Palate morphogenesis

Summary Mesenchymal cells from the palate of mouse embryos at day 14.5 of gestation produce a minor population of stellate cells in culture. These cells are often bipolar and spindle-shaped with long cytoplasmic processes similar to neural-crest cells. Culturing of expiants of palatal mesenchyme enriched for this type of cell. Stellate cells were the first to migrate from the expiants, followed by fibroblast-like cells and then by squamous cells. The majority of the cells in the expiant were fibroblast-like. Squamous cells were present mostly in the anterior and mid-palate and least frequently in those from the posterior palate. They may represent tooth-germ epithelium. When pieces of palate were dissected out and cultured for enrichment of non-muscle contractile systems, most of the migrating cells were stellate. These may represent the highly migratory cells that are, in part, responsible for elevation of the palate shelf. Serotonin was measured in cultured mesenchymal cells from the palate. Its occurrence is consistent with regulation of movement of palate cells.     

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.     

A gradient of responsiveness to the growth-promoting activity of ZPA (zone of polarizing activity) in the chick limb bud

Limb bud mesoderm of stage 22-23 embryos was dissected into four pieces along the anteroposterior axis and dissociated cells of each region were separately cultured under various conditions. When the cells were cultured in medium containing 0.1% fetal calf serum (serum-poor medium) only a slight increase in cell number occurred in the cultures of all four regions. However, when the cells were cultured in medium containing 10% FCS, only cells of two central regions proliferated rapidly, and no growth promotion was observed in cells in the most anterior and posterior regions. Using the serum-poor medium, we examined the growth-promoting effects of cocultured limb bud fragments and of some growth factors on the cells of four regions. Anterior, distal, and proximal fragments promoted cell proliferation and their promotive effect on the cells of each region was equal. On the other hand, posterior fragments (containing ZPA) showed stronger promotive effects on preaxial cells than on postaxial cells. For comparison with the growth-promotive effect of the posterior fragment, fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin, and retinoic acid were tested in cell culture. FGF showed position-dependent growth promotion, while EGF and insulin promoted growth in the cells of all four regions to a similar degree. Retinoic acid showed no effect on cell growth at low concentrations, and was rather toxic at high concentrations. These results suggest that the cells of the posterior region secrete an FGF-like growth factor(s), which controls normal limb development and experimental duplicate formation.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodismH (bpH) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bpH mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

Inhibition and reversion of chondrogenesis by retinoic acid in rat limb bud cell cultures

Summary Mesenchyme cells derived from embryonic rat limb buds cultured at high density differentiated into chondrocytes. The degree of chondrogenesis was assessed by alcian blue staining, a stain specific for cartilage matrix. The addition of retinoic acid on day 1 of culture inhibited chondrogenesis in a dose-dependent fashion. When retinoic acid was added to the cultures on day 5, the cartilage nodules, consisting of newly differentiated cartilage cells, disappeared during the following 6 days. Coinciding with this process the histochemically demonstrable alkaline phosphatase activity, localized in the internodular areas, also disappeared. This indicated that retinoic acid not only inhibited chondrogenesis but also induced resorption of cartilage cells and that at least two cell types were affected, the cartilage cells and the cells bearing alkaline phosphatase.Actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis, suppressed the retinoic acid effect in day 5 limb bud cell cultures. This result indicated that the effect of retinoic acid required RNA and protein synthesis and is compatible with the view that vitamin A may act in a hormone-like way.     

回转器旋转对体外培养的鸡胚神经元的影响

用回转器旋转研究重力改变对体外培养的鸡胚神经元的影响。结果发现在回转器里经过７－９小时的处理后,神经元的形态和行为发生明显改变。只有６．３％的神经元呈现两极型的运动形态,而对照组正常培养的细胞运动形态的神经元占３４．２％。某些细胞的突起出现异常,呈现扭曲的形状。在培养基质上的神经元伸出许多丝状突起,丝状突起的末端锚定在周围的细胞上或锚定在培养基质上。经过重力改变处理的细胞重新放在相差显微镜下观察,结果显示神经元的运动活性降低,许多细胞没有观察到明显运动的迹象。     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Abstract. Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodism^H ( bp ^H ) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bp ^H mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

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.     

