To date, brain imaging has largely relied on X-ray computed tomography and magnetic resonance angiography with limited spatial resolution and long scanning times. Fluorescence-based brain imaging in the visible and traditional near-infrared regions (400–900 nm) is an alternative but currently requires craniotomy, cranial windows and skull thinning techniques, and the penetration depth is limited to 1–2 mm due to light scattering. Here, we report through-scalp and through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy utilizing the intrinsic photoluminescence of single-walled carbon nanotubes in the 1.3–1.4 micrometre near-infrared window. Reduced photon scattering in this spectral region allows fluorescence imaging reaching a depth of >2 mm in mouse brain with sub-10 micrometre resolution. An imaging rate of ~5.3 frames/s allows for dynamic recording of blood perfusion in the cerebral vessels with sufficient temporal resolution, providing real-time assessment of blood flow anomaly in a mouse middle cerebral artery occlusion stroke model.

SUMMARY The canonical Wnt/β-catenin signaling pathway governs diverse developmental, homeostatic and pathologic processes. Palmitoylated Wnt ligands engage cell surface Frizzled (Fzd) receptors and Lrp5/6 co-receptors enabling β-catenin nuclear translocation and Tcf/Lef-dependent gene transactivation1–3. Mutations in Wnt downstream signaling components have revealed diverse functions presumptively attributed to Wnt ligands themselves, although direct attribution remains elusive, as complicated by redundancy between 19 mammalian Wnts and 10 Fzds1 and Wnt hydrophobicity2,3. For example, individual Wnt ligand mutations have not revealed homeostatic phenotypes in the intestinal epithelium4, an archetypal canonical Wnt pathway-dependent rapidly self-renewing tissue whose regeneration is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs)5–9. R-spondin ligands (Rspo1–4) engage distinct Lgr4-6 and Rnf43/Znrf3 receptor classes10–13, markedly potentiate canonical Wnt/β-catenin signaling and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo8,14–17. However, the interchangeability, functional cooperation and relative contributions of Wnt versus Rspo ligands to in vivo canonical Wnt signaling and ISC biology remain unknown. Here, we deconstructed functional roles of Wnt versus Rspo ligands in the intestinal crypt stem cell niche. We demonstrate that the default fate of Lgr5+ ISCs is lineage commitment, escape from which requires both Rspo and Wnt ligands. However, gain-of-function studies using Rspo versus a novel non-lipidated Wnt analog reveal qualitatively distinct, non-interchangeable roles for these ligands in ISCs. Wnts are insufficient to induce Lgr5+ ISC self-renewal, but rather confer a basal competency by maintaining Rspo receptor expression that enables Rspo to actively drive and specify the extent of stem cell expansion. This functionally non-equivalent yet cooperative interplay between Wnt and Rspo ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precision control of tissue regeneration.

Oligodendrocytes myelinate axons in the central nervous system and develop from oligodendrocyte precursor cells (OPCs) that must first migrate extensively during brain and spinal cord development. We show that OPCs require the vasculature as a physical substrate for migration. We observed that OPCs of the embryonic mouse brain and spinal cord, as well as the human cortex, emerge from progenitor domains and associate with the abluminal endothelial surface of nearby blood vessels. Migrating OPCs crawl along and jump between vessels. OPC migration in vivo was disrupted in mice with defective vascular architecture but was normal in mice lacking pericytes. Thus, physical interactions with the vascular endothelium are required for OPC migration. We identify Wnt-Cxcr4 (chemokine receptor 4) signaling in regulation of OPC-endothelial interactions and propose that this signaling coordinates OPC migration with differentiation.

