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Data from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to <i>Vhl</i> Inactivation in the Kidney

<div>Abstract<p>Defining the initial events in oncogenesis and the cellular responses they entrain, even in advance of morphologic abnormality, is a fundamental challenge in understanding cancer initiation. As a paradigm to address this, we longitudinally studied the changes induced by loss of the tumor suppressor gene von Hippel Lindau (<i>VHL</i>), which ultimately drives clear cell renal cell carcinoma. <i>Vhl</i> inactivation was directly coupled to expression of a tdTomato reporter within a single allele, allowing accurate visualization of affected cells in their native context and retrieval from the kidney for single-cell RNA sequencing. This strategy uncovered cell type–specific responses to <i>Vhl</i> inactivation, defined a proximal tubular cell class with oncogenic potential, and revealed longer term adaptive changes in the renal epithelium and the interstitium. Oncogenic cell tagging also revealed markedly heterogeneous cellular effects including time-limited proliferation and elimination of specific cell types. Overall, this study reports an experimental strategy for understanding oncogenic processes in which cells bearing genetic alterations can be generated in their native context, marked, and analyzed over time. The observed effects of loss of <i>Vhl</i> in kidney cells provide insights into VHL tumor suppressor action and development of renal cell carcinoma.</p>Significance:<p>Single-cell analysis of heterogeneous and dynamic responses to <i>Vhl</i> inactivation in the kidney suggests that early events shape the cell type specificity of oncogenesis, providing a focus for mechanistic understanding and therapeutic targeting.</p></div>

Supplementary Figure S2 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to <i>Vhl</i> Inactivation in the Kidney

<p>Biallelic Vhl loss entrains early cell-specific transcriptomic changes in renal tubular cells</p>

Supplementary Table S1 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to <i>Vhl</i> Inactivation in the Kidney

<p>scRNA-seq metrics, cell type markers, and lists of differentially expressed genes</p>

Figure 1 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to <i>Vhl</i> Inactivation in the Kidney

<p>A novel reporter model for <i>Vhl</i> inactivation in the mouse kidney. <b>A,</b> Design and recombination of the cell marking conditional <i>Vhl^pjr</i> allele. Double and single arrows indicate reversible and irreversible processes, respectively. <i>Vhl^pjr.fl, Vhl^pjr.inrec</i>, and <i>Vhl^pjr.KO</i> refer to “floxed,” “incompletely recombined,” and “knockout” forms of the <i>Vhl^pjr</i> allele. P, <i>Vhl</i> promoter; U, untranslated region; E, <i>Vhl</i> exon; I, <i>Vhl</i> intron; pA, polyadenylation site; P2A, porcine teschovirus 2A peptide; SA, splice acceptor. Dashed lines, spliced and translated regions; lightning symbols, excitation and emission wavelengths for tdTomato fluorescence. Red stop sign indicates no interaction between VHL exon 1 fragment and HIFA-1/2 or Elongin B/C. <b>B,</b> Representative tdTomato IHC counterstained with hematoxylin in kidney sections and tdTomato fluorescence-based flow cytometry on renal cells from <i>Vhl^wt/pjr.fl; Pax8-CreERT2</i> mice untreated (top) or given 5 × 2 mg tamoxifen (TMX; bottom) and harvested at the early time point. Scale bar, 250 μm. Magnification, ×20. FACS gates are shown. <b>C,</b> Gel electrophoresis of genomic PCR for <i>Vhl^wt, Vhl^pjr.fl</i>, and <i>Vhl^pjr.KO</i> alleles performed on FAC-sorted tdTomato-positive (left) or tdTomato-negative (right) cells from kidneys of <i>Vhl^wt/pjr.fl; Pax8-CreERT2</i> mice given tamoxifen and harvested at the early time point. <b>D,</b> Representative immunoblots (IB) for HIF1A, HIF2A, or tdTomato protein in tdTomato-negative (−) or tdTomato-positive (+) cells sorted by flow cytometry from dissociated kidneys of <i>Vhl^{jae.KO/pjr.fl}</i> or <i>Vhl^wt/pjr.fl Pax8-CreERT2</i> mice given 5 × 2 mg tamoxifen and harvested at the early time point (<i>n</i> = 3 per genotype).</p>

Figure 1 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to <i>Vhl</i> Inactivation in the Kidney

