Joint loading modality: its application to bone formation and fracture healing.

Sports-related injuries such as impact and stress fractures often require a rehabilitation programme to stimulate bone formation and accelerate fracture healing. This review introduces a recently developed joint loading modality and evaluates its potential applications to bone formation and fracture healing in post-injury rehabilitation. Bone is a dynamic tissue whose structure is constantly altered in response to its mechanical environments. Indeed, many loading modalities can influence the bone remodelling process. The joint loading modality is, however, able to enhance anabolic responses and accelerate wound healing without inducing significant in situ strain at the site of bone formation or fracture healing. This review highlights the unique features of this loading modality and discusses its potential underlying mechanisms as well as possible clinical applications.

In vitro control of organogenesis and body patterning by activin during early amphibian development.

In the process of amphibian development, an embryonic body plan is established through cell division, sequential gene expression, morphogenesis and cell differentiation. The mechanism of body patterning is complex and includes multiple induction events. Activin, a TGF-beta family protein, can induce several kinds of mesodermal and endodermal tissues in animal cap explants in a dose-dependent manner. In a recent study of the role of activin in organogenesis, we succeeded in raising a beating heart by treating animal caps with a high concentration of activin. Activin also participates in kidney organogenesis in combination with retinoic acid. An embryonic kidney induced by activin and retinoic acid in vitro can function in vivo when it is transplanted into a larva in which pronephros rudiments have already been removed. Further, the activin-treated animal caps clearly show organizer actions that are closely related to body patterning along the anteroposterior axis. These experiments will help to serve as a model system for understanding organogenesis and body patterning at the cellular and molecular levels.

Work in progress: the renaissance in amphibian embryology.

Various historical eras in the distant as well as the recent past of amphibian embryology are briefly reviewed. The concepts which emerged from the early years matured, then were laid to rest for several decades. A resurgence, driven by key discoveries with peptide growth factors, and fueled by modern molecular biology methods, is underway. The future for several amphibian research projects should be promising since interest in basic concepts remains strong, and application of frontier methodologies is yielding novel findings.

Future opportunities for life science programs in space.

Most space-related life science programs are expensive and time-consuming, requiring international cooperation and resources with trans-disciplinary expertise. A comprehensive future program in "life sciences in space" needs, therefore, well-defined research goals and strategies as well as a sound ground-based program. The first half of this review will describe four key aspects such as the environment in space, previous accomplishments in space (primarily focusing on amphibian embryogenesis), available resources, and recent advances in bioinformatics and biotechnology, whose clear understanding is imperative for defining future directions. The second half of this review will focus on a broad range of interdisciplinary research opportunities currently supported by the National Aeronautics and Space Administration (NASA), National Institute of Health (NIH), and National Science Foundation (NSF). By listing numerous research topics such as alterations in a diffusion-limited metabolic process, bone loss and skeletal muscle weakness of astronauts, behavioral and cognitive ability in space, life in extreme environment, etc., we will attempt to suggest future opportunities.

Role of activin and other peptide growth factors in body patterning in the early amphibian embryo.

The amphibian body plan is established as the result of a series of inductive interactions. During early cleavage stages cells in the vegetal hemisphere induce overlying animal hemisphere cells to form mesoderm. The interaction represents the first major body-patterning event and is mediated by peptide growth factors. Various peptide growth factors have been implicated in mesoderm development, including most notably members of the transforming growth factor-beta superfamily. Identification of the so-called "natural" inducer from among the several candidate peptide growth factors is being achieved by employing several experimental strategies, including the use of a tissue explant assay for testing potential inducers, cloning of marker genes as indices of early induction events, and microinjection of altered peptide growth factor receptors to disrupt normal embryonic inductions. Activin emerges as the most likely choice for assignment of the role of endogenous mesoderm inducer, because it currently best fulfills the rigorous set of criteria expected of such an important embryonic signaling molecule. Activin, however, may not act alone in mesoderm induction. Other peptide growth factors such as fibroblast growth factor might be involved, especially in the regional patterning of the mesoderm. In addition, several genes (e.g., Wnt and noggin), which are expressed after the mesoderm is initially induced, probably assist in further definition of the mesoderm pattern. Following mesoderm induction, the primary embryonic organizer tissue (first described in 1924 by Spemann) develops and contributes further to body patterning by its action as a neural inducer. Peptide growth factors such as activin may also be involved in the inductive event, either directly (by facilitating gene expression) or indirectly (by serving to constrain pathways).

