Biology subjects

Lim, H.-W.

Publications and source records attributed to Lim, H.-W..

17 recordsLinked to original sources

bioRxiv2026

HNF4A maintains proximal tubule identity and limits injury-associated cell states in the adult mouse kidney

Introduction: HNF4A is required for proximal tubule maturation during kidney development, but its role in maintaining proximal tubule identity in the adult kidney has not been defined. Following acute kidney injury, Hnf4a expression is rapidly suppressed in proximal tubule cells, coinciding with loss of mature epithelial features and activation of injury-associated transcriptional programs. It remains unknown whether HNF4A loss is sufficient to drive injury-associated transcriptional changes. Methods: We conditionally deleted Hnf4a in mature proximal tubules using Slc34a1Cre in mice and analyzed the consequences of Hnf4a loss in adult kidneys. To identify genes directly regulated by HNF4A, we performed proximal tubule-specific transcriptional profiling together with genome-wide mapping of HNF4A binding sites using CUT&RUN. Results: In Hnf4a mutant kidneys, HNF4A protein initially persisted in proximal tubules but was progressively lost from convoluted proximal tubule cells. Loss of Hnf4a resulted in downregulation of proximal tubule-specific transport and metabolic genes, accompanied by reactivation of developmental and injury-associated genes. CUT&RUN analysis revealed that HNF4A directly regulates genes predominantly involved in solute transport and metabolic processes. Conclusions: These findings identify HNF4A as a key regulator of proximal tubule identity and homeostasis in the adult mouse kidney. Genetic loss of Hnf4a in mature proximal tubules leads to loss of mature proximal tubule gene expression and activation of injury-associated transcriptional programs, implicating Hnf4a suppression as a potential contributor to maladaptive repair after kidney injury.

developmental biology↗

bioRxiv2026

Loss of Hnf1b in differentiated proximal tubule cells uncovers nephron segment plasticity

HNF1B is a transcription factor required for proximal tubule (PT) specification during kidney development, but whether it is also required to maintain PT identity after differentiation remains unknown. Using PT-specific genetic deletion in mice, we found that loss of Hnf1b in differentiated PT cells causes cyst formation and early postnatal lethality. PT-specific transcriptomic analysis revealed downregulation of PT-specific gene programs, including Hnf4a and PT-enriched transport and metabolic genes. Strikingly, Hnf1b-deficient PT cells ectopically activated podocyte-specific genes, including Wt1 and Nphs1, demonstrating that PT cells retain the capacity to engage alternative nephron segment programs when identity-stabilizing mechanisms are disrupted. In addition, loss of Hnf1b disrupted epithelial integrity, as evidenced by reduced epithelial adhesion gene expression and induction of mesenchymal markers. Wnt/{beta}-catenin signaling was also aberrantly activated, suggesting broader dysregulation of epithelial homeostasis. These findings establish HNF1B as a critical post-specification regulator of PT identity that sustains PT-specific transcriptional programs and actively suppresses alternative segmental identity programs.

developmental biology↗

bioRxiv2026

Circadian clock regulates intestinal epithelial cell differentiation via NOTCH/Hes1 oscillations

The circadian clock regulates diverse cellular processes, including intestinal epithelial cell (IEC) proliferation. However, mechanisms regulating clock-dependent IEC differentiation remain unknown. We performed a time course RNA-Seq using the mouse small intestine and identified NOTCH signaling, a key mechanism regulating IEC differentiation, as one of the pathways under the control of circadian rhythms. Using mouse enteroids, we discovered that a NOTCH reporter, Hes1-luciferase, exhibits both ultradian or circadian oscillations depending on the stemness of mouse enteroids. Furthermore, single-cell analysis of Hes1-mCherry revealed that the period of Hes1 oscillations varies widely, but circadian rhythms modulate the number of Hes1-mCherry+ cells in the population. Finally, we show that Paneth cell numbers fluctuate over the circadian cycle, suggesting that circadian clock-regulated Hes1 drives the circadian dynamics of IEC composition. Our study provides a deeper insight into circadian regulation of IEC differentiation, which will be critical for applications of chronotherapies for digestive diseases.

molecular biology↗

bioRxiv2026

Combinatorial pioneer transcription factor binding reinforces bivalent epigenetic states to preserve lineage fidelity

