A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate.

VIDEO ABSTRACT: Aging is a complex process that affects multiple organs. Modeling aging and age-related diseases in the lab is challenging because classical vertebrate models have relatively long lifespans. Here, we develop the first platform for rapid exploration of age-dependent traits and diseases in vertebrates, using the naturally short-lived African turquoise killifish. We provide an integrative genomic and genome-editing toolkit in this organism using our de-novo-assembled genome and the CRISPR/Cas9 technology. We mutate many genes encompassing the hallmarks of aging, and for a subset, we produce stable lines within 2-3 months. As a proof of principle, we show that fish deficient for the protein subunit of telomerase exhibit the fastest onset of telomere-related pathologies among vertebrates. We further demonstrate the feasibility of creating specific genetic variants. This genome-to-phenotype platform represents a unique resource for studying vertebrate aging and disease in a high-throughput manner and for investigating candidates arising from human genome-wide studies.

The African Turquoise Killifish Genome Provides Insights into Evolution and Genetic Architecture of Lifespan.

Lifespan is a remarkably diverse trait ranging from a few days to several hundred years in nature, but the mechanisms underlying the evolution of lifespan differences remain elusive. Here we de novo assemble a reference genome for the naturally short-lived African turquoise killifish, providing a unique resource for comparative and experimental genomics. The identification of genes under positive selection in this fish reveals potential candidates to explain its compressed lifespan. Several aging genes are under positive selection in this short-lived fish and long-lived species, raising the intriguing possibility that the same gene could underlie evolution of both compressed and extended lifespans. Comparative genomics and linkage analysis identify candidate genes associated with lifespan differences between various turquoise killifish strains. Remarkably, these genes are clustered on the sex chromosome, suggesting that short lifespan might have co-evolved with sex determination. Our study provides insights into the evolutionary forces that shape lifespan in nature.

A human autoimmune organoid model reveals IL-7 function in coeliac disease.

In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.

Clotting factor genes are associated with preeclampsia in high-altitude pregnant women in the Peruvian Andes.

Preeclampsia is a multi-organ complication of pregnancy characterized by sudden hypertension and proteinuria that is among the leading causes of preterm delivery and maternal morbidity and mortality worldwide. The heterogeneity of preeclampsia poses a challenge for understanding its etiology and molecular basis. Intriguingly, risk for the condition increases in high-altitude regions such as the Peruvian Andes. To investigate the genetic basis of preeclampsia in a population living at high altitude, we characterized genome-wide variation in a cohort of preeclamptic and healthy Andean families (n = 883) from Puno, Peru, a city located above 3,800 meters of altitude. Our study collected genomic DNA and medical records from case-control trios and duos in local hospital settings. We generated genotype data for 439,314 SNPs, determined global ancestry patterns, and mapped associations between genetic variants and preeclampsia phenotypes. A transmission disequilibrium test (TDT) revealed variants near genes of biological importance for placental and blood vessel function. The top candidate region was found on chromosome 13 of the fetal genome and contains clotting factor genes PROZ, F7, and F10. These findings provide supporting evidence that common genetic variants within coagulation genes play an important role in preeclampsia. A selection scan revealed a potential adaptive signal around the ADAM12 locus on chromosome 10, implicated in pregnancy disorders. Our discovery of an association in a functional pathway relevant to pregnancy physiology in an understudied population of Native American origin demonstrates the increased power of family-based study design and underscores the importance of conducting genetic research in diverse populations.

Uterine injury during diestrus leads to placental and embryonic defects in future pregnancies in mice†.

Uterine injury from procedures such as Cesarean sections (C-sections) often have severe consequences on subsequent pregnancy outcomes, leading to disorders such as placenta previa, placenta accreta, and infertility. With rates of C-section at ~30% of deliveries in the USA and projected to continue to climb, a deeper understanding of the mechanisms by which these pregnancy disorders arise and opportunities for intervention are needed. Here we describe a rodent model of uterine injury on subsequent in utero outcomes. We observed three distinct phenotypes: increased rates of resorption and death, embryo spacing defects, and placenta accreta-like features of reduced decidua and expansion of invasive trophoblasts. We show that the appearance of embryo spacing defects depends entirely on the phase of estrous cycle at the time of injury. Using RNA-seq, we identified perturbations in the expression of components of the COX/prostaglandin pathway after recovery from injury, a pathway that has previously been demonstrated to play an important role in embryo spacing. Therefore, we demonstrate that uterine damage in this mouse model causes morphological and molecular changes that ultimately lead to placental and embryonic developmental defects.

PRG2 and AQPEP are misexpressed in fetal membranes in placenta previa and percreta†.

The obstetrical conditions placenta accreta spectrum (PAS) and placenta previa are a significant source of pregnancy-associated morbidity and mortality, yet the specific molecular and cellular underpinnings of these conditions are not known. In this study, we identified misregulated gene expression patterns in tissues from placenta previa and percreta (the most extreme form of PAS) compared with control cases. By comparing this gene set with existing placental single-cell and bulk RNA-Seq datasets, we show that the upregulated genes predominantly mark extravillous trophoblasts. We performed immunofluorescence on several candidate molecules and found that PRG2 and AQPEP protein levels are upregulated in both the fetal membranes and the placental disk in both conditions. While this increased AQPEP expression remains restricted to trophoblasts, PRG2 is mislocalized and is found throughout the fetal membranes. Using a larger patient cohort with a diverse set of gestationally aged-matched controls, we validated PRG2 as a marker for both previa and PAS and AQPEP as a marker for only previa in the fetal membranes. Our findings suggest that the extraembryonic tissues surrounding the conceptus, including both the fetal membranes and the placental disk, harbor a signature of previa and PAS that is characteristic of EVTs and that may reflect increased trophoblast invasiveness.

Structural basis of histone H2A-H2B recognition by the essential chaperone FACT.

Facilitates chromatin transcription (FACT) is a conserved histone chaperone that reorganizes nucleosomes and ensures chromatin integrity during DNA transcription, replication and repair. Key to the broad functions of FACT is its recognition of histones H2A-H2B (ref. 2). However, the structural basis for how histones H2A-H2B are recognized and how this integrates with the other functions of FACT, including the recognition of histones H3-H4 and other nuclear factors, is unknown. Here we reveal the crystal structure of the evolutionarily conserved FACT chaperone domain Spt16M from Chaetomium thermophilum, in complex with the H2A-H2B heterodimer. A novel 'U-turn' motif scaffolded onto a Rtt106-like module embraces the α1 helix of H2B. Biochemical and in vivo assays validate the structure and dissect the contribution of histone tails and H3-H4 towards Spt16M binding. Furthermore, we report the structure of the FACT heterodimerization domain that connects FACT to replicative polymerases. Our results show that Spt16M makes several interactions with histones, which we suggest allow the module to invade the nucleosome gradually and block the strongest interaction of H2B with DNA. FACT would thus enhance 'nucleosome breathing' by re-organizing the first 30 base pairs of nucleosomal histone-DNA contacts. Our snapshot of the engagement of the chaperone with H2A-H2B and the structures of all globular FACT domains enable the high-resolution analysis of the vital chaperoning functions of FACT, shedding light on how the complex promotes the activity of enzymes that require nucleosome reorganization.

Architecture of the human XPC DNA repair and stem cell coactivator complex.

The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair.

Functional and mechanistic studies of XPC DNA-repair complex as transcriptional coactivator in embryonic stem cells.

The embryonic stem cell (ESC) state is transcriptionally controlled by OCT4, SOX2, and NANOG with cofactors, chromatin regulators, noncoding RNAs, and other effectors of signaling pathways. Uncovering components of these regulatory circuits and their interplay provides the knowledge base to deploy ESCs and induced pluripotent stem cells. We recently identified the DNA-repair complex xeroderma pigmentosum C (XPC)-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4/SOX2 transcriptional activation. Here we investigate the role of SCC genome-wide in murine ESCs by mapping regions bound by RAD23B and analyzing transcriptional profiles of SCC-depleted ESCs. We establish OCT4 and SOX2 as the primary transcription factors recruiting SCC to regulatory regions of pluripotency genes and identify the XPC subunit as essential for interaction with the two proteins. The present study reveals new mechanistic and functional aspects of SCC transcriptional activity, and thus underscores the diversified functions of this regulatory complex.

Mouse Surgical Model of Mechanical Uterine Injury and Subsequent Embryo Defects.

Uterine injury from procedures such as Cesarean sections (C-sections) often have severe consequences on subsequent pregnancies, leading to disorders such as uterine placenta previa, placenta accreta spectrum (PAS), and Cesarean scar pregnancy. With rates of C-section at ∼30% of deliveries in the US and projected to continue to climb, an understanding of the mechanisms by which these pregnancy disorders arise and opportunities for intervention are sorely needed. However, there are currently very few animal models of uterine injury and its subsequent impacts on maternal as well as in utero and postnatal fetal outcomes. Here, we describe a procedure for a novel model of surgically induced uterine injury in the genetically tractable laboratory mouse (Mus musculus). We describe preparatory steps for surgery, the induction of uterine injury itself, and post-surgical recovery. We then provide supporting information regarding downstream dissection of pregnant mice. Lastly, we include additional information regarding estrous cycle staging in order to perform surgeries and dissections at the relevant phase in non-pregnant mice. This procedure for incurring uterine injury in a mouse model presents an important step forward in understanding uterine damage and its associated pregnancy disorders. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Preparation for surgery Basic Protocol 2: Surgery and induction of uterine injury Basic Protocol 3: Mating and dissection of pregnant mice as endpoint analyses Support Protocol: Estrous staging of animals.

Cancer organoids 2.0: modelling the complexity of the tumour immune microenvironment.

The development of neoplasia involves a complex and continuous interplay between malignantly transformed cells and the tumour microenvironment (TME). Cancer immunotherapies targeting the immune TME have been increasingly validated in clinical trials but response rates vary substantially between tumour histologies and are often transient, idiosyncratic and confounded by resistance. Faithful experimental models of the patient-specific tumour immune microenvironment, capable of recapitulating tumour biology and immunotherapy effects, would greatly improve patient selection, target identification and definition of resistance mechanisms for immuno-oncology therapeutics. In this Review, we discuss currently available and rapidly evolving 3D tumour organoid models that capture important immune features of the TME. We highlight diverse opportunities for organoid-based investigations of tumour immunity, drug development and precision medicine.

Glaucoma severity and medication adherence in a county hospital population.

OBJECTIVE: To assess the association between disease severity and adherence with glaucoma medications in a county hospital population. DESIGN: Cross-sectional study. PARTICIPANTS: A total of 126 patients diagnosed with glaucoma receiving intraocular pressure (IOP)-lowering medication were recruited from the San Francisco General Hospital Ophthalmology Clinic. METHODS: Subjects completed an oral questionnaire to assess demographic information, knowledge of glaucoma, and perceptions of glaucoma medication adherence. Glaucoma disease severity was classified according to the American Academy of Ophthalmology's Preferred Practice Pattern guidelines. Medication adherence was measured for each patient by obtaining pharmacy refill data and calculating medication possession ratio (MPR), that is, the ratio of total days' supply of medication during a 365-day period. Adherence was measured retrospectively over the 18-month period before study entry. Subjects with an MPR >80% were considered adherent. MAIN OUTCOME MEASURE: Medication adherence. RESULTS: Subjects with mild or moderate glaucoma were more likely to be nonadherent to their prescribed glaucoma medications than those with severe disease (adjusted odds ratio [OR], 1.54; 95% confidence interval [CI], 1.03-2.31; P = 0.04). Age, gender, race, education level, years of glaucoma, number of medications, and glaucoma diagnosis were not found to be statistically significantly associated with adherence. CONCLUSION: Patients with severe glaucoma were more likely to adhere to their topical IOP-lowering medication regimen than those with milder glaucomatous disease. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

A C-terminal motif targets Hedgehog to axons, coordinating assembly of the Drosophila eye and brain.

The developmental signal Hedgehog is distributed to two receptive fields by the photoreceptor neurons of the developing Drosophila retina. Delivery to the retina propagates ommatidial development across a precursor field. Transport along photoreceptor axons induces the development of postsynaptic neurons in the brain. Hedgehog is composed of N-terminal and C-terminal domains that dissociate in an autoproteolytic reaction that attaches cholesterol to the N-terminal cleavage product. Here, we show that the N-terminal domain is targeted to the retina when synthesized in the absence of the C-terminal domain. In contrast to studies that have focused on cholesterol as a determinant of subcellular localization, we find that the C-terminal domain harbors a conserved motif that overrides retinal localization, sending most of the autocleavage products into vesicles bound for growth cones or synapses. Competition between targeting signals at the opposite ends of Hedgehog apparently controls the match between eye and brain development.

Dynamics of CRISPR-Cas9 genome interrogation in living cells.

The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.

The association between compliance with recommended follow-up and glaucomatous disease severity in a county hospital population.

PURPOSE: To assess the association between insufficient follow-up and clinical parameters such as disease severity and medication use among glaucoma patients at a metropolitan county hospital. DESIGN: Cross-sectional study. METHODS: Two-hundred and six patients with established glaucoma were recruited from San Francisco General Hospital. Subjects were classified based on compliance with recommended follow-up examination intervals over the year preceding commencement of the study, as determined by patient medical records. Glaucoma severity was determined based on the American Academy of Ophthalmology Preferred Practice Patterns guidelines. Multivariate logistic regression analysis was used to assess the relationship between adherence with follow-up visits and disease severity. RESULTS: After adjustment for the impact of potential confounding variables, subjects with severe glaucomatous disease were found to have been less adherent to their recommended follow-up than those patients with mild or moderate glaucomatous disease (adjusted OR 1.89, 95% CI 1.21-2.94; P = .01). Subjects who were on glaucoma medications were found to be less adherent to follow-up recommendations (adjusted OR 3.29, 95% CI 1.41-7.65, P = .01). CONCLUSION: Subjects with poor follow-up adherence were significantly more likely to have severe glaucomatous disease, suggesting that poor follow-up may contribute to disease worsening or, alternatively, those with more severe disease are less inclined to follow up at appropriate intervals.

Mapping loci associated with tail color and sex determination in the short-lived fish Nothobranchius furzeri.

The African fish Nothobranchius furzeri is the shortest-lived vertebrate species that can reproduce in captivity, with a median life span of 9-11 weeks for the shortest-lived strain. Natural populations of N. furzeri display differences in life span, aging biomarkers, behavior, and color, which make N. furzeri a unique vertebrate system for studying the genetic basis of these traits. We mapped regions of the genome involved in sex determination and tail color by genotyping microsatellite markers in the F(2) progeny of a cross between a short-lived, yellow-tailed strain and a long-lived, red-tailed strain of N. furzeri. We identified one region linked with the yellow/red tail color that maps close to melanocortin 1 receptor (mc1r), a gene involved in pigmentation in several vertebrate species. Analysis of the segregation of sex-linked markers revealed that N. furzeri has a genetic sex determination system with males as the heterogametic sex and markedly reduced recombination in the male sex-determining region. Our results demonstrate that both naturally-evolved pigmentation differences and sex determination in N. furzeri are controlled by simple genetic mechanisms and set the stage for the molecular genetic dissection of factors underlying such traits. The microsatellite-based linkage map we developed for N. furzeri will also facilitate analysis of the genetic architecture of traits that characterize this group of vertebrates, including short life span and adaptation to extreme environmental conditions.