The cellular landscape of the endochondral bone during the transition to extrauterine life.

The cellular complexity of the endochondral bone underlies its essential and pleiotropic roles during organismal life. While the adult bone has received significant attention, we still lack a deep understanding of the perinatal bone cellulome. Here, we have profiled the full composition of the murine endochondral bone at the single-cell level during the transition from fetal to newborn life and in comparison with the adult tissue, with particular emphasis on the mesenchymal compartment. The perinatal bone contains different fibroblastic clusters with blastema-like characteristics in organizing and supporting skeletogenesis, angiogenesis and hematopoiesis. Our data also suggest dynamic inter- and intra-compartment interactions, as well as a bone marrow milieu that seems prone to anti-inflammation, which we hypothesize is necessary to ensure the proper program of lymphopoiesis and the establishment of central and peripheral tolerance in early life. Our study provides an integrative roadmap for the future design of genetic and cellular functional assays to validate cellular interactions and lineage relationships within the perinatal bone.

Molecular signatures identify immature mesenchymal progenitors in early mouse limb buds that respond differentially to morphogen signaling.

The key molecular interactions governing vertebrate limb bud development are a paradigm for studying the mechanisms controlling progenitor cell proliferation and specification during vertebrate organogenesis. However, little is known about the cellular heterogeneity of the mesenchymal progenitors in early limb buds that ultimately contribute to the chondrogenic condensations prefiguring the skeleton. We combined flow cytometric and transcriptome analyses to identify the molecular signatures of several distinct mesenchymal progenitor cell populations present in early mouse forelimb buds. In particular, jagged 1 (JAG1)-positive cells located in the posterior-distal mesenchyme were identified as the most immature limb bud mesenchymal progenitors (LMPs), which crucially depend on SHH and FGF signaling in culture. The analysis of gremlin 1 (Grem1)-deficient forelimb buds showed that JAG1-expressing LMPs are protected from apoptosis by GREM1-mediated BMP antagonism. At the same stage, the osteo-chondrogenic progenitors (OCPs) located in the core mesenchyme are already actively responding to BMP signaling. This analysis sheds light on the cellular heterogeneity of the early mouse limb bud mesenchyme and on the distinct response of LMPs and OCPs to morphogen signaling.

Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia.

Promiscuous gene expression (PGE) by thymic epithelial cells (TEC) is essential for generating a diverse T cell antigen receptor repertoire tolerant to self-antigens, and thus for avoiding autoimmunity. Nevertheless, the extent and nature of this unusual expression program within TEC populations and single cells are unknown. Using deep transcriptome sequencing of carefully identified mouse TEC subpopulations, we discovered a program of PGE that is common between medullary (m) and cortical TEC, further elaborated in mTEC, and completed in mature mTEC expressing the autoimmune regulator gene (Aire). TEC populations are capable of expressing up to 19,293 protein-coding genes, the highest number of genes known to be expressed in any cell type. Remarkably, in mouse mTEC, Aire expression alone positively regulates 3980 tissue-restricted genes. Notably, the tissue specificities of these genes include known targets of autoimmunity in human AIRE deficiency. Led by the observation that genes induced by Aire expression are generally characterized by a repressive chromatin state in somatic tissues, we found these genes to be strongly associated with H3K27me3 marks in mTEC. Our findings are consistent with AIRE targeting and inducing the promiscuous expression of genes previously epigenetically silenced by Polycomb group proteins. Comparison of the transcriptomes of 174 single mTEC indicates that genes induced by Aire expression are transcribed stochastically at low cell frequency. Furthermore, when present, Aire expression-dependent transcript levels were 16-fold higher, on average, in individual TEC than in the mTEC population.

Epithelial cytoprotection sustains ectopic expression of tissue-restricted antigens in the thymus during murine acute GVHD.

Development of acute graft-versus-host disease (aGVHD) predisposes to chronic GVHD with autoimmune manifestations. A characteristic of experimental aGVHD is the de novo generation of autoreactive T cells. Central tolerance is dependent on the intrathymic expression of tissue-restricted peripheral self-antigens (TRA), which is in mature medullary thymic epithelial cells (mTEC(high)) partly controlled by the autoimmune regulator (Aire). Because TECs are targets of donor T-cell alloimmunity, we tested whether murine aGVHD interfered with the capacity of recipient Aire(+)mTEC(high) to sustain TRA diversity. We report that aGVHD weakens the platform for central tolerance induction because individual TRAs are purged from the total repertoire secondary to a decline in the Aire(+)mTEC(high) cell pool. Peritransplant administration of an epithelial cytoprotective agent, fibroblast growth factor-7, maintained a stable pool of Aire(+)mTEC(high), with an improved TRA transcriptome despite aGVHD. Taken together, our data provide a mechanism for how autoimmunity may develop in the context of antecedent alloimmunity.

MicroRNAs control the maintenance of thymic epithelia and their competence for T lineage commitment and thymocyte selection.

Thymic epithelial cells provide unique cues for the lifelong selection and differentiation of a repertoire of functionally diverse T cells. Rendered microRNA (miRNA) deficient, these stromal cells in the mouse lose their capacity to instruct the commitment of hematopoietic precursors to a T cell fate, to effect thymocyte positive selection, and to achieve promiscuous gene expression required for central tolerance induction. Over time, the microenvironment created by miRNA-deficient thymic epithelia assumes the cellular composition and structure of peripheral lymphoid tissue, where thympoiesis fails to be supported. These findings emphasize a global role for miRNA in the maintenance and function of the thymic epithelial cell scaffold and establish a novel mechanism how these cells control peripheral tissue Ag expression to prompt central immunological tolerance.

The amino terminal end determines the stability and assembling capacity of eukaryotic ribosomal stalk proteins P1 and P2.

