Biology subjects

Kopan, R.

Publications and source records attributed to Kopan, R..

13 recordsLinked to original sources

bioRxiv2025

Bridging the gap of late-gestation nephrogenesis using a non-human primate model

BackgroundHuman nephrogenesis is complete at 34-36 weeks gestation, with 60% of nephrons forming during the third trimester through lateral branch nephrogenesis (LBN). Currently, no mechanism exists for LBN as there are no late gestation human kidney transcriptional datasets. We hypothesized that a differentiated but dividing population of nephron progenitor cells (NPCs) would contribute to the amplification of nephrons in late gestation. We used the rhesus macaque, an established model of LBN, to help identify potential mechanisms. MethodsSingle-cell RNA-sequencing (scRNA-Seq) was performed on cortically-enriched fetal rhesus kidneys (n=9) from late second trimester and third trimester during LBN. This data was integrated with publicly available human scRNA-seq datasets from 8-18 weeks gestation kidneys (n=8) using state-of-the-art bioinformatics pipelines. Differentially expressed genes and ligand-receptor interactions were assessed and validated using RNAScopeTM on human and rhesus archival tissue. ResultsscRNA-Seq of 64,782 rhesus cells revealed 37 transcriptionally distinct cell populations, including 7,879 rhesus NPCs. Pseudotime analyses identified a late gestation-specific lineage branch of differentiated NPC in rhesus that was not observed in mid-gestation humans. Differential expression analyses identified increased SFRP1, FZD4, and TLE2 and decreased FZD7, SHISA2, SHISA3, and TLE4 within the late-gestation rhesus NPC compared to mid-gestation human NPC and increased SEMA3D within the rhesus ureteric bud (UB) tip, suggesting a compositional shift in WNT and SEMA signaling components within the naive NPC population during LBN. ConclusionThe rhesus macaque uniquely enables molecular studies of late-gestation primate nephrogenesis. Our study suggests the hypothesis that a transitional state of self-renewing NPCs supported by compositional shifts in key pathways may underlie the switch from branching phase nephrogenesis to lateral branch nephrogenesis and support ongoing nephron formation in late gestation. Translational statementNo transcriptional data exists for the late-gestation human kidney, during which 60% of the nephron endowment is formed through a primate-specific process called lateral branch nephrogenesis (LBN). In this study, we used single-cell RNA sequencing the late gestation rhesus macaque as a model for this developmental stage. Our findings suggest a potential mechanism for LBN, in which a transitional state of self-renewing nephron progenitor cells (NPCs) is supported by compositional shifts in key pathways, allowing for continued nephron formation in late gestation.

developmental biology↗

bioRxiv2025

A mouse model of Adams-Oliver Syndrome reveals defective Notch1 signaling in endothelial cells as a driver of pathogenesis

Adams-Oliver Syndrome (AOS) is a rare congenital disorder characterized by scalp, limb, and cardiovascular defects. While variants in the NOTCH1 receptor, DLL4 ligand, and RBPJ transcription factor have been implicated in AOS, the driving tissue types and molecular mechanisms by which these variants cause pathogenesis are unknown. Here, we used quantitative binding assays to show that AOS-associated RBPJ missense variants compromise DNA binding but not cofactor binding. These findings suggest that AOS-associated RBPJ variants do not function as loss-of-function alleles but instead act as dominant-negative proteins that sequester cofactors from DNA. Consistent with this idea, mice carrying an AOS-associated Rbpj allele develop dominant phenotypes that include lethality and cardiovascular defects in a Notch1 heterozygous background, whereas Notch1 and Rbpj compound heterozygous null alleles are well-tolerated. To facilitate studies into the key tissues driving AOS pathogenesis, we employed conditional genetics to isolate the contribution of the vasculature to the development of AOS-like phenotypes. Importantly, our studies show that expression of the Rbpj AOS allele in endothelial cells is both necessary and sufficient to cause lethality and cardiovascular defects. These data establish that defective Notch1 signaling in the vasculature is a key driver of pathogenesis in this AOS mouse model.

developmental biology↗

bioRxiv2024

Integrating collecting systems in kidney organoids through fusion of distal nephron to ureteric bud.

