Deposition of collagen type I onto skeletal endothelium reveals a new role for blood vessels in regulating bone morphology.

Recently, blood vessels have been implicated in the morphogenesis of various organs. The vasculature is also known to be essential for endochondral bone development, yet the underlying mechanism has remained elusive. We show that a unique composition of blood vessels facilitates the role of the endothelium in bone mineralization and morphogenesis. Immunostaining and electron microscopy showed that the endothelium in developing bones lacks basement membrane, which normally isolates the blood vessel from its surroundings. Further analysis revealed the presence of collagen type I on the endothelial wall of these vessels. Because collagen type I is the main component of the osteoid, we hypothesized that the bone vasculature guides the formation of the collagenous template and consequently of the mature bone. Indeed, some of the bone vessels were found to undergo mineralization. Moreover, the vascular pattern at each embryonic stage prefigured the mineral distribution pattern observed one day later. Finally, perturbation of vascular patterning by overexpressing Vegf in osteoblasts resulted in abnormal bone morphology, supporting a role for blood vessels in bone morphogenesis. These data reveal the unique composition of the endothelium in developing bones and indicate that vascular patterning plays a role in determining bone shape by forming a template for deposition of bone matrix.

Isometric Scaling in Developing Long Bones Is Achieved by an Optimal Epiphyseal Growth Balance.

One of the major challenges that developing organs face is scaling, that is, the adjustment of physical proportions during the massive increase in size. Although organ scaling is fundamental for development and function, little is known about the mechanisms that regulate it. Bone superstructures are projections that typically serve for tendon and ligament insertion or articulation and, therefore, their position along the bone is crucial for musculoskeletal functionality. As bones are rigid structures that elongate only from their ends, it is unclear how superstructure positions are regulated during growth to end up in the right locations. Here, we document the process of longitudinal scaling in developing mouse long bones and uncover the mechanism that regulates it. To that end, we performed a computational analysis of hundreds of three-dimensional micro-CT images, using a newly developed method for recovering the morphogenetic sequence of developing bones. Strikingly, analysis revealed that the relative position of all superstructures along the bone is highly preserved during more than a 5-fold increase in length, indicating isometric scaling. It has been suggested that during development, bone superstructures are continuously reconstructed and relocated along the shaft, a process known as drift. Surprisingly, our results showed that most superstructures did not drift at all. Instead, we identified a novel mechanism for bone scaling, whereby each bone exhibits a specific and unique balance between proximal and distal growth rates, which accurately maintains the relative position of its superstructures. Moreover, we show mathematically that this mechanism minimizes the cumulative drift of all superstructures, thereby optimizing the scaling process. Our study reveals a general mechanism for the scaling of developing bones. More broadly, these findings suggest an evolutionary mechanism that facilitates variability in bone morphology by controlling the activity of individual epiphyseal plates.

A mechanical Jack-like Mechanism drives spontaneous fracture healing in neonatal mice.

Treatment of fractured bones involves correction of displacement or angulation, known as reduction. However, angulated long-bone fractures in infants often heal and regain proper morphology spontaneously, without reduction. To study the mechanism underlying spontaneous regeneration of fractured bones, we left humeral fractures induced in newborn mice unstabilized, and rapid realignment of initially angulated bones was seen. This realignment was surprisingly not mediated by bone remodeling, but instead involved substantial movement of the two fragments prior to callus ossification. Analysis of gene expression profiles, cell proliferation, and bone growth revealed the formation of a functional, bidirectional growth plate at the concave side of the fracture. This growth plate acts like a mechanical jack, generating opposing forces that straighten the two fragments. Finally, we show that muscle force is important in this process, as blocking muscle contraction disrupts growth plate formation, leading to premature callus ossification and failed reduction.

Muscle force regulates bone shaping for optimal load-bearing capacity during embryogenesis.