<p>A novel reporter model for <i>Vhl</i> inactivation in the mouse kidney. <b>A,</b> Design and recombination of the cell marking conditional <i>Vhl^pjr</i> allele. Double and single arrows indicate reversible and irreversible processes, respectively. <i>Vhl^pjr.fl, Vhl^pjr.inrec</i>, and <i>Vhl^pjr.KO</i> refer to “floxed,” “incompletely recombined,” and “knockout” forms of the <i>Vhl^pjr</i> allele. P, <i>Vhl</i> promoter; U, untranslated region; E, <i>Vhl</i> exon; I, <i>Vhl</i> intron; pA, polyadenylation site; P2A, porcine teschovirus 2A peptide; SA, splice acceptor. Dashed lines, spliced and translated regions; lightning symbols, excitation and emission wavelengths for tdTomato fluorescence. Red stop sign indicates no interaction between VHL exon 1 fragment and HIFA-1/2 or Elongin B/C. <b>B,</b> Representative tdTomato IHC counterstained with hematoxylin in kidney sections and tdTomato fluorescence-based flow cytometry on renal cells from <i>Vhl^wt/pjr.fl; Pax8-CreERT2</i> mice untreated (top) or given 5 × 2 mg tamoxifen (TMX; bottom) and harvested at the early time point. Scale bar, 250 μm. Magnification, ×20. FACS gates are shown. <b>C,</b> Gel electrophoresis of genomic PCR for <i>Vhl^wt, Vhl^pjr.fl</i>, and <i>Vhl^pjr.KO</i> alleles performed on FAC-sorted tdTomato-positive (left) or tdTomato-negative (right) cells from kidneys of <i>Vhl^wt/pjr.fl; Pax8-CreERT2</i> mice given tamoxifen and harvested at the early time point. <b>D,</b> Representative immunoblots (IB) for HIF1A, HIF2A, or tdTomato protein in tdTomato-negative (−) or tdTomato-positive (+) cells sorted by flow cytometry from dissociated kidneys of <i>Vhl^{jae.KO/pjr.fl}</i> or <i>Vhl^wt/pjr.fl Pax8-CreERT2</i> mice given 5 × 2 mg tamoxifen and harvested at the early time point (<i>n</i> = 3 per genotype).</p>

Figure 3 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to <i>Vhl</i> Inactivation in the Kidney

<p>Biallelic <i>Vhl</i> inactivation entrains early cell-specific transcriptomic changes in RTE cells. <b>A,</b> Density plot depicting UMAP distribution of tdTomato-negative and -positive cells from kidneys of Control and KO mice harvested early after recombination. <b>B,</b> Left, UMAP plot depicting cells from Control and KO mice harvested early after recombination colored by UMAP clusters. Right, proportion of cells from each condition belonging to any cluster. <b>C,</b> Scatter plot depicting frequency of expression in tdTomato-negative (top) or tdTomato-positive (bottom) cells from KO mice against log₂-fold change (log₂FC) between cells from KO versus Control mice for all genes at the early time point. Orange, significantly regulated genes. Genes explicitly mentioned in the main text are labeled. <b>D,</b> Scatter plot depicting log₂-fold change between tdTomato-positive cells from KO versus Control for genes significantly regulated in every renal cell identity. Blue, names of HIF target genes. <b>E,</b> PCA of gene expression changes early after <i>Vhl</i> inactivation in different renal cell identities. <b>A–E,</b> scRNA-seq data are shown for <i>n</i> = 3F, 1M for Control negative; <i>n</i> = 3F, 1M mice for Control positive samples; <i>n</i> = 2F, 1M mice for KO negative samples; <i>n</i> = 2F, 2M mice for KO-positive samples.</p>

Supplementary Figure S3 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to <i>Vhl</i> Inactivation in the Kidney

<p>Vhl-null cells specifically undergo time-dependent alterations in gene expression</p>

Future avenues in Drosophila mushroom body research.

How does the brain translate sensory information into complex behaviors? With relatively small neuronal numbers, readable behavioral outputs, and an unparalleled genetic toolkit, the Drosophila mushroom body (MB) offers an excellent model to address this question in the context of associative learning and memory. Recent technological breakthroughs, such as the freshly completed full-brain connectome, multiomics approaches, CRISPR-mediated gene editing, and machine learning techniques, led to major advancements in our understanding of the MB circuit at the molecular, structural, physiological, and functional levels. Despite significant progress in individual MB areas, the field still faces the fundamental challenge of resolving how these different levels combine and interact to ultimately control the behavior of an individual fly. In this review, we discuss various aspects of MB research, with a focus on the current knowledge gaps, and an outlook on the future methodological developments required to reach an overall view of the neurobiological basis of learning and memory.

