The cranial neural crest cells are multipotent cells that provide head skeletogenic mesenchyme and are crucial for craniofacial patterning. We analyzed the chromatin landscapes of mouse cranial neural crest subpopulations in vivo. Early postmigratory subpopulations contributing to distinct mouse craniofacial structures displayed similar chromatin accessibility patterns yet differed transcriptionally. Accessible promoters and enhancers of differentially silenced genes carried H3K27me3/H3K4me2 bivalent chromatin marks embedded in large enhancer of zeste homolog 2–dependent Polycomb domains, indicating transcriptional poising. These postmigratory bivalent chromatin regions were already present in premigratory progenitors. At Polycomb domains, H3K27me3 antagonized H3K4me2 deposition, which was restricted to accessible sites. Thus, bivalent Polycomb domains provide a chromatin template for the regulation of cranial neural crest cell positional identity in vivo, contributing insights into the epigenetic regulation of face morphogenesis.

Providing appropriate positional identity and patterning information to distinct rostrocaudal subpopulations of cranial neural crest cells (CNCCs) is central to vertebrate craniofacial morphogenesis. Hox genes are not expressed in frontonasal and first pharyngeal arch (PA1) CNCCs, whereas a single Hox gene, Hoxa2, is necessary to provide patterning information to second pharyngeal arch (PA2) CNCCs. In frog, chick and mouse embryos, ectopic expression of Hoxa2 in Hox-negative CNCCs induced hypoplastic phenotypes of CNCC derivatives of variable severity, associated or not with homeotic transformation of a subset of PA1 structures into a PA2-like identity. Whether these different morphological outcomes are directly related to distinct Hoxa2 overexpression levels is unknown. To address this issue, we selectively induced Hoxa2 overexpression in mouse CNCCs, using a panel of mouse lines expressing different Hoxa2 ectopic expression levels, including a newly generated Hoxa2 knocked-in mouse line. While ectopic Hoxa2 expression at only 60% of its physiological levels was sufficient for pinna duplication, ectopic Hoxa2 expression at 100% of its normal level was required for complete homeotic repatterning of a subset of PA1 skeletal elements into a duplicated set of PA2-like elements. On the other hand, ectopic Hoxa2 overexpression at non-physiological levels (200% of normal levels) led to an almost complete loss of craniofacial skeletal structures. Moreover, ectopic Hoxa5 overexpression in CNCCs, while also resulting in severe craniofacial defects, did not induce homeotic changes of PA1-derived CNCCs, indicating Hoxa2 specificity in repatterning a subset of Hox-negative CNCCs. These results reconcile some discrepancies in previously published experiments and indicate that distinct subpopulations of CNCCs are differentially sensitive to ectopic levels of Hox expression.

The utilization of dodecaborate boron clusters, [B₁₂X₁₂]²⁻ (X = Cl, Br, or I), as membrane carriers has been demonstrated recently, and their activity is known to be due to their superchaotropic nature. In this work, the mono-alkylation of [B₁₂Br₁₁OH]²⁻ to functionalize it with an aliphatic spacer was developed with a view to expanding the known chemical space of membrane carriers based on [B₁₂Br₁₂]²⁻. A new and improved facile route for the preparation of [B₁₂Br₁₁OH]²⁻, which is an important precursor to other [B₁₂Br₁₁OR]²⁻ species, is reported. Various alkoxylated [B₁₂Br₁₁O(CH₂)₅Z]²⁻ (Z = OH, N(CO)₂C₆H₄, CN and N₃) derivatives were prepared via a divergent synthesis based on [B₁₂Br₁₁O(CH₂)₅Br]²⁻. One of the newly synthesized compounds was utilized as a membrane carrier, and its impact on cell viability was examined.

Mutations of G protein coupled receptors (GPCRs) cause various human diseases, but the mechanistic details are limited. Here we establish p.E303K in the gene encoding the endothelin receptor type A (ET_AR/EDNRA) as a recurrent mutation causing Mandibulofacial dysostosis with alopecia (MFDA), with craniofacial changes similar to those caused by p.Y129F. Mouse models carrying either of these missense mutations exhibit a partial maxillary-to-mandibular transformation, which is rescued by deleting the ligand endothelin 3 (ET3/EDN3). Pharmacological experiments confirmed the causative ET_AR mutations as gain-of-function, dependent on ET3. To elucidate how an amino acid substitution far from the ligand binding site can increase ligand affinity, we used molecular dynamics (MD) simulations. E303 is located at the intracellular end of transmembrane domain 6, and its replacement by a lysine increases flexibility of this portion of the helix, thus favoring G-protein binding and leading to G-protein-mediated enhancement of agonist affinity. The Y129F mutation located under the ligand binding pocket reduces the sodium-water network, thereby affecting the extracellular portion of helices in favor of ET3 binding. These findings provide insight into the pathogenesis of MFDA and into allosteric mechanisms regulating GPCR function, that may provide the basis for drug design targeting GPCRs.

Anionic boron clusters, such as [B₁₂X₁₂]²⁻ (X = Cl, Br, I), have attracted attention in pharmaceuticals due to their unique superchaotropic properties. In particular, [B₁₂Br₁₂]²⁻ (1) has demonstrated strong interactions with biomolecules, facilitating cargo translocation across plasma membranes. In this study, we investigated the effect of covalently attaching chlorinated dodecaborate moiety [B₁₂Cl₁₁O-]²⁻ to 6-carboxyfluorescein (6-FAM) (3) via a PEG3 linker to form conjugate (4). We compared the membrane permeability of this covalent conjugate with that of non-covalent interactions between 6-FAM (3) and [B₁₂Cl₁₂]²⁻ (2). Live-cell fluorescence imaging revealed that the covalent conjugate exhibited enhanced membrane permeability and water solubility while maintaining low cytotoxicity. These results highlight the potential of covalent conjugation with boron clusters for improving the cellular uptake of hydrophilic cargos.

[B₁₂I₁₂]²⁻ exhibits potent membrane transport yet high cytotoxicity. Stepwise hydroxylation moderates chaotropicity, balancing permeability and biocompatibility. Notably, mono-hydroxylation retains transport activity while minimizing toxicity, whereas further hydroxylation ablates permeability. This strategy advances safe, efficient boron cluster-based carriers for drug delivery and related biomedical applications.