Cell lines derived from mouse neural crest are representative of cells at various stages of differentiation

Cell Lines Derived from Mouse Neural Crest are Representative of Cells at Various Stages of Differentiation

Mark Murphy,* Ora Bernard, Kate Reid, and Perry F. Bartlett

t he Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria, 3050, Australia

SUMMARY

I n order to study mammalian neural crest differentiation in vitro, a series of clonal neural crest (NC) cell lines have been generated by infection of migrating mouse neural crest cells with two recombinant retroviruses containing either the c-myc or N-myc proto-oncogenes. Many cell lines were generated which could be subdi- vided into three groups based on their appearance in culture. Eleven of these cell lines representative of each of the morphological groups were characterized for the expression of six antigenic markers expressed by neural cells. In addition, mRNA was prepared from these cell lines and analyzed for the expression of a number of neural specific genes. These analyses show that the cell lines are representative of the following cell types: ( 1) neural crest-like cell lines that do not differentiate in 10%

serum; ( 2 ) progenitor cell lines, some of which can partially differentiate in culture; and (3) mature neuronal cell lines or bipotential cell lines. Southern blot analysis of DNA from these lines indicated that they have multi- ple integration sites for the provirus and suggest that phenotypically different cell types have arisen from a single cell. None of the cell lines showed any proliferative or morphological response to nerve growth factor (NGF), whereas over two-thirds of the lines showed both marked proliferative and morphological responses to fibroblast growth factor (FGF). These data indicate that we have generated a range of cell lines representative of a spectrum of mouse neural crest derivatives.

INTRODUCTION

A large and diverse array of cell types in the verte- brate are developmentally derived from the embryonic neural crest (NC). After migration, the crest cells give rise to the peripheral nervous system (PNS) including sensory and autonomic ganglia and Schwann cells, the adrenal medulla, connec- tive tissues, pigmented cells and muscle, and carti- lage and bone in the head and face. How cells, derived from this transient structure, are capable of following so many distinct pathways of differentia-

Received December 4, 1990; accepted February 22, 1991 Journal of Neurobiology, Vol. 22. No. 5 , pp. 522-535 (1991) 0 1991 John Wiley &Sons, Inc. CCC 0022-3034/91/050522-14$04.00

* To whom correspondence should be addressed.

522

tion is a fundamental problem in developmental biology. The question of whether these cells are multipotential and/ or already committed to a single pathway has been investigated in several systems both at a population level and at a single cell level (Le Douarin, 1986; Weston, 1986; Anderson, 1989). Zn ovo transplantation experiments in which populations of quail cells were grafted into chick embryos support the notion that the environ- ment of the cells determines their differentiated phenotype (Le Douarin, 1986 ). In addition, in vitro experiments show that changes in the growth medium can modulate the phenotypic expression of neural crest cultures. However, these experiments also suggest that the neural crest is not a homogeneous population of pluripotent cells but is comprised of subsets of committed precursor cells (Le Douarin, 1986; Ziller et al., 1987).

Mouse Newal Crest Cell Lines 523

The problem in interpreting results from populations of cells is that changes in the phenotype ofthe cells may result from differential selection of precursor cells rather than true multipotentiality. Thus, attempts have been made to look at the progeny of single cells. In the quail, these cloning experiments have demonstrated that a range of mature cell types can arise from a single cell (see Baroffio, Dupin, and Le Douarin, 1988 for discussion). However, this is an infrequent event and most of the clones contain only a single mature phenotype. This indicates that many of the cells are either committed at this stage or that there is a require- ment for a range of exogeneous factors to reveal the full potentiality of any individual cell. The multipotency of individual neural crest cells can be traced in vivo (Bronner-Fraser and Fraser, 1988), however, the frequency of these multipotent cells is unclear and little is known offactors that may influence the developmental fate of the neural crest.

In mammals, very little is known about cell lineage and commitment of the neural crest. The isolation of large numbers of neural crest cells from mammals is difficult and they have a limited life span in vitro. In order to overcome these restric- tions, we have developed cell lines representative of migratory neural crest cells and their progeny in order to study ccll lineage associations as well as to characterize environmental factors that influence the developmental fate of neural crest cells. Previous work from our laboratory and others has shown that retrovirus-mediated proto-oncogene transduction of the neural precursor cells from mouse neuroepithelium results in the production of stable neuroepithelial cell lines (Bartlett, Reid, Bailey, and Bernard, 1988; Bernard, Reid, and Bartlett, 1989; reviewed in Cepko, 1988, 1989). These cell lines have similar characteristics to primary neuroepithelial cells and, like their primary culture cell counterparts, they differentiate in response to fibroblast growth factor [ FGF] .

