Of the Otic Placode

Developmental Biology 249, 237–254 (2002) doi:10.1006/dbio.2002.0739

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Andrea Streit1 Department of Craniofacial Development, King’s College, Guy’s Hospital, London SE1 9RT, United Kingdom

During development, the vertebrate inner ear arises from the otic placode, a thickened portion of the ectoderm next to the hindbrain. Here, the first detailed fate maps of this region in the chick embryo are presented. At head process stages, placode precursors are scattered throughout a large region of the embryonic ectoderm, where they intermingle with future neural, neural crest, epidermal, and other placode cells. Within the next few hours, dramatic cell movements shift the future otic placode cells toward the midline and ultimately result in convergence to their final position next to rhombomeres 5–6. Individual cells and small cell groups undergo constant cell rearrangements and appear to sort out from nonotic cells. While the major portion of the otic placode is derived from the nonneural ectoderm, the neural folds also contribute cells to the placode at least until the four-somite stage. Comparison of these fate maps with gene expression patterns at equivalent stages reveals molecular heterogeneity of otic precursor cells in terms of their expression of dlx5, msx1, Six4, and ERNI. Although Pax2 expression coincides with the region where otic precursors are found from stage 8, not all Pax2-positive cells will ultimately contribute to the otic placode. © 2002 Elsevier Science (USA) Key Words: chicken; ear development; fate map; placode field.

INTRODUCTION otic vesicle generates the complex architecture of the inner ear. Both initial formation and later patterning of the otic In vertebrates, the inner ear is one of the most complex placode are controlled by inductive interactions with sur- sensory organs comprising the semicircular canals, up to rounding tissues. For example, it has been proposed that the seven different sensory organs or patches, and the cochlea otic placode is induced by sequential signals emanating housing the auditory apparatus. Despite this complexity, from different tissues at different times and that therefore the inner ear (along with other sensory organs and cranial cells become gradually committed to otic fate (Gallagher et ganglia) arises from simple ectodermal thickenings, the al., 1996; Jacobson, 1963a–c; Waddington, 1937; Yntema, cranial placodes, which lie adjacent to the anterior neural 1933, 1950). Indeed, signals from the mesoderm and the plate (for review, see Anniko, 1983; Fritzsch et al., 1998; neural plate play an important role during otic induction Torres and Giraldez, 1998). The otic placode first becomes (Gallagher et al., 1996; Jacobson, 1963a; Mendonsa and morphologically discernible around the 10-somite stage, Riley, 1999; Orts-Llorca and Jimenez-Collado, 1971). In next to rhombomeres 5 and 6 of the hindbrain (Bancroft and addition, recent studies have confirmed that at least two Bellairs, 1977; Haddon and Lewis, 1996; Romanoff, 1960; different signals—FGFs and Wnts—promote the formation Schlosser and Northcutt, 2000). It then invaginates (while of the otic placode (Ladher et al., 2000; Phillips et al., 2001; neuroblasts delaminate to give rise to the cochlear vestibu- Vendrell et al., 2000). However, none of these signals was lar ganglion) and eventually separates from the surface shown to initiate otic development in naive ectoderm, and ectoderm. During subsequent morphogenetic events, the therefore it is still unclear whether they control the first step in otic induction or whether there are more upstream Supplementary data for this article are available on IDEAL signals involved. Therefore, the question of when otic (http://www.idealibrary.com). induction begins still remains open. 1 To whom correspondence should be addressed. Fax: ϩ44-20- Inductive interactions can be revealed by comparing 7955-2704. E-mail: [email protected]. specification maps with fate maps made at the same stages

FIG. 1. Standardization of measurements. DiI- and DiO-labelled positions and gene expression domains were measured by using visible landmarks as described in the text. (A) In stage 5–7 embryos, the position along the anteroposterior axis was expressed as % of the distance between the center of Hensen’s node (0%) and the tip of the prechordal plate (100%; hn-pp). The mediolateral position was calculated as % of the distance between the midline (0%) and the border of the area pellucida (100%; ml-ap). (B) In embryos with two or more somites, the distance between the center of Hensen’s node (0%) and the anterior edge of the ﬁrst somite (100%; hn-som) was measured, and the anteroposterior position of labelled cells was determined as % of hn-som. Mediolateral position is expressed as % of the width of the mediolateral extent of the neural plate (np ϭ 100%). (C, D) Plots to determine the degree of shrinkage along the mediolateral axis suffered by embryos after in situ hybridization. The x-axis shows the position of labels as measured in the living embryo, and the y-axis shows the same labels after in situ hybridization. In both cases, the relative measuring system explained in (A) and (B) above was used. These (and similar plots for other stages) were used to compare the position of gene expression domains to the fate maps. (E) Five cell populations of different sizes were labelled with DiI in a stage 5 embryo. The dye was then photoconverted with DAB and the embryo processed for whole-mount in situ hybridisation with an ERNI antisense probe. (Inset) Section through the same embryo at the level of labelling.

FIG. 2. Examples of DiI-labelled embryos. Small cell populations in the epiblast were labelled with DiI at stages 5–8(AЈ–DЈ) and their fate assessed after the otic placode had developed (A–D). (A, AЈ) Cells from the level of Hensen’s node at 40% ml-ap at stage 5 contributed to the posterior otic placode and adjacent lateral ectoderm (left), while cells from 45% ml-ap gave rise to ventral epidermis (right). (B, BЈ) Cells located in the neural folds (left) and just outside the neural folds (right) at stage 8 contributed to posterior and anterior otic cup, respectively. (C, CЈ) Cells labelled at 30% along the anteroposterior axis and at 40% along the mediolateral axis at stage 7 gave rise to lateral epidermis, including the future geniculate placode (arrow). (D, DЈ) Cells labelled at stage 7 (left: 10% hn-pp, 25% ml-ap; right: 15%hn-pp, 27% ml-ap) generated neural crest cells. The dotted white lines in (A–D) indicate the position of the otic cup. (A–C) Fluorescence images; (D) A pseudocolor overlay of the ﬂuorescence image (red) with the corresponding bright-ﬁeld image (blue–green). (AЈ–DЈ) Diagrams indicating the labelling positions.

