The Batsheva Seminar, Integrative Perspectives on the Development of the Musculoskeletal System,was a unique workshop designed to spark discussions among scientists exploring the various components of the musculoskeletal system (muscle, muscle connective tissue, tendons/ligaments, and cartilage/bone). Participants were encouraged to think beyond the narrow confines of their own research interests, and consider the musculoskeletal system from a more global perspective.
In the last few years, the field of developmental biology has changed dramatically, due to the availability of new and improved genetic and molecular techniques for use in diverse vertebrate models. The subject of this meeting, Integrative Perspectives on the Development of the Musculoskeletal System, constituted a novel approach to the field of developmental biology, pulling together its various aspects by focusing on the morphogenesis of the musculoskeletal system as a whole, rather than viewing the musculoskeletal system and its muscular, connective and skeletal components as separate entities, conceptually as well as physically isolated.
To encourage scientific interactions among those who attended, the size of the meeting was kept relatively small: around 25 principal investigators, plus 30 students and postdoctoral fellows, made up the group. Plenty of time was set aside for both formal and informal discussions. Since the conference organizers felt that fruitful exchanges of ideas were best encouraged not only in formal sessions, but also in the more relaxed atmosphere of social gatherings and tours, several excursions in Jerusalem and the Dead Sea region were included in the program.
The main scientific topics discussed during the meeting focused on the molecular, cellular and developmental aspects of muscle, connective tissue, tendons/ligaments, and cartilage/bone. Recent advances in each of these areas, including new genetic and genomic tools, were presented. One of the themes that emerged from the meeting was that although muscles, tendons and bones in different anatomical locations function in similar ways, it is clear that the developmental programs underlying the genesis of these tissues are strikingly distinct. The meeting also provided a focal point for researchers working in all the major vertebrate model systems (mice, birds, frogs and fish), including some “new” models involving species such as Darwin's finches and cave fish, as well as for those who explore invertebrates (such as Drosophila) for comparison. In addition to the differences between vertebrate and invertebrate model systems, many speakers touched upon the evolutionary aspects of the musculoskeletal system as part of the emerging “Evo-Devo” field, which bridges between evolution and developmental biology.
In their keynote lectures, Cliff Tabin and Benny Shilo set the tone of the conference. Cliff Tabin presented two projects, reflecting the breadth of research carried out in his laboratory. The first part of his talk was directed towards understanding the factors regulating Sonic hedgehog expression in the developing limb bud. The second part focused on the development of a genetic system for studying cave fish evolution. For his part, Benny Shilo discussed the novel concept of morphogen shuttling as a means to generate graded signaling in the early embryo. Benny demonstrated this concept both computationally and experimentally for BMP shuttling in the early Drosophila embryo, and extended it further to BMP signaling in the Xenopus embryo. Finally, he showed that morphogen shuttling could account for scaling in Xenopus embryos, a mechanism which has remained elusive ever since the original experiments by German embryologist Hans Spemann, some 80 years ago.
Scientific summaries of selected talks:
Chaya Kalcheim discussed the mechanisms underlying the development of mural and endothelial cells that contribute to the walls of blood vessels, and compared them with myotomal cells, which appear to be derived from distinct somitic progenitors. In line with the fact that these cell populations are segregated at early developmental stages, she and her colleagues report that Notch signaling stimulates the generation of smooth rather than striated muscle, but has little if any effect on initial specification of the endothelium. BMP signaling does not affect smooth muscle development; yet it does inhibit the differentiation of myotomal myofibers. Thus, they suggest that the initial development of mural and endothelial cells is governed by different mechanisms. In contrast, the development of mural and striated muscle sublineages is a binary choice that depends upon a single signaling system.
Ram Reshef discussed how the treatment of biochemical events with SAT (satisfiability) formalism, a mathematical concept that has been primarily used to solve decision-making problems, can also provide a simple conceptual tool for describing the cause-and-effect relationships in biological phenomena such as myogenesis.
Frederic Relaix has long been interested in the molecular mechanisms underlying skeletal muscle specification during development. His team has designed genetic strategies to investigate the functions of the key upstream transcriptional regulators Pax3/Pax7. In addition, they have shown that regulatory signaling networks downstream of these factors have been functionally conserved throughout evolution.
Andrea Munsterberg presented recent findings on the expression of the muscle-specific microRNAs miR-1/206 and miR-133. The expression of three microRNAs, miR-1, miR-206 and miR-133, is restricted to skeletal myoblasts during embryonic development and muscle cell differentiation, a finding which suggests regulation by muscle regulatory factors (MRFs). Directed expression of MRFs in the neural tube of chick embryos induced ectopic expression of miR-1 and miR-206. Conversely, the lack of Myf5 but not of MyoD resulted in the loss of miR-1 and miR-206 expression. Taken together, these results demonstrate the varying requirements of distinct MRFs for the induction of microRNA gene expression during skeletal myogenesis.
Eyal Schejter focused on how the WASp-Arp2/3 actin polymerization machinery plays an essential role during the process of myoblast fusion, which underlies the formation of multi-nucleated muscle fibers. Actin polymerization appears to enable myoblast fusion by generating the forces necessary to expand nascent fusion pores. In Drosophila, actin polymerization has been shown to play a critical role during both embryonic and adult myogenesis, and the highly conserved nature of the WASp-Arp2/3 system suggests that a similar mechanism is at work during skeletal muscle formation in vertebrates.
