Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/24426161

Dynamicexpressionofneurogenicmarkersinthedevelopingchickolfactoryepithelium

ARTICLEinDEVELOPMENTALDYNAMICS·JUNE2009

ImpactFactor:2.38·DOI:10.1002/dvdy.21966·Source:PubMed

CITATIONS

19

READS

35

2AUTHORS:

EstherMaier

TheUniversityofSheffield

8PUBLICATIONS183CITATIONS

SEEPROFILE

LenaGunhaga

UmeåUniversity

25PUBLICATIONS612CITATIONS

SEEPROFILE

Availablefrom:LenaGunhaga

Retrievedon:03February2016

PATTERNS & PHENOTYPES

Dynamic Expression of Neurogenic Markers inthe Developing Chick Olfactory EpitheliumEsther Maier and Lena Gunhaga*

Neurogenesis in the olfactory epithelium begins in early embryos and proceeds throughout life. Acomparison of neurogenic marker expression at different developmental stages and at different axes of theolfactory epithelium has not been reported in a coordinated way. In this study, we have in detail comparedthe temporal and spatial expression patterns of the precursor markers Hes5, Cash1, Ngn1, and the neuronalmarkers Gap43, HuC/D, Lhx2 in the developing olfactory placode and epithelium in chick embryos fromHH10 to HH34. We show that Hes5 starts to be expressed in cells of the prospective olfactory placode atHH10, 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 olfactoryepithelium. By HH34, expression of the precursor and neuronal markers show the first signs of apical-basalstratification of the epithelium. Developmental Dynamics 238:1617–1625, 2009. © 2009 Wiley-Liss, Inc.

Key words: Hes5; Cash1; Ngn1; Gap43; HuC/D; Lhx2; neurogenesis; olfactory; chick

Accepted 29 March 2009

INTRODUCTION

Neurogenesis of olfactory sensory neu-rons in the olfactory epithelium pro-ceeds throughout life and is often usedas a paradigm to study neurogenesisin general because the simple organi-zation of the olfactory epitheliummakes it easily accessible.

The olfactory epithelium arisesfrom the olfactory placode (Croucherand Tickle, 1989; Kramer et al., 2000).As early as the late gastrula stage,progenitor cells in the anterior neuralplate border region, in between theprospective neuroectoderm and epi-dermis, are specified as olfactory andlens placodal cells (Sjodal et al., 2007).By neural fold stages, Hamburger andHamilton stage (HH) 8 in chick (Ham-burger and Hamilton, 1992) olfactoryand lens placodal progenitor cells are

spatially separated (Bhattacharyya etal., 2004; Sjodal et al., 2007). ByHH14, the olfactory placode becomesvisible as an ectodermal thickening onthe side of the head, which subse-quently invaginates to form a bowl-like structure called the olfactory pitat HH17. At later stages, the olfactoryepithelium extends further inside thehead mesenchyme and distinct olfac-tory turbinates are formed. The ma-ture olfactory epithelium containsstem-like cells, neuronal precursorcells at different maturity stages, ol-factory sensory neurons, and also gli-al-like supporting cells.

The basic helix-loop-helix (bHLH)repressor gene Hes5, the bHLH tran-scriptional regulators chick achaete-scute1 (Cash1) and Neurogenin1(Ngn1), the neuronal markers growth-

associated-protein 43 (Gap43) andHuC/D, and the LIM-homeodomaintranscription factor Lhx2, define cellsat different stages in the olfactory sen-sory neuronal lineage, and will be re-ferred to as neurogenic markersthroughout this study. In the olfactoryepithelium, neurogenesis occurs in anordered fashion; stem-like progenitorcells (Hes5-positive) undergo asym-metric cell division to generate tran-sient amplifying progenitor cells(Cash1-positive), which in their turngenerate the immediate neuronal pre-cursor cells (Ngn1-positive) that di-vide further and finally differentiateinto olfactory sensory neurons(Kawauchi et al., 2004; Beites et al.,2005). Olfactory sensory neurons canbe characterized by the expression ofGap43 (Baizer et al., 1990), members

Umeå Center for Molecular Medicine, Umeå University, Umeå, SwedenGrant sponsor: Medical Faculty of Umeå University, Sweden; Grant sponsor: Royal Swedish Academy of Sciences.*Correspondence to: Lena Gunhaga, Umeå Center for Molecular Medicine, Building 6M, 4th floor, Umeå University, S-901 87Umeå, Sweden. E-mail: lena.gunhaga@ucmm.umu.se

DOI 10.1002/dvdy.21966Published online 15 May 2009 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 238:1617–1625, 2009

© 2009 Wiley-Liss, Inc.

of the Hu class of RNA-binding pro-teins that mark post-mitotic neurons[HuC/D (Fornaro et al., 2003)] andLhx2 (Kolterud et al., 2004).