We have immortalized mouse neural crest cells using retroviruses bearing the c-myc or the N-myc proto-oncogenes. Cloned cell lines derived from such cultures show an array of differentiated phenotypes representative of primary neural crest cells as well as those derived from the neural crest.

MATERIALS AND METHODS

Retrovirus Vectors

The c-myc or N-myc retroviruses used to immortalize the neural crest cells were the same as those used to im-

mortalize neuroepithelial cells; namely the Do1 (c-myc) virus (Bartlett et al., 1988) and later, the MPZenSVNeo (N-myc) virus (Bernard et al., 1989).

Preparation of Neural Crest Cells

CBA mouse embryos at embryonic day 9 (E9) (average 16-22 somites) were removed from the uterus and placed in a petri dish containing Hepes-buffered Eagles Medium(HEMjwith l%fetal bovineserum(FBS).The head and tail were removed using 26-gauge syringe needles with the aid of a dissecting microscope leaving a trunk segment with 8-12 somites on each side of the neural tube. These trunk segments were placed in a fresh petri dish with HEM/lG FBS, and the somites, and surrounding tissue were carefully removed from the neural tube using 26-gauge needles. One or two tubes were then placed in each well of a 24-well plate (Linbro) that had been previously coated with fibronectin [ 5 F g / mL] . Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS was then carefully run down the side of each well, so that it almost covered the bottom of the well. This enabled the neural tubes to associate and ad- here to the fibronectin substratum. The plates were incubated at 37°C in 10% C0,/90% air for 24 h after which time the tubes were carefully removed, leaving a layer of migratory neural crest cells. One ml, of DMEM/10% FBS was added and the cells were further incubated at 37°C. Figure I shows a typical neural crest culture.

Generation and Cloning of Neural Crest Cell Lines

E9 neural tubes prepared as described above, were plated in 24-well costar plates onto irradiated (2800 rad) monolayers of an equal mixture ( lo4 cells total) of CBA astroglia prepared from postnatal day 2 (P2) cerebella (Bartlett et al., 198 1 ) and the +, cells producing either Do1 (c -my) or MPZenSVNeo (N-myc) viruses. Neural tubes were rigorously removed after 24 h at which time the cultures appeared the same as uninfected cultures. This was found to be the most efficient way to infect the cells and resulted in infection and retention in the cultures only of migrated cells. Selection medium containing G418 (400 pg/mI,) in DMEM/ 10% FBS (Southern and Berg, 1982) was added 4 or 5 days later and was changed twice a week until the cultures became confluent with infected neural crest cells (at least 10 days) but were depleted of the irradiated astroglia and two pro- ducer cells that died during the culture period. Individ- ual wells were then trypsinised, and the cells were re- plated in DMEM/ 10% FBS onto twice the surface area on plastic. Initially, cultures were never split from con- fluence more than twofold because this sometimes resulted in the culture entering a crisis phase from which it did not recover.

Cell lines were cloned by limiting dilution in 96-well plates (Linbro j in DMEM/ 10% FBS either directly or

Figure 1 Culture of mouse neural crest. Neural tubes were prepared from E9 CBA embryos and placed in culture for 24 h as described in Materials and Methods. ( a ) Low magnification view of part of the neural tube with migrated neural crest cells. Scale bar = 200 l m . (b) Higher magnification view of migrated neural crest cells. Scale bar = 100 pm.

onto irradiated monolayers of the bulk cultures of cells from which they were derived. Cell lines plated directly onto plastic gavc rise to clones of flat, apparently undifferentiated cells, whereas cell lines cloned on monolayers of immortalized neural crest cells contained, in addition to these flat cells, all other phenotypes described. Clones were picked and either transferred to 96-well plates or 24-well plates and split 1:2 when confluent. In some cases, this took at least 1 month; however, when cell lines became established in culture they normally lost some of their sensitivity to splitting more than 1:2. The first number in the name of the neural crest cell lines is the experi- ment number. The clonality of the cell lines was con- firmed by Southern blot analysis of DNA from subclones of the cell lines hybridized to the c-myc probe in case of the Do1 (c-myc)-infected cell lines or N-myc probe when the lines were generated by infection with MPZenSVNeo (N-myc) virus. The subclones were derived from micro- manipulated single cells.