Cooperative and collaborative modes of transcription factor (TF) binding are central to eukaryotic gene activation, providing regulatory specificity and robustness in cell fate control. However, gene activation is only half the story of transcriptional regulation, and cells also require mechanisms to repress inappropriate gene programs. It remains largely unknown how broadly expressed repressive TFs and epigenetic regulators achieve context-specific repression required for proper cell differentiation. Here, we show that the repressive TF PRDM1 cooperates with lineage- and stage-specific pioneer TFs during human endoderm differentiation. Notably, the number of diverse pioneer TFs bound at enhancers determines the strength of PRDM1-mediated enhancer repression. Mechanistically, collaborative binding by multiple pioneer TFs synergistically promotes local nucleosome remodeling and stabilizes PRDM1 occupancy, culminating in the formation of hyper-bivalent enhancers that are characterized by strongly elevated levels of H3K4me1 and Polycomb-associated modifications. These enhancers reinforce the repression of alternative-lineage, and past and future developmental programs. Together, our findings redefine the roles of TF cooperation and chromatin accessibility beyond the prevailing activation-centric view. We propose a collaborative repression model in which diverse pioneer TFs establish focal chromatin accessibility that reinforces the context-specific assembly of repressive regulators, thereby safeguarding lineage and developmental fidelity.

genomics↗

bioRxiv2026

Esophageal epithelial cell-state transitions underlie the severity of pediatric eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a leading cause of chronic esophageal dysfunction driven by immune-mediated inflammation. Peak eosinophil count (PEC) in esophageal biopsies is routinely used to assess disease activity, and its associated molecular mechanisms have been well studied. However, PEC only partially captures overall disease severity, which is comprehensively captured by the Index of Severity for Eosinophilic Esophagitis (I-SEE). In contrast to PEC, the molecular and cellular programs associated with the I-SEE-defined disease severity, particularly in children, remain poorly understood. We integrated bulk transcriptomic profiling of pediatric esophageal biopsies with clinical severity metrics and a matched single-cell transcriptomic reference. Increasing severity was associated with a shift from type 2 inflammatory activation toward epithelial stress, cytoskeletal and junctional disruption, metabolic dysfunction, and extracellular matrix remodeling. Single-cell-informed analyses identified that proliferating and transitional epithelial cell states were strongly associated with higher I-SEE scores and exhibited impaired differentiation, heightened metabolic and oxidative stress responses, and structural remodeling programs not captured by bulk transcriptomic analyses alone. These findings reposition epithelial remodeling, rather than eosinophil burden alone, as a central molecular correlate of disease severity in pediatric EoE and provide a framework for improved disease stratification and therapeutic intervention.

immunology↗

bioRxiv2026

A knock-in Six2Cre line reveals transient interstitial potential in nephron progenitors

The developmental relationship between nephron progenitors and the renal interstitium remains unresolved, in part due to limitations of existing lineage tracing tools. The widely used transgenic Six2TGC line, which is routinely employed to target the nephron lineage, exhibits mosaic recombination and altered progenitor dynamics. To overcome these shortcomings, we generate a knock-in Six2Cre mouse allele that faithfully recapitulates endogenous Six2 expression, preserves nephron endowment, and achieves near-complete, non-mosaic recombination. Side-by-side lineage tracing with Six2Cre and Six2TGC, combined with RNA velocity analysis of single-cell RNA-sequencing datasets, reveals a brief interval around embryonic day 11 during which Six2-expressing mesenchymal nephron progenitors contribute to the renal interstitium. This contribution is transient and stage-restricted. These findings reveal an early dual potential within nephron progenitors and define a precise developmental window for dissecting mechanisms that coordinate nephron-interstitium integration.

developmental biology↗

bioRxiv2025

Constitutive Yap activation in distal nephron segments disrupts epithelial identity and nephron patterning

