Reaction-diffusion microtubule concentration patterns occur during biological morphogenesis.

Reaction-diffusion processes can lead to a macroscopic concentration pattern from an initially homogeneous solution, and thus provide a physical-chemical mechanism for biological pattern formation and morphogenesis. The central prediction of reaction-diffusion theory is that the patterns contain periodic concentration variations in some of the reactives. Microtubules assembled in vitro spontaneously self-organise and form stationary striped macroscopic structures. In agreement with reaction-diffusion theory. Here we show, in agreement with reaction-diffusion theory, that these preparations contain substantial microtubule concentration variations. Similar striped microtubule patterns arise during Drosophila embryogenesis. A characteristic of these patterns is their dependence on sample dimensions. In Drosophila eggs shortened by ligation, we found that the microtubule pattern varied with egg fragment length in the same way as the in vitro microtubule pattern varied with sample length, and as expected from theory. This is evidence that reaction-diffusion structures occur during Drosophila morphogenesis.     

Self-organization in biological systems

Biological systems are considered that are capable of dynamic self-organization, i.e., spontaneous emergence of spatio-temporal order with the formation of various spatio-temporal patterns. A cell is involved in the organization of ontogenesis of all stages. Embryonic cells exhibit coordinated social behavior and generate ordered morphological patterns displaying variability and equifinality of development. Physical and topological patterns are essential for biological systems as an imperative that restricts and directs biological morphogenesis. Biological self-organization is directed and fixed by natural selection during which selection of the most sustainable, flexible, modular systems capable of adaptive self-organization occurs.     

Can morphogenesis be understood in terms of physical rules?

Because the morphogenesis of biological systems is not fully understood, researches from various points of view are necessary. The present author has recently made computer simulations with his colleagues to construct branching systems of human organs, such as the lung airway and the liver blood vessels. In the simulations certain rules are assumed to govern bifurcating processes of the systems. These rules are expressed in terms of physical and geometrical concepts, such as minimum energy consumption and uniform filling of branches in the space of organs. Results of computer simulation are quite similar to real structures. However, actual mechanisms of morphogenesis, i.e. effects of genes or proteins, are not considered in these studies. In this article, the present work is discussed in relation to the concept of biological pattern formation by Meinhardt and a recent study by Miura and Shiota on lung growth.     

Dissipative structures in morphogenetic models of the Turing type

We investigate the general conditions for morphogenesis. It is found that not only the Turing condition shall be satisfied, but also that some other requirements must be fulfilled. Their physical meaning is that the system should have more than one stationary state. In biological terms this means that the competence of differentiation is necessary in order to finish the morphogenesic process. These ideas are illustrated by a simple mathematical model. For this model the bifurcation analysis and computer results are discussed. Furthermore the parametric regulation of the morphogenesis is considered in connection with the role played by the genetic information.     

The Theory of Tensegrity and Spatial Organization of Living Matter

There is still no consensus on the mechanisms regulating the formation and maintenance of morphological structures in the individual development of living organisms. The hypothesis that the mechanical forces are important for biological morphogenesis was put forward more than 100 years ago. In recent decades, studies indicating the regulatory role of mechanical stresses at different levels of organization of life have appeared. The signaling mechanisms that are responsible for the reception of mechanical influences and reprogramming of the properties of cells and tissues are studied. Since the mid-1970s, the principles of selfstressed structures or the tensegrity (tensional integrity) theory have been applied to understand the structure and functions of living structures in statics and dynamics. According to this standpoint, the cell can be represented as a self-stressed structure in which microtubules function as rigid rods and microfilaments serve as elastic threads. Such a system is anchored to extracellular matrix through cellular contacts, since it is adjusted to the external patterns of mechanical stresses. The notion of living systems as self-stressed structures provides a fresh look at the mechanotransduction, developing organism integrity, and biological morphogenesis. Although the application of the ideas of tensegrity to biological systems has not yet received broad support among biologists, the influence of these ideas on the formation of modern mechanobiology and understanding the crucial role of cytoskeletal structures in cellular processes should be mentioned.     

