A novel model to study the dorsolateral migration of melanoblasts

Alice Beauvais-Jouneaua, Patrick Plaa, Florence Bernexb, Sylvie Dufourc, Jean Salameroc,Reinhard FaÈsslerd, Jean-Jacques Panthierb, Jean Paul Thieryc, Lionel Laruea,*

aDevelopmental Genetics of Melanocytes, UMR 146 CNRS-Institut Curie, Bat. 110, 91405 Orsay Cedex, FrancebURA-INRA de GeÂneÂtique MoleÂculaire, Ecole Nationale VeÂteÂrinaire d'Alfort, 7 avenue du GeÂneÂral-de-Gaulle, 94704 Maisons-Alfort Cedex, France

cSubcellular Structure and Cellular Dynamics, UMR 144 CNRS-Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, FrancedDepartment of Experimental Pathology, Lund University Hospital, 22185 Lund, Sweden

Received 1 June 1999; accepted 30 July 1999

Abstract

Melanocytes derived from pluripotent neural crest cells migrate initially in the dorsolateral pathway between the ectoderm and dermo-

myotome. To understand the role of speci®c proteins involved in this cell migration, we looked for a cellular model that mimics the in vivo

behavior of melanoblasts, and that allows functional studies of their migration. We report here that wild-type embryonic stem (ES) cells are

able to follow the ventral and dorsolateral neural crest pathways after being grafted into chicken embryos. By contrast, a mutant ES cell line

de®cient for b1 integrin subunits, proteins involved in cell±extracellular interactions, had a severely impaired migratory behavior. Inter-

estingly, ES cells de®cient for Kit, the tyrosine kinase receptor for the stem cell factor (SCF), behaved similarly to wild-type ES cells. Thus,

grafting mouse ES cells into chicken embryos provides a new cellular system that allows both in vitro and in vivo studies of the molecular

mechanisms controlling dorsolateral migration. q 1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Melanocyte; Pigmented cell; Embryonic stem cell; Embryo; b1 integrin; Kit; Xenograft; Neural crest

1. Introduction

The neural crest of vertebrates is comprised of a transient

population of cells and is localized in the dorsal part of the

closing neural tube. The neural crest cells are morphologi-

cally indistinguishable from each other and are highly plur-

ipotent, capable of giving rise to many cell types (Le

Douarin, 1982; Thiery et al., 1982; Serbedzija et al., 1994;

Anderson, 1997). The cells proliferate extensively and

follow different routes to reach their ®nal destinations. Initi-

ally, truncal neural crest cell derivatives are able to follow

two loosely organized streams of cells. Cells in the ventral

pathway give rise to the neurons and glia of the peripheral

nervous system. In the dorsolateral stream determined

neural crest cells migrate to and subsequently penetrate

the epidermis where they differentiate into melanocytes.

In the mouse, the full population of melanocytes is

derived from only thirty-four founders, as determined by

genetic analysis (Mintz, 1967). The melanoblasts derived

from these founders proliferate actively before and during

their migration. Melanoblasts migrating in the dorsolateral

pathway have been studied using different approaches such

as cultures of cells derived from the neural crest, transplan-

tation of the neural tube from a quail or a mouse embryo into

a chick embryo, and analysis of melanocyte development of

mice carrying a coat color mutation.

In vertebrates, melanoblasts express different markers,

including transcription factors such as Mitf, the micro-

phthalmia-associated transcription factor (Mitf); enzymes

such as dopachrome tautomerase (encoded at the Dct

locus, formerly Trp2/slaty), and cell surface receptors

such as Kit (Kit/W), the receptor for the stem cell factor

(SCF) (Pavan and Tilghman, 1994; Motro et al., 1991; Jack-

son et al., 1992; Nakayama et al., 1998). In the absence of

one of these proteins, or of the ligand for c-kit (Steel), mice

have a partial or total lack of hair pigmentation (Steel et al.,

1992; Murphy et al., 1992; Hodgkinson et al., 1993).

However, none of these genes are expressed at the time of

their determination. In addition, speci®c perturbation of

dorsolateral migration is not easy to achieve in vivo, due

to the poor accessibility of mouse embryos and the absence

of antibodies for many chicken melanoblast proteins.

We looked for a cellular model that can reproduce the in

vivo behavior of melanoblasts, and that would allow func-

Mechanisms of Development 89 (1999) 3±14

0925-4773/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved.

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* Corresponding author. Tel.:133-1-6986-7107; fax:133-1-6907-4525.

E-mail address: lionel.larue@curie.fr (L. Larue)

tional studies of their migration, differentiation, and prolif-

eration. The mouse sarcoma cell line S180 are able to follow

the ventral pathway of neural crest cells after being grafted

into chicken embryos, but S180 cells do not migrate along

the dorsolateral pathway (Erickson et al., 1980). Only S180

cells that ectopically expressed the integrins containing a

b1-chain, a4b1 and a5b1, are able to follow the dorsolat-

eral pathway (Beauvais et al., 1995). However, murine

sarcoma S180 cells do not offer a convenient system since

these cells are not related to neural crest cell derivatives, are

not able to migrate spontaneously in the dorsolateral path-

way, and genes cannot easily be inactivated in these aneu-

ploid cells.

Therefore, we decided to test the migratory behavior of

cells developmentally upstream of the melanocytic lineage.

We chose a cell line that could mimic this migration, murine

embryonic stem (ES) cells. These cells are totipotent and are

known to express a very large spectrum of genes (Keller et

al., 1993). Several studies involving xenografts between

mouse and chicken have shown that the main environmental

cues are conserved between mouse and chick embryos

(Fontaine-Perus et al., 1995, 1997). We thus postulated

that ES cells would be responsive to chicken environmental

factors. In this study, we show that grafted ES cells follow

both the ventral and dorsolateral, neural crest pathways.

Furthermore, mutant ES cell line de®cient for b1-chain of

the ®bronectin receptor had a severely impaired migratory

behavior, whereas Kit-de®cient ES cells behaved similarly

to the wild-type ES cells. ES cells thus offer a new cellular

system for both in vitro and in vivo studies of the molecular

mechanisms controlling dorsolateral migration.

2. Results

2.1. Murine ES cells are able to follow both classical

migration pathways of neural crest cells

ES cells were labeled with a ¯uorescent marker (CFSE)

and grafted into early trunk neural crest cell migratory path-

ways (Fig. 1A,B). The migratory behavior of the cells was

assessed by ®xing the host embryos 18 h after receiving the

graft and then observing them under a confocal scanning

¯uorescence microscope. Murine ES cells were able to

migrate in both the ventral and dorsolateral pathways simul-

taneously (Fig. 1C). In some cases, an integration of the

cells into the ectoderm was observed (data not shown).

