EGF antisense oligodeoxynucleotides block murine odontogenesis in vitro

DEVELOPMENTAL

BIOLOGY

147,485-488 (1991)

EGF Antisense Oligodeoxynucleotides Block Murine Odontogenesis in Vitro J.E. KRONMILLER, Department

of BioStructure

and Functimz,

School

W.B. UPHOLT,AND

of Dental Medicine, Accepted

June

University

E.J. KOLLAR of Connecticut,

Farmington,

Connecticut 06032

24, 1991

The initiation of odontogenesis depends on the site-specific proliferation of mandibular epithelium beginning at Day 11 in embryonic mice. We have previously reported that the local expression of epidermal growth factor mRNA in the murine mandible is developmentally regulated, expressed at Days 9 and 10 immediately prior to the initiation of tooth bud formation at Day 11. Exposure of Day 9 mandibular explants to antisense oligomers of epidermal growth factor blocks the initiation of odontogenesis. These results are the first demonstration of the involvement of epidermal growth factor in the inductive specification of a complex epithelial derivative. o 1~1 Academic PRSS, he.

small amounts of sterile water, and stock solutions were prepared by dilution with plain media. The oligomers were designed to overlap the initiation codon of the EGF mRNA (Fig. 1).

INTRODUCTION

At Gestational Days 9 and 10, coincident with EGF mRNA expression (Kronmiller et al, 1991), the mandibular epithelium plays an important role in the induction of odontogenesis (Mina and Kollar, 1987). The first morphological sign of odontogenesis, the formation of the dental lamina, occurs at Day 11-12 in the mouse as discrete epithelial proliferations in the incisor and molar regions (Mina and Kollar, 1987). The exact nature of the inductive signal and the importance of this site-specific proliferation in initiating odontogenesis are not known. Odontogenic epithelium expresses EGF receptors (Partanen and Thesleff, 1987), and EGF causes proliferation of dental epithelium (Partanen and Thesleff, 1985). In an effort to further study the role of EGF in the initiation of odontogenesis, we used antisense oligodeoxynucleotides to block the effects of in situ EGF production in organ cultures of embryonic mandibles. This treatment resulted in the inhibition of odontogenesis. MATERIALS

Preparation

Organ Cultures Mandibles were dissected from embryonic CD-l Swiss mice (Charles Rivers) at Day 9 of gestation (Mina and Kollar, 1987). Four mandibles were placed in each Falcon organ culture dish and were supported by a Millipore filter (13-mm diameter) on a stainless steel grid. Cultures were incubated for 14 days at 37°C in a humidified atmosphere of 95% air and 5% COZ. BGJb (FittonJackson modification, GIBCO) culture medium was supplemented with 100 U/ml penicillin/streptomycin (Sigma), 50 pg/ml ascorbic acid (Sigma), and 10% fetal calf serum (Hyclone). Media were changed every other day. Five treatment groups were used: control (water), EAS-1, EAS-2, ES, and EAS-1 + EGF. All oligomers, control reagents, and EGF (Sigma) were added to the organ cultures at the beginning of the culture period and were present only for the first 3 days with one change of media after 2 days. Serum was excluded from the media during these first 3 days. Oligomers were added at a final concentration of 2 piV, and EGF was present at a final concentration of 20 rig/ml of media. The EGF stock solution was prepared in HBSS and diluted to the final volume with media. Following the first 3 days in culture the mandibles were maintained in media containing serum for an additional 11 days. At the end of the culture period explants were prepared for histologic examination (Mina and Kollar, 1987). All se-

AND METHODS

of Oligomers

Two antisense (EAS-1 and EAS-2) and one sense (ES) 15-base oligomers were synthesized by standard P-phosphoramidite chemistry using a Cyclone DNA synthesizer (Biosearch Inc.). The oligomers were purified by ethanol precipitation and repeated washes with 70% ethanol (Anfossi et aL, 1989) and quantified by optical density at 260 nm. All oligomers were redissolved in 485

001%1606/91$3.00 Copyright All rights

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

486

DEVELOPMENTALBIOLOGY

Met 5’-AAA 3’-TTT

TAA ATT

AAG TTC

3’3’-TTT 5’-

ATT

TTC

Pro CCC GGG

Trp TGG ACC

01~

-g

AT0 TAC

GGC CCG

CGA-3 GCT-5’

TAC

GGG

ACC

CC0

GCT-5’

TAC

GGG

ATG

CCC

EAS-1

-5’ TGG

GGC

CGA-3’

Em-2 ES

FIG. 1. Sequences of oligomers used. EAS-1, EGF antisense-1; EAS2, EGF antisense 2; ES, EGF sense. The DNA sequence shown is for nucieotides 301-324, (Gray et al, 1983).

rial sections of each sample were evaluated of odontogenesis. RESULTS

for evidence

AND DISCUSSION

Control explants from Day 9 mice grown in organ culture for 14 days contained well-formed molars in the bell stage of development (Fig. 2A). These teeth showed well-defined cuspal outlines and mesenchymal condensations forming the dental papilla and dental follicle. Meckel’s cartilage and bone as well as salivary gland and tongue epithelium are present in these explants after this period of organ culture. Well-formed molars (similar to those observed in the control explants) were found in the explants exposed to the sense oligomer (ES) (Fig. 2B). These mandibles also contained well-formed Meckel’s cartilage and bone. The development of the tongue and salivary glands was similar to that in the control explants. Exposure to antisense oligomer EAS-1 resulted in a total inhibition of odontogenesis (Fig. 2C). Dental lamina did not form in any of the explants exposed to this oligomer. The keratinized epithelium formed a smooth junction devoid of any epithelial invasion into the underlying mesenchyme. Bone formed in these explants but Meckel’s cartilage development was impaired or entirely abolished. Small remnants of tongue epithelium were found very infrequently. There was no evidence of cellular damage in the EAS-l-treated explants. The second antisense oligomer (EAS-2) was used to confirm the specificity of the effects of EAS-1. This oligomer also inhibited odontogenesis in a manner similar to that of the EAS-l-exposed explants (Fig. 2D). One explant in the EAS-2 group contained one poorly formed epithelial bud in the molar region of one side of the mandible (Table 1). Flat keratinizing epithelium predominated in these explants as in the explants in the EAS-1 group. Although bone was present, cartilage formation was impaired as in the EAS-1 explants. The

