Extracellular matrix environment influences chondrogenic pattern formation in limb bud micromass culture: Experimental verification of theoretical models

Abstract

Various theoretical models have been proposed to explain the periodic-ity in the pattern of limb chondrogenesis, but experimental comparison of these models have seldom been performed properly. In the present study, micromass culture of limb bud mesenchyme cells was undertaken to test the validity of three theoretical models: the reaction-diffusion model, the cell sorting model, and the mechanochemical model. Computer simulations were undertaken to predict the factors that can affect the coarseness of the chondrogenic pattern. According to the predictions, we performed micro-mass culture of limb mesenchyme in collagen and agarose gel. Then we carried out time-lapse observation to analyze the cell movement during pattern formation. From computer simulations it was theoretically predicted that changes in the surrounding extracellular matrix should alter the periodicity of the chondrogenic pattern in vitro, and we found that pattern changes actually occurred under different culture conditions. When compared with the culture in a liquid medium, the chondrogenic pattern became less coarse when the cells were cultured in collagen or agarose gel, and the pattern change appeared to be independent of the cell differentiation. Time-lapse observation revealed a decrease in cell motility when the cells were cultured in gel. It was found that both the reaction-diffusion and cell sorting models fit the pattern change produced and that the mechanochemi-cal model is not primarily important in the chondrogenic pattern formation in vitro.

Existence of solutions to a new model of biological pattern formation

“…System (1.1)-(1.8) exhibits Turing-type instabilities, consistent with an earlier suggestion that vertebrate limb development is governed by this class of mechanisms [6]. Recent experiments [17,18] have provided evidence that chondrogenic patterning in cultures of isolated limb cells self-organizes by a Turing-like process involving TGF-β and computational modeling has confirmed the plausibility of this mechanism [19]. In the system under consideration the prepattern of activator concentration is transferred to the subsystem describing the dynamics of moving cells by using the coefficient k 12 (c, c a ) [1].…”

The Morphostatic Limit for a Model of Skeletal Pattern Formation in the Vertebrate Limb

“…Beyond this, the following experimental findings, count in favor of the relevance of a reaction-diffusion mechanism for limb pattern formation: (i) The pattern of precartilage condensations in limb mesenchyme in vitro changes in a fashion consistent with reaction-diffusion mechanism (and not with an alternative mechanochemical mechanism) when the density of the surrounding matrix is varied (Miura and Shiota, 2000b); (ii) exogenous FGF perturbs the kinetics of condensation formation by limb precartilage mesenchymal cells in vitro in a fashion consistent with a role for this factor in regulating inhibitor production in a reaction-diffusion model (Miura and Maini, 2004); (iii) the "thick-thin" pattern of digits in the Doublefoot mouse mutant can be accounted for by the assumption of that the normal pattern is governed by a reaction-diffusion process the parameters of which are modified by the mutation ; and (iv) the scale-dependence of reaction-diffusion systems (i.e., the addition or loss of pattern elements when the tissue primordium has variable size), sometimes considered to count against such mechanisms for developmental processes, may actually represent the biological reality in the developing limb. Experiments show, for example, that the number of digits that arise is sensitive to the anteroposterior (thumb-to-little finger breadth) of the developing limb bud, and will increase (Cooke and Summerbell, 1981) or decrease (Alberch and Gale, 1983) over typical values if the limb is broadened or narrowed.…”

Gene Expression Time Delays and Turing Pattern Formation Systems

“…However, while morphogens play significant roles in many pattern forming events during development, there is no definitive evidence for a set of Turing morphogens that generate pattern by undergoing a Turing bifurcation in a biological system. Nonetheless, there has been speculation that a Turing type mechanism may be involved in vertebrate limb development (Miura and Shiota, 2000;Glimm et al, 2004;Miura and Maini, 2004) and avian feather bud formation (Jung et al, 1998). In addition, a number of experiments have indicated that the secreted TGF-β proteins Nodal and Lefty behave as an activator-inhibitor reaction diffusion system in zebrafish mesendodermal induction Schier, 2001, 2002;Yost, 2002, 2004;Solnica-Krezel, 2003;Chen and Shen, 2004).…”