Characterization of developmental stage and neuronal potential of the rat PNS-derived stem cell line, RT4-AC

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 94 (1996) 67-80

Research report

Characterization of developmental stage and neuronal potential of the rat PNS-derived stem cell line, RT4-AC Laurel M. Donahue *, Penelope W. Coates, Adam J. Reinhart Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA Accepted 16 January 1996

Abstract

RT4 is a family of cell lines derived from a rat peripheral neurotumor and consists of a multipotential stem cell line that spontaneously gives rise to three derivative cell types: one glial-like and two neuronal-like. Previous studies have established that the RT4 glial derivative expresses many properties of Schwann cells; however, the neuronal designation of the other RT4 derivatives is less well substantiated. To further characterize the developmental stage and lineages represented by the RT4 stem cell and its derivatives we examined the expression of 16 marker genes whose expression is either specific to neurons or in some cases, neural tissue. Taken together our results indicate that (i) the RT4 neuronal-like derivatives express only immature neuronal properties, (ii) the RT4 cell lines most closely resemble neural crest derivatives from embryonic day 10 to 12 in the rat, (iii) treatment with cAMP and steroids, although capable of promoting process extension by the RT4 neuronal-like derivatives, did not affect the expression of any of the 16 marker genes examined, and (iv) when compared to other neural stem cell systems, RT4-AC generates the most immature neuronal derivatives. Keywords: Neural crest; Neuroblastoma; Stern cell; RT4; Rat; cAMP; Differentiation; Peripheral nervous system

1. Introduction

We are interested in the molecular mechanisms underlying the ability of a neural stem cell to give rise to both neurons and glia. Lineage tracing studies demonstrate that neurons and glia arise from a common precursor in vivo both in the central nervous system (CNS [50,66]) and in the peripheral nervous system (PNS [6,17,31]). A number of immortalized cell line systems [4,27,41,43,49,67] and primary cultures of neural crest cells [56] are used to model the differentiation of neurons and glia. We have focused our studies on the RT4 family of four cell lines isolated from an ethylnitrosourea-induced rat peripheral neurotumor [32]. The multipotential stem cell line (RT4-AC) expresses some properties of both neuronal and glial cells, and spontaneously gives rise in culture to the other three cell types of the RT4 family (RT4-B, RT4-D and RT4-E) at a frequency of 1 0 - 6 - 1 0 -5 in a process called cell type conversion ([32]; Fig. 1). Upon conversion of the stern cell, the neuronal and glial properties segregate. RT4-D has been classified as glial based on

the expression of properties generally confined to glial cells, including S100/3 protein [20,32], glial fibrillary acidic protein (GFAP [19,20]), and a glycoprotein component of peripheral nerve myelin (P0 [24,30]). RT4-B and RT4-E have been classified as neuronal-like based on the expression of properties which are generally confined to neuronal cells, such as the presence of voltage-sensitive Na ÷ and K ÷ channels, the ability to fire action potentials [12,61,62,68] (D. Spray, personal communication) and the lack of expression of glial markers. The two neuronal-like derivatives differ morphologically and in their expression of the low affinity NGF receptor (LNGFR [30]; Fig. 1). Conversion to the derivative cell lines is permanent, reproducible and is not correlated with any detectable change in karyotype [28]. All of the derivative cell types can also be induced to further differentiate under appropriate culture conditions. For example, upon addition of dibutyryl-cAMP (db-cAMP) and testosterone, RT4-E and RT4-B cells extend long neurite-like processes and RT4-D acquires a glial-specific high affinity uptake mechanism for y-aminobutyric acid (GABA [13]). Addition of db-cAMP alone, or activation of adenylyl cyclase by forskolin treatment, can

L.M. Donahueet al./ DevelopmentalBrain Research94 (1996)67-80

68

In this study we sought to better define the lineage and developmental stage(s) represented by the RT4 cell lines, The glial classification of RT4-D is well substantiated,

all of the RT4 cell lines express markers of immature neural tissue such as the intermediate filament proteins nestin and vimentin, and the 70 kDa form of MAP2

However, the classification of RT4-E and RT4-B as neuronal is weaker, because the electrical excitability properties exhibited by RT4-E and RT4-B are not by themselves definitive neuronal properties since they are properties of muscle cells as well. Therefore, we examined the expression of 16 marker proteins which are either expressed by neurons or by neural tissue. We examined by either Immunoblot or RNase protection assay analysis the expression of each marker in the RT4 cell lines both treated with db-cAMP and 5c~-Dihydrotestosterone (DHT) and untreated. We did not detect markers characteristic of more mature neurons, such as the neurofilament triplet proteins, peripherin, tau, or choline acetyltransferase. However, the stem cell line RT4-AC ~md the derivative line RT4-B were found to express SCG10, a neuronal-specific protein found in all embryonic CNS and PNS neurons [1,55]. In addition,

(MAP2c). Comparison of the pattern of marker genes expressed by the RT4 cell lines with similar published marker studies using both primary cultures and established neural cell lines suggests that RT4-B and RT4-E represent very early stages of neuronal maturation. Indeed the pattern of marker gene expression observed matches that seen in a variety of rat neural crest derivatives at E10-E12. Somewhat surprisingly, although treatment with cAMP and DHT caused characteristic morphological changes in the RT4 cell lines (consistent with what has been reported previously [13]), no significant effect on expression of any of the 16 marker genes was observed. In this study we have demonstrated that the RT4 neuronal derivatives have only begun to differentiate along the neuronal pathway and that treatment with cAMP and DHT causes a very modest level of neuronal maturation. Taken

