Differential growth of facial primordia in chick embryos: responses of facial mesenchyme to basic fibroblast growth factor (bFGF) and serum in micromass culture

Differential growth of the three major facial primordia, the frontonasal mass, maxilla and mandible, results in a characteristic face shape. Abnormal growth of any of the primordia can lead to facial defects. In order to dissect out the factors that control growth, we developed a functional assay for cell proliferation using micromass culture and defined medium. Cell number was determined over a 4 day period and BrdU incorporation was used to determine the percentage of cells in S-phase. In defined medium, cell number progressively decreases and proliferation is very reduced in cultures of cells from all three primordia. When foetal calf serum was added, frontonasal mass cell number triples, mandible doubles and maxilla increases by half. The number of cells in S-phase increased in every case but the final cell number reflects a balance between proliferation and cell loss from the culture. The addition of basic fibroblast growth factor (bFGF) to defined medium leads to an increase in cell number in the frontonasal mass, while the cell number of mandibular and maxillary cultures is relatively unaffected. The percentage of cells in S-phase is highest in frontonasal mass cultures. Serum and bFGF both increase chondrogenesis in frontonasal mass cultures when compared to defined medium. In contrast in mandibular cultures, serum does not change the amount of cartilage and with bFGF chondrogenesis is reduced. The coordination of the changes in proliferation and differentiation in frontonasal mass cultures suggest that either these two processes are independently stimulated to the same extent or a single subpopulation of cells is stimulated to divide and differentiate into chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Selective stimulation of in vitro limb-bud chondrogenesis by retinoic acid

Embryonic exposure to pharmacologic doses of vitamin A analogs (retinoids) is a well-known cause of limb-skeletal deletions, limb truncation and other skeletal malformations. The exclusively inhibitory effect of retinoic acid (RA) on chondrogenesis in standard serum-containing cultures of limb-bud mesenchymal cells is equally well known and has provided a means to explore the cellular basis for RA-mediated skeletal teratogenesis. Recent studies showing that lower RA concentrations can cause skeletal duplication when applied directly to the anterior border of a developing limb, suggest that RA may have a role in normal limb development as a diffusible morphogen capable of regulating skeletal pattern. While RA treatment causes both, skeletal deletions and duplications are clearly different (if not opposing) effects, the latter of which is difficult to reconcile with RA's heretofore exclusively inhibitory effect on in vitro chondrogenesis. In the present study. RA's effects on chondrogenesis and myogenesis were examined in serum-free cultures of chick limb-bud mesenchymal cells and compared with its effects on similar cultures grown in serum-containing medium. When added to serum-free medium, concentrations of RA known to cause skeletal duplication in vivo dramatically enhanced in vitro chondrogenesis (to over 200% of control values) as judged by both Alcian-blue staining and [35S]sulfate incorporation, while having little effect on myogenesis. Higher concentrations inhibited both chondrogenesis and myogenesis. The results indicate that at physiological concentrations. RA can selectively modulate chondrogenic expression and suggest that at higher concentrations, RA's inhibitory effects are less specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ascorbate on myogenesis in micromass culture

Summary Micromass cultures from stage 23 and 24 chick wing mesenchyme were grown in serum-containing medium with or without additional ascorbic acid. It was found that ascorbic acid administered as a single pulse or present continuously throughout culture, in concentrations as low as 25 μg/ml, was sufficient to abolish 80% of myogenesis as assessed by immunolocalization using muscle-specific antibodies. This effect was not significantly altered when cultures were maintained in a serum-free medium that promotes myogenesis. In contrast to the above findings, spectrophotometric analysis of accumulated sulphated glycosaminoglycans, an indicator of chondrogenesis, was elevated by ascorbate treatment. Furthermore, a similar level of glycosaminoglycan stimulation was found in ascorbate treated stage 23 distal-tip limb cultures that were essentially free of myogenic cells. We conclude, therefore, that the presence of myoblasts in whole-limb cultures has no appreciable inhibitory effects on chondrogenesis. This work was supported by the Nuffield Foundation, England.     

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.     

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.     

