Ethanol Exposure Stimulates Cartilage Differentiation by Embryonic Limb Mesenchyme Cells

Studies of neural, hepatic, and other cells have demonstrated thatin vitroethanol exposure can influence a variety of membrane-associated signaling mechanisms. These include processes such as receptor-kinase phosphorylation, adenylate cyclase and protein kinase C activation, and prostaglandin production that have been implicated as critical regulators of chondrocyte differentiation during embryonic limb development. The potential for ethanol to affect signaling mechanisms controlling chondrogenesis in the developing limb, together with its known ability to promote congenital skeletal deformitiesin vivo,prompted us to examine whether chronic alcohol exposure could influence cartilage differentiation in cultures of prechondrogenic mesenchyme cells isolated from limb buds of stage 23–25 chick embryos. We have made the novel and surprising finding that ethanol is a potent stimulant ofin vitrochondrogenesis at both pre- and posttranslational levels. In high-density cultures of embryonic limb mesenchyme cells, which spontaneously undergo extensive cartilage differentiation, the presence of ethanol in the culture medium promoted increased Alcian-blue-positive cartilage matrix production, a quantitative rise in³⁵SO₄incorporation into matrix glycosaminoglycans (GAG), and the precocious accumulation of mRNAs for cartilage-characteristic type II collagen and aggrecan (cartilage proteoglycan). Stimulation of matrix GAG accumulation was maximal at a concentration of 2% ethanol (v/v), although a significant increase was elicited by as little as 0.5% ethanol (approximately 85 mM). The alcohol appears to directly influence differentiation of the chondrogenic progenitor cells of the limb, since ethanol elevated cartilage formation even in cultures prepared from distal subridge mesenchyme of stage 24/25 chick embryo wing buds, which is free of myogenic precursor cells. When limb mesenchyme cells were cultured at low density, which suppresses spontaneous chondrogenesis, ethanol exposure induced the expression of high levels of type II collagen and aggrecan mRNAs and promoted abundant cartilage matrix formation. These stimulatory effects were not specific to ethanol, since methanol, propanol, and tertiary butanol treatments also enhanced cartilage differentiation in embryonic limb mesenchyme cultures. Further investigations of the stimulatory effects of ethanol onin vitrochondrogenesis may provide insights into the mechanisms regulating chondrocyte differentiation during embryogenesis and the molecular basis of alcohol's teratogenic effects on skeletal morphogenesis.     

The enhancement of in vitro survival and chondrogenesis of limb bud cells by cartilage conditioned medium

Dissociated stage 21–28 chick embryo limb bud cells showed an increasing ability to produce cartilage colonies in vitro with in vivo maturation. In addition dissociated stage 21–28 chick embryo limb bud cells exposed to cartilage conditioned medium continuously or only for 48 hr prior to subculture showed an enhanced (as much as 15-fold) ability to form differentiated cartilage colonies. By this criterion, cells were more responsive to conditioned medium prior to stage 25. Conditioned medium from fibroblast cultures caused an inhibition of cartilage colony formation, suggesting that the effect is cell-type specific. Besides increasing cartilage colony formation by enhanced cell survival, the incorporation of S³⁵O₄ into isolated glycosaminoglycans is also stimulated when limb bud cells are exposed to cartilage conditioned medium. The results support a model for cell differentiation which involves the enhancement of a particular differentiated capacity by a diffusible cell-type-specific macromolecule.     

Characterization of bone-derived chondrogenesis-stimulating activity on embryonic limb mesenchymal cells in vitro