The kidney maintains homeostasis through an array of parallel nephrons, which all originate in development as isolated epithelial structures that later fuse through their distal poles to a system of collecting ducts (CD). This connection is required to generate functional nephrons by providing a pathway for excretion of metabolic waste and byproducts. Currently, methods for differentiating human pluripotent stem cells into kidney organoids generate nephrons that lack CDs and instead terminate as blind-ended tubules. Here we describe a developmentally inspired system that addresses this deficiency through assembly of induced nephrogenic mesenchyme with ureteric bud (UB) tissues, the embryonic building blocks of the kidneys collecting system. The UB progenitors grow and develop into a network of CDs within the organoid, and importantly, they functionally integrate with the nephrons through recapitulating fusion between the distal tubule and CD to create a continuous epithelial lumen. We further showed that proximal-distal nephron specification, fusion frequency, and maturation of the CD can be augmented through temporal manipulation of developmental signaling pathways. This work provides a platform for interrogating the principles and mechanisms underlying nephron-UB fusion and a framework for engineering unobstructed nephrons with patterned collecting systems, an important step toward the de novo generation of functional kidney tissue.

developmental biology↗

bioRxiv2024

Notch dimerization provides robustness against environmental insults and is required for vascular integrity.

The Notch intracellular domain (NICD) regulates gene expression during development and homeostasis in a transcription factor complex that binds DNA either as monomer, or cooperatively as dimers. Mice expressing Notch dimerization-deficient (NDD) alleles of Notch1 and Notch2 have defects in multiple tissues that are sensitized to environmental insults. Here, we report that cardiac phenotypes and DSS (Dextran Sodium Sulfate) sensitivity in NDD mice can be ameliorated by housing mice under hypo-allergenic conditions (food/bedding). However, compound heterozygote NDD mice (N1RA/-; N2RA/-) in hypo-allergenic conditions subsequently develop severe hydrocephalus and hemorrhages. Further analysis revealed multiple vascular phenotypes in NDD mice including leakage, malformations of brain vasculature, and vasodilation in kidneys, leading to demise around P21. This mouse model is thus a hypomorphic allele useful to analyze vascular phenotypes and gene-environment interactions. The possibility of a non-canonical Notch signal regulating barrier formation in the gut, skin, and blood systems is discussed.

developmental biology↗

bioRxiv2024

Notch dimerization contributes to maintenance of intestinal homeostasis by a mechanism involving HDAC2.

A tri-protein complex containing NICD, RBPj and MAML1 binds DNA as monomer or, cooperatively, as dimers to regulate transcription. Mice expressing Notch dimerization-deficient (DD) alleles of Notch1 and Notch2 (NDD) are sensitized to environmental insults but otherwise develop and age normally. Transcriptomic analysis of colonic spheroids uncovered no evidence of dimer-dependent target gene miss-regulation, confirmed impaired stem cell maintenance in-vitro, and discovered an elevated signature of epithelial innate immune response to symbionts, the likely underlying cause for heightened sensitivity in NDD mice. TurboID followed by quantitative nano-spray MS/MS mass-spectrometry analyses in a human colon carcinoma cell line expressing either NOTCH2DD or NOTCH2 revealed an unbalanced interactome, with reduced interaction of NOTCH2DD with the transcription machinery but relatively preserved interaction with the HDAC2 interactome suggesting modulation via cooperativity. To ask if HDAC2 activity contributes to Notch loss-of-function phenotypes, we used the HDAC2 inhibitor Valproic acid (VPA) and discovered it could prevent the intestinal consequences of gamma secretase inhibitor (DBZ or DAPT) treatment in mice and spheroids, suggesting synergy between HDAC activity and pro-differentiation program in intestinal stem cells.

developmental biology↗

bioRxiv2023

The unique function of Runx1 in skeletal muscle differentiation and regeneration is mediated by an ETS interaction domain

The conserved Runt-related (RUNX) transcription factor family are well-known master regulators of developmental and regenerative processes. Runx1 and Runx2 are both expressed in satellite cells (SC) and skeletal myotubes. Conditional deletion of Runx1 in adult SC negatively impacted self-renewal and impaired skeletal muscle maintenance. Runx1- deficient SC retain Runx2 expression but cannot support muscle regeneration in response to injury. To determine the unique molecular functions of Runx1 that cannot be compensated by Runx2 we deleted Runx1 in C2C12 that retain Runx2 expression and established that myoblasts differentiation was blocked in vitro due in part to ectopic expression of Mef2c, a target repressed by Runx1. Structure-function analysis demonstrated that the Ets-interacting MID/EID region of Runx1, absent from Runx2, is critical to regulating myoblasts proliferation, differentiation, and fusion. Analysis of in-house and published ChIP-seq datasets from Runx1 (T-cells, muscle) versus Runx2 (preosteoblasts) dependent tissue identified enrichment for a Ets:Runx composite site in Runx1-dependent tissues. Comparing ATACseq datasets from WT and Runx1KO C2C12 cells showed that the Ets:Runx composite motif was enriched in peaks open exclusively in WT cells compared to peaks unique to Runx1KO cells. Thus, engagement of a set of targets by the RUNX1/ETS complex define the non-redundant functions of Runx1.