RT4-AC _ ( s t e m cell t y p e ) + S1001~ +P0 + GFAP + CNPase + SCIP + LNGFR + Na + influx (SkM2'~ + K +efflux + action p o t e n t i a l ~

j (glial type)

. B

+ + + + + +

S100[~ Po GFAP CNPase SCIP LNGFR

- Na + influx

K+ e f f l u x - action p o t e n t i a l

~

(neuronal

(neuronal

t y p e 1)

- sl0o~

- sl0o~

- P0

- P0

- GFAP - CNPase SCIP + LNGFR + Na* influx (SkM2) + K+ e f f l u x + action p o t e n t i a l

+ + +

I+

GABAuptake MBP processes

t y p e 2)

GFAP CNPase SCIP LNGFR Na + influx (SkM2) K + efflux action potential

~

cAMP

cAMP

cAMP

GABA uptake MBP processes

RT4-E

RT4-B

RT4-D

1 processes

Fig. 1. Properties of the RT4 cell lines. The RT4-AC cell line exhibits properties characteristic of both glial and neuronal cells (i.e. expression of GFAP, P0 and voltage-sensitive sodium and potassium channels). Upon conversion of the stem cell to RT4-D, only glial properties are expressed, whereas upon conversion to RT4-B or RT4-E only neuronal properties are expressed. Some phenotypes have been shown to be enhanced by the addition of cAMP to the growth medium (S100/3 and GFAP) and others have been demonstrated to be newlv induced (GARA uotake M'RP and nroc-e~ e~xt~n~inn~ Thi~ ell. . . . . i~

L.M. Donahue et al. / Developmental Brain Research 94 (1996) 67-80

together these observations indicate that the RT4 system is an ideal in vitro system within which to (i) study the mechanisms whereby a stem cell becomes committed to a neuronal cell fate and (ii) characterize the factors which enable immature neurons to further differentiate.

2. Materials and methods

2.1. Cell lines and tissue culture methods The RT4 cell lines used in this study were RT4-D6-5-1 (RT4-D), RT4-B8 (RT4-B), and RT4-E5-1 (RT4-E) [12]. The RT4 variant subline, RT4-AC36A (RT4-AC), which does not undergo cell-type conversion at a detectable frequency, but retains all other properties of RT4-AC (N. Sueoka and T. Leighton, unpublished), was used in all experiments described. The RT4 cell lines were propagated as described [12]. RT4 cells which were treated with cAMP and testosterone ( C / T ; maturation conditions) were first plated in high glucose DMEM with 2.5% FBS at 1.50 × 105 cells per 10 cm Primaria Falcon tissue culture dish. The plating media was removed 24 h later and replaced with fresh plating media containing 1.0 mM dibutyryl cAMP and 100 nM 5~-dihydrotestosterone (DHT). After an additional 72 h, the cells were harvested for protein lysates or RNA. 3T3-L1 adipoblasts (American Type Culture Collection, Rockville, MD; ATCC) were grown to confluence and differentiated into adipocytes as described [22]. PC12 pheochrom~cytoma cells (ATCC) were grown on collagen coated tissue culture dishes in DMEM, supplemented with 10% horse serum, 5% FBS, 100 units/ml penicillin and 1 0 0 / z g / m l streptomycin. The immortalized CNS precursor cell line, ST15A was a gift from Ronald D.G. McKay (NINDS, Bethesda, MD) and was cultured as described [18]. 2.2. Electron microscopy Standard procedures for electron microscopy were followed. Briefly, cell cultures were rinsed with serum-free medium that had been warmed to 37°C, and cells were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer, then washed in 0.1 M phosphate buffer, and post-fixed in 1% osmium tetroxide. For scanning electron microscopy (SEM), fixed cultures were processed by punching out pieces from the plastic cultures dishes, followed by dehydration in a graded series of ethanol. The specimens were critical-point dried, sputter coated with gold, and examined in a Hitachi S-500 electron microscope. For transmission electron microscopy (TEM), cultures were stained en bloc with 2% uranyl acetate, dehydrated in a graded series of ethanol and embedded with Epon. The polymerized plastic was separated from culture dishes by immersion in liquid nitrogen. Sections were cut at approximately 50-60 /xm, picked up on copper grids, post-stained with 4% aqueous

69

uranyl acetate and lead citrate, and examined in a Hitachi H-600 electron microscope. 2.3. RNA isolation Total RNA from rat tissues was isolated by using RNAzol B (Biotecx Laboratories, Houston, TX) and following the protocol supplied by Biotecx. Rat tissues were placed into RNAzol B and immediately homogenized with a polytron homogenizer. Poly (A) + RNA was isolated from the cell lines exactly as previously described [12]. Determination of RNA concentration was by both optical density and RNase protection assays using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference standard (see below). GAPDH is expressed at relatively equal levels in most cell types and is a commonly used internal reference standard. 2.4. RNase protection assay 2.4.1. Assay RNase protection assays were performed with the RPA II kit from Ambion (Austin, TX) using the protocol provided by the manufacturer. Briefly, 10 /~g of poly (A) + RNA or 20 /zg of total RNA from cells or tissues was hybridized to 1 × 104-1 X 105 cpm of the 32p-labeled antisense RNA probe of interest, digested with RNase and electrophoresed on a 6% acrylamide/8M urea sequencing type gel. 2.4.2. Hybridization probes Antisense RNA probes were prepared using Promega's Riboprobe kit (Madison, WI) and [a-32p]CTP (DuPont/NEN, Boston, MA). Probes were synthesized immediately prior to their use and were not gel purified. The GAPDH template was purchased from Ambion. The cDNA clone, SCG10-8.6 (gift from D.J. Anderson, CalTech, Pasadena, CA; [55]; Fig. 5e) was used to synthesize an SCG10 antisense RNA probe. HindIII digestion of the SCG10-8.6 plasmid, followed by transcription using SP6 polymerase generated a probe of ~ 409 nucleotides (nt) and upon hybridization a protected fragment of ~ 399 nt. The template for ChAT was obtained by Sau3A1 linearization of RatChAT (gift from L. Hersh, University of Kentucky, Lexington, KY), followed by transcription with T7 polymerase, which generated a probe of ~ 312 nt and a protected fragment of ~ 253 nt. 2.5. Reverse transcription polymerase chain reaction The GeneAmp RNA PCR kit (Perkin Elmer Cetus, Norwalk, CT) was used to prepare cDNAs from 0.5 /zg poly (A) + RNA according to the manufacturers protocol. Following the reverse transcription reaction, SCG10 cDNA was specifically amplified using 25 pmol of each of the forward and reverse primers and 0.025 U / / z l Taq DNA

