A single cell transcriptional atlas of early synovial joint development

Abstract

42 43 2 SUMMARY 44 45 Synovial joint development begins with the formation of the interzone, a region of condensed 46 mesenchymal cells at the site of the prospective joint. Recently, lineage tracing strategies have 47 55 transcriptional atlas will serve as a resource for the community to uncover transcriptional 56 programs and cell interactions that regulate synovial joint development. 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 100 to laser-capture micro-dissected regions of the interzone (Jenner et al. 2014). While these 101 investigations have yielded new insights into the genetic programs underpinning limb and joint 102 morphogenesis, they provide limited resolution of the expression states for individual cell types 103 due to the heterogenous nature of the samples profiled. With the advent of single cell profiling, it 104 is now possible to detect transient populations of cells, to reconstruct developmental 105 4 transcriptional programs, and to identity new cell populations (Guo et al. 2010; Kumar et al. 106 2017). For example, Feng et al revealed molecular signatures and lineage trajectories of an 107 interzone related Lgr5 + population in the murine E14.5 knee joint that contributes to the 108 formation of cruciate ligaments, synovial membrane, and articular chondrocytes (Feng et al. 109 2019). 110 111 Here, we applied single-cell RNA-sequencing on Gdf5-lineage cells of the murine hindlimb to 112 determine the transcriptional programs of early synovial joint development. In contrast to the 113 recent study of Feng et al 2019, which focused on lineage divergence of a specific Lrg5 + 114interzone population, we sought to characterize formation of the entire IZ and to discover the 115 extent to which heterogeneity in the nascent interzone is resolved into the distinct lineages that 116 are apparent later at cavitation. Therefore, we sequenced Gdf5-lineage cells from the 117 presumptive joint of the hindlimb from E12.5 (prior to frank IZ formation) through E15.5 118 (coinciding with cavitation). We combined computational analytics and in situ hybridization to 119 infer the lineage relationships of joint progenitors and to identify the combinatorial transcriptional 120 programs that mediate the elaboration of the interzone into the major synovial joint lineages. We 121 found that early Gdf5-lineage enriched cells consist of sub-populations with chondrogenic or 122 fibrous-lineage bias. Furthermore, we discovered within the chondrogenic-biased population 123 were two distinct sub-populations that followed similar trajectories to de-differentiate into IZ 124 cells, supporting a model of regionally and temporally complex IZ specification (Shwartz et al. 125 2016). To aid the community in discovering additional transcriptional programs and in inferring 126 cell interactions that contribute to synovial joint development, we have made this data freely and 127 easily accessible with a web application at http://www.cahanlab.org/resources/joint_ontogeny. 128 129 Results 130 131 Gdf5Cre + cells in the hind limb ...

Extracellular matrix deposition precedes muscle-tendon integration during murine forelimb morphogenesis

“…NID2) (Figure S1). Indeed, cartilage-related proteins were significantly elevated (ACAN, COL2A1, COL9A1&3, COL11A1&2, COMP), or exclusively found (COL8A2, COL27A1), in the Pax3 Cre/Cre limb (Bian et al, 2020;Boot-Handford et al, 2003;Ocken et al, 2020), which can be explained by the relative increase of cartilage content in the absence of muscle (Figure 2E).…”

Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage

“…The visualization of the transcriptome landscape at the single-cell level in a heterogeneous musculoskeletal cell population is technically challenging but was previously achieved for the developing knee joint and growth plate cartilage ( 8 , 9 , 10 ). Here, we successfully applied scRNA-Seq analysis to mature cartilage from 1-month-old TW helicase K320E transgenic mice.…”

“…In joint biology, scRNA-Seq was used to study cellular heterogeneity and lineage specification during development. Tissue-specific cell clusters of hind limb development were identified, distinct transcriptome signatures of joint progenitor cells were described ( 8 , 9 ), and transcriptional profiles of the major developmental paths for joint progenitors were determined ( 10 ). scRNA-Seq analysis could also confirm the cluster-specific expression of genes in growth plate cartilage ( 11 ) originally defined by oligoarray-transcriptome profiling ( 12 ).…”

Turning Nature’s own processes into design strategies for living bone implant biomanufacturing: a decade of Developmental Engineering

“…Recent work on the development of limbs in murine and chick models presents trajectories of lineage specification during embryonic limb development. This can serve as a useful resource for DE studies by dissecting subsets of the limb population related to bone development [ 38 , [55] , [56] , [57] ] and identifying central cell-cell communication networks [ [58] , [59] , [60] ]. These can be used in turn to more adequately tune growth factor supplementation of the differentiation media.…”