Development and cell fate in interspecific (Mus musculus/Mus caroli) orthotopic transplants of mouse molar tooth germs detected by in situ hybridization.

Interpretation of results from previous tooth germ transplantation studies is limited by the inability to distinguish between donor and host cells unequivocally. Furthermore, ectopic transplantation sites have generally been used and the relevance of this to tooth development in situ is uncertain. The aim here was to determine cell fate in orthotopic tooth germ transplants using an interspecific mouse marker system. Mandibular first molar tooth germs were dissected from Mus musculus (CD1) and Mus caroli mice (age range 15-19 day embryo) and transplanted interspecifically into the alveolar crypt of extirpated first mandibular molars in neonatal M. musculus (CD1) and M. caroli hosts. Grafts were recovered at intervals up to 4 weeks postoperatively. Paraffin wax-embedded sections were examined using routine histological techniques and in situ hybridization with a biotinylated DNA probe (pmSat5) specific for M. musculus, to distinguish between donor and host cells. Development of M. musculus tooth germs in M. caroli mandibles and vice versa was similar and transplants progressed to incipient root formation. Vascularization of transplants was chimaeric, being donor-derived in the pulp and host-derived more peripherally. The investing soft tissues comprised a mixture of donor and host cells, predominantly donor. Donor cells were also found in the soft tissue of intertrabecular spaces in the surrounding bone, but alveolar osteocytes were almost entirely host-derived. Long-term survival of grafts was limited and few donor cells were present after 2 weeks. This study provides an unequivocal demonstration of the origin of all cells present in transplanted tooth germs.

Development and cell fate in interspecific (Mus musculus/Mus caroli) intraocular transplants of mouse molar tooth-germ tissues detected by in situ hybridization.

Mandibular first molar tooth germs were dissected from Mus musculus (CDI) and Mus caroli (age range: 14-day embryo to 1-day postnatal). Most of the tooth germs were separated enzymically into epithelial and mesenchymal components. Interspecific tissue recombinations and intact M. caroli tooth germs were grown in the anterior chamber of the eye of adult CDI mice for 24 weeks. Recombinations of M. caroli enamel-organ epithelium with M. musculus, dental papilla and follicle mesenchyme developed into normal teeth with advanced root, periodontal ligament and bone formation, thereby confirming extensive epithelial-mesenchymal interactions across the species barrier. Labelling sections by in situ hybridization with a M. musculus-specific DNA probe (pMSat5) showed that almost all cells in the pulp, periodontal ligament and bone were M. musculus, including cementoblasts. Reduced enamel epithelium and epithelial cell rests derived from donor M. caroli enamel organ were unlabelled. This indicates that any cementogenic role of Hertwig's epithelial root sheath must be short-lived. The immunological privilege of the intraocular transplantation site in M. musculus CDI mice did not extend to grafts including xenogeneic M. caroli dental mesenchyme. Thus, intact M. caroli tooth germs and recombinations of M. musculus enamel organ with M. caroli dental papilla and follicle showed limited development, with no root formation, and were populated almost exclusively with labelled host M. musculus lymphocytes.

Target control of collateral extension and directional axon growth in the mammalian brain.

Individual neurons in the brain send their axons over considerable distances to multiple targets, but the mechanisms governing this process are unresolved. An amenable system for studying axon outgrowth, branching, and target selection is the mammalian corticopontine projection. This major connection develops from parent corticospinal axons that have already grown past the pons, by a delayed interstitial budding of collateral branches that then grow directly into their target, the basilar pons. When cocultured with explants of developing cortex in three-dimensional collagen matrices, the basilar pons elicits the formation and directional growth of cortical axon collaterals across the intervening matrix. This effect appears to be target-specific and selectively influences neurons in the appropriate cortical layer. These in vitro findings provide evidence that the basilar pons becomes innervated by controlling at a distance the budding and directed ingrowth of cortical axon collaterals through the release of a diffusible, chemotropic molecule.

Chemotropic guidance of developing axons in the mammalian central nervous system.