developmental biology↗

bioRxiv2023

Reduced nephron endowment in the common Six2-TGCtg mouse line is due to Six3 misexpression by aberrant enhancer-promoter interactions in the transgene

Lifelong kidney function relies on the complement of nephrons generated during mammalian development from a mesenchymal nephron progenitor cell (NPC) population. Low nephron endowment confers increased susceptibility to chronic kidney disease. We asked whether reduced nephron numbers in the popular Six2TGC transgenic mouse line1 was due to disruption of a regulatory gene at the integration site or to ectopic expression of a gene(s) contained within the transgene. Targeted locus amplification identified integration of the Six2TGC transgene within an intron of Cntnap5a on chr1. We generated Hi-C datasets from NPCs isolated from the Six2TGCtg/+ mice, the Cited1CreERT2/+control mice, and the Six2TGCtg/+;Tsc1+/Flox,2 mice that exhibited restored nephron number compared with Six2TGCtg/+mice, and mapped the precise integration of Six2TGC and Cited1CreERT2transgenes to chr1 and chr14, respectively. No changes in topology, accessibility, or expression were observed within the 50-megabase region centered on Cntnap5a in Six2TGCtg/+ mice compared with control mice. By contrast, we identified an aberrant regulatory interaction between a Six2 distal enhancer and the Six3 promoter contained within the transgene. Increasing the Six2TGCtg to Six2 locus ratio or removing one Six2 allele in Six2TGCtg/+ mice, caused severe renal hypoplasia. Furthermore, CRISPR disruption of Six3 within the transgene (Six2TGC{Delta}Six3CT) restored nephron endowment to wildtype levels and abolished the stoichiometric effect. Data from genetic and biochemical studies together suggest that in Six2TGC, SIX3 interferes with SIX2 function in NPC renewal through its C-terminal domain. SignificanceUsing high-resolution chromatin conformation and accessibility datasets we mapped the integration site of two popular transgenes used in studies of nephron progenitor cells and kidney development. Aberrant enhancer-promoter interactions drive ectopic expression of Six3 in the Six2TGCtgline which was correlated with disruption of nephrogenesis. Disruption of Six3 within the transgene restored nephron numbers to control levels; further genetic and biochemical studies suggest that Six3 interferes with Six2-mediated regulation of NPC renewal.

developmental biology↗

bioRxiv2021

Revisiting The Role of Notch in Nephron Segmentation: Notch is required for proximal, not distal fate selection during mouse and human nephrogenesis

Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human iPSC to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the ability of Nicastrin-deficient hiPSCs-derived organoids to differentiate into TFA2B+ distal tubule and CDH1 connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors. SummaryNotch signaling acts in nephron segmentation to select early proximal, but not distal tubule fate downstream of a global role promoting epithelial growth and maturation in mouse and human.

developmental biology↗

bioRxiv2020

Runx1 Shapes the Chromatin Landscape Via a Cascade of Direct and Indirect Targets

Runt-related transcription factor 1 (Runx1) can act as both an activator and a repressor. Here we show that CRISPR-mediated deletion of Runx1 in an embryonic kidney-derived cell (mK4) results in large-scale genome-wide changes to chromatin accessibility and gene expression. Open chromatin regions near down-regulated loci are enriched for Runx sites, remain bound by Runx2, but lose chromatin accessibility and expression in Runx1 knockout cells. Unexpectedly, regions near upregulated genes are depleted of Runx sites and are instead enriched for Zeb transcription factor binding sites. Re-expressing Zeb2 in Runx1 knockout cells restores suppression. These data confirm that Runx1 activity is uniquely needed to maintain open chromatin at many loci, and demonstrate that genome-scale derepression is an indirect consequence of losing Runx1-dependent Zeb expression.

genomics↗

bioRxiv2020

Age-Dependent Changes in the Progenitor Translatome Coordinated in part by Tsc1 Increase Perception of Signaling Inputs to End Nephrogenesis

Mammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that in young niches, cellular Wnt agonists are poorly translated, Fgf20 levels are high and R-spondin levels are low, resulting in a pro self-renewal environment. By contrast, older niches are low in Fgf20 and high in R-spondin, with increased cellular translation of Wnt agonists, including the signalosome-promoting Tmem59. This suggests a hypothesis that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving differentiation. We show Tsc1 hemizygosity differentially promoted translation of Wnt antagonists over agonists, expanding a transitional (Six2+, Cited1+, Wnt4+) state and delaying the tipping point. As predicted by these findings, reducing Rspo3 dosage in nephron progenitors or Tmem59 globally increased nephron numbers in vivo.

developmental biology↗

bioRxiv2020

Exposure to Mites Sensitizes Intestinal Stem Cell Maintenance, Splenic Marginal Zone B Cell Homeostasis, And Heart Development to Notch Dosage and Cooperativity.

Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence paired sites (SPS) located near many Notch-regulated genes. While most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer-deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromising viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% DSS. The most striking phenotypes - gender imbalance and splenic marginal zone B cell lymphoma - emerged in combination with dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis, and is consistent with Notch-dependent anti-parasite immune responses being compromised in the dimer deficient animals. HighlightsO_LINotch dimerization has an in vivo role in contributing to intestinal homeostasis C_LIO_LILoss of cooperativity can manifest as Notch gain or loss of function phenotypes C_LIO_LIMite infestation exacerbates all phenotypes, triggers MZB hyperproliferation in mutant animals C_LIO_LIMite-infested mutant mice develop SMZL with age C_LI

developmental biology↗

bioRxiv2019

Notch signaling regulates Akap12 expression and primary cilia length during renal tubule morphogenesis.

Alagille syndrome patients present with loss of function mutations in either JAG1 or NOTCH2. About 40-50% of patients have kidney abnormalities, with multi-cystic, dysplastic kidneys being one of the more frequent kidney defects. Additionally, gain-of-function mutations in NOTCH2 are associated with cystic kidneys in Hajdu-Cheney syndrome patients. Conditional inactivation of Notch1, Notch2, or RBPJ within the nephrogenic lineage impairs nephrogenesis and produces proximal tubule cysts in mice. How perturbations in Notch signaling cause renal tubular cysts remains unclear. Here we have determined that inhibition of Notch signaling in the kidney increases Akap12 expression. Ectopic expression of Akap12 in renal epithelia results in abnormally long primary cilia similar to those observed in Notch-signal-deficiency. Both loss of Notch signaling and elevated Akap12 expression disrupt the ability of renal epithelial cells to form spherical structures with a single lumen when grown embedded in matrix. We conclude that Notch signaling regulates Akap12 expression to ensure normal primary cilia length and renal epithelial morphogenesis, and suggest that diseases associated with defective Notch signaling, such as Alagille syndrome, maybe mechanistically related to ciliopathies.\n\nTranslational StatementThe current study examines how a reduction in Notch signaling results in abnormal renal tubule formation, as occurs in Alagille Syndrome patients with mutations in JAG1 or NOTCH2. The finding that reduced Notch signaling results in abnormally long cilia is suggestive that some of the clinical manifestations in Alagille Syndrome, such as small cystic kidneys, may originate due to defective cilia function. Linking Notch to primary cilia also opens up the possibility that coinheritance of mutations in ciliopathy genes along with a mutation in JAG1 or NOTCH2 may enhance the severity of the clinical phenotypes such as cystic kidney disease and may explain the variable occurrence and onset of kidney disease among Alagille Syndrome patients.

cell biology↗

bioRxiv2019

Enhancer architecture sensitizes cell specific responses to Notch gene dose via a bind and discard mechanism

Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene dosage. Here, we show that inserting a single enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can unexpectedly induce a subset of Drosophila Notch haploinsufficiency phenotypes in an animal with wild type Notch gene dose. Mechanistically, this enhancer couples Notch DNA binding to degradation in a Cdk8-dependent, transcription-independent manner. Using mathematical modeling combined with quantitative trait and expression analysis, we show that tissues requiring long duration Notch signals are more sensitive to perturbations in Notch degradation compared to tissues relying upon short duration processes. These findings support a novel \"bind and discard\" mechanism in which enhancers with specific binding sites promote rapid Notch turnover, reduce Notch-dependent transcription at other loci, and thereby sensitize tissues to gene dose based upon signal duration.

developmental biology↗