Identification of Molecular Mechanisms Mediating TWIST-1 Regulation of Mesenchymal Stem Cell Proliferation and Differentiation

Bone marrow-derived mesenchymal stem/ stromal cells (BMSC) are self-renewing, multipotent cells that can give rise to multiple lineages including osteoblasts (bone), chondrocytes (cartilage) and adipocytes (fat). Interestingly, various pathways that promote BMSC osteogenesis/chondrogenesis simultaneously suppress adipogenesis and vice versa. The basic Helix-Loop-Helix (bHLH) transcription factor, TWIST-1 is highly expressed by BMSC and plays an important role in BMSC proliferation, lifespan, differentiation and commitment. Enforced expression of TWIST-1 enhances proliferation potential and lifespan of BMSC. It also enhances the adipogenic potential of BMSC yet inhibits chondrogenesis and osteogenesis. However, the underlying mechanisms mediating TWIST-1 regulation of BMSC growth and differentiation are not fully understood. In order to identify novel TWIST-1 gene targets involved in BMSC proliferation and osteogenic differentiation, previous studies from our laboratory have compared the gene expression profile of BMSC, which express either endogenous or enforced expression of TWIST-1 during either normal growth conditions or osteogenic inductive conditions, using microarray analysis. Two novel differentially expressed genes were identified, HOPX and CMTM8, as being suppressed by TWIST-1. The aim of this thesis is to determine whether HOPX and CMTM8 are novel targets of TWIST-1 in BMSC and whether they are involved in mediating the effects of TWIST-1 on cell proliferation and lineage commitment. To assess the functional role of HOPX and CMTM8 in the context of BMSC biology, expression of HOPX and CMTM8 were overexpressed using retroviral transduction and supressed using siRNA. The present thesis demonstrated that HOPX counteracts TWIST-1/EZH2 regulation of BMSC cell fate determination via suppression of adipogenic genes such as C/EBPα, ADIPOQ, FABP4, PLIN1 and PLIN4, while HOPX is also a promoter of BMSC proliferation. This thesis also reported that CMTM8 is a suppressor of BMSC osteogenic differentiation and promoter of proliferation and cell migration via the EGFR signalling pathway. This thesis provides better understanding of the downstream molecular mechanisms of TWIST-1 in bone development and post-natal homeostasis and therefore, provides insight into possible future therapeutic strategies that will alter the function of TWIST-1 targets.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201

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

Peer reviewed: TrueAcknowledgements: We thank Edwina McGlinn, Jan Manent, Jan Kaslin and all members of the Rosello-Diez lab for interesting discussions about mTOR in development and regeneration. We also thank the reviewers for their suggestions to improve the manuscript.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

Compensatory growth and recovery of cartilage cytoarchitecture after transient cell death in fetal mouse limbs.

Acknowledgements: We thank Jonathan Gleadle, Vincent Wong and Darling Rojas-Canales for inspiring discussions about mTORC1 and the balance between proliferation and cell size in compensatory responses. We also acknowledge the Monash Bioinformatics Platform (especially Kirill Tsyganov), and Trevor Wilson, at the Medical Genomics Facility, Monash Health Translation Precinct, for their excellent technical help. This study is funded by HFSP CDA00021/2019-C (to A.R-D.) and NHMRC Ideas grant 2002084 (to C.H.H. and A.R-D.). The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.A major question in developmental and regenerative biology is how organ size and architecture are controlled by progenitor cells. While limb bones exhibit catch-up growth (recovery of a normal growth trajectory after transient developmental perturbation), it is unclear how this emerges from the behaviour of chondroprogenitors, the cells sustaining the cartilage anlagen that are progressively replaced by bone. Here we show that transient sparse cell death in the mouse fetal cartilage is repaired postnatally, via a two-step process. During injury, progression of chondroprogenitors towards more differentiated states is delayed, leading to altered cartilage cytoarchitecture and impaired bone growth. Then, once cell death is over, chondroprogenitor differentiation is accelerated and cartilage structure recovered, including partial rescue of bone growth. At the molecular level, ectopic activation of mTORC1 correlates with, and is necessary for, part of the recovery, revealing a specific candidate to be explored during normal growth and in future therapies

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