70

L.M. Donahueet al./ DevelopmentalBrain Research94 (1996) 67-80

polymerase (Promega, Madison, WI) in a total reaction volume of 100 /~1. 'The primer sequences were: 5'C G C A A C A T C A A C A T C T A C A C C (nucleotides 176-197; forward primer [55]) and 3 ' - T G G A G A A G C T A G A G T T C G T G G (nucleotides 289-310; reverse primer [55]). Amplification conditions were: 95°C for 2 min, followed by 30 cycles of 94°C four 1 min, 57°C for 1.5 min, 72°C for 1 min. The 135 bp SCG10-specific amplified product was separated on a 2% agarose gel, visualized with ethidium bromide and photographed. 2.6. Immunoblotting

1.0 mM EDTA, 1.0 mM PMSF, 10 /xM E-64 and 0.1 /.tg/ml each of chymostatin, leupeptin, antipain and pepstatin). Tissues were homogenized with a polytron homogenizer; cultured cells were scraped from the plate and were passed 5 X through a 25 gauge needle. Protein concentrations of the lysates were determined using the BCA protein assay (Pierce, Rockford, IL) and the lysates were stored in aliquots at - 8 0 ° C . Individual samples were prepared for electrophoresis by addition of 1 / 4 volume of sample buffer (500 mM Tris, pH 6.8, 8% SDS, 80 mM DTT, 40% glycerol and 0.04% bromophenol blue) and incubation at 55°C for 10 min. prior to loading.

2.6.1. Lysate preparation Both tissues and cell[ monolayers were washed briefly with 4°C TBS (150 mM NaC1, 20 mM Tris, pH 7.4) and lysed in SDS lysis buffi~r (50 mM Tris, pH 7.4, 1% SDS,

2.6.2. Gel electrophoresis, electroblotting and antigen detection Proteins were separated by SDS-PAGE on 4% stacking and 5% or 9% running gels. The proteins were transferred

a. Virnentin

d. N F - L

AbPbP4L1 1 2 3 4 5 6 7 8

Ad P1 L1 1 2 3 4 5 6 7 8

150-

20211898-

1189869-

47-

b. Nestin

e. N F - M

P4St st' L1 1 2 3 4 5 6 7 8

Ad P1 kl 1 2 3 4 5 6 7 8 202118"98-

202-

6947-

150-

c. P e r i p h e r i n

f. N F - H

AdP1L11

AdP1L1 1 2 3 4 5 6 7 8

2 3 4 5 6 78

20211898-

11898-

69-

69-

47-

"- o

47-

Fig. 2. Intermediate filament immunoblots. In each of the six blots (a-f), lanes designated 1-8 are: 1, RT4-AC; 2, RT4-AC C/T; 3, RT4-D; 4, RT4-D C/T; 5, RT4-B; 6, RT4-B C/T; 7, RT4-E; 8, RT4-E C/T. C/T indicates that the cells were grown in the presence of cAMP and DHT (maturation conditions; see Section 2. In all cases 3T3-L1 adipoblasts (L1) were used as a negative control. Positive control samples include: postnatal day 1 and day 4 rat cerebellum (P1 and P4 respectively), adult rat cerebellum (Ad), adult and postnatal day 12 rat brain (Ab and Pb respectively) and ST15A cells (St). The lanes designated 1-8 in a and St' in b had 2 /.tg of protein loaded as opposed to the usual 20 p.g/lane. Positions for molecular weight standards are shown on the left hand side of each immunoblot.

LM Oonahueetal,/DevelopmentalBrainResearch94(1996)67-80 to Immobilon P membranes (Millipore, Bedford, MA) by electroblotting overnight at 40 mA in blot buffer (12.5 mM Tris, 96 mM glycine, 0.01% SDS, 10% methanol) and antigen-antibody binding was visualized by chemiluminescence. Briefly, after electroblotting, filters were blocked in 5% nonfat dry milk in TBS for 30 min. at room temperature (RT), washed 1 X 30 min in TBS at RT, exposed to primary antibody in 1% milk in TBS overnight at 4°C, washed 2 X 30 rain in TBS at RT, exposed to horseradish peroxidase (HRP) conjugated secondary antibody in 1% milk in TBS for 30 min at RT and then washed 3 X 30 min in TBS at RT. After the final TBS wash, filters were placed into Renaissance chemiluminescence reagents (DuPont/NEN), and then exposed to X-ray film for 2-60 min.

