A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling
Abstract
Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signalling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signalling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here we describe a set of CRISPR based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.
Data availability
Full genome sequencing for isogenized Attp40 Stock submitted to NCBI (BioProject ID PRJNA606147). Full genome sequencing for S2R+ cells submitted to NCBI (Bioproject ID PRJNA606149). Images for PI signaling genetic screen saved at Open Source Frame https://osf.io/pt7zu/?view_only=14642fc3a5d74e408fb3766c2555393f
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Full genome sequencing for isogenized Attp40 StockNCBI, BioProject ID PRJNA606147.
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Full genome sequencing for S2R+ cellsNCBI, BioProject ID PRJNA606149.
Article and author information
Author details
Funding
Department of Biotechnology, Ministry of Science and Technology, India (BT/PRJ3748/GET/l 19/27/2015)
- Deepti Trivedi
- Vinitha CM
- Karishma Bisht
- Vishnu Janardan
- Padinjat Raghu
Wellcome-DBT India Alliance (IA/S/14/2/501540)
- Vinitha CM
- Karishma Bisht
- Vishnu Janardan
- Bishal Basak
- Padinjat Raghu
National Centre for Biological Sciences (core)
- Deepti Trivedi
- Vinitha CM
- Karishma Bisht
- Vishnu Janardan
- Awadhesh Pandit
- Bishal Basak
- Padinjat Raghu
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Jiwon Shim, Hanyang University, Republic of Korea
Publication history
- Received: February 6, 2020
- Accepted: December 14, 2020
- Accepted Manuscript published: December 15, 2020 (version 1)
- Version of Record published: December 29, 2020 (version 2)
Copyright
© 2020, Trivedi et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Developmental Biology
Bone homeostasis is regulated by hormones such as parathyroid hormone (PTH). While PTH can stimulate osteo-progenitor expansion and bone synthesis, how the PTH-signaling intensity in progenitors is controlled is unclear. Endochondral bone osteoblasts arise from perichondrium-derived osteoprogenitors and hypertrophic chondrocytes (HC). We found, via single-cell transcriptomics, that HC-descendent cells activate membrane-type 1 metalloproteinase 14 (MMP14) and the PTH pathway as they transition to osteoblasts in neonatal and adult mice. Unlike Mmp14 global knockouts, postnatal day 10 (p10) HC lineage-specific Mmp14 null mutants (Mmp14ΔHC) produce more bone. Mechanistically, MMP14 cleaves the extracellular domain of PTH1R, dampening PTH signaling, and consistent with the implied regulatory role, in Mmp14ΔHC mutants, PTH signaling is enhanced. We found that HC-derived osteoblasts contribute ~50% of osteogenesis promoted by treatment with PTH 1–34, and this response was amplified in Mmp14ΔHC. MMP14 control of PTH signaling likely applies also to both HC- and non-HC-derived osteoblasts because their transcriptomes are highly similar. Our study identifies a novel paradigm of MMP14 activity-mediated modulation of PTH signaling in the osteoblast lineage, contributing new insights into bone metabolism with therapeutic significance for bone-wasting diseases.
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- Biochemistry and Chemical Biology
- Developmental Biology
In mammals, interactions between the bone marrow (BM) stroma and hematopoietic progenitors contribute to bone-BM homeostasis. Perinatal bone growth and ossification provide a microenvironment for the transition to definitive hematopoiesis; however, mechanisms and interactions orchestrating the development of skeletal and hematopoietic systems remain largely unknown. Here, we establish intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) modification as a posttranslational switch that dictates the differentiation fate and niche function of early BM stromal cells (BMSCs). By modifying and activating RUNX2, O-GlcNAcylation promotes osteogenic differentiation of BMSCs and stromal IL-7 expression to support lymphopoiesis. In contrast, C/EBPβ-dependent marrow adipogenesis and expression of myelopoietic stem cell factor (SCF) is inhibited by O-GlcNAcylation. Ablating O-GlcNAc transferase (OGT) in BMSCs leads to impaired bone formation, increased marrow adiposity, as well as defective B-cell lymphopoiesis and myeloid overproduction in mice. Thus, the balance of osteogenic and adipogenic differentiation of BMSCs is determined by reciprocal O-GlcNAc regulation of transcription factors, which simultaneously shapes the hematopoietic niche.