Search bioRxiv⌕ Search

bioRxiv · 10.1101/2024.03.08.584197

Exploration of phosphoproteomic association during epimorphic regeneration

Abstract

Unravelling the intricate patterns of site-specific protein phosphorylation during Epimorphic regeneration holds the key to unlocking the secrets of tissue complexity. Understanding these precise modifications and their impact on protein function could shed light on the remarkable regenerative capacity of tissues, with potential implications for therapeutic interventions. In this study we have systematically mapped the global phosphorylation modifications within regenerating tissue of zebrafish caudal fins, elucidating the intricate landscape of signalling pathway associate with the regeneration process. A total of 74 and 440 proteins were found undergoing differentially phosphorylated during the process of regeneration from 12hpa to 7dpa against control based on TiO2 column enrichment and immuno precipitation using phosphoserine, phosphothreonine and phosphotyrosine antibodies respectively. Interestingly 95% of the proteins identified from TiO2 enrichment method were also found to be identified through the phosphoprotein antibody pull down method impacting the high accuracy and significance of the methods and greater association of the 70 proteins undergoing differential phosphorylation during the process of regeneration. Whole mount immunohistochemistry analysis reveals high association of phosphorylation at 1dpa, 2dpa and 3 dpa regeneration time points. Based on network pathway analysis it was evident that Fc Receptor-mediated Phagocytosis in Macrophages and Monocytes, Actin cytoskeleton signaling, HGF signaling and Insulin receptor signaling are the most highly associated network pathways for regeneration through differential phosphorylation. This research enhances our comprehension on protein post-translational modification in the context of zebrafish caudal fin tissue regeneration, shedding light on its prospective application in the field of regenerative medicine.

Source connections

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Banu, S., V, A. P., Mandal, K., Idris, M. M.. 2024-03-13. Exploration of phosphoproteomic association during epimorphic regeneration. https://doi.org/10.1101/2024.03.08.584197

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

A bicistronic Aldh1a3-P2A-TagBFP knock-in reporter mouse line for studying genitourinary tract development

Aldehyde dehydrogenase 1a3 (Aldh1a3) is an enzyme involved in retinoic acid synthesis with dynamic expression patterns during development, including in the urogenital system. Here, we generated a bicistronic Aldh1a3-P2A-TagBFP knock-in mouse using CRISPR/Cas9 genome editing, inserting TagBFP immediately upstream of the endogenous Aldh1a3 stop codon. Correct targeting was confirmed by Oxford Nanopore long-read sequencing, and heterozygous and homozygous mice were viable and fertile without overt morphological abnormalities. TagBFP fluorescence faithfully overlapped with endogenous Aldh1a3 immunoreactivity and reproduced established expression domains in the developing craniofacial region, intestine, kidney, and broader urogenital system. Extensive characterization of the urogenital system revealed dynamic, spatially restricted BFP reporter activity in Aldh1a3-expressing domains across several key structures, including the ureteric bud and collecting duct lineage, seminal vesicles, caput epididymis, and developing uterine horns. The Aldh1a3-P2A-TagBFP mouse provides a fluorescent resource for visualizing Aldh1a3 expression across development and in adult tissues, including for the characterization of Aldh1a3-expressing domains in the urogenital system. The relatively low fluorescence intensity of TagBFP should be considered when assessing low-level reporter expression.

developmental biology↗

Translation of a small upstream open reading frame functions as a rheostat for the regulation of lin-41 by the Let-7 microRNA in Caenorhabditis elegans

MicroRNAs have been likened to the "dark matter" of eukaryotic genomes, reflecting their pervasive regulatory influence. MicroRNAs were first identified through genetic studies of developmental timing in the nematode Caenorhabditis elegans. Let-7 was the first microRNA recognized to be broadly conserved. The principal target of Let-7 in the developmental timing pathway is the TRIM-NHL RNA-binding protein LIN-41. During the L4 larval stage, Let-7 represses lin-41 translation by binding to two Let-7 complementary sites in the lin-41 3'UTR. Despite the importance of microRNA-based translational regulation, the underlying molecular mechanisms are incompletely understood. Through genetic analysis, we discovered an unrecognized feature of the mechanism by which Let-7 controls lin-41 translation. This mechanism requires a 5'-regulatory exon containing a seven-amino acid upstream open reading frame (uORF) and conserved sequence elements. Genome editing indicates that the specific uORF amino acid sequence itself is not important. Our data suggest that uORF translation and 5'UTR structure limit initiation at the downstream lin-41 start codon, enabling tight control by Let-7. Without this mechanism, the Let-7 microRNA is unable to properly regulate lin-41 to enable proper development.

developmental biology↗

Ductal myofibroblasts reactivate contractile program to stabilize alveolar architecture during lung regeneration

The alveolar sac architecture is essential for efficient gas exchange and must be precisely maintained throughout life; however, how this delicate structure is preserved during adult regeneration remains poorly understood. Using a mouse pneumonectomy model, we found that Lgr6+ Hhip+ ductal myofibroblasts, a poorly characterized mesenchymal population, are indispensable for maintaining alveolar integrity during lung regrowth. Comprehensive characterization using single-cell transcriptomics, mouse genetics, and pharmacological assays demonstrated that these ductal myofibroblasts secrete myogenic factors, most notably CCN4, to reactivate a myogenic program that converts them into contractile PA-DMFs, thereby preserving alveolar architecture. Lineage-tracing further revealed that these ductal myofibroblasts originate from embryonic MCAM- SMA+ distal progenitors via subepithelial TGF-{beta} signaling, serving as a lifelong guardian of alveolar structural integrity. Notably, cross-species analysis identified an analogous population of LGR6+ fibromyocytes in human respiratory bronchioles. Together, these findings indicate ductal myofibroblasts as a developmentally programmed cell population that reactivate a contractile program to structurally support the regeneration of adult lungs.

developmental biology↗