(Slack, 1983): any differences between the two are likely to different stages suggests that extensive cell movements indicate the regions and times at which tissue interactions accompany placodal development. This was confirmed by are required for cells to become determined to specific fates. time-lapse microscopy, which revealed for the first time Recently, Groves and Bronner-Fraser (2000) have reported that scattered cells and cell groups converge to the placode that chick ectoderm isolated from the otic region at or later while constantly undergoing cell rearrangements. Even at than stage 8 retains Pax2 expression, but not BMP7, Sox3, fairly late stages, cells arising from the ectoderm along most or Notch (which are specified at stage 9–10). However, most of the hindbrain and from the neural folds contribute to the fate maps produced to date that include the otic territory placode. Finally, a detailed comparison is made between have concentrated on early stages of development (primi- otic fate and the expression patterns of several genes at the tive streak stages), when otic precursors are distributed over border of the neural plate, including the prospective otic a large area of the epiblast and appear to be intermingled region. This comparison reveals that otic cells are recruited with precursors for other tissues. No detailed maps are from a molecularly heterogeneous cell population, through available between the primitive streak stage and the onset extensive migration. of expression of the first otic marker Pax2, which might allow us to determine at what stage otic precursors first become confined to a discrete domain. MATERIALS AND METHODS Here, a detailed fate map of the chick otic placode between head process and four-somite stages is presented. Embryo Techniques Our data reveal that, even after primitive streak stages, otic Fertile hen eggs (Winter Farm, Hertfordshire, UK) were incu- precursors continue to be interspersed with future neural, bated at 38°C for 20–44 h to obtain embryos at stages 5–10 neural crest, epidermal, and epibranchial placode cells. (Hamburger and Hamilton, 1951). Stage 5 and 6 embryos were Although the otic domain gradually condenses, cells of explanted in Pannett Compton saline and maintained in New different fates remain mixed within it even after the onset culture (New, 1955) modified after Stern and Ireland (1981), while of Pax2 expression. Comparison of fate maps made at older embryos were cultured in ovo. For video time-lapse analysis,

embryos were cultured dorsal side up on agar–albumin plates Whole-Mount in Situ Hybridization and Histology (Chapman et al., 2001). For fate-mapping studies, small groups of epiblast cells were labelled by using the fluorescent dyes DiI and/or cDNAs for dlx5 (Ferrari et al., 1995), msx1 (Liem et al., 1995), DiO as described previously (Stern, 1998). Briefly, stocks of 0.5% Six4 (Esteve and Bovolenta, 1999), and Sox2 (Kamachi et al., 1995; DiI or 0.25% DiO in absolute alcohol were diluted 1:10 in 0.3 M Uwanogho et al., 1995) were kind gifts from R. A. Kosher, T. M. sucrose at 50°C and injected by air pressure using a micropipette Jessell, P. Bovolenta, P. Scotting, and R. Lovell-Badge, respectively. made from 50-␮l borosilicate glass capillaries. The labelled position ERNI has been described previously (Streit et al., 2000). Whole- was determined in relation to other landmarks (see below), and the mount in situ hybridization using DIG- and FITC-labelled anti- embryos were then cultured until the otic placode or cup could be sense RNA-probes was performed as previously described (Stern, identified by morphological criteria (stage 12–15). The fate of the 1998; Streit et al., 1997; The´ry and Stern, 1996). To detect a single labelled cells was assessed in whole mounts, in 40-␮m vibratome transcript, embryos were developed by using NBT/BCIP as a ␮ sections, or after photo-oxidation and wax sectioning. Photo- substrate (Sigma), embedded in paraffin, and sectioned (10 m). For in situ oxidation using 3,3Ј-diaminobenzidine (DAB; Sigma) was per- double hybridization, NBT/BCIP (Sigma) and int-BCIP (Roche) were used as substrates and 40-␮m sections were cut with formed as described previously (Izpisua-Belmonte et al., 1993). a vibratome (Leitz).

Standardization of the Position of Labelled Cells Determination of Gene Expression Domains The mediolateral extent of the expression domains of dlx5, The anteroposterior and mediolateral positions of DiI- and/or ERNI, msx1, and Six4 and the position of the Pax2 domain were DiO-labelled cells were measured by using an eyepiece graticule determined after whole-mount in situ hybridization by using the immediately after injection. In stage 5–7 embryos, distances were same criteria as described for the fate map (see Figs. 1A and 1B). For determined from the centre of Hensen’s node (primitive pit) to the each stage and transcript, 5–10 embryos were measured. However, ϭ tip of the prechordal plate (hn-pp 100%; see Fig. 1A) and to the embryos subjected to in situ hybridization undergo considerable labelled cells, respectively. The position of the label was calculated shrinkage, particularly along the mediolateral axis (Figs. 1C and as a percentage of the total length hn-pp. To standardise the 1D). To compensate for this shrinkage so that direct comparisons mediolateral position, the distance between the midline and the could be made between fate map results and gene expression labelled cells was expressed as % of the distance between the domains, a calibration curve was drawn: spots of DiI were placed on midline and the edge of the area pellucida (ml-ap ϭ 100%; see Fig. the epiblast of 32 embryos (stage 5–8), the position of each spot was 1A). measured, and the embryos were immediately fixed, after which In embryos with two somites or more, distances were measured the dye was photoconverted by using DAB. They were then from the primitive pit to the anterior edge of the first somite processed for whole-mount in situ hybridization by using Six4, (hn-som ϭ 100%; see Fig. 1B) and to the labelled cells. The position dlx5, ERNI,orPax2 probes, and both the expression domains and of the label was standardized as a percentage of the total distance the position of the label were measured (Fig. 1E). The positions of hn-som. The mediolateral position of the labelled cells was ex- the DiI label before and after hybridization were used to construct pressed as a percentage of the distance between the midline and the graphs like those in Figs. 1C and 1D. In embryos with 2 somites or lateral edge of the neural plate (np ϭ 100%). more, no significant difference was found between both sets of Note that, using this system, these measurements are relative measurements. However, in younger embryos, there was signifi- such that the 100% value differs considerably between the medio- cant shrinkage, which was greater in lateral regions than close to lateral and anteroposterior axes, as well as between different stages. the midline (see Figs. 1C and 1D). These graphs were used to For example, at stage 5, the ml-ap distance (100%) averages 1012 Ϯ extrapolate the position of gene expression domains to their 97 ␮m and the hn-pp distance (100%) corresponds to 1250 Ϯ 46 ␮m. presumed positions before in situ hybridization and to relate them At the two-somite stage, the hn-som distance (100%) averages directly to the fate map data. 434 Ϯ 46 ␮m, while the np distance (100%) is 200 Ϯ 20 ␮m.