Peptide growth factors in amphibian embryogenesis: intersection of modern molecular approaches with traditional inductive interaction paradigms.

Recent discoveries of the role peptide growth factors (PGFs) play in regulating embryonic patterning and differentiation have profoundly influenced research on the molecular biology of early amphibian embryogenesis. Several PGFs have been recognized to be present as endogenous components of amphibian eggs and early embryos, while other PGFs -- which are known from heterologous systems (e.g., Drosophila) -- exert remarkable effects when injected as either protein or mRNA into eggs/embryos or when added to cultured embryonic tissue. For a variety of reasons (reviewed herein) optimism abounds that an understanding in molecular terms of the classical Spemann and Nieuwkoop tissue interactions which are generally believed to drive embryonic patterning is within reach. A critical assessment of the interpretations of some of the contemporary data on PGFs (included herein) should, however, temper some of that optimism. Likely, multiple rather than single PGFs act in a combinatorial fashion to contribute to individual patterning events. As well, substantial redundancy in PGF regulatory circuits probably exists, so the heavy reliance on tissue culture assays and overexpression studies which characterize much recent research needs to be circumvented. Potential experimental approaches for "next generation" experiments are discussed.

An essay on the similarities and differences between inductive interactions in anuran and urodele embryos.

As a first step towards providing a conceptual approach to understanding similarities and differences in the mechanisms which guide inductive interactions among related organisms (e.g. various amphibia), a set of five principles is offered here. These principles were formulated by analyzing literature examples of classical embryological phenomena and by performing experiments with activin, a peptide growth factor which is currently suspected to play for a role in mesoderm induction. Mechanisms which account, at least in part, for the observed differences between anuran and urodele inductive processes can be derived from these principles.

Activin treated urodele ectoderm: a model experimental system for cardiogenesis.

The tissue interactions which comprise the inductive phenomena associated with urodele heart morphogenesis are relatively well understood. In order to take full advantage of the experimental potential of this system formulation of an in vitro tissue culture system would be very helpful. Herein are described conditions for culturing Cynops pyrrhogaster early gastrula ectoderm tissue in the presence of the peptide growth factor activin. Two-week old explant cultures frequently displayed beating heart-like rudiments within. The beating frequency was measured and the extent to which cytodifferentiation mimicked normal heart differentiation assessed. Both measurements provided optimistic assessments which should encourage further exploitation of this model system.

Cloning and expression of the axolotl proto-oncogene ski.

In vitro and in vivo overexpression studies have demonstrated that the c-ski proto-oncogene can influence proliferation, morphological transformation and myogenic differentiation. We report the isolation and expression of an axolotl (Ambystoma mexicanum) c-ski (aski) gene. Sequence analysis revealed a high degree of nucleotide and predicted amino acid (AA) homology with mammalian and anuran c-ski, showing the highest conservation to Xenopus laevis c-ski (74% nucleotide and 87% AA). Northern analysis showed that axolotl c-ski is expressed in unfertilized eggs and at increasing levels in embryos from blastula to tadpole stage. c-ski expression was also detected in larval limb muscle and in several stages of regenerating limb blastemas. These data indicate that axolotl c-ski is highly conserved among amphibians and mammals and suggests that it plays a role in urodele embryogenesis and limb regeneration.

Expression of the axolotl homologue of mouse chaperonin t-complex protein-1 during early development.

