Stimulatory effect of transferrin on the proliferation of embryonic mouse molar pre-odontoblasts and pre-ameloblasts in organ culture

Arrhs oral Bid. Vol. 34, No. 3, pp. 153-159. Printed in Greal Britain. All rights reserved

1989 Copyright

STIMULATORY

EFFECT

PROLIFERATION

OF TRANSFERRIN

OF EMBRYONIC

PIRE-ODONTOBLASTS

AND

IN ORGAN

de Biologie

Mtdicale,

0003-9969/89 $3.00 + 0.00 1989 Pergamon Press plc

ON THE MOLAR

PRE-AMELOBLASTS

CULTURE

Y. CAM, A. BOUKARI and Institut

MOUSE

0

J. V. RUCH

INSERM CJF No. 88-08, Faculte 67085 Strasbourg Cedex, France

de M&de&e,

II, rue Humann,

(Accepted 28 September 1988) Summary-The effects of transferrin on the proliferation kinetics of these cells from day-14 lower first molars, cultured for 2-6 days in a chemically defined medium supplemented with 5 and 50 pg/ml of human diferric transferrin, were studied. Transferrin stimulated the mitotic and [)H]-thymidine labelling indices. These data were correlated with immunolocalization of the transferrin receptor using indirect immunofluorescence and specific monoclonal antibodies. The presence of specific transferrin receptors in pre-odontob:.asts and pre-ameloblasts, and in ameloblasts of older teeth (day-18 to day-21), was also assessed by indirect immunofluorescence and binding experiments using iodinated transferrin.

However, previous studies on the effects and possible role of Tf in mouse tooth development have not considered in detail the kinetics of pre-odontoblasts (PO) and pre-ameloblasts (PA) and the localization of specific Tf receptors in vim We have therefore focussed our attention on the cytokinetics of dental cells of cultured tooth germs in the presence of Tf and looked for Tf receptors in these cells in vice. We have sought to determine the mitotic and the [3H]-thymidine labelling indices of PO and PA in mouse first lower molars cultivated in the presence of Tf, and also to measure the binding of [‘*‘I]-Tf to epithelial and mesenchymal cells of molar components freshly separated by trypsin.

INTRODUCTION

respective roles of genetic and environmental factors in tooth development are still controversial. The crown size and morphology of a specific tooth is a function mainly of the number of post-mitotic odontoblasts and ameloblasts and of their spatial distribution. The metabolic activities of these cells, which control the amount of dentine and enamel, also play important roles. Several experimental results (Ruth, 1985; Ahmad and Ruth, 1987) have led to the hypothesis that the odontoblast and ameloblast genetic programmes may determine a minimal number of cell cycles before overt differentiation can be triggered by specific, epigenetic, matrix-mediated interactions. Overt odontoblast differentiation is triggered by a stage- and space-related basement membrane (Ruth, 1985). The terminal differentiation of ameloblasts occurs in the presence of the predentinedentine complex ( Karcher-Djuricic et al., 1985). Circulating, paracrine and/or autocrine factors might intervene in the epigenetic control of odontogenesis. Epidermal growth factor (EGF), associated with serum and collagen or non-viable dental mesenchyme, has Ibeen shown to enhance the proliferative potential of odontogenic epithelial cells (Brownell and Rovero, 1980; Steidler and Reade, 1981). Partanen, Ekblom and Thesleff (1985) have described an inhibitory effect of EGF on tooth morphogenesis during the early cap stage that might be mediated by the dental mesenchyme. Partanen, Thesleff and Ekblom (1984) and Partanen and Thesleff (1987a, b:l found that transferrin (Tf), an iron-binding serum protein (Ward, 1987) was necessary for the in vitro completion of the bud and early cap stages of mouse molar teeth. Tf is an essential component of chemically defined media for the growth of cells in culture (Barnes and Sato, 1980) and is considered to be a fetal growth factor by Ekblom et nl. (1981, 1983). The

