bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.03.16.643512

Chronic morphine treatment leads to a global DNA hypomethylation via active and passive demethylation mechanisms in mESCs

Araolaza, M.·Munoa-Hoyos, I.·Urizar-Arenaza, I.·Calzado, I.·Subiran, N.
View original source ↗Export metadata ↓

Abstract

Epigenetic changes are essential for normal development and ageing, but there is still limited understanding of how environmental factors can cause epigenetic changes that leads to health problems or diseases. Morphine is known to pass through the placental barrier and impact normal embryo development by affecting the neural tube, frontal cortex and spinal cord development, and, as a consequence, delaying nervous system development. In fact, in-utero morphine exposure has shown alterations in anxiety-like behaviours, analgesic tolerance, synaptic plasticity and the neuronal structure of offspring. However, how morphine leads to abnormal neurogenesis and other physiological consequences during embryo development is still unknown. Considering that DNA methylation is a key epigenetic factor crucial for embryo development, our aim is to elucidate the role of methylation in response to morphine. Chronic morphine treatment (24h, 10M) induces a global hypomethylation in mESC. WGBSeq identifies 16,808 sensitive to morphine which are involved in embryo development, signalling pathways, metabolism and/or gene expression, suggesting that morphine might impact methylation levels at developmental genes. Integrative analyses between WGBSeq and RNASeq identified Tet1 as morphine-sensitive gene. Morphine increased the gene expression of Tet1, modifying the methylation levels at the promoter. On the other hand, RNASeq and qRT-PCR analyses revealed that Dnmt1 gene expression decreased after morphine treatment, without altering the methylation patter at its promoters. By MS/MS approaches confirms a decrease in DNA methylation after chronic morphine treatment, together with an increase in hydroxymethylation global levels in mESCs. In conclusion, morphine induces a global hypomethylation in mESC through different mechanisms that involves passive demethylation and a self-regulatory mechanism via active demethylation. One Sentence SummaryUnderstanding how environmental epigenetics impact on embryo development aids to unravel changes that leads to health problems or diseases in the adulthood. Graphical abstractChronic morphine treatment induces a global hypomethylation in mESC through different mechanisms that involves passive demethylation and a self-regulatory mechanism via active demethylation in genes involved in embryo development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/643512v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@e9d286org.highwire.dtl.DTLVardef@fc7066org.highwire.dtl.DTLVardef@1374039org.highwire.dtl.DTLVardef@598f05_HPS_FORMAT_FIGEXP M_FIG C_FIG

Source connections

Explore related subjects

developmental biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Araolaza, M., Munoa-Hoyos, I., Urizar-Arenaza, I., Calzado, I., Subiran, N.. 2025-03-16. Chronic morphine treatment leads to a global DNA hypomethylation via active and passive demethylation mechanisms in mESCs. https://doi.org/10.1101/2025.03.16.643512

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

KEEP EXPLORING

Related preprints

bioRxiv2026

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↗
bioRxiv2026

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↗
bioRxiv2026

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↗