Retinoic acid enhances the displacement of newly synthesized hyaluronate from cell layer to culture medium during early phases of chondrogenesis

Chondrogenic differentiation in mouse limb bud mesenchymal cells cultured at high density was suppressed by supplementation of the medium with retinoic acid (1 microgram/ml or 3.3 X 10(-6) M). Since in control medium overt chondrogenesis begins on day 3, retinoic acid was introduced on day 2 so that the relationship between initial biosynthetic changes and inhibition of chondrogenesis could be studied. During the first 24 h of exposure the treated cells remained viable but suffered 10% inhibition in growth and synthesized [3H]glucosamine-labeled glycosaminoglycan at a level 24% below untreated cells. The amount of labeled hyaluronic acid released into the culture medium by the treated cells was, however, 2-fold greater, on a per cell basis, than that in the untreated cultures. It is suggested that the displacement of hyaluronate may play a role in the disruption of mesenchymal cell differentiation and of limb morphogenesis as observed in other systems.     

Evidence for sequential appearance of cartilage matrix proteins in developing mouse limbs and in cultures of mouse mesenchymal cells

The initiation of synthesis and the accumulation of four cartilage matrix proteins (type II collagen and three noncollagenous proteins, one of Mr 148, one of Mr 59, and an oligometric protein of Mr above 500 with 100-kDa subunits, respectively) were studied in developing mouse limbs and in cultures of limb bud mesenchyme by means of immunolocalization. On day 13 of gestation, type II collagen was observed throughout the entire humerus, whereas the 148-kDa protein was localized only in the central portion. Neither the 100-kDa-subunit protein nor the 59-kDa protein could be demonstrated in the humerus at that stage. On day 14 1/2, type II collagen and the 148-kDa protein were codistributed throughout the humerus. The 100-kDa-subunit protein was detectable in the periphery of the humerus, whereas little 59-kDa protein could yet be demonstrated. On day 18, all four proteins being studied could be detected immunologically in the developing mouse humerus. They differed in immunolocalization. Type Ii collagen, the 148-kDa protein, and the 100-kDa-subunit protein were codistributed throughout the distal and proximal parts of the cartilage. However, the 148-kDa protein could no longer be detected immunochemically in the outermost part of the cartilage in the proximal shoulder joint. The 148-kDa protein codistributed with type II collagen and the 100-kDa-subunit protein in the distal cartilaginous region, where joint development was less advanced. On the other hand, the 59-kDa protein was not demonstrated directly within the hyaline cartilaginous structures, but surrounded the entire structure. This protein was also present in the same part of the proximal joint region as that in which the 148-kDa protein was not detected. To develop an in vitro model for studies of skeletogenesis, mesenchymal cells prepared from mouse limb buds were cultured as micromass cultures at high initial cell density to favor chondrogenesis. On day 3 of culture, type II collagen was the only protein that could be detected immunochemically in the cultures, whereas on day 6 the 148-kDa protein was demonstrated, and a few chondrocytes in the central portion of each cartilaginous nodule were associated with the 100-kDa-subunit protein. The 59-kDa protein could not yet be immunochemically detected.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lack of chondrogenic expression in mouse limb bud micromass cultures exposed to exogenous beta galactosidase or N-acetyl-beta-glucosaminidase