RESULTS Video Time-Lapse Filming The otic placode forms in the ectoderm adjacent to the Four different positions of the epiblast on each side of stage 6–8 neural plate just anterior to the first somite and can first be embryos were labelled with DiI (0.025% in 0.3 M sucrose; diluted identified morphologically around the 10- to 14-somite 1:20 from a 0.5% stock solution in absolute alcohol). The embryos stage (Bancroft and Bellairs, 1977; Haddon and Lewis, 1996; were cultured in a heated chamber placed around a Zeiss Axioscope Romanoff, 1960; Schlosser and Northcutt, 2000). In the with fluorescence optics. A transmitted light and a fluorescence primitive streak stage chick embryo, future otic cells are ϫ image were captured with a 5 objective every 10 min by using a distributed over a fairly large region of the epiblast, appar- Princeton Instruments cooled CCD camera and Metamorph soft- ently interspersed with precursors for other tissues (Garcia- ware at a resolution of 658 ϫ 517 (24-bit color). The two images Martinez et al., 1993; Schoenwolf and Sheard, 1990). To were overlaid by encoding the fluorescence image in the red channel and the transmitted light image in both the green and blue understand how cells from this broad area converge to form channels. The resulting time-lapse series was saved as a stack the otic placode, a fate map was constructed from head typically consisting of 150 color-encoded images. For posting as process to the 4-somite stage by injecting the fluorescent supplementary material, the stacks were converted to AVI files by dyes DiI and DiO to label small cell populations of the using Cinepak (compressed by radius). chick epiblast. The position of labelled cells was analyzed

© 2002 Elsevier Science (USA). All rights reserved. Formation of the Otic Placode 241 after the otic placode or vesicle had formed (stage 11–15). In contribute to a large region of the neural tube and neural total, 398 embryos were analyzed, most of which had crest, extending from the posterior midbrain to the anterior received one DiI and one DiO injection on each side of the spinal cord. After this, they begin to condense at the level of midline. Figure 2 shows some examples of DiI-labelled future rhombomeres 2–7. However, even at later stages embryos. (stage 8, 4 somites), some future otic cells are still found at the same level as cells that will contribute as far anteriorly Origin of the Otic Placode as rhombomere 1 and as far posteriorly as the rostral spinal cord. Otic precursors converge from a broad region of the Regional subdivision of the otic placode. From around ectoderm to form the placode. At the head process stage stage 14, several molecular markers become restricted to (stage 5), otic placode precursors are distributed over a large subdomains of the otic placode, such as Pax2 (Herbrand et region of the epiblast lateral to Hensen’s node and are al., 1998; Hidalgo-Sanchez et al., 2000; Nornes et al., 1990), intermingled with future neural tube, neural crest, epi- Gbx2 (Hidalgo-Sanchez et al., 2000), and BMP7 (Groves and branchial placode, and epidermis cells (Fig. 3A). They span Bronner-Fraser, 2000) medially, and SOHo (Kiernan et al., about one-third of the distance between the midline and the 1997), Otx2 (Hidalgo-Sanchez et al., 2000; Morsli et al., lateral edge of the area pellucida (see Table 1; total width 1999), Hmx3 (Herbrand et al., 1998), and GH6 (Kiernan et ml-ap, 1012 ␮m; width of otic territory, 324 ␮m) and extend al., 1997) laterally (for review, see Brigande et al., 2000). both anteriorly and posteriorly from Hensen’s node (about These domains of gene expression correlate approximately 25% of the distance between the node and the prechordal with areas destined to form the cochlear (ventromedial) and plate in both directions; hn-pp, 1250␮m). By head-fold vestibular (lateral) portions of the inner ear (see Li et al., stages (stage 6/7; Fig. 3B), otic precursors are still inter- 1978). Do these subdomains of the otic cup arise from spersed with cells of other fates, but have moved, together separate groups of precursors? To address this, it was with future neural and neural crest cells, towards the determined whether specific positions of the early fate midline (see Table 1; width of otic territory, 276 ␮m). They maps give rise to cells restricted to specific quadrants of the are now located anterior to Hensen’s node and spread about otic vesicle (Fig. 5). Although there is a general tendency for 500 ␮m along the anteroposterior axis (about 30% of hn-pp). more posteriorly located precursors to contribute to the Precursors for the epibranchial placodes are mixed with posterior portion of the otic vesicle, and similarly for the future otic cells but extend more into the lateral ectoderm mediolateral axis, there is no clear subdivision of the fate (ml-ap position 30–60%, 295 ␮m). map into individual compartments at any of the stages While they have largely separated from hindbrain precur- investigated. Even at later (4-somite) stages, cell groups that sors at the two-somite stage (except at the edge of the neural will contribute progeny to opposite poles of the otic vesicle plate; Fig. 3C), many future otic cells are found in close are often found adjacent to each other. These findings are in association with neural crest precursors at the border of the agreement with a recent study in Xenopus, showing that neural plate. The otic domain, however, extends further cells continue to mix extensively even after formation of laterally into the ectoderm (to np position 175%, 170 ␮m the otic vesicle (Kil and Collazo, 2001). lateral to the border of the neural plate) than the neural In summary, our results indicate that the otic placode crest territory (to np position 125%, 57 ␮m from the border arises from a very broad region of the embryonic epiblast. of the neural plate) and overlaps laterally with precursors Up to the one-somite stage, its precursors are intermingled for the epibranchial placodes (np position 130%–220%, with cells that contribute to the epidermis, the epibranchial width 180 ␮m). The density of otic precursors close to the placodes, the central nervous system, and neural crest cells region where the placode will form has increased. at all levels between the anterior and posterior hindbrain. During subsequent stages (Figs. 3D and 3E), the relative There is no early regional subdivision of the presumptive position of the future otic placode remains fairly constant otic territory. Otic precursors accumulate at the border of and extends from the level of the future anterior hindbrain the neural plate and even at fairly late stages cells from the to the level of the first somite. Otic precursors have not yet inner neural folds are recruited into the placode. Compari- separated from future epibranchial or neural crest cells. The son of the position of otic precursors at different stages neural folds continue to give rise to neural, neural crest, and suggests that extensive cell movements accompany the otic cells; even at the four-somite stage, the inner neural formation of the otic placode. folds contribute cells to the otic placode. Otic precursors do not arise in register with rhom- Extensive Cell Movements Lead to the Formation bomeres 5 and 6. Once the otic cup becomes visible, it of the Otic Placode lies opposite rhombomeres 5 and 6 of the hindbrain. How early is this alignment fixed? To address this, the rostro- The results presented above led to the possibility that caudal position of cells contributing to various levels of the formation of the otic placode is accompanied by massive neural tube and neural crest was analyzed and compared cell movements. To confirm this and to visualize these with the position of otic precursors (Fig. 4). At early stages movements directly, we used time-lapse video analysis. (stage 5–6), otic precursors are mixed with cells that will Three to four cell groups on each side of the embryo (stage