Genetic studies in mice have ad-dressed the role of some of these tran-scription factors in olfactory neuro-genesis. Mice mutant for Mash1 (themouse homologue of chicken Cash1)fail to generate olfactory progenitorsand thereby also olfactory sensoryneurons, among other neurogenic de-fects (Cau et al., 2002; Murray et al.,2003). In Ngn1 null mutant mice, dif-ferentiation of neural progenitors inthe olfactory epithelium is blocked(Cau et al., 2002), whereas Lhx2-defi-cient mice display a relatively normalorganization of the olfactory epithe-lium, but show a perturbed generationof olfactory sensory neurons (Hirotaand Mombaerts, 2004; Kolterud et al.,2004). Consequently, the expression ofseveral neurogenic markers has beenseparately described in the olfactorypit in mouse (Cau et al., 1997, 2000,2002; Hirota and Mombaerts, 2004;Kolterud et al., 2004). However, acomparison of neurogenic expressionpatterns between species has not beenreported, and the expression of neuro-genic markers has not been examinedin the chick olfactory epithelium.Thus, whether a conserved pattern ofneurogenesis in the olfactory epithe-lium exists among vertebrates still re-mains to be determined. Moreover, acollective temporal and spatial analy-sis of these markers in any species hasnot been examined. Such analyseswould define anterior-posterior andsuperior-inferior positions of precur-sors, immature and mature neuronsat various time points in the olfactoryepithelium.

In this study, we have analysed thetemporal and spatial expression ofHes5, Cash1, Ngn1, Gap43, HuC/D,and Lhx2 during the early develop-ment of the chick olfactory placodeand the olfactory epithelium by anal-ysing HH10, before the placode ismorphologically apparent, to HH34when distinct olfactory turbinates areformed. The selected markers definecells at different stages in the olfac-tory sensory neuronal lineage, whichenables us to visualize the progressionfrom stem and progenitor cells to dif-ferentiated neurons. When possible,we compare our results with pub-

lished mouse data, and we have corre-lated chick and mouse developmentalstages based on comparisons of olfac-tory epithelial/head morphologythroughout the study (Kaufman,1998; Bellairs and Osmond, 1998).

RESULTS AND DISCUSSION

Onset of Hes5 Expression inProspective OlfactoryPlacodal Cells

Hes-genes are known targets of theDelta-Notch signalling pathway (Jar-riault et al., 1995). The role of Hes-genes in olfactory epithelial develop-ment is dual; at early stages ofdevelopment, Hes-genes are impli-cated as pre-patterning genes definingthe domain of neurogenesis within theolfactory placodal region, whereas atlater stages, Hes-genes are involved incontrolling the number of neural pro-genitors emerging in this domain bynegatively regulating neurogenesis(Cau et al., 2000). Previous studies inmouse have shown that Hes5 is ex-pressed in the olfactory epithelium atE10.5 (�HH18 in chick) (Cau et al.,2000). In chick, three Hes5-like genes(Hes5-1, Hes5-2, Hes5-3) have beendescribed (Fior and Henrique, 2005).Since Hes5-1, Hes5-2, and Hes5-3show similar expression patterns inthe olfactory epithelium (data notshown), in this study the Hes5 expres-sion is represented by Hes5-1, and re-ferred to as Hes5. Our results showthat Hes5 can be detected in the ante-rior-lateral head ectoderm in chick al-ready at HH10 (Fig. 1B1–2). Based onfate maps in chick at HH10, whichhave shown that progenitor cells ofthe olfactory placode are located at themost anterior ectoderm (Bhatta-charyya et al., 2004), we conclude thatthe Hes5-positive region correspondsto prospective placodal cells. The earlyexpression domain of Hes5 is uniform,suggesting that in chick Hes5 is in-volved in pre-patterning of the pro-spective olfactory placode. Moreover,at this stage no Cash1, Ngn1, GAP43,HuC/D, or Lhx2 expression is detected(data not shown). Thus, at HH10 pro-spective olfactory placodal cells ex-press Hes5, which appears to be theearliest marker associated with neu-ronal determination in the chick olfac-tory placode.