The effect of two exoglycosidases, beta-galactosidase and N-acetyl-beta-glucosaminidase (GlcNAc-ase) on chondrogenic expression of stage 19 mouse limb bud micromass cultures was investigated. Chondrogenic expression was monitored by Alcian blue staining and immunofluorescent localization of cartilage-specific proteoglycan and type II collagen. Chondrogenesis was inhibited by exposure to 0.1 U/ml beta-galactosidase or 0.025 U/ml GlcNAc-ase for 24 h or longer in culture. The effect of both enzymes was concentration and time dependent. Exoglycosidic hydrolysis of galactose or N-acetylglucosamine was substantiated by treatment with HRP-conjugated peanut agglutinin and succinylated wheat germ agglutinin, respectively. Cells treated with beta-galactosidase appeared to be flattened with a stellate morphology, whereas GlcNAc-ase-treated cells were bipolar forming ridge-like mounds that had a directional orientation. The antichondrogenic effect was not alleviated when the cells were induced to assume a spherical shape upon treatment with cytochalasin D. DNA measurements indicated that the lack of chondrogenic expression was not related to cell attachment or cell proliferation. These data support the hypothesis that the expression of specific terminal sugars on cell surface glycoconjugates of limb bud cells represents an important component of the chondrogenic process.     

Proteoglycan synthesis in organ cultures from regions of bovine tendon subjected to different mechanical forces.

Synthesis of proteoglycans by morphologically and chemically distinct regions of bovine flexor tendon was investigated in explant cultures. Proximal regions of the flexor tendon which experience only tensile forces and have low contents of proteoglycans initially exhibited relatively low rates of proteoglycan synthesis but high rates of collagen synthesis. The predominant proteoglycan produced by all proximal explants was of small hydrodynamic size and appeared similar to that extracted from proximal tissue. In contrast, explants derived from the distal tendon region, which experiences frictional and compressive forces in addition to tensile forces, and has a high content of proteoglycans, showed relatively high initial rates of proteoglycan synthesis and lower rates of collagen synthesis. These distal explants produced primarily large proteoglycans on the first day in culture. Turnover of newly synthesized proteoglycans was not detectable in proximal tissue, and was low in distal tissue. Loss of unlabelled proteoglycan from proximal and distal explants was not detected during the 12 days of culture. These observations suggest that the increase in specific types of proteoglycans in regions of tendon subjected to frictional and compressive forces is the result of elevated synthesis rates in this tissue. Two alterations in proteoglycan synthesis occurred during the 12-day culture period. (1) The rate of proteoglycan synthesis by all explants increased with time in culture. (2) The proportion of small proteoglycans synthesized by distal explants increased from 32% of the total proteoglycan produced on day 1, to 80% of that produced on day 12. Explants from proximal tendon continued to produce only small proteoglycans throughout the 12 days in culture. This switch in proteoglycan phenotype, resulting in decreased synthesis of large proteoglycans by the distal tissue, may be due to a lack of compressive forces on the cultured explants.     

Regenerative responses in cultured hindlimb stumps of larval Xenopus laevis.

The regenerative capacity of larval Xenopus laevis hindlimbs amputated through the tarsalia at different stages of development and explanted in vitro was tested. In the first experimental series hindlimb stumps from stage 53, 54, 55, and 57 larvae (according to Nieuwkoop and Faber, '56) were cultured in Leibovitz's L-15 medium supplemented with 10% FCS, and 0.04 U of insulin and 10(-8) mg of L-thyroxine per ml of medium. Results showed that the distal part of the limb stumps from stages 53, 54, and 55 formed a regeneration blastema composed of proliferating mesenchymal cells beneath a typical apical cap. No blastema was formed in the proximal part of the stump. In limb stumps from stage 57, a regeneration blastema did not form either in the proximal or in the distal part of the stump. In a second experimental series, hindlimb stumps from stage 55 larvae, denervated 5 days prior to amputation in order to eliminate any residual neurotrophic factor, were cultured in a simplified L-15 medium containing 2% FCS and lacking insulin and thyroxine. Results showed that also in these experimental conditions the stumps from stage 55 formed a conical regeneration blastema at the distal tip. The blastema cells duplicated their own DNA and divided. At the proximal extremity no regeneration blastema was formed. In the same culture medium, the stumps of larvae at stage 57 did not form a regeneration blastema.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activated stellate (Ito) cells possess voltage-activated calcium current

