Activator-Inhibitor Dynamics of Vertebrate Limb Pattern Formation Stuart A. Newman* and Ramray Bhat The development of the vertebrate limb depends on an interplay of cel- lular differentiation, pattern formation, and tissue morphogenesis on multiple spatial and temporal scales. While numerous gene products have been described that participate in, and influence, the generation of the limb skeletal pattern, an understanding of the most salient feature of the developing limb—its quasiperiodic arrangement of bones, requires additional organizational principles. We review several such principles, drawing on concepts of physics and chemical dynamics along with mo- lecular genetics and cell biology. First, a ‘‘core mechanism’’ for precarti- lage mesenchymal condensation is described, based on positive auto- regulation of the morphogen transforming growth factor (TGF)-b, induc- tion of the extracellular matrix (ECM) protein fibronectin, and focal accumulation of cells via haptotaxis. This core mechanism is shown to be part of a local autoactivation-lateral inhibition (LALI) system that ensures that the condensations will be regularly spaced. Next, a ‘‘bare- bones’’ model for limb development is described in which the LALI-core mechanism is placed in a growing geometric framework with prediffer- entiated ‘‘apical,’’ differentiating ‘‘active,’’ and irreversibly differentiated ‘‘frozen’’ zones defined by distance from an apical source of a fibroblast growth factor (FGF)-type morphogen. This model is shown to account for classic features of the developing limb, including the proximodistal (PD) emergence over time of increasing numbers of bones. We review earlier and recent work suggesting that the inhibitory component of the LALI system for condensation may not be a diffusible morphogen, and propose an alternative mechanism for lateral inhibition, based on syn- chronization of oscillations of a Hes mediator of the Notch signaling pathway. Finally, we discuss how viewing development as an interplay between molecular-genetic and dynamic physical processes can provide new insight into the origin of congenital anomalies. Birth Defects Research (Part C) 81:305–319, 2007. V C 2008 Wiley-Liss, Inc. INTRODUCTION Defects of the limb skeleton are among the most frequent human congenital anomalies. Mutations and teratogens can dramatically affect the structure of the limb skeleton without otherwise impair- ing survival, reproduction, and other bodily functions. This has led to the presence of a wide-ranging set of limb variations throughout the human population as well as providing the opportunity, using experimental systems, to explore the bases of normal and abnormal limb formation. The limb skeleton, an array of jointed bone or carti- lage elements, has a stereotypical pattern that (as Charles Darwin noted) is only modestly altered by adaptations for functions as varied as walking, swimming, flying, and grasping (Darwin, 1859). The transmission and molecular genet- ics of limb variations have been extensively studied in humans and mice, while the developing appen- dages in embryos of egg-laying species have lent themselves to experimental analysis by surgical manipulation. In fishes and amphibians the paired limbs, or related structures, exist with vari- ant anatomical characteristics and regenerative properties, enabling informative comparative studies. Finally, limb bud mesenchymal cells from avian and mammalian species can be grown in culture, where they undergo differentiation and pattern formation, though simplified, with a time-course and on a spatial scale similar to that in the respective embryos. These features have made the limb a highly favorable system for study- ing the generation of multicellular form. Over the past three decades the protein products of scores of genes have been identified as par- ticipating in patterning of the limb skeleton (reviewed in Tickle, 2003; Newman and Mu ¨ller, 2005). Genes and the interactive net- works in which they participate, however, are only part of the story in the generation of biological structures (Nijhout, 1990; New- man and Comper, 1990; Newman, REVIEW V C 2008 Wiley-Liss, Inc. Birth Defects Research (Part C) 81:305–319 (2007) Stuart A. Newman and Ramray Bhat are from the Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York. Grant sponsor: National Science Foundation. *Correspondence to: Stuart A. Newman, Department of Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10595. E-mail: newman@NYMC.edu Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.20112