lmmunohistochemistry For immunocytochemistry, cell lines were plated at 5 X lo3 cells/well on glass microscope slides containing 12

wells of 8 mm in diameter (Flow 10805) and incubated for 2-3 days in DMEM/lO% FBS. For staining with A2B5 antibodies (Eisenbarth et al., 1979), cells were incubated for 30 rnin at room temperature with A2B5 ascites fluid diluted 1:lOO in HEM/l% FBS. The cells were then washed in the same medium and incubated for 30 min with a fluorescein isothiocyanate-conjugated (FITC) sheep anti-mouse antibody fragment (Silenus, Australia) diluted 1:50 in HEM/ 1% FBS, washed an ad- ditional three times in HEM/ 1% FBS, and fixed in eth- anol/acetic acid (19:l) for 15 rnin at -20°C. The cells were then washed in water, air dried, and the coverslips were mounted in 2.6%: I .4-diazobicyclo-( 2,2,2)-octane in phosphate-buffered saline/glycerol ( 1:9) (Merc, Aus- tralia) to prevent bleaching. For staining with antibodies to vimentin, glial fibrillary acidic protein (GFAP), or neurofilament, the cells were first fixed in methanol for 30 rnin at ~ 2O"C, washed three times in PBS, and incubated for 30 rnin with either a mouse anti-vimentin antibody (Serotec, U.K.) diluted 1: 100 in HEM/ 1% FBS, a rabbit anti-GFAP antibody (Dakopatt, Denmark) diluted 1:50 in HEM/ 1% FBS, or an anti-neurofilament antibodies, a rabbit anti- 150-kD ncurofilament antibody (Chemicon, CA) or monoclonal RT97 antibody (Law- son et al., 1984: Anderton et al.. 1982) diluted 1 : l O in HEM/ 1% FBS. The wells were then washed and incubated with an FITC sheep anti-mouse F(ab)2 antibody fragment (for the vimentin and RT97 antibodies) or with an FITC sheep anti-rabbit antibody (Silenus, for the GFAP and anti- 150-kD neurofilament antibodies) diluted 1 :50 in PBS/ 1% FBS, washed and mounted as described above.

Analysis of RNA and DNA

PolyA RNA was prepared as described by Gonda et al. ( 1982). Samples of 2 fig were fractionated on formaldehyde/agarose gels and transferred onto nitrocellulose filters as described previously (Thomas, 1980). DNA was prepared from 5 X 1 O6 cells by lysis with guanidine hydrochloride (Bowtell, 1987) and analyzed by South- ern blot hybridization (Southern, 1975). The c-myc probe was the XhuI fragment of the c-myc cDNA clone pM-c-myc 54 (Stanton et al.. 1983); the N-myc probe was the I-lincII/SacII fragment containing exons 2 and 3 of the N-myc genomic clone (DePinho et al., 1986). The NGF probe was a 1-kb PstI/SmaI fragment of the mouse cDNA clone (Scott et al., 1983). The NGF receptor probe was a 3.4-kb EcuRI/HindIII fragment of the rat cDNA clone (Radeke, Misko. Hsu, Herzenberg, and Shooter, 1987). The superior cervical ganglion (SCG) 10 probe was a 1.8-kb fragment isolated from the SCG10.3 cDNA clone (Anderson and Axel, 1985). The MBP probe was the 2.4-kb EcoRI fragment containing the full-length MBP cDNA (Roach et al., 1983). The PL.P cDNA probe was the 3.2-kb BamHI fragment of p27 (Milner et al.. 1985).

Mouse Neural Crest Cell Lines 525

RESULTS

Generation and Morphological Subgrouping of Neural Crest Cell Lines

In initial experiments infection of neural crest cells with the Do1 (c-myc) and MPZenSVNeo (N-myc) retroviruses resulted in rapid proliferation during the first 5 days of culture. The morphology of the crest cells remained undifferentiated, that is, the cells were flat and rhombohedra1 to triangular, looking like freshly isolated neural crest cells. After 5 days, G4 18 was added to the cultures to select for retrovirus-infected cells. In mock-infected cultures, all the neural crest cells died within 7 days of G4 18 addition, whereas the infected neural crest cultures contained healthy proliferating cells, the number of which was far greater in the MPZenSV- neo ( N-myc)-infected cultures. Later studies with MPZenSVNeo (c-myc) viruses showed essentially the same patterns of infection as the N-myc virus (data not shown). During the first 2 weeks after addition of G4 18 to the infected cultures, many of the cells changed their morphology and appeared to differentiate into Schwann-like cells and gangli- onic-like clusters of neurons as well as other morphologically unidentifiable cell types.

Cloning the cells by limiting dilution in 96-well plates resulted in clones containing broad flat cells that looked undifferentiated and probably represent immortalized neural crest cells or mesenchy- ma1 cells. Subsequently, the infected neural crest cultures were cloned by plating the cells on irradiated monolayers of uncloned cultures of them- selves. These cells could later be grown on plastic. This strategy proved particularly successful for cells infected with the MPZenSVNeo (N-myc) virus, and many clones of varying morphology were isolated in this way. The different clones could be broadly classified into three subgroups that are represented in Figure 2. Group 1 contained flat adherent cells, round to oblong, which form a cobblestone pattern at high density [Fig. 2( A,B)] . Mostly, but not always, cell lines in this group looked the same as primary neural crest cells [Fig. 2 ( A,B), compare with Fig. 11. Some cell lines in this group gave rise to small phase-bright round cells arising at higher densities [Fig. 2( C)] . Group 2 contained flat cells at low density, a proportion of which at higher density and longer time in culture, tended to become stellate with one to four processes that could be ruffled or branched [Fig. 2 (C,D)] . Group 3 cells grew initially as flat cells but after a relatively short time in culture, most of the cells elongated and put out processes [Fig.