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

By HH14 (�22 somites), the olfactoryplacodal epithelium starts to thicken(Fig. 2A). Previous studies in chickhave monitored the first migratorycells derived from the olfactory pla-code, termed epithelioid cells(Croucher and Tickle, 1989; Drapkinand Silverman, 1999), around HH14by analyzing HuC/D-positive cells, de-tecting early post-mitotic neurons(Fornaro et al., 2001). However, theexpression pattern of Hes5, Cash1,Ngn1, Gap43, or Lhx2 have not beenexamined in either chick or mouse atthis stage. Our results show that atHH14, apart from Hes5-expression,expression of Cash1, Ngn1, Gap43,and HuC/D can be detected in a fewcells in the olfactory placodal region(Figs. 2B–F). No Lhx2 expression isdetected in or around the olfactoryplacode at this stage (data not shown).Hes5 is expressed in a broad domainspanning almost the entire olfactoryplacodal region (Fig. 2B1–4). In con-trast, Cash1, which marks transientamplifying progenitor cells, can onlybe detected in a few cells in the ante-rior part of the olfactory placode (Fig.2C1–4). Moreover, at this stage theneuronal markers Ngn1, Gap43, andHuC/D are expressed in a few cells inthe anterior-medial region of the olfac-tory placode (Fig. 2D2,3, E2,3, F1–3),but excluded from the most posteriorpart of the placode (Fig. 2D4, E4, F4).Furthermore, at this stage, a fewNgn1, Gap43, and HuC/D-positiveneurons can be detected in the headmesenchyme close to the placodal ep-ithelium (Fig. 2D2, E2, F1 arrow-heads), showing the first cells migrat-ing away from the olfactory placode.The function of these early-delaminat-ing neurons remains unclear, al-though they have been suggested toperforate the olfactory epithelial basallamina, creating openings later uti-lized by emerging olfactory axons(Drapkin and Silverman, 1999), andto build up the olfactory nerve tractand migrate towards the anterior tel-encephalon (Fornaro et al., 2003). Pre-vious studies in mouse have describedthe earliest expression of Mash1 andNgn1 in the olfactory epitheliumaround E10.5 (Cau et al., 1997, 2000).

1618 MAIER AND GUNHAGA

Our results show that in the develop-ing chick olfactory placode, Cash1,Ngn1, and Gap43 are expressed atHH14 (�E9.5 in mouse). The differ-ence in onset of Mash1/Cash1 andNgn1 expression in chick and mouse ismost likely due to the lack of expres-sion analyses at earlier stages inmouse, rather than a species differ-ence, but remains to be determined.

Lhx2 Expression Is FirstDetected in the OlfactoryPlacode at HH17

We continued to analyze the expres-sion of neurogenic markers in the ol-factory placode at HH17 (�30somites). By this stage, the placodalepithelium has thickened consider-

Fig. 1. Expression of Hes5 detected by in situ hybridization (B) in the prospective olfactory placodein 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 thetelencephalon. The arrows outline the prospective olfactory region. B: At HH10 (10 somites), expressionof Hes5 is detected in the head ectoderm in the region of the prospective olfactory placode. Hes5expression can also be detected in the telencephalon. T, telencephalon. Scale bar � 100 �m.

Fig. 2. Expression of Hes5, Cash1, Ngn1, and Gap43 detected by in situ hybridization (B–E) and HuC/D detected by immunohistochemistry (F) in theolfactory placode of HH14 chick embryos. Schematic chick embryo to the left indicates the transversal plane of sections and the sectioned area. A: Theboxed area in the DAPI-stained section indicates the newly formed olfactory placode close to the telencephalon. The arrows mark the olfactoryplacodal region. Sections are represented from anterior at the top to posterior at the bottom. B: At HH14 (22 somites), Hes5 is expressed in theolfactory 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 detectedin a few migratory cells (arrowheads). Hes5 expression can also be detected in the telencephalon. T, telencephalon. Scale bar � 100 �m.

