Developmental Cell

Short Article

Time of Exposure to BMP Signals Plays a Key Rolein the Specification of the Olfactoryand Lens Placodes Ex VivoMy Sjodal,1 Thomas Edlund,1 and Lena Gunhaga1,*1 Umea Center for Molecular Medicine, Building 6M, 4th floor, Umea University, S-901 87 Umea, Sweden

*Correspondence: Lena.Gunhaga@ucmm.umu.se

DOI 10.1016/j.devcel.2007.04.020

SUMMARY

Spatial gradients of extracellular signals areimplicated in the patterning of many differenttissues. Much less is known, however, abouthow differences in time of exposure of progen-itor cells to patterning signals can influencedifferent cell fates. Bone morphogenetic pro-tein (BMP) signals are known to pattern embry-onic ectoderm. The olfactory and lens placodesare ectodermal structures of the vertebratehead. By using an explant assay of placodalcell differentiation, we now provide evidencethat BMP signals are required and sufficient toinduce olfactory and lens placodal cells fromprogenitor cells located at the anterior neuralplate border. We also provide evidence thattime of exposure of these progenitor cells toBMP signals plays a key role in the differentialspecification of olfactory and lens placodalcells.

INTRODUCTION

BMPs play important roles during patterning of embryonic

ectoderm. In blastula stage chick embryos, BMP signals

promote the generation of epidermal ectoderm at the

expense of neural tissue (Wilson et al., 1997; Wilson and

Edlund, 2001). By neurula stages, BMP signals act on

neural progenitor cells in the caudal spinal cord to pro-

mote the generation of distinct classes of dorsal interneu-

rons (Chizhikov and Millen, 2005). BMP signals are also

required for the induction and patterning of neural crest

cells (Raible, 2006). The olfactory and lens placodes are

ectodermal structures of the vertebrate head. The olfac-

tory placode forms in the vicinity of the anterior forebrain

and will subsequently invaginate into the nasal pit, while

the lens placode forms next to the optic vesicle and differ-

entiates into the lens vesicle. Fate maps of chick embryos

at late gastrula and head-fold stages (stage 4/6) show that

prospective olfactory and lens placodal cells are inter-

mingled in a domain of the anterior border region between

the neural plate and the future epidermis, and, by the neu-

ral fold stage (stage 8), olfactory and lens progenitor cells

Devel

have become spatially separated (Bhattacharyya et al.,

2004; Couly and Le Douarin, 1985). In a stage 17 (E2.5)

chick embryo the olfactory epithelium and the lens are dis-

tinct morphological structures. The role, if any, of BMP

signals in the generation of the olfactory and lens placodes

remains unknown.

To examine whether BMP signals are important for the

generation of olfactory and/or lens placodal cells, we

established assays of placodal cell differentiation in the

chick embryo and analyzed a panel of markers expressed

in differentiated olfactory and lens placodal cells. Here we

show that BMP signals are required and sufficient to

induce olfactory and lens placodal cells from progenitor

cells located at the anterior border region, and that time

of exposure of these progenitor cells to BMP signals plays

a key role in the differential specification of olfactory and

lens placodal cells.

RESULTS

Olfactory and Lens Placodal Cells Are Specified

at the Late Gastrula Stage

In stage 8 chick embryos, prospective placodal cells

express Six1 and Dlx5 (Figure S1A) (Litsiou et al., 2005;

Streit, 2002). By E2.5, cells in the olfactory placode

express Raldh3, a retinoic acid-producing enzyme (Blen-

tic et al., 2003); Dlx3, Dlx5, herein detected by a pan-Dlx

antibody (Pera and Kessel, 1999); Cytokeratin (Keratin);

a nonneural ectodermal marker (Comte et al., 2004); and

a subset of cells express HuC/D, an early marker for post-

mitotic neurons (Fornaro et al., 2003) (Figures 1A and 4A).