Microtiter micromass cultures of limb-bud mesenchymal cells

Summary A method is described for growing high-density micromass cultures of chick and mouse limb mesenchyme cells in 96-well microtiter plates (μTμM cultures). Rapid quantitative estimates of chondrogenic expression were obtained by automated spectrophotometric analysis of Alcian-blue-stained cartilage matrix extracts performed in the wells in which the cells had been grown. Quantitative estimates of myogenic expression were obtained similarly using anti-sarcomere myosin monoclonal antibody and modified ELISA techniques. This μTμM-ELISA method may be adapted for use with other antigens for which specific antibodies are available. These methods were used to compare cartilage and muscle differentiation in 1 to 4 d μTμM cultures grown in serum-containing (SCM) and defined (DM) media. The DM contains minimal additives (insulin, hydrocortisone, and in some cases, ascorbate or transferrin) and supports both chondrogenesis and myogenesis. The colorimetric analyses agree well with the morphologic appraisal of chondrogenesis and myogenesis. Similar numbers of cartilage nodules formed in all cultures, but in DM the nodules failed to enlarge; explaining the reduced matrix synthesis in DM as compared with SCM, and suggesting that nodule enlargement is a discrete, serum-dependent step. Studies of selected additives to DM show that transferrin enhances myogenesis, ascorbic acid enhances chondrogenesis, and retinoic acid inhibits chondrogenesis. Together, the μTμM system, in situ colorimetric assays of chondrogenesis and myogenesis, and DM will allow rapid prescreening of teratogens and screening of various bioactive compounds (e.g., hormones, growth factors, vitamins, adhesion factors) for effects on limb mesenchymal cell differentiation. This work was supported by grants RR08006-13 (DFP) and HD05505 and HD18577 (MS) from the National Institutes of Health, Bethesda, MD. MF-20 hybridoma supernatant was obtained from the Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa, Iowa City, Iowa 52242 (maintained by NIH grant NO1-HD62915).     

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.     

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.     

Nonuniformity within Embryonic Somites: Differental Response to Retinoic Acid in Vitro

Recent studies have shown that in the developing chick embryo, at physiological level retinoic acid (RA) causes mirror-image duplication of limb skeletal elements. This has led to the suggestion that RA could be the endogenous morphogen or isgnal substance. In this study, in order to explore the effect of RA on somite chondrogenesis, we have standardized a serum-free chemically defined medium that supports the growth of somite explants in vitro. The results indicate that in somites RA at 10 ng/ml level induces cell proliferation, DNA and sulfated proteoglycan synthesis, and at higher concentrations is toxic. The results further show that RA induced stimulation of somite chondrogenesis is sclerotomal specific and the dermamyotemal portion of the somites does not exihibit a similar response. Retinoic acid also increases heparan sulfate synthesis and aggregation of isolated sclerotomal cells in culture. These results thus suggest that in amplifying chondrogenesis, RA acts at all phases such as cell proliferation (may increase cell viability) and aggregation, increased DNA synthesis and increased synthesis of matrix components. In otherwords, RA seems to initiate a chain of inter-related events.     

Inhibition of chondrogenesis by retinoic acid in limb mesenchymal cells in vitro: Effects on PGE2 and cyclic AMP concentrations

Effects of retinoic acid (RA) on prostaglandin E₂ (PGE₂) and cyclic AMP (cAMP) concentrations were investigated in high density, micromass cultures of mesenchymal cells derived from chick limb buds. Exposure of cells during the initial 24 h of culture to RA concentrations between 0.05–1.0 μg/ml inhibited chondrogenesis in a dose-dependent manner with 1.0 μg/ml totally inhibiting cartilage formation. Concentrations of PGE₂ and cAMP increased during the prechondrogenic period in control cells in a closely related way and remained elevated throughout the six-day period examined. Addition of RA (0.05 and 0.5 μg/ml) did not significantly alter cAMP concentrations at any time point, but significantly elevated PGE₂ levels relative to control cells in six-day cultures in a concentration-dependent manner. Addition of dibutyryl cAMP enhanced chondrogenesis in control cells between days 3 and 4, but failed to alter the inhibitory effect of RA on chondrogenesis. The results indicate that while PGE₂ and cAMP are important signals in cartilage differentiation, the inhibitory effects of RA on this process are mediated through some other mechanism.     

Evaluation of the sensitive step of inhibition of chondrogenesis by retinoids in limb mesenchymal cells in vitro

The sensitive step of inhibition of chondrogenesis in vitro by retinoids was investigated in modified micromass cultures of limb bud mesenchymal cells from mouse embryos of day 11 and 12. Evaluation of chondrogenesis was performed after alcian blue staining, using a simple random hit counting of cartilage nodules. All-trans-retinoic acid, 13-cis-retinoic acid, and a newly developed arotinoid, RO 13-6298, were tested for their ability to inhibit chondrogenesis. We found that inhibition of chondrogenesis depended on the dosage and the duration of treatment with the different retinoids. Further analysis showed that chondrogenesis in limb bud mesenchymal cells from the proximal part was irreversibly inhibited after one hour of treatment, whereas distal cells showed a reduction of cartilage development only after a treatment period of 12 and more hours. In respect to the doses of the retinoids, proximal cells were about one magnitude more vulnerable than distal cells. These proximo-distal differences were obtained with 13-cis-retinoic acid at 10 micrograms/ml, with all-trans-retinoic acid at 1 microgram/ml and with arotinoid RO 13-6298 with 10 ng/ml. It is supposed that the late blastemal stage of chondrogenic differentiation before the onset of matrix synthesis is the step which is most vulnerable to retinoid treatment.     