2.7. Antibodies The following antibodies were used for immunoblotting at the dilutions indicated. Vimentin: 1:40000 of mouse monoclonal anti-vimentin (V9, Sigma); secondary Ab = 1:10 000. Nestiu: 1:10 000 of mouse monoclonal anti-nestin (Rat-401) developed by Ronald D.G. McKay, obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore MA 21205, and the Department of Biology, University of Iowa, Iowa City, IA 52242; secondary A b = 1:10000. Peripherin: 1:10000 of rabbit anti-peripherin (gift from Edward Ziff, New York University Medical Center, New York, NY); secondary Ab = 1 : 10 000. N e u r o f i l a m e n t (NF-L): 1:4000 of mouse monoclonal anti-neurofilament 68 (NR4, Sigma, St. Louis, MO); secondary Ab = 1:20 000. N e u r o f i l a m e n t (NF-M): 1:4000 of mouse monoclonal anti-neurofilament 160 (NN18, Sigma); secondary Ab = 1:20000. N e u r o f i l a m e n t (NF-H): 1:4000 of mouse monoclonal anti-neurofilament 200 (NE14, Sigma); secondary Ab = 1:20000. MAP2:1:5000 of mouse monoclonal anti-MAP2 (HM-2, Sigma); secondary A b = 1:10000. /~-tubulin III: 1:4000 of mouse monoclonal anti-/3-tubulin III (SDL.3D10, Sigma); secondary Ab = 1:10000. Tau: 1:20000 of mouse monoclonal anti-tan (anti-Tan-l, Boehringer Mannheim, Indianapolis, IN); secondary Ab = 1:20000. N-CAM: 1:10000 of rabbit anti-N-CAM (AB1505, Chemicon, Temecula, CA); secondary Ab = 1:10000. GAP-43:1:1000 of mouse rnc~nc~elnnal anti-CTAP-43 (91E12. #1379011. Boehrin~er

7l

3. R e s u l t s

3.1. Intermediate filaments Intermediate filament protein expression was assayed by immunoblot analysis (Fig. 2). For all six immunoblots, the lanes designated numbers 1 through 8 are: 1, RT4-AC; 2, RT4-AC C / T ; 3, RT4-D; 4, RT4-D C / T ; 5 RT4-B; 6, RT4-B C / T ; 7, RT4-E; 8, RT4-E C / T where C / T indicates that the cells were grown in the presence of cAMP and DHT (maturation conditions; see Section 2. In all cases 3T3-L1 adipoblasts (L1) were used as a negative control and positive controls included: postnatal day 1 and day 4 rat cerebellum (P1 and P4 respectively), adult rat cerebellum (Ad), adult and postnatal day 12 rat brain (Ab and Pb respectively) and STI5A cells (St, an immortalized CNS neuronal precursor cell line [18]).

3.1.1. Vimentin Immunoblotting revealed that vimentin was abundantly expressed at relatively equal levels in all of the RT4 cell lines, whether they were C / T treated or untreated (Fig. 2a). Although vimentin has a molecular weight of ~ 54 kDa it may run at a higher apparent molecular weight on SDS-PAGE due to differences in the phosphorylation state of the protein [37]. Note that 2 /~g of total protein extract was loaded in each of the RT4 lanes (1-8), whereas 20/~g was loaded in both the positive (Ab, Pb, P4) and negative (L1) control lanes. 3.1.2. Nestin As shown in Fig. 2b, nestin is expressed in all the RT4 cell lines at approximately equal levels and this level of expression did not change appreciably upon C / T treatment. The calculated molecular weight of nestin is ~ 200 kDa [37]; however it appears significantly larger on SDSPAGE and is often visualized on immunoblots as a cluster of 2 - 4 high molecular weight bands [59,60]. Note that 20 /~g of protein was loaded into the positive control lane designated St and that only 2 /~g was loaded in the lane designated St'. We also observed the expression of nestin mRNA in all of the RT4 cell lines by RNase protection assay analysis (data not shown). 3.1.3. Peripherin The apparent molecular weight of peripherin as determined on SDS-PAGE is ~ 57-58 kDa [37]. We did not

72

L,M,Donahueet al,/ DevelopmentalBrainResearch94 (1996)67-80

a, MAP2a,b

c, ClassIII B.tubulin

AbPbP4L112 3 4 5 6 78

P1P4AdL112345678 1189869-

202,15098.-

4730-

b. MAP2c

d. T a u

AbPbP4L11 2 3 4 5 6 7 8

PbP4Pc L l l

2 3 4 5 6 7 8

202-

~o..

~ ~ii~i~ii~i~i

98-

~'~

~. - ~ i ~ i

.....

~

20215098" 69"

47.. 4730Fig. 3. Microtubule and associated protein immunoblots. All lane designations are the same as in Fig. 2 except for the addition of PC12 cells (Pc).

a. SCG10 M

P

t

c. N-CAM Rb Pc AC

500400 -

D

E

B

AdP1L1 1 2 3 4 5 6 7 8

2021189869-

300200-

47-

d. GAP-43 PbP4PcL1 1 2 3 4 5 6 7 8 b. SCG10 c

AC f D M E B

2029869-

47*30118p-

L,M,Donahueet al,/ DevelopmentalBrainResearch94 (1996)67-80 indicate light, middle and high molecular weights, respectively [37]. We were unable to detect expression of any of the neurofilament triplet proteins in any of the RT4 cell lines, even upon C / T treatment (Fig. 2d-f). Identical results were obtained by immunocytochemistry (data not shown).

