The effects of retinoids on cartilage differentiation in micromass cultures of chick facial primordia and the relationship to a specific facial defect

Retinoids produce facial defects in chicken embryos. Outgrowth of the frontonasal mass with accompanying cartilage differentiation and pattern formation is inhibited. In contrast, the development of the mandibular primordia that give rise to the lower beak proceeds normally. To investigate whether the upper beak defect is based on the inhibition of cartilage differentiation specifically in the frontonasal mass, the effects of retinoids on chondrogenesis in micromass (high density) cultures of cells from facial primordia have been studied. When either 10(-6) M retinoic acid or 10(-8) M (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl-1- propenyl]benzoic acid (TTNPB; a stable retinoid) is added to the culture medium, cartilage differentiation is inhibited. Both frontonasal mass and mandible cultures are equally affected. The concentration of TTNPB found in both facial primordia in vivo, after a treatment that produces the defect, is also about 10(-8) M. This rules out preferential accumulation of the retinoid by the frontonasal mass as an explanation for the defect. In fact, the concentration of retinoid found in vivo, should, from the culture studies, be sufficient to markedly inhibit chondrogenesis in both the frontonasal mass and mandibles. The effects of exposure to retinoids in the intact face appear to be different to those in culture. Furthermore, when cells from retinoid-treated facial primordia are cultured in micromass, the extent and pattern of chondrogenesis in frontonasal mass cultures is identical to that of cells from untreated primordia. Cartilage differentiation in mandible cultures is slightly affected. These findings suggest that retinoids do not produce the specific facial defect by directly interfering with cartilage differentiation.     

The patterns on chondrogenesis of cells from facial primordia of chick embryos in micromass culture

Chondrogenesis of mesenchymal cells from the frontonasal mass, mandibles and maxillae of stage-24 chick embryos has been investigated in micromass (high-density) cultures. Distinct differences in the amount and pattern of cartilage differentiation are found. In cultures of frontonasal mass cells, a central sheet of cartilage develops; in cultures of mandible cells, less cartilage differentiates and nodules form; while in cultures of maxillae cells, virtually no chondrogenesis takes place. The same patterns of cartilage are found in cultures established from stage-20 embryos. At stage 28, frontonasal mass cultures form cartilage nodules and the number of nodules in mandible cultures is markedly decreased. There are striking parallels between the chondrogenic patterns of cells from the face and limb buds in micromass culture. The frontonasal mass cell cultures of stage-20 and -24 chick embryos resemble those established from the progress zone of limb buds. The progress zone is an undifferentiated region of the limb in which positional cues operate. Cultures established from the frontonasal mass of stage-28 chick embryos and from the mandibles of all stages resemble cultures of whole limb buds. These contain a mixture of committed and uncommitted cells. Ectoderm from facial primordia locally inhibits chondrogenesis in micromass cultures and this could provide a positional cue. The differences in chondrogenic potential of cells from facial primordia may underlie the specific retinoid effects on the frontonasal mass.     

Chondrogenesis and myogenesis in micromass cultures of mesenchyme from mouse facial primordia

Summary The face develops from small buds of tissue positioned around the primitive mouth. The chondrogenic and myogenic cell populations contained within these facial primordia in mouse embryos have been investigated in short-term micromass culture. Chondrogenesis occurred in frontonasal mass mesenchyme from E11-E13 embryos, in maxillary mesenchyme from E12.5 embryos and was absent in mandibular mesenchyme. Myogenesis was greatest in mandibular mesenchyme, moderate in maxillary mesenchyme and low in the frontonasal mass. When compared with chick embryos the mouse facial primordia have lower chondrogenic potential, which in the case of the frontonasal mass may be related to the relative outgrowth of the primordia in the two species. Chondrogenesis in the mouse mandibular mesenchyme may be affected by the presence of a large population of odontogenic mesenchyme. The behavior of myogenic cell populations is related to the pattern of the musculature of the face, as the mandible contains the most muscle, the maxilla some, and the frontonasal mass none. However, the presence of myoblasts in early mesenchyme of all primordia may indicate that, as with chick, facial primordia are initially seeded with muscle cells and that the size of the cell population is subsequently controlled according to the development of the musculature within the primordia.     