NEUROGENIC MARKERS IN CHICK OLFACTORY EPITHELIUM 1619

ably compared to HH14 and is visibleas an indention in an anterior-lateralposition of the head. Hes5 is stillstrongly expressed in nearly all cellsof the olfactory placode (Fig. 3B1–4),and only the most posterior part of theolfactory placode has lower levels ofHes5 expression (Fig. 3B5). At thisstage, Cash1 is expressed only in a fewcells in the medial part of the placode,whereas strong expression of Cash1 is

restricted to cells located in the basalpart of the epithelium adjacent to themesenchyme and to the rim of the pla-code (Fig. 3C2–4). Similar to the Cash1expression pattern, the olfactory neu-ronal markers Ngn1, Gap43, andHuC/D are all expressed in cells lo-cated in the medial part of the pla-code, and the majority of Ngn1-,Gap43-, and HuC/D-positive cells arefound close to the basal lamina (Fig.

3D2,3, E2,3, F2–4). At HH17, Lhx2 ex-pression can only be detected in themedial part of the placode in a fewround-shaped cells close to the basallamina (Fig. 3G2,3, arrow). In addi-tion, Ngn1, Gap43, HuC/D, and Lhx2expression is also detected in neuronsmigrating away from the placode,termed the migratory mass, towardsthe anterior telencephalon (Fig. 3D2,E2–4, F2,3, G2,3, arrowheads). Thus,

Fig. 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 fromanterior 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 partof the olfactory placode has lower levels of Hes5 expression (B5). C: Cash1 expression is restricted the medial areas of the olfactory placode and cellsexpressing 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) andHuC/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 themesenchyme (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-upin 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). Thebroken 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 inthe telencephalon. M, mesenchyme; OP, olfactory placode; T, telencephalon. Scale bars � 100�m.

1620 MAIER AND GUNHAGA

the relatively few Lhx2-positive cellsin the medial part of the olfactory ep-ithelium, compared to the numbers ofLhx2-positive cells in the migratorymass, indicate that at HH17 the ma-jority of the differentiated neurons inthe olfactory epithelium leave the pla-code and migrate away. This is inagreement with previous studies, sug-

gesting that the first-born neurons inthe olfactory epithelium leave the pla-code, and build up and broaden theolfactory nerve (Croucher and Tickle,1989; De Carlos et al., 1995). In addi-tion, previous studies have demon-strated that the olfactory nerve is ap-parent at HH18, providing a pathalong which several different olfactory

epithelium-derived cells migrate to-wards the anterior telencephalon(Croucher and Tickle, 1989; De Carloset al., 1995; Drapkin and Silverman,1999). Our results show that at HH17,all Lhx2-positive migratory cells ex-press HuC/D, but not all HuC/D cellsare Lhx2 positive (Fig. 3H), indicatingthat at this stage there might be at

Fig. 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-likeolfactory pit. A: The boxed area in the DAPI-stained section indicates the invaginated olfactory pit close to the telencephalon. Sections are representedfrom 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 posteriorinferior 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, withthe 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 cellsin 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 expressingHes5, 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 �m.

NEUROGENIC MARKERS IN CHICK OLFACTORY EPITHELIUM 1621

Fig. 5.

Fig. 6.

1622 MAIER AND GUNHAGA

least two subgroups of HuC/D-positiveneurons migrating from the olfactoryplacode; however, the fate of theseneurons remains to be determined.Taken together, we show that neuro-genesis is ongoing in the olfactory pla-code prior to invagination of the olfac-tory epithelium.

Neurogenesis in theOlfactory Pit Proceeds AlongDistinct Axes

By HH18, the olfactory placode beginsto invaginate and by HH22 (�E12.25 inmouse) the olfactory pit has formed in abowl-like shape (Fig. 4). The rim of theolfactory pit is termed the inferior re-gion, while the invaginated part of theepithelium is called the superior region(Fig. 4). In mouse, E10.5–E12 are theearliest stages in which separate neuro-genic markers in the olfactory epithe-lium have been studied (Cau et al.,1997, 2000, 2002). However, these stud-ies have not analyzed whether the ex-pression of various neurogenic markersdiffers at different anterior-posteriorlevels of the olfactory epithelium. To an-alyze this issue in detail, we collectivelyexamined the expression pattern ofHes5, Cash1, Ngn1, Gap43, HuC/D, andLhx2 at HH22. Similar to earlier stagesof development, Hes5 is expressed in amajority of cells in the olfactory pit, andexcluded from the inferior regions of thepit (Fig. 4B1–6). At HH22, the expres-sion domain of Hes5 in the olfactory pitappears to be patchier (Fig. 4B2–6), in