been widely used as promising agents for multifunctional blood vessel imaging and tumor imaging. All these agents with well-defined surface chemistry performed good stability and high fluorescence in physiological environment and can be used for NIR-II imaging in vivo. However, due to the large hydrodynamic size, most of inorganic nanoparticles still cannot be excreted rapidly by kidney. The accumulation of these materials in body may induce potential liver toxicity, which prevents their further applications in clinical medicine. Moreover, the organic materials, such as conjugated polymer fluorophores [7] and small molecules, [8] have improved biocompatibility, showing great potential in clinical translation. Nevertheless, the fluorescence quantum yield (QY) of these materials is still far from ideal. Thus, it is desirable to design an NIR-II agent with high QY as well as high efficiency in renal clearance for wide biological and clinical applications. Here, we present a bright Au 25 cluster with the unique cage-like structure that can emit NIR-II fluorescence at 1100-1350 nm by the charge transfer between ligand and gold core. [9] Metal doping further increases fluorescence QY of Au 25 clusters. The time-resolved brain blood flow shows significant differences between healthy and injured brain, which allow us to distinguish the lipopolysaccharides (LPS) induced brain injury and stroke in vivo. Meanwhile, real-time cancer metastasis is monitored by NIR-II imaging. Importantly, the ultrasmall hydrodynamic size of 3.2 nm allows the gold clusters to cross the glomerular filtration and be excreted fast by Near-infrared II (NIR-II) imaging at 1100-1700 nm shows great promise for medical diagnosis related to blood vessels because it possesses deep penetration and high resolution in biological tissue. Unfortunately, currently available NIR-II fluorophores exhibit slow excretion and low brightness, which prevents their potential medical applications. An atomic-precision gold (Au) cluster with 25 gold atoms and 18 peptide ligands is presented. The Au 25 clusters show emission at 1100-1350 nm and the fluorescence quantum yield is significantly increased by metal-atom doping. Bright gold clusters can penetrate deep tissue and can be applied in in vivo brain vessel imaging and tumor metastasis. Time-resolved brain blood-flow imaging shows significant differences between healthy and injured mice with different brain diseases in vivo. High-resolution imaging of cancer metastasis allows for the identification of the primary tumor, blood vessel, and lymphatic metastasis. In addition, gold clusters with NIR-II fluorescence are used to monitor highresolution imaging of kidney at a depth of 0.61 cm, and the quantitative measurement shows 86% of the gold clusters are cleared from body without any acute or long-term toxicity at a dose of 100 mg kg −1 .

Wnts modulate cell proliferation, differentiation and stem cell self-renewal, by inducing β-catenin dependent signaling through Frizzled (Fzd) and Lrp5/6 to regulate cell fate decisions, and the growth and repair of a multitude of tissues1. The 19 mammalian Wnts interact promiscuously with the 10 Fzds, which has complicated the attribution of specific Fzd/Wnt subtype interactions to distinct biological functions. Furthermore, Wnts are post-translationally modified by palmitoylation, which is essential for Wnt secretion and functions as a critical site of interaction with Fzd 2–4. As a result of their acylation, Wnts are very hydrophobic proteins requiring detergents for purification, which presents major obstacles for the preparation and application of recombinant Wnts. This has hindered the delineation of the molecular mechanisms of Wnt signaling activation, understanding of the functional significance of Fzd subtypes, and the use of Wnts as therapeutics. Here we developed surrogate Wnt agonists, water-soluble Fzd-Lrp5/6 heterodimerizers, consisting of Fzd5/8-specific and broadly Fzd-reactive binding domains, that elicit a characteristic β-catenin signaling response in a Fzd-selective fashion, enhance osteogenic lineage commitment of primary mesenchymal stem cells (MSCs), and support the growth of a broad range of primary human organoid cultures comparably to Wnt3a. Furthermore, we demonstrate that the surrogates can be systemically expressed and exhibit Wnt activity in vivo, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signaling can be activated simply through bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced non-lipidated Wnt surrogate agonists offer a new avenue to facilitate functional studies of Wnt signaling and the exploration of Wnt agonists for translational applications in regenerative medicine.

Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N4-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.

Compared to imaging in the visible and near-infrared regions below 900 nm, imaging in the second near-infrared window (NIR-II, 1000-1700 nm) is a promising method for deep-tissue high-resolution optical imaging in vivo mainly owing to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single-walled carbon nanotubes with large diameters were used for in vivo fluorescence imaging in the long-wavelength NIR region (1500-1700 nm, NIR-IIb). With this imaging agent, 3-4 μm wide capillary blood vessels at a depth of about 3 mm could be resolved. Meanwhile, the blood-flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR-IIb tumor imaging of a live mouse was explored. NIR-IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700 nm for high-performance in vivo optical imaging.

Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo-spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel-specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia-inducible factor (HIF)-1a levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo. Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.