This intercalation into the ectoderm have certainly been

stabilized by homophilic interactions mediated by L-

CAM, the chick E-cadherin homologue, present at the cell

surface of ectodermal cells of the host, and E-cadherin,

expressed on the cytoplasmic membrane of ES cells. There-

fore, ES cells seemed to be a suitable model to study neural

crest cell migration and, more speci®cally, dorsolateral

migration.

To further characterize this model, we examined the fate

of the grafted cells in the embryo after a longer period.

CFSE was hardly detectable 42 h after the graft. Thus, the

lacZ marker was used to follow ES cells in the embryo.

Wild-type ES cells were transfected with the p62 plasmid

containing lacZ, selected with G418, and ES cells expres-

sing a high level of b -galactosidase were isolated. As a

second screening, ES cells were in vitro differentiated and

stained with Xgal, and ES cell lines expressing a high

amount of b -galactosidase before and after differentiation

were kept. The resulting ES cells were grafted into chick

embryos and incubated at 388C for 18, 42 and 66 h. lacZ-

expressing ES cells were recovered at all stages in the chick

embryo in the ventral as well as in the dorsolateral pathways

(Fig. 2A±C). These cells migrated farther along the dorso-

lateral pathway. No cell proliferation was seen in this path-

way.

We looked for the ability of grafted genuine ES to differ-

entiate into pigment cell precursors in chick embryos 18 h

after the graft. Mitf, the microphthalmia-associated tran-

scription factor, was used as a marker to evaluate the differ-

entiation stage of the grafted ES cells. In situ hybridization

experiments revealed that the ES cells started to express at a

signi®cant level the Mitf gene in the dorsolateral pathway 18

h after the graft (unpublished results).

2.2. ES cells express several molecular markers of

melanoblasts

To better understand the molecular basis of the migratory

behavior of ES cells, we examined the expression of various

melanoblast markers in non-differentiated ES cell culture.

Mitf RNA was expressed at a very low level in ES cells.

After a long exposure period, Mitf RNA could be detected

by northern blot analysis using polyA mRNA (Fig. 3A), but

the protein could not be detected (Fig. 3D). A 10 nM forsko-

lin treatment has been shown to enhance the expression of

Mitf in melanoma cells and melanocytes (Bertolotto et al.,

1998). This treatment was thus applied to the ES cells, but

we were still unable to detect the Mitf protein. This result

suggested that there is only a small amount of Mitf protein

in these cells, if any. The tyrosine kinase receptor Kit was

expressed in genuine ES cells and was phosphorylated in the

presence of exogenous SCF, its ligand (Fig. 3E). In contrast,

there was no endogenous expression of Steel (Fig. 3B,F).

Finally, expression of the genes encoding for the endothelin

receptor type B (Ednrb), the tyrosinase related protein 1

(Tyrp1), the dopachrome tautomerase (Dct), the tyrosinase

(Tyr), and the Slug protein (Slugh) was found by reverse

transcription±polymerase chain reaction (RT±PCR) in ES

cells, while no transcripts corresponding to the gene encod-

ing endothelin 3 (Edn3) or the paired box 3 protein (Pax3)

were detected (data not shown).

2.3. Kit-de®cient ES cells migrate in the dorsolateral

pathway

Analysis of Kit mutant mice suggested that this tyrosine-

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±144

kinase receptor played a role in migration, survival and/or

proliferation of pigment cell precursors throughout their

development. However, a direct role of the Kit receptor in

early dorsolateral migration was not clearly established. The

KitW-lacZ mutation, which was created by replacing the ®rst

exon of Kit with the Escherichia coli lacZ gene using gene

targeting technology, is a null allele at the Kit locus (Bernex

et al., 1996). To isolate Kit-de®cient ES cell lines, blasto-

cysts, recovered from matings between KitW-lacZ/ 1 mice,

were cultured in vitro. Twelve independent embryo-derived

ES cell lines were derived: 4, 6 and 2 were KitW-lacZ/KitW-lacZ,

KitW-lacZ/ 1 and 1 / 1 , respectively (data not shown). Two

cell lines, KitW-lacZ/KitW-lacZ and KitW-lacZ/ 1 , respectively

(designated Kit2/2 and Kit1/2 hereafter), were chosen for

the following experiments. Kit2/2 and Kit1/2 ES cells were

labeled with CSFE and grafted into chick embryos. Eigh-

teen hours later, we examined the ¯uorescence. Kit2/2 and

Kit1/2 ES cells were also revealed by histochemistry after

staining for b-galactosidase (b -gal) activity (Fig. 4A,B,

data not shown). The distribution and number of CSFE-

and b-gal-positive cells were identical in embryos that

were grafted either with Kit2/2 or Kit1/2 ES cells. No differ-

ence was found between the Kit2/2 or Kit1/2 ES cells and

the Kit1/1 ES cells. These results suggest that the Kit recep-

tor is not necessary for the initiation of dorsolateral migra-

tion. To further study the role of Kit, we directly visualized

the b -gal activity of Kit2/2 ES cells 18, 42 and 66 h after the

embryos had been grafted (Fig. 2D±F). The number of Kit2/

2 ES cells was identical to the wild-type ES cells. Further-

more, the Kit2/2 ES cells had migrated farther along the

dorsolateral pathway. As quoted with the wild-type ES

cells, no obvious cell proliferation was observed.

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±14 5

Fig. 1. Murine ES cells are able to migrate in the ventral and dorsolateral pathways of neural crest cells when grafted into the chicken embryo. (A) Scheme of a

22-somite-old chicken embryo (E2.5). The horizontal arrow indicates the level at which the graft was performed. (B) Schematic view of a transverse section. A

small aggregate of cells (g) is inserted between the neural tube and the somite. The migratory pathways for neural crest cells: one goes ventrally (1); the other

one goes dorsolaterally (2). (C) Cells were labeled with a green ¯uorescent marker (CFSE) prior to the graft. Embryos were ®xed 18 h after receiving the graft,

immunostained with an anti-laminin antibody that recognizes the basal lamina of epithelia of the embryo (red), and analyzed by optical scanning using a

confocal scanning ¯uorescence microscope. OmA, omphalo-mesenteric artery; S, somite; He, heart; NT, neural tube; No, notochord; Dm, dermomyotome.

Scale bar: 15 mm.

