Response to Maarten Van Dyck’s commentary

In response to Maarten Van Dyck’s commentary, I present a translation of Vailati’s original paper with a short introductory note.     

Synthesis of Fine Chemicals by the Conjugate Addition of Nitroalkanes to Electrophilic Alkenes

It is well known the ability of primary and secondary nitroalkanes to generate carbanions （under basic conditions） strongly stabilized by the electron-withdrawing effect of the nitro group. Thus, the main use of nitroalkanes is devoted to the generation of new carbon-carbon bonds through two principal approaches （Scheme 1） ： （i） reaction with carbonyl derivatives （nitroaldol-Henry-reaction）, and （ii） Michael addition to electron poor alkenes.     

Chromatin remodelling and epigenetic features of germ cells

Germ cells have the unique capacity to start a new life upon fertilization. They are generated during a sex-specific differentiation programme called gametogenesis. Maturation of germ cells is characterized by an impressive degree of cellular restructuring and gene regulation that involves remarkable genomic reorganization. These events are finely tuned, but are also susceptible to the introduction of various types of error. Because stable genetic transmission to future generations is essential for life, understanding the control of these processes has far-reaching implications for human health and reproduction.     

Gli3 and Plzf cooperate in proximal limb patterning at early stages of limb development

The vertebrate limb initially develops as a bud of mesenchymal cells that subsequently aggregate in a proximal to distal (P-D) sequence to give rise to cartilage condensations that prefigure all limb skeletal components. Of the three cardinal limb axes, the mechanisms that lead to establishment and patterning of skeletal elements along the P-D axis are the least understood. Here we identify a genetic interaction between Gli3 (GLI-Kruppel family member 3) and Plzf (promyelocytic leukaemia zinc finger, also known as Zbtb16 and Zfp145), which is required specifically at very early stages of limb development for all proximal cartilage condensations in the hindlimb (femur, tibia, fibula). Notably, distal condensations comprising the foot are relatively unperturbed in Gli3(-/-);Plzf(-/-) mouse embryos. We demonstrate that the cooperative activity of Gli3 and Plzf establishes the correct temporal and spatial distribution of chondrocyte progenitors in the proximal limb-bud independently of known P-D patterning markers and overall limb-bud size. Moreover, the limb defects in Gli3(-/-);Plzf(-/-) embryos correlate with the transient death of a specific subset of proximal mesenchymal cells that express bone morphogenetic protein receptor, type 1B (Bmpr1b) at the onset of limb development. These findings suggest that the development of proximal and distal skeletal elements is distinctly regulated early during limb-bud formation. The initial division of the vertebrate limb into two distinct molecular domains is consistent with fossil evidence indicating that the upper and lower extremities of the limb have different evolutionary origins.     

Generation of functional multipotent adult stem cells from GPR125+ germline progenitors

Adult mammalian testis is a source of pluripotent stem cells. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125-beta-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125-LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125-LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.