Francesca V. Mariani

Functions of FGF signalling from the apical ectodermal ridge in limb development

To determine the role of fibroblast growth factor (FGF) signalling from the apical ectodermal ridge (AER), we inactivated Fgf4 and Fgf8 in AER cells or their precursors at different stages of mouse limb development. We show that FGF4 and FGF8 regulate cell number in the nascent limb bud and are required for survival of cells located far from the AER. On the basis of the skeletal phenotypes observed, we conclude that these functions are essential to ensure that sufficient progenitor cells are available to form the normal complement of skeletal elements, and perhaps other limb tissues. In the complete absence of both FGF4 and FGF8 activities, limb development fails. We present a model to explain how the mutant phenotypes arise from FGF-mediated effects on limb bud size and cell survival.

Genetic evidence that FGFs have an instructive role in limb proximal–distal patterning

Half a century ago, the apical ectodermal ridge (AER) at the distal tip of the tetrapod limb bud was shown to produce signals necessary for development along the proximal–distal (P–D) axis, but how these signals influence limb patterning is still much debated. Fibroblast growth factor (FGF) gene family members are key AER-derived signals, with Fgf4, Fgf8, Fgf9 and Fgf17 expressed specifically in the mouse AER. Here we demonstrate that mouse limbs lacking Fgf4, Fgf9 and Fgf17 have normal skeletal pattern, indicating that Fgf8 is sufficient among AER-FGFs to sustain normal limb formation. Inactivation of Fgf8 alone causes a mild skeletal phenotype; however, when we also removed different combinations of the other AER-FGF genes, we obtained unexpected skeletal phenotypes of increasing severity, reflecting the contribution that each FGF can make to the total AER-FGF signal. Analysis of the compound mutant limb buds revealed that, in addition to sustaining cell survival, AER-FGFs regulate P–D-patterning gene expression during early limb bud development, providing genetic evidence that AER-FGFs function to specify a distal domain and challenging the long-standing hypothesis that AER-FGF signalling is permissive rather than instructive for limb patterning. We discuss how a two-signal model for P–D patterning can be integrated with the concept of early specification to explain the genetic data presented here.

Deciphering skeletal patterning: clues from the limb

Even young children can distinguish a Tyrannosaurus rex from a Brontosaurus by observing differences in bone size, shape, number and arrangement, that is, skeletal pattern. But despite our extensive knowledge about cartilage and bone formation per se, it is still largely a mystery how skeletal pattern is established. Much of what we do know has been learned from studying limb development in chicken and mouse embryos. Based on the data from such studies, models for how limb skeletal pattern is established have been proposed and continue to be hotly debated.

The Neural Plate Specifies Somite Size in the Xenopus laevis Gastrula

The organizer has traditionally been considered the major source of somite-inducing signals. We show here that signaling from the neural plate specifies somite tissue and regulates somite size in the Xenopus gastrula. Ectopic undifferentiated neural tissue induces massive somite expansion at the expense of intermediate and lateral plate mesoderm. Although the early expanded somite expresses muscle-specific markers, only a portion terminally differentiates, suggesting that myotome development requires additional signals. Explant assays demonstrate that neural tissue induces somite-specific marker expression even in the absence of the organizer. Finally, we demonstrate that neural tissue is required for proper somite development because elimination of neural precursors results in pronounced somite reduction. Thus, an important reciprocal interaction exists between somite and neural tissue that is mutually reinforcing and critical for normal embryonic patterning.

Proximal to distal patterning during limb development and regeneration: a review of converging disciplines

Regeneration of lost structures typically involves distinct events: wound healing at the damaged site, the accumulation of cells that will be used as future building blocks and, finally, the initiation of molecular signaling pathways that dictate the form and pattern of the regenerated structures. Amphibians and urodeles in particular, have long been known to have exceptional regenerative properties. For many years, these animals have been the model of choice for understanding limb regeneration, a complex process that involves reconstructing skin, muscle, bone, connective tissue and nerves into a functional 3D structure. It appears that this process of rebuilding an adult limb has many similarities with how the limb forms in the first place--for example, in the embryo, all the components of the limb need to be formed and this requires signaling mechanisms to specify the final pattern. Thus, both limb formation and limb regeneration are likely to employ the same molecular pathways. Given the available tools of molecular biology and genetics, this is an exciting time for both fields to share findings and make significant progress in understanding more about the events that dictate embryonic limb pattern and control limb regeneration. This article focuses particularly on what is known about the molecular control of patterning along the proximal-distal axis.

Ihha induces hybrid cartilage-bone cells during zebrafish jawbone regeneration.