2.4. b1 integrin-de®cient ES cells cannot ef®ciently migrate

in the dorsolateral pathway of neural crest cells

Cell motility is controlled by the receptors that mediate

cell-substrate adhesion. Integrins are the main family of

extracellular matrix receptors (Hynes, 1992). As many as

11 integrin subunits associate with the b1 integrin subunit.

In vivo, b1 integrin is expressed in migrating neural crest

cells (Duband et al., 1986). We have shown previously that

a4b1 or a5b1 are important for dorsolateral migration

(Beauvais et al., 1995). ES cells express a5b1, a3b1,

a6b1 and avb1 integrin subunits (FaÈssler et al., 1995;

Coppolino et al., 1997). To further examine the role of b1

integrins in cell migration, nullizygous (ItgbI2/ItgbI2 desig-

nated hereafter b12=b12 � b12=2) and heterozygous

(b11/2) ES cells were tested for their ability to migrate in

chicken neural crest cell pathways. The b11/2 ES cells (Fig.

5A) behaved similarly to the wild-type cells (Fig. 1C). Posi-

tive grafted embryos were de®ned as those embryos in

which the cells were still present when observed under the

¯uorescent microscope. Surprisingly, the percentage of

positive grafted embryos was greater for the heterozygous

(86%) ES cells than for the nullizygous (60%) ES cells (Fig.

5D). One potential explanation is that nullizygous cells are

less able to integrate into the host tissues and thus graft is

more easily expulsed by the embryo.

In positive grafted embryos, three types of in vivo ES cell

behavior were usually observed: no migration, migration

along the ventral pathway and migration along both the

ventral and dorsolateral pathways. Non-migrating cells

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±146

Fig. 2. ES cells migrate for at least 66 h in the chicken embryo. Dorsal view of the trunk of Xgal-stained embryos, after 18 h (A,D), 42 h (B,E), and 66 h (C,F).

The lacZ-expressing cells are bgeo-transfected wild-type ES cells (A±C) and Kit-de®cient ES cells (D±F). The cells located in the ventral pathway of neural

crest cells are not visible. Vertical white bars indicate the graft site. NT, neural tube.

generally remained as tightly aggregated clusters at the graft

site (Fig. 5B).

The main result was that the ability of b12/2 ES cells to

migrate in the dorsolateral pathway was severely impaired.

Indeed, only 6% of the embryos grafted with nullizygous

cells contained cells migrating in the dorsolateral pathway,

compared to 43% for embryos grafted with heterozygous ES

cells (Fig. 5D). However, b12/2 ES cells were still able to

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±14 7

Fig. 4. Kit2/2 ES cells can migrate in the dorsolateral pathway. Embryos were ®xed 18 h after receiving the graft and analyzed by (A) labeling with CFSE

labeling then visualizing the cells by epi¯uorescence optical sectioning, or (B) by histological sectioning after Xgal staining. NT, neural tube; Dm, dermo-

myotome. Scale bar: 25 mm.

Fig. 3. ES cells expressed several melanoblast markers. The expression of Mitf (A,D), SCF (B,F), Kit (E) and Gapd (C) were followed in ES cells by Northern

blot (A±C) and Western blot (D±F). The ®broblast cell line (NIH-3T3, 3T3) and the melanoblast cell line (melb-a), were used as negative and positive controls.

The blots were loaded either with 20 mg of total RNA or with about 8 mg of polyA RNA. PolyA RNA was used when the signal was not detectable on total RNA

and is indicated with an asterisk located on the top right of the band. The size of Mitf, Kit and SCF mRNAs are 5.5, 8.2 and 6.5 kb, respectively. The molecular

weights of Mitf and Kit proteins are 55±62 kDa and 145 kDa, respectively. Kit2/2 ES cells were used as a negative control. The phosphorylation of Kit was

followed (E, lane c) after immunoprecipitation of this protein using anti Kit antibodies, and incubation of the membrane with anti-phosphotyrosine 4G10. SCF

proteins have molecular weights of 28 and 45 kDa. COS-KLM1 10£ and 1£ correspond to cell extracts (2 different loading volumes) derived from COS cells

expressing the transmembrane form of Steel (45 kDa). COS-KLS corresponds to the supernatant of COS cells expressing a soluble form of SCF (28 kDa).

These two proteins are indicated by the two arrowheads. Melb-a cells were used as a negative control.

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±148

Fig. 5. b1 integrin-de®cient ES cells cannot ef®ciently migrate in the dorsolateral pathway. Embryos were ®xed 18 h after receiving the graft and analyzed by

epi¯uorescence optical sectioning. (A) Heterozygous (b11/2) ES cells migrating in the dorsolateral and ventral pathways of the neural crest cells, (B)

Aggregate of homozygous (b12/2) ES cells that do not migrate in the chicken embryo. (C) Homozygous (b12/2) ES cells migrating in the ventral pathway.

(D) The diameter of the pie charts represents the percentage of positive grafted embryos. The number of positive grafted embryos was lower for b12/2 ES cells

than for b11/2 ES cells. The embryos were classi®ed as followed: embryos with no migrating cells (pink areas), embryos containing cells migrating in the

ventral and dorsolateral pathway (green areas) and embryos containing cells migrating solely in the ventral pathway (purple areas). NT, neural tube; No,

notochord; D, dermomyotome Ec, ectoderm. Scale bar: 25 mm.

migrate ventrally in vivo, although with a lower ef®ciency

than the heterozygous ES cells (Fig. 5C). Two parameters

explain this lower ef®ciency. First, 53% of positive grafted

embryos grafted with nullizygous ES cells contained non-

migrating cells, compared to 35% for the grafts of hetero-

zygous cells (Fig. 5D). Second, although the number of

grafted cells were similar, the average number of migrating

cells in embryos grafted with b11/2 ES cells was about 8

compared to 3 for the b12/2 ES cells.

In conclusion, the behavior of b12/2 integrin and Kit2/2

ES cells presented above illustrates the interest of the

system for studying the mechanisms controlling the migra-

tion of neural crest cells.

2.5. b l integrin-de®cient ES cells bind to ®bronectin

through RGD-dependent integrins

Fibronectin is the major component of the extracellular

matrix in the migration pathways of neural crest cells.

Fibronectin binds to the cells through different b1 and

non-b1 integrins. The non-b1 integrins are av-containing

integrins such as avb3, avb5, and avb6. To further under-

stand the differential migration requirement of b1 integrins

for the ventral and dorsolateral pathway, we performed

adhesion assays with ®bronectin. The same number of

b11/2 and b12/2 ES cells were incubated on 10 and 50

mg/ml ®bronectin (Fig. 6 and data not shown, respectively).

b 12/2 ES cells were able to interact with ®bronectin (Fig.