The distal nephron segments play a critical role in maintaining electrolyte balance, yet the mechanisms that preserve epithelial identity and segmental organization within this region remain poorly defined. Yes-associated protein (Yap), a key effector of Hippo signaling, is essential for kidney development, but its function in distal nephron epithelia is unknown. Using a genetic gain-of-function approach to activate Yap selectively in distal nephron segments, we found that sustained Yap activity profoundly disrupts epithelial organization and nephron patterning. Lineage tracing revealed that both distal convoluted tubule and connecting tubule cells originate from Slc12a3-expressing cells, and Yap activation in these segments led to increased proliferation, displacement of lineage-labeled cells beyond expected segment boundaries, and loss of segment-specific gene expression. These changes were accompanied by defects in apicobasal polarity and junctional integrity, consistent with epithelial plasticity. Unexpectedly, Yap activation in distal nephron segments also suppressed proximal tubule gene expression, indicating non-cell-autonomous effects on nephron differentiation. Together, these findings identify Yap as a critical regulator of epithelial identity in the distal nephron segments and reveal a previously unrecognized role for Hippo signaling in coordinating intersegmental organization during kidney development.

developmental biology↗

bioRxiv2025

Epithelial-intrinsic nitric oxide synthase 2 sustains host-microbiota dynamics that promote colitis

The microbiota influence disease pathogenesis and treatment, however we have limited ability to assess patient status in relation to the microbiota. Here we find that the nitric oxide generating enzyme, nitric oxide synthase 2 (Nos2), is transcriptionally primed in intestinal epithelial cells (IECs), as opposed to immune cells, in inflammatory bowel disease (IBD) patients. Generation of IEC-specific Nos2 knockout mice revealed that epithelial Nos2 activity promoted susceptibility to intestinal disease and sustained a colitogenic microbiota. Epithelial Nos2 increased levels of nitric oxide-derived nitrates and nitrate-metabolizing bacteria in the intestine. Unexpectedly, extra-intestinal nitrates also reflected IEC-intrinsic Nos2 expression, and systemic nitrate concentrations in patients paralleled intestinal Nos2 activation. In fact, temporally inhibiting epithelial Nos2 was sufficient to alter intestinal nitrate homeostasis and inflammation in mice, as well as restrict nitrate production by human intestinal organoids. These data reveal that epithelial nitric oxide metabolism directs host-microbiota dynamics that can alter disease and that monitoring and targeting this axis may benefit patients with IBD.

immunology↗

bioRxiv2025

MAPK/ERK signaling blocks ectopic H3K9me3 heterochromatin formation to confer mesoderm and endoderm developmental competence

During gastrulation, dynamic interplay among cell signaling pathways dictates cell fate decisions. While extensive studies have elucidated their critical roles in morphological regulation, how these signals orchestrate the epigenome to confer developmental competence remains unclear. In this study, we demonstrate that H3K9me3-marked facultative heterochromatin domains undergo global reorganization during differentiation of human pluripotent stem cells into mesoderm and endoderm, which arise through epithelial-mesenchymal transition (EMT), but not into ectoderm, which retains epithelial state. We identify the MAPK/ERK pathway, acting downstream of FGF signaling, as a key mediator of this reorganization. Specifically, the MAPK/ERK pathway prevents ectopic formation of H3K9me3 domains at EMT- and lineage-specific gene loci whose expression is necessary for mesoderm and endoderm differentiation. Collectively, our findings reveal a previously unrecognized role for MAPK/ERK signaling in reorganizing the H3K9me3 landscape to enable mesoderm and endoderm differentiation, bridging a critical gap in our knowledge of how cell signaling pathways shape the epigenetic landscape during development.

developmental biology↗

bioRxiv2025

Primitive Hepatoblasts Driving Early Liver Development

The embryonic development of the liver is initiated by the emergence of hepatoblasts, originating from the ventral foregut endoderm adjacent to the heart. Here, we identify and characterize a previously unrecognized population of early hepatoblasts at the ventroposterior part of the emerging liver bud, traced from Cdx2-positive endoderm progenitors, which we term primitive hepatoblasts. Mouse and human single-cell transcriptomics reveals the expression of both canonical hepatoblast markers TBX3, FGB, and KRT8/18 and primitive-specific mesenchymal markers ID3, VIM, and GATA4. Lineage tracing revealed the notable contribution up to 12.6% of LIV2+ hepatoblasts at E11.5 but diminishes in late fetal and postnatal development. Epigenetic and functional perturbation studies further uncover that primitive hepatoblast emergence is primed by WNT-suppression on RA-permissive CDX2+FOXA2+ progenitors. Furthermore, human pluripotent stem cell-derived primitive hepatoblast-like cells secrete pleiotrophin and midkine to amplify hepatoblast populations and develop epithelial-mesenchymal hybrid tissues in vivo. Our results provide a new framework for understanding lineage heterogeneity during early hepatogenesis and offer revised insights into strategies to model normal and abnormal liver development.