2(E,F)]. There was a tendency for these cell lines to lose their ability to “morphodifferentiate” in culture after many passages, and only early freez- ings showed 100% of the cells with a bipolar phenotype. As we demonstrate below, these morphological groupings probably reflect the degree of differentiation that these cell lines could achieve in tissue culture.

Expression of c-myc and N-myc in the Neural Crest Lines

A number of the cell lines were examined for the expression of c-myc and N-myc mRNA. All the immortalized cell lines expressed high levels of the proviral transcripts (Fig. 3). As expected, the Do1 (c-myc) virus-infected lines (NC 4.10.1 C and NC 4.10.1H) expressed only one proviral c-myc tran- script while all the MPZenSVNeo N-myc virus-infected cell lines expressed proviral transcripts of 6 kb and a smaller 5.6-kb subgenomic N-myc mRNA (Bernard et al., 1989). The expression of the cellular c-myc mRNA was suppressed in most of the cell lines regardless of the nature of the proviral insertion (Fig. 3 ). High levels of myc proteins have previously been shown to result in suppres- sion of the cellular c-myc mRNA (Bernard et al., 1989). Only two of the cell lines examined, NC 15.8.Sl andNC 15.2.10H, expressedquitehighlev- els of endogenous c-myc mRNA, possibly because they expressed only low amounts of viral N-myc protein.

Expression of Vimentin and S100

In the mouse there is no definitive marker for neural crest cells. The standard marker for neural crest cells in the chick, HNK-1, stains few if any mouse neural crest cells (Holley and Yu, 1987). However, the cell lines were tested for the presence of vimentin and S 100, both of which are expressed by the neural crest or crest-derived cells. Vimentin is expressed predominantly in mesenchymal cells as well as migrating neural crest cells (Ziller, Du- pin, Brazeau, Paulin, Le Douarin, 1983). SlOO is expressed principally in the central and peripheral nervous systems and probably at highest levels in astrocytes, Schwann cells, and enteric glia as well as in neuronal cells ( Zomzely-Neurath and Walker, 1980; Fem et al., 1982). All cell lines tested were vimentin positive [Fig. 4(A)] and most of them were also S- 100 positive [Table 1, Fig. 4( B)] . In both cases, every cell was positively stained. Thus, thesc findings are consistent with the cell lines be-

Figure 2 Morphology of neural crest cell lines. Shown are phase-contrast views of cells from cell line (a) NC 15.2.lOH and (b) NC 15.8.1 1. which are in group 1; (c) NC 14.9.1 and (d) NC 14.4.1 ID which are in group 2: ( e ) NC 14.4.9D and ( f ) NC 14.4.6E from group 3: (g) NC 15.8.51 from group 1; and (h) NC 4.10.1C from group 3. Scale bar = 200 fim except for (e), which = 80 km.

Moure A'ewal Crest Cell Lines 527

0 000 00000000 z z z z z z z z z z z z

N - rnyc -

c - myc -

GAPDH -

Figure 3 Northern blot analysis of RNA isolated from the different NC cell lines with myc Poly A+ RNA (2 pg) was separated on a 1% agarose/formaldehyde gel and transferred onto a nitrocellulose filter. The filter was hybridized to c-myc and N-nzyr probes. The band labelled N-myc is the retrovirally derived RNA. In the particular cases of cell lines NC 4.10. IC and NC 4.10. IH. which were generated with the Do1 c-mvc retrovirus, this RNA species is thus retrovirally derived c - m j ~ The band labelled c-myc is the endogenous cellular c-myc. The filter was stripped off and hybridized to a glyceraldehyde phosphate dehydrogenase probe (GAPDH) for quantita- tion of the RNA.

ing of neural crest origin, but do not exclude ori- gins from the neural tube.