MATERIALS

AND METHODS

Collection and preparation of tissues Day-14, - 18, - 19, -20 and -2 1 lower first molars and day-12 and -14 heads of laboratory-raised Swiss mouse embryos and postnates were dissected free in Hanks’ balanced salts solution (HBSS; Gibco, Paisley, Scotland; vaginal plug: day 0). Trypsin dissocation Dental papillae and enamel organs of 18-day-old embryonic molars were isolated by incubation for 90 min at 4°C in 1% trypsin (Difco I : 250) in HBSS. After washing in a HBSSfetal calf serum (FCS) mixture (1: 1, v/v), the dental components were mechanically dissected. Tissue culture Four to six day-14 molars were placed upon Millipore filters (150 pm HAPB, 0.45 pm pore size) and incubated for 2-6 days in Petri dishes, as described by Boukari and Ruth (1981). Control medium was made of RPM1 1640 (Gibco) containing 2mM glutamine (Gibco), 740pm ascorbic acid (Merck, Darmstadt, F.R.G.), penicillin (5 IU/ml) and streptomycin 153

154

Y. CAM et al.

(50 pgg/ml) (Gibco). Experimental cultures were carried out with supplements of either human Tf (5 and 50 pg/ml) loaded with ferric ions [diferric Tf: Tf (Fe),], according to the procedure described by Karin and Mintz (1981), or 50 pg/ml of bovine serum albumin (BSA). Radiolabelling, histology and autoradiography In order to determine the labelling indices of dental cells, medium was changed on the second or the sixth day, and explants were incubated for 60 min at 37°C in their respective media containing 1 pCi/ml of [methyl-3H]-thymidine (CEA, Gif-sur-Yvette, France; sp. act. 45 Ci/mmol). Incubation was stopped by transferring filters to unlabelled HBSS. Three additional washes in the same solution were carried out before fixation. Cultured germs were fixed in tooth Bouin-Holland’s solution, embedded in paraffin, serially sectioned at 5 pm and counterstained with Masson’s trichrome. Slides were then deparaffinized and coated with Kodak AR10 stripping film. The slides were dried, exposed for 10 days, developed for 5 min at 25°C in Kodak D19 developer and fixed for 3 min in 30% (w/v) sodium thiosulphate solution. After rinsing in water, slides were stained with Mayer’s haemalum solution (Merck). Determination of mitotic indices (MI) A minimum of 1000 PO and PA was counted for each control and experimental culture on alternate sections of at least 3 teeth, using a Leitz microscope equipped with a Planapo x 100 objective for oil immersion. Cells in late prophase, metaphase, anaphase and early telophase were counted. The percentages of mitotic cells or MI were then calculated for both types of cells. Determination of labelling indices (LI) Alternate sections of serial sections of samples prepared for autoradiography were analysed; each labelled cell was counted. At least 3 teeth were examined for each culture condition and the percentages (LI) of labelled PO and PA were determined. Background was determined by counting the grains corresponding to the metaphases (Wright and Alison, 1984).

incubated for 30min at room temperature in a humidified atmosphere with samples of supernatants of hybridoma culture media. After three washes in phosphate-buffered saline (PBS), the slides were further incubated for 30min at room temperature with diluted (I:40 in PBS) solutions of fluoroisothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulins (Cappel Laboratories, Cochranville, Penn., U.S.A.). After three more washes in PBS, the preparations were mounted, examined and photographed in a Leitz Orthoplan microscope equipped for fluorescence with an Orthomat automatic microphotograph device. Binding 0j”[‘~~1]-Tf(Fe), to day- 18 molar components Five to eight dental papillae and enamel organs were used per time point. Experiments were run in duplicate. Preincubation. Trypsin-isolated papillae and enamel organs of day- 18 molars were preincubated at 4°C for 20min with soybean trypsin-inhibitor (Sigma, St Louis, MO., U.S.A.) in order to inactivate residual trypsin (1 mg inhibitor inactivating I mg trypsin). Tooth components were then washed and preincubated for 2 h at 37°C in the binding medium of RPM1 1640 (Gibco), devoid of bicarbonate, and containing 0.1% (w/w) BSA and 25 mM 4-(2-)hydroxyethyl-I-piperazine-ethane-sulphonic acid (Hepes pH 7.7). Incubation. Dental papillae and enamel organs were incubated for 3-15 min at 30°C in the binding medium (Text Fig. 1) supplemented with [‘*‘I]-Tf(Fe), (NEN, Du Pont de Nemours, Paris, France, sp. act. 0.3-l.OpCi/pg) at a final concentration of 0.6 pg/ml. Incubation was stopped by putting the samples on ice. They were quickly rinsed (4 x ) in PBS (pH 7.4). In order to know the amount of labelled Tf bound to plasma membranes, tissues were treated on ice for 2 x 10 min with a stripping 0.2 M acetic acid solution containing 0.5 M NaCl. The samples were finally dissolved for 2 h in 1 M NaOH. Samples of stripping solutions and samples dissolved in NaOH were counted in a Packard 5230 autogamma scintillation spectrometer. The sum of these two counts for each

.