line with a role for Hes-genes in Notch-mediated lateral inhibition in the neu-rogenic area of the olfactory pit (Cau etal., 2000). At HH22, Cash1 expressionis excluded from the most anterior re-gion of the olfactory pit (Fig. 4C1). In themedial part of the pit, Cash1 expressionis located in the inferior region of theolfactory epithelium (Fig. 4C2,3),whereas in the posterior part of the ol-factory pit Cash1-positive cells are de-tected in the superior region of the epi-thelium (Fig. 4C4–6). At HH22, Ngn1,Gap43, and HuC/D are expressed inscattered patterns throughout the ante-rior-posterior axis of the olfactory pit,with the majority of expressing cells inthe superior region at anterior to me-dial levels (Fig. 4D2–4, E2–4, F2–4). Inthe posterior part of the olfactory pit,the numbers of Ngn1-positive cells arehigher (Fig. 4D5,6), compared to Gap43-and HuC/D-positive neurons (Fig.4E5,6, F5,6). Ngn1 expression is detectedin both the apical and basal part of theolfactory epithelium evenly distributedalong the anterior-posterior axis (Fig.4D1–6). In contrast, Gap43- and HuC/D-positive cells are located in the basalpart of the epithelium adjacent to themesenchyme and the strongest Gap43and HuC/D expression is detected in themedial-superior region of the olfactorypit (Fig. 4E2–4, F2–4). Furthermore, atHH22, Lhx2 is expressed in a subset ofHuC/D-positive cells in the medial partof the olfactory pit in the most superiorregion 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, com-pared to the broader Cash1 and Ngn1expression, clearly resembles the factthat Cash1 and Ngn1 are expressed byneuronal precursor cells, while Gap43,HuC/D, and Lhx2 are expressed by ol-factory sensory neurons. At this stage,there is little obvious divergence in theexpression patterns of Hes5, Cash1,Ngn1, Gap43, and Lhx2 in the middle ofthe anterior-posterior axis of the olfac-tory pit in chick compared to publishedmouse expression (Cau et al., 1997,2000, 2002; Hirota and Mombaerts,2004; Kolterud et al., 2004). However,our results show that neurogenesis inthe olfactory region proceeds along dis-tinct axes in the olfactory pit, with themost differentiated neurons being lo-cated at medial-superior positions. Inaddition, at this stage Ngn1, Gap43,HuC/D, and Lhx2 expression are alsodetected in migratory neurons thathave emerged from the olfactory epithe-lium (Fig. 4D1,2, E1,2, F1,2, G1–3). Previ-ous studies in chick have shown that,around HH22, a small fraction of themigratory cells are GnRH (gonadotro-pin-releasing hormone) neurons thatwill migrate via the olfactory nerve tothe hypothalamus (Mulrenin et al.,1999).

By HH25, the expression patternsof Hes5, Cash1, Ngn1, Gap43, HuC/D,and Lhx2 are similar to the expressionat HH22, but the numbers of neuronshave increased (data not shown),which is in agreement with progres-sive neurogenesis in the olfactory epi-thelium.

By HH29, Hes5 Is Expressedin a Scattered Manner inthe Olfactory Epithelium

Around HH29 (�E14 in mouse), themorphology of the nasal cavity beginsto change and the nasal conchaestarts to form (Croucher and Tickle,1989; Leibovici et al., 1996). The olfac-tory and the respiratory epitheliumcan now be distinguished from one an-other by their different morphology(Croucher and Tickle, 1989; Leiboviciet al., 1996). The olfactory epitheliumis thicker and located further insidethe head mesenchyme (Fig. 5A), but atthis stage stratification has not yetbegun. Compared to mouse, where theolfactory epithelium begins to attain a

Fig. 5. Expression of Hes5, Cash1, Ngn1, and Gap43 detected by in situ hybridization (B–E) andHuC/D and Lhx2 detected by immunohistochemistry (F–H) in the olfactory epithelium of an HH29chick embryo. Schematic chick embryo to the left indicates the transversal plane of sections andthe sectioned area. A: The boxed area in the DAPI-stained section indicates the invaginated andextended olfactory epithelium inside the head mesenchyme, and the thinner respiratory epitheliumlocated more distally in the nasal passage. Sections are represented from anterior at the top toposterior at the bottom. B: Hes5 is expressed in a salt-and-pepper-like pattern in nearly the entireolfactory epithelium (B1-4). C: A few Cash1-expressing cells can be detected at apical and basalpositions in the epithelium (C1–4). D: Ngn1 are expressed at high levels in a scattered patternthroughout the olfactory epithelium (D1–4). E–G: Gap43 (E1–4), HuC/D (F1–4), and Lhx2 (G1–4) areexpressed in the majority of cells in the olfactory epithelium. The olfactory nerve is visualised by theexpression of Gap43 (arrowhead in E2). No expression of Hes5, Cash1, Ngn1, Gap43, HuC/D, orLhx2 can be detected in the respiratory epithelium. OE, olfactory epithelium; Re, respiratoryepithelium. Scale bar � 100 �m.