Differential Expression of Antigenic and Molecular Neural Markers

In order to classify these cell lines as being linked to a particular neural crest lineage, the cell lines were examined for the expression of some lineage-specific or -related antigenic markers that are found on neural crest-derived cells. We have used antibodies to glial fibrillary acidic protein, neurofilament (NF), and the A2B5 antibody. GFAP is specific for mature glia in the central nervous system and has also been found in the glia of the enteric nervous system (Jessen and Mirsky, 1983) as well as in a subpopulation of nonmyelinating Schwann cells (Yen and Fields, 198 1 ). One of the NF antibodies used is a polyclonal antibody directed against 150-kD NF, and binds to peripheral neurons as early as El0 (Cochard and Paulin, 1984). The other NF antibody, RT97, recognizes a phos- phorylation-dependent epitope on the 150-kD and 200-kD NF, and is expressed only in mature neu-

rons (Kai-Kai. Anderson, and Keen, 1986; Law- son et al.. 1984). A2B5 reacts with a ganglioside present on neurons and probably their precursors in avian neural crest cultures (Girdlestone and Weston, 1985). It also recognizes some glial cells and their precursors in the rat central nervous system (Raffet a]., 1983, 1984). Representative staining of particular neural crest cell lines with each of these antibodies is shown in Figure 5.

The results of this analysis showed patterns of staining consistent with individual cell lines being at different stages of differentiation. Eleven cell lines were selected as being representative of the different types of cells that emerged from the neural crest immortalization. RNA from these 11 cell lines was analyzed for the expression of neural- specific mRNAs. We have examined the expression of nerve growth factor (NGF) and its receptor (NGF-R), which are expressed by cells in the peripheral nervous system, myelin basic protein (MBP), and the proteolipid protein (PLP) of myelin, which, in the peripheral nervous system, are specific to Schwann cells (Puckett et al., 1986). A neuron-specific gene, SCG10 was also used in the analysis (see below). The results of the analyses of these 1 1 cell lines are shown in Table 1. In summary, the different groups of neural crest cell lines show the following phenotypes discussed below.

Group 1. Neural Crest-Like Cell Lines. The cell lines examined in this group did not express any of the markers specific for terminally differentiated neurons or glia. However, one of the lines was positive for A2B5 (Table 1 ). Four of these cell lines, NC 14.9.10, NC 15.2.10H, NC 14.3.36, and NC 15.256 were examined for the expression of the neural-specific mRNAs described above (Figs. 6 and 7). One of these cell lines, NC 14.3.36, expressed NGF-R mRNA, and cell line NC 15.2.1OH expressed low levels of NGF mRNA. However, these cell lines did not express transcripts for any of the other neural genes. These data are consistent with the morphological characterization of these cell lines as being "crest-like.''

Group 2. Cells Lines "Partially Differentiating" in Culture. Most of the cell lines in this group expressed A2B5; in these lines A2B5 was expressed on between 5% and 50% of the cells (Table 1). Furthermore, some cells in many of these cell lines expressed 150-kD NF, and of these cell lines some also bound RT97. PolyA+ RNA was prepared from high-density stationary-phase cultures of four of these cell lines, and was analyzed by Northern

Figure 4 Representative staining of neural crest cell lines. Cell lines were plated onto 12-well microscope slides and stained with the particular antibodies as described in Materials and Methods. Shown are representative fluorescence photographs of staining by each antibody used in this study. (a ) Staining of NC 14.4.9D with an anti-vimentin antibody. (b) Staining of NC 4.10.2F with S 100. Scale bar = 30 pm.

blot analysis for the expression of neural-specific genes (Table 1, Figs. 6 and 7 ). All these cell lines expressed NGF and NGF-R mRNA (Fig. 6 ) , and each of the cell lines also expressed high levels of PLP mRNA (Fig. 7). NC 14.4.8 also expressed high levels of MBP and, in addition, a small proportion of the cells bound GFAP antibodies. Thus, at high cell density, these cells have the hallmarks of differentiated Schwann cells. Likewise, NC 14.9.8 expressed high levels ofMBP. The other two cell lines in this group both expressed low levels of MBP, and in addition, a small proportion of cells in both lines bound both RT97 and the 150-kD NF polyclonal antibody.

Group 3. “Difierentiated” Cell Lines. The characteristic of the cell lines in this group are that most of the cells are process-bearing cells, however, individual cell lines in the group had different morphological characteristics in the processes and cell bodies. A2B5 was found on at least 50% of the cells in cell lines from this group, and, in cell lines NC

14.4.6E and NC 14.4.9D, most of the cells bound anti-150-kD NF and RT97. Northern blot analysis demonstrated that these two cell lines expressed NGF, but only NC 14.4.9D expressed NGF-R mRNA (Fig. 6).

One of the genes that is expressed only in developing and mature neurons is SCGIO; this gene is first expressed in embryonic (ElO-El 1 in the rat), central, and peripheral neurons (Stein et al., 1988). Hybridization ofthe SCG10 probe to RNA isolated from the neural crest cell lines showed that only lines NC 14.4.6E and NC 14.4.9D were positive (Fig. 6) . In particular, cell line NC 14.4.6E has the morphological [ fine processes emanating from a round cell body; Fig. 2 ( F)] , antigenic, and molecular characteristics of a neuronal cell. However, cell line NC 14.4.9D also expressed PLP mRNA (Fig. 7 ), showing that this line has both glial and neuronal characteristics. In this case, it cannot be determined whether expression is in the same cells or in mutually exclusive subpopulations. Neither of these cell lines expressed MBP mRNA.

