this stage, a few Ngn1, Gap43, and HuC/D-positive neurons can be detected in the head mesenchyme close to the placodal epithelium (Fig. 2D2, E2, F1 arrowheads), showing the first cells migrating away from the olfactory placode. The function of these early-delaminating neurons remains unclear, although they have been suggested to perforate the olfactory epithelial basal lamina, creating openings later utilized by emerging olfactory axons (Drapkin and Silverman,1999), and to build up the olfactory nerve tract and migrate towards the anterior telencephalon (Fornaro et al.,2003). Previous studies in mouse have described the earliest expression of Mash1 and Ngn1 in the olfactory epithelium around E10.5 (Cau et al.,1997,2000). Our results show that in the developing chick olfactory placode, Cash1, Ngn1, and Gap43 are expressed at HH14 (∼E9.5 in mouse). The difference in onset of Mash1/Cash1 and Ngn1 expression in chick and mouse is most likely due to the lack of expression analyses at earlier stages in mouse, rather than a species difference, but remains to be determined.

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Figure 2. Expression of Hes5, Cash1, Ngn1, and Gap43 detected by in situ hybridization (B–E) and HuC/D detected by immunohistochemistry (F) in the olfactory placode of HH14 chick embryos. Schematic chick embryo to the left indicates the transversal plane of sections and the sectioned area. A: The boxed area in the DAPI-stained section indicates the newly formed olfactory placode close to the telencephalon. The arrows mark the olfactory placodal region. Sections are represented from anterior at the top to posterior at the bottom. B: At HH14 (22 somites), Hes5 is expressed in the olfactory placode. C:Cash1 is expressed in a few cells in the anterior part of the olfactory placode. D–F:Ngn1 (D1,2), Gap43 (E1,2), and HuC/D (F1–3) are expressed in a few cells mostly in the medial part of the olfactory placode, and Ngn1 (D2), Gap43 (E2), and HuC/D (F1) expression can be detected in a few migratory cells (arrowheads). Hes5 expression can also be detected in the telencephalon. T, telencephalon. Scale bar = 100 μm.

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Lhx2 Expression Is First Detected in the Olfactory Placode at HH17

We continued to analyze the expression of neurogenic markers in the olfactory placode at HH17 (∼30 somites). By this stage, the placodal epithelium has thickened considerably compared to HH14 and is visible as an indention in an anterior-lateral position of the head. Hes5 is still strongly expressed in nearly all cells of the olfactory placode (Fig. 3B1–4), and only the most posterior part of the olfactory placode has lower levels of Hes5 expression (Fig. 3B5). At this stage, Cash1 is expressed only in a few cells in the medial part of the placode, whereas strong expression of Cash1 is restricted to cells located in the basal part of the epithelium adjacent to the mesenchyme and to the rim of the placode (Fig. 3C2–4). Similar to the Cash1 expression pattern, the olfactory neuronal markers Ngn1, Gap43, and HuC/D are all expressed in cells located in the medial part of the placode, and the majority of Ngn1-, Gap43-, and HuC/D-positive cells are found close to the basal lamina (Fig. 3D2,3, E2,3, F2–4). At HH17, Lhx2 expression can only be detected in the medial part of the placode in a few round-shaped cells close to the basal lamina (Fig. 3G2,3, arrow). In addition, Ngn1, Gap43, HuC/D, and Lhx2 expression is also detected in neurons migrating away from the placode, termed the migratory mass, towards the anterior telencephalon (Fig. 3D2, E2–4, F2,3, G2,3, arrowheads). Thus, the relatively few Lhx2-positive cells in the medial part of the olfactory epithelium, compared to the numbers of Lhx2-positive cells in the migratory mass, indicate that at HH17 the majority of the differentiated neurons in the olfactory epithelium leave the placode and migrate away. This is in agreement with previous studies, suggesting that the first-born neurons in the olfactory epithelium leave the placode, and build up and broaden the olfactory nerve (Croucher and Tickle,1989; De Carlos et al.,1995). In addition, previous studies have demonstrated that the olfactory nerve is apparent at HH18, providing a path along which several different olfactory epithelium-derived cells migrate towards the anterior telencephalon (Croucher and Tickle,1989; De Carlos et al.,1995; Drapkin and Silverman,1999). Our results show that at HH17, all Lhx2-positive migratory cells express HuC/D, but not all HuC/D cells are Lhx2 positive (Fig. 3H), indicating that at this stage there might be at least two subgroups of HuC/D-positive neurons migrating from the olfactory placode; however, the fate of these neurons remains to be determined. Taken together, we show that neurogenesis is ongoing in the olfactory placode prior to invagination of the olfactory epithelium.