6D,E), though to a lesser extent than b11/2 ES cells (20%

reduction, Fig. 6A,B). In addition, b12/2 ES cells were

much less spread, and thus appeared smaller than

b11/2 ES ES cells (Fig. 6B,E).

In the presence of RGDS peptides which compete for

binding to the RGD site normally present on ®bronectin

(Pierschbacher and Ruoslahti, 1984), attachment of b12/2

cells to ®bronectin was nearly completely inhibited (Fig.

6D,F). By contrast, no change in binding ef®ciency was

observed for b11/2 cells (Fig. 6A,C). Fibronectin also

binds to cells through cellular proteoglycans (Woods et

al., 1993). One hundred mg/ml heparin was used to compete

for binding to proteoglycans. Heparin had no effect on the

adhesion of b12/2 ES cells, suggesting that this adhesion

was not mediated only by proteoglycans (data not shown).

Altogether, these experiments suggested that b12/2 cell

adhesion to ®bronectin involved RGD-dependent receptors

such as the avb3 and avb5 integrins. Furthermore, these

integrins were suf®cient to allow ventral migration of b1-

de®cient cells, whereas the dorsolateral migration was more

dependent upon b1 integrins.

3. Discussion

Melanocytes are derived from the neural crest. The

pigment cell precursors follow the dorsolateral pathway

between the ectoderm and the dermomyotome. This path-

way is only taken by cells already determined as melano-

blasts (Mintz, 1967; Erickson and Goins, 1995). Before the

migration, the pigment cell progenitors proliferate and accu-

mulate in a region close to the dorsal neural tube, the migra-

tion staging area (Weston, 1991). The earliest known

murine markers, Mitf (Opdecamp et al., 1997), Dct (Steel

et al., 1992), and Kit (Motro et al., 1991), are expressed in

cells located in this zone. The limited number of cells and

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±14 9

Fig. 6. b1 integrin-de®cient ES cells attach to ®bronectin through RGD-dependent integrins. b11/2 (A±C) and b12/2 (D±F) ES cells were seeded on

®bronectin at a concentration of 10 mg/ml, in the presence (C,F) or absence (A,B,D,E) of 1 mg/ml RGDS peptides. The enlargements are similar in A,C,D and F

(scale bars in D,F: 100 mm), and in B and E (scale bar in E: 15 mm).

the absence of known markers make it dif®cult to study the

melanocyte founders, and the molecular mechanisms under-

lying their determination, proliferation, migration, and

differentiation are still poorly understood. In the present

study, an indirect approach has been adopted, using cultured

cells grafted into the chicken neural crest cell migratory

pathways, to better understand these fundamental cellular

mechanisms.

Sarcoma S180 cells, NIH-3T3 ®broblasts, and cells

derived from the somite or the dorsal root ganglia, either

do not migrate or migrate only ventrally but not in the

dorsolateral pathway (Erickson et al., 1980). To identify

cells that could migrate in the dorsolateral pathway, the in

vivo behavior of several cell lines were tested. In a ®rst

attempt, we grafted a skin melanoblast cell line derived

from mouse newborn skin, the melb-a cells (Sviderskaya

et al., 1995). The cells migrated only ventrally, showing

that during their development they lost the transient ability

of the melanoblast precursors to migrate in the dorsolateral

pathway (data not shown). The migratory behavior

displayed by ES cells appears to be unique among the differ-

ent cells tested. Wild-type ES cells were able to migrate

simultaneously in the ventral and the dorsolateral pathways

for at least 66 h. The activation of the bHLH transcription

factor Mitf suggested that the grafted wild-type ES cells

were differentiating in the chicken embryo.

Mutations in the genes encoding Mitf, Kit or Dct are

responsible for complete or partial lack of skin and hair

melanocytes. While Kit is expressed at a high level in ES

cells, Mitf and Dct are expressed at very low levels. Never-

theless, grafted ES cells migrate along the dorsolateral path-

way. This suggests that expression of Mitf and Dct is not

crucial at least for the onset of dorsolateral migration. This

conclusion agrees with previous data showing that Kit-posi-

tive pigment cell precursors are seen along the dorsolateral

migration pathway in Mitf-de®cient mice (Opdecamp et al.,

1997). It has been suggested that Mitf is responsible for an

increase of Kit expression in pigment cell precursors while

they migrate; this increase would stimulate their prolifera-

tion. If this hypothesis is correct, the lack of proliferation of

the grafted ES cells in the chick embryo could result from

the low level of Mitf expression in the ES cells. Alterna-

tively, the phosphorylated status of Mitf may not be appro-

priate in ES cells. The use of ES cells carrying an

overexpressed Mitf allele could possibly lead to the prolif-

eration of grafted ES cells in the chick embryo. This hypoth-

esis is currently under investigation.

SCF, the Kit ligand, is not expressed in ES cells, thereby

eliminating the possibility of an autocrine loop. In the

chicken embryo, the dorsolateral migration is delayed 18

h compared to the ventral migration (Erickson et al.,

1992). The onset of endogenous Steel expression in the

ectoderm occurs concomitant to the dorsolateral migration

of endogenous crest cells (Lecoin et al., 1995). However, it

has been suggested that neural crest cells themselves could

transiently synthesize SCF gene during their stay in the

migration staging area (Guo et al., 1997). Therefore, it is

not clear if SCF is available for ES cells at the time of their

initial migration. Moreover, whether the chicken SCF is

able to induce a identical response following its interaction

with a murine Kit receptor is unknown. Anyhow, the beha-

vior of Kit-de®cient cells suggests that Kit is not necessary

for the migration of neural crest cell at this stage. Thus, ES

cells may already possess the correct set of molecules, or at

least an acceptable combination of proteins, required to

migrate in the ventral and dorsolateral pathways.