Cells in the lens placode express Keratin, L-Maf, which is

an upstream regulator of d-crystallin (Reza and Yasuda,

2004), and lens fiber cells express d-crystallin (Sullivan

et al., 1998) (Figures 1B and 4B). Cells of the epidermal

ectoderm express Keratin (Comte et al., 2004). Forebrain

cells express L5, Sox1, Otx2, and HuC/D, and cells in

the ventral forebrain express, in addition, Nkx2.1 (Fornaro

et al., 2003; Gunhaga et al., 2000; Nordstrom et al., 2006)

(see Figure S2A in the Supplemental Data available with

this article online).

To examine when ectodermal cells become specified as

lens and olfactory placodal cells, guided by fate maps

(Bhattacharyya et al., 2004; Rudnick, 1944), we isolated

ectodermal explants of the anterior border region from

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BMP Signals Induce the Olfactory and Lens Placodes

Figure 1. Olfactory and Lens Placodal Cells Are Specified at the Gastrula Stage

(A and B) Schematic drawing depicting an E2.5 chick embryo. The lines indicate the level of the transverse sections shown in the corresponding

panel. In the olfactory placode, cells express Raldh3 and Keratin, and HuC/D is expressed in postmitotic neurons (A). In the lens placode, cells ex-

press d-crystallin and Keratin (B).

(C) Ectodermal explants were isolated, separated from the mesoderm and cultured in vitro to the developmental equivalent time point of E2.5, before

fixating, freezing, and sectioning.

(D and E) Stage 4 OLP explants (n = 30) (D) and stage 6 OLP explants (n = 25) (E) cultured alone generated a distinct region of Raldh3+, HuC/D+,

and Keratin+ cells, and a separate region of cells expressed d-crystallin and Keratin. Data are represented as mean ± SEM. Scale bar, 100 mm

(D and E).

stage 3, 4, and 6 embryos, denoted olfactory/lens placo-

dal (OLP) explants. The OLP explants were cultured for

42–50 hr, which in intact embryos would correspond to

E2.5, processed, and serially sectioned (Figure 1C). Stage

3 explants generated L5+ neural cells and Keratin+ epider-

mal cells in distinct domains of the explants, but no

142 Developmental Cell 13, 141–149, July 2007 ª2007 Elsevie

Raldh3+ or d-crystallin+ cells (data not shown). In contrast,

and consistent with fate maps, stage 4 and stage 6 OLP

explants generated Raldh3+, HuC/D+, and Keratin+ cells

characteristic of the olfactory placode and d-crystallin+

and Keratin+ cells characteristic of the lens placode in

distinct nonoverlapping regions of the explants (Figures

r Inc.

Developmental Cell

BMP Signals Induce the Olfactory and Lens Placodes

1D and 1E). Thus, cells in the anterior border region are

specified as olfactory and lens placodal cells at stage 4.

BMP Activity Is Required and Sufficient to Induce

Olfactory and Lens Placodal Cells

At gastrula stages, Bmp2 and Bmp4 are expressed in the

ectoderm of the anterior border region (Chapman et al.,

2002), domains where pSmad-1 indicative of activated

BMP signaling also is detected (Faure et al., 2002). To

examine whether BMP signals are required to generate

olfactory and/or lens placodal cells, we cultured stage

4 OLP explants in the presence of Noggin, a known

BMP inhibitor (Lamb et al., 1993), or with soluble domi-

nant-negative BMP receptor 1a/b isoforms (dnBMPR1a/b).

Under these conditions, the generation of Raldh3+, d-

crystallin+, and Keratin+ cells was blocked, and most or

all cells acquired a neural forebrain character (Figure 2B

and 2C; Figure S2C), which is consistent with BMP signals

that block neural fate (Wilson et al., 1997; Wilson and

Edlund, 2001). Moreover, stage 4 OLP explants cultured

for 15 hr, which in intact embryos would correspond to

�stage 8, generated Six1+ and Dlx5+ placodal progenitor

cells, and, under these conditions, Noggin blocked the

generation of Six1+ and Dlx5+ cells (Figures S1B and

S1C). Thus, at stage 4, BMP signals are required for the

specification of placodal progenitor cells, and in the

absence of BMP activity, prospective placodal cells

acquire forebrain character.