Simon Hughes presented an overview of zebrafish somite development, including the discovery that the external layer of the somitic cell has parallels to amniote dermomyotomes, the generative cells of later muscle growth marked by Pax3/Pax7 genes. Analysis of Pax3 function in zebrafish revealed the lack of a specific type of pigment cells, due to failure of neural crest development. Finally, mef2 genes were shown to control sarcomere assembly.
James Hanken presented a fate map derived from a new transgenic line of Xenopus laevis (clawed frog), capable of evaluating the derivation of the bony adult skull from embryonic neural crest cells. The extent and pattern of neural crest derivation – and overall cranial segmentation — in this species, differs from that seen in other tetrapods. Hence, even basic features of vertebrate cranial development and organization are evolutionarily labile, and susceptible to modification associated with the origins of specialized life histories, developmental modes, and other types of adaptive change.
Elazar Zelzer focused on the mechanism by which muscle contraction regulates joint formation. His lab studies the role of muscle contraction in regulating the cell fate of joint progenitors in several murine models with defective limb musculature, as well as in mice that lack muscle contraction. He demonstrated that muscle contraction is necesery to maintain the genetic program controlling the differentiation sequence of joint progenitor cells. Recent findings in the Zelzer lab point to a mechanical contribution of the musculature in the developmental regulation of skeletal structures.
Véronique Lefebvre spoke about the HMG box transcription factors Sox9, Sox5 and Sox6 in chondrogenesis. She and her group have identified a conserved 359bp sequence 10kb upstream of aggrecan, a proteoglycan essential to cartilage development, and a key marker of chondrocyte differentiation. In the mouse, this sequence directs gene expression in both embryonic and adult cartilage. The chondrogenic trio is required and sufficient to mediate this enhancer activity. Their data facilitate an understanding of the mechanisms whereby the Sox trio directs chondrogenesis and, in particular, the expression of aggrecan, which plays a critical role in cartilage development.
Peleg Hasson (M. Logan’s lab) presented the lab’s findings in a mouse model, and showed that deletion of Tbx4 and Tbx5 in the hindlimb and forelimb, respectively, leads to a novel phenotype affecting all aspects of muscle and tendon patterning, without, however, disrupting other stages of their development. Significantly, the patterning of the skeleton remained unaffected, demonstrating that the two processes are independent of each other. Their results identify the transcription factors Tbx4 and Tbx5 as belonging to a new class of musculoskeletal patterning organizers which act in muscle connective tissue to regulate N-Cadherin.
Randy Johnson described mechanisms that coordinate the patterning of the musculoskeleton. Using conditional gene targeting strategies, he and his team have identified autonomous and non-autonomous roles for an essential transcription factor, lmx1b, that controls musculoskeletal patterning along the dorsal-ventral limb axis. These studies highlight the critical role of lmx1b in dorsal-ventral limb patterning, and suggest an underlying developmental logic for the integration of signals that orchestrate the formation of complex musculoskeletal morphologies.
Gabrielle Kardon is interested in the role played by muscle and connective tissue interactions in the proper development and regeneration of skeletal muscle. She and her colleagues have recently identified a transcription factor, Tcf4, which constitutes the first molecular marker of connective tissue fibroblasts, and developed Tcf4GPCre and Tcf4CreERT2 mouse lines to genetically label and manipulate connective tissue fibroblasts. Using these mouse lines, they confirmed that the Tcf4 lineage gives rise to connective tissue fibroblasts, and also found that Tcf4 function is critical to the development of proper muscle attachment to bone, and the correct muscle fiber types.
Arhat Abzhanov focused on craniofacial development, from early mesenchymal cell differentiation decisions, to the later morphogenesis of cranial skeletal elements. These researchers have direct access to bird species from the Galapagos Islands, enabling them to study the patterning and integration of the jaw, skeleton, and musculature. In order to explain the enormous diversity of vertebrate cranial structures, new methodologies for the elucidation of ontogenetic processes across species were presented.
Rich Schneider focused on the molecular and cellular mechanisms that pattern the musculoskeletal system in the avian jaw. Data were presented demonstrating that the stage-specific and species-specific size and shape of cartilage is inherent in the neural crest-derived chondrocytes. Additionally, neural crest-derived connective tissues pattern the associated musculature.
Eldad Tzahor described the processes underlying head muscle development in chick and mouse embryonic models. He discussed recent studies showing that the development of the head musculature differs profoundly from trunk myogenesis. Consistent with the theme of the meeting, his group has identified extrinsic signaling pathways that regulate, both positively and negatively, the patterning and differentiation of muscle progenitors in the head. Other findings that he presented highlighted the fact that head muscles are developmentally linked to cardiac formation.
Eran Hornstein spoke about the role played by miRNAs during craniofacial development. miRNAs are a newly discovered set of genes. These small RNAs regulate gene expression at the post-transcriptional level. Using loss-of-function techniques in a mouse model, the Hornstein group has demonstrated the involvement of miRNAs in the development of structures such as Meckle's cartilage.
The final scientific session was devoted to integrative discussions. It was led by Gabrielle Kardon, who first presented evolutionary perspectives on the musculoskeletal system.
Overall, participants expressed the need to share reagents and experimental protocols; in particular, mouse genetic Cre lines that could be used to manipulate gene expression in distinct components of the musculoskeletal system. Accordingly, it was suggested that a website be set up for sharing these resources. Together with the many scientific and social ties that have formed between the members of our small community and were strengthened during this meeting, these steps are expected to further advance this field of research.