J. C. de Vicente 1, R. Cabo 2, E. Ciriaco 3, R. Laur/t 3, F. J. Naves 4, I. Silos-Santiago s, and J. A. Vega 4' a
Departamentos de 1 Cirugfa y Especialidades M6dico-Quirfirgicas, Escuela de Estomato- logfa, and 4 Departamento de Morfologfa y Biologfa Celular, Universidad de Oviedo,
C/Juli~in Claverfa, s/n, E-33006 Oviedo, Spain 2 Departamento de Anatomfa, Universidad de Valladolid, Spain
3 Istituto di Anatomia degli Animali Domestici, Facolt~ di Medicina Veterinaria, Universit~ di Messina, Italy
5 Department of Neurobiology, Millennium Pharmaceutics Inc., Cambridge, MA, USA
Summary. Glial cell line-derived neurotrophic factor pro- motes the survival of multiple neuron types in the central and peripheral nervous system. Moreover, it plays a key role in the development of the enteric nervous system and in the kidney organogenesis. Glial cell line-derived neurotrophic factor and their receptors are expressed in the developing tooth as well as in the trigeminal ganglion. However, the precise role of this growth factor in tooth morphogenesis and cell differentiation, or in the develop- ment of trigeminal ganglion cells, is still elusive. Using structural and ultrastructural techniques we analyzed in detail the first molar tooth germ of glial cell line-derived neurotrophic factor deficient mice as well as the neuronal density in trigeminal ganglion. The length and width of first molar tooth germ in knockout deficient animals showed no differences in the knockout animals in com- parison with age-matched heterozygous or wild-type lit- termates. Nevertheless, in mice lacking glial cell line- derived neurotrophic factor, both ameloblasts and odon- toblasts failed to fully develop and differentiate, and the enamel, matrix and predentin layers were absent. On the other hand, the number of trigeminal sensory neurons and the structure of the nerves supplying first molar tooth germ were largely normal. Present results suggest a new non-neuronal role for glial cell line-derived neurotrophic factor in tooth development. Glial cell line-derived neu- rotrophic factor seems not to be involved in tooth initia-
tion and morphogenesis, whereas it seems essential for cytodifferentiation. Conversely, neither development of trigeminal neuron nor nerve fibers supplying teeth are di- rectly dependent on glial cell line-derived neutrophic fac- tor.
The mammalian teeth develop as a result of sequential and reciprocal morphogenetic interactions between the ectodermally derived epithelium covering the craniofacial processes and the underlying neural crest-derived mes- enchymal cells (see for a review Thesleff et al. 1995 a, b; Thesleff and Nieminen 1996; Dassule and McMahon 1998). In these events a wide range of trophic factors are involved regulating cell differentiation and survival (see Thesleff and Sharpe 1997). They include: neurotrophins (Luukko et al. 1997 a, b, 1998; Luukko, 1998; Nosrat et al. 1998), bone morphogenetic proteins (Helder et al. 1998), hepatocyte growth factor (Tabata et al. 1996), transform- ing growth factor-[~ (Toyono et al. 1997), basic fibroblastic growth factor (Russo et al. 1998), and glial-cell line de- rived neurotrophic factor (GDNF) among other related factors (Hellmich et al. 1996; Nostrat et al. 1996, 1997, 1998; Sanchez et al. 1996; Luukko et al. 1997 b; Golden et al. 1999).
The developing murine teeth express m R N A for differ- ent members of the G D N F family of growth factors (see Saarma and Sariola 1999; Baloh et al. 2000) as well as for their cognate receptors (see Rosenthal 1999; Hellmich et al. 1996; Nosrat et al. 1996, 1997, 1998; Sanchez et al. 1996; Luukko et al. 1997 b; Widenfalk et al. 1997; Golden et al. 1999). Nevertheless, al though these data suggest a role of these growth factors during tooth formation, their precise role remains still elusive since incisors and molars develop normally in GDNF- or Ret-deficient mice (Schu- chard et al. 1994; Granholm et al. 1997). However these studies used conventional structural techniques (hematox- ylin-eosin), which do not lend themselves to detailed ana- lysis of the impact of GDNF-deficiency in tooth cell maturat ion and differentiation.