A unique role for 6-O sulfation modification in zebrafish vascular development

Heparan sulfate proteoglycans are important modulators of growth factor signaling in a variety of patterning processes. Secreted growth factors that play critical roles in angiogenesis bind to heparan sulfate, and this association is affected by 6-O-sulfation of the heparan sulfate chains. Addition of 6-O-sulfate is catalyzed by a family of sulfotransferases (HS6STs), and genetic manipulation of their function permits an assessment of their contribution to vascular assembly. We report on the biochemical activity and expression patterns of two zebrafish HS6ST genes. In situ hybridization reveals dynamic and distinct expression patterns of these two genes during development. Structural analysis of heparan sulfate from wild-type and morpholino antisense 'knockdown' embryos suggests that HS6ST-1 and HS6ST-2 have similar biochemical activity. HS6ST-2, but not HS6ST-1, morphants exhibit abnormalities in the branching morphogenesis of the caudal vein during embryonic development of the zebrafish. Our finding that HS6ST-2 is required for the branching morphogenesis of the caudal vein is the first in vivo evidence for an essential role of a gene encoding a heparan sulfate modifying enzyme in vertebrate angiogenesis. Our analysis of two zebrafish HS6ST genes suggests that a wide range of biological processes may be regulated by an array of sulfation-modifying enzymes in the vertebrate genome.     

Embryonic mouse testis development: role of platelet derived growth factor (PDGF-BB)

Platelet-derived growth factors (PDGFs) are paracrine growth factors mediating epithelial-mesenchymal interactions and exerting multiple biological activities which include cell proliferation, motility, and differentiation. As previously demonstrated, PDGFs act during embryonic development and recently, by culturing male genital ridges, we have demonstrated that PDGF-BB is able to support in vitro testicular cord formation. In the present paper, we report that PDGF-BB is present during embryonic testis development and, in organ culture, induces cord formation although with reduced diameters compared with the cords formed in the genital ridges cultured in the presence of HGF. Moreover we have analyzed the roles exerted by this growth factor during the morphogenesis of the testis. We demonstrate by immunohistochemical experiments that PDGF-BB and its receptors are synthesized by the male UGRs isolated from 11.5 and 13.5 dpc embryos and by Western blot that the factor is secreted in a biologically active form by testicular cells isolated from 13.5 dpc embryos. The biological roles of the factor have also been studied and we demonstrate that PDGF-BB acts as a migratory factor for male mesonephric cells whose migration is a male specific event necessary for a normal testicular morphogenesis. In addition we demonstrate that during testicular development, PDGF-BB induces testicular cell proliferation being in this way responsible for the increase in size of the testis. Finally we demonstrate that PDGF-BB is able to reorganize dissociated testicular cells inducing the formation of large cellular aggregates. However the structures formed in vitro under PDGF-BB stimulation never had a cord-like morphology similar to the cord-like structures formed in the presence of HGF (Ricci et al., 2002, Mech Dev 118:19-28), suggesting that this factor does not act as a morphogenetic factor during testicular development. All together the data presented in this paper demonstrate that PDGF-BB and its receptors (alpha- and beta-subunits) are present during the crucial ages of embryonic mouse testis morphogenesis and indicate the multiple roles exerted by this factor during the development of the male gonad.     

Development of the limb neuromuscular system

Appendages, such as wings of a fly or limbs of a vertebrate, are excellent models to study the principles of patterning and morphogenesis. In the adult these structures are used for a variety of behaviors, including locomotion. Although support structures of the adult vertebrate limb are generated within the limb bud, its dynamic elements are derived from the somitic mesoderm and neural tube. Recent studies show that regional patterns set up in the mesenchyme-filled limb bud guide muscle precursors and developing motor axons to their proper location within the limb. Subsequent development of the neuromuscular system is regulated by cell surface interactions between pre-specified muscle fibers and motor axons.     

A large multigene family codes for the polypeptides of the crystalline trichocyst matrix in Paramecium.

