Embryogenesis and Adult Life in the Absence of Intrinsic Apoptosis Effectors BAX, BAK, and BOK.

Intrinsic apoptosis, reliant on BAX and BAK, has been postulated to be fundamental for morphogenesis, but its precise contribution to this process has not been fully explored in mammals. Our structural analysis of BOK suggests close resemblance to BAX and BAK structures. Notably, Bok-/-Bax-/-Bak-/- animals exhibited more severe defects and died earlier than Bax-/-Bak-/- mice, implying that BOK has overlapping roles with BAX and BAK during developmental cell death. By analyzing Bok-/-Bax-/-Bak-/- triple-knockout mice whose cells are incapable of undergoing intrinsic apoptosis, we identified tissues that formed well without this process. We provide evidence that necroptosis, pyroptosis, or autophagy does not substantially substitute for the loss of apoptosis. Albeit very rare, unexpected attainment of adult Bok-/-Bax-/-Bak-/- mice suggests that morphogenesis can proceed entirely without apoptosis mediated by these proteins and possibly without cell death in general.

The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.

Author Correction: The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Macrophage and neutrophil death programs differentially confer resistance to tuberculosis.

Apoptosis can potently defend against intracellular pathogens by directly killing microbes and eliminating their replicative niche. However, the reported ability of Mycobacterium tuberculosis to restrict apoptotic pathways in macrophages in vitro has led to apoptosis being dismissed as a host-protective process in tuberculosis despite a lack of in vivo evidence. Here we define crucial in vivo functions of the death receptor-mediated and BCL-2-regulated apoptosis pathways in mediating protection against tuberculosis by eliminating distinct populations of infected macrophages and neutrophils and priming T cell responses. We further show that apoptotic pathways can be targeted therapeutically with clinical-stage compounds that antagonize inhibitor of apoptosis (IAP) proteins to promote clearance of M. tuberculosis in mice. These findings reveal that any inhibition of apoptosis by M. tuberculosis is incomplete in vivo, advancing our understanding of host-protective responses to tuberculosis (TB) and revealing host pathways that may be targetable for treatment of disease.

Suv39h-catalyzed H3K9me3 is critical for euchromatic genome organization and the maintenance of gene transcription.

H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes in differentiation and development. Here, we investigate the molecular consequences of heterochromatin loss in cells deficient in both SUV39H1 and SUV39H2 (Suv39DKO), the major mammalian histone methyltransferase enzymes that catalyze heterochromatic H3K9me3 deposition. We reveal a paradoxical repression of protein-coding genes in Suv39DKO cells, with these differentially expressed genes principally in euchromatic (Tn5-accessible, H3K4me3- and H3K27ac-marked) rather than heterochromatic (H3K9me3-marked) or polycomb (H3K27me3-marked) regions. Examination of the three-dimensional (3D) nucleome reveals that transcriptomic dysregulation occurs in euchromatic regions close to the nuclear periphery in 3D space. Moreover, this transcriptomic dysregulation is highly correlated with altered 3D genome organization in Suv39DKO cells. Together, our results suggest that the nuclear lamina-tethering of Suv39-dependent H3K9me3 domains provides an essential scaffold to support euchromatic genome organization and the maintenance of gene transcription for healthy cellular function.

Loss of PHF6 causes spontaneous seizures, enlarged brain ventricles and altered transcription in the cortex of a mouse model of the Börjeson-Forssman-Lehmann intellectual disability syndrome.

Börjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked intellectual disability and endocrine disorder caused by pathogenic variants of plant homeodomain finger gene 6 (PHF6). An understanding of the role of PHF6 in vivo in the development of the mammalian nervous system is required to advance our knowledge of how PHF6 mutations cause BFLS. Here, we show that PHF6 protein levels are greatly reduced in cells derived from a subset of patients with BFLS. We report the phenotypic, anatomical, cellular and molecular characterization of the brain in males and females in two mouse models of BFLS, namely loss of Phf6 in the germline and nervous system-specific deletion of Phf6. We show that loss of PHF6 resulted in spontaneous seizures occurring via a neural intrinsic mechanism. Histological and morphological analysis revealed a significant enlargement of the lateral ventricles in adult Phf6-deficient mice, while other brain structures and cortical lamination were normal. Phf6 deficient neural precursor cells showed a reduced capacity for self-renewal and increased differentiation into neurons. Phf6 deficient cortical neurons commenced spontaneous neuronal activity prematurely suggesting precocious neuronal maturation. We show that loss of PHF6 in the foetal cortex and isolated cortical neurons predominantly caused upregulation of genes, including Reln, Nr4a2, Slc12a5, Phip and ZIC family transcription factor genes, involved in neural development and function, providing insight into the molecular effects of loss of PHF6 in the developing brain.

SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

SARS-CoV-2 mouse adaptation selects virulence mutations that cause TNF-driven age-dependent severe disease with human correlates.

The diversity of COVID-19 disease in otherwise healthy people, from seemingly asymptomatic infection to severe life-threatening disease, is not clearly understood. We passaged a naturally occurring near-ancestral SARS-CoV-2 variant, capable of infecting wild-type mice, and identified viral genomic mutations coinciding with the acquisition of severe disease in young adult mice and lethality in aged animals. Transcriptomic analysis of lung tissues from mice with severe disease elucidated a host antiviral response dominated mainly by interferon and IL-6 pathway activation in young mice, while in aged animals, a fatal outcome was dominated by TNF and TGF-ß signaling. Congruent with our pathway analysis, we showed that young TNF-deficient mice had mild disease compared to controls and aged TNF-deficient animals were more likely to survive infection. Emerging clinical correlates of disease are consistent with our preclinical studies, and our model may provide value in defining aberrant host responses that are causative of severe COVID-19.

Mutant TRP53 exerts a target gene-selective dominant-negative effect to drive tumor development.

Mutations in Trp53, prevalent in human cancer, are reported to drive tumorigenesis through dominant-negative effects (DNEs) over wild-type TRP53 function as well as neomorphic gain-of-function (GOF) activity. We show that five TRP53 mutants do not accelerate lymphomagenesis on a TRP53-deficient background but strongly synergize with c-MYC overexpression in a manner that distinguishes the hot spot Trp53 mutations. RNA sequencing revealed that the mutant TRP53 DNE does not globally repress wild-type TRP53 function but disproportionately impacts a subset of wild-type TRP53 target genes. Accordingly, TRP53 mutant proteins impair pathways for DNA repair, proliferation, and metabolism in premalignant cells. This reveals that, in our studies of lymphomagenesis, mutant TRP53 drives tumorigenesis primarily through the DNE, which modulates wild-type TRP53 function in a manner advantageous for neoplastic transformation.

DaXi-high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy.

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 µm axially over an imaging volume of 3,000 × 800 × 300 µm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development - up to nine embryos at a time.

BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage.

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.

The histone acetyltransferase HBO1 promotes efficient tip cell sprouting during angiogenesis.

Blood vessel growth and remodelling are essential during embryonic development and disease pathogenesis. The diversity of endothelial cells (ECs) is transcriptionally evident and ECs undergo dynamic changes in gene expression during vessel growth and remodelling. Here, we investigated the role of the histone acetyltransferase HBO1 (KAT7), which is important for activating genes during development and for histone H3 lysine 14 acetylation (H3K14ac). Loss of HBO1 and H3K14ac impaired developmental sprouting angiogenesis and reduced pathological EC overgrowth in the retinal endothelium. Single-cell RNA sequencing of retinal ECs revealed an increased abundance of tip cells in Hbo1-deficient retinas, which led to EC overcrowding in the retinal sprouting front and prevented efficient tip cell migration. We found that H3K14ac was highly abundant in the endothelial genome in both intra- and intergenic regions, suggesting that HBO1 acts as a genome organiser that promotes efficient tip cell behaviour necessary for sprouting angiogenesis. This article has an associated 'The people behind the papers' interview.

Real-time multi-angle projection imaging of biological dynamics.

We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on the fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.

Downregulation of the GHRH/GH/IGF1 axis in a mouse model of Börjeson-Forssman-Lehman syndrome.

Börjeson-Forssman-Lehmann syndrome (BFLS) is an intellectual disability and endocrine disorder caused by plant homeodomain finger 6 (PHF6) mutations. Individuals with BFLS present with short stature. We report a mouse model of BFLS, in which deletion of Phf6 causes a proportional reduction in body size compared with control mice. Growth hormone (GH) levels were reduced in the absence of PHF6. Phf6-/Y animals displayed a reduction in the expression of the genes encoding GH-releasing hormone (GHRH) in the brain, GH in the pituitary gland and insulin-like growth factor 1 (IGF1) in the liver. Phf6 deletion specifically in the nervous system caused a proportional growth defect, indicating a neuroendocrine contribution to the phenotype. Loss of suppressor of cytokine signaling 2 (SOCS2), a negative regulator of growth hormone signaling partially rescued body size, supporting a reversible deficiency in GH signaling. These results demonstrate that PHF6 regulates the GHRH/GH/IGF1 axis.