Abstract. Demineralized bone matrix contains factors which stimulate chondrogenesis and osteogenesis in vivo. A water-soluble extract of bone has been shown to stimulate chondrogenesis in vitro in embryonic limb mesenchymal cells (Syftestad, Lucas & Caplan, 1985). The aim of this study was to analyse the cellular mechanism of the bone-derived chondrogenesis-stimulating activity, with particular attention on how normal requirements for chondrogenesis may be altered. The effects of bovine bone extract (BBE) on chondrogenesis in vitro were studied using micromass cultures of chick limb bud mesenchyme isolated from embryos at Hamburger-Hamilton (HH) stage 23/24, an experimental system which is capable of undergoing chondrogenic differentiation. Bovine diaphyseal long bones were demineralized and extracted with guanidine-HCl to prepare BBE (Syftestad & Caplan, 1984). High-density mesenchyme cultures (30 times 10⁶ cells/ml) were exposed to different doses of BBE (0–01-1-0 mg ml^-1) and chondrogenesis was quantified based on cartilage nodule number and [³⁵S]sulphate incorporation. BBE was tested on micromass cultures of varying plating densities (2–30 times 10⁶ cells/ml), on cultures of ‘young’ limb bud cells (HH stage 17/18), and on cultures enriched with chondroprogenitor cells obtained from subridge mesoderm. Since poly-L-lysine (PL) has recently been shown (San Antonio & Tuan, 1986) to promote chondrogensis, PL and BBE were introduced together in different doses, in the culture medium, to determine if their actions were synergistic. Our results show that BBE stimulates chondrogenesis in a dose-dependent manner and by a specific, direct action on the chondroprogenitor cells but not in normally non-chondrogenic, low density or ‘young’ limb bud cell cultures. The effects of PL and BBE are additive and these agents appear to act by separate mechanisms to stimulate chondrogenesis; PL primarily enhances nodule formation, and BBE appears to promote nodule growth.     

Patterns of cell polarity in the developing mouse limb

Most cells have a morphological polarity with the centrioles and Golgi apparatus occupying one pole of the cell and the nucleus the other. This structural polarity often correlates with functional polarity as in secretory epithelia where the Golgi apparatus moves to the pole of the cell from which secretory materials are exreted. In limb development an interaction of unknown mechanism occurs between the epithelium and mesenchyme. We have evaluated the pattern of cell polarity using silver impregnation of the Golgi apparatus in limb epithelium and mesenchyme of mouse embryos from day 9.5, when limbs are first visible, to day 15, when cartilage formation is complete. Cells in the epithelium almost always have the Golgi apparatus in the apex of the cell, i.e., oriented away from the basement membrane. The layer of mesenchyme cells just beneath the basement membrane initially has only 16 to 25% of the cells oriented toward the basement membrane. A marked shift in orientation occurs between days 12 and 13 so that from days 13 to 15 up to 53% of the mesenchyme cells are oriented toward the basement membrane. This shift in orientation occurs more slowly in the mesenchyme at a depth of four cells below the basement membrane. This changing pattern of mesenchymal cell polarity occurs at a time when there is an apparent increase in the amount of extracellular matrix, especially in the region just below the basement membrane.     

The Golgi apparatus in the endomembrane-rich gastric parietal cells exist as functional stable mini-stacks dispersed throughout the cytoplasm

Background information. Acid‐secreting gastric parietal cells are polarized epithelial cells that harbour highly abundant and specialized, H⁺, K⁺ ATPase‐containing, tubulovesicular membranes in the apical cytoplasm. The Golgi apparatus has been implicated in the biogenesis of the tubulovesicular membranes; however, an unanswered question is how a typical Golgi organization could regulate normal membrane transport within the membrane‐dense cytoplasm of parietal cells. Results. Here, we demonstrate that the Golgi apparatus of parietal cells is not the typical juxta‐nuclear ribbon of stacks, but rather individual Golgi units are scattered throughout the cytoplasm. The Golgi membrane structures labelled with markers of both cis‐ and trans‐Golgi membrane, indicating the presence of intact Golgi stacks. The parietal cell Golgi stacks were closely aligned with the microtubule network and were shown to participate in both anterograde and retrograde transport pathways. Dispersed Golgi stacks were also observed in parietal cells from H⁺, K⁺ ATPase‐deficient mice that lack tubulovesicular membranes. Conclusions. These results indicate that the unusual organization of individual Golgi stacks dispersed throughout the cytoplasm of these terminally differentiated cells is likely to be a developmentally regulated event.     

Cellular analysis of limb development in the mouse mutant Hypodactyly

The limb defect in the mouse Hypodoctyly (Hd) affects only the distal structures. Heterozygotes (Hd/+) lack all or part of the distal phalanx and the terminal claw of digit 1 on the hindlimbs; mice homozygous (Hd/Hd) for the mutation have just one digit on each of the four limbs. Early limb development in the mutant appears normal and a change in morphology can only be detected later. Limb buds of Hd/+ and Hd/Hd embryos become reduced in width, with Hd/Hd buds becoming very pointed instead of rounded. This change in bud shape is correlated with an increase in cell death anteriorly in Hd/+ hindlimbs and both anteriorly and posteriorly in Hd/Hd fore- and hindlimb buds. The apical ectodermal ridge is very pronounced in pointed Hd/Hd limb buds. Mesenchyme cells from the Hd/Hd mutant in culture show a cell-autonomous change in behaviour and less cartilage differentiates. © 1996 Wiley-Liss, Inc.     