The secretory granules (trichocysts) of Paramecium are characterized by a highly constrained shape that reflects the crystalline organization of their protein contents. Yet the crystalline trichocyst content is composed not of a single protein but of a family of related polypeptides that derive from a family of precursors by protein processing. In this paper we show that a multigene family, of unusually large size for a unicellular organism, codes for these proteins. The family is organized in subfamilies; each subfamily codes for proteins with different primary structures, but within the subfamilies several genes code for nearly identical proteins. For one subfamily, we have obtained direct evidence that the different members are coexpressed. The three subfamilies we have characterized are located on different macronuclear chromosomes. Typical 23-29 nucleotide Paramecium introns are found in one of the regions studied and the intron sequences are more variable than the surrounding coding sequences, providing gene-specific markers. We suggest that this multigene family may have evolved to assure a microheterogeneity of structural proteins necessary for morphogenesis of a complex secretory granule core with a constrained shape and dynamic properties: genetic analysis has shown that correct assembly of the crystalline core is necessary for trichocyst function.     

Chemical morphogenesis: Turing patterns in an experimental chemical system

Patterns resulting from the sole interplay between reaction and diffusion are probably involved in certain stages of morphogenesis in biological systems, as initially proposed by Alan Turing. Self-organization phenomena of this type can only develop in nonlinear systems (i.e. involving positive and negative feedback loops) maintained far from equilibrium. We present Turing patterns experimentally observed in a chemical system. An oscillating chemical reaction, the CIMA reaction, is operated in an open spatial reactor designed in order to obtain a pure reaction-diffusion system. The two types of Turing patterns observed, hexagonal arrays of spots and parallel stripes, are characterized by an intrinsic wavelength. We identify the origin of the necessary difference of diffusivity between activator and inhibitor. We also describe a pattern growth mechanism by spot splitting that recalls cell division.     

Akt activation disrupts mammary acinar architecture and enhances proliferation in an mTOR-dependent manner

Activation of the serine/threonine kinase Akt/PKB positively impacts on three cellular processes relevant to tumor progression: proliferation, survival, and cell size/growth. Using a three-dimensional culture model of MCF-10A mammary cells, we have examined how Akt influences the morphogenesis of polarized epithelial structures. Activation of a conditionally active variant of Akt elicits large, misshapen structures, which primarily arise from the combined effects of Akt on proliferation and cell size. Importantly, Akt activation amplifies proliferation during the early stages of morphogenesis, but cannot overcome signals suppressing proliferation in late-stage cultures. Akt also cooperates with oncoproteins such as cyclin D1 or HPV E7 to promote proliferation and morphogenesis in the absence of growth factors. Pharmacological inhibition of the Akt effector, mammalian target of rapamycin (mTOR), with rapamycin prevents the morphological disruption elicited by Akt activation, including its effect on cell size and number, and the cooperative effect of Akt on oncogene-driven proliferation, indicating that mTOR function is required for the multiple biological effects of Akt activation during morphogenesis.     

GRK5 promotes F-actin bundling and targets bundles to membrane structures to control neuronal morphogenesis

Neuronal morphogenesis requires extensive membrane remodeling and cytoskeleton dynamics. In this paper, we show that GRK5, a G protein-coupled receptor kinase, is critically involved in neurite outgrowth, dendrite branching, and spine morphogenesis through promotion of filopodial protrusion. Interestingly, GRK5 is not acting as a kinase but rather provides a key link between the plasma membrane and the actin cytoskeleton. GRK5 promoted filamentous actin (F-actin) bundling at the membranes of dynamic neuronal structures by interacting with both F-actin and phosphatidylinositol-4,5-bisphosphate. Moreover, separate domains of GRK5 mediated the coupling of actin cytoskeleton dynamics and membrane remodeling and were required for its effects on neuronal morphogenesis. Accordingly, GRK5 knockout mice exhibited immature spine morphology and deficient learning and memory. Our findings identify GRK5 as a critical mediator of dendritic development and suggest that coordinated actin cytoskeleton and membrane remodeling mediated by bifunctional actin-bundling and membrane-targeting molecules, such as GRK5, is crucial for proper neuronal morphogenesis and the establishment of functional neuronal circuitry.     

A dynamic programming approach for finding common patterns in RNAs.