Statistical method Means of mitotic and labelling indices were compared by using the parametric Student’s t-test.

l

Immunocytochemical study of Tf receptors We used monoclonal antibodies (IgG 2a class) directed against mouse Tf receptors present ar the surface of a Friend erythro-leukaemia cell line. These antibodies were produced according to Lesley et al. (1984) by a hybridoma cell line (TIB 219) commercialized by the American Type Culture Collection (Rockeville, Md, U.S.A.). Mouse bone marrow cells, smeared on slides and immediately frozen, served as a positive control for our antigen-monoclonal antibody reaction. Frozen frontal sections (8 pm) of day-12 and day14 mouse fetal heads and sagittal sections of mouse lower molars from day-18, -19, -20 and -21 were

B

0

.

0 0

I

0 *

Incubation

time (mini

Fig. 1, Specific binding of [‘251]-transferrin to dental papillae (0) and enamel organs (A) freshly dissociated from day- I8 mouse molars.

Effects of transferrin on mouse molar growth

sample equals the total (external plus internalized, specific plus non-specific) binding of the sample. Determination of the spec$c binding. A dosedependent curve was obtained by incubating tissues for 10 min with a fixed quantity of [‘*‘I]-Tf(Fe), (0.6 pg/ml) and increasing amounts of unlabelled Tf(Fe), (6600pg/ml). From this curve we deduced the minimal concentration of unlabelled Tf(Fe), which renders minimal the amount of Tf(Fe), bound, that is the non-specific binding. By incubating as described above, except that we added the minimal concentration of unlabelled Tf, we obtained the non-specific bindin for each sample in every condition of incubation.

155

treated with 50pgg/ml Tf and those treated with 50 pg/ml BSA. Most LI were enhanced at both stages of culture in the presence of 5 pg/ml Tf in comparison with control medium. However, these increases were not significant. Increasing Tf concentration from 5 to 50pgg/ml resulted in dose-dependent effects after 6 days in culture; both MI and LI of PO and PA were significantly increased. After 2 days in culture no significant effect of Tf on MI and LI was found. Nevertheless, both MI and LI of PO were significantly enhanced in the presence of 50 pg/ml Tf when compared with control medium (Tables 1 and 2). Immunolocalization of transferrin receptors

RESULTS

Mouse bone-marrow cells showed an intense pericellular positive reaction (Plate Fig. 5), in agreement with previous reports (Lebmann et al., 1982; Lesley et al., 1984). On the twelfth day of gestation there was a heavier positive reaction of the anti-Tf receptor monoclonal antibody on epithelial cells than on mesenchymal cells of mouse tooth buds (not shown). Mesenchymal, as well as epithelial cells of day-14 mouse molars, showed positive immunofluorescence reactions (Plate Fig. 6). Day-19 molars had positive reactions for both PO and PA (Plate Fig. 7). In day-20 and day-21 molars PO still showed faint fluorescent staining while odontoblasts were negative. At the same stages intense fluorescence reactions were observed around PA and on the apical pole of polarized ameloblasts (not shown).

Effects of Tf(Fe), on the morphogenesis and cell proliferation of day-14 molars in organ culture Tooth morphogenesis. Day-14 first mandibular molars were at the early cap stage. Whatever the culture medium, no polarized (post-mitotic) odontoblast was observed after 6 days in culture. When cultures were performed for 6 days in the chemically defined medium (RPMI, vitamin C and glutamine) day-14 molars did not form cusps (Plate Fig. 2). Addition of 5 and 50 pg/ml Tf(Fe), to culture media resulted respectively in slight (Plate Fig. 3) and pronounced (Plate Fig. 4) cusp formation after 6 days in culture. Crown morphogenesis in the presence of 50 pgg/ml BSA was similar to that observed in the chemically defined medium. No apparent effect of Tf(Fe), (5 and 50 {[g/ml) was found after 2 days in culture. MI and LZ. When compared to the control medium, the presence of BSA in the media caused a non-significant inc:rease in MI. There were no significant differences, except in the LI of PO after 6 days in culture. in MI and LI between teeth

Binding of [‘251]-TF(Fe)2 to dental components No acid-strippable counts of [‘2SI]-Tf(Fe)2 were found in freshly dissociated components of day-l 8 tooth germs. Non-specific binding was obtained by adding 70 pg/ml of unlabelled Tf(Fe), to the binding medium at the beginning of incubation. Mean values of 5%