Fig. 6. Expression of Hes5, Cash1, Ngn1, and Gap43 detected by in situ hybridization and HuC/Dand 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 superiorconcha. 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 theolfactory epithelium. Broken lines in B–G indicate the position of the basal lamina. M, mesenchyme;OE, olfactory epithelium. Scale bar � 100 �m.

NEUROGENIC MARKERS IN CHICK OLFACTORY EPITHELIUM 1623

layered morphology around E12.5(Cau et al., 2000), the development ofthe olfactory epithelium in chick ap-pears to proceed much slower. The re-spiratory epithelium is a thin layer ofcells and is located more distally inthe nasal passage (Fig. 5A). No Hes5,Cash1, Ngn1, Gap43, HuC/D, or Lhx2expression is detected in the respira-tory epithelium (Fig. 5B–G), consis-tent with the fact that the respiratoryepithelium does not give rise to olfac-tory sensory neurons.

By HH29, although Hes5 is ex-pressed in nearly the entire neuro-genic region, levels of expression aremuch lower than at earlier stages anda salt-and-pepper-like pattern isclearly distinguishable (Fig. 5B1–4), inaccordance with a role of Hes5 in lat-eral inhibition (Cau et al., 2000). Sim-ilar to Hes5 expression, a few scat-tered Cash1-positive cells can bedetected in the entire neurogenic re-gion (Fig. 5C1–4). At this stage, Ngn1is expressed in a salt-and-pepper pat-tern throughout the olfactory epithe-lium (Fig. 5D1–4). Moreover, a major-ity of the cells within the olfactoryepithelium express Gap43 and HuC/D(Fig. 5E1–4, F1–4), and a subset ofHuC/D-positive cells expresses Lhx2(Fig. 5G1–4). At HH29, migrating neu-rons that follow the olfactory nervetowards the telencephalon expressGap43, HuC/D, and Lhx2 (Fig. 5E2

and data not shown).

Stratification of the ChickOlfactory Epithelium Is FirstDetected at HH34

At HH34, Hes5 is expressed in theapical part of the olfactory epitheliumof the superior concha (Fig. 6A). Themajority of the Cash1- and Ngn1-pos-itive cells are also present in the api-cal region, and a few Cash1- andNgn1-expressing cells are detected inthe middle area of the olfactory epi-thelium (Fig. 6B, C). At this stage,Gap43, HuC/D, and Lhx2 are ex-pressed predominately in the basal re-gion of the epithelium, but Gap43-,HuC/D-, and Lhx2-positive cells aredetected along the entire apical-basalaxis of the olfactory epithelium (Fig.6D–F). Thus, by HH34, the initialstratification of the chick olfactory ep-ithelium is apparent.

In summary, in this study we de-

scribe in detail the temporal and spa-tial expression of Hes5, Cash1, Ngn1,Gap43, HuC/D, and Lhx2, duringearly stages of neural determinationand neurogenesis in the olfactory epi-thelium in chick. Hes5 is expressed inthe prospective olfactory placode al-ready at HH10, earlier than previ-ously reported and before the placodeis morphologically apparent, indicat-ing that Hes5 may be involved in de-termining the neurogenic area of theepithelium. Moreover, our resultsshow that neurogenesis is ongoing inthe olfactory placode prior to invagi-nation of the olfactory epithelium.During olfactory pit stages, both ananterior-posterior and a superior-infe-rior distinction in the expression ofthe investigated neurogenic factorsbecomes apparent, with the most dif-ferentiated neurons being located atmedial-superior positions. In contrast,at HH29 the neuronal markersGap43, HuC/D, and Lhx2 are ex-pressed evenly throughout the entireolfactory epithelium. The different ex-pression patterns of the neuronalmarkers between HH22 to HH29 mostlikely reflect the spatial expansion ofneurogenesis from the early restrictedmedial region of the pit at HH22 to theentire olfactory epithelium at HH29.By HH34, stratification of the chickolfactory epithelium is visible, inwhich the precursor markers Hes5,Cash1, and Ngn1 are expressed in theapical part of the epithelium, whilethe neuronal markers Gap43, HuC/D,and Lhx2 are expressed preferentiallyin the basal part of the olfactory epi-thelium. Our results of expressionpatterns of neurogenic markers in theolfactory epithelium in chick corrobo-rate the previous published mousedata, and indicate that there is a con-served pattern of neurogenesis in theolfactory epithelium among verte-brates.

