Notch signaling enhances bone regeneration in the zebrafish mandible.

Loss or damage to the mandible caused by trauma, treatment of oral malignancies, and other diseases is treated using bone-grafting techniques that suffer from numerous shortcomings and contraindications. Zebrafish naturally heal large injuries to mandibular bone, offering an opportunity to understand how to boost intrinsic healing potential. Using a novel her6:mCherry Notch reporter, we show that canonical Notch signaling is induced during the initial stages of cartilage callus formation in both mesenchymal cells and chondrocytes following surgical mandibulectomy. We also show that modulation of Notch signaling during the initial post-operative period results in lasting changes to regenerate bone quantity one month later. Pharmacological inhibition of Notch signaling reduces the size of the cartilage callus and delays its conversion into bone, resulting in non-union. Conversely, conditional transgenic activation of Notch signaling accelerates conversion of the cartilage callus into bone, improving bone healing. Given the conserved functions of this pathway in bone repair across vertebrates, we propose that targeted activation of Notch signaling during the early phases of bone healing in mammals may both augment the size of the initial callus and boost its ossification into reparative bone.

Programmed conversion of hypertrophic chondrocytes into osteoblasts and marrow adipocytes within zebrafish bones.

Much of the vertebrate skeleton develops from cartilage templates that are progressively remodeled into bone. Lineage tracing studies in mouse suggest that chondrocytes within these templates persist and become osteoblasts, yet the underlying mechanisms of this process and whether chondrocytes can generate other derivatives remain unclear. We find that zebrafish cartilages undergo extensive remodeling and vascularization during juvenile stages to generate fat-filled bones. Growth plate chondrocytes marked by sox10 and col2a1a contribute to osteoblasts, marrow adipocytes, and mesenchymal cells within adult bones. At the edge of the hypertrophic zone, chondrocytes re-enter the cell cycle and express leptin receptor (lepr), suggesting conversion into progenitors. Further, mutation of matrix metalloproteinase 9 (mmp9) results in delayed growth plate remodeling and fewer marrow adipocytes. Our data support Mmp9-dependent growth plate remodeling and conversion of chondrocytes into osteoblasts and marrow adipocytes as conserved features of bony vertebrates.

Screen for Slit/Robo signaling in trunk neural cells reveals new players.

Slits ligands and their Robo receptors are involved in quite disparate cell signaling pathways that include axon guidance, cell proliferation, cell motility and angiogenesis. Neural crest cells emerge by delamination from neural cells in the dorsal neural tube, and give rise to various components of the peripheral nervous system in vertebrates. It is well established that these cells change from a non-migratory to a highly migratory state allowing them to reach distant regions before they differentiate. However, but the mechanism controlling this delamination and subsequent migration are still not fully understood. The repulsive Slit ligand family members, have been classified also as true tumor suppressor molecules. The present study explored in further detail what possible Slit/Robo signals are at play in the trunk neural cells and neural crest cells by carrying out a microarray after Slit2 gain of function in trunk neural tubes. We found that in addition to molecules known to be downstream of Slit/Robo signaling, there were a large set of molecules known to be important in maintaining cells in non-motile, epithelia phenotype. Furthermore, we found new molecules previously not associated with Slit/Robo signaling: cell proliferation markers, Ankyrins and RAB intracellular transporters. Our findings suggest that neural crest cells use and array of different Slit/Robo pathways during their transformation from non-motile to highly motile cells.

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.

Chicken trunk neural crest migration visualized with HNK1.

The development of the nervous system involves cells remaining within the neural tube (CNS) and a group of cells that delaminate from the dorsal neural tube and migrate extensively throughout the developing embryo called neural crest cells (NCC). These cells are a mesenchymal highly migratory group of cells that give rise to a wide variety of cell derivatives: melanocytes, sensory neurons, bone, Schwann cells, etc. But not all NCC can give rise to all derivatives, they have fate restrictions based on their axial level of origin: cranial, vagal, trunk and sacral. Our aim was to provide a thorough presentation on how does trunk neural crest cell migration looks in the chicken embryo, in wholemount and in sections using the unique chicken marker HNK1. The description presented here makes a good guideline for those interested in viewing trunk NCC migration patterns. We show how before HH14 there are few trunk NCC delaminating and migrating, but between HH15 through HH19 trunk NCC delaminate in large numbers. Melanocytes precursors begin to enter the dorsolateral pathway by HH17. We found that by HH20 HNK1 is not a valid good marker for NCC and that HNK1 is a better marker than Sox10 when looking at neural crest cells morphology and migration details.

Slit molecules prevent entrance of trunk neural crest cells in developing gut.

Neural crest cells emerge from the dorsal neural tube early in development and give rise to sensory and sympathetic ganglia, adrenal cells, teeth, melanocytes and especially enteric nervous system. Several inhibitory molecules have been shown to play important roles in neural crest migration, among them are the chemorepulsive Slit1-3. It was known that Slits chemorepellants are expressed at the entry to the gut, and thus could play a role in the differential ability of vagal but not trunk neural crest cells to invade the gut and form enteric ganglia. Especially since trunk neural crest cells express Robo receptor while vagal do not. Thus, although we know that Robo mediates migration along the dorsal pathway in neural crest cells, we do not know if it is responsible in preventing their entry into the gut. The goal of this study was to further corroborate a role for Slit molecules in keeping trunk neural crest cells away from the gut. We observed that when we silenced Robo receptor in trunk neural crest, the sympathoadrenal (somites 18-24) were capable of invading gut mesenchyme in larger proportion than more rostral counterparts. The more rostral trunk neural crest tended not to migrate beyond the ventral aorta, suggesting that there are other repulsive molecules keeping them away from the gut. Interestingly, we also found that when we silenced Robo in sacral neural crest they did not wait for the arrival of vagal crest but entered the gut and migrated rostrally, suggesting that Slit molecules are the ones responsible for keeping them waiting at the hindgut mesenchyme. These combined results confirm that Slit molecules are responsible for keeping the timeliness of colonization of the gut by neural crest cells.

Slits affect the timely migration of neural crest cells via Robo receptor.

BACKGROUND: Neural crest cells emerge by delamination from the dorsal neural tube and give rise to various components of the peripheral nervous system in vertebrate embryos. These cells change from non-motile into highly motile cells migrating to distant areas before further differentiation. Mechanisms controlling delamination and subsequent migration of neural crest cells are not fully understood. Slit2, a chemorepellant for axonal guidance that repels and stimulates motility of trunk neural crest cells away from the gut has recently been suggested to be a tumor suppressor molecule. The goal of this study was to further investigate the role of Slit2 in trunk neural crest cell migration by constitutive expression in neural crest cells. RESULTS: We found that Slit gain-of-function significantly impaired neural crest cell migration while Slit loss-of-function favored migration. In addition, we observed that the distribution of key cytoskeletal markers was disrupted in both gain and loss of function instances. CONCLUSIONS: These findings suggest that Slit molecules might be involved in the processes that allow neural crest cells to begin migrating and transitioning to a mesenchymal type.