To address whether BMP signals are sufficient to

induce olfactory and/or lens placodal character, we

exposed stage 4 prospective forebrain (FB) explants

(Figure 2D; Figure S1D) to BMP4. BMP4 (35 ng/ml)

blocked the generation of neural cells and induced olfac-

tory and lens placodal cells in distinct nonoverlapping

regions of the explants (Figure 2E). By labeling the medial

edge of the explants with DiI crystals to keep track of the

orientation of the explants during culture, the results

showed that olfactory and lens placodal cells were not

induced by BMP4 in any distinct spatial order with respect

to the rostrocaudal and mediolateral axes of the explants

(Figures S3A and S3B). These results excluded the possi-

bility that BMP signals act on prepatterned neural progen-

itor cells, and indicated sorting of olfactory and lens placo-

dal cells within the explants. To provide further evidence

that BMP signals directly induce placodal progenitors,

we exposed stage 4 FB explants to BMP4 (50 ng/ml) for

only 2 hr and 4 hr. After 2 hr of culture, no Six1+ or Dlx5+

cells were detected (data not shown). However, after 4

hr, most cells expressed Six1 and Dlx5, indicating that

placodal progenitor cells are directly induced by BMP

signals and not indirectly by interactions between neural

and epidermal cells (Figures S1E and S1G). After 15 hr of

exposure to BMP4, cells were still Six1+ and Dlx5+, but no

Raldh3+, HuC/D+, or d-crystallin+ cells were detected,

mimicking the temporal generation of placodal progeni-

tors (Figures S1D and S1F, and data not shown). Taken to-

gether, these results provide evidence that BMP signals

are required and sufficient to induce a pool of progenitor

Deve

cells that later differentiate into olfactory and lens placodal

cells.

Levels of BMP Signals Do Not Differentially Induce

Olfactory or Lens Placodal Cells

We next examined whether the differential induction of

olfactory and lens placodal cells depends on levels of

BMP signals by exposing stage 4 FB explants to different

concentrations of BMP4 (10–50 ng/ml). Under these con-

ditions, no significant change of induced olfactory (p =

0.30) or lens (p = 0.32) placodal cells was observed (Fig-

ures S4A and S4B; Figure 2E). However, in agreement

with previous results (Wilson and Hemmati-Brivanlou,

1995; Wilson et al., 2000), high levels of BMP4 (>50 ng/ml)

induced Keratin+ epidermal cells (Figure 2F). Thus, cranial

placodal and epidermal cells are induced at different

levels of BMP signals. In contrast, levels of BMP signals

appear not to affect the differential induction of olfactory

and lens placodal cells.

FGF Signals Prevent Prospective Placodal Cells

from Acquiring Epidermal Character

A recent study has suggested that Fibroblast Growth

Factor (FGF) signals promote the generation of olfactory

placodal cells (Bailey et al., 2006). To examine whether

FGF activity affected the differential specification of olfac-

tory and lens placodal cells, we first cultured stage 4 OLP

explants together with SU5402 (5 mM), an inhibitor of FGF

receptor signaling (Mohammadi et al., 1997). Under these

conditions, the generation of placodal cells was blocked,

and most or all cells expressed Keratin (Figure S4D), indi-

cating that cells have acquired epidermal character.

Second, we exposed stage 4 OLP explants to FGF8

(0.1–1 mg/ml). At 0.1–0.2 mg/ml, FGF8 did not significantly

change the generation of olfactory (p = 0.25) or lens

(p = 0.49) placodal cells (Figures S4E and Figure 2A).

However, at 0.5–1 mg/ml of FGF8, the generation of

placodal cells was blocked, and L5+ and HuC/D+ neural

cells were induced (Figure S4F). Consistently, in the pres-

ence of FGF8 (0.5–1 mg/ml), stage 4 FB explants still

generated cells of forebrain character (Figures S1H and

S4C). Together, these results provide evidence that at

stage 4, FGF activity prevents prospective placodal cells

from acquiring epidermal fate but do not contribute

to the differential specification of olfactory and lens placo-

dal cells.

Continued Exposure to BMP Signals Promotes

the Generation of Lens Cells while Inhibiting

Olfactory Placodal Cells

Since levels of BMP signals did not promote the differen-

tial specification of olfactory and lens placodal cells, we

examined whether time of exposure to BMP signals could

play a role. To address this issue, we first cultured stage 4

OLP explants alone and FB explants together with BMP4

(35 ng/ml) for 12–15 hr to generate placodal progenitors.

