The pre-dimerization of endosome-localized RNA sensor Toll-like receptor 3 (TLR3) is required for its innate recognition, yet how TLR3 pre-dimers are formed and precisely primed for innate activation remains unclear. Here, we demonstrate that endosome-localized self RNA Rmrp directly binds to TLR3 and induces TLR3 dimerization in the early endosome but does not interact with endosome-localized TLR7, TLR8, TLR9 or cytoplasmic RNA sensor RIG-I under homeostatic conditions. Cryo-EM structure of Rmrp-TLR3 complex reveals a novel lapped conformation of TLR3 dimer engaged by Rmrp, which is distinct from the activation mechanism by dsRNA and the specific structural feature at the 3'-end of Rmrp is critical for its functional interaction with TLR3. Furthermore, K42 residue of TLR3 is essential for binding to Rmrp and subsequent dimerization. Rmrp dissociates from TLR3 following endosomal acidification, generating a matured TLR3 dimer which is primed for innate recognition and activation. Myeloid-cell deficiency of Rmrp reduces TLR3 dimerization and attenuates TLR3-mediated antiviral responses against influenza A both in vitro and in vivo. These findings elucidate the structural mode of self RNA Rmrp-primed TLR3 dimerization and ready for efficient innate recognition on endosomal membrane, extending our knowledge of how membrane-associated TLRs pre-dimerize and suggesting a new function of subcellular localized self RNAs in empowering innate activation.

The absence of stem cells capable of efficiently generating both trophoblast and epiblast lineages has hindered precise recapitulation of embryonic development. Through high-content chemical screening, we established an (AS and LY)AL medium to generate mouse bidirectional pluripotent stem cells (BPSCs) characterized by concurrent expression of OCT4 and CDX2. Mouse BPSCs demonstrated highly plastic differentiation into trophoblast, epiblast and primitive endoderm (PrE) lineages in vitro within 48 h without exogenous inducing factors and efficiently contributed to embryonic and extraembryonic tissues in vivo. Mechanistically, hyperactivation of the Wnt signaling pathway breaks the early lineage differentiation barrier by initiating a Lef1-dependent bypass. Remarkably, integration of BPSCs with PrE induction system enables high-efficiency generation of E8.5-stage embryo models. These advanced models complete gastrulation and recapitulate definitive developmental milestones including brain morphogenesis, neural tube closure, cardiac contraction, somite patterning, and primordial germ cell specification. Moreover, human cells cultured under AL conditions acquire an OCT4 and CDX2 double-positive state and corresponding gene expression profiles, revealing conserved functionality of this culturing platform across species. These findings highlight BPSCs as a powerful tool for investigating early lineage specification and post-gastrulation embryonic development.

Pluripotent stem cells (PSCs) have been derived from various species, but most culture systems stabilize only a single PSC type. By contrast, epiblast cells in vivo exist along a continuum and interact dynamically with both embryonic and extraembryonic cells, interactions missing in standard PSC cultures. This absence limits the self-organizing potential of PSCs and leads to disorganized tissue formation in teratomas. To address this, we developed a unified culture system that supports the stable differentiation of epiblast-like cells into multiple key human gastrulating cell types, collectively called human gastrulating stem cells (hGaSCs). hGaSCs, composed of endoderm-like, mesoderm-like, ectoderm-like, amnion ectoderm-like, and primordial germ cell-like cells, maintain a stable balance during long-term culture. In 3D culture, hGaSCs self-assemble into gastruloid-like structures (hGaSC-gastruloids) that model aspects of a Carnegie Stage 7 human embryo, including gastrulation and germ layer specification. Using hGaSC-gastruloids, we modeled the effects of valproic acid (VPA) on human gastrulation and uncovered molecular pathways underlying VPA-induced malformations. When transplanted into the seminiferous tubules, hGaSCs formed embryo-like structures, progressing through fetal tissue and organ development, unlike the disorganized growth seen in teratomas. In conclusion, hGaSCs provide a versatile platform to study human gastrulation, early organogenesis, developmental defects, and drug teratogenicity, with promising applications in tissue and organ generation from cultured stem cells.

KCNQ1 potassium channels are essential for physiological processes such as cardiac rhythm and intestinal chloride secretion. KCNE-family subunits (KCNE1–5) associate with KCNQ1, conferring distinct properties across various tissues. KCNQ1 activation requires membrane depolarization and phosphatidylinositol 4,5-bisphosphate (PIP2) whose cellular levels are controlled by Gαq-coupled GPCR activation. While modulation of KCNQ1’s voltage-dependent activation by KCNE1/3 is well-characterized, their effects on PIP2-dependent gating of KCNQ1 via GPCR signaling remain less understood. Here we resolved structures of KCNQ1–KCNE1 and reassessed reported KCNQ1-KCNE3 structures with and without PIP2. We revealed that KCNQ1–KCNE1/3 complexes feature two PIP2-binding sites, with KCNE1/3 contributing to a previously overlooked, uncharacterized site involving residues critical for voltage sensor and pore domain coupling. Via this site, KCNE1 and KCNE3 distinctly modulate the PIP2-dependent gating, in addition to the voltage sensitivity, of KCNQ1. Consequently, KCNE3 converts KCNQ1 into a voltage-insensitive PIP2-gated channel governed by GPCR signaling to maintain ion homeostasis in non-excitable cells. KCNE1, by significantly enhancing KCNQ1’s PIP2 affinity and resistance to GPCR regulation, forms predominantly voltage-gated channels with KCNQ1 for conducting the slow-delayed rectifier current in excitable cardiac cells. Our study highlights how KCNE1/3 modulates KCNQ1 gating in different cellular contexts, providing insights for tissue-specifically targeting multi-functional channels.