Molecular chaperones assist in the folding of proteins, but their role during development is not well understood. Here we report the temporal and spatial expression pattern of the axolotl homologue of mouse chaperonin TCP-1 during normal amphibian embryogenesis and in several models of abnormal embryogenesis. A partial axolotl TCP-1 cDNA (646 bp; 519 coding bp) isolated by 3' RACE PCR shows considerable homology to mouse TCP-1. Developmental Northerns and RT-PCR analyses of whole axolot1 embryos revealed a low level of maternal TCP-1 transcripts in fertilized eggs. The maternal transcripts were down-regulated to a non-detectable level in early gastrulae. Zygotic TCP-1 transcripts first appeared during gastrulation. They were mainly expressed in mid-neurula and later stage embryos. Whole-mount in situ hybridization studies showed abundant TCP-1 transcripts in the blastopore at the mid-gastrula stage and in the brain and spinal cord beginning at the neurula stage, and in the somites (myotomes) at the tailbud stage. RT-PCR analysis of TCP-1 expression in axolotl embryos treated with either high salt (causing exogastrulation) or ultraviolet (UV) irradiation (causing ventralization) substantiated the correlation between TCP-1 expression and neural and somitic development. In high salt-induced exogastrulated embryos TCP-1 mRNA was detectable in the ectoderm part (with neural tissues) but not in its exogastrulated endoderm part. Lower levels of TCP-1 expression were detected in UV-irradiated, ventralized embryos with smaller head and reduced neural and somitic tissues. Normal levels of TCP-1 expression were detected in embryos with double axes/heads. These studies provide strong evidence that at the transcript level axolotl chaperonin TCP-1 is regulated both temporally and spatially during embryogenesis, especially in neural and somitic development.

Overexpression of XMyoD or XMyf5 in Xenopus embryos induces the formation of enlarged myotomes through recruitment of cells of nonsomitic lineage.

The myogenic regulatory factors (MRFs) MyoD and Myf5 are the earliest described muscle-specific genes to be expressed in Xenopus development. To study the in vivo effects of overexpressing Xenopus MyoD and Myf5, synthetic RNAs were microinjected into single blastomeres of 2- to 32-cell stage Xenopus embryos. In vivo overexpression of these MRFs initiates the precocious and ectopic expression of actin and myosin. The effects of unilateral injection of either mRNA were indistinguishable; embryos injected at the 2-cell stage showed ipsilaterally enlarged cranial and anterior trunk myotomes composed of increased numbers of primary myotome myocytes. In addition, formation of ectopic muscle in lateral plate and neural tissue was observed. The MRF-induced effects persist through secondary myogenesis, with the enlarged cranial myotomes failing to undergo the normal program of degeneration. Experiments combining MRF RNA and lineage tracer injections showed that myotomal enlargement is due in part to the contribution of cells of nonsomitic lineage to the myotome, rather than to an increase in muscle precursor cell division. Overexpression of XMyoD and XMyf5 also affected the morphogenesis of the skin and the nervous system. These results reveal that overexpression of XMyoD or XMyf5 in vivo clearly influences the regulation of early myogenesis and the morphogenesis of skin and nervous tissue.

The location of the third cleavage plane of Xenopus embryos partitions morphogenetic information in animal quartets.

Analysis of the developmental potential of animal quartets (the set of four animal blastomeres isolated from the 8-cell stage Xenopus embryo) provided insight into the manner in which morphogenetic information is distributed along the animal-vegetal axis. Gravity treatments were employed to alter the partitioning plane. Animal quartets isolated from embryos exposed to simulated weightlessness had larger animal blastomeres, and they formed structures such as a groove and a protrusion more often than 1g-control animal quartets. Animal quartets with an unusual non-horizontal third cleavage plane were also found to have a higher frequency of protrusion formation than animal quartets with a typical horizontal cleavage plane. The increase in the frequency seen in simulated weightlessness animal quartets was not due to their increased size. Fusing two animal quartets isolated from hypergravity (3g) exposed embryos (small blastomeres and low incidence of protrusions) did not affect the frequency of protrusion formation. Molecular analyses revealed that a partial induction was associated with the protrusion formation. Transcripts of the dorsal lip specific homeobox gene, goosecoid, and alpha-cardiac actin were detectable by PCR amplification in the animal quartet with a protrusion, and alpha-cardiac actin mRNA was found by whole-mount in situ hybridization to be localized in the protrusion. Taken together, all these results are consistent with the notion that both animal and vegetal information is necessary for normal development and the partitioning of morphogenetic information into animal quartets results in gravity-dependent differential morphogenesis and gene regulation.