Thereafter, we blocked BMP activity by adding Noggin

or exposed the cells to additional BMP4 (35 ng/ml) and

cultured the explants for 30 hr. Inhibition of BMP activity

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Developmental Cell

BMP Signals Induce the Olfactory and Lens Placodes

Figure 2. BMP Signals Are Required and Sufficient to Induce the Olfactory and Lens Placodes

(A) Stage 4 OLP explants (n = 30) cultured alone generated a region of olfactory placodal cells and a separate region of lens cells.

(B and C) In stage 4 OLP explants cultured together with Noggin (n = 30) (B) or BMPR1a/b (n = 15) (C), the generation of placodal cells was blocked,

and L5+, HuC/D+, and Nkx2.1+ neural cells were generated.

(D) Stage 4 FB explants (n = 30) cultured alone generated L5+, HuC/D+, and Nkx2.1+ neural cells.

(E) In stage 4 FB explants (n = 30), BMP4 (35 ng/ml) blocked the generation of neural cells and induced a distinct region of olfactory placodal cells and

a separate region of lens cells.

(F) Stage 4 FB explants (n = 25) cultured together with BMP4 (100 ng/ml) generated epidermal cells. Data are represented as mean ± SEM. Scale bar,

100 mm.

after 12–15 hr blocked the generation of lens cells, and

most cells acquired olfactory placodal character (Figures

3A and 3B). Thus, the generation of lens cells, but not

olfactory placodal cells, requires prolonged exposure to

BMP signals, and when BMP signals are blocked after

144 Developmental Cell 13, 141–149, July 2007 ª2007 Elsevie

12–15 hr, prospective lens cells acquire olfactory placodal

character. Conversely, exposure to BMP activity after 12–

15 hr blocked the generation of olfactory placodal cells

and most cells acquired lens character (Figures 3C and

3D), indicating that BMP signals promote the generation

r Inc.

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BMP Signals Induce the Olfactory and Lens Placodes

Figure 3. Time of Exposure to BMP Signals Controls the Generation of the Olfactory and Lens Placodes

(A and B) In stage 4 OLP explants (n = 15) (A), and stage 4 FB explants (n = 18) exposed to BMP4 (B) cultured for 12–15 hr, exposure of Noggin blocked

the generation of lens cells, and olfactory placodal cells were generated.

(C and D) In stage 4 OLP explants (n = 15) (C), and stage 4 FB explants (n = 18) exposed to BMP4 (D) cultured for 12–15 hr, addition of BMP blocked the

generation of olfactory placodal cells, and lens cells were generated. Data are represented as mean ± SEM. Scale bar, 100 mm.

(B, D) The statistical significance is calculated versus FB + BMP4 (35 ng/ml).

of lens cells while inhibiting olfactory placodal cells. No

significant differences in cell proliferation or apoptosis

were detected in control explants compared to explants

exposed to Noggin or BMP4 (Figure S5). Taken together,

these results provide evidence that time of exposure in

combination with levels of BMP signals mediate the differ-

ential specification of olfactory and lens placodal cells

from prospective placodal cells.

