A single-cell transcriptomic atlas of the developing chicken limb.

BACKGROUND: Through precise implementation of distinct cell type specification programs, differentially regulated in both space and time, complex patterns emerge during organogenesis. Thanks to its easy experimental accessibility, the developing chicken limb has long served as a paradigm to study vertebrate pattern formation. Through decades' worth of research, we now have a firm grasp on the molecular mechanisms driving limb formation at the tissue-level. However, to elucidate the dynamic interplay between transcriptional cell type specification programs and pattern formation at its relevant cellular scale, we lack appropriately resolved molecular data at the genome-wide level. Here, making use of droplet-based single-cell RNA-sequencing, we catalogue the developmental emergence of distinct tissue types and their transcriptome dynamics in the distal chicken limb, the so-called autopod, at cellular resolution. RESULTS: Using single-cell RNA-sequencing technology, we sequenced a total of 17,628 cells coming from three key developmental stages of chicken autopod patterning. Overall, we identified 23 cell populations with distinct transcriptional profiles. Amongst them were small, albeit essential populations like the apical ectodermal ridge, demonstrating the ability to detect even rare cell types. Moreover, we uncovered the existence of molecularly distinct sub-populations within previously defined compartments of the developing limb, some of which have important signaling functions during autopod pattern formation. Finally, we inferred gene co-expression modules that coincide with distinct tissue types across developmental time, and used them to track patterning-relevant cell populations of the forming digits. CONCLUSIONS: We provide a comprehensive functional genomics resource to study the molecular effectors of chicken limb patterning at cellular resolution. Our single-cell transcriptomic atlas captures all major cell populations of the developing autopod, and highlights the transcriptional complexity in many of its components. Finally, integrating our data-set with other single-cell transcriptomics resources will enable researchers to assess molecular similarities in orthologous cell types across the major tetrapod clades, and provide an extensive candidate gene list to functionally test cell-type-specific drivers of limb morphological diversification.

Single-cell RNA sequencing analysis of the temporomandibular joint condyle in 3 and 4-month-old human embryos.

BACKGROUND: The temporomandibular joint (TMJ) is a complex joint consisting of the condyle, the temporal articular surface, and the articular disc. Functions such as mastication, swallowing and articulation are accomplished by the movements of the TMJ. To date, the TMJ has been studied more extensively, but the types of TMJ cells, their differentiation, and their interrelationship during growth and development are still unclear and the study of the TMJ is limited. The aim of this study was to establish a molecular cellular atlas of the human embryonic temporomandibular joint condyle (TMJC) by single-cell RNA sequencing, which will contribute to understanding and solving clinical problems. RESULTS: Human embryos at 3 and 4 months of age are an important stage of TMJC development. We performed a comprehensive transcriptome analysis of TMJC tissue from human embryos at 3 and 4 months of age using single-cell RNA sequencing. A total of 16,624 cells were captured and the gene expression profiles of 15 cell clusters in human embryonic TMJC were determined, including 14 known cell types and one previously unknown cell type, "transition state cells (TSCs)". Immunofluorescence assays confirmed that TSCs are not the same cell cluster as mesenchymal stem cells (MSCs). Pseudotime trajectory and RNA velocity analysis revealed that MSCs transformed into TSCs, which further differentiated into osteoblasts, hypertrophic chondrocytes and tenocytes. In addition, chondrocytes (CYTL1high + THBS1high) from secondary cartilage were detected only in 4-month-old human embryonic TMJC. CONCLUSIONS: Our study provides an atlas of differentiation stages of human embryonic TMJC tissue cells, which will contribute to an in-depth understanding of the pathophysiology of the TMJC tissue repair process and ultimately help to solve clinical problems.