On the other hand, it is well known that innervation can modulate tooth development, although it does not seem to be necessary for tooth initiation and morphogen- esis (see Lumsden et al. 1986; Luukko 1998). In any case evidence exists that some factor needed for neuronal de- velopment may also be involved in the regulation of tooth formation. This seems to be the case for G D N F which acts as a target-derived neurotrophic factor regulating de- veloping tooth innervation (Luukko et al. 1997b) and tooth morphogenesis, although controversia exist (Lille- saar et al. 1999). In this way embryonic and postnatal tri- geminal ganglia express Ret, GFRc~-I, GFRa-2, GFRa-3 and GDNFR-[~ (Luukko et al. 1997 a, b; Widenfalk et al. 1997, 1998; Naveilhan et al. 1998), as well as immunoreac- tivity for G D N F (Quartu et al. 1999). Thus, it could be speculated that a deficit in G D N F might alter the devel- opment of trigeminal neurons and tooth innervation.
To investigate in vivo the role of G D N F in tooth devel- opment we analyzed in detail the ultrastructure of the first molar tooth germ (FMTG) in newborn mice carrying a null-mutation in the gene codifying for G D N F (Sanchez et al. 1996). Furthermore, to study the impact of this mu- tation in trigeminal ganglion, we also studied the number of neurons in this ganglion and the structure of the nerve supplying that tooth. The tooth of GDNF-deficient mice showed ultrastructural changes both in the ameloblasts and the odontoblasts as well as an absence of enamel ma- trix and predentin tissues. However, the ultrastructure of the nerves as well as the number of the trigeminal sensory neurons was basically unvaried.
Materials and methods
Animals and treatment of tissues. Mice with a targeted mutation of the GDNF gene were obtained from the colony of the Depart- ment of Molecular Biology, Bristol-Myers-Squibb, Princeton, NJ. These animals were bred out over the C57B1/6 background and genotyped by polymerase chain reaction (Sanchez et al. 1996). Newborn (Postnatal day -Pd- 0) wild-type (n = 3), heterozygous (n = 5), and homozygous (n = 7) mice, were included in the study. Animals were deeply anaesthesized with ether and perfused
transcardially with a cold solution of 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Then the head was separated from the body and divided into two parts sagittally. One half was used for light microscopy studies; in the other half the upper and lower jaws were removed. The pieces were immediately immersed into the same fixative at 4 °C, and then processed for routine paraffin-em- bedding (the entire half head), or Durcapan ® ACM (Fluka) re- sin-embedding (the upper and lower jaw segments containing FMTG).
Structural and ultrastructural studies. The pieces embedded in paraffin were cut in serial sections 10 gm thick, mounted on gela- tin-coated microscope slides, and used for hematoxylin-eosin staining. For the ultrastructural analysis the pieces were washed repeatedly in 0.1 M PBS (pH 7.4) for two days, and posffixed in 1% osmium tetroxide in 0.1 M PBS for 2 hours. The tissues were then dehydrated with increasing acetone concentrations, in mix- tures of anhydrous acetone and resin (3:1, 1:1, 1:3), and then in pure resin, each step for i h. Sections were obtained with an ultramicrotome, and semi-thin sections (1 ~tm) were stained with Toluidin Blue and examined with a light microscope. Ultra-thin sections (400 A_) were obtained from selected areas, stained with uranyl acetate and lead citrate, and examined and photographed with a JEOL-JEM-T8 electron microscope.
Quantitative analysis. The width and length of FMTG were measured in hematoxylin-eosin stained or semithin sections using an automatic image analysis system (Quantimet 550, Leika, QWIN Program, Servicio de Analisis de Imagenes, University of Oviedo). Furthermore, the same image analysis system was used to establish the neuronal density in the trigeminal ganglion. Counts were made on 10 semithin sections per specimen, 150 gm apart to avoid counting the same neuron twice, evaluating in the entire section to include all the cell bodies with apparent nuclei. Also, the density of Schwann cell nucleus profiles were counted on 10 sections of the nerve localized at the basis of the first mo- lar tooth of the inferior jaw. Because axons forming this nerve were still immature, axonal counting was not carried out. Values are expressed as mean values+ standard error of the mean (SEM) for each group of animals and differences between wild type and GDNF-mutated mice for the parameters analyzed were tested using a one-tailed Student's t-test, and values of p _< 0.05 were considered as significant.
Results
In all the animals examined, F M T G were regularly ob- served (data not shown). Their gross morphology was si- milar in the three groups of mice, and no significant differences among groups were found for length and width or the first molar tooth (Table 1).