At the Neural Fold Stage, BMP Signals Induce Lens

Cells at the Expense of Olfactory Placodal Cells

At stage 8, olfactory and lens placodal progenitor cells are

spatially separated, which prompted us to directly test

whether exposure of placodal progenitors to BMP activity

beyond stage 8 affects the differential specification of lens

and olfactory placodal cells. To address this issue, we cul-

tured stage 8 prospective olfactory placode (OP) and lens

Deve

placode (LP) explants alone or in the presence of Noggin

or BMP4. Noggin blocked the generation of L-Maf+ and

d-crystallin+ lens cells in stage 8 LP explants, and under

these conditions, Raldh3+, Dlx+, HuC/D+, and Keratin+

cells characteristic of the olfactory placode were gener-

ated (Figures 4C and 4D). In contrast, stage 8 OP explants

cultured in the presence of Noggin still generated olfactory

placodal cells (Figures 4E and 4F). Moreover, BMP4 (35

ng/ml) induced L-Maf+ and d-crystallin+ lens cells at the

expense of olfactory placodal cells in stage 8 OP explants

(Figure 4G). Together, these results suggest that by stage

8, the generation of lens but not olfactory placodal cells re-

quires prolonged exposure to BMP signals. In the absence

of BMP activity, prospective lens cells acquire olfactory

placodal character, whereas exposure of placodal pro-

genitors to BMP activity inhibits olfactory placodal fate

and promotes the generation of lens cells. In agreement

lopmental Cell 13, 141–149, July 2007 ª2007 Elsevier Inc. 145

Developmental Cell

BMP Signals Induce the Olfactory and Lens Placodes

Figure 4. Time of Exposure to BMP Signals Promotes the Generation of Lens Cells at the Expense of Olfactory Placodal Cells

(A) The pan-Dlx antibody detects cells in the olfactory placode.

(B) Cells in the lens express L-Maf.

(C) Stage 8 LP explants (n = 25) cultured alone generated L-Maf+, d-crystallin+, and Keratin+ cells.

(D) In stage 8 LP explants (n = 20) cultured together with Noggin, the generation of lens cells was blocked, while Raldh3+, Dlx+, and HuC/D+/Keratin+

cells were increased.

(E and F) Stage 8 OP explants cultured alone (n = 25) (E) or together with Noggin (n = 20) (F) generated Raldh3+, Dlx+, HuC/D+, and Keratin+ cells.

(G) In stage 8 OP explants (n = 20) cultured together with BMP4 (35 ng/ml), the generation of olfactory placodal cells was blocked, while L-Maf+ and

d-crystallin+ cells were increased.

(E–G) Most stage 8 OP explants also generated L5+, HuC/D+, and Nkx2.1+ cells in a distinct region of the explants, however, only Keratin+ nonneural

cells were included in the statistical analysis. Data are represented as mean ± SEM. Scale bar, 100 mm.

with these results, at stage 8/9 Bmp4 and pSmad1/5/8 are

preferentially expressed in the prospective lens ectoderm

compared to the prospective olfactory placodal region

(Figures S6A and S6B). Thus, time of exposure to BMP

signals appears to play a major role in the differential

specification of olfactory and lens placodal cells.

Finally, we tested the differential requirement for BMP

signals in intact embryos by electroporating stage 8 chick

embryos in ovo in the anterior neural fold region, including

the prospective olfactory and lens placodes to transfer

i/ a control vector ii/ a Noggin vector (Timmer et al., 2002)

iii/ a constitutively active BMP receptor, Alk6, vector

(James and Schultheiss, 2005) and cultured to approxi-

mately E2.5. Control embryos developed normally (n =

10) (Figures S7A and S8A). All Noggin-transfected

146 Developmental Cell 13, 141–149, July 2007 ª2007 Elsevier

embryos (n = 11) lacked a lens, and no d-crystallin expres-

sion could be detected on the electroporated side

(Figure S7B), whereas the olfactory placode appeared

normal (Figure S7C). In addition, in embryos cultured to

approximately E2 (n = 3), no L-Maf+ lens placodal cells

were generated on the Noggin electroporated side (data

not shown). In all Alk6-transfected embryos (n = 4), no

Raldh3 and greatly reduced Dlx expression could be

detected in the olfactory placodal region on the electro-

porated side (Figure S8B), whereas the lens placode

appeared normal (data not shown). Thus, these results

support the idea that around neural fold stages, BMP sig-

nals are required for the generation of the lens but not the

olfactory placode, and that olfactory placodal character is

inhibited by exposure to BMP signals.

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Developmental Cell

BMP Signals Induce the Olfactory and Lens Placodes

DISCUSSION

When and how cells in the anterior border region become

specified as olfactory and lens placodal cells has

remained poorly understood. In this study, we provide

evidence that cells become specified as olfactory and

lens placodal cells at the late gastrula stage. We also pro-

vide evidence that BMP signals are required and sufficient

to induce both olfactory and lens placodal cells, and that

time of exposure to BMP signals plays a key role in the

differential specification of these two cranial placodes.