Table 1. Mean values ( + SD) of MI and LI indices of PO and PA of day-14 mouse embryo first lower molars, cultured for 2 and 6 days 14 + 2 days in culture MI

Medium

0.92 + 0.23 (4) 1.68 + 0.35 (3) *1.93+0.17(3) 1.ss + 0.39 (4)

14 + 6 days in culture 0.76 0.61 + 0.28 0.13 (4) (4) *1.32+0.09(3) 1.16+0.39(3)

MI

PO

PO

LI

MI

29.35 + 24.46 + a33.75 + 29.40 +

1.20 (3) 8.93 (4) 0.35 (3) 1.55 (3j

a b c

1.23 + 0.38 (4) 1.49+0.14(3) 1.83 + 0.38 (3) 1.44 + 0.50 (4)

32.40 + 32.75 + ‘53.90 + 28.66 +

1.27 (3) 0.63 (3j 1.55 (3) 7.30 (4j

0.64 0.98 + 0.20 0.22 (4) (5) *1.29+0.18(3) 0.90 + 0.11 (3)

L1

MI

PA

PA

LI

23.0 + 22.00 + 20.83 + 22.35 +

7.6 (4) 0.14 (3) 1.10(4) 0.35 (3)

; G

21.95 21.75 + 0.07 0.35 (3) (3) ‘33.30 + 2.96 (3) 25.80 + 7.35 (3)

LI

(a): Control cultures; (b), (c) and (d): experimental cultures with supplements of 5 pg/ml Tf, SOfig/ml Tf and SOpg/ml BSA respectively. Numbers in parentheses are numbers of teeth examined. *Indicates a significant difference (p < 0.05).

Y. CAMel al

156

Table 2. Statistical comparison (values of p) of the MI and LI indices of PO and PA of day- 14 mouse embryo first lower molars cultured for

2 and 6 days 14 + 2 days in culture

Media compared

0.07 0.01; 0.07 0.45 0.73 0.23

b:d c:d

0.28 0.04* 0.30 0.01* 0.12 0.62

LI

0.41 0.03’ 0.97 0.17 0.41 0.06

a:b a:c a:d b:c b:d c:d

0.76 0.004* 0.45 0.003’ 0.40 O.Olf

MI

0.37 0.18 0.61 0.36 0.87 0.39

a:b a:c a:d b:c b:d c:d

0.09 0.03 0.17 0.14 0.59 0.12

0.51

a:b

0.44

a:c

0.51 0.03*

0.53 0.16 0.32 0.11

a:d b:c b:d c:d

0.53 0.03: 0.51 0.31

MI

a:b a:c

14 + 6 days in culture

a:d b:c

M1

PO

PO

L1

MI

PA

PA

LI

L1

(a): Control cultures; (b), (c) and (d): experimental cultures with supplements of 5 pg/ml Tf, 50pg/ml Tf and 50pg/ml BSA respectively. *Indicates a significant difference (p < 0.05). of total binding were found for non-specific binding of [‘251]-Tf(Fe), to freshly dissociated dental papillae and enamel organs. Results expressed as specific binding per tooth component versus incubation time are shown in Text Fig. 1. Both dental components showed specific binding of [‘251]-Tf(Fe)z; the binding reaction to dental papillae equilibrated more rapidly than the one to enamel organs.

DISCUSSION

Partanen et al. (1984) and Partanen and Thesleff (1987a, b) in their work on the role of Tf in mouse tooth morphogenesis, have emphasized the requirement for Tf in its early stages up to the fourteenth day of gestation, and they have demonstrated specific binding of iodinated Tf and retention of Tf by mouse molar teeth in organ culture. They also measured DNA content and [‘HI-thymidine incorporation and concluded that there was a correlation between the stimulation of growth and the ability of Tf to support tooth morphogenesis in organ culture. However, “the small day-14 and day-15 explants contain some surrounding non-dental mesenchyme” (Partanen et al., 1985). Thus, their DNA measurement in early stages of tooth development included DNA of peridental as well as dental cells. Wright and Alison (1984) have emphasized the necessity of defining the target popu-