FIG. 3. Fate map of the otic placode between head process and 4-somite stages. Small cell groups in the epiblast were labelled with DiI and DiO and their fate determined after the otic placode or vesicle could be identified by morphology. (A) At stage 5, otic precursors are distributed among neural, neural crest, epidermal, and epibranchical placode precursors in a large region of the epiblast. (B) By stage 6–7, they have converged towards the midline to become located in the ectoderm, first anterior to Hensen’s node (stage 6), and later anterior to the first somite (stage 7). (C) By stage 7ϩ, the prospective otic territory has almost completely separated from future neural plate cells (except at the neural folds), but still contains precursors for other tissues. (D, E) During the 3- (D) and 4-somite (E) stages, the neural folds still contribute cells to the otic placode; otic precursors are also found in the epidermis adjacent to most of the hindbrain. The circles represent labels that gave rise to a cells in a single location, while squares represent those where progeny was found in more than one tissue. Crossed circles represent positions labelled in the filmed embryo shown in Fig. 6 (see Supplementary Material). The dotted line in (C–E) indicates the visible edge of the neural plate. The grey oval in (D) and (E) approximately encircles the Pax2 expression domain, and the horizontal grey line shows the posterior boundary of Pax2 in the CNS. The mediolateral extents of the expression domains of msx1, dlx5, Six4, and ERNI are indicated by horizontal bars. nraStreit Andrea Formation of the Otic Placode 243

FIG. 4. Otic precursors are not aligned with R5 and R6 precursors. Diagrams showing the position of future otic cells (black circles) at stages 5–8 (A, stage 5; B, stage 6/7; C, stage 7ϩ; D, stage 8Ϫ; E, stage 8), in relation to precursors for the neural tube (circles) and neural crest (squares) at different positions along the rostrocaudal axis. R1–R7, rhombomeres 1–7. The origin of neural crest cells was scored according to their ﬁnal position based on data available in the literature (Noden, 1975; Lumsden and Keynes, 1989; Birgbauer et al., 1995); numbers in brackets reﬂect minor contribution of crest from these rhombomeres. Otic precursors arise from a very large rostrocaudal territory spanning the entire length of the hindbrain. The grey oval in (D) and (E) approximately encircles the Pax2 expression domain, and the horizontal grey line corresponds to the posterior limit of Pax2 in the CNS. The dotted line in (C–E) marks the position of the neural fold. FIG. 5. Regionalisation of the otic vesicle. Diagrams showing the position of precursors for anterior, posterior, medial, and lateral otic placode at stages 5–8 (A, stage 5; B, stage 6/7; C, stage 7ϩ; D, stage 8Ϫ; E, stage 8). The circles indicate labels that contributed cells to an entire half of the otic vesicle; the squares mark those that contributed to less than half (e.g., anterior–median quadrant). In no case did any label contribute to more than half of the otic cup. Note that cells contributing to different portions of the otic vesicle are extensively intermixed, even as late as stage 8 (4 somites).

TABLE 1 Comparison between the Position of Otic Precursors and Gene Expression Domains

Medial border Lateral border

Stage msx1 dlx5 Six4 ERNI otic msx1 dlx5 Six4 ERNI otic

Stage 5 384 Ϯ 14.4 404 Ϯ 15.2 455 Ϯ 25.3 475 Ϯ 19.2 283 516 Ϯ 24.3 617 Ϯ 20.2 678 Ϯ 16.1 738 Ϯ 25.3 607 Stage 6/7 276 Ϯ 18.7 342 Ϯ 14.4 353 Ϯ 11.0 385 Ϯ 22.1 276 419 Ϯ 16.5 574 Ϯ 19.8 618 Ϯ 24.3 607 Ϯ 18.7 552 2 somites 190 Ϯ 4.0 200 Ϯ 6.0 230 Ϯ 5.1 240 Ϯ 5.0 190 250 Ϯ 10.0 350 Ϯ 8.2 366 Ϯ 9.8 374 Ϯ 10.4 365

Note. The mediolateral position of the expression domains of msx1, dlx5, Six4, and ERNI were measured at stages 5, 6/7, and 7ϩ in 7–10 embryos of each stage. The average distance between the midline and the medial and lateral edges, respectively, of the expression domains is given in ␮m Ϯ standard deviation (ml-ap in Fig. 1). The most medial and most lateral position in which otic precursors were found at different stages is also given in ␮m. At stage 5, none of the expression domains matches the mediolateral limits of the otic territory; from stage 6/7 onwards the medial border of the otic region closely correlates with the medial limit of msx1 expression, while its lateral border coincides with the lateral limit of Six4 expression.