Estimating the selective advantage of mutant p53 tumour cells to repeated rounds of hypoxia

The tumour suppressor gene, p53, plays an important role in tumour development. Under low levels of oxygen (hypoxia), cells expressing wild-type p53 undergo programmed cell death (apoptosis), whereas cells expressing mutations in the p53 gene may survive and express angiogenic growth factors that stimulate tumour vascularization. Given that cells expressing mutations in the p53 gene have been observed in many forms of human tumour, it is important to understand how both wild-type and mutant cells react to hypoxic conditions. In this paper a mathematical model is presented to investigate the effects of alternating periods of hypoxia and normoxia (normal oxygen levels) on a population of wild-type and mutant p53 tumour cells. The model consists of three coupled ordinary differential equations that describe the densities of the two cell types and the oxygen concentration and, as such, may describe the growth of avascular tumours in vitro and/or in vivo. Numerical and analytical techniques are used to determine how changes in the system parameters influence the time at which mutant cells become dominant within the population. A feedback mechanism, which switches off the oxygen supply when the total cell density exceeds a threshold value, is introduced into the model to investigate the impact that vessel collapse (and the associated hypoxia) has on the time at which the mutant cells become dominant within vascular tumours growing in vivo. Using the model we can predict the time it takes for a subpopulation of mutant p53 tumour cells to become the dominant population within either an avascular tumour or a localized region of a vascular tumour. Based on independent experimental results, our model suggests that the mutant population becomes dominant more quickly in vivo than in vitro (12 days vs 17 days).     

Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis

The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin^−/− (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation of mpcs, the EOMCD34 cells, that are retained in significantly higher percentages in normal, mdx and DKO mice EOM, appear to be resistant to elevated levels of oxidative stress and toxins, and actively proliferate throughout life. Current studies are focused on further defining the EOMCD34 cell subtype molecularly, with the hopes that this may shed light on a cell type with potential therapeutic use in patients with sarcopenia, cachexia, or muscular dystrophy.     

Global comparative transcriptome analysis of cartilage formation in vivo

Background  

During vertebrate embryogenesis the initial stages of bone formation by endochondral ossification involve the aggregation and proliferation of mesenchymal cells into condensations. Continued growth of the condensations and differentiation of the mesenchymal cells into chondrocytes results in the formation of cartilage templates, or anlagen, which prefigure the shape of the future bones. The chondrocytes in the anlagen further differentiate by undergoing a complex sequence of maturation and hypertrophy, and are eventually replaced by mineralized bone. Regulation of the onset of chondrogenesis is incompletely understood, and would be informed by comprehensive analyses of in vivo gene expression.     

A model generating the pattern of cartilage skeletal elements in the embryonic chick limb.

The development of the vertebrate limb involves the production of a specific external form arising from cell division and other growth processes at the cellular level, and the origin within it of specific patterns of tissues arising by cellular differentiation, of which the pattern of cartilages which pre-figure the limb skeleton is the most striking.In this paper we propose a model for the differentiation (or the preceding determination) process that, using only localized cell to cell interactions, can approximate the cartilage pattern in any limb shape. The model requires cells to modify their metabolism irreversibly at critical threshold levels of a diffusible morphogen which may be made or destroyed by these cells. Restrictions inherent in the successful development of a total limb pattern using this system lead to the prediction that the process is confined to a distal band which has no significant interaction with more proximal regions but within this band the characteristic features of the anterior-posterior axis of the limb develop without additional interactions. Cartilage elements are initiated as single “cells” and expand centrifugally to their final size; these elements developing sequentially along the anterior-posterior axis, showing a distinct polarity of size. The model also predicts that equivalent cartilage elements in all vertebrate limbs will be roughly the same relative size at determination, the extensive range of adult structures arising by differential growth and fusion, possibly controlled by global aspects of the model.It must be emphasized that this model only satisfactorily simulates the anterior-posterior patterning of cartilage elements, the disto-proximal pattern being externally imposed.The final cartilage pattern is shown to be a function of (1) the developing shape of the limb, (2) the position of an initiator region that starts the patterning process and (3) the rate of production of the diffusible morphogen. Using parameters selected with as much realism as possible the model gives a good approximation to the pattern of c cartilages found in the normal chick limb; modifying the shape of the limb to that of the talpid³ mutant produces the characteristics features of the cartilage pattern found in that mutant and modifying the rate of morphogen production simulates patterns resembling those found in some ancestral vertebrate fossil forms.     