lation precisely and warned of the potential pitfalls and artefacts when assaying DNA synthesis. We investigated the mitotic and thymidine labelling indices of specific cells, PO and PA, in tooth germs cultivated in the presence of Tf. Our study, like that of Partanen et al. (1984), shows a morphogenetic effect of Tf (50 pgg/ml) on the in vitro development of the lower first molar of l4-day-old mouse embryo which, after 6 days of culture, reaches the equivalent of day-17 tooth germs in vivo (Ahmad and Ruth, 1987). Our mitotic indices for day-14 molars showed a doubling of the number of mitotic PO after a short period (2 days) in the presence of Tf (50 pg/ml). Enhancement of MI in PO was slightly reduced after 6 days in culture, while the MI of PA has doubled at this stage compared to molars in control medium. DNA synthesis, expressed as the percentage of cells having incorporated [‘HI-thymidine (LI), was also enhanced in the presence of Tf (50 pg/ml) earlier and to a greater extent in PO than in PA. Addition of BSA (mol. wt = 66 kdalton), used in our studies as a substitute for Tf (mol. wt = 80 kdalton) for protein intake of cultured teeth, led to non-significant increases in MI. Unless BSA has intrinsic mitogenic activity for dental cells, one possible explanation for this finding is that our BSA batch was contaminated by residual serum Tf; such contamination has been reported before (Stein and Sussman, 1986). Taken together, Partanen’s and our results reveal a significant effect of Tf, when used at higher concen-

Effects of transferrin

on mouse molar growth

tration (50 pg/ml), on the kinetic parameters of both types of cells. The fact that PO were more responsive than PA after 2 days in culture and the opposite after 6 days in culture might be related to the genetically determined time-dependent proliferation rates of both types of cells, observed in vivo (Ahmad and Ruth, 1987). During the whole of odontogenesis, PO and PA (cells able to divide) of lower first molars have specific Tf receptors, as documented by our immunofluorescence study. This finding is corroborated by the fact that day-18 mouse molars specifically bind and internalize [‘*‘I]-Tf. Kay and Benzie (1986) have suggested that the function of the Tf receptor is directly to provide iron for DNA synthesis itself rather than to act a:< the receptor for a general signal required to initiate entry into S phase: ironcontaining ribonucleotide reductase catalyses the reduction of ribonucleotides to deoxyribonucleotides (Ward, 1987). Polarized functional odontoblasts were no longer stained in the presence of anti-Tf receptor antibodies, while polarized ameloblasts had staining of their apical pole. This late expression of Tf receptors in post-mitotic amelobtasts might be related to functional differentiation (McKee et al., 1987). Given that iron is also required for collagen synthesis (Ryhanen and Kivirikko, 1974), it is difficult to establish a specific correlation between the permissive effects of Tf on cell division :and tooth morphogenesis, which is controlled by epithelio-mesenchymal matrixmediated interactions (reviewed by Ruth, 1984). We conclude that Tf receptor-mediated iron uptake by odontogenic cells; is a prerequisite for the achievement of the genetically determined proliferation kinetics involved in tooth morphogenesis. Furthermore, iron uptake night also be required for terminal differentiation of amelobtasts. Further investigations should include the developmental regulation of Tf receptor gene expression during odontogenesis. Acknowledgements-We wish to thank A. Ackermann, G. Cadiou and P. Meyer for their technical assistance. This work benetitted from the financial support of INSERM and ARC.

REFERENCES

Ahmad N. and Ruth .I. V. (1987) Comparison of growth and cell proliferation kinetics during mouse molar odontogenesis in viva and in vitro. Cell Tiss. Kinet. 20,319-329. Barnes D. and Sato G. (1980) Serum-free cell culture: a unifying approach. Cell 22, 649-655. Boukari A. and Ruth J. V. (1981) Comportement d’ebauches dentaires d’embryons de souris in vitro: maintien de la morphologie coronaire, mintralisation. J. Biol. buccale 10, 263-269. Brownell A. G. and Rover0 L. J. (1980) DNA synthesis of enamel organ epithelium in vitro is enhanced by co-