6–8) were labelled with DiI to follow their movements over Relationship between Genetic Markers of the a 15- to 24-h period. Figure 6 shows frames from one Border of the Neural Plate and the Future Otic representative movie (a compressed version of which can be Region seen in Supplementary material: http://www. . . ). In agree- At head process stages, several genes, including dlx5 (Pera ment with the fate map data (see Fig. 3), of the four cell et al., 1999), msx1 (Streit and Stern, 1999), Six4 (Esteve and groups labelled on the left side, three contributed to the otic Bovolenta, 1999), and ERNI (Streit et al., 2000), are ex- placode; while on the right, only progeny from the most pressed at the anterior lateral border of the neural plate, a medial cell group does. The diagram in Fig. 7A illustrates region that is thought to give rise to sensory placodes and the movements of these four cell groups at 3-h intervals. cranial neural crest (for review, see Baker and Bronner- The most dramatic movements are observed within the Fraser, 2001). To investigate which of these genes, if any, first 6–9 h, when lateral cells rapidly converge to the best reflects the mediolateral position of the future otic midline (see groups 1, 2, and 4 in Fig. 7A; see also Fig. 7C, placode in the newly generated fate maps, their expression groups 1 and 2). Afterwards, medially directed movements between stages 5 and 8 was compared to the fate map of the continue, but at a much slower rate. For example, by 9 h otic region by using single and double whole-mount in situ (Fig. 7A; and inset in Fig. 7A, turquoise outline), group 1 hybridization followed by histological sections. (mediolateral start position 41%) has fused with group 2 Five to ten embryos of each stage were processed for in (mediolateral start position 28%) and during the next 6 h situ hybridization for each gene, and the domains of expres- there is little directional movement; however, smaller sion were measured by using the same criteria that were groups of cells constantly change neighbors and eventually employed for the fate map (but correcting for shrinkage as split off the main group to give rise to epidermis and the explained in Materials and Methods; see Fig. 1). The results otic placode. are summarized in Table 1 and Figs. 8 and 9. Cells from position 3 (Fig. 7A—shown in more detail in At stage 5, dlx5, Six4, and ERNI transcripts are detected Fig. 7B) initially move as a coherent group towards the in a horseshoe shape surrounding the anterior neural plate midline. After 12 h, the cells move away from the midline with a caudal limit just posterior to, or at the level of, Hensen’s node (Fig. 8A, a, e, and i). In contrast, msx1 and split up into several groups (14 h). Figure 7B illustrates expression is broad posteriorly, extending anteriorly along the movements of one such group over the next 360 min: the border of the neural plate with a rostral limit just after splitting off, the cells stall for about 100 min before anterior to the node (Fig. 8A, m). During stages 6–7, dlx5, they slowly begin to migrate away from the midline (during Six4, and ERNI domains maintain their posterior boundary 180 min) and then rapidly (within 100 min) join the otic relative to the regressing node (Fig. 8A, b, c, f, g, j, and k; Fig. vesicle. 8B, a, b, f, g, p, and q), while msx1 expression moves These results confirm that otic precursors converge to anteriorly to surround the neural plate (Fig. 8A, n and o; Fig. their final position from a large region of the embryonic 8B, k and l). Beginning at the two-somite stage, both ERNI ectoderm. During this process, extensive cell mixing occurs and dlx5 are down-regulated in a posterior-to-anterior di- and some cell clusters derived from a coherent group of rection (Fig. 8A, d and h; Fig. 8B, c–e and h–j) and ERNI labelled cells split into smaller groups. Of these, some will transcripts disappear completely by stage 9Ϫ, while dlx5 contribute to the otic placode, while others will give rise to becomes concentrated in the most anterior neural folds and different tissues. the adjacent ectoderm (Fig. 8A, h). Six4 and msx1 remain

© 2002 Elsevier Science (USA). All rights reserved. Formation of the Otic Placode 245 expressed at the border of the neural plate (Fig. 8A, l and p; ling, photo-oxidized, and processed for Pax2 in situ hybrid- Fig. 8B, m–o, r, s). Medially, all four genes overlap with the ization (Figs. 10J and 10K). Of these, 34/38 labels (90%) expression domains of the neural markers Sox2 (Fig. 8A, were found within the Pax2 domain. In another 10 em- q–t; Fig. 8B, u–y) and Sox3 (not shown) until stage 7ϩ/8, bryos, the fate of cells labelled as above was assessed after when only msx1 coincides with Sox2 in the inner neural the otic cup had formed. Most (9/10; 90%) showed DiO- and folds. Thus, the expression of all four genes begins to DiI-labelled cell groups in each otic vesicle (Figs. 10M–10S). overlap at node levels and subsequently spreads to encircle Often, red and green cells were found in overlapping re- the entire anterior neural plate. gions, confirming that, even at these fairly late stages These expression domains appear very similar; to estab- (stages 8–10), cell mixing occurs frequently among cells lish whether they are identical, we performed double in situ that will contribute to the placode. However, some of the hybridization with various combinations of these markers cells labelled within the Pax2 domain contribute to the (Fig. 9). The msx1 territory is narrowest and lies most epidermis, including the future epibranchial placodes (Fig. medial, followed laterally by dlx5, Six4, and ERNI, all of 10N; see also Fig. 3E). Together, these results suggest that, which overlap with msx1 to different extents. Throughout although cells derived from the entire Pax2-positive region all stages, this mediolateral sequence of expression is main- can contribute to the otic placode, not all of them do. tained. Since the expression domains of these genes are not quite identical to each other, which, if any, correlates with the DISCUSSION otic territory established in the fate maps? At stage 5, none of the gene expression domains can be related to the Dual Origin of the Otic Placode: Contribution of position of the otic precursors (see Table 1; Fig. 3A). Neural and Nonneural Ectoderm However, like the otic territory, the msx1-, dlx5-, Six4-, and In this study, a detailed analysis of the cellular move- ERNI-positive regions shift towards the midline between ments that accompany the formation of the otic placode in stages 5 and 6 (Fig. 8A, b, c, f, and g), while Sox2 expression the chick embryo is presented. Evidence is provided that narrows (Fig. 8A, r). From stage 6/7, the mediolateral cells fated to give rise to the otic placode originate from a location of future otic cells corresponds approximately to large region of the epiblast at head process stages and the expression domain of Six4 except most medially (where converge towards their final position whilst they separate Six4 does not overlap with msx1), while both ERNI and from other cell populations. In agreement with previous dlx5 are down-regulated from the otic territory after stage fate maps of primitive streak stage embryos (Garcia- 7ϩ. Although some cells from the msx1 domain contribute Martinez et al., 1993; Schoenwolf and Sheard, 1990), this to the otic placode, this domain never encompasses all otic study shows that, at head process stages, otic precursors are precursors. In conclusion, therefore, the expression do- intermingled with future neural, neural crest, epidermis, mains of genes that mark the border of the neural plate are and epibranchial placodes. There is no clearly defined not quite identical, and none of them relates directly to the domain within which all cells are fated to become otic, as position of precursors of the otic vesicle at stages 5–8 along suggested by Rudnick (1944) based on her transection and either the mediolateral or the anteroposterior axis. explantation experiments. By stage 6/7, otic precursors are largely separated from future neural tube cells, but they continue to be mixed with prospective crest, epidermal, and Correlation of Pax2 Expression and Otic Fate epibranchial cells. Based on morphological criteria, it has Shortly after dlx5 and ERNI are downregulated (stage 7ϩ), been suggested that a proportion of otic cells is recruited the transcription factor Pax2 begins to be expressed, first as from the neural plate and that, even at late stages of a salt-and-pepper pattern within a domain that corresponds development, individual cells leave the neural plate to approximately to the otic region of our fate maps (cf. Figs. contribute to the placode (Mayordomo et al., 1998). Our 10A–10D and 10L with Fig. 3). Subsequently, more cells results show that, while the major portion of the otic within this domain begin to express Pax2 (Fig. 10E) until placode is derived from lateral ectoderm, a minor portion is expression becomes uniform at stage 10 (Figs. 10F–10I). Do indeed recruited from the inner portion of the neural folds cells from the entire Pax2 domain contribute to the otic (coexpressing msx1 and the neural marker Sox2) at least up placode? To predict the Pax2 domain reliably, whole-mount to the four-somite stage, suggesting that the neural folds in situ hybridization was performed on embryos at stages contain either multipotent precursors or mixed populations 8–10 (5–7 embryos at each stage), and the domain was of cells with different fates. Indeed, it has previously been measured with respect to other landmarks (see Fig. 1A). reported that single cells (Bronner-Fraser and Fraser, 1988, Using these measurements, we then labelled 3–4 small cell 1989; Frank and Sanes, 1991; Selleck and Bronner-Fraser, groups at different anteroposterior and mediolateral levels 1995) in the caudal neural folds can give rise to neural tube, within the Pax2 domain with DiI and DiO (n ϭ 17; Figs. 10J, neural crest, and epidermal progeny. Likewise, small cell 10K, and 10M–10S). To confirm that the labelled cells were groups well within the caudal neural plate of embryos at indeed contained within the Pax2-positive region, 7 em- stage 6–7 can contribute to both neural tube and epidermis bryos (38 labels total) were fixed immediately after label- (Brown and Storey, 2000). The present results reveal that