b1 integrin is expressed in vivo in migrating neural crest

cells (Duband et al., 1986; Krotoski et al., 1986). In our

study, the behavior of b1 integrin-de®cient ES cells showed

that b1 integrins are differentially involved in ventral and

dorsolateral migration. Although their motility was reduced,

b l2/2 ES cells were still able to migrate in the ventral path-

way and this migration was qualitatively not different from

that of b1-expressing cells. In contrast, the dorsolateral

migration was severely impaired. This is in agreement

with previous results from chimeras obtained from normal

mouse blastocysts injected with b l2/2 ES cells. Some b l2/2

cells were found in ventral derivatives of neural crest cells,

such as the peripheral nervous system and the adrenal

medulla (FaÈssler and Meyer, 1995). b12/2 ES cells were

originally derived from Tyr1/Tyr1 mice. No pigmented

melanocytes derived from b l2/2 ES cells were found once

injected into albino blastocysts (unpublished result). Alto-

gether, this indicates that b1 integrin is required for the

melanocyte lineage. b1 integrins bind to different matrix

proteins, such as ®bronectin, laminin, collagen and vitronec-

tin. All these substrates are present along the crest migratory

pathways (Krotoski et al., 1986; Perris et al., 1993). b1-

de®cient ES cells could still attach to ®bronectin, though

less ef®ciently than b1 expressing cells. This attachment

was almost completely dependent on binding to the RGD

site of ®bronectin. a3b1, a5b1 and different av-containing

integrins (avb1, avb3, avb5, avb6) bind to this RGD

site. However, a5b1 binding also requires a synergistic

site located nearby (Akiyama et al., 1995). In addition, it

was recently shown that a5b1 could bind to another site

located in the amino-terminal of ®brin domains (Hocking et

al., 1998). This could explain the difference between the

b11/2 and b12/2 cells in the inhibitory action of the RGD

peptide.

Our experiments show that the integrins required for the

ventral and dorsolateral migration are different. The migra-

tion into the ventral pathway can be mediated by non-b1

integrin, such as av containing integrins, and the migration

into the dorsolateral pathway requires b1 integrins. Among

these integrins, a5b1 is probably important, as suggested

previously (Beauvais et al., 1995). Until now, no obvious

differences have been observed in the distribution of matrix

proteins along the two pathways. However, local variations

in the orientation of the ®brils and/or concentrations of the

components may not have been detected. Moreover, in addi-

tion to their adhesion properties, integrins cooperate with

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±1410

growth factors to regulate cell growth, and with cadherins to

modulate cell adhesion properties.

The usefulness of combining murine ES cells with avian

neural crest cells in vivo offers an attractive system because

transformed ES cells carrying subtle mutations may be

obtained. Indeed, several hundreds of murine ES cell lines

heterozygous for null-mutations are available. If the corre-

sponding homozygous ES cells is not available, the deriva-

tion of ES cell lines homozygous for the mutant allele is

obtained using either the heterozygous ES cell line or homo-

zygous blastocysts as starting point material. Here, we illu-

strated this strategy by analyzing the behavior of Kit- and

b1 integrin-de®cient ES cells in chick embryos. Our analy-

sis brought new insights into the role of Kit and b1 integrin

in neural crest cells migration. Although, we used ES cells

carrying loss-of-function mutations, the analysis of ES cells

carrying gain-of-function mutations should be possible. In

particular, ES cells overexpressing various proteins possibly

involved in the migration of neural crest cells should be

performed. One can even think of grafting gene-trapped

ES cells as a screening method.

To our knowledge, we have provided the ®rst information

about the speci®city of the dorsolateral pathway, and its

restrictive access. The fate of the grafted cells has been

examined after 2 and 3 days in the chicken embryo. The

number of cells did not increased, although cells, found

mainly in the dorsolateral pathway, were able to migrate

and differentiate further. Therefore, we have encountered

the present limit of the model. This novel way of using

wild-type and mutant ES cells has already proven to be

useful, and further studies will bring new information

about the role of various proteins involved in the neural

crest cell lineages. At the very least, these experiments

shed some light on the molecular mechanisms underlying

the dorsolateral migration of melanoblasts.

4. Experimental procedures

4.1. Antibodies and reagents

The CS hybridoma supernatant against Mitf was a

gift from D.E. Fisher (Harvard Medical School, Boston,

MA; Weilbaecher et al., 1998). C19 polyclonal antibodies

against Kit were purchased from Santa-Cruz (cat. no. SC-

168). Rabbit antiserum against SCF, and supernatants

and cell extracts of SCF-expressing COS cells were kindly

provided by B. Wehrle-Haller (Centre Medical Universi-

taire, Geneva). Rabbit antiserum against laminin (AndreÂ

et al., 1994) was a gift from H. Feracci (CNRS-Institut

Curie, Paris, France). RGDS peptides (Sigma, A9041) and

heparin (Laboratoire Choay, Paris, France) were used

according to the manufacturers' recommendations. CFSE

is a ¯uorescent dye that was purchased from Molecular

Probes.

4.2. Cell culture and cell lines

G119 and G201 ES cell clones are heterozygous and

homozygous for the b1 integrin null allele, respectively.

They were previously generated by transfection of D3 ES

cells with a gene trap type vector containing a bgeo (lacZ-

neo) fusion gene to abolish the expression of b1 integrin

(FaÈssler and Meyer, 1995; Doetschman et al., 1985).

KitW-lacZ/KitW-lacZ, KitW-lacZ/1 ES cells were derived from

blastocysts according to standard procedures (Robertson,

1987; Bernex et al., 1996). Resulting KitW-lacZ/KitW-lacZ,

KitW-lacZ/1 and 1/1 ES clones were tested for the presence

of the lacZ gene by PCR using speci®c oligonucleotides

(Hanley and Merlie, 1991). The presence of the ®rst exon

of Kit (De Sepulveda et al., 1995; GenBank no. X86451),

which is missing in the KitW-lacZ allele, was detected by

ampli®cation of a 159-bp fragment using two primers

(5 0TCC TGT TGG TCC TGC TCC3 0 and 5 0CCA CCT

TCG AGG TGG TAG G3 0, respectively). ES cells expres-

sing constitutively b -galactosidase were generated by elec-

troporation of the 62 plasmid containing the bgeo chimeric

(b -galactosidase±neomycin) gene under the control of the

PGK promoter (Larue et al., 1996). Various clones were

produced. The clone used in the present study expressed a

high amount of b -galactosidase and continued to express

uniformly this marker after the differentiation of the ES

cells.

The various ES cells were grown on gelatin in high-

glucose DMEM supplemented with 15% heat inactivated

FCS, 2 mM glutamine (GIBCO-BRL, Gaithersburg, MD),

2000 U/ml mouse LIF (GIBCO-BRL), and 0.1 mM b -

mercaptoethanol (Sigma) in the presence of penicillin and

streptomycin.

The melb-a cell line was kindly provided by D. Bennett

(St. George's Hospital Medical School, London, UK). This

cell line was derived from melanoblasts isolated from

newborn mouse skin (Sviderskaya et al., 1995). The melb-

a cells were grown in RPMI supplemented with 10% FCS,

20 ng/ml SCF (Serotec, UK), and 40 pM basic FGF (a gift

from H. Prats, Toulouse, France). NIH-3T3 cells were clas-

sically grown in DMEM medium containing 5% heat-inac-

tivated FCS in the presence of penicillin and streptomycin.