Using markers of both differentiated lens and olfactory

placodal cells on serially sectioned explants, we provide

evidence that in stage 4 and 6 embryos, cells located at

the anterior border region are specified as both olfactory

and lens placodal cells, which is consistent with estab-

lished fate maps (Bhattacharyya et al., 2004; Rudnick,

1944). In contrast, it was recently suggested that lens

specification is the ground state of all sensory placodes

(Bailey et al., 2006). However, in this study, prospective

olfactory/lens explants isolated from stage 6 chick embryos

were cultured for 38–42 hr and expression of only lens but

no olfactory placodal markers was monitored by whole

mount in situ hybridization (Bailey et al., 2006). Thus, the

use of only lens markers and of whole mount analysis

may have prohibited the detection of both lens and olfac-

tory placodal cells in individual explants.

The molecular mechanism by which cells in the anterior

border region become specified as olfactory and lens

placodal cells has also remained unresolved. Bailey et al.

(2006) proposed that FGF signaling represses lens spe-

cification and induces olfactory fate. However, in their

study, inhibition of FGF signaling only reduced the number

of olfactory placodal cells but did not convert prospective

olfactory placodal cells to lens cells, and FGF8 did not

induce olfactory character in prospective lens cells. Our

results are consistent with these observations: stage 8

lens progenitor cells still generated L-Maf+ and d-crystal-

lin+ cells when exposed to FGF8 (1 mg/ml), and inhibition

of FGF signaling did not induce lens cells in stage 8 pro-

spective olfactory cells (data not shown). In addition, our

results provide evidence that during the initial specifica-

tion of cranial placodal cells, at the late gastrula stage,

FGF8 did not induce placodal progenitors or promote

olfactory cells at the expense of lens cells. However, at

this stage, a balance of FGF and BMP signals is required

for the generation of placodal progenitor cells, in part by

preventing the generation of cells of epidermal and neural

character, respectively, which is in agreement with previ-

ous studies (Wilson et al., 2001). Similar results are

observed in Xenopus embryos, where nonneural ectoder-

mal Six1 expression is induced by a combination of FGF8

and low levels of BMP activity, but not by FGF8 alone

(Ahrens and Schlosser, 2005). It remains possible, how-

ever, that at neural fold stages, FGF signaling plays

a role in maintenance and/or proliferation rather than in

specification of olfactory placodal cells.

Previous results in Xenopus, zebrafish, and chick have

suggested that the generation of border region cells,

Deve

which later can generate different placodal cell types

and neural crest cells, requires suppression or intermedi-

ate levels of BMP activity (Raible, 2006; Schlosser, 2006).

It has also been suggested that the generation of placodal

progenitor cells depends on an interaction between epi-

dermal and neural cells (Raible, 2006; Schlosser, 2006).

However, our results indicate that BMP signals can

directly induce Six1+ and Dlx5+ placodal progenitors in

prospective neural cells. Moreover, our results provide

evidence that at stage 4, BMP activity is required and suf-

ficient to induce both olfactory and lens placodal cells, but

that levels of BMP signals do not mediate the differential

induction, implying that other molecular events may dis-

criminate between lens and olfactory placodal progenitors

at this stage. By the neural fold stage, stage 8, the gener-

ation of olfactory placodal cells has become independent

of further exposure to BMP signals, whereas the genera-

tion of lens cells requires continued exposure to BMP

signals. Moreover, at this stage, cells can switch between

an olfactory and lens placodal fate in response to changes

in BMP activity, providing evidence that at stage 8 BMP

signals promote the generation of lens cells at the expense

of olfactory placodal cells. These results indicate that time

of exposure of cranial placodal progenitors to BMP signals

plays a key role in the differential specification of olfactory

and lens placodal cells. In agreement with our results, at

stage 4 pSmad-1/5/8 is detected in a domain where

both prospective olfactory and lens placodal cells are po-

sitioned (Faure et al., 2002), while at stage 8/9 pSmad-1/5/

8 is preferentially detected in the prospective lens ecto-

derm compared to the prospective olfactory placodal

region. Consistently in intact embryos, ectopic BMP activ-

ity blocks the generation of olfactory placodal cells. Con-

versely, blockade of BMP signaling inhibits the generation

of lens cells, but under these conditions no ectopic olfac-

tory placodal cells were generated, which may indicate

that when BMP signaling is suppressed, prospective

lens ectoderm respond to other signals that inhibit the

generation of olfactory placodal cells.