FIG. 6. Extensive cell movements and rearrangements during the formation of the otic placode. Four separate cell groups were labelled with DiI on each side of a stage 7 (0 h) embryo, and their movements were followed by using time-lapse video filming for 21 h. The panels illustrate the embryo at 1 h intervals. An AVI movie of this embryo can be found as Supplementary Material at: http://www. . . Note: due to the culture method, some DiI crystals adhere to the vitelline membrane where they remain throughout the culture. The movie in Supplementary Material shows that dye is not transferred from these acellular deposits to cells during their migration. the otic placode has a dual origin: it arises from both neural from the amphibian embryo, showing that no such subdi- and nonneural portions of the ectoderm. vision can be demonstrated before stage 8 in the chick. The first molecular signs of regionalization of the chick otic Regional Subdivision of the Otic Placode placode appear around stage 14/15 (for review, see Brigande et al., 2000; Fekete, 1996), when, for example, Pax2 (Her- In fish, the prospective otic territory seems to be subdi- brand et al., 1998; Hidalgo-Sanchez et al., 2000; Nornes et vided into anterior, posterior, medial, and lateral domains al., 1990), Gbx2 (Hidalgo-Sanchez et al., 2000), and BMP7 at midgastrula stages (50% epiboly; Kozlowski et al., 1997), (Groves and Bronner-Fraser, 2000) begin to be restricted to whilst a recent study in Xenopus showed that extensive cell the medial placode, while SOHo (Kiernan et al., 1997), Otx2 movements occur between different quadrants of the otic (Hidalgo-Sanchez et al., 2000; Morsli et al., 1999), Hmx3 vesicle even after its closure (Kil and Collazo, 2001). The (previously Nkx5.1; Herbrand et al., 1998), and GH6 (Kier- data presented here are more consistent with the results nan et al., 1997) are expressed in its lateral portion. Rota-

FIG. 7. Movements of selected cell groups from filmed embryos. (A) Traces of the contours of four groups of labelled cells (numbered 1–4) from the filmed embryo shown in Fig. 6 (also shown in Supplementary Material), at 3- to 6-h intervals. Note that the most rapid movements occur in lateral regions during the first 9 h (e.g., group 1 was labelled at position 41% ml-ap; after 9 h [turquoise] it has fused with group 2, which started at ml-ap position 28%, see inset). Red, 0 h; blue, 3 h; turquoise, 9 h; purple, 12 h; yellow, 18 h; orange, 21 h. (B) In the same film, a small group of cells (white) has separated from group 3 at 14 h (grey outline); thereafter, it hardly moves for the first 1.5 h, then it slowly starts to move away from the midline for about 3 h, and finally moves rapidly towards the otic placode in the final 1.5 h of this sequence. The outlines of this cell group are overlaid onto the final frame of the film, dotted line showing the position of the otic cup. Grey, 0 min; light orange, 20 min; dark green, 80 min; pink, 120 min; blue, 140 min; purple, 160 min; yellow, 220 min; red, 240 min; light green, 260 min; dark orange, 280 min; brown, 300 min; turquoise, 320 min; white, 340 min; black, 360 min. (C) Movements of three cell groups in a different filmed embryo, which had been labelled at stage 6. Two groups (1, 3) contribute to the otic vesicles, while the third does not (2). Red, 0 h; blue, 3 h; turquoise, 9 h; purple, 12 h; brown, 15 h. Dotted lines in (A) and (C) demarcate the midline and the position of the otic cup at the final stage. tion experiments revealed that, while the anteroposterior Neural crest cells migrating from particular rhom- axis of the otic vesicle is set around stage 12, determination bomeres follow unique routes and have defined fates (Chan of the mediolateral axis begins shortly after asymmetric and Tam, 1988; Lumsden and Keynes, 1989; Lumsden et al., gene expression is observed but is not completed until 1991; Noden, 1975). Likewise, it has been suggested that much later (after embryonic day 3.5; Hutson et al., 1999; cranial ectoderm may be subdivided into segments (“ecto- Wu et al., 1998). meres”), aligned with the rhombomeres of the neighbouring hindbrain, and coordinated with specific neural crest migration routes (Couly and Le Douarin, 1990; Noden, 1993). Coordinated Development of Neural and Placodal This hypothesis predicts that boundaries exist that prevent Structures? cells in each ectomere from crossing into neighbouring During sensory organ development, peripheral structures ectomeres. The broad distribution of otic precursors along often arise in close association with the targets to which the entire hindbrain found in the present study does not they later project, e.g., the future olfactory placode initially support this idea. As late as the six- to eight-somite stage, abuts the future olfactory bulb region before they become otic precursors converge from different rhombomere levels separated from each other by morphogenetic movements to reach their final position adjacent to rhombomeres 5 and (Couly and Le Douarin, 1985, 1987, 1988; Whitlock and 6, suggesting that the ectoderm is not subdivided into Westerfield, 2000). Neuroblasts delaminating from the otic lineally separate domains at this stage. placode give rise to the cochlear–vestibular ganglion, whose axons project to the auditory and vestibular nuclei of the A Common Placodal Field? brain stem. During development, these nuclei arise from rhombomeres 4–7 (Cramer et al., 2000; Marin and Puelles, It has been suggested (Jacobson, 1963c, 1966; for review, 1995; Tan and Le Douarin, 1991). Otic precursors, however, see Torres and Giraldez, 1998; Baker and Bronner-Fraser, are found in a much broader region of the ectoderm, from 2001; but, Graham and Begbie, 2000; Schlosser and North- the posterior midbrain to the rostral spinal cord, indicating cutt, 2000) that all cranial placodes are derived from a that they begin to align with the future auditory and single, common region—the “placodal field”—and that vestibular nuclei only around the time that the placode their initial induction involves a common molecular becomes morphologically distinct. mechanism. In fact, a continuous ectodermal thickening