4.3. Northern blot

Total RNA was prepared from cell cultures using the

Roti-Quick RNA puri®cation kit (Roth). PolyA mRNA

was isolated with the PolyATract mRNA isolation kit

(Promega). Twenty mg of total RNA (melb-a and NIH-

3T3 cells) or polyA RNA puri®ed from 800 mg of total

RNA (ES cells) was separated on a 1% agarose formalde-

hyde gel under denaturing conditions, transferred onto a

nylon membrane (Hybond-N1, Amersham) and hybridized

with a 32P-labeled probe (Maniatis et al., 1982). The blots

were hybridized with a speci®c probe for Mitf (nucleotides

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±14 11

84±1278), SCF (nucleotides 1±823), or GAPDH (exons 5-

8).

4.4. Western blot

To prepare total cell extracts, ES cells and melb-a cells

were directly lysed on a Petri dish with RIPA buffer (10 mM

Tris pH 7.5, 1% Na-deoxycholate, 1% NP-40, 150 mM

NaCl, 0.1% SDS, 1 mg/ml leupeptin, 100 IU aprotinin, 1

mM NaVO4). The lysates were centrifuged for 5 min at

14 000 rpm and the supernatants were mixed with 1/5

volume of 5 £ Laemmli buffer. Proteins were separated on

a 10% SDS±polyacrylamide gel and electrotransferred to a

nitrocellulose membrane. The membrane was then incu-

bated in blocking buffer (5% low fat milk, 0.25% Tween-

20 in Tris-buffered saline). The Mitf, Kit and SCF proteins

were detected with the CS monoclonal antibody (1/10 dilu-

tion in blocking buffer), C19 polyclonal antibodies (2 mg/ml

diluted in blocking buffer) and a rabbit antiserum directed

against SCF (1/100 dilution in blocking buffer), respec-

tively. These proteins were visualized using secondary anti-

bodies coupled to peroxidase and the enhanced

chemiluminescence system (ECL) from Amersham.

4.5. Fluorescence labeling of cells

On day 0, the cells were trypsinized and 5 £ 105 ES cells

were plated on a Petri dish of 60 mm diameter. On day 1, the

cells were detached from the plastic with a PBS solution

containing 0.25% trypsin and 1 mM EDTA (GIBCO-BRL),

rinsed in ES cell culture medium and centrifuged for 5 min

at 130 £ g. The cell pellet was resuspended at a density of

106 cells/ml in ES cell culture medium containing 1 m1

CFSE (10 mM stock solution in DMSO; Molecular Probes)

and incubated for 45 min at 378C. The cells were then

centrifuged for 5 min at 130 £ g, resuspended in 1 ml

fresh culture medium and kept at 378C under 10% CO2 in

a humid atmosphere. During the grafting experiments, 50-

ml aliquots of the cell suspension were transferred to bacter-

iological Petri dishes (diameter 35 mm, coated with 3 mg/ml

BSA in PBS for 30 min at room temperature) containing 1.5

ml DMEM equilibrated at 378C. The cells were stained with

10 ml of 0.2% (w/v) Neutral Red (Sigma). The survival of

the cells was routinely checked by taking samples from the

bacteriological Petri dishes and plating them on a tissue

culture dish containing culture medium. Cells were able to

attach to the plastic and grew with normal ef®ciency. The

viability of the cells was monitored for a couple of days and

under these conditions, no toxicity of the CFSE-stained cells

was observed.

4.6. Grafting experiments

The grafting experiments were performed essentially as

previously described (Beauvais et al., 1995), except that in

this case the grafts were not only performed in vitro but also

in ovo. Brie¯y, chicken embryos were incubated until they

had developed 20±25 somite pairs. For in ovo experiments,

1 to 2 ml of albumin was aspirated through the shell with a

syringe (gauge 25) and a window of about 2 cm in diameter

was created above the blastoderm. The blastoderm was

stained with a drop of Neutral Red and the vitelline

membrane on the top of the embryo was opened using tung-

sten needles. Aggregates of approximately 20±40 ES cells

were pipetted from the Petri dish onto the embryo and

gently inserted between a somite and the neural tube

through a slit in the ectoderm. The graft was made at the

axial level where neural crest cells had just migrated off the

dorsal surface of the neural tube (i.e. the ®fth last somite

pairs). After grafting, the eggs were sealed with tape and

incubated at 388C for about 18, 42 or 66 h. At 18 h, no

difference was observed between in vitro or in ovo graftings.

4.7. Analysis of grafted embryos: confocal scanning

¯uorescence microscopy

For confocal scanning ¯uorescence analysis, grafted

embryos were dissected and those containing ¯uorescent

cells were selected using an epi¯uorescence microscope.

Embryo pieces around the graft site were ®xed for 1 h in

methanol at 2208C, 1 h in methanol at 48C and rehydrated.

The pieces were rinsed three times in PBS, incubated over-

night at 48C in PBS containing 5% FCS, then for 5 h at 48Cin the presence of antibodies directed against laminin (1:500

dilution in PBS containing 5% FCS). The pieces were

washed overnight in PBS at 48C, incubated for 5 h with

secondary antibodies (1/50 dilution of Texas Red-conju-

gated anti-rabbit Ig (Amersham)) and then washed again

overnight at 48C in PBS. Embryo pieces were shortened

around the graft to reach a length of 2 somites. These pieces

were positioned transversally on slides in a drop of mowiol.

They were optically sectioned transversally with a confocal

scanning ¯uorescence microscope (Leica Lasertechnic,

Heidelberg, Germany). Immuno¯uorescence analysis was

performed using a TCS4D confocal microscope based on

a DM microscope interfaced with an argon/krypton laser.

Simultaneous double ¯uorescence acquisitions were

performed using the 488 nm and the 568 nm laser lines

using a 16£ (numeric aperture � 0:5) or a 40£ (numeric

aperture � 1 oil immersion) objectives. The ¯uorescence

was selected with appropriate double ¯uorescence dichroic

mirror and band pass ®lters and measured with blue-green-

sensitive and red-side-sensitive-one photomultipliers.

Depths up to 160 mm can be scanned in this way, and it is

therefore possible to completely examine the region of the

graft and follow the migration of the grafted cells by optical

slicing. This method allows the rapid screening and analysis

of a large number of embryos.