EXPERIMENTALPROCEDURES

Chick Embryos

Fertilized white Leghorn chicken eggswere obtained from Agrisera AB,UmeÅ, Sweden. The use of chick em-bryos in this study was approved bythe Ethical Committee on Animal Ex-periments for Northern Sweden.

Chick embryos were staged accordingto the protocol of Hamburger andHamilton (Hamburger and Hamilton,1992).

Cloning of Chick Gap43

A cDNA fragment corresponding to thenucleotides 1,371–1,841 of the chickGAP43 sequence (GenBank AccessionNumber XM425527) was obtained byRT-PCR using total RNA isolated fromE5 chick embryos as template and thefollowing primers: 5�-CAGCTTCCGTG-GACACATA-3� (forward) and 5�-TTG-GCAGTAGCATCCTCTG-3� (reverse).The PCR products were cloned intopGEMTeasy vector and the sequenceconfirmed through sequencing. TheGap43 riboprobe was synthesized bylinearizing the plasmid with SpeI andusing the T7-polymerase.

In Situ Hybridization andImmunohistochemistry

For in situ RNA hybridization and im-munohistochemistry, chick embryoswere fixed at 4°C 1–4 hr in 4% para-formaldehyde (PFA) in phosphatebuffered saline (PBS), dehydrated,frozen, and serially cryosectioned at10 �m. In situ RNA hybridization us-ing chick digoxigenin-labeled Hes5 (ElWakil et al., 2006), Cash1 (Jasoni etal., 1994), Ngn1 (Perez et al., 1999),and Gap43 probes was performed es-sentially as described (Wilkinson andNieto, 1993). Immunohistochemistrywas performed using an anti-Lhx2rabbit antibody (1:8,000) (Lee et al.,1998) and an anti-HuC/D monoclonalmouse antibody (1:200) (MolecularProbes). Nuclei were stained usingDAPI (Sigma). Secondary antibodiesused 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).

ACKNOWLEDGMENTSWe are grateful to T. Jessell for theLhx2 antibody. Probes were kindlyprovided by J. Nardelli (Hes5), C. Ja-soni (Cash1), and D. Anderson (Ngn1).We thank Matthew Marklund andBarnabas Kolumban for help withcloning the chick Gap43, andJonathan Gilthorpe for helpful com-ments on the manuscript.

1624 MAIER AND GUNHAGA

REFERENCES

Baizer L, Alkan S, Stocker K, Ciment G.1990. Chicken growth-associated protein(GAP)-43: primary structure and regu-lated expression of mRNA during embry-ogenesis. Brain Res Mol Brain Res 7:61–68.

Beites CL, Kawauchi S, Crocker CE, CalofAL. 2005. Identification and molecularregulation of neural stem cells in the ol-factory epithelium. Exp Cell Res306:309–316.

Bellairs R, Osmond, M. 1998. The atlas ofchick development. New York: AcademicPress, 323p.

Bhattacharyya S, Bailey AP, Bronner-Fraser M, Streit A. 2004. Segregation oflens and olfactory precursors from a com-mon territory: cell sorting and reciproc-ity of Dlx5 and Pax6 expression. Dev Biol271:403–414.

Cau E, Gradwohl G, Fode C, Guillemot F.1997. Mash1 activates a cascade of bHLHregulators in olfactory neuron progenitors.Development 124:1611–1621.

Cau E, Gradwohl G, Casarosa S,Kageyama R, Guillemot F. 2000. Hesgenes regulate sequential stages of neu-rogenesis in the olfactory epithelium.Development 127:2323–2332.

Cau E, Casarosa S, Guillemot F. 2002.Mash1 and Ngn1 control distinct steps ofdetermination and differentiation in theolfactory sensory neuron lineage. Devel-opment 129:1871–1880.

Croucher SJ, Tickle C. 1989. Characteriza-tion of epithelial domains in the nasalpassages of chick embryos: spatial andtemporal mapping of a range of extracel-lular matrix and cell surface moleculesduring development of the nasal placode.Development 106:493–509.

