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A major cause of human deafness and vestibular dysfunction is permanent loss of the mechanosensory hair cells of the inner ear. In non-mammalian vertebrates such as zebrafish, regeneration of missing hair cells can occur throughout life. While a comparative approach has the potential to reveal the basis of such differential regenerative ability, the degree to which the inner ears of fish and mammals share common hair cells and supporting cell types remains unresolved. Here, we perform single-cell RNA sequencing of the zebrafish inner ear at embryonic through adult stages to catalog the diversity of hair cells and non-sensory supporting cells. We identify a putative progenitor population for hair cells and supporting cells, as well as distinct hair and supporting cell types in the maculae versus cristae. The hair cell and supporting cell types differ from those described for the lateral line system, a distributed mechanosensory organ in zebrafish in which most studies of hair cell regeneration have been conducted. In the maculae, we identify two subtypes of hair cells that share gene expression with mammalian striolar or extrastriolar hair cells. In situ hybridization reveals that these hair cell subtypes occupy distinct spatial domains within the three macular organs, the utricle, saccule, and lagena, consistent with the reported distinct electrophysiological properties of hair cells within these domains. These findings suggest that primitive specialization of spatially distinct striolar and extrastriolar hair cells likely arose in the last common ancestor of fish and mammals. The similarities of inner ear cell type composition between fish and mammals validate zebrafish as a relevant model for understanding inner ear-specific hair cell function and regeneration.

Whereas no known living vertebrate possesses gills derived from the jaw-forming mandibular arch, it has been proposed that the jaw arose through modifications of an ancestral mandibular gill. Here, we show that the zebrafish pseudobranch, which regulates blood pressure in the eye, develops from mandibular arch mesenchyme and first pouch epithelia and shares gene expression, enhancer utilization, and developmental gata3 dependence with the gills. Combined with work in chondrichthyans, our findings in a teleost fish point to the presence of a mandibular pseudobranch with serial homology to gills in the last common ancestor of jawed vertebrates, consistent with a gill origin of vertebrate jaws.

The specification of cartilage requires Sox9, a transcription factor with broad roles for organogenesis outside the skeletal system. How Sox9 and other factors gain access to cartilage-specific cis-regulatory regions during skeletal development was unknown. By analyzing chromatin accessibility during the differentiation of neural crest cells into chondrocytes of the zebrafish head, we find that cartilage-associated chromatin accessibility is dynamically established. Cartilage-associated regions that become accessible after neural crest migration are co-enriched for Sox9 and Fox transcription factor binding motifs. In zebrafish lacking Foxc1 paralogs, we find a global decrease in chromatin accessibility in chondrocytes, consistent with a later loss of dorsal facial cartilages. Zebrafish transgenesis assays confirm that many of these Foxc1-dependent elements function as enhancers with region- and stage-specific activity in facial cartilages. These results show that Foxc1 promotes chondrogenesis in the face by establishing chromatin accessibility at a number of cartilage-associated gene enhancers.

The vertebrate skull varies widely in shape, accommodating diverse strategies of feeding and predation. The braincase is composed of several flat bones that meet at flexible joints called sutures. Nearly all vertebrates have a prominent ‘coronal’ suture that separates the front and back of the skull. This suture can develop entirely within mesoderm-derived tissue, neural crest-derived tissue, or at the boundary of the two. Recent paleontological findings and genetic insights in non-mammalian model organisms serve to revise fundamental knowledge on the development and evolution of this suture. Growing evidence supports a decoupling of the germ layer origins of the mesenchyme that forms the calvarial bones from inductive signaling that establishes discrete bone centers. Changes in these relationships facilitate skull evolution and may create susceptibility to disease. These concepts provide a general framework for approaching issues of homology in cases where germ layer origins have shifted during evolution.

Most bones in mammals display a limited capacity for natural large-scale repair. The ribs are a notable exception, yet the source of their remarkable regenerative ability remains unknown. Here, we identify a Sox9-expressing periosteal subpopulation that orchestrates large-scale regeneration of murine rib bones. Deletion of the obligate Hedgehog co-receptor, Smoothened, in Sox9-expressing cells prior to injury results in a near-complete loss of callus formation and rib bone regeneration. In contrast to its role in development, Hedgehog signaling is dispensable for the proliferative expansion of callus cells in response to injury. Instead, Sox9-positive lineage cells require Hh signaling to stimulate neighboring cells to differentiate via an unknown signal into a skeletal cell type with dual chondrocyte/osteoblast properties. This type of callus cell may be critical for bridging large bone injuries. Thus despite contributing to only a subset of callus cells, Sox9-positive progenitors play a major role in orchestrating large-scale bone regeneration.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

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.

Cranial sutures separate the skull bones and house stem cells for bone growth and repair. In Saethre-Chotzen syndrome, mutations in TCF12 or TWIST1 ablate a specific suture, the coronal. This suture forms at a neural-crest/mesoderm interface in mammals and a mesoderm/mesoderm interface in zebrafish. Despite this difference, we show that combinatorial loss of TCF12 and TWIST1 homologs in zebrafish also results in specific loss of the coronal suture. Sequential bone staining reveals an initial, directional acceleration of bone production in the mutant skull, with subsequent localized stalling of bone growth prefiguring coronal suture loss. Mouse genetics further reveal requirements for Twist1 and Tcf12 in both the frontal and parietal bones for suture patency, and to maintain putative progenitors in the coronal region. These findings reveal conservation of coronal suture formation despite evolutionary shifts in embryonic origins, and suggest that the coronal suture might be especially susceptible to imbalances in progenitor maintenance and osteoblast differentiation.

Synovial joints are the lubricated connections between the bones of our body that are commonly affected in arthritis. It is assumed that synovial joints first evolved as vertebrates came to land, with ray-finned fishes lacking lubricated joints. Here, we examine the expression and function of a critical lubricating protein of mammalian synovial joints, Prg4/Lubricin, in diverse ray-finned fishes. We find that Prg4 homologs are specifically enriched at the jaw and pectoral fin joints of zebrafish, stickleback, and gar, with genetic deletion of the zebrafish prg4b gene resulting in the same age-related degeneration of joints as seen in lubricin-deficient mice and humans. Our data support lubricated synovial joints evolving much earlier than currently accepted, at least in the common ancestor of all bony vertebrates. Establishment of the first arthritis model in the highly regenerative zebrafish will offer unique opportunities to understand the aetiology and possible treatment of synovial joint disease.