Differential effects of physiological concentrations of retinoic acid in vitro on chondrogenesis and myogenesis in chick craniofacial mesenchyme

Recent studies have shown that in the developing limb bud retinoic acid is a skeletal morphogen at physiological levels, but a potent teratogen at higher levels. Retinoic acid has also been shown to be teratogenic during facial development, but very low levels may have an as yet unspecified role in normal development. In the present study the effects of retinoic acid on chondrogenesis and myogenesis by craniofacial cells grown in micromass cell culture were investigated. Retinoic acid, at concentrations of 0.01-100 ng/ml, was supplied to cells derived from day-4 (H.H stage 23/24) chick embryo mandibular, maxillary and frontonasal processes, grown in micromass cultures for 4 days in both serum-containing and defined media. Based on Alcian-blue-staining, concentrations of retinoic acid of 0.1-1 ng/ml were found to enhance chondrogenesis by mandibular cells grown in defined medium, while greater concentrations up to 100 ng/ml inhibited chondrogenesis. By contrast, chondrogenesis was generally retarded by all concentrations of retinoic acid applied to frontonasal cells grown in defined medium and when applied to both mandibular and frontonasal cells when grown in serum-containing medium. Cells from stage-23/24 maxillae did not display any significant chondrogenic activity in either medium under these culture conditions. Unlike chondrogenesis, myogenesis in mandibular, frontonasal and maxillary cultures was greater in defined than serum-containing medium, based on the appearance of immunologically detectable muscle myosin, and was reduced considerably less in defined medium by all concentrations of retinoic acid tested. In the presence of serum however, myogenesis was retarded with increasing concentrations of retinoic acid beyond 1 ng/ml in micromass cultures from all three facial regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of myogenic cells in micromass cultures of cells from chick facial primordia

Antibodies to the myosin heavy chains of striated muscle were used to trace myogenic differentiation in the developing face and in cultures of cells from the facial primordia of chick embryos. In the intact face, myogenic cells differentiate first in the mandibular primordia and can be detected at stage 28. The early muscle blocks contain both fast and slow classes of myosin heavy chains. At stages 20 and 24, no myogenic cells are found in any of the facial primordia. However, when the cells are placed in micromass (high density) cultures, myogenic cells differentiate, revealing the presence of potentially myogenic cells in all the facial primordia. The number of myogenic cells bears no consistent relationship to the extent and pattern of chondrogenesis. Therefore the ability of the cell populations of the facial primordia to differentiate into cartilage when placed in culture is independent of the muscle cell lineage. The facial primordia represent a mixed cell population of neural crest and mesodermal cells from at least as early as stage 18.     

MEK-ERK signaling plays diverse roles in the regulation of facial chondrogenesis

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, also known as the MEK-ERK cascade, has been shown to regulate cartilage differentiation in embryonic limb mesoderm and several chondrogenic cell lines. In the present study, we employed the micromass culture system to define the roles of MEK-ERK signaling in the chondrogenic differentiation of neural crest-derived ectomesenchyme cells of the embryonic chick facial primordia. In cultures of frontonasal mesenchyme isolated from stage 24/25 embryos, treatment with the MEK inhibitor U0126 increased type II collagen and glycosaminoglycan deposition into cartilage matrix, elevated mRNA levels for three chondrogenic marker genes (col2a1, aggrecan, and sox9), and increased expression of a Sox9-responsive collagen II enhancer-luciferase reporter gene. Transfection of frontonasal mesenchyme cells with dominant negative ERK increased collagen II enhancer activation, whereas transfection of constitutively active MEK decreased its activity. Thus, MEK-ERK signaling inhibits chondrogenesis in stage 24/25 frontonasal mesenchyme. Conversely, MEK-ERK signaling enhanced chondrogenic differentiation in mesenchyme of the stage 24/25 mandibular arch. In mandibular mesenchyme cultures, pharmacological MEK inhibition decreased cartilage matrix deposition, cartilage-specific RNA levels, and collagen II enhancer activity. Expression of constitutively active MEK increased collagen II enhancer activation in mandibular mesenchyme, while dominant negative ERK had the opposite effect. Interestingly, MEK-ERK modulation had no significant effects on cultures of maxillary or hyoid process mesenchyme cells. Moreover, we observed a striking shift in the response of frontonasal mesenchyme to MEK-ERK modulation by stage 28/29 of development.     

Epithelial-mesenchymal interactions in the outgrowth of limb buds and facial primordia in chick embryos.

The facial primordia in the chick embryo begin as rounded swellings that surround the primitive mouth and these grow out to form the beak. The control of proximodistal outgrowth is not well understood but may involve similar mechanisms to the limb bud. In order to test this hypothesis, combinations were made between epithelium and mesenchyme from facial primordia and limb buds. Signals from all three types of facial mesenchyme (frontonasal mass, mandibular, and maxillary) maintained the thickened apical ectodermal ridge of limb epithelium for up to 48 h. Combinations of tissues from the frontonasal mass mesenchyme and limb epithelium underwent substantial and correct morphogenesis. In contrast, poor development was observed in combinations with mandibular mesenchyme. Signals from frontonasal mass epithelium promoted outgrowth and morphogenesis of limb mesenchyme whereas mandibular and maxillary epithelium did not support joint morphogenesis. The results suggest that signals employed in the epithelial-mesenchymal interactions in facial primordia are similar but not identical to those signals used in the limb bud.     