In the developing nervous system, axons project considerable distances along stereotyped pathways to reach their targets. Axon guidance depends partly on the recognition of cell-surface and extracellular matrix cues derived from cells along the pathways. It has also been proposed that neuronal growth cones are guided by gradients of chemoattractant molecules emanating from their intermediate or final cellular targets. Although there is evidence that the axons of some peripheral neurons in vertebrates are guided by chemotropism and the directed growth of some central axons to their targets is consistent with such a mechanism, it remains to be determined whether chemotropism operates in the central nervous system. During development of the spinal cord, commissural axons are deflected towards a specialized set of midline neural epithelial cells, termed the floor plate, which could reflect guidance by substrate cues or by diffusible chemoattractant molecules. Here we provide evidence in support of chemotropic guidance by demonstrating that the rat floor-plate cells secrete a diffusible factor(s) that influences the pattern and orientation of commissural axon growth in vitro without affecting other embryonic spinal cord axons. These findings support the hypothesis that chemotropic mechanisms guide developing axons to their intermediate targets in the vertebrate CNS.

Spatial organization of the epithelium and the role of neural crest cells in the initiation of the mammalian tooth germ.

Teeth develop from composite organ rudiments that are formed through the interaction of oral epithelium and mesenchyme of the first branchial arch; cells of the former differentiate into enamel-secreting ameloblasts whereas those of the latter differentiate into dentine-secreting odontoblasts. Experimental analysis of odontogenic tissue interactions in mammalian embryos has focused on the late developmental stages of morphogenesis and cytodifferentiation; little is known about initial pattern-forming events, during which presumptive tooth-forming cells are specified and the sites of tooth initiation become established. It requires to be shown, for example, whether the mesenchymal cells of mammalian teeth are derived, like those of amphibians, from the cranial neural crest, and if so, whether these form a specified subpopulation in the neural folds. Alternatively, are they specified after migration into the mandibular arch, possibly by interaction with the oral epithelium? The developmental potentials of mouse embryo premigratory cranial neural crest cells (CNC - explanted from the caudal mesencephalic and rostral metencephalic neural folds) have been studied in intraocular homograft recombinations with various regions of embryonic surface ectoderm. Cartilage, bone and neural tissue developed in all combinations of CNC and epithelium. Teeth formed in combinations of CNC with mandibular arch epithelium but not in combinations of CNC with limb bud epithelium. Teeth also formed in combinations of mandibular arch epithelium with neural crest explanted from the trunk level. These results indicate that mammalian neural crest has an odontogenic potential but that this is not restricted to the crest of presumptive tooth-forming levels. Normal migration appears not to be a prerequisite for expression of odontogenic potential but this does require an interaction with region-specific epithelium. It is reasonable to infer that during normal development the neural crest that enters the mandibular arch is odontogenically unspecified before or during migration and that the oral epithelium is the earliest known site of tooth pattern.

Development of periodontal ligament and alveolar bone in homografted recombinations of enamel organs and papillary, pulpal and follicular mesenchyme in the mouse.

Mandibular first-molar tooth germs were dissected from 16-day-embryo and new-born CD1 mice. By incubation in collagenase they were separated into enamel organ, papilla and follicle. Dental pulp was obtained from mandibular first molars of 3-, 7- and 10-day-old mice. Various combinations of epithelial and mesenchymal tissues were grown for periods up to four weeks in the anterior chamber of the eye of homologous adult male mice. Recombinations of enamel organ and papilla formed teeth with regeneration of the investing layer of follicle and a root-related periodontal ligament, but no formation of alveolar bone. Bone only formed in those grafts which also included follicle. Recombinations of enamel organ and pulp produced dysplastic dentine with no enamel formation or proper tooth development. It was impossible, therefore, to assess whether the potential to regenerate an investing layer extends to the pulp later in development. At an earlier stage, however, the papillary mesenchyme has the ability to regenerate investing-layer cells which lack the capacity to form bone.

Neural crest-derived proprioceptive neurons express nerve growth factor receptors but are not supported by nerve growth factor in culture.

The neural crest-derived, first-order, sensory neurons of the embryonic chick trigeminal mesencephalic nucleus were grown in dissociated, glia-free culture. Whereas brain-derived neurotrophic factor promoted the survival and growth of the majority of these neurons (over 70% after 48 h incubation), nerve growth factor had no effect on their survival. The percentage survival in cultures supplemented with nerve growth factor at concentrations ranging from 0.2 to 625 ng/ml was only 2%, the same percentage survival as in control cultures. Furthermore, nerve growth factor did not change the dose-response of these neurons to brain-derived neurotrophic factor. Although nerve growth factor did not influence the survival of trigeminal mesencephalic neurons in culture, nerve growth factor specifically bound to the great majority of neurons growing in the presence of brain-derived neurotrophic factor. Autoradiographs of cultures incubated with iodinated nerve growth factor showed that the perikarya and processes of neurons were heavily labelled with silver grains. These findings demonstrate the existence of a population of neural crest-derived sensory neurons which express nerve growth factor receptors but are not supported by nerve growth factor in culture.

