Cellular taxonomy of Hic1+ mesenchymal progenitor derivatives in the limb: from embryo to adult.

Tissue development and regeneration rely on the cooperation of multiple mesenchymal progenitor (MP) subpopulations. We recently identified Hic1 as a marker of quiescent MPs in multiple adult tissues. Here, we describe the embryonic origin of appendicular Hic1+ MPs and demonstrate that they arise in the hypaxial somite, and migrate into the developing limb at embryonic day 11.5, well after limb bud initiation. Time-resolved single-cell-omics analyses coupled with lineage tracing reveal that Hic1+ cells generate a unique MP hierarchy, that includes both recently identified adult universal fibroblast populations (Dpt+, Pi16+ and Dpt+ Col15a1+) and more specialised mesenchymal derivatives such as, peri and endoneurial cells, pericytes, bone marrow stromal cells, myotenocytes, tenocytes, fascia-resident fibroblasts, with limited contributions to chondrocytes and osteocytes within the skeletal elements. MPs endure within these compartments, continue to express Hic1 and represent a critical reservoir to support post-natal growth and regeneration.

Regulation of Active DNA Demethylation through RAR-Mediated Recruitment of a TET/TDG Complex.

Retinoic acid (RA) plays important roles in development, growth, and homeostasis through regulation of the nuclear receptors for RA (RARs). Herein, we identify Hypermethylated in Cancer 1 (Hic1) as an RA-inducible gene. HIC1 encodes a tumor suppressor, which is often silenced by promoter hypermethylation in cancer. Treatment of cells with an RAR agonist causes a rapid recruitment of an RAR/RXR complex consisting of TDG, the lysine acetyltransferase CBP, and TET 1/2 to the Hic1 promoter. Complex binding coincides with a transient accumulation of 5fC/5caC and concomitant upregulation of Hic1 expression, both of which are TDG dependent. Furthermore, conditional deletion of Tdg in vivo is associated with Hic1 silencing and DNA hypermethylation of the Hic1 promoter. These findings suggest that the catalytic and scaffolding activities of TDG are essential for RA-dependent gene expression and provide important insights into the mechanisms underlying targeting of TET-TDG complexes.

Geminin is required for the maintenance of pluripotency.

Pluripotency requires the expression of the three core transcriptions factors Oct4, Sox2 and Nanog, as well as further, complementary proteins. The geminin protein is part of this network, and was shown to play a role in the regulation of DNA replication, the control of the cell cycle, and the acquisition of neural fate. It is highly expressed in the early embryo, in particular the epiblast and the early neural ectoderm, and also in pluripotent embryonic stem cells. The genetic inactivation of geminin resulted in lethality after the first few cell divisions, and thus prohibited the outgrowth of pluripotent cells. We established embryonic stem cells allowing the deletion of the geminin gene by induction of of Cre-recombinase with tamoxifen. Here, we show that geminin deficiency quickly leads to a loss of pluripotency, and to differentiation into the mesendodermal direction with high Oct4/low Sox2 levels. Simultaneous loss of geminin and induction of the neural lineage resulted in immediate apoptosis. These results suggested that in early development geminin functions via the co-expressed Sox2 gene. We found that the stem cell enhancer SRR2 of Sox2 is occupied by the activating esBAF complex in the presence of geminin, but becomes epigenetically repressed in its absence by the Polycomb repressive complex PRC2. The importance of geminin for Sox2 expression also explains the absolute requirement for geminin during the induction of pluripotency by OSKM viruses. In summary, geminin is required for Sox2 expression, and thus for the maintenance of totipotency, pluripotency and the early neural lineage.

Nidogens-Extracellular matrix linker molecules.

Nidogens/entactins are a family of highly conserved, sulfated glycoproteins. Biochemical studies have implicated them as having a major structural role in the basement membrane. However despite being ubiquitous components of this specialized extracellular matrix and having a wide spectrum of binding partners, genetic analysis has shown that they are not required for the overall architecture of the basement membrane. Rather in development they play an important role in its stabilization especially in tissues undergoing rapid growth or turnover. Nidogen breakdown has been implicated as a key event in the basement membrane degradation occurring in mammary gland involution. A number of studies, most compellingly those in C. elegans, demonstrated that nidogens may have other nonstructural roles and be involved in axonal pathfinding and synaptic transmission.

Binding of netrin-4 to laminin short arms regulates basement membrane assembly.

Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks. Here we demonstrate that netrin-4 is a component of basement membranes and is integrated into the laminin polymer via interactions with the laminin gamma1 andgamma3 short arms. The binding is mediated through the laminin N-terminal domain of netrin-4. In contrast to netrin-4, other members of the netrin family do not bind to these laminin short arms. Moreover, a truncated form of netrin-4 completely inhibits laminin-111 self-assembly in vitro, and full-length netrin-4 can partially disrupt laminin self-interactions. When added to explant cultures, netrin-4 retards salivary gland branching morphogenesis.

Loss of nidogen-1 and -2 results in syndactyly and changes in limb development.

Nidogens are two ubiquitous basement membrane proteins produced mainly by mesenchymal cells. Nidogen-mediated interactions, in particular with laminin, collagen IV, and perlecan have been considered important in the formation and maintenance of the basement membrane. However, whereas mice lacking both nidogen isoforms or carrying mutations in the high affinity nidogen-binding site upon the laminin gamma1 chain have specific basement membrane defects in certain organs, particularly in the lung, characterization of these mice has also shown that basement membrane formation per se does not need nidogens or the laminin-nidogen interaction. Limb development requires the complex interplay of numerous growth factors whose expression is dependent upon the apical ectodermal ridge. Here, we show that lack of nidogen-1 and -2 results in a specific and time-limited failure in the ectodermal basement membrane of the limb bud. The absence of this basement membrane leads to aberrant apical ectodermal ridge formation. It also causes altered distribution of growth factors, such as fibroblast growth factors and leads to a fully penetrant soft tissue syndactyly caused by the dysregulation of interdigital apoptosis. Further, in certain animals more severe changes in bone formation occur, providing evidence for the interplay between growth factors and the extracellular matrix.