3.2. Microtubule and microtubule associated proteins The expression of four microtubule and microtubule associated proteins was assayed by immunoblotting. Tau and /3Ha-tubulin are neuron-specific while the MAP2 proteins are expressed in both neurons and glia (see Section 4). Lanes designated 1-8 and the negative and positive control samples are the same as described above for the intermediate filament immunoblots (Fig. 2) except that PC12 cells (Pc) were also used as a positive control.

3.2.1. MAP2 MAP2 has three isoforms: MAP2a, MAP2b and MAP2c, which on SDS-PAGE are 280 kDa, 270 kDa and 70 kDa respectively [37]. We were unable to detect the higher molecular weight MAP2a or MAP2b in any of the RT4 cell lines with or without C / T treatment (Fig. 3a), but observed expression of the smaller, more embryonic MAP2c [52] in all of the RT4 cell lines (Fig. 3b). MAP2c was expressed at similar levels in all of the RT4 cell lines (although possibly 2 fold less in RT4-B and RT4-E compared to RT4-AC or RT4-D) and its relative abundance was unaffected by C / T treatment. 3.2.2. ~in-tubulin All /3-tubulin isoforms are ~ 55 kDa [37]. By immunoblot analysis we were unable to detect ~iii in any of the RT4 cell lines even upon C / T treatment (Fig. 3c).

73

RT-PCR, the RT4 cell lines are designated as above and negative control samples consisted of no RNA (C) and NIH 3T3 fibroblast RNA (f). M designates HaeIII digested q~X174 DNA size markers. For the immunoblots, all lane designations are the same as previously described.

3.3.1. SCGIO Superior cervical ganglion clone 10 (SCG10) is a small growth-associated protein [55]. Since SCG10 is one of only a small number of proteins that are expressed early in development and are neuron-specific, it is an important marker. Fig. 4a is an RNase protection assay using an SCG10 probe made from the plasmid, SCG10-8.6 (see Section 2). The probe is ~ 409 nt and the size of the SCG10-specific protected fragment is ~ 399 nt. Fig. 4a demonstrates that SCG10 was expressed in PC12 cells (Pc) as predicted, and also in RT4-B. A longer exposure of the gel reveals that SCG10 was also expressed in the stem cell line, RT4-AC (lower panel of Fig. 4a), but not by either RT4-D or RT4-E. Treatment with cAMP and DHT caused little or no effect on SCG10 mRNA levels (data not shown). Using RT-PCR we confirmed the presence of SCG10 mRNA in RT4-AC and RT4-B by amplification of a 135 bp SCG10 specific fragment. This fragment was not amplified from RT4-D and RT4-E (Fig. 4b). 3.3.2. N-CAM There are three major isoforms of the neural cell adhesion molecule (N-CAM) with apparent molecular weights

a. T H Pc L1 1 2 3 4 5 6 7 8 20298-

3.2.3. Tau Tau purified from mammalian brain resolves as four to six isoforms ranging in molecular mass on SDS-PAGE from 35-65 kDa [37] and two higher molecular weight forms of 103-125 kDa are found in the PNS [9]. We were unable to detect tau expression in any of the RT4 cell lines irrespective of C / T treatment (Fig. 3d).

6947-

b. C h A T

3.3. Neural lineage associated markers SCG10 expression was analyzed by RNase protection assay and RT-PCR, while N-CAM and GAP43 expression were examined on immunoblots (Fig. 4). For the RNase protection assays, lanes designated AC, D, E and B refer to poly (A) + mRNA from the RT4 cell lines, probe alone is designated P, and PC12 cell poly (A) + mRNA (Pc) and postnatal day 5 total rat brain RNA (Rb) were used as positive control samples. A tRNA control sample (t) was added to ensure that the RNase digestion went to completion and M designates molecular weight markers. For

M

P

Pc 1

2

3

4

5

6

7 8

t

500400300; 200-

Fig. 5. Neurotransmitterexpression. (a) TH immunoblot and (b) ChAT RNase protection assay. In both cases the lane designations are the same as in Fig. 2, Fig. 3, and Fig. 4.

74

L.M. Donahue et al. / Developmental Brain Research 94 (1996) 67-80

L.M. Donahue et al. / Developmental Brain Research 94 (1996) 67-80

on SDS-PAGE of 120, 140 and 180 kDa respectively [36]. We analyzed N-CAM expression by immunoblotting using an antibody directed against the protein sequence of the extracellular domain common to all three isoforms [51]. As shown in Fig. 4c, all four RT4 cell lines, untreated as well as C / T treated, express the 140 and 180 kDa isoforms of N-CAM. Expression of the 120 kDa isoform is readily detected in RT4-E (lanes 7 and 8) and although less distinct, is probably also present in RT4-B, -D and AC. Treatment with cAMP and DHT did not cause any detectable change in either (i) isoform expression or (ii) relative abundance of any individual isoform. 3.3.3. G A P - 4 3

Growth associated protein-43 (GAP-43) migrates with an apparent molecular weight of 4 3 - 5 7 kDa on SDS-PAGE [53]. Although we detected GAP-43 in the positive control samples of P I 2 rat brain and P4 rat cerebellum, immunoblotting did not reveal the presence of GAP-43 in any of the RT4 cell lines even upon C / T treatment (Fig. 4d). 3.4. Neurotransmitters

Tyrosine hydroxylase (TH) expression was assayed by immunoblot analysis while choline acetyltransferase (CHAT) expression was assayed by RNase protection assay analysis (Fig. 5). For both the immunoblot and RNase protection assay gels, all lane designations are the same as previously described. 3.4.1. T H