Epithelia are interchangeable between facial primordia of chick embryos and morphogenesis is controlled by the mesenchyme

The embryonic chick face is composed of a series of facial primordia, epithelium-covered buds of mesenchyme, which surround the presumptive mouth. The protruding adult upper beak containing the prenasal cartilage is formed from the frontonasal mass, the paired maxillary primordia form the sides of the face, while the lower beak is derived from the paired mandibular primordia which contain the two Meckel's cartilages. When grafted to a host wing bud, the frontonasal mass and the mandibular primordia both form elongated outgrowths, whereas the maxillary primordium forms a ball of tissue. Facial epithelium is required for growth and morphogenesis of all primordia. Recombinations between epithelium and mesenchyme from different primordia show that the epithelia are interchangeable and appear to be equivalent. Even the epithelium from the maxillary primordium that does not grow out in a polarized fashion can support outgrowth of the frontonasal mass and mandibular mesenchyme. The form of the recombined graft is determined by the mesenchymal component.     

Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro

Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1-1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1-1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.     

Epithelial-mesenchymal interactions in the development of chick facial primordia and the target of retinoid action

The development of the chick face involves outgrowth of buds of tissue, accompanied by the differentiation of cartilage and bone in spatially defined patterns. To investigate the role of epithelial-mesenchymal interactions in facial morphogenesis, small fragments of facial tissue have been grafted to host chick wing buds to continue their development in isolation. Fragments of the frontonasal mass give rise to typical upper-beak-like structures: a long central rod of cartilage, the prenasal cartilage and an egg tooth. Meckel's cartilage, characteristic of the lower beak, develops from fragments of the mandible. Removal of the ectoderm prior to grafting leads to truncated development. In fragments of frontonasal mass mesenchyme only a small spur of cartilage differentiates and there is no outgrowth. The mandible is less affected; a rod of cartilage still forms but the amount of outgrowth is reduced. Retinoid treatment of chick embryos specifically affects the development of the upper beak and outgrowth and cartilage differentiation in the frontonasal mass are inhibited. The mandibles, however, are unaffected and develop normally. In order to investigate whether the epithelium or the mesenchyme of the frontonasal mass is the target of retinoid action, recombinations of retinoid-treated and untreated facial tissue have been grafted to host wing buds. Recombinations of retinoid-treated frontonasal mass ectoderm with untreated mesenchyme develop normally whereas recombinations of untreated ectoderm with retinoid-treated mesenchyme lead to truncations. The amount of outgrowth in fragments of mandibular tissue is slightly reduced when either the ectoderm or the mesenchyme has been treated with retinoids. These recombination experiments demonstrate that the mesenchyme of the frontonasal mass is the target of retinoid action. This suggests that retinoids interfere with the reciprocal epithelial-mesenchymal interactions necessary for outgrowth and normal upper beak development.     

Retinoic Acid Treatment Induces Cell Death and the Protein Expression of Retinoic Acid Receptor β in the Mesenchymal Cells of Mouse Facial Primordia in Vitro

We isolated mesenchymal cells from individual facial primordia of mouse embryos on 11 days post coitum and examined the effects of retinoic acid (RA) on chondrogenesis, induction of cell death, and the protein expression of retinoic acid receptor (RAR) β and γ in micromass culture. Under the control condition, cells of both medial and lateral nasal prominences (MNP and LNP) displayed high chondrogenic potential, while those of maxillary and mandibular prominences (Mx and Md) had constant growth activity and low chondrogenic potential. Though none of the cells expressed detectable levels of the RAR β protein, RAR γ was expressed in the cells of all the facial primordia. One μM RA inhibited the chondrogenesis, and induced cell death accompanied with the induction of the RAR β protein in LNP, MX and Md cells within 6 hr. On the contrary, both cell death and RAR β protein induction were detected in the MNP cells treated with RA for 24 hr. These results suggest that the RAR β is involved in the process of the cell death induced by the RA treatment in the mesenchymal cells of the mouse facial primordia.     