Figure 5 Representative staining of neural crest eel1 lines. Cell lines were stained as in Figure 4. Phase-contrast photos of the same field are also shown when only a proportion of the cells were positively stained by the particular antibody. (a) A2B5 staining of NC 14.4.9D. (b) Phase-contrast view of the same field as in (a). (e) GFAP staining of NC 14.4.8. (d) Phase-eon- trast view of the same field as in (c). the arrow indicatcs the brightly stained cell in (c). (e) Staining of NC 14.4.6E with the anti- 150-kD NF antibody. ( f ) Phase-contrast view of same field as in (e). (8) Staining of NC 14.4.9D with the RT97 antibody. Scale bar = 30 pm.

Mouse hTeiiral Cresr Cell Lines 529

530 Murphy et al.

Table 1 Classification of Neural Crest Cell Lines with Antigenic and Molecular Markers

Antigenic marker

NF Molecular Marker

Cell Line Sl00 A2B5 G F A P 1 5 0 k D RT97 NGF NGF-R SCG10 MBP PLP

“Undifferentiated” Neural crest-like cells

NC 14.9.10 NC 15.2.10H N C 14.3.3G NC 15.2.56

culture NC 14.9.8 NC 14.4.8 NC 14.9.1 NC 4.10. lH

NC 14.4.6E NC 14.4.9D NC4.10 . lC

Cells “differentiating” in

“Differentiated” cells

+ + + + +

-

-

-

+ + + + + + +

-

+ + + +

-

+

t + +

+ + + +

+ + + + +

- -

~

- + + + + + + + +

- + + + + -

- +/2 i - f +

Cells were stained or RNA was analyLed by Northern blot analysis as described in Materials and Methods. +/2 faint transcripts were detected by Northern blot analysis; f indicates very low levels of transcripts.

o x D W 0 T: ‘9 r $ ? ; 5 z l ? l q e m ? ? m + O l G r % . $ P +

expression of NF was limited to a small percentage - r 7 Z 2 - i 4 - . - : z

The other cell line examined in this group, NC 4.10.1C. binds both NF antibodies, although the * * * of cells. In addition, this cell line expresses NGF and NGF-R mRNA as well as PLP and MBP mRNA. These data indicate that this cell line has glial characteristics but that a small number of cells express neuronal markers.

NGF-R -

Integration Patterns of Proviral DNA

Can Give Rise to Different Phenotypes Indicate that a Single Immortalized Cell NGF -

Since the neural crest cell lines were generated by retroviral infection of myc retroviruses, each cloned cell line should have a specific integration pattern of the viral rnyc genes. Because EcoRI cuts only once within the proviral DNA, the number of EcoRI fragments hybridizing to the myc probe in Southern blots indicates the number of retroviral integrations within the cellular DNA. Figure 8 de- picts Southern blots of DNA isolated from the cell lines and hybridized to the N-rnyc probe. As can be seen, some of the cell lines have identical band patterns. For example, the cell lines NC 15.2.10H and NC 15.2.56 most probably originated from a single cell since they have identical viral integration patterns. However, while NC 15.2.10H appeared always as a classical neural crest-like cell line and

L- SCGlO

Figure 6 Northern blot analysis. RNA from the different NC cell lines was transferred onto a nitrocellulose filter that was hybridized first to the NGF-R probe fol- lowed by hybridization to the N G F probe. The filter was then stripped off and hybridized to the SCG- 10 probe.

expressed cellular c-myc mRNA and some NGF mRNA, NC 15.2.5G gave rise to many round cells in culture, and did not express NGF or cellular c-myc mRNA. A more striking example can be

Mouse Neurul Crmt Cell Lines 531

Table 2 Responses of Neural Crest Cell Lines to FGF

C + d , . . : % % % ~ % 2 Response Response

I 0 n g E X $ $ ' " r ' C z $ c g : d T f , , - 7 , - m

Cell Line to FGF Cell Line to FGF

P L P -

MBP -

GAPDH -

Group 1 NC 15.2.IOG N C 15.1.2 N C 15.2.8H NC 15.8.3 NC 15.8.4 N C 14.4.14 NC 14.9.10 N C 15.2.10H N C 1 5 . 2 3 3 NC 15.8.1 1

Group 2 N C 14.9.1

Figure 7 Northern blot analysis of RNA isolated from NC 4.10.1B the different NC lines and adult mouse brain. The filter NC 4.10.1G was first hybridized to the MBP probe, then stripped off, NC 4.10.2F and hybridized to the PLP probe. The lane correspond- N C 14.4.1 I ing to brain hybridized very strongly to the PLP probe NC 14.4.9H masking the bands in the adjacent lane and was therefore NC 14.9.8 excised. NC 14.4.8

NC 4.10.1H

found with lines NC 14.9.10 and NC 14.4.8, which also have the same viral N-mvc integration pattern. Whereas line NC 14.9.10 is a typical undifferentiated neural crest-like line, NC 14.4.8 differen- tiates into mature Schwann cells in culture and expresses NGF, NGF-R, MBP, and PLP mRNA. These integration patterns were also shared by cell lines NC 14 .9 .8 and NC 14.9.1, which both have characteristics of Schwann cells and neuronal cells.