Fasciculation in the early mouse trigeminal nerve is not ordered in relation to the emerging pattern of whisker follicles.

Detailed reconstructions of lengths of the embryonic mouse maxillary nerve were made from serial light and electron microscope sections to determine whether there is any correspondence between the arrangement of fasciculi in the developing nerve and the emerging pattern of whisker follicles on the snout. There was neither correspondence nor obvious pattern in the arrangement of fasciculi at either E11, when trigeminal nerve fibers first contact the presumptive whisker pad, or at E12, when the pattern of developing whisker follicles becomes apparent. Fasciculi merged and branched to form an intricate plexus. Furthermore, the ratio of the number of nerve fibers in one fasciculus to the number in another prior to their merger differed significantly from the ratio of fiber numbers in the two fasciculi after their separation, which indicates that nerve fibers are freely exchanged between fasciculi. Our findings suggest that the somatotopic representation of the whisker follicle pattern in the brainstem does not develop by nerve fiber growth augmenting an initially ordered pattern of fasciculi.

Chemotropic effect of specific target epithelium in the developing mammalian nervous system.

Developing nerve fibres are guided to their targets by specific directional cues which are thought to be expressed in the tissues along the route and may involve the extracellular matrix. Another possibility, that directional cues emanate from the target itself, is consistent with the recent demonstration of homing behaviour by ectopic retinal ganglion axons and our previous demonstration that early trigeminal neurites grow directly to their virgin peripheral target in vitro. Here we show that this chemotropic effect is precisely limited to the trigeminal system; trigeminal ganglion neurites grow directly to their own target field but not to the adjoining field, normally innervated by the geniculate ganglion; furthermore, the trigeminal field does not influence the growth of geniculate neurites. Also, when trigeminal ganglia are co-cultured with isolated tissue layers of their target, neurites grow only towards the epithelial and not the mesenchymal component. These findings suggest that trigeminal epithelium is specified to attract correct innervation and that pathway mesenchyme, in which preformed guidance cues have been postulated, may provide favourable conditions for nerve fibre growth but not govern its direction.

An experimental study of timing and topography of early tooth development in the mouse embryo with an analysis of the role of innervation.

The putative involvement of the innervation in determining the sites of dental development was investigated by intra-ocular homografting and organ culture methods. Mandibular arches were dissected from 9 and 10-day-old (E9-E10) mouse embryos and grafted to the anterior eye chambers of homologous adult mice for 12-14 days. There was no significant difference in the incidence of tooth formation between grafts in which the trigeminal ganglion was included (n = 82, 68 per cent) or excluded (n = 72, 65 per cent). A parallel in vitro study in which E9 and E10 mouse embryo mandibular arches were cultured in the absence of trigeminal innervation showed that definitive tooth germs were formed during the 7-9 day culture period. It is concluded that innervation plays no part in determination of tooth development. In further experiments with E9 and E10 material, the complete mandibular arch, the hemimandibular arch and the ventral midline region of the mandibular arch (including the median epithelial isthmus) were each grafted for a period of 21 days. Alizarin-red whole-mount staining of recovered grafts revealed that bone had been deposited and mineralized in the majority of grafts of all types. Incisor and molar teeth with near normal crown shapes developed in grafts of complete mandibular arches. Hemimandibular arches gave rise almost exclusively to molars. Grafts of the E10 ventral midline region gave rise exclusively to incisors, but the same graft performed at E9 did not produce teeth. It is concluded that incisor primordia are localized in or near the median epithelial isthmus (and are thus destroyed or damaged when the mandibular arch is hemisected) and that incisors are not determined until E10. At E9 the odontogenic neural crest has yet to complete its ventrad migration into proximity with presumptive incisor epithelium. By this time, however, odontogenic crest and presumptive molar epithelium have already reached juxtaposition and molar primordia are fully competent. In grafts of the frontonasal and maxillary processes made at E10, prior to merging of their respective mesenchymes, frontonasal processes gave rise exclusively to incisors, whereas maxillary processes gave rise exclusively to molars.