TH has an apparent molecular weight of ~ 60 kDa [23]. While we were able to detect abundant expression in PC12 cells (Pc), we were unable to detect TH immunoreactivity in any of the RT4 cell lines. Treatment of the RT4 cells with cAMP and DHT did not induce TH expression (Fig. 5a),

75

3.5. Ultrastructure

Because a greater percentage of RT4-E cells, compared to RT4-B cells, respond to C / T treatment by extension of long neurite-like processes we examined the morphology of C / T treated RT4-E in greater detail (Fig. 6). Phase contrast microscopy of RT4-E cells treated with cAMP and DHT suggested that two distinct cell populations were present (Fig. 6A). Most cells were phase-dull and fiat with broad processes that were relatively featureless. The other population (less than 10%) were phase-bright, rounded multipolar cells with thin processes that branched occasionally and resembled neurites. Some processes extended for considerable distances ( > 1 mm) over the fiat cells or plastic substrate. Scanning electron microscopy confirmed the presence of two morphologically different cell populations: numerous broad, flat cells that were smooth-surfaced except for occasional microvilli, and less frequent rounded, multipolar cells that had neurite-like, smooth processes and abundant microvilli on their cell bodies (Fig. 6B). Some processes showed varicosities along their length (arrow, Fig. 6B), Transmission electron microscopy revealed epitheliallike cells with large euchromatic nuclei (Fig. 6C) that were occasionally infolded. Some cells contained abundant polyand single ribosomes (although not organized into an extensive rough endoplasmic reticulum), perinuclear Golgi complexes, and mitochondria. Centrioles were regularly discerned. Microtubules and intermediate filaments were seen in profiles of processes. No evidence of specialized junctional complexes or synapses were observed, although RTg-E cells grown in the presence of cAMP and DHT demonstrate gap junctional coupling (I. Martinez, L. Donahue, D. Webster and D. Spray, unpublished observation).

3.4.2. C h A T

4. Discussion

ChAT expression was assayed by RNase protection assay (Fig. 5b). The ChAT probe was ~ 312 nt in length and the predicted length of the protected fragment was ~ 253 nt (see Section 2). Although we could detect expression of ChAT in PC12 cells (Pc) we were unable to detect expression in any of the RT4 cell lines even upon C / T treatment. In addition, using RT-PCR we were unable to detect ChAT expression in any of the RT4 cell lines (data not shown).

This study was undertaken to better document the developmental stages and lineages represented by the RT4 cell line family. The expression of 16 different marker proteins was analyzed in the RT4 cell lines. The marker proteins were chosen because they were either (i) nervous system-specific or neuron-specific in their expression or (ii) are widely used by neurobiologists. While in some cases their expression may not be confined solely to the

Fig. 6. cAMP and DHT induce an immature neuronal morphology in some RT4-E cells. (A) Low power phase contrast micrograph shows two different kinds of cells, one of which appears neuronal-like with long, thin, sometimes branched processes. Non-neuronal appearing cells are flat and more numerous. Bar = 50 /~m. (B) Scanning electron micrograph. Three neuronal-like multipolar cells that are rounded and have long thin processes are present, as well as non-neuronalappearing fiat cells. Note the varicosity(arrow) on a process. Bar = 10/~m. (C) Transmission electron micrograph of a portion of a RT4-E cell. The cell appears immature, although features that could be interpreted as neuronal include a large euchromatic nucleus (Nu), abundant perinuclear Golgi complexes (arrowheads), ribosomes and mitochondria. Bar = 5 /~m.

L.M. Donahue et aL / Developmental Brain Research 94 (1996) 67-80

76

Table 1 Neural m a r k e r expression in the R T 4 cell line family Class

Marker

RT4-AC RT4-D RT4-B RT4-E stem glial neuronal neuronal

Intermediatefilaments Vimentin Nesti~ Periph erin NFs

+ + . .

. .

. .

. .

Microtubule a n d associated proteins

.

.

.

.

MAP2c + /3m-tubulin . Tan .

. .

. .

. .

Neural lineage associated

Neurotransmitter

MAPTa, b

+ +

+ +

+

SCG10

+

-

N-CAM GAP-Z-3

+ .

.

.

.

TH ChAT

. .

. .

. .

. .

+

+ +

+

+

+

-

+

+

S u m m a t i o n o f m a r k e r expre~,;sion studies. All markers were also e x a m ined in e a c h R T 4 cell line after treatment with c A M P and D H T ( C / T treatment). In all cases, m a r k e r expression w a s u n c h a n g e d by C / T treatment.

nervous system, these proteins are considered to be traditional neural markers whose cell type a n d / o r developmental expression patterns in the nervous system are well documented.

4.1. Lineages represented by RT4-B and RT4-E Results from our study, discussed below and summarized in Table 1, establish that RT4-B is an immature neuronal cell line based primarily on its expression of SCG10, a marker of early neuronal differentiation [1,55]. In contrast, the classification of RT4-E remains more difficult. RT4-E does not express SCG10 and only expresses markers of immature neural tissue such as nestin, vimentin, MAP2c and N-CAM. The lack of neuronalspecific markers expressed by RT4-E indicates that it may be either poised to become neuronal or it may represent another potential neural crest lineage.