The vertebrate skeleton consists of over 200 individual bones, each with its own unique shape, size and function. We study the role of intrauterine muscle-induced mechanical loads in determining the three-dimensional morphology of developing bones. Analysis of the force-generating capacity of intrauterine muscles in mice revealed that developing bones are subjected to significant and progressively increasing mechanical challenges. To evaluate the effect of intrauterine loads on bone morphogenesis and the contribution of the emerging shape to the ability of bones to withstand these loads, we monitored structural and mineral changes during development. Using daily micro-CT scans of appendicular long bones we identify a developmental program, which we term preferential bone growth, that determines the specific circumferential shape of each bone by employing asymmetric mineral deposition and transient cortical thickening. Finite element analysis demonstrates that the resulting bone structure has optimal load-bearing capacity. To test the hypothesis that muscle forces regulate preferential bone growth in utero, we examine this process in a mouse strain (mdg) that lacks muscle contractions. In the absence of mechanical loads, the stereotypical circumferential outline of each bone is lost, leading to the development of mechanically inferior bones. This study identifies muscle force regulation of preferential bone growth as the module that shapes the circumferential outline of bones and, consequently, optimizes their load-bearing capacity during development. Our findings invoke a common mechanism that permits the formation of different circumferential outlines in different bones.

Diversity of sponge mitochondrial introns revealed by cox 1 sequences of Tetillidae.

BACKGROUND: Animal mitochondrial introns are rare. In sponges and cnidarians they have been found in the cox 1 gene of some spirophorid and homosclerophorid sponges, as well as in the cox 1 and nad 5 genes of some Hexacorallia. Their sporadic distribution has raised a debate as to whether these mobile elements have been vertically or horizontally transmitted among their hosts. The first sponge found to possess a mitochondrial intron was a spirophorid sponge from the Tetillidae family. To better understand the mode of transmission of mitochondrial introns in sponges, we studied cox 1 intron distribution among representatives of this family. RESULTS: Seventeen tetillid cox 1 sequences were examined. Among these sequences only six were found to possess group I introns. Remarkably, three different forms of introns were found, named introns 714, 723 and 870 based on their different positions in the cox 1 alignment. These introns had distinct secondary structures and encoded LAGLIDADG ORFs belonging to three different lineages. Interestingly, sponges harboring the same intron form did not always form monophyletic groups, suggesting that their introns might have been transferred horizontally. To evaluate whether the introns were vertically or horizontally transmitted in sponges and cnidarians we used a host parasite approach. We tested for co-speciation between introns 723 (the introns with the highest number of sponge representatives) and their nesting cox 1 sequences. Reciprocal AU tests indicated that the intron and cox 1 tree are significantly different, while a likelihood ratio test was not significant. A global test of co-phylogeny had significant results; however, when cnidarian sequences were analyzed separately the results were not significant. CONCLUSIONS: The co-speciation analyses thus suggest that a vertical transmission of introns in the ancestor of sponges and cnidarians, followed by numerous independent losses, cannot solely explain the current distribution of metazoan group I introns. An alternative scenario that includes horizontal gene transfer events appears to be more suitable to explain the incongruence between the intron 723 and the cox 1 topologies. In addition, our results suggest that three different intron forms independently colonized the cox 1 gene of tetillids. Among sponges, the Tetillidae family seems to be experiencing an unusual number of intron insertions.

Putative cross-kingdom horizontal gene transfer in sponge (Porifera) mitochondria.

BACKGROUND: The mitochondrial genome of Metazoa is usually a compact molecule without introns. Exceptions to this rule have been reported only in corals and sea anemones (Cnidaria), in which group I introns have been discovered in the cox1 and nad5 genes. Here we show several lines of evidence demonstrating that introns can also be found in the mitochondria of sponges (Porifera). RESULTS: A 2,349 bp fragment of the mitochondrial cox1 gene was sequenced from the sponge Tetilla sp. (Spirophorida). This fragment suggests the presence of a 1143 bp intron. Similar to all the cnidarian mitochondrial introns, the putative intron has group I intron characteristics. The intron is present in the cox1 gene and encodes a putative homing endonuclease. In order to establish the distribution of this intron in sponges, the cox1 gene was sequenced from several representatives of the demosponge diversity. The intron was found only in the sponge order Spirophorida. A phylogenetic analysis of the COI protein sequence and of the intron open reading frame suggests that the intron may have been transmitted horizontally from a fungus donor. CONCLUSION: Little is known about sponge-associated fungi, although in the last few years the latter have been frequently isolated from sponges. We suggest that the horizontal gene transfer of a mitochondrial intron was facilitated by a symbiotic relationship between fungus and sponge. Ecological relationships are known to have implications at the genomic level. Here, an ecological relationship between sponge and fungus is suggested based on the genomic analysis.