Cellular transitions during cranial suture establishment in zebrafish.

Cranial sutures separate neighboring skull bones and are sites of bone growth. A key question is how osteogenic activity is controlled to promote bone growth while preventing aberrant bone fusions during skull expansion. Using single-cell transcriptomics, lineage tracing, and mutant analysis in zebrafish, we uncover key developmental transitions regulating bone formation at sutures during skull expansion. In particular, we identify a subpopulation of mesenchyme cells in the mid-suture region that upregulate a suite of genes including BMP antagonists (e.g. grem1a) and pro-angiogenic factors. Lineage tracing with grem1a:nlsEOS reveals that this mid-suture subpopulation is largely non-osteogenic. Moreover, combinatorial mutation of BMP antagonists enriched in this mid-suture subpopulation results in increased BMP signaling in the suture, misregulated bone formation, and abnormal suture morphology. These data reveal establishment of a non-osteogenic mesenchyme population in the mid-suture region that restricts bone formation through local BMP antagonism, thus ensuring proper suture morphology.

Ligament injury in adult zebrafish triggers ECM remodeling and cell dedifferentiation for scar-free regeneration.

After traumatic injury, healing of mammalian ligaments is typically associated with fibrotic scarring as opposed to scar-free regeneration. In contrast, here we show that the ligament supporting the jaw joint of adult zebrafish is capable of rapid and complete scar-free healing. Following surgical transection of the jaw joint ligament, we observe breakdown of ligament tissue adjacent to the cut sites, expansion of mesenchymal tissue within the wound site, and then remodeling of extracellular matrix (ECM) to a normal ligament morphology. Lineage tracing of mature ligamentocytes following transection shows that they dedifferentiate, undergo cell cycle re-entry, and contribute to the regenerated ligament. Single-cell RNA sequencing of the regenerating ligament reveals dynamic expression of ECM genes in neural-crest-derived mesenchymal cells, as well as diverse immune cells expressing the endopeptidase-encoding gene legumain. Analysis of legumain mutant zebrafish shows a requirement for early ECM remodeling and efficient ligament regeneration. Our study establishes a new model of adult scar-free ligament regeneration and highlights roles of immune-mesenchyme cross-talk in ECM remodeling that initiates regeneration.

Nuclear receptor Nr5a2 promotes diverse connective tissue fates in the jaw.

Organ development involves the sustained production of diverse cell types with spatiotemporal precision. In the vertebrate jaw, neural-crest-derived progenitors produce not only skeletal tissues but also later-forming tendons and salivary glands. Here we identify the pluripotency factor Nr5a2 as essential for cell-fate decisions in the jaw. In zebrafish and mice, we observe transient expression of Nr5a2 in a subset of mandibular postmigratory neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells that would normally form tendons generate excess jaw cartilage. In mice, neural-crest-specific Nr5a2 loss results in analogous skeletal and tendon defects in the jaw and middle ear, as well as salivary gland loss. Single-cell profiling shows that Nr5a2, distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene expression that is essential for tendon and gland fates. Thus, repurposing of Nr5a2 promotes connective tissue fates to generate the full repertoire of derivatives required for jaw and middle ear function.

Ligament injury in adult zebrafish triggers ECM remodeling and cell dedifferentiation for scar-free regeneration.

After traumatic injury, healing of mammalian ligaments is typically associated with fibrotic scarring as opposed to scar-free regeneration. In contrast, here we show that the ligament supporting the jaw joint of adult zebrafish is capable of rapid and complete scar-free healing. Following surgical transection of the jaw joint ligament, we observe breakdown of ligament tissue adjacent to the cut sites, expansion of mesenchymal tissue within the wound site, and then remodeling of extracellular matrix (ECM) to a normal ligament morphology. Lineage tracing of mature ligamentocytes following transection shows that they dedifferentiate, undergo cell cycle re-entry, and contribute to the regenerated ligament. Single-cell RNA sequencing of the regenerating ligament reveals dynamic expression of ECM genes in neural-crest-derived mesenchymal cells, as well as diverse immune cells expressing the endopeptidase-encoding gene legumain . Analysis of legumain mutant zebrafish shows a requirement for early ECM remodeling and efficient ligament regeneration. Our study establishes a new model of adult scar-free ligament regeneration and highlights roles of immune-mesenchyme cross-talk in ECM remodeling that initiates regeneration. Highlights: Rapid regeneration of the jaw joint ligament in adult zebrafishDedifferentiation of mature ligamentocytes contributes to regenerationscRNAseq reveals dynamic ECM remodeling and immune activation during regenerationRequirement of Legumain for ECM remodeling and ligament healing.

Lifelong single-cell profiling of cranial neural crest diversification in zebrafish.