Figure 8 Southern blot analysis of DNA isolated from NIH-3T3 cells (3T3) and from the NC cell lines. DNA ( 10 p g ) were digested with Xbal ( X ) or EcoRI (RI) and separated on 0.7% agarose gel. After transfer. the nitrocellulose filters were hybridized to the N-myc probe. Arrow heads indicate the cellular N-myc fragment.

Group 3 + NC 14.4.9D ++ ++ NC 14.4.6E -

++ NC 14.4.9 + + NC 4.10. IC + + NC 14.3.10C ++ +

+ + + + + + + +

Cells were plated in triplicate at both 50 and 100 cells/well in 6O-w,cll H L - 4 plates (Lux) in DMEM/l% FBS and in the presence or abscnce of 50 ng/mL bFGF (Amersham). After 7 days the cells in each well were counted by phase-contrast micros- copy. A minus sign indicates no increase in cell number over control wclls. A plus sign indicates up to a tenfold increase over control and a double-plus sign indicatcs ovcr a tenfold increase over control.

The molecular clonality of these cell lines was con- firmed by Southern blots of DNA isolated from subclones that had been grown from single micro- manipulated cells. In these cases, each subclone had an identical integration pattern (data not shown),

The Effect of Growth Factors on the Neural Crest Lines

All the cell lines were tested for their response to two factors, NGF and basic FGF (bFGF), both of which are trophic for neural crest-derived cells ( Levi-Montalcini and Angeletti, 1968; Halaban et al., 1988; Stemple et al., 1988; Kalcheim, 1989). The assays were done at lon7 cell numbers (50 and 100 cells/well) in 60-well HL-A plates. In most cases, cells would survive at either or both of these cell densities, and either proliferate to some extent or not at all in medium containing 1% FRS alone (Table 2) . NGF had no observable effect on any of

532 Mirrphy el a1

the neural crest cell lines, suggesting that NGF does not stimulate proliferation or differention of neural crest cells. However, the lack of effect could also be explained by the fact that any of the effects of NGF may have been masked by endogenous production of NGF in many of the cell lines. In contrast, bFGF had marked survival or proliferative effects on many of the cell lines tested (Table 2) . Of the 3 1 cell lines tested, approximately two- thirds responded significantly to bFGF, showing a moderate to strong response. Cell lines in each sub- group responded to bFGF. bFGF also had marked morphological effects on almost all of the cell lines. In general. the response was a rounding up of the cells that was sometimes accompanied with some outgrowth of processes, either uni- or bipolar (data not shown). In some cases the bFGF caused the cells to elongate and become spindle shaped.

DISCUSSION

We have generated cell lines from infection of mouse neural crest cultures with retroviruses containing the c-myc or the N-myc proto-oncogenes. These cell lines are most probably of neural crest origin for the following reasons. (1) Our neural crest cultures, when plated onto a fibronectin sub- strate. show no visible contamination with either somites or with neural tube-derived epithelial cells. (2) All cell lines expressed both vimentin and S100, consistent with them being of neural origin. (3 ) The majority of the cell lines isolated looked like primary neural crest cells or their differentiated progeny. (4) The expression of all other markers described in this paper is consistent with the cell lines being of neural crest origin. However, it cannot be totally excluded that some of the lines were derived from the neural tube as there are no definitive mouse neural crest markers currently available.

A major question arising from the establishment of the neural crest cell lines is whether they possess characteristic features of migrating crest cells and their differentiated progeny. Morphologi- cally, the cell lines had a variety of appearances from flat cells resembling migrating neural crest cells through to multiprocessed cells reminiscent of neurons or Schwann cells. The cell lines in group 1 not only morphologically resembled migrating neural crest cells, but this group also was largely devoid of phenotypic markers, both antigenic and mRNA, expressed by mature neural cells. These observations are consistent with the idea that some

of the migrating neural crest cells are not yet committed to a single developmental pathway and probably represent stem cells.