Effect of unilateral partial facial paralysis on periosteal growth at the muscle-bone interface of facial muscles and facial bones

In a previous study, the influence of the midfacial musculature upon growth and development of the maxilla and mandible was established macroscopically. Dry skull measurements revealed a reduced premaxillary, maxillary, mandibular, and anterior corpus length with a simultaneous increase in mandibular ramal height on the paralyzed side. It was demonstrated that these reduced premaxillary and maxillary lengths were among others the result of reduced nasofrontal growth, whereas the increased ramal height was accompanied by condylar growth alterations. This study investigated whether the growth alterations at the mandibular corpus region could be explained by altered periosteal growth at the muscle-bone interface of the zygomatico-auricular muscle and the mandibular corpus, caused by altered muscle activity acting upon the periosteal sleeve. Fifty-six 12-day-old New Zealand White rabbits were randomly assigned to either a control or an experimental group. In the experimental group, left-sided partial facial paralysis was induced surgically when the animals were 12 days old. To study the muscle-bone interface, seven follow-up time intervals were defined between 3.5 and 60 days following the surgery. At these time intervals, four randomly selected control animals and four randomly selected experimental animals were killed. The anterior mandibular corpus region with the muscle-bone interface of the left control hemimandible and the left and right experimental hemimandibles was processed for undecalcified tissue preparation. Quantitative analysis of the total bone area at the muscle-bone interface revealed no significant differences between the left control hemimandible and the left and right experimental hemimandibles. Also, qualitative study of the histologic sections showed no major changes in the appearance or development of the trabecular pattern between the groups. However, slight differences in the distribution pattern of osteoblasts and osteoclasts along the bony surface were found between the left control hemimandible and the left and right experimental hemimandibles, which seemed to explain the alterations in mandibular corpus shape between these groups. It was suggested that these changes in the distribution pattern of osteoblasts and osteoclasts were the result of changes in the loading distribution pattern acting upon the mandible, caused by an altered neuromuscular recruitment pattern of the remaining functionally intact, mandibularly attached muscles. The latter was probably the result of adaptive mandibular positioning in response to an altered occlusal relationship, which was induced by the abnormal maxillary growth as a result of the unilateral partial facial paralysis.     

Effect of TGF-beta 1, TGF-beta 2, and bFGF on chick cartilage and muscle cell differentiation

In insulin containing defined medium TGF-beta 1, TGF-beta 2, and bFGF all stimulate chondrogenic differentiation in high-density micromass cultures of distal limb bud mesenchyme cells of chick embryos. In addition bFGF inhibits myogenic differentiation, while TGF-beta 1 and TGF-beta 2 appear to have no effect. TGF-beta 1 and bFGF together act additively to enhance chondrogenesis, while TGF-beta blocks the bFGF inhibitory action on myoblasts, thus allowing them to differentiate. In the absence of insulin, the inhibitory effect of bFGF on muscle cell differentiation is reduced; cartilage differentiation in the presence of the above growth factors is also slightly reduced.     

FGF signals from the nasal pit are necessary for normal facial morphogenesis

Fibroblast growth factors (FGFs) are required for brain, pharyngeal arch, suture and neural crest cell development and mutations in the FGF receptors have been linked to human craniofacial malformations. To study the functions of FGF during facial morphogenesis we locally perturb FGF signalling in the avian facial prominences with FGFR antagonists, foil barriers and FGF2 protein. We tested 4 positions with antagonist-soaked beads but only one of these induced a facial defect. Embryos treated in the lateral frontonasal mass, adjacent to the nasal slit developed cleft beaks. The main mechanisms were a block in proliferation and an increase in apoptosis in those areas that were most dependent on FGF signaling. We inserted foil barriers with the goal of blocking diffusion of FGF ligands out of the lateral edge of the frontonasal mass. The barriers induced an upregulation of the FGF target gene, SPRY2 compared to the control side. Moreover, these changes in expression were associated with deletions of the lateral edge of the premaxillary bone. To determine whether we could replicate the effects of the foil by increasing FGF levels, beads soaked in FGF2 were placed into the lateral edge of the frontonasal mass. There was a significant increase in proliferation and an expansion of the frontonasal mass but the skeletal defects were minor and not the same as those produced by the foil. Instead it is more likely that the foil repressed FGF signaling perhaps mediated by the increase in SPRY2 expression. In summary, we have found that the nasal slit is a source of FGF signals and the function of FGF is to stimulate proliferation in the cranial frontonasal mass. The FGF independent regions correlate with those previously determined to be dependent on BMP signaling. We propose a new model whereby, FGF-dependent microenvironments exist in the cranial frontonasal mass and caudal maxillary prominence and these flank BMP-dependent regions. Coordination of the proliferation in these regions leads ultimately to normal facial morphogenesis.     