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Figure 3. 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 placode of HH17 chick embryos. Schematic chick embryo to the left indicates the transversal plane of sections and the sectioned area. A: The boxed area in the DAPI stained section indicates the olfactory placode beginning to invaginate close to the telencephalon. Sections are represented from anterior at the top to posterior at the bottom. B: At HH17 (31 somites), Hes5 is expressed throughout the placodal region, and only the most posterior part of the olfactory placode has lower levels of Hes5 expression (B5). C:Cash1 expression is restricted the medial areas of the olfactory placode and cells expressing high levels of Cash1 are located in the basal part of the epithelium and close to the rim of the placode (C2–4). D–F:Ngn1 (D2,3), Gap43 (E2,3) and HuC/D (F2–4) are expressed predominantly in medial-superior areas of the olfactory placode and in cells of the migratory mass (arrowheads in D2, E2, F2). G, H: Only a few Lhx2 positive cells can be detected in the medial-superior part of the invaginating placode in the basal part of the epithelium adjacent to the mesenchyme (arrows in G2,3, H1,2), and in the migratory mass (arrowheads in G2, H1,2,). The boxed area in G3 indicates the selection used for the blow-up in H1,2. All Lhx2 positive migratory cells express HuC/D (yellow cells, arrowhead in H2), but some HuC/D positive cells do not express Lhx2 (red cells). The broken line in H1,2 indicates the border of the olfactory placode close to the mesenchyme. Cells expressing Hes5, Cash1 and Lhx2 can also be detected in the telencephalon. M, mesenchyme; OP, olfactory placode; T, telencephalon. Scale bars = 100μm.

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Neurogenesis in the Olfactory Pit Proceeds Along Distinct Axes

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).

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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.

By HH29, Hes5 Is Expressed in a Scattered Manner in the Olfactory Epithelium

Around HH29 (∼E14 in mouse), the morphology of the nasal cavity begins to change and the nasal conchae starts to form (Croucher and Tickle,1989; Leibovici et al.,1996). The olfactory and the respiratory epithelium can now be distinguished from one another by their different morphology (Croucher and Tickle,1989; Leibovici et al.,1996). The olfactory epithelium is thicker and located further inside the head mesenchyme (Fig. 5A), but at this stage stratification has not yet begun. Compared to mouse, where the olfactory epithelium begins to attain a layered morphology around E12.5 (Cau et al.,2000), the development of the olfactory epithelium in chick appears to proceed much slower. The respiratory epithelium is a thin layer of cells and is located more distally in the nasal passage (Fig. 5A). No Hes5, Cash1, Ngn1, Gap43, HuC/D, or Lhx2 expression is detected in the respiratory epithelium (Fig. 5B–G), consistent with the fact that the respiratory epithelium does not give rise to olfactory sensory neurons.

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Figure 5. 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 epithelium of an HH29 chick embryo. Schematic chick embryo to the left indicates the transversal plane of sections and the sectioned area. A: The boxed area in the DAPI-stained section indicates the invaginated and extended olfactory epithelium inside the head mesenchyme, and the thinner respiratory epithelium located more distally in the nasal passage. Sections are represented from anterior at the top to posterior at the bottom. B:Hes5 is expressed in a salt-and-pepper-like pattern in nearly the entire olfactory epithelium (B1-4). C: A few Cash1-expressing cells can be detected at apical and basal positions in the epithelium (C1–4). D:Ngn1 are expressed at high levels in a scattered pattern throughout the olfactory epithelium (D1–4). E–G:Gap43 (E1–4), HuC/D (F1–4), and Lhx2 (G1–4) are expressed in the majority of cells in the olfactory epithelium. The olfactory nerve is visualised by the expression of Gap43 (arrowhead in E2). No expression of Hes5, Cash1, Ngn1, Gap43, HuC/D, or Lhx2 can be detected in the respiratory epithelium. OE, olfactory epithelium; Re, respiratory epithelium. Scale bar = 100 μm.

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By HH29, although Hes5 is expressed in nearly the entire neurogenic region, levels of expression are much lower than at earlier stages and a salt-and-pepper-like pattern is clearly distinguishable (Fig. 5B1–4), in accordance with a role of Hes5 in lateral inhibition (Cau et al.,2000). Similar to Hes5 expression, a few scattered Cash1-positive cells can be detected in the entire neurogenic region (Fig. 5C1–4). At this stage, Ngn1 is expressed in a salt-and-pepper pattern throughout the olfactory epithelium (Fig. 5D1–4). Moreover, a majority of the cells within the olfactory epithelium express Gap43 and HuC/D (Fig. 5E1–4, F1–4), and a subset of HuC/D-positive cells expresses Lhx2 (Fig. 5G1–4). At HH29, migrating neurons that follow the olfactory nerve towards the telencephalon express Gap43, HuC/D, and Lhx2 (Fig. 5E2 and data not shown).