Early development of Xenopus embryos is affected by simulated gravity.

Early amphibian (Xenopus laevis) development under clinostat-simulated weightlessness and centrifuge-simulated hypergravity was studied. The results revealed significant effects on (i) "morphological patterning" such as the cleavage furrow pattern in the vegetal hemisphere at the eight-cell stage and the shape of the dorsal lip in early gastrulae and (ii) "the timing of embryonic events" such as the third cleavage furrow completion and the dorsal lip appearance. Substantial variations in sensitivity to simulated force fields were observed, which should be considered in interpreting spaceflight data.

Early amphibian (anuran) morphogenesis is sensitive to novel gravitational fields.

Anuran amphibian embryos (Xenopus laevis and Rana dybowskii) are sensitive to novel gravitational fields. Under simulated weightlessness, (i) the location of the first horizontal cleavage furrow was shifted toward the vegetal pole at the eight-cell stage; (ii) the position of the blastocoel was more centered, and the number of cell layers in the blastocoel roof was increased at the blastula stage; (iii) the dorsal lip appeared closer to the vegetal pole at the gastrula stage; and (iv) head and eye dimensions were enlarged at the hatching tadpole stage. Effects of simulated hypergravity were opposite to those of simulated weightlessness, except that hypergravity, unlike simulated weightlessness, reduced the number of primordial germ cells in feeding tadpoles. Despite those dramatic differences in the early embryogenesis, tadpoles at the feeding stage are largely indistinguishable from controls.

Altering the position of the first horizontal cleavage furrow of the amphibian (Xenopus) egg reduces embryonic survival.

The animal/vegetal cleavage ratio (AVCR), defined as the ratio of the height of the animal blastomere to the height of the Xenopus embryo at the 8 cell stage, can be shifted by placing embryos in novel gravitational fields: clinostating (microgravity simulation) increases AVCR, and centrifugation (hypergravity simulation) reduces AVCR. This report contributes to an understanding of the subcellular mechanism responsible for the furrow relocation and assesses its significance. Embryo inversion and D2O immersion were found to increase AVCR, and cold shock was found to reduce AVCR. Based on the additive or antagonistic effects of combined treatments, it is postulated that the primary cause of AVCR changes is an alteration in the distribution of yolk platelets and the rearrangement of microtubule arrays. Embryos with a decreased AVCR exhibited reduced survival in early developmental stages, indicating serious difficulties in cleavage, blastulation and/or gastrulation. Cold-shocked embryos with a reduced AVCR could be rescued by D2O pretreatment or clinostating, an observation which supports the notion that changes accompanying AVCR modifications represent the primary cause of the reduction in percent survival.

Understanding the organization of the amphibian egg cytoplasm: gravitational force as a probe.

A combination of hypergravity (centrifugation) and hypogravity (clinostat) studies have been carried out on amphibian (frog, Xenopus) eggs. The results reveal that the twinning caused by centrifugation exhibits substantial spawning to spawning variation. That variation can be attributed to the apparent viscosity of the egg's internal cytoplasm. Simulated hypogravity results in a relocation of the egg's third (horizontal) cleavage furrow, towards the equator. Substantial egg-to-egg variation is also observed in this "cleavage effect". For interpreting spaceflight data and for using G-forces as probes for understanding the egg's architecture the egg variation documented herein should be considered.