Although every cell contains the same genes, not all genes are active at any given time. Gene regulation is a fundamental process that ensures only the necessary genes are expressed in each cell type. This is why, for example, neurons differ in structure and function from muscle cells. Precise fine-tuning of gene regulation is especially critical during development. Timed waves of transcriptional activity ensure that an embryo develops into a healthy organism with properly positioned and formed limbs, organs, and tissues. This process is driven by specialised genes and controlled by regulatory elements in the genome.
Many such gene regulatory elements have been identified. Over the past decade, scientists have increasingly turned their attention to the non-coding regions of the genome – that is, genetic sequences that do not encode functional proteins.
“Long non-coding RNAs have moved into the focus of research in recent years,” says Dr Alessa Ringel, postdoctoral researcher at the Max Planck Institute for Molecular Genetics and the first author of the study. “They are highly abundant but often expressed at low levels, and they are increasingly implicated in gene regulation and various human diseases.”
The research team previously identified a lncRNA locus they named MAENLI and demonstrated that it activates the developmental gene En1, which is known to regulate brain development and proper limb patterning. EN1 mutations can result in limb and brain malformations, while MAENLI deletionsresult only in severe limb abnormalities.
“Even after the paper was published, we still had many questions,” Ringel says. “We noticed that after a certain developmental stage, MAENLI activity drops, but En1 transcription stays high.” These observations suggested that additional regulatory elements might be involved. The researchers used a range of techniques – from CRISPR/Cas9 gene editing and epigenetic profiling to functional reporter assays – to gain a clearer understanding of how En1 is controlled. “That made us wonder whether, in our model, deleting the lncRNA locus also removed other nearby regulatory DNA elements” says Alessa.
“Indeed, we found two waves of En1 transcription. An early wave that was controlled by MAENLI, and a late wave that was controlled by two newenhancers that we identified close to MAENLI and named LSEE1&2”, says Lila Allou, Head of the Genomic Variation and Disease group at the LMS andone of the corresponding authors of the study, “What was very interesting is that by controlling En1 waves of transcription, these genetic elements regulated distinct aspects of limb development.”
Their findings further show that loss of either early or late En1 regulation leads to distinct limb defects in mice. This has important implications for human congenital limb malformations, which often present with subtle differences. These are difficult to pinpoint genetically, as diagnostic approachesoften focus on coding sequences.
“Our work indicates that the slight variability in phenotypes we see for some isolated birth defects is likely explained by differences in when genes are turned on rather than caused by different broken genes”, says Lila.
This study was funded by: Deutsche Forschungsgemeinschaft (DFG) (MU 880/16-1) for Stefan Mundlos, UCL Award 156782/ Project 580721 and the Medical Research Council Laboratory of Medical Sciences (MC-A652-5QA34) for Lila Allou.
Read the full publication: https://genesdev.cshlp.org/content/early/2026/04/15/gad.353542.125.short?rss=1
This article was written by Sándor Fülöp, Communications Officer at the Max Planck Institute for Molecular Genetics in Berlin, Germany.
About the image: Skeletal limb abnormalities associated with engrailed-1 mouse mutants. Credit: Lila Allou and Alessa Ringel.