Chick endogenous lectin enhances chondrogenesis of cultured chick limb bud cells

We have studied the effect of β-d-galactoside-specific lectin purified from 14-day-old chick embryos on the differentiation of the mesenchymal cells dissociated from the limb buds of stage 24 chick embryos, using the micro-mass culture method described previously. When the cells were incubated with the lectin during the initial 12 hr of culture, cell proliferation became slightly activated. The lectin-treated cells formed a greater number of cartilage nodules and incorporated about twice as much as [³⁵S]sulfate per cell than the control cultures. The results of this study show that the chick endogenous lectin promotes cartilage differentiation in vitro and that endogenous lectin may possibly be involved in chondrogenesis in vivo.     

Cell Density and Cell Division in the Early Morphogenesis of the Chick Wing

THE early development of the chick wing involves cell differentiation, pattern formation and growth¹. In general terms, its morphogenesis can be seen in terms of how the growth of the mesenchyme and ectoderm is controlled, so that the very simple initial protrusion is transformed into an elongated paddle-like structure. At the same time a spatial pattern of differentiation must be specified within the mesenchyme, the cells forming cartilage and muscle so that the major skeletal and muscular features are laid down. In a previous paper² we described the changing pattern of the distribution of mitoses in the mesenchyme. We found that there was an overall fall in mitotic index from early stages (18–19 Hamburger-Hamilton) but that this occurred more rapidly in the proximal regions after about stage 23 so that a graded proximo-distal increase in mitotic index became established. We also suggested that the overall form was determined by the ectoderm and not the mesenchyme. This raised specific problems about the control of growth of the mesenchyme: we could not account for the observed distribution of mitoses but wondered whether this involved a temporal programme or was related to positional information. Our investigations of the so-called mesenchymal condensations which are supposed to be the prelude to cartilage formation³ have led to detailed analysis of cell density during early morphogenesis. We show here that the cell density varies in a very regular manner and is closely correlated with mitotic index: mitotic index is inversely proportional to cell density. This finding is not only important in its own right because it may be the first demonstration of density dependent growth control⁴in vivo, but because it provides a new mechanism for the control of growth and pattern formation in limb morphogenesis.     

Involvement of Frzb-1 in mesenchymal condensation and cartilage differentiation in the chick limb bud

In developing limb bud, mesenchymal cells form cellular aggregates called "mesenchymal condensations". These condensations show the prepattern of skeletal elements of the limb prior to cartilage differentiation. Roles of various signaling molecules in chondrogenesis in the limb bud have been reported. One group of signaling factors includes the Wnt proteins, which have been shown to have an inhibitory effect on chondrogenesis in the limb bud. Therefore, regulation of Wnt activity may be important in regulating cartilage differentiation. Here we show that Frzb-1, which encodes a secreted frizzled-related protein that can bind to Wnt proteins and can antagonize the activity of some Wnts, is expressed in the developing limb bud. At early stages of limb development, Frzb-1 is expressed in the ventral core mesenchyme of the limb bud, and later Frzb-1 expression becomes restricted to the central core region where mesenchymal condensations occur. At these stages, a chondrogenic marker gene, aggrecan, is not yet expressed. As limb development proceeds, expression of Frzb-1 is detected in cartilage primordial cells, although ultimately Frzb-1 expression is down-regulated. Similar results were obtained in the recombinant limb bud, which was constructed from dissociated and re-aggregated mesenchymal cells and an ectodermal jacket with the apical ectodermal ridge. In addition, Frzb-1 expression preceded aggrecan expression in micromass cultures. These results suggest that Frzb-1 has a role in condensation formation and cartilage differentiation by regulating Wnt activity in the limb bud.     