Appreciation of Sir Tom Blundell.

Chronic insomnia, REM sleep instability and emotional dysregulation: A pathway to anxiety and depression?

The world-wide prevalence of insomnia disorder reaches up to 10% of the adult population. Women are more often afflicted than men, and insomnia disorder is a risk factor for somatic and mental illness, especially depression and anxiety disorders. Persistent hyperarousals at the cognitive, emotional, cortical and/or physiological levels are central to most theories regarding the pathophysiology of insomnia. Of the defining features of insomnia disorder, the discrepancy between minor objective polysomnographic alterations of sleep continuity and substantive subjective impairment in insomnia disorder remains enigmatic. Microstructural alterations, especially in rapid eye movement sleep ("rapid eye movement sleep instability"), might explain this mismatch between subjective and objective findings. As rapid eye movement sleep represents the most highly aroused brain state during sleep, it might be particularly prone to fragmentation in individuals with persistent hyperarousal. In consequence, mentation during rapid eye movement sleep may be toned more as conscious-like wake experience, reflecting pre-sleep concerns. It is suggested that this instability of rapid eye movement sleep is involved in the mismatch between subjective and objective measures of sleep in insomnia disorder. Furthermore, as rapid eye movement sleep has been linked in previous works to emotional processing, rapid eye movement sleep instability could play a central role in the close association between insomnia and depressive and anxiety disorders.

MARCO in alveolar macrophages negatively regulates Ace expression and aldosterone production

Dopamine encoding of novelty facilitates efficient uncertainty-driven exploration.

When facing an unfamiliar environment, animals need to explore to gain new knowledge about which actions provide reward, but also put the newly acquired knowledge to use as quickly as possible. Optimal reinforcement learning strategies should therefore assess the uncertainties of these action-reward associations and utilise them to inform decision making. We propose a novel model whereby direct and indirect striatal pathways act together to estimate both the mean and variance of reward distributions, and mesolimbic dopaminergic neurons provide transient novelty signals, facilitating effective uncertainty-driven exploration. We utilised electrophysiological recording data to verify our model of the basal ganglia, and we fitted exploration strategies derived from the neural model to data from behavioural experiments. We also compared the performance of directed exploration strategies inspired by our basal ganglia model with other exploration algorithms including classic variants of upper confidence bound (UCB) strategy in simulation. The exploration strategies inspired by the basal ganglia model can achieve overall superior performance in simulation, and we found qualitatively similar results in fitting model to behavioural data compared with the fitting of more idealised normative models with less implementation level detail. Overall, our results suggest that transient dopamine levels in the basal ganglia that encode novelty could contribute to an uncertainty representation which efficiently drives exploration in reinforcement learning.

Many paths lead to immunology.

While some people pore over the textbook and train through the classics of the field, many scientists come to immunology when they discover it intersecting with their "first love" interests. Five of these "accidental immunologists" tell us how they found their way to a fascination with the immune system.

TopOMetry systematically learns and evaluates the latent dimensions of single-cell atlases

OCULAR NECESSITIES: A NEUROETHOLOGICAL PERSPECTIVE ON VERTEBRATE VISUAL DEVELOPMENT.

BACKGROUND: By examining species-specific innate behaviours, neuroethologists have characterised unique neural strategies and specializations from throughout the animal kingdom. Simultaneously, the field of evolutionary developmental biology (informally, "evo-devo") seeks to make inferences about animals' evolutionary histories through careful comparison of developmental processes between species, because evolution is the evolution of development. Yet despite the shared focus on cross-species comparisons, there is surprisingly little cross-talk between these two fields. Insights can be gleaned at the intersection of neuroethology and evo-devo. Every animal develops within an environment, wherein ecological pressures advantage some behaviours and disadvantage others. These pressures are reflected in the neurodevelopmental strategies employed by different animals across taxa. SUMMARY: Vision is a system of particular interest for studying the adaptation of animals to their environments. The visual system enables a wide variety of animals across the vertebrate lineage to interact with their environments, presenting a fantastic opportunity to examine how ecological pressures have shaped animals' behaviours and developmental strategies. Applying a neuroethological lens to the study of visual development, we advance a novel theory that accounts for the evolution of spontaneous retinal waves, an important phenomenon in the development of the visual system, across the vertebrate lineage. KEY MESSAGES: We synthesise literature on spontaneous retinal waves from across the vertebrate lineage. We find that ethological considerations explain some cross-species differences in the dynamics of retinal waves. In zebrafish, retinal waves may be more important for the development of the retina itself, rather than the retinofugal projections. We additionally suggest empirical tests to determine whether Xenopus laevis experiences retinal waves.