Stage-related chondrogenic potential of avian mandibular ectomesenchymal cells

We have examined the in vitro stage-related chondrogenic potential of avian mandibular ectomesenchymal cells using micromass cultures. Our results indicate that mandibular ectomesenchymal cells as early as stage 16, soon after the formation of the mandibular arches and well before the initiation of in vivo chondrogenesis, have chondrogenic potential which is expressed in micromass culture. There is an increase in the total area of the cultures occupied by cartilage when cells from increasing stages of development are used. The nodular pattern of chondrogenesis in these cultures indicates that mandibular ectomesenchymal cells are a heterogenous population from the time of mandibular arch formation. In addition, we studied the temporal expression of the genes for extracellular matrix proteins during in vitro chondrogenesis and correlated the morphological changes with the pattern of gene expression. Low levels of type II collagen mRNA are present in the cultures prior to detection of any stainable cartilage matrix and increase 5 fold just before the onset of chondrogenesis in vitro. On the other hand mRNA for cartilage proteoglycan core protein was not detected until the second day of culture when stainable cartilage matrix was present and progressively increased thereafter. Messenger RNA for type I collagen was present at the time of initiation of cultures and continuously increased during the culture period. Our experiments also indicated that embryonic epithelia can inhibit the in vitro chondrogenesis of mandibular ectomesenchymal cells and that the inhibitory effect of embryonic epithelia is independent of its age and site of origin.     

Retinoic acid promotes proliferation and chondrogenesis in the distal mesodermal cells of chick limb bud

Distal and proximal mesoderm of chick limb bud was respectively dissociated and cultured in the medium containing various concentrations of retinoic acid (RA). At low concentrations (5-50 ng/ml), RA promoted proliferation and chondrogenesis in the distal mesodermal cells. The distal cells of stage 20-24 limb buds were responsive to RA, although those of stages 25-27 were unresponsive. Both the cells of anterior and posterior regions of the distal mesoderm were responsive to RA, while the cells of proximal mesoderm were unresponsive. At higher concentrations, the growth-promoting effect of RA was reduced and chondrogenesis in the distal cells was rather inhibited. These results were discussed in relation to the role of RA as the morphogen in normal limb development and experimental duplicate formation.     

Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. In this study, we have used multipotential murine C3H10T1/2 cells to analyze the effect and mechanism of action of bone morphogenetic protein 2 (BMP-2) on chondrogenesis. C3H10T1/2 cells have been previously shown to undergo multiple differentiation pathways. While chondrogenesis, osteogenesis, myogenesis and adipogenesis have been observed, chondrocytes appear significantly less frequently than the other cell types, and the appearance of chondrocytes exclusive of the other cell types has not been observed. We report here that the appearance of chondrocytes in C3H10T1/2 cells is markedly enhanced as a result of culture under conditions favorable for chondrogenesis, i.e. plating as high-density micromass and treatment with BMP-2. Such cultures contain chondrocyte-like cells, elaborate an Alcian blue stained cartilage-like matrix, express link protein and type II collagen, both cartilage matrix markers, and show increased [35S]sulfate incorporation. The appearance of Alcian blue positive material and increased sulfate incorporation are dependent on the dose of BMP-2, culture time, and cell plating density of the micromass cultures. Differentiation of cells within the micromass was specific to the chondrogenic lineage, as alkaline phosphatase staining revealed only faint staining in the micromass at the highest BMP-2 concentration. The importance of enhanced cell-cell interaction in the chondroinductive effects of BMP-2 on high-density C3H10T1/2 cultures was further implicated by the additional promotion of chondrogenesis in the presence of the polycationic compound, poly-L-lysine, which has been previously reported to enhance cellular interactions and chondrogenesis in embryonic limb mesenchymal cells. Taken together, these findings suggest that chondrogenesis in C3H10T1/2 cells is inducible by BMP-2 and requires cell-cell interaction.