4.2. Developmental stage represented by the RT4 cell line family Although it is impossible to pinpoint the in vivo developmental stage represented by the RT4 cell lines, taken together, the data from this study give a strong indication of relative developmenlal timing. In this study we found that all of the RT4 cell lines expressed only markers of immature neural tissue such as nestin, vimentin, MAP2c and N-CAM, and RT4-B and RT4-AC were found to express SCG10. However, none of the RT4 cell lines expressed markers of more mature neural tissue, such as the neurofilament triplet proteins, peripherin, MAP2a, MAP2b, tau, or CHAT. The combination of markers that is

expressed, in addition to those that could not be detected, most closely resembles those expressed by neural crest derivatives at E10-E12, suggesting that the RT4 cell lines are immature. These findings are discussed in greater detail below.

4.2.1. Intermediate filament expression Because neither the neurofilament triplet proteins nor peripherin were expressed by the RT4 cell lines, we thought it possible that they were developmentally immature. Previous reports had demonstrated that (i) neurons that originate from migrating rat neural crest cells do not express NF-L or peripherin until they have reached their final destination [14] and (ii) cultured trunk neural crest stem cells from El0.5 rat embryos do not express NF-M or peripherin [56]. Therefore, we thought that the RT4 cells might express nestin a n d / o r vimentin as their IF protein(s). Immunocytochemical analysis in the rat indicates that vimentin is expressed in embryonic CNS and PNS neurons, but only transiently between embryonic days 12 and 14 [3] and is not usually expressed in adult neurons [7]. Vimentin is expressed by Schwann cells throughout their development and remains expressed even in the adult [7]. We found abundant vimentin expression in all of the RT4 cell lines. Nestin is expressed by mammalian CNS precursor cells and myoblasts transiently during embryogenesis [42,59,69] and has been demonstrated to co-localize with vimentin [7,37]. In the rat neural crest, nestin is expressed transiently by neural precursors and is extinguished in neuronal cells [56]. However, nestin expression in Schwann cells, like vimentin expression, remains throughout development and persists in the adult [21,56]. We found nestin expression in all of the RT4 cell lines. 4.2.2. Microtubule and microtubule-associated proteins In rat and quail, the expression of the three MAP2 proteins is developmentally regulated [52,65]. In rat brain, MAP2c is first detected in embryos, but its expression is difficult to detect later in the adult. MAP2b and MAP2a appear perinatally and between 10-15 days postnatally respectively [52]. High levels of both MAP2a and MAP2b persist in the adult where they are expressed only by neurons [63]. MAP2c however, is found in both neurons and glia (reviewed in [11,25,47]). We were unable to detect MAP2a or MAP2b expression in any of the RT4 cell lines. However, we did observe MAP2c expression in all of the RT4 cell lines, including the glial derivative RT4-D. In the PNS, both the low molecular weight (LMW) and the high molecular weight (HMW) taus are present early in development, while in the mature PNS the HMW forms are expressed almost exclusively [26]. We were unable to detect expression of LMW or HMW tau in any of the RT4 cell lines. The observation that the RT4 cell lines express MAP2c but not tan is significant in terms of determining develop-

L.M. Donahue et al. / DeuelopmentalBrain Research 94 (1996) 67-80

mental stage. This same pattern was seen in quail neural crest cells at day 3 of embryonic development ([64]; by day 3.5 tau was also expressed). Day 3 of quail development corresponds to a stage 18 embryo with ~ 30 somites which is seen at approximately E 1 1 - E l 2 in the rat. In nervous tissue, class III /3-tubulin is expressed solely by neurons and preferentially by neurons which are in the later stages of neuritic and axonal outgrowth [16,34]. We were unable to detect expression of /3iirtubulin in any of the RT4 cell lines. 4.2.3. Neural lineage associated markers

Superior cervical ganglion clone 10 (SCG10) is a member of a family of small (22 kDa), developmentally regulated, growth associated proteins that was isolated as a neuronal marker of neural crest [55]. By in situ hybridization and Northern blot analysis, expression of SCG10 appears to be strictly neuronal and has been found in both the developing CNS and PNS [1,55]. The earliest stage that SCG10 could be detected in the rat PNS was at E l l . 5 in the cranial ganglia primordia. Later at E13.5 SCG10 was found in both sympathetic and parasympathetic ganglia [ 1]. We were able to detect low levels of SCG10 mRNA in the RT4 stem cell line, RT4-AC and much higher levels in the neuronal derivative, RT4-B. We did not detect SCG10 mRNA in either RT4-D or RT4-E cells. Neural cell adhesion molecule (N-CAM) is one of the most abundant adhesion molecules of neural cells [reviewed in 36]. N-CAM is a membrane glycoprotein encoded by a single gene, however multiple isoforms exist due to differential mRNA splicing and extensive posttranslational modification [36]. There are three major forms of N-CAM: 120, 140 and 180 kDa [36]. In vertebrates, N-CAM is first detected during gastrulation, continues to be expressed throughout the development of the CNS and PNS, and remains expressed at reduced levels in the adult [33]. N-CAM is expressed by neurons, glia, muscle, and hematopoetic cells [reviewed in [39,54]]. All of the RT4 cell lines express the three major N-CAM isoforms with some variation in abundance. GAP-43, a membrane-bound growth associated protein, is widely expressed in the adult rat PNS ([57] and references therein). We were unable to detect GAP-43 expression in any of the RT4 cell lines. 4.2.4. Neurotransmitters

TH is the rate limiting enzyme in the synthesis of norepinephrine and is found in noradrenergic neurons, dopaminergic neurons and adrenal chromaffin cells [36]. TH was first detected by immunocytochemical analysis in the rat PNS on E l l . 5 - E 1 2 . 5 in both the sympathetic ganglia primordia and in scattered cells in the gut [2,8]. By immunoblot analysis we were unable to detect TH in any of the RT4 cell lines. Interestingly, it was previously observed that after thirty days of culture in defined medium, RT4-AC cells expressed norepinephrine [5]. Our studies