Structure and ultrastructure o f the first molar tooth. The structure of F M T G in Pd0 wild-type mice consisted of an external layer of ameloblasts and a layer of odontoblasts separated into a free cell layer of enamel matrix and pre- dentin (Fig. 1A). Abundant images of mitosis were ob- served in the odontoblasts layer (data not shown). This pattern of normal morphogenesis and cell arrangement was strongly altered in the GDNF-deficient mice (Fig. 1 B). Both the ameloblasts and odontoblasts were re-
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Table 1. Length and width of the first molar tooth germ, neuro- nal density in TG and number of Schwann cell nucleus profiles of Pd0 wild-type-, heterozygous- and homozygous-GDNF defi- cient mice
(1) antero-posterior distance measured between the most distal cuspal zones (measured in 5 sections per animal) in mm
(2) transversal distance measured between the most distal cuspal zones (measured in 5 sections per animal) in mm
H&E = hematoxylin-eosin staining; TB = toluidin blue staining; TG = trigeminal ganglion All differences were not statistically significant.
duced in size, but apparent ly increased in number and do not attain its typical morphology and a r rangement as a pseudostratif ied epithelial forming a proper ly stratified epithelium. Interestingly, the enamel matr ix and preden- tin layer, al though present, were irregular in thickness and showed either weak or no staining (Fig. 1 B).
The ultrastructural analysis largely confirmed the above observations. Light and dark ameloblasts (corresponding to secreting and differentiating ameloblasts) were clearly differentiated in the wild-type (Fig. 2 A) but not in the G D N F - k n o c k o u t (Fig. 2B) mice. Moreover , ameloblasts in the G D N F deficient mice lacked Tome's processes, their typical organelles being poorly developed, and no m e m b r a n e junctions were observed. The odontoblasts also showed a poor deve lopment of cytoplasmic orga- nelles, especially the rough endoplasmic ret iculum and Golgi 's apparatus (Fig. 2 B).
But the most striking finding in the G D N F deficient mice was the absence of predent in and the collagen net- work that forms the enamel matrix (Figs. 2 and 3). The space occupied normally by these tissues existed in the G D N F knockout mice, but it was empty or contained cell debris (Fig. 3 B).
Analysis of the trigeminal ganglion and the first molar tooth nerve. The tr igeminal ganglion of the heterozygous and knockout mice showed no significant reduction in the number of neurons with respect to the wild-type or the heterozygous ones (Fig. 4; see Table 1). However , the size of the ganglia was reduced in the GDNF-def ic ien t mice (Fig. 4 E), thus suggesting a decrease in the neuron size or in the number of non-neuronal ganglionic cells, i.e. satel- lite glial cells (Fig. 4). On the other hand, the nerve at the base of the F M T G showed no differences be tween the groups regarding the structure of the developing unmyeli- nated axons or the number of Schwann cell nucleus pro- files (Figs. 1 C and D; Table 1).
D i s c u s s i o n
Fig. 1. Structure of the first molar tooth (A, B) and the nerve supplying it (C, D) of the lower jaw in the wild-type (A, C) and GDNF-knockout mice (B, D). am = ameloblasts; dp = dental pulp; od = odontoblasts; sr = stellate reticulum. * denote the enamel-dentine layer. Scale bar = 8.5 gm
The mammal i an developing teeth express both growth factors of the G D N F family and their receptors (Ro- senthal 1999; Hel lmich et al. 1996; Nosra t et al. 1996, 1997, 1998; Sanchez et al. 1996; Luukko et al. 1997b; Widenfalk et al. 1997; Golden et al. 1999). The present study was designed to establish the in vivo role of G F D N in the development , morphogenesis and cytodifferentia- tion of teeth (using F M T G as a model) analyzing the GDNF-def ic ien t mice. Fur thermore , the impact of a G D N F deficiency in the tr igeminal ganglion and the nerve supplying F M T G was investigated. Da ta were ob- tained form newborn mice because mice lacking G D N F die soon after birth.
Previous studies have repor ted that tooth normal ly de- velop in mice deficient for G D N F (Granho lm et al. 1997) or its Re t receptor (Schuchard et al. 1994). Our data re-
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garding the presence of FMTG in GDNF deficient mice, as well as in the morphometric parameters, agree well with those by Granholm et al. (1997). Nevertheless, when analyzed in depth FMTG from the GDNF deficient mice showed strong alterations in the developmental differen- tiation of ameloblasts and odontoblasts, resulting in the absence of enamel matrix and predentin.