© 2002 Elsevier Science (USA). All rights reserved. Formation of the Otic Placode 249 surrounding the anterior neural plate from which placodes before the first Pax2-positive cells appear and otic cells arise has been observed in mouse (Verwoerd and van begin to be specified (Groves and Bronner-Fraser, 2000). Oostrom, 1979), fish (Miyake et al., 1997), and amphibians Therefore, if a common placodal territory exists, inductive (Knouff, 1935; Platt, 1896). The molecular network of Pax, interactions or other mechanisms must exist to direct Six, eya, and dac genes plays an important role in eye specific subgroups of cells to specific placodal fates from development, and members of these families are also ex- within this territory. pressed in other sensory structures, like the inner ear, the olfactory placode, and the cranial ganglia (for review, see Correlation between Pax2 Expression and Otic Wawersik and Maas, 2000). The preplacodal region ex- Fate and Possible Mechanisms of Placode presses Six1 and -4 (Esteve and Bovolenta, 1999; Ghanbari Specification et al., 2001; Kobayashi et al., 2000; Pandur and Moody, 2000) and eya1 (Mishima and Tomarev, 1998; Sahly et al., The transcription factor Pax2 is generally regarded as an 1999), while dach1 is expressed in the neural plate as well early otic-specific marker, although a direct comparison of as in part of the placodal field (unpublished observations). In its expression domain with regions containing future otic contrast, none of the Pax genes has been found in this cells has never been made. Otic placode specification is region; instead their expression begins only in regions thought to begin around the time when Pax2 is first where placodes ultimately form, like Pax2 in the otic expressed (Groves and Bronner-Fraser, 2000). Here, it is (Groves and Bronner-Fraser, 2000; Hidalgo-Sanchez et al., shown that, just like otic precursors, the earliest Pax2- 2000; Nornes et al., 1990; Pfeffer et al., 1998), Pax6 in the positive cells to appear at stage 8 are scattered in a salt-and- lens and olfactory (Hirsch and Harris, 1997; Li et al., 1994; pepper pattern within the region containing most, if not all, Walther and Gruss, 1991), and Pax3 in the trigeminal cells fated to contribute to the otic vesicle (see Fig. 10L). placode (Baker et al., 1999; Stark et al., 1997). This raises However, this territory also contains cells that do not the possibility that a common placodal region is set up by contribute to this structure. It is conceivable that these assembling part of the molecular network (Six, eya, and early Pax2-positive cells correspond to cells specified to dach)—a molecular mechanism common to all placodes— become otic. If so, the extensive cell movements described and is then further refined by more localized signals that in this study could reflect an active process of cell sorting. initiate Pax gene expression to establish placode identity. Unfortunately, techniques to determine this directly are A number of genes, among them dlx5 (Pera et al., 1999), not yet available, particularly because shortly afterwards, msx1 (Streit and Stern, 1999), Six4 (Esteve and Bovolenta, Pax2 expression within this domain becomes uniform, 1999), and ERNI (Streit et al., 2000) are all coexpressed at which precludes the use of the Pax2 promoter to lineage- the lateral border of the anterior neural plate and have label the earliest expressing cells. variously been referred to as markers for the border, the At the five- to eight-somite stage, otic precursors still neural crest (msx1), or placodal (Six4) region or the nonneu- arise from the entire Pax2 domain; however, nonotic cells ral ectoderm (dlx5). Is the expression domain of any of these also continue to originate from this territory. They subse- consistent with the idea of a common placodal field? Here, quently move to the epibranchial placodes, where they it is shown that the domains of expression of these genes maintain or re-express Pax2, or they become epidermis and are not identical, that they change over time, and that none down-regulate expression. Thus, although the Pax2 expres- of the genes ever encompasses the entire otic territory. All sion domain correlates very well with the position of the four transcripts overlap, but only partially, in a large portion prospective otic territory, not all cells from this domain of the ectoderm containing cells destined for many different will contribute to the otic region. structures. When otic precursors begin to accumulate at The findings presented here leave two main possible their final position anterior to the first somite, dlx5 and mechanisms by which otic placode specification could ERNI are down-regulated from this region, just a few hours occur. Either individual cells are specified at an early stage

FIG. 8. Expression domains of putative placodal markers. (A) Whole-mount in situ hybridization of stage 5/6 (a, e, i, m, q), 6/7 (b, f, j, n, r), 7/8 (c, g, k, o, s), and 8/8ϩ (d, h, l, p, t) embryos, showing expression of ERNI (a–d), dlx5 (e–h), Six4 (i–l), msx1 (m–p), and Sox2 (q–t). (B) Histological sections of embryos at stage 6/7 (a, b, f, g, k, l, p, q, u, v) or stage 8/8ϩ (c–e, h–j, m–o, r–t, w–y) after whole-mount in situ hybridization with dlx5 (a–e), ERNI (f–j), msx1 (k–o), Six4 (p–t), and Sox2 (u–y). At stage 6/7, expression of dlx5 and ERNI is very similar (a, b, f, g), being excluded from the neural plate (compare with u, v); at stage 8, dlx5 expression (c–e) medially overlaps with msx1 (m–o), while ERNI is expressed more lateral (h–j) and does not overlap with msx1. The distance between the lateral edge of the neural plate and ERNI expression increases from anterior to posterior (h–j). Msx1 is expressed in two separate domains in the ectoderm (k–o): a thin stripe of cells along the border of the neural plate and in posterior lateral ectoderm (l). Ectodermal Six4 expression (p–t) abuts the neural plate; Six4 transcripts are also found in mesoderm and endoderm underlying the neural plate and the head mesenchyme (r). At stage 6/7, Sox2 expression is conﬁned to the neural plate in anterior regions (u), while it is more widely expressed posteriorly (v). At stage 8, Sox2 is expressed in the forming neural tube, including the neural folds where it overlaps with msx1 expression (w); more posteriorly it is excluded from the folds (x).