4.8. Analysis of grafted embryos: histological analysis

Embryos grafted with lacZ-expressing ES cells were

®xed in PBS containing 0.25% glutaraldehyde (Sigma,

cat. no. G5882), at 48C for 1 h, incubated in permeabiliza-

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±1412

tion buffer (2 mM MgCl2, 0.01% Na-deoxycholate, 0.02%

NP-40 in PBS) for 30 min at RT, and then incubated over-

night at 308C with slight agitation in X-gal staining solution

(5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 0.01%

Na-deoxycholate, 0.02% NP-40 in PBS, with 0.04% X-gal).

The embryos were washed three times in PBS, post®xed in

4% paraformaldehyde at 48C for 2 h, washed overnight in

PBS, and then dehydrated in graded alcohol, embedded in

paraplast, and cut into 7-mm sections.

4.9. Adhesion assay

Bacterial dishes were coated with 50-ml drops of 10 or 50

mg/ml bovine ®bronectin diluted in PBS (Gibco) and then

incubated at 378C for 1 h. The plastic was then saturated

with a 3 mg/ml solution of BSA in PBS for 30 min at 378C,

and then rinsed with PBS. The solution of BSA had been

heat-inactivated at 808C for 3 min.

ES cells were harvested with 0.05% trypsin and 0.02%

EDTA in PBS and then incubated in suspension in low-

calcium medium (SMEM with 10% dialyzed FCS

(GIBCO-BRL)) for 1 h at 378C under 10% CO2 to allow

recovery from trypsin treatment and to avoid aggregation.

The number of cells was determined and then centrifuged at

1000 rpm for 5 min. The pellet of cells was resuspended in

DMEM in the presence or absence of RGDS peptides or

heparin. Droplets of 5000 cells suspended in DMEM were

seeded onto the substrates and incubated for 1 h at 378C.

RGDS peptides (1 mg/ml) and heparin (100 mg/ml) were

added to the suspension and immediately deposited on the

substrates. Attached cells were ®xed in 3.7% formaldehyde

in PBS. Three independent experiments were performed and

gave similar results.

Acknowledgements

We wish to thank B. Wehrle-Haller, H. Feracci and D.E.

Fisher for anti-SCF, anti-laminin and anti-Mitf antisera,

respectively. We also thank D. Bennett for the melb-a cell

line and H. Prats for the recombinant basic FGF. We thank

B. Wehrle-Haller for helpful discussions and C. Jeanney for

technical assistance. This work was supported by grants

from Ligue Nationale contre le Cancer and from ARC.

A.J. was supported by a fellowship from Ligue Departemen-

tale contre le Cancer (Essonnes).

References

Akiyama, S.K., Aota, S., Yamada, K.M., 1995. Function and receptor

speci®city of a minimal 20 kilodalton cell adhesive fragment of ®bro-

nectin. Cell Adhes. Commun. 3, 13±25.

Anderson, D.J., 1997. Cellular and molecular biology of neural crest cell

lineage determination. Trends Genet. 13, 276±280.

AndreÂ, F., Filippi, P., Feracci, H., 1994. Merosin is synthesized by thyroid

cells in primary culture irrespective of cellular organization. J. Cell Sci.

107, 183±193.

Beauvais, A., Erickson, C.A., Goins, T., Craig, S.E., Humphries, M.J.,

Thiery, J.P., Dufour, S., 1995. Changes in the ®bronectin-speci®c integ-

rin expression pattern modify the migratory behavior in the embryonic

environment. J. Cell Biol. 128, 699±713.

Bernex, F., De Sepulveda, P., Kress, C., Elbaz, C., Debuis, C., Panthier, J.J.,

1996. Spatial and temporal patterns of c-kit-expressing cells in WlacZ/

1 and WlacZ/WlacZ mouse embryos. Development 122, 3023±3033.

Bertolotto, C., Abbe, P., Hemesath, T.J., Bille, K., Fisher, D.E., Ballotti, R.,

1998. Microphthalmia gene product as a signal transducer in cAMP-

induced differentiation of melanocytes. Mol. Cell Biol. 142, 827±835.

Coppolino, M.G., Woodside, M.J., Demaurex, N., Grinstein, S., St-Arnaud,

R., Dedhar, S., 1997. Calreticulin is essential for integrin-mediated

calcium signalling and cell adhesion. Nature 386, 843±847.

De Sepulveda, P., Salaun, P., Maas, N., Andre, C., Panthier, J.-J., 1995.

SARs do not impair position-dependent expression of a KIT/LACZ

transgene. Biochem. Biophys. Res. Commun. 191, 893±901.

Doetschman, T., Eistetter, H., Katz, M., Schmidt, W., Kemler, R., 1985.

The in vitro development of blastocyst-derived embryonic stem cell

lines: formation of visceral yolk sac, blood islands and myocardium.

J. Embryol. Exp. Morphol. 87, 27±45.

Duband, J.-L., Rocher, S., Chen, W.-T., Yamada, K.M., Thiery, J.-P., 1986.

Cell adhesion and migration in the early vertebrate embryo: Location

and possible role of the putative ®bronectin receptor complex. J. Cell

Biol. 102, 160±178.

Erickson, C.A., Goins, T.L., 1995. Avian neural crest cells can migrate in

the dorsolateral path only if they are speci®ed as melanocytes. Devel-

opment 121, 915±924.

Erickson, C.A., Tosney, K.W., Weston, J.A., 1980. Analysis of migratory

behavior of neural crest and ®broblastic cells in embryonic tissues. Dev.

Biol. 77, 142±156.

Erickson, C.A., Duong, T.D., Tosney, K.W., 1992. Descriptive and experi-

mental analysis of the dispersion of neural crest cells along the dorso-

lateral path and their entry into ectoderm in the chick embryo. Dev.

Biol. 151, 251±272.

FaÈssler, R., Meyer, M., 1995. Consequences of lack of b1 integrin gene

expression in mice. Genes Dev. 9, 1896±1908.

FaÈssler, R., Pfaff, M., Murphy, J., Noegel, A.A., Johansson, S., Timpl, R.,

Albrecht, R., 1995. Lack of b1 integrin gene in embryonic stem cells

affects morphology, adhesion, and migration but not integration into the

inner cell mass of blastocysts. J. Cell Biol. 128, 979±988.

Fontaine-PeÂrus, J., Jarno, V., Fournier Le Ray, C., Li, Z., Paulin, D., 1995.

Mouse chick chimera: a new model to study the in ovo developmental

potentialities of mammalian somites. Development 121, 1705±1718.