It is now well established that reciprocal and sequential
ectodermal-mesenchymal interactions occur for teeth de- velopment (Thesleff et al. 1995; Thesleff and Nieminen 1996; Dassule and McMahon 1998). Trophic factors, pre- sumably including GDNF, participate in regulating cell differentiation (see Thesleff and Sharpe 1997). Present re- sults demonstrate that the absence of GDNF results in abnormal cytodifferentiation of both ameloblasts (ecto- dermic in origin) and odontoblasts (regarded as derived
l~g. 2. Ultrastructure of the enamel-dentine layer in the first molar tooth of the lower jaw in the wild-type (A) and GDNF-knockout mice (B). am = ameloblasts; od = odontoblasts. * = Thomes's processes. Small arrows in A denote bundles of collagen fibrils, and large arrows indicate electron-dense particles of enamel. Original enlargement = x 2 000
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from the neural crest-derived mesenchymal cells). Whether the primary failure is in the ectodermic or mes- enchymal cells remains to be clarified in future studies, because both epithelial and mesenchymal cells express GDNF mRNA during tooth development (Nosrat et al. 1998). GDNF is involved during the inductive epithelial- mesenchymal interactions that accompany kidney organo- genesis and its absence results in renal agenesis due to a lack of an inductive signal from the ureteric bud to the metanephric kidney (Moore et al. 1996; Pichel et al. 1996; Sanchez et al. 1996). But because teeth develop in ab- sence of GDNF, our results suggest that G D N F acts as a local secreted factor (Nosrat et al. 1998) for dental epithelium cell differentiation rather than in tooth induc- tion and morphogenesis. Presumably in these phases
other growth factors participate which are capable of sub- stituting GDNF or it does not participate at this point. In supporting a role for GDNF in tooth cytodifferentiation the wild-type mice showed both secreting and differen- tiating ameloblasts whereas the GDNF-deficient animals do not. Also, ultrastructurally mature odontoblasts were presents at Pd0 in normal pups but not in the GDNF knockout ones (for a review on the normal development of ameloblasts and odontoblasts see Luukko, 1998).
The second main goal of this study was the analysis of the trigeminal ganglion and the nerve fibers supplying FMTG in the absence of GDNF. GDNF promotes the survival of multiple neuron types in the peripheral ner- vous system including sensory neurons in the trigeminal ganglion (see Fritzsch et al. 1997; Saarma and Sariola
Fig. 3. Ultrastructure of the enamel-dentine layer in the first molar tooth of the lower jaw in the wild-type (A) and GDNF-knockout mice (B). am = ameloblasts; od = odontoblasts. White x denote dentine stores. Original enlargement = x 8 000
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Fig. 4. Parasagittal section of the trigeminal ganglion (TG) in wild-type (A, B), heterozygous (C, D) and knockout (E, F) GDNF mice. Scale bar = 90 ktm for A, C, E; scale bar = 20 ~tm for B, D, E
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1999). In these neurons both immunoreactivity for G D N F (Quartu et al. 1999) and m R N A for its putative receptors have been found (Luukko et al. 1997 a, b; Winderfalk et al. 1997, 1998; Naveilhan et al. 1998). In spite of this, no significant losses of trigeminal neurons were found in mice lacking G D N F (Moore et al. 1996; present results) or in its receptor GFRa-1 (Cacalano et al. 1998), and the neurons absent in G D N F deficient mice are those that are GFRa-1 positive (Naveilhan et al. 1998). Neverthe- less, the size of the trigeminal ganglion in the G D N F knockout mice was clearly reduced and the number of neurons lost in the G D N F deficient animals is not high enough to explain this. Therefore, either neurons in these animals are hypoplastic or atrophic, or this reduction in the ganglion size is due to a decrease in the density of non-neuronal cells, i.e. satellite glial cells. Preliminary studies f rom our laboratory do not reveal significant re- duction of the neuronal size, but the number of cells dis- playing S100 protein immunoreactivity seems to be reduced (JC de Vicente et al. unpublished). This finding requires further investigations but was not surprising since G D N F m R N A is expressed by satellite glial cells in the dorsal root ganglion (Hammarberg et al. 1996).
The role of nerve fibers in triggering the development of the teeth or in the onset of the dentin and enamel for- mation is still controversial (see Luukko 1998) although evidences suggest that tooth morphogenesis and innerva- tion are independent (Lumsden et al. 1986; Luukko 1998). In our study, the F M T G nerves were almost all identical in structure and density of Schwann cells in G D N F deficient and wild type mice. This suggests that GDNF, like other members of the G D N F family, i.e. neurturin (Luukko et al. 1998), is not involved in the guidance or maintenance of the structure of nerves to the developing tooth nor in the survival of Schwann cells (see Jessen and Mirsky 1999). If G D N F has any role in these biological processes its absence could be supplied by GDNF-l ike molecules (see Widenfalk et al. 1998, 1999).
Taken together, the present results suggest that G D N F plays a key role in the mechanisms of cytodifferentiation, but not induction or morphogenesis of the mammalian tooth. Conversely, it lacks significant relevance in the maintenance of trigeminal neurons and tooth innervation.
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