Jag1 insufficiency alters liver fibrosis via T cell and hepatocyte differentiation defects.

Fibrosis contributes to tissue repair, but excessive fibrosis disrupts organ function. Alagille syndrome (ALGS, caused by mutations in JAGGED1) results in liver disease and characteristic fibrosis. Here, we show that Jag1Ndr/Ndr mice, a model for ALGS, recapitulate ALGS-like fibrosis. Single-cell RNA-seq and multi-color flow cytometry of the liver revealed immature hepatocytes and paradoxically low intrahepatic T cell infiltration despite cholestasis in Jag1Ndr/Ndr mice. Thymic and splenic regulatory T cells (Tregs) were enriched and Jag1Ndr/Ndr lymphocyte immune and fibrotic capacity was tested with adoptive transfer into Rag1-/- mice, challenged with dextran sulfate sodium (DSS) or bile duct ligation (BDL). Transplanted Jag1Ndr/Ndr lymphocytes were less inflammatory with fewer activated T cells than Jag1+/+ lymphocytes in response to DSS. Cholestasis induced by BDL in Rag1-/- mice with Jag1Ndr/Ndr lymphocytes resulted in periportal Treg accumulation and three-fold less periportal fibrosis than in Rag1-/- mice with Jag1+/+ lymphocytes. Finally, the Jag1Ndr/Ndr hepatocyte expression profile and Treg overrepresentation were corroborated in patients' liver samples. Jag1-dependent hepatic and immune defects thus interact to determine the fibrotic process in ALGS.

Jag1 represses Notch activation in lateral supporting cells and inhibits an outer hair cell fate in the medial cochlea.

Notch signaling patterns the cochlear organ of Corti, and patients with the JAG1/NOTCH2-related genetic disorder Alagille syndrome can thus experience hearing loss. We investigated the function of Jag1 in cochlear patterning and signaling using Jag1Ndr/Ndr mice, a model of Alagille syndrome. Jag1Ndr/Ndr mice exhibited expected vestibular and auditory deficits, a dose-dependent increase in ectopic inner hair cells, and a reduction in outer hair cells. Single cell RNA sequencing of the organ of Corti demonstrated a global dysregulation of genes associated with inner ear development and deafness. Analysis of individual cell types further revealed that Jag1 represses Notch activation in lateral supporting cells and demonstrated a function for Jag1 in gene regulation and development of outer hair cells. Surprisingly, ectopic "outer hair cell-like" cells were present in the medial compartment and pillar cell region of Jag1Ndr/Ndr cochleae, yet they exhibited location-dependent expression of the inner hair cell fate-determinant Tbx2, suggesting Jag1 is required for Tbx2 to drive inner hair cell commitment. This study thus identifies new roles for Jag1 in supporting cells, and in outer hair cell specification and positioning.

Host-pathogen interactions in the Plasmodium-infected mouse liver at spatial and single-cell resolution.

Upon infecting its vertebrate host, the malaria parasite initially invades the liver where it undergoes massive replication, whilst remaining clinically silent. The coordination of host responses across the complex liver tissue during malaria infection remains unexplored. Here, we perform spatial transcriptomics in combination with single-nuclei RNA sequencing over multiple time points to delineate host-pathogen interactions across Plasmodium berghei-infected liver tissues. Our data reveals significant changes in spatial gene expression in the malaria-infected tissues. These include changes related to lipid metabolism in the proximity to sites of Plasmodium infection, distinct inflammation programs between lobular zones, and regions with enrichment of different inflammatory cells, which we term 'inflammatory hotspots'. We also observe significant upregulation of genes involved in inflammation in the control liver tissues of mice injected with mosquito salivary gland components. However, this response is considerably delayed compared to that observed in P. berghei-infected mice. Our study establishes a benchmark for investigating transcriptome changes during host-parasite interactions in tissues, it provides informative insights regarding in vivo study design linked to infection and offers a useful tool for the discovery and validation of de novo intervention strategies aimed at malaria liver stage infection.

Jagged-mediated development and disease: Mechanistic insights and therapeutic implications for Alagille syndrome.

Notch signaling controls multiple aspects of embryonic development and adult homeostasis. Alagille syndrome is usually caused by a single mutation in the jagged canonical Notch ligand 1 (JAG1), and manifests with liver disease and cardiovascular symptoms that are a direct consequence of JAG1 haploinsufficiency. Recent insights into Jag1/Notch-controlled developmental and homeostatic processes explain how pathology develops in the hepatic and cardiovascular systems and, together with recent elucidation of mechanisms modulating liver regeneration, provide a basis for therapeutic efforts. Importantly, disease presentation can be regulated by genetic modifiers, that may also be therapeutically leverageable. Here, we summarize recent insights into how Jag1 controls processes of relevance to Alagille syndrome, focused on Jag1/Notch functions in hepatic and cardiovascular development and homeostasis.

Spatially segregated defects and IGF1-responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome.

BACKGROUND & AIMS: Alagille syndrome (ALGS) manifests with peripheral intrahepatic bile duct (IHBD) paucity, which can spontaneously resolve. In a model for ALGS, Jag1Ndr/Ndr mice, this occurs with distinct architectural mechanisms in hilar and peripheral IHBDs. Here, we investigated region-specific IHBD characteristics and addressed whether IGF1, a cholangiocyte mitogen that is downregulated in ALGS and in Jag1Ndr/Ndr mice, can improve biliary outcomes. METHODS: Intrahepatic cholangiocyte organoids (ICOs) were derived from hilar and peripheral adult Jag1+/+ and Jag1Ndr/Ndr livers (hICOs and pICOs, respectively). ICOs were grown in Matrigel or microwell arrays, and characterized using bulk RNA sequencing, immunofluorescence, and high throughput analyses of nuclear sizes. ICOs were treated with IGF1, followed by analyses of growth, proliferation, and death. CellProfiler and Python scripts were custom written for image analyses. Key results were validated in vivo by immunostaining. RESULTS: Cell growth assays and transcriptomics demonstrated that Jag1Ndr/Ndr ICOs were less proliferative than Jag1+/+ ICOs. IGF1 specifically rescued survival and growth of Jag1Ndr/Ndr pICOs. Jag1Ndr/Ndr hICOs were the least proliferative, with lower Notch signalling and an enrichment of hepatocyte signatures and IGF uptake/transport pathways. In vitro (Jag1Ndr/Ndr hICOs) and in vivo (Jag1Ndr/Ndr hilar portal tracts) analyses revealed ectopic HNF4a+ hepatocytes. CONCLUSIONS: Hilar and peripheral Jag1Ndr/Ndr ICOs exhibit differences in Notch signalling status, proliferation, and cholangiocyte commitment which may result in cholangiocyte-to-hepatocyte transdifferentiation. While Jag1Ndr/Ndr pICOs can be rescued by IGF1, hICOs are unresponsive, perhaps due to their hepatocyte-like state and/or expression of IGF transport components. IGF1 represents a potential therapeutic for peripheral bile ducts.

Efficient 3'-pairing renders microRNA targeting less sensitive to mRNA seed accessibility.

MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression by binding to specific sites in mRNAs. Site recognition is primarily mediated by the seed region (nucleotides g2-g8 in the miRNA), but pairing beyond the seed (3'-pairing) is important for some miRNA:target interactions. Here, we use SHAPE, luciferase reporter assays and transcriptomics analyses to study the combined effect of 3'-pairing and secondary structures in mRNAs on repression efficiency. Using the interaction between miR-34a and its SIRT1 binding site as a model, we provide structural and functional evidence that 3'-pairing can compensate for low seed-binding site accessibility, enabling repression of sites that would otherwise be ineffective. We show that miRNA 3'-pairing regions can productively base-pair with nucleotides far upstream of the seed-binding site and that both hairpins and unstructured bulges within the target site are tolerated. We use SHAPE to show that sequences that overcome inaccessible seed-binding sites by strong 3'-pairing adopt the predicted structures and corroborate the model using luciferase assays and high-throughput modelling of 8177 3'-UTR targets for six miRNAs. Finally, we demonstrate that PHB2, a target of miR-141, is an inaccessible target rescued by efficient 3'-pairing. We propose that these results could refine predictions of effective target sites.

RNA:RNA interaction in ternary complexes resolved by chemical probing.

RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.

Sex differences and risk factors for bleeding in Alagille syndrome.