Fontaine-PeÂrus, J., Halgand, P., Cheraud, Y., Rouaud, T., Velasco, M.E.,

Diaz, C.C., Rieger, F., 1997. Mouse chick chimera: a developmental

model of murine neurogenic cells. Development 124, 3025±3036.

Guo, C.S., Wehrle-Haller, B., Rossi, J., Ciment, G., 1997. Autocrine regu-

lation of neural-crest cell development by steel factor. Dev. Biol. 184,

61±69.

Hanley, T., Merlie, J.P., 1991. Transgene detection in unpuri®ed mouse tail

DNA by polymerase chain reaction. Biotechniques 10, 56.

Hocking, D.C., Sottile, J., McKeown-Longo, P.J., 1998. Activation of

distinct alpha5beta1-mediated signaling pathways by ®bronectin's

cell adhesion and matrix assembly domains. J. Cell Biol. 141, 241±253.

Hodgkinson, C.A., Moore, K.J., Nakayama, A., Steingrimsson, E., Cope-

land, N.G., Jenkins, N.A., Arnheiter, H., 1993. Mutations at the mouse

microphthalmia locus are associated with defects in a gene encoding a

novel basic-helix-loop-helix-zipper protein. Cell 74, 395±404.

Hynes, R.O., 1992. Integrins: versatility, modulation, and signaling in cell

adhesion. Cell 68, 11±25.

Jackson, I., Chambers, D., Tsukamoto, K., Copeland, N., Gilbert, D.,

Jenkins, N., Hearing, V., 1992. A second tyrosinase-related protein,

TRP-2, maps to and is mutated at the mouse slaty locus. EMBO J.

11, 527±535.

Keller, G., Kennedy, M., Papayannopoulou, T., Wiles, M.V., 1993. Hema-

topoietic commitment during embryonic stem cell differentiation in

culture. Mol. Cell. Biol. 13, 473±486.

Krotoski, D.M., Domingo, C., Bronner-Fraser, M., 1986. Distribution of a

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±14 13

putative cell surface receptor for ®bronectin and laminin in the avian

embryo. J. Cell Biol. 103, 1061±1071.

Larue,L.,Antos,C.,Butz,S.,Huber,O.,Delmas,V.,Dominis,M.,Kemler,R.,

1996. A role for cadherins in tissue formation. Development 122, 3185±

3194.

Le Douarin, N.M., 1982. The Neural Crest, Cambridge University Press,

Cambridge.

Lecoin, L., Lahav, R., Martin, F.H., Teillet, M.A., Le Douarin, N.M., 1995.

Steel and C-Kit in the development of avian melanocytes: a study of

normally pigmented birds and of the hyperpigmented mutant silky fowl.

Dev. Dyn. 203, 106±118.

Maniatis, T., Fritsch, E.F., Sambrook, J., 1982. Molecular Cloning: A

Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring

Harbor, NY.

Mintz, B., 1967. Gene control of mammalian pigmentary differentiation.

I.clonal origin of melanocytes. Proc. Natl. Acad. Sci USA 58, 344±351.

Motro, B., Van der Kooy, D., Rossant, D., Reith, A., Bernstein, A., 1991.

Contiguous patterns of c-kit and steel expression: analysis of mutations

at the W and Sl loci. Development 113, 1207±1221.

Murphy, M., Reid, K., Williams, D., Lyman, S., Bartlett, P., 1992. Steel

factor is required for maintenance, but not differentiation, of melano-

cyte precursors in the neural crest. Dev. Biol. 153, 396±401.

Nakayama, A., Minh-Thanh, T.N., Chen, C.C., Opdecamp, K., Hodgkin-

son, C.A., Arnheiter, H., 1998. Mutations in microphthalmia, the mouse

homolog of the human deafness gene MITF, affect neuroepithelial and

neural crest-derived melanocytes differently. Mech. Dev. 70, 155±

166.

Opdecamp, K., Nakayama, A., Nguyen, M.-T.T., Hodgkinson, C.A., Pavan,

W.J., Arnheiter, H., 1997. Melanocyte development in vivo in neural

crest cell cultures crucial dependence on the Mitf basic-helix-loop-

helix-zipper transcription factor. Development 124, 2377±2386.

Pavan, W.J., Tilghman, S.M., 1994. Piebald lethal (sl) acts early to disrupt

the development of neural crest-derived melanocytes. Proc. Natl. Acad.

Sci. USA 91, 7159±7163.

Perris, R., Kuo, H.J., Glanville, R.W., Bronner-Fraser, M., 1993. Collagen

type IV in neural crest development: distribution in situ and interaction

with cells in vitro. Dev. Dyn. 198, 135±149.

Pierschbacher, M.D., Ruoslahti, E., 1984. Variants of the cell recognition

site of ®bronectin that retain attachment-promoting activity. Proc. Natl.

Acad. Sci. USA 81, 5985±5988.

Robertson, E.J., 1987. Teratomas and Embryonic Stem Cells: Practical

Approach, IRL Press, Oxford.

Serbedzija, G.N., Bronner-Fraser, M., Fraser, S.E., 1994. Developmental

potential of trunk neural crest cells in the mouse. Development 120,

1709±1718.

Steel, K.P., Davidson, D.R., Jackson, I.J., 1992. TRP-2/DT, a new early

melanoblast marker, shows that steel growth factor (c-kit ligand) is a

survival factor. Development 115, 1111±1119.

Sviderskaya, E.V., Wakeling, W.F., Bennet, D.C., 1995. A cloned, immor-

tal line of murine melanoblasts inducible to differentiate to melano-

cytes. Development 121, 1547±1557.

Thiery, J.P., Duband, J.L., DelouveÂe, A., 1982. Pathways and mechanisms

of avian trunk neural crest cell migration and localization. Dev. Biol.

93, 324±343.

Weilbaecher, K.N., Hershey, C.L., Takemoto, C.M., Horstmann, M.A.,

Hemesath, T.J., Tashjian, A.H., Fisher, D.E., 1998. Age-resolving

osteopetrosis: a rat model implicating microphthalmia and the related

transcription factor TFE3. J. Exp. Med. 187, 775±785.

Weston, J.A., 1991. Sequential segregation and fate of developmentally

restricted intermediate cell populations in the neural crest lineage.

Curr. Top. Dev. Biol. 25, 133±153.

Woods, A., McCarthy, J.B., Furcht, L.T., Couchman, J.R., 1993. A synthetic

peptide from the COOH-terminal heparin-binding domain of ®bronectin

promotes focal adhesion formation. Mol. Biol. Cell 4, 605±613.

A. Beauvais-Jouneau et al. / Mechanisms of Development 89 (1999) 3±1414