THYMOSIN B4 MEDIATES VASCULAR PROTECTION VIA REGULATION OF LOW DENSITY LIPOPROTEIN RECEPTOR RELATED PROTEIN 1 (LRP1)

THYMOSIN β4-A NOVEL REGULATOR OF LOW DENSITY LIPOPROTEIN RECEPTOR RELATED PROTEIN 1 (LRP1) IN VASCULAR DISEASE

Thymosin β4 mediates vascular protection via regulation of Low Density Lipoprotein Related Protein 1 (LRP1): Supplemental Figures 1-10

Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). However, injury-induced growth factors stimulate a contractile-synthetic phenotypic switch which promotes atherosclerosis and susceptibility to abdominal aortic aneurysm (AAA). As a regulator of embryonic VSMC differentiation, we hypothesised that Thymosin β4 may additionally function to maintain healthy vasculature and protect against disease throughout postnatal life. This was supported by identification of an interaction with Low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of PDGF-BB signalling and VSMC proliferation. LRP1 variants have been identified by GWAS as major risk loci for AAA and coronary artery disease. Tβ4-null mice display aortic VSMC and elastin defects, phenocopying LRP1 mutants and suggesting compromised vascular integrity. We confirmed predisposition to disease in models of atherosclerosis and AAA. Diseased vessels and plaques were characterised by accelerated contractile-synthetic VSMC switching and augmented PDGFRβ signalling. In vitro, enhanced sensitivity to PDGF-BB, upon loss of Tβ4, coincided with dysregulated endocytosis, leading to increased recycling of LRP1-PDGFRβ and reduced lysosomal targeting. Our study identifies Tβ4 as a key regulator of LRP1 for maintaining vascular health, providing insight which may reveal useful therapeutic targets for modulation of VSMC phenotypic switching and disease progression.

Thymosin β4 preserves vascular smooth muscle phenotype in atherosclerosis via regulation of low density lipoprotein related protein 1 (LRP1).

Atherosclerosis is a progressive, degenerative vascular disease and a leading cause of morbidity and mortality. In response to endothelial damage, platelet derived growth factor (PDGF)-BB induced phenotypic modulation of medial smooth muscle cells (VSMCs) promotes atherosclerotic lesion formation and destabilisation of the vessel wall. VSMC sensitivity to PDGF-BB is determined by endocytosis of Low density lipoprotein receptor related protein 1 (LRP1)-PDGFR β complexes to balance receptor recycling with lysosomal degradation. Consequently, LRP1 is implicated in various arterial diseases. Having identified Tβ4 as a regulator of LRP1-mediated endocytosis to protect against aortic aneurysm, we sought to determine whether Tβ4 may additionally function to protect against atherosclerosis, by regulating LRP1-mediated growth factor signalling. By single cell transcriptomic analysis, Tmsb4x, encoding Tβ4, strongly correlated with contractile gene expression and was significantly down-regulated in cells that adopted a modulated phenotype in atherosclerosis. We assessed susceptibility to atherosclerosis of global Tβ4 knockout mice using the ApoE-/- hypercholesterolaemia model. Inflammation, elastin integrity, VSMC phenotype and signalling were analysed in the aortic root and descending aorta. Tβ4KO; ApoE-/- mice develop larger atherosclerotic plaques than control mice, with medial layer degeneration characterised by accelerated VSMC phenotypic modulation. Defects in Tβ4KO; ApoE-/- mice phenocopied those in VSMC-specific LRP1 nulls and, moreover, were underpinned by hyperactivated LRP1-PDGFRβ signalling. We identify an atheroprotective role for endogenous Tβ4 in maintaining differentiated VSMC phenotype via LRP1-mediated PDGFRβ signalling.

Computed Cardiopulmonography for the Detection of Early Smoking-Related Changes in the Lungs of Young Individuals Who Smoke.