Stratification of the Chick Olfactory Epithelium Is First Detected at HH34

At HH34, Hes5 is expressed in the apical part of the olfactory epithelium of the superior concha (Fig. 6A). The majority of the Cash1- and Ngn1-positive cells are also present in the apical region, and a few Cash1- and Ngn1-expressing cells are detected in the middle area of the olfactory epithelium (Fig. 6B, C). At this stage, Gap43, HuC/D, and Lhx2 are expressed predominately in the basal region of the epithelium, but Gap43-, HuC/D-, and Lhx2-positive cells are detected along the entire apical-basal axis of the olfactory epithelium (Fig. 6D–F). Thus, by HH34, the initial stratification of the chick olfactory epithelium is apparent.

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Figure 6. Expression of Hes5, Cash1, Ngn1, and Gap43 detected by in situ hybridization and HuC/D and Lhx2 detected by immunohistochemistry in the olfactory epithelium of an HH34 chick embryo. A: The boxed area in the DAPI-stained section indicates parts of the OE located in the superior concha. B–G:Hes5 (B), Cash1 (C), and Ngn1 (D) are expressed in the apical part of the epithelium, while Gap43 (E), HuC/D (F), and Lhx2 (G) are expressed preferentially in the basal part of the olfactory epithelium. Broken lines in B–G indicate the position of the basal lamina. M, mesenchyme; OE, olfactory epithelium. Scale bar = 100 μm.

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In summary, in this study we describe in detail the temporal and spatial expression of Hes5, Cash1, Ngn1, Gap43, HuC/D, and Lhx2, during early stages of neural determination and neurogenesis in the olfactory epithelium in chick. Hes5 is expressed in the prospective olfactory placode already at HH10, earlier than previously reported and before the placode is morphologically apparent, indicating that Hes5 may be involved in determining the neurogenic area of the epithelium. Moreover, our results show that neurogenesis is ongoing in the olfactory placode prior to invagination of the olfactory epithelium. During olfactory pit stages, both an anterior-posterior and a superior-inferior distinction in the expression of the investigated neurogenic factors becomes apparent, with the most differentiated neurons being located at medial-superior positions. In contrast, at HH29 the neuronal markers Gap43, HuC/D, and Lhx2 are expressed evenly throughout the entire olfactory epithelium. The different expression patterns of the neuronal markers between HH22 to HH29 most likely reflect the spatial expansion of neurogenesis from the early restricted medial region of the pit at HH22 to the entire olfactory epithelium at HH29. By HH34, stratification of the chick olfactory epithelium is visible, in which the precursor markers Hes5, Cash1, and Ngn1 are expressed in the apical part of the epithelium, while the neuronal markers Gap43, HuC/D, and Lhx2 are expressed preferentially in the basal part of the olfactory epithelium. Our results of expression patterns of neurogenic markers in the olfactory epithelium in chick corroborate the previous published mouse data, and indicate that there is a conserved pattern of neurogenesis in the olfactory epithelium among vertebrates.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Chick Embryos

Fertilized white Leghorn chicken eggs were obtained from Agrisera AB, UmeÅ, Sweden. The use of chick embryos in this study was approved by the Ethical Committee on Animal Experiments for Northern Sweden. Chick embryos were staged according to the protocol of Hamburger and Hamilton (Hamburger and Hamilton,1992).

Cloning of Chick Gap43

A cDNA fragment corresponding to the nucleotides 1,371–1,841 of the chick GAP43 sequence (GenBank Accession Number XM425527) was obtained by RT-PCR using total RNA isolated from E5 chick embryos as template and the following primers: 5′-CAGCTTCCGTGGACACATA-3′ (forward) and 5′-TTGGCAGTAGCATCCTCTG-3′ (reverse). The PCR products were cloned into pGEMTeasy vector and the sequence confirmed through sequencing. The Gap43 riboprobe was synthesized by linearizing the plasmid with SpeI and using the T7-polymerase.

In Situ Hybridization and Immunohistochemistry

For in situ RNA hybridization and immunohistochemistry, chick embryos were fixed at 4°C 1–4 hr in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS), dehydrated, frozen, and serially cryosectioned at 10 μm. In situ RNA hybridization using chick digoxigenin-labeled Hes5 (El Wakil et al.,2006), Cash1 (Jasoni et al.,1994), Ngn1 (Perez et al.,1999), and Gap43 probes was performed essentially as described (Wilkinson and Nieto,1993). Immunohistochemistry was performed using an anti-Lhx2 rabbit antibody (1:8,000) (Lee et al.,1998) and an anti-HuC/D monoclonal mouse antibody (1:200) (Molecular Probes). Nuclei were stained using DAPI (Sigma). Secondary antibodies used were goat anti-rabbit Alexa 488 (1:400), goat anti-mouse Alexa 488 (1:400), and goat anti-mouse Alexa 594 (1:400) (Molecular Probes).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We are grateful to T. Jessell for the Lhx2 antibody. Probes were kindly provided by J. Nardelli (Hes5), C. Jasoni (Cash1), and D. Anderson (Ngn1). We thank Matthew Marklund and Barnabas Kolumban for help with cloning the chick Gap43, and Jonathan Gilthorpe for helpful comments on the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES