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
-
Full genome sequencing for isogenized Attp40 StockNCBI, BioProject ID PRJNA606147.
-
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.
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.
Metrics
-
- 3,307
- views
-
- 367
- downloads
-
- 16
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Developmental Biology
Notch signaling has been identified as a key regulatory pathway in patterning the endocardium through activation of endothelial-to-mesenchymal transition (EMT) in the atrioventricular canal (AVC) and proximal outflow tract (OFT) region. However, the precise mechanism underlying Notch activation remains elusive. By transiently blocking the heartbeat of E9.5 mouse embryos, we found that Notch activation in the arterial endothelium was dependent on its ligand Dll4, whereas the reduced expression of Dll4 in the endocardium led to a ligand-depleted field, enabling Notch to be specifically activated in AVC and OFT by regional increased shear stress. The strong shear stress altered the membrane lipid microdomain structure of endocardial cells, which activated mTORC2 and PKC and promoted Notch1 cleavage even in the absence of strong ligand stimulation. These findings highlight the role of mechanical forces as a primary cue for endocardial patterning and provide insights into the mechanisms underlying congenital heart diseases of endocardial origin.
-
- Developmental Biology
- Evolutionary Biology
Seahorses, pipefishes, and seadragons are fishes from the family Syngnathidae that have evolved extraordinary traits including male pregnancy, elongated snouts, loss of teeth, and dermal bony armor. The developmental genetic and cellular changes that led to the evolution of these traits are largely unknown. Recent syngnathid genome assemblies revealed suggestive gene content differences and provided the opportunity for detailed genetic analyses. We created a single-cell RNA sequencing atlas of Gulf pipefish embryos to understand the developmental basis of four traits: derived head shape, toothlessness, dermal armor, and male pregnancy. We completed marker gene analyses, built genetic networks, and examined the spatial expression of select genes. We identified osteochondrogenic mesenchymal cells in the elongating face that express regulatory genes bmp4, sfrp1a, and prdm16. We found no evidence for tooth primordia cells, and we observed re-deployment of osteoblast genetic networks in developing dermal armor. Finally, we found that epidermal cells expressed nutrient processing and environmental sensing genes, potentially relevant for the brooding environment. The examined pipefish evolutionary innovations are composed of recognizable cell types, suggesting that derived features originate from changes within existing gene networks. Future work addressing syngnathid gene networks across multiple stages and species is essential for understanding how the novelties of these fish evolved.