We developed a dynamic programming approach of computing common sequence structure patterns among two RNAs given their primary sequences and their secondary structures. Common patterns between two RNAs are defined to share the same local sequential and structural properties. The locality is based on the connections of nucleotides given by their phosphodiester and hydrogen bonds. The idea of interpreting secondary structures as chains of structure elements leads us to develop an efficient dynamic programming approach in time O(nm) and space O(nm), where n and m are the lengths of the RNAs. The biological motivation is given by detecting common, local regions of RNAs, although they do not necessarily share global sequential and structural properties. This might happen if RNAs fold into different structures but share a lot of local, stable regions. Here, we illustrate our algorithm on Hepatitis C virus internal ribosome entry sites. Our method is useful for detecting and describing local motifs as well. An implementation in C++ is available and can be obtained by contacting one of the authors.     

Emergence of community structure in terrestrial mammal-dominated ecosystems

We present a paper that combines empirical and theoretical research about the trophic organization of biological communities. Some regularities are observed in the analysis of the relationship between the trophic structure (how the species are distributed among a set of feeding groups) of a number of African large mammal communities and the type of ecosystem. Different types of ecosystems are characterized by specific patterns in the trophic structure of the mammal community. In order to explain the origin of these patterns, we propose a model defining the underlying dynamic of mammal-dominated ecosystems. The main aim of this study is to show that it is possible to obtain a dynamic explanation of those patterns. The model is shown to spontaneously define different types of structures in community organization, related to those observed. We propose a model that could help to explain the correlation between different environmental factors and the abundance or diversity of herbivores, and which establishes a general mechanism that makes it possible to understand how some rules constrain the assembly of the communities. In addition, the proposed model leads us to see how biological communities can operate in an integrated way, which allows for the acceptance of their changes on large time-scales as evolutionary. In summary, we suggest that communities are unitary structures with coherent properties that result from the self-organizing dynamic of the whole system.     

Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.     

Turing pattern with proportion preservation

Although Turing pattern is one of the most universal mechanisms for pattern formation, in its standard model the number of stripes changes with the system size, since the wavelength of the pattern is invariant. It fails to preserve the proportionality of the pattern, i.e. the ratio of the wavelength to the size, that is often required in biological morphogenesis. To get over this problem, we show that the Turing pattern can preserve proportionality by introducing a catalytic chemical whose concentration depends on the system size. Several plausible mechanisms for such size dependence of the concentration are discussed. Following this general discussion, two models are studied in which arising Turing patterns indeed preserve the proportionality. Relevance of the present mechanism to biological morphogenesis is discussed from the viewpoint of its generality, robustness, and evolutionary accessibility.     

Rho1 regulates Drosophila adherens junctions independently of p120ctn

During animal development, adherens junctions (AJs) maintain epithelial cell adhesion and coordinate changes in cell shape by linking the actin cytoskeletons of adjacent cells. Identifying AJ regulators and their mechanisms of action are key to understanding the cellular basis of morphogenesis. Previous studies linked both p120catenin and the small GTPase Rho to AJ regulation and revealed that p120 may negatively regulate Rho. Here we examine the roles of these candidate AJ regulators during Drosophila development. We found that although p120 is not essential for development, it contributes to morphogenesis efficiency, clarifying its role as a redundant AJ regulator. Rho has a dynamic localization pattern throughout ovarian and embryonic development. It preferentially accumulates basally or basolaterally in several tissues, but does not preferentially accumulate in AJs. Further, Rho1 localization is not obviously altered by loss of p120 or by reduction of core AJ proteins. Genetic and cell biological tests suggest that p120 is not a major dose-sensitive regulator of Rho1. However, Rho1 itself appears to be a regulator of AJs. Loss of Rho1 results in ectopic accumulation of cytoplasmic DE-cadherin, but ectopic cadherin does not accumulate with its partner Armadillo. These data suggest Rho1 regulates AJs during morphogenesis, but this regulation is p120 independent.     

Cytomechanical control of morphogenesis

The role of mechanically strained state of cells and multicellular structures in morphogenesis regulating in vertebrate embryos is discussed. Regular changes in patterns of mechanical strain during embryonic development are described. Artificial relaxation of mechanical strain performed on definite developmental stages and retension of embryonic tissues in arbitrary directions considerably affects morphogenesis and cell differentiation patterns. Cytomechanical models of morphogenesis are reviewed and a concept of hyperrestoration of mechanical strain as a possible driving force of morphogeneiss is suggested.