The cranial neural crest generates a huge diversity of derivatives, including the bulk of connective and skeletal tissues of the vertebrate head. How neural crest cells acquire such extraordinary lineage potential remains unresolved. By integrating single-cell transcriptome and chromatin accessibility profiles of cranial neural crest-derived cells across the zebrafish lifetime, we observe progressive and region-specific establishment of enhancer accessibility for distinct fates. Neural crest-derived cells rapidly diversify into specialized progenitors, including multipotent skeletal progenitors, stromal cells with a regenerative signature, fibroblasts with a unique metabolic signature linked to skeletal integrity, and gill-specific progenitors generating cell types for respiration. By retrogradely mapping the emergence of lineage-specific chromatin accessibility, we identify a wealth of candidate lineage-priming factors, including a Gata3 regulatory circuit for respiratory cell fates. Rather than multilineage potential being established during cranial neural crest specification, our findings support progressive and region-specific chromatin remodeling underlying acquisition of diverse potential.

The developing mouse coronal suture at single-cell resolution.

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. The coronal suture is most commonly fused in monogenic craniosynostosis, yet the unique aspects of its development remain incompletely understood. To uncover the cellular diversity within the murine embryonic coronal suture, we generated single-cell transcriptomes and performed extensive expression validation. We find distinct pre-osteoblast signatures between the bone fronts and periosteum, a ligament-like population above the suture that persists into adulthood, and a chondrogenic-like population in the dura mater underlying the suture. Lineage tracing reveals an embryonic Six2+ osteoprogenitor population that contributes to the postnatal suture mesenchyme, with these progenitors being preferentially affected in a Twist1+/-; Tcf12+/- mouse model of Saethre-Chotzen Syndrome. This single-cell atlas provides a resource for understanding the development of the coronal suture and the mechanisms for its loss in craniosynostosis.

Development and maintenance of tendons and ligaments.

Tendons and ligaments are fibrous connective tissues vital to the transmission of force and stabilization of the musculoskeletal system. Arising in precise regions of the embryo, tendons and ligaments share many properties and little is known about the molecular differences that differentiate them. Recent studies have revealed heterogeneity and plasticity within tendon and ligament cells, raising questions regarding the developmental mechanisms regulating tendon and ligament identity. Here, we discuss recent findings that contribute to our understanding of the mechanisms that establish and maintain tendon progenitors and their differentiated progeny in the head, trunk and limb. We also review the extent to which these findings are specific to certain anatomical regions and model organisms, and indicate which findings similarly apply to ligaments. Finally, we address current research regarding the cellular lineages that contribute to tendon and ligament repair, and to what extent their regulation is conserved within tendon and ligament development.

A C. elegans Thermosensory Circuit Regulates Longevity through crh-1/CREB-Dependent flp-6 Neuropeptide Signaling.

Sensory perception, including thermosensation, shapes longevity in diverse organisms, but longevity-modulating signals from the sensory neurons are largely obscure. Here we show that CRH-1/CREB activation by CMK-1/CaMKI in the AFD thermosensory neuron is a key mechanism that maintains lifespan at warm temperatures in C. elegans. In response to temperature rise and crh-1 activation, the AFD neurons produce and secrete the FMRFamide neuropeptide FLP-6. Both CRH-1 and FLP-6 are necessary and sufficient for longevity at warm temperatures. Our data suggest that FLP-6 targets the AIY interneurons and engages DAF-9 sterol hormone signaling. Moreover, we show that FLP-6 signaling downregulates ins-7/insulin-like peptide and several insulin pathway genes, whose activity compromises lifespan. Our work illustrates how temperature experience is integrated by the thermosensory circuit to generate neuropeptide signals that remodel insulin and sterol hormone signaling and reveals a neuronal-endocrine circuit driven by thermosensation to promote temperature-specific longevity.

Purge-and-trap ion chromatography for the determination of trace ammonium ion in high-salinity water samples.

It has always been assumed that purge-and-trap (P&T) method is only used for the analysis of volatile organic compounds (VOCs) in aqueous samples. In this paper, a novel P&T preconcentrator has been developed for the determination of trace amounts of ammonium ion in high-salinity water samples by ion chromatography (IC). Method performance is evaluated as a function of concentration of assistant purging material, purging time, and flow rate. Under the optimum P&T conditions with the purified nitrogen gas at flow rate 40 mL/min for 15.0 min at 40 degrees C, the overall collection efficiency is independent of the concentration of ammonium over the range 1.2-5.9 microM. The enrichment factor (EF) of ammonium correlates the ratio of the sample volume to the acceptor solution volume in the trap vessel, providing potentially unlimited increase of the ammonium signal. Our results indicate that environmental samples with low levels of ammonium in matrices with high concentrations of sodium can be easily analyzed and the detection limit down to 75 nM (1.35 ppb) level, corresponding to picomole of ammonia in the injected sample. Calibration graph was constructed with ammonium standards ranging from 0.05 to 6.0 microM and the linearity of the present method was good as suggested by the square of correlation coefficients being better than 0.997. Thus, we have demonstrated that the P&T-IC method allows the routine determination of ammonium ion in seawater samples without cation interferences.