Artificial intelligence takes center stage: exploring the capabilities and implications of ChatGPT and other AI-assisted technologies in scientific research and education.

The emergence of large language models (LLMs) and assisted artificial intelligence (AI) technologies have revolutionized the way in which we interact with technology. A recent symposium at the Walter and Eliza Hall Institute explored the current practical applications of LLMs in medical research and canvassed the emerging ethical, legal and social implications for the use of AI-assisted technologies in the sciences. This paper provides an overview of the symposium's key themes and discussions delivered by diverse speakers, including early career researchers, group leaders, educators and policy-makers highlighting the opportunities and challenges that lie ahead for scientific researchers and educators as we continue to explore the potential of this cutting-edge and emerging technology.

Monosodium Urate Crystals Generate Nuclease-Resistant Neutrophil Extracellular Traps via a Distinct Molecular Pathway.

Neutrophil extracellular traps (NETs) and the cell death associated with it (NETosis) have been implicated in numerous diseases. Mechanistic studies of NETosis have typically relied on nonphysiological stimuli, such as PMA. The human disease of gout is caused by monosodium urate (MSU) crystals. We observed that DNA consistent with NETs is present in fluid from acutely inflamed joints of gout patients. NETs also coat the crystals found in uninflamed tophi of chronic gout patients. We developed a quantitative, live cell imaging assay, which measures the key features of NETosis, namely, cell death and chromatin decondensation. We show that MSU and other physiologically relevant crystals induce NETosis through a molecular pathway that is distinct from PMA and Candida hyphae. Crystals interact with lysosomes to induce NADPH oxidase-independent cell death, with postmortem chromatin decondensation mediated by neutrophil elastase. The resulting MSU-induced NETs are enriched for actin and are resistant to serum and DNase degradation. These findings demonstrate a distinct physiological NETosis pathway in response to MSU crystals, which coats MSU crystals in DNA that persists in tissues as gouty tophi.

Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner.

Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1ß. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1ß secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1ß release, was not essential for MLKL-dependent death or IL-1ß secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1ß cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.

Apoptosis regulates endothelial cell number and capillary vessel diameter but not vessel regression during retinal angiogenesis.

The growth of hierarchical blood vessel networks occurs by angiogenesis. During this process, new vessel growth is accompanied by the removal of redundant vessel segments by selective vessel regression ('pruning') and a reduction in endothelial cell (EC) density in order to establish an efficient, hierarchical network. EC apoptosis has long been recognised for its association with angiogenesis, but its contribution to this process has remained unclear. We generated mice in which EC apoptosis was blocked by tissue-specific deletion of the apoptosis effector proteins BAK and BAX. Using the retina as a model, we found that apoptosis made a minor contribution to the efficiency of capillary regression around arteries where apoptosis was most concentrated, but was otherwise dispensable for vessel pruning. Instead, apoptosis was necessary for the removal of non-perfused vessel segments and the reduction in EC density that occurs during vessel maturation. In the absence of apoptosis, increased EC density resulted in an increase in the diameter of capillaries, but not arteries or veins. Our findings show that apoptosis does not influence the number of vessels generated during angiogenesis. Rather it removes non-perfused vessel segments and regulates EC number during vessel maturation, which has vessel-specific consequences for vessel diameter.

Constitutive overexpression of TNF in BPSM1 mice causes iBALT and bone marrow nodular lymphocytic hyperplasia.

BPSM1 (Bone phenotype spontaneous mutant 1) mice develop severe polyarthritis and heart valve disease as a result of a spontaneous mutation in the Tnf gene. In these mice, the insertion of a retrotransposon in the 3' untranslated region of Tnf causes a large increase in the expression of the cytokine. We have found that these mice also develop inducible bronchus-associated lymphoid tissue (iBALT), as well as nodular lymphoid hyperplasia (NLH) in the bone marrow. Loss of TNFR1 prevents the development of both types of follicles, but deficiency of TNFR1 in the hematopoietic compartment only prevents the iBALT and not the NLH phenotype. We show that the development of arthritis and heart valve disease does not depend on the presence of the tertiary lymphoid tissues. Interestingly, while loss of IL-17 or IL-23 limits iBALT and NLH development to some extent, it has no effect on polyarthritis or heart valve disease in BPSM1 mice.