De Carlos JA, Lopez-Mascaraque L,Valverde F. 1995. The telencephalic ves-icles are innervated by olfactory placode-derived cells: a possible mechanism toinduce neocortical development. Neuro-science 68:1167–1178.

Drapkin PT, Silverman AJ. 1999. Develop-ment of the chick olfactory nerve. DevDyn 214:349–360.

El Wakil A, Francius C, Wolff A, Pleau-Varet J, Nardelli J. 2006. The GATA2transcription factor negatively regulatesthe proliferation of neuronal progenitors.Development 133:2155–2165.

Fior R, Henrique D. 2005. A novel hes5/hes6 circuitry of negative regulation con-trols Notch activity during neurogenesis.Dev Biol 281:318–333.

Fornaro M, Geuna S, Fasolo A, Giacobini-Robecchi MG. 2001. Evidence of veryearly neuronal migration from the olfac-tory placode of the chick embryo. Neuro-science 107:191–197.

Fornaro M, Geuna S, Fasolo A, Giacobini-Robecchi MG. 2003. HuC/D confocal imag-ing points to olfactory migratory cells asthe first cell population that expresses apost-mitotic neuronal phenotype in thechick embryo. Neuroscience 122:123–128.

Hamburger V, Hamilton HL. 1992. A se-ries of normal stages in the developmentof the chick embryo. 1951. Dev Dyn 195:231–272.

Hirota J, Mombaerts P. 2004. The LIM-homeodomain protein Lhx2 is requiredfor complete development of mouse olfac-tory sensory neurons. Proc Natl Acad SciUSA 101:8751–8755.

Jarriault S, Brou C, Logeat F, SchroeterEH, Kopan R, Israel A. 1995. Signallingdownstream of activated mammalianNotch. Nature 377:355–358.

Jasoni CL, Walker MB, Morris MD, RehTA. 1994. A chicken achaete-scute ho-molog (CASH-1) is expressed in a tempo-rally and spatially discrete manner inthe developing nervous system. Develop-ment 120:769–783.

Kaufman MH. 1998. The atlas of mousedevelopment, revised ed. New York: Ac-ademic Press. 525 p.

Kawauchi S, Beites CL, Crocker CE, WuHH, Bonnin A, Murray R, Calof AL.2004. Molecular signals regulating pro-liferation of stem and progenitor cells in

mouse olfactory epithelium. Dev Neuro-sci 26:166–180.

Kolterud A, Alenius M, Carlsson L, BohmS. 2004. The Lim homeobox gene Lhx2 isrequired for olfactory sensory neuronidentity. Development 131:5319–5326.

Kramer PR, Guerrero G, KrishnamurthyR, Mitchell PJ, Wray S. 2000. Ectopicexpression of luteinizing hormone-re-leasing hormone and peripherin in therespiratory epithelium of mice lackingtranscription factor AP-2alpha. MechDev 94:79–94.

Lee KJ, Mendelsohn M, Jessell TM. 1998.Neuronal patterning by BMPs: a re-quirement for GDF7 in the generation ofa discrete class of commissural interneu-rons in the mouse spinal cord. Genes Dev12:3394–3407.

Leibovici M, Lapointe F, Aletta P, Ayer-LeLievre C. 1996. Avian olfactory recep-tors: differentiation of olfactory neuronsunder normal and experimental condi-tions. Dev Biol 175:118–131.

Mulrenin EM, Witkin JW, Silverman AJ.1999. Embryonic development of the go-nadotropin-releasing hormone (GnRH)system in the chick: a spatio-temporalanalysis of GnRH neuronal generation,site of origin, and migration. Endocrinol-ogy 140:422–433.

Murray RC, Navi D, Fesenko J, LanderAD, Calof AL. 2003. Widespread defectsin the primary olfactory pathway causedby loss of Mash1 function. J Neurosci23:1769–1780.

Perez SE, Rebelo S, Anderson DJ. 1999.Early specification of sensory neuronfate revealed by expression and functionof neurogenins in the chick embryo. De-velopment 126:1715–1728.

Sjodal M, Edlund T, Gunhaga L. 2007.Time of exposure to BMP signals plays akey role in the specification of the olfac-tory and lens placodes ex vivo. Dev Cell13:141–149.

Wilkinson DG, Nieto MA. 1993. Detectionof messenger RNA by in situ hybridiza-tion to tissue sections and whole mounts.Methods Enzymol 225:361–373.

NEUROGENIC MARKERS IN CHICK OLFACTORY EPITHELIUM 1625