Plate

I57

cultivation with non-viable mesenchyme cells. J. dent. Res. 59, 1075-1080. Ekblom P., Thesleff I., Miettinen A. and Saxen L. (1981) Organogenesis in a defined medium sunolemented with transfer&. Cell Difirenf. 10, 281-288.’ . Ekblom P., Thesleff I., Saxen L., Miettinen A. and Timnl R. (1983) Transferrin as a fetal growth factor: acquisition of responsiveness related to embryonic induction. Proc. natn. Acad. Sci. U.S.A. 80, 2651-2655. Karcher-Djuricic V., Staubli A., Meyer J. M. and Ruth J. V. (1985) Acellular dental matrices promote functional differentiation of ameloblasts. Dt@renriafion 29, 169-175. Karin M. and Mintz B. (1981) Receptor-mediated endocytosis of transferrin in developmentally totipotent mouse teratocarcinoma stem cells. J. biol. Chem. 256,324s3252. Kay J. E. and Benzie C. R. (1986) The role of the transferrin receptor in lymphocyte activation. Immun. Left. 12, 55-58. Lebmann D., Trucco M., Bottero L., Lange B., Pessano S. and Rovera G. (1982) A monoclonal antibody that detects expression of transferrin receptor in human erythroid precursor cells. Blood 59, 671-678. Lesley J., Hyman R., Schulte R. and Trotter J. (1984) Expression of transferrin receptor on murine hematopoietic progenitors. Cell Immun. 83, 1425. McKee M. D., Zerounian C., Martineau-Doize B. and Warshawsky H. (1987) Specific binding sites for transferrin on ameloblasts of the enamel maturation zone in the rat incisor. Anat Rec. 218, 123-127. Partanen A. M. and ThesletT I. (1987a) Levels and patterns of ‘251-labeled transferrin binding in mouse embryonic teeth and kidneys at various developmental stages. Dtfirentiation 34, 18-24. Partanen A. M. and Thesleff I. (1987b) Transferrin and tooth morphogenesis: retention of transferrin by mouse embryonic teeth in organ culture. Differentialion 34, 25-3 1. Partanen A. M., Thesleff I. and Ekblom P. (1984) Transferrin is required for early tooth morphogenesis. Dtjjerentiafion 27, 59-66. Partanen A. M., Ekblom P. and Thesleff I. (1985) Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth. Deul Biol. 111, 84-94. Ruth J. V. (1984) Tooth morphogenesis and differentiation. In: Dentin and Dentinogenesis (Edited by Linde A.), pp. 47-79. CRC Press, Boca Raton, Fla. Ruth J. V. (1985) Odontoblast differentiation and the formation of the odontoblast layer. J. dent. Res. 64, 489-498. Ryhanen L. and Kivirikko K. I. (1974) Developmental changes in protocollagen lysyl hydroxylase activity in the chick embryo. Biochim. biophys. Acta 343, 121-128. Steidler N. E. and Reade P. C. (1981) Epidermal growth factor and proliferation of odontogenic cells in culture. J. dent. Res. 60, 1977-1982. Stein 8. S. and Sussman M. M. (1986) Demonstration of two distinct transferrin receptor recycling pathways and transferrin independent receptor internalization in K562 cells. J. biol. Chem. 261, 10,319-10,331. Ward J. H. (1987) The structure, function and regulation of transferrin receptors. Invest. Radiol. 22, 7+83. Wright N. and Alison M. (1984) The Biology of Epithelial Cell Populations, Vol. I, Chap. 3, p. 123. Oxford Science Publications, Clarendon Press, Oxford.

1 overleaf

Y. CAMet al.

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Plate Fig. 2. Sagittal section of day-14 is seen. Masson’s trichrome.

1

molar cultured for 6 days in control medium. EO = enamel organ; PA = pre-ameloblasts; DP = dental papilla. x 325

No cusp morphogenesis PO = pre-odontoblasts;

Fig. 3. Frontal section of day-14 molar cultured for 6 days in medium supplemented with Spg/ml of transferrin. The arrows point to the very beginning of cusp formation. Masson’s trichrome. x 325 Fig. 4. Frontal section of day-14 molar cultured for 6 days in medium supplemented with 50pg/ml of transferrin. There is cuspal morphogenesis (arrows). Masson’s trichrome. MF = Millipore filter. x 325 Fig. 5. Immunofluorescent

localization

of transferrin

receptors x 350

on cells of a smear of mouse bone marrow.

Fig. 6. Immunofluorescent localization of transferrin receptors on a 8 pm thick frozen section of a day-14 mouse molar. The cells of the enamel organ (EO) and of the dental papilla (DP) are immunostained. x 350 Fig. 7. Immunofluorescent localization of transferrin receptors on a 8 pm thick frozen section of a day-19 mouse molar. The PA, PO and dental papilla cells (DP) are immunostained. The cells of the stellate reticulum (SR) appear negative. x 350

Effects of transferrin

on mouse

Plate

1

molar

growth

159