developmental biology↗

bioRxiv2025

The thin descending limb of the loop of Henle originates from proximal tubule cells during mouse kidney development

BackgroundThe thin descending limb of the loop of Henle is crucial for urine concentration, as it facilitates passive water reabsorption. Despite its importance, little is known about how this nephron segment forms during kidney development. MethodsWe assembled a large single-cell RNA sequencing (scRNA-seq) dataset by integrating multiple datasets of non-mutant developing mouse kidneys to identify developing thin descending limb cells. To test whether those cells originate from proximal tubule cells, we generated a proximal tubule-specific Cre line, Slc34a1eGFPCre, and conducted lineage tracing. Additionally, given that the transcription factor Hnf4a directly binds to the Aqp1 gene, we examined whether the loss of Hnf4a affects Aqp1 expression in thin descending limb cells. ResultsFrom our scRNA-seq dataset, we identified a small cluster of cells distinct from both the proximal tubule and the thick ascending limb of the loop of Henle. Those cells exhibited high expression of thin descending limb marker genes, including Aqp1 and Bst1. Notably, a subset of proximal tubule cells also expressed thin descending limb marker genes, suggesting that proximal tubule cells may give rise to thin descending limb cells. Using lineage tracing with the Slc34a1eGFPCre line, we found that, at least, a subset of thin descending limb cells are descendants of proximal tubule cells. Furthermore, the loss of Hnf4a, a transcription factor essential for mature proximal tubule cell formation, disrupted proper Aqp1 expression in thin descending limb cells, providing additional evidence of a developmental link between proximal tubule cells and thin descending limb cells. ConclusionOur findings shed new light on the developmental origin of thin descending limb cells and highlight the importance of Hnf4a in regulating their formation. Key PointsO_LIReference single cell RNA-seq dataset of the developing mouse kidney was assembled and used to identify the thin descending limb of the loop of Henle. C_LIO_LILineage analysis of proximal tubules in the mouse kidney shows that proximal tubule cells give rise to the thin descending limb of the loop of Henle. C_LIO_LIDeletion of Hnf4a disrupts the expression of Aqp1 in the thin descending limb of the loop of Henle, highlighting a developmental link between proximal tubules and the loop of Henle. C_LI

developmental biology↗

bioRxiv2023

The widely used Ucp1-CreEvdr transgene elicits complex developmental and metabolic phenotypes.

Bacterial artificial chromosome transgenic models, including most Cre-recombinases, enable potent interrogation of gene function in vivo but require rigorous validation as limitations emerge. Due to its high relevance to metabolic studies, we performed comprehensive analysis of the Ucp1-CreEvdr line which is widely used for brown fat research. Hemizygotes exhibited major brown and white fat transcriptomic dysregulation, indicating potential altered tissue function. Ucp1-CreEvdrhomozygotes also show high mortality, growth defects, and craniofacial abnormalities. Mapping the transgene insertion site revealed insertion in chromosome 1 accompanied by large genomic alterations disrupting several genes expressed in a range of tissues. Notably, Ucp1-CreEvdr transgene retains an extra Ucp1 gene copy that may be highly expressed under high thermogenic burden. Our multi-faceted analysis highlights a complex phenotype arising from the presence of the Ucp1-CreEvdr transgene independently of the intended genetic manipulations. Overall, comprehensive validation of transgenic mice is imperative to maximize discovery while mitigating unexpected, off-target effects. HighlightsO_LIHemizygous Ucp1-CreEvdr mice exhibit substantial brown and white fat tissue dysregulation. C_LIO_LIHomozygous Ucp1-CreEvdr mice display high mortality, growth defects, and craniofacial abnormalities. C_LIO_LIThe Ucp1-CreEvdr transgene integration resulted in major genomic disruptions affecting multiple genes. C_LIO_LIThe Ucp1-CreEvdr transgene retains a possibly functional extra Ucp1 copy. C_LI

molecular biology↗

bioRxiv2023

Hedgehog signaling is required for the maintenance of mesenchymal nephron progenitors