VOLIJME147,1991

tongue epithelium in this group was better formed than that in the EAS-1 group but not developed as well as that in the control groups. Exogenous EGF was added to cultures that contained EAS-1 oligomer to further verify the specificity of EGF inhibition (Fig. 2E). As in the control explants and those exposed to the sense oligomer, well-formed molar buds developed in these mandibles. Cartilage, bone, tongue epithelium, and salivary gland formation were also present as in the control samples. The results of the present experiments demonstrate the involvement of EGF during the initiation of odontogenesis. We have previously demonstrated the expression of EGF mRNA immediately prior to the beginning of dental lamina formation in the mouse mandible (Kronmiller et ah, 1991). EGF mRNA is expressed only at Days 9 and 10 and not at Days 11-14 when tooth buds have already formed. The inhibition of odontogenesis by exposure to antisense oligomers for only 3 days at this critical stage of development confirms the developmental regulation of local EGF production (Days 9 and 10 only) as it relates to tooth formation (initiation at Day 11) because the presence of serum after the first 3 days did not restore normal development. It is not clear whether EGF acts as a paracrine or autocrine factor in the initiation of odontogenesis since it is present in both epithelium and mesenchyme (Kronmiller et aL, 1991). The EAS-1 oligomer may be slightly more effective in the inhibition of odontogenesis than EAS-2 (Table 1). However, only one poorly developed molar bud was found out of all 14 explants exposed to either antisense oligomer (l/28 possible molar buds). Differences in effectiveness have been reported for other oligomers designed against different regions of the same message (Colman, 1990). We verified the specificity of EGF inhibition in three ways: A sense oligomer, the complement of EAS-1, had no effect on odontogenesis; A second antisense oligomer which overlaps EAS-1 by 6 nucleotides and contains an additional 9 nucleotides of the 5’ untranslated region of the mRNA; and, by adding EGF protein to cultures containing EAS-1 antisense oligomer. All three treatments confirm the specificity of the EGF inhibition. TABLE 1 PROPORTIONOFEXPLANTSWITI-IMOLARBUDS Treatment

Control

EAS-1

EAS-2

ES

EAS-1 + EGF

Positive explants (Number of molars)

7/7

O/7

l/7

7/7

7/7

14/14

o/14*

1/14*

14/14

14/14

* Statistically

significant at P < .OOl (x2 analysis).

BRIEF

FIG. 2. Effects of EGF antisense oligomers on odontogenesis. Mandibular explants after 14 days in organ culture and an initial exposure for 3 days to (A) water (control), (B) ES sense oligomer, (C) EAS-1 antisense oligomer, (D) EAS-2 antisense oligomer, and (E) EAS-1 antisense oligomer + EGF. m, molar; c, cartilage; b, bone; and t, tongue. Magnifications at 100X. Bar = 100 ym.

In addition to demonstrating the importance of EGF in the initiation of odontogenesis, we also observed its effects on other tissues in the developing mandible. Cartilage and tongue epithelium development was impaired by EGF antisense oligomers. Cartilage formation requires proliferation of mesenchymal cells prior to the condensation phase. Inhibition of local EGF production may impair cartilage formation by decreasing the proliferative signal. The patterning of the dentition depends on the sitespecific proliferation of mandibular epithelium and we have demonstrated the need for EGF during the initial event of odontogenesis. However, since the addition of a uniform concentration of exogenous EGF did not cause

any abnormal development, the patterning could be established by further control at the level of EGF-receptor expression. We are currently studying the mechanisms involved in the regulation of the pattern of the dentition. This study was supported by NIH Grant DE00286-02 (JEK).

REFERENCES ANFOSSI, G., GEWIRTZ, A., and CALABRET~A, B. (1989). An oligomer complementary to c-myb-encoded mRNA inhibits proliferation of human myeloid leukemia cell lines. Proc. Natl. Acad Sci USA 86, 3379-3383.

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DEVELOPMENTAL BIOLOGY

COLMAN, A. J. (1990). Antisense strategies in cell and developmental biology. Cell Sti 97, 399-409. GRAY, A., DULL, T., and ULLRICH, A. (1983). Nucleotide sequence of epidermal growth factor cDNA predicts a 128,000 molecular weight protein precursor. Nature 303,722-726. KRONMILLER, J., UPHOLT, W. B., and KOLLAR, E. (1991). Expression of epidermal growth factor in RNA in the developing mouse mandibular process. Arch. Oral BioL 36,405-410. MINA, M., and KOLLAR, E. (1987). The induction of odontogenesis in

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non-dental mesenchyme combined with early murine mandibular arch epithelium. Arch. Oral Viol. 32,1213-127. PARTANEN, A., and THESLEFF, I. (1985). Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth. Dev. BioL 111,84-94. PARTANEN, A., and THESLEFF, I. (1987). Localization and quantification of 12SI-epidermal growth factor binding in mouse embryonic tissues and other embryonic tissues at different developmental stages. Dev. BioL 120,186-197.