Differential regulation of cerebroside sulfotransferase and 2',3'-cyclic nucleotide 3'-phosphodiesterase by basic fibroblast growth factor in relation to proliferation in rat oligodendrocyte cultures.

Previous results (Fressinaud, C., Sarliève, L.L., and Labourdette, G. J. J. Cell. Physiol., 141:667-674, 1989b) have shown that cerebroside sulfotransferase (CST; EC 2.8.2.11) is enriched in pure rat oligodendrocyte (OL) cultures and that its activity is increased by factors mitogenic for OL precursors and galactocerebroside (GC) expressing OL, such as basic fibroblast growth factor (bFGF), platelet-derived growth factor, and high insulin concentrations. In contrast, transforming growth factor beta or low insulin concentrations were found to be ineffective in this culture system. As bFGF mainly enhanced the proliferation of OL precursors (GC negative cells) rather than that of differentiated (GC+) cells, a relationship between OL precursor proliferation and CST increase was suggested. This hypothesis was first tested in 20-day-old OL cultures grown in chemically defined medium. The dose-response curve of [125I] Iododeoxyuridine ([125I]dUrd) incorporation toward bFGF was parallel to that of CST specific activity, and maximal stimulation was reached at 5 ng/ml bFGF for both. In contrast, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) specific activity decreased after bFGF treatment. To determine if CST increase was linked to the proliferation of OL precursors induced by bFGF, cell proliferation was blocked by cytosine arabinoside (ARA-C). From 10(-8) to 10(-5) M ARA-C there was a dose-dependent inhibition of cell proliferation and a decrease in CST specific activity, whereas CNP specific activity was enhanced. When the cells were treated with bFGF and 10(-6) M ARA-C together, the proliferation was completely blocked and CST activity decreased by 72% below control values, whereas CNP activity was not significantly decreased. Immunocytochemical studies showed that the number of sulfatide-expressing cells and the number of cycling cells were increased after bFGF treatment, but that there was no overlapping between these two populations. Taken together these results suggest that CST activity and sulfatide expression appear shortly after the arrest of OL precursor division.     

Fibroblast growth factor stimulates the gene expression and production of tissue inhibitor of metalloproteinase-1 in bovine granulosa cells

Summary The hormonal control of tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression and production by growth factors, gonadotrophins, and serum factors in cultured bovine granulosa cells (BGC) were investigated. Confluent cultures of BGC were exposed to various factors in a defined medium and levels of TIMP-1 in the conditioned medium were determined by enzyme immunoassay. Basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) showed potent stimulation of cell proliferation and TIMP-1 production by BGC, while insulin stimulated growth but not TIMP-1 production. Basic FGF stimulated TIMP-1 production and BGC cell proliferation in a dose-dependent manner. A time course of TIMP-1 production showed substantially increased levels between 18 and 24 h in both control and bFGF-stimulated BGC cultures with bFGF-stimulated cultures having markedly higher TIMP-1 production at all time points. Consistent with the TIMP-1 production data, bFGF and aFGF increased the expression of TIMP-1 mRNA as determined by northern blot analysis, while insulin, inhibited TIMP-1 mRNA levels. These results indicate that FGF-induced TIMP-1 production by BGC may support bovine embryo development in vitro.     

Cell proliferation during formation of the embryonic facial primordia.

Cell proliferation of mesenchyme in the developing primary palate of the chick embryo was analyzed by tritiated thymidine autoradiography. Pulse labeling, repeated labeling, and label dilution techniques were employed to determine generation times, transit times, growth fractions, and other parameters of the cell cycle. In vivo and in vitro studies were performed to evaluate the role of tissue interactions during outgrowth of the facial primordia. These studies indicated that initially, during early stages of primary palate formation, virtually all mesenchymal cells are in the division cycle with relatively short generation times. As development proceeds, mesenchymal cell populations in the facial primordia, such as the maxillary process, retain cycle characteristics comparable to those of the progenitor cell populations. In regions adjacent to the facial primordia, such as the roof of the stomodeum, cell cycle times become more heterogeneous and result in removal of cells from rapidly cycling cell populations into subpopulations that are cycling more slowly and that, in some instances, become quiescent. Regional analysis of cell proliferation in the maxillary process indicated that growth rates of mesenchyme differ based on proximity to the overlying epithelium. Correlative in vitro studies of epithelial-mesenchymal separation and recombination experiments in organ culture revealed that the viability of mesenchyme was dependent on the presence of epithelium and that this effect was strongly stage-dependent. These and other results lead us to the conclusion that epithelial-mesenchymal interaction is significant to the maintenance of growth rates in the facial primordia and that the effects observed are mediated, at least in part, by developmental signals at the epithelial-mesenchymal interface.