Department of Physiology, Anatomy and Genetics (DPAG)

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

<div>AbstractDefining 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 (VHL), which ultimately drives clear cell renal cell carcinoma. Vhl 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 Vhl 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 Vhl in kidney cells provide insights into VHL tumor suppressor action and development of renal cell carcinoma.Significance:Single-cell analysis of heterogeneous and dynamic responses to Vhl inactivation in the kidney suggests that early events shape the cell type specificity of oncogenesis, providing a focus for mechanistic understanding and therapeutic targeting.</div>

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

Vhl-null cells specifically undergo time-dependent alterations in gene expression

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

scRNA-seq metrics, cell type markers, and lists of differentially expressed genes

Supplementary Figure S1 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney

Single-cell RNA sequencing on flow-sorted renal cells

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

Biallelic Vhl inactivation entrains early cell-specific transcriptomic changes in RTE cells. A, Density plot depicting UMAP distribution of tdTomato-negative and -positive cells from kidneys of Control and KO mice harvested early after recombination. 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. C, 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. D, 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. E, PCA of gene expression changes early after Vhl inactivation in different renal cell identities. A–E, scRNA-seq data are shown for n = 3F, 1M for Control negative; n = 3F, 1M mice for Control positive samples; n = 2F, 1M mice for KO negative samples; n = 2F, 2M mice for KO-positive samples.

Supplementary Figure S1 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney

Single-cell RNA sequencing on flow-sorted renal cells

Figure 6 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney

Vhl-null cells exhibit time-dependent proliferation and association with ccRCC-like gene expression. A, Representative dual IHC for tdTomato (brown) and Ki67 (purple) counterstained with hematoxylin in kidneys of KO mice harvested early after recombination. Scale bar, 25 μm. Magnification, ×40. Black arrow, dual-positive cell; red arrow, tdTomato-negative Ki67-positive cell. B, Proportion of tdTomato-positive (top) or tdTomato-negative (bottom) cells that are positive for Ki67 by dual IHC in kidneys of Control and KO mice harvested early and late after recombination (n = 2F, 6M for Control early; n = 4F, 2M for KO early; n = 4F, 5M for Control late; n = 1F, 6M for KO late). Pairwise comparisons by Kruskal–Wallis test with Dunn correction. C, UMAP plot depicting RTE cells from Control and KO mice at the early and late time points. Orange, cells expressing Mki67. D, Proportion of tdTomato-positive (top) or tdTomato-negative (bottom) RTE cells that express Mki67 in different conditions. Pairwise comparisons tested by one-way ANOVA with Holm–Šídák correction. E, Proportion of tdTomato-positive cells of different PT identities that express Mki67 in different conditions. Pairwise comparisons tested by two-way ANOVA with Holm–Šídák correction. F, Violin plot overlaid with boxplot depicting expression score for genes upregulated in ccRCC cells known to be HIF targets (left) and not known to be HIF targets (right) in tdTomato-positive cells from Control and KO mice harvested at early or late time points. G, Scatter plot depicting changes in mean expression scores for HIF-target (top) and non-HIF-target (bottom) genes specifically upregulated in ccRCC, in tdTomato-positive cells of different PT identities from different conditions when compared with those from Control mice at the early time point. B, D, and E, Median and interquartile range plotted. Only significant (P < 0.05) comparisons shown. C–G, scRNA-seq data shown for n = 3F, 1M mice for tdTomato-positive and tdTomato-negative Control early and Control late samples; n = 2F, 1M mice for tdTomato-negative KO early samples; n = 2F, 2M mice for tdTomato-positive KO early samples; n = 2F, 2M mice for tdTomato-positive and tdTomato-negative KO late samples.

Figure 4 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney

Renal cortex, but not the papilla, is permissive to long-term survival of Vhl-null cells. A, Representative tdTomato IHC counterstained with hematoxylin in different renal anatomical regions of Control or KO mice harvested at the early or late time points. Scale bar, 100 μm. Magnification, ×20. B, Proportion of cells that are tdTomato-positive in different regions of the kidney as quantified by tdTomato IHC in kidneys from Control or KO mice harvested at different intervals after recombination. n = 7F, 16M for all regions for KO; n = 9F, 15M; 9F, 15M; 9F, 14M; 8F, 14M for cortex, outer medulla, inner medulla, and papilla, respectively, for Control. Line denotes linear regression. Significance testing performed for slope and intercept of linear regression by t test.

Figure 2 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney

scRNA-seq on flow-sorted renal cells. A, UMAP plot of tdTomato-negative (left) or tdTomato-positive cells (right) from kidneys of Control mice harvested at the early time point. Cells are colored by inferred cell type. LoH, loop of Henle; DCT, distal convoluted tubule; CD, collecting duct; PC, principal cell; IC, intercalated; PEC, parietal epithelial cell; VSMC, vascular smooth muscle cell; NK, natural killer cell. B, Proportion of sequenced cells inferred to be of each cell type in tdTomato-positive or tdTomato-negative populations from kidneys of Control mice harvested at the early time point. Median and interquartile range plotted. C, UMAP plot depicting expression of PT Module A (left) and PT Module B (right) genes in PT cells from Control mice. D, Representative in situ RNA hybridization exhibiting spatially distinct expression of Neat1 (blue) and Fxyd2 (red) mRNA in FFPE kidney cortex from Control mice harvested at the early time point. Scale bar, 10 μm. Magnification, ×40. E, UMAP plot depicting cells from Control mice at the early time point. Cells are colored by assigned PT Class. A–E, scRNA-seq data shown for n = 3 female (3F) and n = 1 male (1M) Control mice.