The healing of bone often involves a cartilage intermediate, yet how such cartilage is induced and utilized during repair is not fully understood. By studying a model of large-scale bone regeneration in the lower jaw of adult zebrafish, we show that chondrocytes are crucial for generating thick bone during repair. During jawbone regeneration, we find that chondrocytes co-express genes associated with osteoblast differentiation and produce extensive mineralization, which is in marked contrast to the behavior of chondrocytes during facial skeletal development. We also identify the likely source of repair chondrocytes as a population of Runx2+/Sp7− cells that emanate from the periosteum, a tissue that normally contributes only osteoblasts during homeostasis. Analysis of Indian hedgehog homolog a (ihha) mutants shows that the ability of periosteal cells to generate cartilage in response to injury depends on a repair-specific role of Ihha in the induction as opposed to the proliferation of chondrocytes. The large-scale regeneration of the zebrafish jawbone thus employs a cartilage differentiation program distinct from that seen during development, with the bone-forming potential of repair chondrocytes potentially due to their derivation from osteogenic cells in the periosteum. Highlighted article: The analysis of zebrafish jawbone regeneration reveals important differences between how the skeleton is formed in the embryo and repaired in adults.

Iroquois Proteins Promote Skeletal Joint Formation by Maintaining Chondrocytes in an Immature State

An early event in skeletal joint development is the specification of articular chondrocytes at the joint surface. Articular chondrocytes are distinct in producing lower levels of cartilage matrix and not being replaced by bone, yet how they acquire these properties remains poorly understood. Here, we show that two members of the Iroquois transcriptional repressor family, Irx7 and Irx5a, function to block chondrocyte maturation at the developing hyoid joint of zebrafish. These Irx factors suppress the production of cartilage matrix at the joint in part by preventing the activation of a col2a1a enhancer by Sox9a. Further, both zebrafish Irx7 and mouse IRX1 are able to repress cartilage matrix production in a murine chondrogenic cell line. Iroquois proteins may therefore have a conserved role in keeping chondrocytes in an immature state, with the lower levels of cartilage matrix produced by these immature cells contributing to joint flexibility.

Atypical PKC-iota Controls Stem Cell Expansion via Regulation of the Notch Pathway.

Summary The number of stem/progenitor cells available can profoundly impact tissue homeostasis and the response to injury or disease. Here, we propose that an atypical PKC, Prkci, is a key player in regulating the switch from an expansion to a differentiation/maintenance phase via regulation of Notch, thus linking the polarity pathway with the control of stem cell self-renewal. Prkci is known to influence symmetric cell division in invertebrates; however a definitive role in mammals has not yet emerged. Using a genetic approach, we find that loss of Prkci results in a marked increase in the number of various stem/progenitor cells. The mechanism used likely involves inactivation and symmetric localization of NUMB, leading to the activation of NOTCH1 and its downstream effectors. Inhibition of atypical PKCs may be useful for boosting the production of pluripotent stem cells, multipotent stem cells, or possibly even primordial germ cells by promoting the stem cell/progenitor fate.

Natural Large‐Scale Regeneration of Rib Cartilage in a Mouse Model

The clinical need for methods to repair and regenerate large cartilage and bone lesions persists. One way to make new headway is to study skeletal regeneration when it occurs naturally. Cartilage repair is typically slow and incomplete. However, an exception to this observation can be found in the costal cartilages, where complete repair has been reported in humans but the cellular and molecular mechanisms have not yet been characterized. In this study, we establish a novel animal model for cartilage repair using the mouse rib costal cartilage. We then use this model to test the hypothesis that the perichondrium, the dense connective tissue that surrounds the cartilage, is a tissue essential for repair. Our results show that full replacement of the resected cartilage occurs quickly (within 1 to 2 months) and properly differentiates but that repair occurs only in the presence of the perichondrium. We then provide evidence that the rib perichondrium contains a special niche that houses chondrogenic progenitors that possess qualities particularly suited for mediating repair. Label‐retaining cells can be found within the perichondrium that can give rise to new chondrocytes. Furthermore, the perichondrium proliferates and thickens during the healing period and when ectopically placed can generate new cartilage. In conclusion, we have successfully established a model for hyaline cartilage repair in the mouse rib, which should be useful for gaining a more detailed understanding of cartilage regeneration and ultimately for developing methods to improve cartilage and bone repair in other parts of the skeleton.

Evidence that the limb bud ectoderm is required for survival of the underlying mesoderm.

The limb forms from a bud of mesoderm encased in a hull of ectoderm that grows out from the flank of the embryo. Coordinated signaling between the limb mesoderm and ectoderm is critical for normal limb outgrowth and patterning. The apical ectodermal ridge (AER), found at the distal tip, is a rich source of signaling molecules and has been proposed to specify distal structures and maintain the survival of cells in the underlying distal mesoderm. The dorsal and ventral non-AER ectoderm is also a source of signaling molecules and is important for dorsal-ventral patterning of the limb bud. Here we determine if this ectoderm provides cell survival signals by surgically removing the dorsal or ventral ectoderm during early chicken limb bud development and assaying for programmed cell death. We find that, similar to the AER, removal of the dorsal or ventral non-AER ectoderm results in massive cell death in the underlying mesoderm. In addition, although a re-epithelialization occurs, we find perturbations in the timing of Shh expression and, for the case of the dorsal ectoderm removal, defects in soft tissue and skeletal development along the proximal-distal axis. Furthermore, ectoderm substitution experiments show that the survival signal produced by the dorsal limb ectoderm is specific. Thus, our results argue that the non-AER ectoderm, like the AER, provides a specific survival signal to the underlying mesoderm that is necessary for normal limb development and conclusions drawn from experiments in which the non-AER ectoderm is removed, need to take into consideration this observation.