The blood-brain barrier (BBB) is composed of tightly bound endothelial cells (ECs) and perivascular astrocytes that regulate central nervous system (CNS) homeostasis. We showed that astrocytes secrete Sonic hedgehog and that BBB ECs express Hedgehog (Hh) receptors, which together promote BBB formation and integrity during embryonic development and adulthood. Using pharmacological inhibition and genetic inactivation of the Hh signaling pathway in ECs, we also demonstrated a critical role of the Hh pathway in promoting the immune quiescence of BBB ECs by decreasing the expression of proinflammatory mediators and the adhesion and migration of leukocytes, in vivo and in vitro. Overall, the Hh pathway provides a barrier-promoting effect and an endogenous anti-inflammatory balance to CNS-directed immune attacks, as occurs in multiple sclerosis.

In the spinal cord, sonic hedgehog (Shh) is secreted by the floor plate to control the generation of distinct classes of ventral neurons along the dorsoventral axis. Genetic and in vitro studies have shown that Shh also later acts as a midline-derived chemoattractant for commissural axons. However, the receptor(s) responsible for Shh attraction remain unknown. Here we show that two Robo-related proteins, Boc and Cdon, bind specifically to Shh and are therefore candidate receptors for the action of Shh as an axon guidance ligand. Boc is expressed by commissural neurons, and targeted disruption of Boc in mouse results in the misguidance of commissural axons towards the floor plate. RNA-interference-mediated knockdown of Boc impairs the ability of rat commissural axons to turn towards an ectopic source of Shh in vitro. Taken together, these data suggest that Boc is essential as a receptor for Shh in commissural axon guidance.

Serum-to-2i interconversion of mouse embryonic stem cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum-grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon their further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary, but not sufficient, to establish these interactions, as confirmed by Capture Hi-C on Eed(-/-) serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation.

The mammalian HoxD cluster lies between two topologically associating domains (TADs) matching distinct enhancer-rich regulatory landscapes. During limb development, the telomeric TAD controls the early transcription of Hoxd genes in forearm cells, whereas the centromeric TAD subsequently regulates more posterior Hoxd genes in digit cells. Therefore, the TAD boundary prevents the terminal Hoxd13 gene from responding to forearm enhancers, thereby allowing proper limb patterning. To assess the nature and function of this CTCF-rich DNA region in embryos, we compared chromatin interaction profiles between proximal and distal limb bud cells isolated from mutant stocks where various parts of this boundary region were removed. The resulting progressive release in boundary effect triggered inter-TAD contacts, favored by the activity of the newly accessed enhancers. However, the boundary was highly resilient, and only a 400-kb deletion, including the whole-gene cluster, was eventually able to merge the neighboring TADs into a single structure. In this unified TAD, both proximal and distal limb enhancers nevertheless continued to work independently over a targeted transgenic reporter construct. We propose that the whole HoxD cluster is a dynamic TAD border and that the exact boundary position varies depending on both the transcriptional status and the developmental context.

Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics, and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra-and interchromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral lamina-associated domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type-specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active, nuclear environments.Hox genes | Polycomb | long-range chromatin contacts | nuclear organization

Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons towards the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning towards floor plate tissue, suggesting that additional guidance cues are present. Here, we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, while Flk1-blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand / receptor pair controlling commissural axon guidance.

The mammalian HoxD cluster lies between two topologically associating domains (TADs) matching distinct, enhancer-rich regulatory landscapes. During limb development, the telomeric TAD controls the early transcription of Hoxd gene in forearm cells, whereas the centromeric TAD subsequently regulates more posteriorHoxd genes in digit cells. Therefore, the TAD boundary prevents the terminal Hoxd13 gene to respond to forearm enhancers, thereby allowing proper limb patterning. To assess the nature and function of this CTCF-rich DNA region in embryo, we compared chromatin interaction profiles between proximal and distal limb bud cells isolated from mutant stocks where various parts or this boundary region were removed. The resulting progressive release in boundary effect triggered inter-TAD contacts, favored by the activity of the newly accessed enhancers. However, the boundary was highly resilient and only a 400kb large deletion including the whole gene cluster was eventually able to merge the neighboring TADs into a single structure. In this unified TAD, both proximal and distal limb enhancers nevertheless continued to work independently over a targeted transgenic reporter construct. We propose that the whole HoxD cluster is a dynamic TAD border and that the exact boundary position varies depending on both the transcriptional status and the developmental context.

The pattern of contralaterally and ipsilaterally projecting retinal ganglion cell (RGC) axons at the optic chiasm is essential for the establishment of binocular vision. Contralateral axons cross the chiasm midline as they progress from the optic nerve to the optic tract. In contrast, ipsilateral axons deviate from the chiasm and continue in the ipsilateral optic tract, avoiding the chiasm midline. The molecular mechanism underlying this phenomenon is not completely understood. Here we show that the Sonic Hedgehog (Shh) receptor Boc is enriched in ipsilateral RGCs of the developing retina. Together with the presence of Shh at the midline, this complementary expression pattern led us to hypothesize that Shh might repel ipsilateral RGC axons at the chiasm. Consistent with this hypothesis, we found that only Boc-positive RGC axons retract in vitro in response to Shh and that this response is lost in Boc mutant RGCs. In vivo, we show that Boc is required for the normal segregation of ipsilateral axons at the optic chiasm and, conversely, that Boc expression in contralateral RGCs prevents their axons from crossing the optic chiasm. Together, these results suggest that Shh repels ipsilateral RGC axons at the optic chiasm via its receptor Boc. This work identifies a novel molecular pathway required for the segregation of axons at the optic chiasm.