Spontaneous bleeds are a leading cause of death in the pediatric JAG1-related liver disease Alagille syndrome (ALGS). We asked whether there are sex differences in bleeding events in patients, whether Jag1Ndr/Ndr mice display bleeds or vascular defects, and whether discovered vascular pathology can be confirmed in patients non-invasively. We performed a systematic review of patients with ALGS and vascular events following PRISMA guidelines, in the context of patient sex, and found significantly more girls than boys reported with spontaneous intracranial hemorrhage. We investigated vascular development, homeostasis, and bleeding in Jag1Ndr/Ndr mice, using retina as a model. Jag1Ndr/Ndr mice displayed sporadic brain bleeds, a thin skull, tortuous blood vessels, sparse arterial smooth muscle cell coverage in multiple organs, which could be aggravated by hypertension, and sex-specific venous defects. Importantly, we demonstrated that retinographs from patients display similar characteristics with significantly increased vascular tortuosity. In conclusion, there are clinically important sex differences in vascular disease in ALGS, and retinography allows non-invasive vascular analysis in patients. Finally, Jag1Ndr/Ndr mice represent a new model for vascular compromise in ALGS.

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting >95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. Following this protocol, it is possible to achieve transduction of >95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the mouse liver.

Reconstruction of heterogeneity through single cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we apply Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology.

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis.

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or "DUCT". DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Highly efficient manipulation of nervous system gene expression with NEPTUNE.

Genetic loss and gain of function in mice have typically been studied by using knockout or knockin mice that take months to years to generate. To address this problem for the nervous system, we developed NEPTUNE (NEural Plate Targeting by in Utero NanoinjEction) to rapidly and flexibly transduce the neural plate with virus prior to neurulation, and thus manipulate the future nervous system. Stable integration in >95% of cells in the brain enabled long-term overexpression, and conditional expression was achieved by using cell-type-specific MiniPromoters. Knockdown of Olig2 by using NEPTUNE recapitulated the phenotype of Olig2 -/- embryos. We used NEPTUNE to investigate Sptbn2, mutations in which cause spinocerebellar ataxia type 5. Sptbn2 knockdown induced dose-dependent defects in the neural tube, embryonic turning, and abdominal wall closure, previously unreported functions for Sptbn2. NEPTUNE thus offers a rapid and cost-effective technique to test gene function in the nervous system and can reveal phenotypes incompatible with life.

Single-cell transcriptomics of human embryos identifies multiple sympathoblast lineages with potential implications for neuroblastoma origin.

Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.

Mouse Models for Diseases in the Cholangiocyte Lineage.

Cholangiopathies are an important group of liver diseases affecting the biliary system, and the purpose of this review is to describe how diseases in the biliary system can be studied in mouse models. A particular focus is placed on mouse models for Alagille syndrome, a cholangiopathy with a strong genetic link to dysfunctional Notch signaling. Recently, a number of different genetic mouse models based on various manipulations of the Notch signaling pathway have been generated to study Alagille syndrome, and we discuss the resulting phenotypes, and possible causes for the phenotypic heterogeneity among the various models. In the final section, we provide a more general discussion on how well mouse models can be expected to mimic human liver disease, as well as an outlook toward the need for new technologies that can help us to gain new insights from mouse models for liver disease.

Do as I say, Not(ch) as I do: Lateral control of cell fate.

Breaking symmetry in populations of uniform cells, to induce adoption of an alternative cell fate, is an essential developmental mechanism. Similarly, domain and boundary establishment are crucial steps to forming organs during development. Notch signaling is a pathway ideally suited to mediating precise patterning cues, as both receptors and ligands are membrane-bound and can thus act as a precise switch to toggle cell fates on or off. Fine-tuning of signaling by positive or negative feedback mechanisms dictate whether signaling results in lateral induction or lateral inhibition, respectively, allowing Notch to either induce entire regions of cell specification, or dictate binary fate choices. Furthermore, pathway activity is modulated by Fringe modification of receptors or ligands, co-expression of receptors with ligands, mode of ligand presentation, and cell surface area in contact. In this review, we describe how Notch signaling is fine-tuned to mediate lateral induction or lateral inhibition cues, and discuss examples from C.elegans, D. melanogaster and M. musculus. Identifying the cellular machinery dictating the choice between lateral induction and lateral inhibition highlights the versatility of the Notch signaling pathway in development.