OSTEIL, P. | Search bioRxiv

bioRxiv2026

Netrin-1 Acts as a Guardian of Naive Pluripotency in Human Embryonic Stem Cells

We investigated the role of Netrin-1 (NTN1) in human naive pluripotency using complementary loss- and gain-of-function approaches. In primate embryos and human embryonic stem cells (hESCs), Netrin-1 expression is associated with the naive pluripotent state. Disruption of NTN1 had no detectable effect on hESCs maintained on murine embryonic fibroblasts. However, under sub-optimal culture conditions, NTN1-knockout cells exhibited compromised naive pluripotency, which was rescued with feeder cells overexpressing Netrin-1. Netrin-1 overexpression in hESCs accelerated acquisition of the naive state and markedly increased resistance to differentiation. These effects were accompanied by extensive epigenetic remodeling, including H3K27ac and H2K27me3. Proteomic and phospho-proteomic analyses further revealed rapid Netrin-1-dependent alterations in pathways controlling cell adhesion, signaling, and chromatin regulation. Together, these findings extend the role of Netrin-1 beyond its established functions and identify it as a coordinator of extracellular cues, intracellular signaling, and nuclear regulatory mechanisms that support human naive pluripotency.

cell biology↗

bioRxiv2024

CTCF-DEPENDENT INSULATION OF Hoxb13 AND THE HETEROCHRONIC CONTROL OF TAIL LENGTH

In mammals, tail length is controlled by several genetic determinants, amongst which Hox13 genes located at the posterior extremities of Hox clusters, whose main function are to terminate the extension of the body axis. In this view, the precise timing in the transcriptional activation of these genes may impact upon body length. Unlike other Hox clusters, HoxB lacks all posterior genes between Hoxb9 and Hoxb13, two genes separated by a ca. 70 kb large DNA segment containing an unusually high number of CTCF sites, suggesting it isolates Hoxb13 from the rest of the cluster, thereby delaying its negative impact on trunk extension. We deleted the spacer DNA to induce a potential heterochronic gain of function of Hoxb13 at physiological concentration and observed a shortening of the tail as well as other abnormal phenotypes, which were all rescued by inactivating Hoxb13 in-cis with the deletion. A comparable gain of function was observed in mutant ES cells grown as pseudo-embryos in vitro, which allowed us to examine in details the importance of both the number and the orientation of CTCF sites in the insulating activity of the DNA spacer. A short cassette containing all the CTCF sites was sufficient to insulate Hoxb13 from the rest of HoxB and additional modifications of this CTCF cassette showed that two CTCF sites in convergent orientations are already capable of importantly delaying Hoxb13 activation in these conditions. We discuss the relative importance of genomic distance versus number and orientation of CTCF sites in preventing Hoxb13 to be activated too early during trunk extension and hence to modulate tail length.

developmental biology↗