Earliest sensory nerve fibres are guided to peripheral targets by attractants other than nerve growth factor.

Recent studies have shown that developing nerve fibres grow directly to their targets and are guided by specific cues, but the nature of these cues and the mechanism of guidance remain unknown. The growth of sympathetic axons towards an artificial source of nerve growth factor (NGF) in vivo and of sensory neurites up a concentration gradient of NGF in vitro has supported the hypothesis that NGF, produced by target tissues, acts as a chemotactic attractant for these nerve fibres during development. Both these studies and those of the influence of NGF or target tissues on neurite growth in vitro were conducted late in development when, following target encounter, the neurones had become dependent on NGF or target for survival. Here we have co-cultured embryonic mouse sensory neurones and their peripheral target tissue at a stage preceding their contact in vivo. Neurites grew directly and exclusively towards their own target but not to regionally inappropriate peripheral tissue. Antiserum to isogeneric NGF did not reduce this outgrowth but did reduce undirected neurite outgrowth which occurred in co-cultures of older neurones with denervated target tissue. These results demonstrate that agents other than NGF guide neurites of NGF-responsive neurones in development.

Neurite outgrowth from murine sensory ganglia cultured in a serumless medium.

Trigeminal and dorsal root ganglia were excised from mouse embryos of 10 and 18 days in utero age, respectively, and grown in tissue culture. A quantitative method was used to assess the extent of neurite outgrowth from the explants after 24 and 48 h in culture. Outgrowth from ganglia grown in a serumless medium was compared with that from ganglia in a medium supplemented with serum. There was no significant difference between the extent of outgrowth in either medium after 24 h in culture; however, after 48 h there was significantly greater outgrowth in the serum-supplemented medium.

Influence of nerve growth factor on the embryonic mouse trigeminal ganglion in culture.

Developing trigeminal ganglia have been excised from mouse embryos of 9, 10, 11 and 12 days gestation and grown in tissue culture. A quantitative method was used to assess the effect of nerve growth factor (NGF) and an antiserum to NGF (anti-NGF) on fiber outgrowth from the explanted ganglia. Fiber outgrowth from 9-day ganglia (E9 ganglia) appeared to be unaffected by the presence of NGF. However, the ganglia became increasingly responsive to NGF from embryonic day 10 through 12. The increased responsiveness to NGF in vitro coincided with the establishment of fiber contacts with peripheral target tissues in vivo. Anti-NGF produced no significant reduction of fiber outgrowth from E9 ganglia, but from later ganglia significant reduction were observed.

Tooth morphogenesis: the role of the innervation during induction and pattern formation.

The hypothesis is discussed that the innervation of the early mandibular and maxillary processes influences the initiation and patterning of tooth germs. Silver staining of embryonic mouse tissue supports the notion that the innervation is present before tooth buds appear. Data from other experimental studies is discussed in support of this hypothesis. The importance of the early events of tooth morphogenesis in initiating and patterning the dentition is discussed in the context of tooth morphogenesis as a whole. The processes involved in odontogenesis are categorized as Phase I: Initiating events; Phase II: Histogenetic events; and Phase III: Cytodifferentiative events. The complications of the interrelationship of these three seemingly discrete segments of tooth development are discussed in the context of inductive tissue interaction.

Pattern formation in the molar dentition of the mouse.

The developing molar tissues of 10 to 16 day mouse embryos have been dissected from the lower jaws and cultured for periods of up to four weeks in the anterior eye chambers of homologous adult hosts. Grafts of 10 and 11 day presumptive molar tissues developed two complete tooth crowns identifiable as M1 and M2. Grafts of 12 day (dental lamina) and later stages produced similar results but in many cases all three molar teeth were developed, in their normal sequence and with normal crown shapes and proportions. From 12 days and probably earlier the complete molar dentition is represented by a discrete group of cells whose later growth and differentiation are already determined. The fact that individual primordia for the second and third molars are not yet present at this stage suggests that neither their appearance nor their normal differentiation in the jaws requires a morphogenetic gradient field, an extrinsic control which has been proposed and is often assumed to exist. The results are consistent with developmental theories which propose that gradations of shape and size in the individual sequentially initiated elements of a series are expressions of intrinsic time-dependent alterations in the growing cell population which forms them.