Look who's TORking: mTOR-mediated integration of cell status and external signals during limb development and endochondral bone growth.

The balance of cell proliferation and size is key for the control of organ development and repair. Moreover, this balance has to be coordinated within tissues and between tissues to achieve robustness in the organ's pattern and size. The tetrapod limb has been used to study these topics during development and repair, and several conserved pathways have emerged. Among them, mechanistic target of rapamycin (mTOR) signaling, despite being active in several cell types and developmental stages, is one of the least understood in limb development, perhaps because of its multiple potential roles and interactions with other pathways. In the body of this review, we have collated and integrated what is known about the role of mTOR signaling in three aspects of tetrapod limb development: 1) limb outgrowth; 2) chondrocyte differentiation after mesenchymal condensation and 3) endochondral ossification-driven longitudinal bone growth. We conclude that, given its ability to interact with the most common signaling pathways, its presence in multiple cell types, and its ability to influence cell proliferation, size and differentiation, the mTOR pathway is a critical integrator of external stimuli and internal status, coordinating developmental transitions as complex as those taking place during limb development. This suggests that the study of the signaling pathways and transcription factors involved in limb patterning, morphogenesis and growth could benefit from probing the interaction of these pathways with mTOR components.

The Nestin neural enhancer is essential for normal levels of endogenous Nestin in neuroprogenitors but is not required for embryo development.

Enhancers are vitally important during embryonic development to control the spatial and temporal expression of genes. Recently, large scale genome projects have identified a vast number of putative developmental regulatory elements. However, the proportion of these that have been functionally assessed is relatively low. While enhancers have traditionally been studied using reporter assays, this approach does not characterise their contribution to endogenous gene expression. We have studied the murine Nestin (Nes) intron 2 enhancer, which is widely used to direct exogenous gene expression within neural progenitor cells in cultured cells and in vivo. We generated CRISPR deletions of the enhancer region in mice and assessed their impact on Nes expression during embryonic development. Loss of the Nes neural enhancer significantly reduced Nes expression in the developing CNS by as much as 82%. By assessing NES protein localization, we also show that this enhancer region contains repressor element(s) that inhibit Nes expression within the vasculature. Previous reports have stated that Nes is an essential gene, and its loss causes embryonic lethality. We also generated 2 independent Nes null lines and show that both develop without any obvious phenotypic effects. Finally, through crossing of null and enhancer deletion mice we provide evidence of trans-chromosomal interaction of the Nes enhancer and promoter.

A New Pipeline to Automatically Segment and Semi-Automatically Measure Bone Length on 3D Models Obtained by Computed Tomography.

The characterization of developmental phenotypes often relies on the accurate linear measurement of structures that are small and require laborious preparation. This is tedious and prone to errors, especially when repeated for the multiple replicates that are required for statistical analysis, or when multiple distinct structures have to be analyzed. To address this issue, we have developed a pipeline for characterization of long-bone length using X-ray microtomography (XMT) scans. The pipeline involves semi-automated algorithms for automatic thresholding and fast interactive isolation and 3D-model generation of the main limb bones, using either the open-source ImageJ plugin BoneJ or the commercial Mimics Innovation Suite package. The tests showed the appropriate combination of scanning conditions and analysis parameters yields fast and comparable length results, highly correlated with the measurements obtained via ex vivo skeletal preparations. Moreover, since XMT is not destructive, the samples can be used afterward for histology or other applications. Our new pipelines will help developmental biologists and evolutionary researchers to achieve fast, reproducible and non-destructive length measurement of bone samples from multiple animal species.

CMTM8 Is a Suppressor of Human Mesenchymal Stem Cell Osteogenic Differentiation and Promoter of Proliferation Via EGFR Signaling.

Multipotent bone marrow-derived mesenchymal stem/stromal cells (BMSCs) exhibit a finite life span after ex vivo expansion leading to cellular senescence. Many factors can contribute to this. Recently, our group has identified for the first time expression of the chemokine-like factor superfamily 8 (CMTM8) gene in cultured human BMSCs. In this study, we examine the role of CMTM8 in BMSC proliferation, migration, and differentiation. Functional studies using siRNA-mediated knockdown of CMTM8 in human BMSCs resulted in decreased capacity to undergo proliferation and migration and an increased capacity for osteogenic differentiation in vitro. Furthermore, reduced CMTM8 levels led to a decrease in the epidermal growth factor receptor (EGFR) signaling pathway during BMSC proliferation and migration, respectively. Supportive studies using retroviral mediated enforced expression of CMTM8 in BMSC resulted in an increased capacity for proliferation and migration but a decreased osteogenic differentiation potential. Collectively, these data suggest that CMTM8 promotes BMSC proliferation and BMSC migration through the EGFR/ERK1/2 pathway. This study provides insight into novel regulatory mechanisms of human BMSC growth and cell fate determination.