Hemopoietic progenitor cells of W anemic mice studied in vivo and in vitro

The proliferation and differentiation of hemopoietic cells from genetically anemic W^v/W^x,W/W^v, and W^v/W^v mice, and from nonanemic carrier W/+, W^b/+, and W^v/+ mice have been evaluated in vivo by transplantation techniques and in vitro by the agar gel culture method. Marrow from anemic and carrier mice contained progenitor cells which were decreased in number and formed small, often rudimentary, colonies in the spleens of irradiated recipient mice. Proliferation and differentiation of both erythropoietic and leukopoietic progenitor cells were delayed and reduced, but erythropoiesis was more severely affected than leukopoiesis. The severity of the hemopoietic impairment was gene-dose dependent. The W gene effect on leukopoietic progenitor cells was not secondary to anemia or to abnormal erythropoiesis. The marrow cells of anemic and carrier mice which form colonies of granulocytic and mononuclear cells in vitro were neither decreased in number nor impaired in proliferation and differentiation. Hypertransfusion of red blood cells increased the frequency of in vitro colony-forming cells, but not that of in vivo progenitor cells. The data demonstrate that colony-forming cells which proliferate in the agar gel cultures in vitro are distinct from the in vivo colony-forming cells and suggest that the former are primitive members of the granulocytic cell line. Perhaps in vitro CFU are in an intermediate stage of differentiation between in vivo CFU and myeloblasts, analogous to that which has been suggested for the erythropoietin-sensitive cell in the red cell series. W mutant alleles appear to act, therefore, at or very near the beginning of hemopoietic differentiation.     

A fraction from extracts of demineralized adult bone stimulates the conversion of mesenchymal cells into chondrocytes

Demineralized adult bone contains factors which stimulate nonskeletal mesenchymal cells to undergo a developmental progression resulting in de novo endochondral ossification. In this study, isolated embryonic stage 24 chick limb bud mesenchymal cells maintained in culture were utilized as an in vitro assay system for detection of specific bioactive components solubilized from adult chicken bone matrix. Guanidinium chloride extracts (4 M) of demineralized-defatted bone were fractionated and tested in limb mesenchymal cell cultures for possible effects upon growth and chondrogenesis. Two low-molecular-weight fractions were found to be active in these cultures. A cold water-insoluble, but warm Trisbuffered saline-soluble fraction provoked a dose-dependent increase in the amount of cartilage formed after 7 days of continuous exposure as evidenced by an increased number of chondrocytes observed in living cultures, elevated cell-layer-associated ³⁵S incorporation per microgram DNA, and greater numbers of toluidine blue-staining foci (i.e., cartilage nodules). Growth inhibitory substances were detected in a low-molecular-weight, water-soluble fraction; 7 days of continuous exposure to this material resulted in less cartilage formation and reduced cell numbers (accumulated DNA) on each plate. These observations demonstrate the usefulness of stage 24 chick limb bud cell cultures for identifying bioactive factors extracted from adult bone matrix. In addition, the action of these factors on mesenchymal cells may now be studied in a cell culture system.     

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix: I. Ultrastructural aspects

Previous studies have demonstrated that embryonic skeletal muscle is competent to form hyaline cartilage when cultured in vitro on demineralized bone matrix (Nogami, H., and Urist, M. R. (1970). Exp. Cell Res.63, 404–410; Nathanson, M. A., et al. (1978). Develop. Biol.64, 99–117). The present experiments were undertaken to determine the nature of the morphological alterations which attend this phenotypic transformation and to investigate the ultrastructural characteristics of the myoblasts and fibroblasts of skeletal muscle during the transformation. Nineteen-day embryonic rat limb muscles were minced and the tissue fragments explanted to bone matrix or collagen gels. The trauma of excision and mincing causes syncytial myotubes to degenerate and the nuclei of mononucleate cells to enter a heterochromatic “resting stage.” In culture, nuclei of mononucleate cells rapidly regain euchromasia. No myoblast or fibroblast cell death can be detected. On bone matrix, the entire mononucleate population transforms into fibroblast-like cells. Myoblasts are the major contributor to this population; they dissociate from the degenerate myotubes and begin to acquire endoplasmic reticulum by 24 h in vitro. The fibroblast-like morphology persists through 4 days in vitro. By 6 days in vitro some of these fibroblast-like cells acquire the phenotypic characteristics of chondrocytes, and by 10 days masses of hyaline cartilage are found. In control explants of skeletal muscle onto collagen gels, the heterochromatic nuclei of the mononucleated cells expand after 24 hr in vitro, but the mononucleated cells remain as myoblasts and fibroblasts and begin to regenerate skeletal muscle by 4 days in vitro. No cartilage forms. The results indicate that both myoblasts and fibroblasts have chondrogenic potential when grown on demineralized bone. It is tempting to conclude that the embryonic mesenchymal cells which give rise to skeletal muscle, cartilage, and other connective tissue of the limb have similar developmental potentials and that local influences, rather than separate cell lineages, account for the final pattern of differentiation.     

