Osteoclast activity sculpts craniofacial form to permit sensorineural patterning in the zebrafish skull.

Efforts to understand the morphogenesis of complex craniofacial structures have largely focused on the role of chondrocytes and osteoblasts. Along with these bone-creating cells, bone-resorbing osteoclasts are critical in homeostasis of adult skeletal structures, but there is currently limited information on their role in the complex morphogenetic events of craniofacial development. Fundamental aspects of skull formation and general skeletal development are conserved from zebrafish to mammals. Using a cathepsinK reporter, we documented osteoclast location in the developing zebrafish skull over several weeks, from 5.18 mm to 9.6 mm standard length (approximately 15 to 34 days post fertilization). While broad distribution of osteoclasts is consistent across individuals, they are sparse and the exact locations vary among fish and across developmental time points. Interestingly, we observed osteoclasts concentrating at areas associated with neuromasts and their associated nerves, in particular the hyomandibular foramina and around the supraorbital lateral line. These are areas of active remodeling. In contrast, other areas of rapid bone growth, such as the osteogenic fronts of the frontal and parietal bones, show no particular concentration of osteoclasts, suggesting that they play a special role in shaping bone near neuromasts and nerves. In csf1ra mutants lacking functional osteoclasts, the morphology of the cranial bone was disrupted in both areas. The hyomandibular foramen is present in the initial cartilage template, but after the initiation of ossification, the diameter of the canal is significantly smaller in the absence of osteoclasts. The diameter of the supraorbital lateral line canals was also reduced in the mutants, as was the number of pores associated with neuromasts, which allow for the passage of associated nerves through the bone. Our findings define important and previously unappreciated roles for osteoclast activity in shaping craniofacial skeletal structures with a particular role in bone modeling around peripheral cranial nerves, providing a scaffold for wiring the sensioneural system during craniofacial development. This has important implications for the formation of the evolutionarily diverse lateral line system, as well understanding the mechanism of neurologic sequelae of congenital osteoclast dysfunction in human craniofacial development.

Tipping the Scales With Zebrafish to Understand Adaptive Tumor Immunity.

The future of improved immunotherapy against cancer depends on an in-depth understanding of the dynamic interactions between the immune system and tumors. Over the past two decades, the zebrafish has served as a valuable model system to provide fresh insights into both the development of the immune system and the etiologies of many different cancers. This well-established foundation of knowledge combined with the imaging and genetic capacities of the zebrafish provides a new frontier in cancer immunology research. In this review, we provide an overview of the development of the zebrafish immune system along with a side-by-side comparison of its human counterpart. We then introduce components of the adaptive immune system with a focus on their roles in the tumor microenvironment (TME) of teleosts. In addition, we summarize zebrafish models developed for the study of cancer and adaptive immunity along with other available tools and technology afforded by this experimental system. Finally, we discuss some recent research conducted using the zebrafish to investigate adaptive immune cell-tumor interactions. Without a doubt, the zebrafish will arise as one of the driving forces to help expand the knowledge of tumor immunity and facilitate the development of improved anti-cancer immunotherapy in the foreseeable future.

Cx3cr1-deficient microglia exhibit a premature aging transcriptome.

CX3CR1, one of the highest expressed genes in microglia in mice and humans, is implicated in numerous microglial functions. However, the molecular mechanisms underlying Cx3cr1 signaling are not well understood. Here, we analyzed transcriptomes of Cx3cr1-deficient microglia under varying conditions by RNA-sequencing (RNA-seq). In 2-mo-old mice, Cx3cr1 deletion resulted in the down-regulation of a subset of immune-related genes, without substantial epigenetic changes in markers of active chromatin. Surprisingly, Cx3cr1-deficient microglia from young mice exhibited a transcriptome consistent with that of aged Cx3cr1-sufficient animals, suggesting a premature aging transcriptomic signature. Immunohistochemical analysis of microglia in young and aged mice revealed that loss of Cx3cr1 modulates microglial morphology in a comparable fashion. Our results suggest that CX3CR1 may regulate microglial function in part by modulating the expression levels of a subset of inflammatory genes during chronological aging, making Cx3cr1-deficient mice useful for studying aged microglia.

Initiation and early growth of the skull vault in zebrafish.

The zebrafish offers powerful advantages as a model system for examining the growth of the skull vault and the formation of cranial sutures. The zebrafish is well suited for large-scale genetic screens, available in large numbers, and continual advances in genetic engineering facilitate precise modeling of human genetic disorders. Most importantly, zebrafish are continuously accessible for imaging during critical periods of skull formation when both mouse and chick are physically inaccessible. To establish a foundation of information on the dynamics of skull formation, we performed a longitudinal study based on confocal microscopy of individual live transgenic zebrafish. Discrete events occur at stereotyped stages in overall growth, with little variation in timing among individuals. The frontal and parietal bones initiate as small clusters of cells closely associated with cartilage around the perimeter of the skull, prior to metamorphosis and the transition to juvenile fish. Over a period of ~30 days, the frontal and parietal bones grow towards the apex of the skull and meet to begin suture formation. To aid in visualization, we have generated interactive three-dimensional models based on the imaging data, with annotated cartilage and bone elements. We propose a framework to conceptualize development of bones of the skull vault in three phases: initiation in close association with cartilage; rapid planar growth towards the apex of the skull; and finally overlapping to form sutures. Our data provide an important framework for comparing the stages and timing of skull development across model organisms, and also a baseline for the examination of zebrafish mutants affecting skull development. To facilitate these comparative analyses, the raw imaging data and the models are available as an online atlas through the FaceBase consortium (facebase.org).