We previously reported stellate (Ito) cells possess voltage-activated Ca2+ current. The activation of stellate cells has been indicated to contribute to liver fibrosis and the regulation of hepatic hemodynamics. The aim of this study was to investigate the relationship between voltage-activated Ca2+ current and activation of stellate cells. Voltage-activated Ca2+ current in stellate cells isolated from rats were studied using whole-cell patch clamp technique. L-type voltage-activated Ca2+ current was hardly detected in stellate cells cultured for less than 9 days. Ca2+ current was detected 12.5 and 69% of cells at the 10th and 14th day of culture, respectively. BrdU incorporation indicated cell proliferation was recognized over 50% of cells at the 3rd and 5th day of culture, respectively, then decreased significantly in a time-dependent manner. On the other hand, the expression of alpha-smooth muscle actin indicated cell activation increased from 7th day of culture and collagen type I mRNA appeared remarkably in cells cultured for more than 10 days. In this study, we concluded L-type voltage-activated Ca2+ current was recognized in activated stellate (myofibroblast-like) cells.     

Positional identity of adult stem cells in salamander limb regeneration

Limb regeneration in larval and adult salamanders proceeds from a mound of mesenchymal stem cells called the limb blastema. The blastema gives rise just to those structures distal to its level of origin, and this property of positional identity is reset to more proximal values by treatment with retinoic acid. We have identified a cell surface protein, called Prod1/CD59, which appears to be a determinant of proximodistal identity. Prod1 is expressed in an exponential gradient in an adult limb as determined by detection of both mRNA and immunoreactive protein. Prod1 protein is up-regulated after treatment of distal blastemas with RA and this is particularly marked in cells of the dermis. These cells have previously been implicated in pattern formation during limb regeneration.     

The newt ortholog of CD59 is implicated in proximodistal identity during amphibian limb regeneration

The proximodistal identity of a newt limb regeneration blastema is respecified by exposure to retinoic acid, but its molecular basis is unclear. We identified from a differential screen the cDNA for Prod 1, a gene whose expression in normal and regenerating limbs is regulated by proximodistal location and retinoic acid: Prod 1 is the newt ortholog of CD59. Prod 1/CD59 was found to be located at the cell surface with a GPI anchor which is cleaved by PIPLC. A proximal newt limb blastema engulfs a distal blastema after juxtaposition in culture, and engulfment is specifically blocked by PIPLC, and by affinity-purified antibodies to two distinct Prod 1/CD59 peptides. Prod 1 is therefore a cell surface protein implicated in the local cell-cell interactions mediating positional identity.     

Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins

The mechanisms by which cells obtain instructions to precisely re-create the missing parts of an organ remain an unresolved question in regenerative biology. Urodele limb regeneration is a powerful model in which to study these mechanisms. Following limb amputation, blastema cells interpret the proximal-most positional identity in the stump to reproduce missing parts faithfully. Classical experiments showed the ability of retinoic acid (RA) to proximalize blastema positional values. Meis homeobox genes are involved in RA-dependent specification of proximal cell identity during limb development. To understand the molecular basis for specifying proximal positional identities during regeneration, we isolated the axolotl Meis homeobox family. Axolotl Meis genes are RA-regulated during both regeneration and embryonic limb development. During limb regeneration, Meis overexpression relocates distal blastema cells to more proximal locations, whereas Meis knockdown inhibits RA proximalization of limb blastemas. Meis genes are thus crucial targets of RA proximalizing activity on blastema cells.     

The effects of cytohalasin B and dimethyl sulfoxide on the movement of mouse kidney cells. I. Effect on the outgrowth of cells

The following experiments were undertaken to determine the effect of cytochalasin B (CB) on the movement of mouse kidney epitheliocytes in primary culture. Fragments of mouse kidney were cultured in Sykes-Moore chambers with control medium (McCoy's 5a, modified), CB in dimethyl sulfoxide (DMSO), and DMSO. CB, 10 μg/ml, in 1% DMSO completely inhibited the outgrowth of cells from the kidney fragments. These effects were reversible. Unexpectedly, 1% DMSO suppressed the outgrowth of the kidney cells. A myriad of phenomenological effects of CB on mammalian cells have been reported in the recent literature. Most investigators report no effect of DMSO on the cells. These results suggest that DMSO is affecting the movement of mouse kidney cells.