Figure 2 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to Vhl Inactivation in the Kidney

scRNA-seq on flow-sorted renal cells. A, UMAP plot of tdTomato-negative (left) or tdTomato-positive cells (right) from kidneys of Control mice harvested at the early time point. Cells are colored by inferred cell type. LoH, loop of Henle; DCT, distal convoluted tubule; CD, collecting duct; PC, principal cell; IC, intercalated; PEC, parietal epithelial cell; VSMC, vascular smooth muscle cell; NK, natural killer cell. B, Proportion of sequenced cells inferred to be of each cell type in tdTomato-positive or tdTomato-negative populations from kidneys of Control mice harvested at the early time point. Median and interquartile range plotted. C, UMAP plot depicting expression of PT Module A (left) and PT Module B (right) genes in PT cells from Control mice. D, Representative in situ RNA hybridization exhibiting spatially distinct expression of Neat1 (blue) and Fxyd2 (red) mRNA in FFPE kidney cortex from Control mice harvested at the early time point. Scale bar, 10 μm. Magnification, ×40. E, UMAP plot depicting cells from Control mice at the early time point. Cells are colored by assigned PT Class. A–E, scRNA-seq data shown for n = 3 female (3F) and n = 1 male (1M) Control mice.

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

<div>AbstractDefining 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 (VHL), which ultimately drives clear cell renal cell carcinoma. Vhl 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 Vhl 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 Vhl in kidney cells provide insights into VHL tumor suppressor action and development of renal cell carcinoma.Significance:Single-cell analysis of heterogeneous and dynamic responses to Vhl inactivation in the kidney suggests that early events shape the cell type specificity of oncogenesis, providing a focus for mechanistic understanding and therapeutic targeting.</div>

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

Biallelic Vhl loss entrains early cell-specific transcriptomic changes in renal tubular cells

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

scRNA-seq metrics, cell type markers, and lists of differentially expressed genes

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

A novel reporter model for Vhl inactivation in the mouse kidney. A, Design and recombination of the cell marking conditional Vhlpjr allele. Double and single arrows indicate reversible and irreversible processes, respectively. Vhlpjr.fl, Vhlpjr.inrec, and Vhlpjr.KO refer to “floxed,” “incompletely recombined,” and “knockout” forms of the Vhlpjr allele. P, Vhl promoter; U, untranslated region; E, Vhl exon; I, Vhl 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, Representative tdTomato IHC counterstained with hematoxylin in kidney sections and tdTomato fluorescence-based flow cytometry on renal cells from Vhlwt/pjr.fl; Pax8-CreERT2 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. C, Gel electrophoresis of genomic PCR for Vhlwt, Vhlpjr.fl, and Vhlpjr.KO alleles performed on FAC-sorted tdTomato-positive (left) or tdTomato-negative (right) cells from kidneys of Vhlwt/pjr.fl; Pax8-CreERT2 mice given tamoxifen and harvested at the early time point. D, Representative immunoblots (IB) for HIF1A, HIF2A, or tdTomato protein in tdTomato-negative (−) or tdTomato-positive (+) cells sorted by flow cytometry from dissociated kidneys of Vhljae.KO/pjr.fl or Vhlwt/pjr.fl Pax8-CreERT2 mice given 5 × 2 mg tamoxifen and harvested at the early time point (n = 3 per genotype).

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

A novel reporter model for Vhl inactivation in the mouse kidney. A, Design and recombination of the cell marking conditional Vhlpjr allele. Double and single arrows indicate reversible and irreversible processes, respectively. Vhlpjr.fl, Vhlpjr.inrec, and Vhlpjr.KO refer to “floxed,” “incompletely recombined,” and “knockout” forms of the Vhlpjr allele. P, Vhl promoter; U, untranslated region; E, Vhl exon; I, Vhl 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, Representative tdTomato IHC counterstained with hematoxylin in kidney sections and tdTomato fluorescence-based flow cytometry on renal cells from Vhlwt/pjr.fl; Pax8-CreERT2 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. C, Gel electrophoresis of genomic PCR for Vhlwt, Vhlpjr.fl, and Vhlpjr.KO alleles performed on FAC-sorted tdTomato-positive (left) or tdTomato-negative (right) cells from kidneys of Vhlwt/pjr.fl; Pax8-CreERT2 mice given tamoxifen and harvested at the early time point. D, Representative immunoblots (IB) for HIF1A, HIF2A, or tdTomato protein in tdTomato-negative (−) or tdTomato-positive (+) cells sorted by flow cytometry from dissociated kidneys of Vhljae.KO/pjr.fl or Vhlwt/pjr.fl Pax8-CreERT2 mice given 5 × 2 mg tamoxifen and harvested at the early time point (n = 3 per genotype).

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

Biallelic Vhl inactivation entrains early cell-specific transcriptomic changes in RTE cells. A, Density plot depicting UMAP distribution of tdTomato-negative and -positive cells from kidneys of Control and KO mice harvested early after recombination. 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. C, 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. D, 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. E, PCA of gene expression changes early after Vhl inactivation in different renal cell identities. A–E, scRNA-seq data are shown for n = 3F, 1M for Control negative; n = 3F, 1M mice for Control positive samples; n = 2F, 1M mice for KO negative samples; n = 2F, 2M mice for KO-positive samples.

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

Vhl-null cells specifically undergo time-dependent alterations in gene expression

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.

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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

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

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

Supplementary Figure 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

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

Supplementary Figure 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

Figure 6 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to <i>Vhl</i> Inactivation in the Kidney

Figure 4 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to <i>Vhl</i> Inactivation in the Kidney

Figure 2 from Oncogenic Cell Tagging and Single-Cell Transcriptomics Reveal Cell Type–Specific and Time-Resolved Responses to <i>Vhl</i> Inactivation in the Kidney