The eukaryotic ribosomal proteins P1 and P2 bind to protein P0 through their N-terminal domain to form the essential ribosomal stalk. A mutational analysis points to amino acids at positions 2 and 3 as determinants for the drastic difference of Saccharomyces cerevisiae P1 and P2 half-life, and suggest different degradation mechanisms for each protein type. Moreover, the capacity to form P1/P2 heterodimers is drastically affected by mutations in the P2ß four initial amino acids, while these mutations have no effect on P1ß. Binding of P2ß and, to a lesser extent, P1ß to the ribosome is also seriously affected showing the high relevance of the amino acids in the first turn of the NTD α-helix 1 for the stalk assembly. The negative effect of some mutations on ribosome binding can be reversed by the presence of the second P1/P2 couple in the ribosome, indicating a stabilizing structural influence between the two heterodimers. Unexpectedly, some mutations totally abolish heterodimer formation but allow significant ribosome binding and, therefore, a previous P1 and P2 association seems not to be an absolute requirement for stalk assembly. Homology modeling of the protein complexes suggests that the mutated residues can affect the overall protein conformation.

Ontogenic Identification and Analysis of Mesenchymal Stromal Cell Populations during Mouse Limb and Long Bone Development.

Bone-derived mesenchymal stromal cells (MSCs) differentiate into multiple lineages including chondro- and osteogenic fates and function in establishing the hematopoietic compartment of the bone marrow. Here, we analyze the emergence of different MSC types during mouse limb and long bone development. In particular, PDGFRαposSCA-1pos (PαS) cells and mouse skeletal stem cells (mSSCs) are detected within the PDGFRαposCD51pos (PαCD51) mesenchymal progenitors, which are the most abundant progenitors in early limb buds and developing long bones until birth. Long-bone-derived PαS cells and mSSCs are most prevalent in newborn mice, and molecular analysis shows that they constitute distinct progenitor populations from the earliest stages onward. Differential expression of CD90 and CD73 identifies four PαS subpopulations that display distinct chondro- and osteogenic differentiation potentials. Finally, we show that cartilage constructs generated from CD90pos PαS cells are remodeled into bone organoids encompassing functional endothelial and hematopoietic compartments, which makes these cells suited for bone tissue engineering.

GLI3 constrains digit number by controlling both progenitor proliferation and BMP-dependent exit to chondrogenesis.

Inactivation of Gli3, a key component of Hedgehog signaling in vertebrates, results in formation of additional digits (polydactyly) during limb bud development. The analysis of mouse embryos constitutively lacking Gli3 has revealed the essential GLI3 functions in specifying the anteroposterior (AP) limb axis and digit identities. We conditionally inactivated Gli3 during mouse hand plate development, which uncoupled the resulting preaxial polydactyly from known GLI3 functions in establishing AP and digit identities. Our analysis revealed that GLI3 directly restricts the expression of regulators of the G(1)-S cell-cycle transition such as Cdk6 and constrains S phase entry of digit progenitors in the anterior hand plate. Furthermore, GLI3 promotes the exit of proliferating progenitors toward BMP-dependent chondrogenic differentiation by spatiotemporally restricting and terminating the expression of the BMP antagonist Gremlin1. Thus, Gli3 is a negative regulator of the proliferative expansion of digit progenitors and acts as a gatekeeper for the exit to chondrogenic differentiation.

Clearing the AIRE: on the pathophysiological basis of the autoimmune polyendocrinopathy syndrome type-1.

Autoimmune polyendocrine syndrome type-1 clinically manifests as the triad of hypoparathyroidism, primary adrenocortical insufficiency, and chronic mucocutaneous candidiasis. Mutations in the gene that encodes the autoimmune regulator protein, AIRE, have been identified as the cause of the autoimmune polyendocrine syndrome type-1. The loss of immunologic tolerance to tissue-restricted antigens consequent to an absence of AIRE expression in the thymus results in the thymic export of autoreactive T cells that initiate autoimmunity. In this article, we discuss the role of AIRE in autoimmune polyendocrine syndrome type-1 and identify issues that still need to be addressed to fully understand the molecular pathophysiology of this complex syndrome.

Identification of CMS as a cytosolic adaptor of the human pTalpha chain involved in pre-TCR function.

The T-cell receptor beta (TCRbeta)/pre-TCRalpha (pTalpha) pre-TCR complex (pre-TCR) signals the expansion and differentiation of de-veloping thymocytes. Functional pro-perties of the pre-TCR rely on its unique pTalpha chain, which suggests the participation of specific intracellular adaptors. However, pTalpha-interacting molecules remain unknown. Here, we identified a polyproline-arginine sequence in the human pTalpha cytoplasmic tail that interacted in vitro with SH3 domains of the CIN85/CMS family of adaptors, and mediated the recruitment of multiprotein complexes involving all (CMS, CIN85, and CD2BP3) members. Supporting the physiologic relevance of this interaction, we found that 1 such adaptor, CMS, interacted in vivo with human pTalpha, and its expression was selectively up-regulated during human thymopoiesis in pre-TCR-activated thymocytes. Upon activation, pre-TCR clustering was induced, and CMS and polymerized actin were simultaneously recruited to the pre-TCR activation site. CMS also associated via its C-terminal region to the actin cytoskeleton in the endocytic compartment, where it colocalized with internalized pTalpha in traffic to lysosomal degradation. Notably, deletion of the pTalpha CIN85/CMS-binding motif impaired pre-TCR-mediated Ca(2+) mobilization and NFAT transcriptional activity, and precluded activation induced by overexpression of a CMS-SH3 N-terminal mutant. These results provide the first molecular evidence for a pTalpha intracellular adaptor involved in pre-TCR function.