Cell-cell interaction by mouse limb cells during in vitro chondrogenesis: Analysis of the brachypod mutation

This paper contains observations and experiments which collectively demonstrate a requirement for cell-cell interactions among limb bud mesenchyme cells during chondrogenic differentiation. Limb bud cells isolated from brachypodism^H (bp^H) and wild-type mouse embyros between Thieler stage 16–17 and midstage 21 were compared with respect to their abilities to undergo chondrogenic differentiation in high-density micromass cultures. Nodules formed by dissociated Day 12 (stage 20) bp^H limb bud cells have been reported previously to be abnormally reduced in size and number, and delayed in formation. We corroborate these results, but find that bp^H cultures prepared from earlier-stage limb buds (between stages 16–17 and early stage 21) are progressively more like wild-type cultures. Stage 16–17 bp^H cultures at 72 hr actually contain normal numbers of and size nodules, while stage 18 bp^H cultures are intermediate between stages 16–17 and stage 21 in nodule formation. On the other hand, we also find that the initial rate of aggregate formation is normal even in bp^H cultures prepared from stage 20 cultures in which nodule formation is not normal. Preparation of cultures composed primarily of early stage 21 bp^H limb bud cells mixed with small quantities (e.g., 5%) of stage 16–17 wild-type limb bud cells showed significant increases in cartilage nodule formation over control cultures composed only of early stage 21 bp^H cells. Greater proportions of wild-type cells obtained from embryos older than stages 16–17 were required for the same degree of normalization, supporting the hypothesis that a specific cell type, whose proportion decreases normally in the limb bud over time, is required to increase in vitro chondrogenesis by bp^H cells. Additionally, cultures containing stage 23 chick limb cells and early stage 21 bp^H cells at a ratio of 1:20 contained wild-type levels of nodules per square millimeter of culture. Thus, bp^H cells appear to respond to chondrogenic inductive signals from normal limb mesenchyme cells. In order to test for the ability of bp^H limb bud mesenchyme to induce chondrogenesis, stage 16–17 bp^H and wild-type limb bud cells, which form identical numbers of aggregates and nodules in culture, were each mixed with early stage 21 bp^H cells at ratios of 1:20, 1:10, and 1:3. Although low proportions of wild-type stage 17 cells significantly increased the number of aggregates and nodules in these mixed cultures, low proportions of bp^H stage 16–17 cells did not. It is, therefore, concluded that the primary defect of the bp^H mutation is likely to reside in the reduced ability of a specific mesenchyme cell subpopulation to provide an inductive stimulus for chondrogenesis.     

The H-2KbtsA58 transgenic mouse: A new tool for the rapid generation of novel cell lines

The ability to generate expanded populations of individual cell types able to undergo normal differentiationin vitro andin vivo is of critical importance in the investigation of the mechanisms that underly differentiation and in studies on the use of cell transplantation to repair damaged tissues. This review discusses the development of a strain of transgenic mice that allows the direct derivation of conditionally immortal cell lines from a variety of tissues, simply by dissociation of the tissue of interest and growth of cells in appropriate conditions. In these mice the tsA58 mutant of SV40 large T antigen is controlled by the interferon-inducible Class I antigen promoter. Cells can be grown for extended periodsin vitro simply by growing them at 33°C in the presence of interferon, while still retaining the capacity to undergo normal differentiationin vivo andin vitro. In addition, it appears that cell lines expressing mutant phenotypes can readily be generated by preparing cultures from appropriate offspring of matings between H-2K^btsA58 transgenic mice and mutant mice of interest.     

Hyaluronan in limb morphogenesis

Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself. Has2 expression and HA production are downregulated in the proximal central core of the limb bud during the formation of the precartilage condensations of the skeletal elements, suggesting that downregulation of HA may be necessary for the close juxtaposition of cells and the resulting cell-cell interactions that trigger cartilage differentiation during condensation. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements that do form in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately. We also demonstrate that sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that downregulation of HA is indeed necessary for formation of the precartilage condensations that trigger cartilage differentiation. Taken together these results suggest involvement of HA in various aspects of limb morphogenesis.