77

were conducted with cells grown in serum containing medium. In addition, it has also been reported that the presence of exogenous cAMP analogs inhibited TH expression in quail neural crest cultures [45]. Therefore, it is possible that rather than being too developmentally immature to express TH, our culture conditions may have prevented us from seeing the adrenergic phenotype (see below). Choline acetyltransferase (CHAT) is the enzyme that catalyzes the acetylation of choline to form acetylcholine (ACh). ChAT is first detectable immunocytochemically in ciliary ganglia of 5.5 day old (stage 28, > 44 somites) quail embryos [10], which although difficult to directly compare to rat is at least older than an El3 (40 somite) rat embryo. In vitro studies demonstrated that ChAT immunoreactivity was first detected four days after E 9 . 5 - E I 0 mouse neural crest was put into culture [44]. However, the timing of appearance of ChAT in neural crest explants depends upon the axial level of the crest examined. For example, ChAT was detected two days earlier in quail cranial crest cultures compared to trunk cultures (5 versus 7 days respectively [40]). RNase protection assay analysis showed no ChAT expression in any of the RT4 cells. RT-PCR to examine ChAT expression confirmed our RNase protection assay findings (data not shown). Care must be taken in the interpretation of the lack of TH and ChAT expression in the RT4 cell lines. Culture conditions have a dramatic effect on which neurotransmitters are expressed, particularly in sympathetic neurons (reviewed in [29]), probably reflecting interactions between neighboring cells, including target selection [38]. Therefore, although it is possible that the RT4 cell lines are developmentally immature and have not made a neurotransmitter choice, it is also possible that our culture conditions do not allow for the expression of TH or CHAT. However, it should be noted that the observed lack of neurotransmitter expression would not be unexpected if the RT4 neuronal derivatives are developmentally poised around E10-E12 as we propose. 4.3. Effects o f cAMP and D H T on maturation in the RT4 system

Previously, treatment with cAMP and testosterone derivatives was shown to cause process extension in RT4-B and RT4-E, as well as induce synthesis of both myelin basic protein and a glial-specific high affinity uptake mechanism for GABA in RT4-D cells [13,30]. In contrast to that previous work, we did not observe any effects upon the expression of the 16 marker genes examined in this study. None of the markers we examined were either newly induced or appreciably up- or down-regulated by treatment with cAMP and DHT. We did however observe morphological changes consistent with what had been reported previously [13]. Our detailed analysis examining the morphology of RT4-E cells treated with cAMP and

78

L.M. Donahue et al. / Developmental Brain Research 94 (1996) 67-80

Table 2 Comparison of neural marke.r expression in three stem cell systems and their neuronal derivatives Marker

P19 Neural RT4-AC RT4-B,-E P19 Neural stem crest stem neuronal neuronal crest cell stem cell cell neuronal

Nestin MAP2c LNGFR NFs Peripherin Tan MAP2a, b TH/catechol ChAT/ACh

-

+

+ + + . -

+ . . . -

.

. -

.

. .

+ + ___

+ -

. .

. -

+ +

very similar to that expressed by neural crest derived tissues from E10-E12 rat embryos, (iii) conditions used to cause maturation of the RT4 derivative cell types (cAMP and DHT) have only modest effects on the RT4 neuronal derivatives, and (iv) when compared to other neural stem cell systems RT4-AC generates the most immature neuronal derivatives.

+ +

. -

+

Compared to other stem cell systems, RT4 generates the most immature neuronal derivatives. Expression of the neural marker genes listed in the left-most column is compared between the stem cells and neuronal derivatives of RT4, P19 embryonal carcinoma cells [15,35,46,48,58] and primary cultures of neural crest [56]. + indicates expression was detected, - indicates expression was not detected, and blank indicates that this information is unknown.

Acknowledgements This study was supported by grants from the Muscular Dystrophy Association and the National Institutes of Health (R29HD29400). The authors wish to thank Dr. Kurt Droms for valuable discussions and critical reading of the manuscript. We also wish to thank Darrell Eubank, Matthew Laverdiere, Pattye Staub and Marilyn Tnrnbow for technical assistance.

References DHT indicates that RT4-E has some features that are consistent with the hypothesis that these cells are immature neurons. However, it is clear from this study that cAMP and DHT treatment, while causing significant maturation in the glial lineage of RT4, has more modest effects in the other RT4 cell types. 4.4. Comparison of RT4 to other in vitro neural stem cell systems

Table 2 is a compari son of neural marker expression in two multipotential cell lines and one primary culture system: RT4-AC, P19 embryonal carcinoma cells [15,35,46,48,58] and primary cultures of neural crest stem (NCS) cells [56]. In thi,; comparison, it is apparent that the RT4 neuronal derivatives are immature compared to the neuronal cell types derived from either P19 cells or NCS cells. P19 and NCS cell-derived neurons expressed markers such as NF, peripherin, tau, MAP2a, MAP2b and cholinergic properties. As discussed above, these markers are all expressed by more mature neuronal cell types and are not expressed by the RT4 neuronal derivatives, RT4-E and RT4-B. This is an important observation since it indicates that the RT4 system may allow for the study of early decisions in neuronal maturation that other systems do not. 4.5. Conclusions

The data from this study has led to four important conclusions concerning the RT4 system: (i) the previously designated RT4 'neuronal-like' derivatives express properties of immature neurons, (ii) the combination of markers expressed by the RT4 stem cell and neuronal derivatives is

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