FIG. 9. Comparison of the expression domains of dlx5, ERNI, Six4, Sox2, and msx1 by double in situ hybridization. (A–F) Double in situ hybridization showing the expression of dlx5 (red) and msx1 (blue) in whole mount (A, D) and sections (B, C, E, F) at stages 6 (A–C) and 8 (D–F). (G–I) Comparison of ERNI and msx1 at stage 6 (ERNI in red and msx1 in blue in G, vice versa in H and I). (J–O) Comparison of Six4 (blue) and msx1 (red) at stages 6 (J–L) and 8 (M–O). (P–R) ERNI (red) and Sox2 (blue) at stage 7. Note that posteriorly at stages 6–7, msx1 expression overlaps with dlx5, ERNI, and Six4 (arrowheads), with msx1 extending medially from this region and the other genes laterally; at this stage, msx1 does not surround the most anterior region of the neural plate and separates from the other genes at the level of sections B, H, and K (arrows). More medially, Sox2 expression marks the neural plate, overlapping with ERNI at its lateral edge (Q, arrowheads; therefore, also with all other genes except Six4).

(just before stage 8) through a widespread otic-inducing distinguish between these possibilities, it will be necessary signal to which only some cells respond; this response is both to unravel the molecular cascade leading to otic accompanied by the initial expression of Pax2 and is fol- induction as well as a molecular dissection of the regulatory lowed by the sorting out of these cells from their neigh- elements controlling Pax2 expression. bours and migration to the site of the otic vesicle. Alterna- By the four- to five-somite stage, fate (this study) and tively, otic-inducing signals are localized, cell movements specification maps (Groves and Bronner-Fraser, 2000) for in the epiblast occur randomly, and only those cells that the otic placode overlap. At the same stage, FGF-19 and reach the vicinity of the signal will be specified as otic. To Wnt-8c, two signals that have been implicated in otic

FIG. 10. Pax2 expression demarcates the position of future otic cells. (A–D) Pax2 begins to be expressed in the otic territory in a salt-and-pepper pattern at stage 8. (E) The number of Pax2-expressing cells increases during stage 9 and the domain enlarges. (F–I) At stage 10, the otic expression of Pax2 is uniform. (J, K) DiI labelling of several cell groups (arrow heads: 6 groups in J, 8 in K) within the Pax2 domain: to ensure that the Pax2 domain could be labelled reliably, embryos were ﬁxed immediately after labelling and the positions of the labelled cells measured. The dye was then photo-oxidized (brown; arrowheads) and the embryos processed for in situ hybridization with Pax2 (purple). All cell groups labelled are within the Pax2 domain. (L) Pax2 expression overlaps with the otic territory at stage 8. The dotted line shows the outline of Pax2 expression; the purple circles correspond to the labelling positions in Fig. 3E that contributed to the otic placode. (M–S) Three to four cell groups (numbered 1–7) in the Pax2 domain on each side of the embryo were labelled with DiI (red) or DiO (green). (M, O, R) Embryos immediately after labelling. (N, P, Q, S) Embryos after 21 h. (P, Q) The left and right otic cups, respectively, of embryo (O). Note that most of the cell groups (the only exception being groups 1 and 4 in M and N) labelled within the Pax2 domain contributed to the otic cup, but that extensive cell mixing has occurred between groups (e.g., arrows in S, where cells of group 2 have impinged into and split group 3). Some cell groups contributed to both the otic placode (arrow heads in N) and the adjacent epidermis (groups 2, 5, and 7 in M and N; group 1 in O and P; and groups 1 and 3 in R and S). induction (Ladher et al., 2000), become localized in the to contain at least some cells with an otic fate, and the mesoderm underlying and the neural tube adjacent to the possibility therefore remains that FGF and Wnt are only future otic placode. Together, these observations suggest permissive, rather than instructive. It will now be important that the latter possibility is more likely and that otic induc- to test whether these signals can induce otic fate and expres- tion is a late event. On the other hand, based on the present sion of otic-speciﬁc genes in regions that do not contain these fate maps, all of the test tissues used in this study are likely cells.

Cell Movements and Their Coordination Baker, C. V., Stark, M. R., Marcelle, C., and Bronner-Fraser, M. (1999). Competence, specification and induction of Pax-3 in the The results presented here reveal a very considerable trigeminal placode. Development 126, 147–156. amount of cell mixing and cell movement accompanying Baker, C. V. H., and Bronner-Fraser, M. (2001). Vertebrate cranial formation of the otic placode in the chick. In contrast, the placodes. Part I. Embryonic induction. Dev. Biol. 232, 1–61. otic placode in zebrafish (Kozlowski et al., 1997) and in Bancroft, M., and Bellairs, R. (1977). Placodes of the chick embryo amphibians (Carpenter, 1937; Ro¨ hlich, 1931; Schlosser and studied by SEM. Anat. Embryol. (Berl.) 151, 97–108. Northcutt, 2000) is already confined to a fairly small region Birgbauer, E., Sechrist, J., Bronner-Fraser, M., and Fraser, S. (1995). by the neurula stage or shortly thereafter and is well Rhombomeric origin and rostrocaudal reassortment of neural separated from precursors of the neural plate and other crest cells revealed by intravital microscopy. Development 121, placodes. On the other hand, cell movements comparable to 935–945. those described here have been observed for olfactory pla- Boilly, B., Vercoutter-Edouart, A. S., Hondermarck, H., Nurcombe, V., and Le Bourhis, X. (2000). FGF signals for cell proliferation code precursors in zebrafish (Whitlock and Westerfield, and migration through different pathways. Cytokine Growth 2000). Here, olfactory cells arise from a broad region of the Factor Rev. 11, 295–302. outer rim of the neural plate and converge to their final Brigande, J. V., Kiernan, A. E., Gao, X., Iten, L. E., and Fekete, D. M. position adjacent to the future olfactory bulb. However, (2000). 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