BMP4�/� mouse embryos lack lens placodes, but ex-

pression of Six3 and Pax6 is detected in the prospective

lens ectoderm and the olfactory placode appears normal

(Furuta and Hogan, 1998). These results suggest that pla-

codal progenitor cells are induced in BMP4 null mice,

which may reflect functional redundancy between BMP

family members, as Bmp2 also is expressed in the border

region at the late gastrula stage (Chapman et al., 2002).

These results also indicate that BMP4 activity is required

for the differentiation of lens but not olfactory placodal

cells after the initial specification of placodal progenitors,

which supports our finding that time of exposure of placo-

dal progenitors to BMP activity plays a key role in the

differential specification of olfactory and lens placodal

cells. Sustained BMP signaling also appears to play a sig-

nificant role in the specification of the zebrafish cloaca

by maintaining cloaca progenitor fate (Pyati et al., 2006).

Moreover, previous studies have suggested that the dura-

tion of Sonic hedgehog signaling plays an important role in

the specification of different digits in the posterior half of

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Developmental Cell

BMP Signals Induce the Olfactory and Lens Placodes

the limb (Harfe et al., 2004). Thus, these and our results

support the emerging idea that time of exposure of pro-

genitor cells to patterning signals plays an important role

during cell fate specification in different tissues of verte-

brate embryos.

EXPERIMENTAL PROCEDURES

Isolation and Culture of Tissue Explants

Ectodermal explants of the prospective olfactory/lens placodal region

were isolated from Hamburger and Hamilton (HH) stage 3, 4, and 6

chick embryos, and explants from the prospective forebrain were iso-

lated from stage 4 chick embryos (Bhattacharyya et al., 2004; Gunhaga

et al., 2000; Rudnick, 1944). Prospective lens and olfactory placodal

explants were isolated from stage 8 chick embryos (Bhattacharyya

et al., 2004; Couly and Le Douarin, 1985). The explants were cultured

in vitro in serum-free OPTI-MEM (GIBCO) containing N2 supplement

(Invitrogen) and fibronectin (Sigma).

In Ovo Electroporation

Coinjection of a green fluorescent protein vector (pCaggs-GFP) (An-

dersson et al., 2006) was used to monitor the transfection efficiency.

In Situ Hybridization and Immunohistochemistry

For the use of in situ RNA hybridization and immunohistochemistry,

embryos were fixed in 4% paraformaldehyde (PFA) in phosphate buff-

ered saline (PBS) for 2 hr and explants for 25 min. In situ RNA hybrid-

ization was performed essentially as described (Schaeren-Wiemers

and Gerfin-Moser, 1993).

Statistical Analysis

To quantify the percentage of antigen-expressing cells in each explant,

the explants were sectioned at 10 mm, and the consecutive sections

from the same explants were stained in multiple ways. The antigen-

positive cells were counted and compared with the total number of

cells, determined by counting the number of nuclei using DAPI (Boeh-

ringer Mannheim). The graphs represent mean number ± SEM as a

percentage of total cell number.

Supplemental Data

Supplemental Data include detailed Supplemental Experimental

Procedures, Supplemental References, and eight figures and are

available at http://www.developmentalcell.com/cgi/content/full/13/1/

141/DC1/.

ACKNOWLEDGMENTS

We thank G. Boekhoff-Falk, J. Ericson, R. Harland, T.M. Jessell, M.

Kessel, H. Ohuchi, L. Niswander, J. Piatigorsky, T. Schultheiss, A.

Streit, and C. Tabin for kindly providing us antibodies, plasmids, and

cell lines. Special thanks to M. Marklund for electroporation assis-

tance. We are grateful to members of the T. Edlund laboratory for help-

ful discussions. T.E. is supported by the Swedish Medical Research

Council, the Foundation for Strategic Research, and European Union

research program.

Received: October 19, 2006

Revised: March 15, 2007

Accepted: April 27, 2007

Published: July 2, 2007

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