A major characteristic of the neural crest is its plastic and multipotential nature. There is evidence that some of the cell lines may be multipotential. Cell line NC 14.9.1 appeared to be bipotential since in a cloned population these cells expressed neurofilament as well as MBP and PLP. Likewise. NC 14.4.9D expressed both PLP mRNA and SCG 10 mRNA, and all the cells express neurofilament. Similarly, multipotent neural cell lines have been isolated from the newborn brain (Fred- eriksen, Jat, Levy, and McKay, 1988; Ryder et al., 1990). These cell lines also share some other characteristics of our cell lines in that some of the antigenic markers examined were expressed on a small proportion of cells in particular cell lines.

The multipotential nature of the neural crest cells that were originally infected with either c- myc- or N-myc-containing viruses was also demonstrated by the observation that cell lines that have the same myc integration pattern, and thus must have originated from the same cell, can have quite different phenotypes. It is possible that an immor- tilized multipotential cell divided a number of times before differentiation of the progeny cells into the different phenotypes took place. Thus, a single crest cell can rise to a neural crest-like line, Schwann cell progenitor, and a bipotential cell line.

In addition to stem-like activities, there is evidence that some of the lines also represent progenitor populations that can differentiate in culture. Cell lines from group 2 appear to contain such lines. For example, NC 14.4.8 cells contained cells that differentiated after 1-2 weeks in culture into Schwann-like cells. Furthermore, these older cultures expressed mRNA for MBP, PLP, NGF, and NGF-R. All these observations are consistent with this cell line comprising Schwann cell progenitors.

Finally, one of the cell lines appears to represent differentiated neuronal cells. These cells (NC 14.4.6E cells) have fine processes that contain neurofilament. In addition, these cells express mRNA for the neuronal protein SCG10, as well as for NGF, but not NGF-R or other nonneuronal markers. The cells do express vimentin, but this is not surprising since vimentin is present in neural crest cells up until neuronal differentiation.

Although cell lines representative of both neurons and Schwann cells have been characterized there is no evidence that other neural crest-derived cells are represented. None ofthe lines contain mel-

Mouse Neural Crest Cell Lines 533

anin pigments or resembled melanocytes, and this is in contrast to the finding in quail neural crest transformed by Rous sarcoma virus (RSV) in which the cell lines gave rise to melanocytes (Pes- sac, Ziller, Vautrion, Girard, and Calothy, 1985). However, avian and mouse neural crest appear to differ markedly in their capacity to give rise to mature melanocytes: the mouse neural crest appears to require melanocyte stimulating hormone (Ito and Takeuchi, 1984) or phorbol ester (P. F. Bart- lett, unpublished observations) to induce melanin formation in vitro, whereas melanin formation oc- curs spontaneously in primary avian neural crest cultures. Preliminary experiments using phorbol ester on our cell lines have shown no evidence for melanocyte differentiation. The particular viruses and oncogenes used in these experiments must also have an influence on the phenotypes of the resul- tant cell lines (Cepko, 1989).

Recently, Fauquet, Stehelin. and Saule, (1990) reported that v-myc (MC29) and c-myc retroviruses induce the expression of catecholaminergic traits in quail neural crest cells. We have also found that most of our cell lines stain positively with antibodies that recognize tyrosine hydroxlase and in this way the cell lines share a characteristic or the myc-infected quail cells (M. Murphy and K. Reid, unpublished observations).

Many of the neuroepithelial cell lines responded to growth factors, such as FGF (Bartlett et al., 1988), which is known to affect neural proliferation and differentiation of freshly isolated neuroepithelial cells (Murphy et al., 1990). Likewise, most of the neural crest cell lines in this study respond to FGF by proliferating and by changes in morphology, including the group 1 crest-like cells. The sig- nificance of these morphological changes in term$ of differentiation is currently being investigated. In addition. high cell density and fetal calf serum are often required to ensure continued growth of some of the immortalized lines. Thus, these cell lines may be very useful in assaying for factors that are important in differentiation, for example, FGF, serum factors (Ziller et al., 1983). It is possible, therefore, that the cells could be pushed down one differentiative pathway or another by the addition of particular factors in a similar manner to that shown for the neural crest-derived sympathoadre- nal cell types (Doupe et al., 1985a,b; Anderson and Axel, 1986). In addition, mixing experiments between the cell lines and other cell types may help to uncover the importance of cell-cell interaction in neural crest development. Such experiments are ef- fectively impossible to do with primary neural crest

cultures because they rapidly become a heteroge- neous population of cells.

We thank K. Wycherley, M. Costa, S. Baudinette, and S. Ganiatsas for excellent technical assistance, D. Newgreen for critical assessment of this manuscript, D. Anderson, and F. Alt for generous gifts of SCGlO and N-myc probes, and I. Collins for typing this manuscript. The work was supported by the National Health and Medical Research Council of Australia.

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Cell lines derived from mouse neural crest are representative of cells at various stages of differentiation