Mesenchymal nephron progenitors (mNPs) give rise to all nephron tubules in the mammalian kidney. Since premature depletion of these cells leads to low nephron numbers, high blood pressure, and various renal diseases, it is critical that we understand how mNPs are maintained. While Fgf, Bmp, and Wnt signaling pathways are known to be required for the maintenance of these cells, it is unclear if any other signaling pathways also play roles. In this report, we explored the role of Hedgehog signaling in mNPs. We found that loss of either Shh in the collecting duct or Smo from the nephron lineage resulted in premature depletion of mNPs. Transcriptional profiling of mNPs with different Smo dosages suggested that Hedgehog signaling inhibited Notch signaling and upregulated the expression of Fox transcription factors such as Foxc1 and Foxp4. Consistent with these observations, we found that ectopic expression of Jag1 caused the premature depletion of mNPs as seen in the Smo mutant kidney. We also found that Foxc1 was capable of binding to mitotic condensed chromatin, a feature of a mitotic bookmarking factor. Our study demonstrates a previously unappreciated role of Hedgehog signaling in preventing premature depletion of mNPs by repressing Notch signaling and likely by activating the expression of Fox factors. TRANSLATIONAL STATEMENTPremature depletion of nephron progenitors results in low nephron endowment, leading to high blood pressure and various renal diseases. Sound understanding of the molecular mechanisms underlying the maintenance of nephron progenitors is required for intervention. Although defective Hedgehog signaling is known to cause Pallister-Hall syndrome, its activity in nephron progenitors has been elusive. Here we report that Hedgehog signaling plays an important role in maintaining nephron progenitors. Our findings suggest that Hedgehog signaling pathway is a potential target for enhancing nephron endowment.

developmental biology↗

bioRxiv2023

Single cell transcriptomics identifies adipose tissue CD271+ progenitors for enhanced angiogenesis in limb ischemia

Therapeutic angiogenesis using mesenchymal stem/stromal cell grafts have shown modest and controversial effects in preventing amputation for patients with critical limb ischemia. Through single-cell transcriptomic analysis of human tissues, we identified CD271+ progenitors specifically from subcutaneous adipose tissue (AT) as having the most prominent pro-angiogenic gene profile distinct from other stem cell populations. AT-CD271+ progenitors demonstrated robust in vivo angiogenic capacity, over conventional adipose stromal cell grafts, characterized by long-term engraftment, augmented tissue regeneration, and significant recovery of blood flow in a xenograft model of limb ischemia. Mechanistically, the angiogenic capacity of CD271+ progenitors is dependent on functional CD271 and mTOR signaling. Notably, the number and angiogenic capacity of CD271+ progenitors was strikingly reduced in insulin resistant donors. Our study highlights the identification of AT-CD271+ progenitors with in vivo superior efficacy for limb ischemia. Furthermore, we showcase comprehensive single-cell transcriptomics strategies for identification of suitable grafts for cell therapy. HIGHLIGHTSO_LIAdipose tissue stromal cells have a distinct angiogenic gene profile among human cell sources. C_LIO_LICD271+ progenitors in adipose tissue have a prominent angiogenic gene profile. C_LIO_LICD271+ progenitors show superior therapeutic capacities for limb ischemia. C_LIO_LICD271+ progenitors are reduced and functionally impaired in insulin resistant donors. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/527726v2_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1b5e1aorg.highwire.dtl.DTLVardef@bf2c34org.highwire.dtl.DTLVardef@ef0e68org.highwire.dtl.DTLVardef@9aee1f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Pioneer transcription factors coordinate active and repressive gene expression states to regulate cell fate

Pioneer transcription factors (TFs) regulate cell fate by establishing transcriptionally primed and active states. However, cell fate control requires the coordination of both lineage-specific gene activation and repression of alternative lineage programs, a process that is poorly understood. Here, we demonstrate that the pioneer TF Forkhead box A (FOXA), required for endoderm lineage commitment, coordinates with the PR domain zinc finger 1 (PRDM1) TF to recruit Polycomb repressive complexes, which establish bivalent enhancers and repress alternative lineage programs. Similarly, the pioneer TF OCT4 coordinates with PRDM14 to repress cell differentiation programs in pluripotent stem cells, suggesting this is a common feature of pioneer TFs. We propose that pioneer and PRDM TFs coordinate recruitment of Polycomb complexes to safeguard cell fate. One-Sentence SummaryPioneer and PRDM transcription factors repress alternative lineage programs.

cell biology↗

bioRxiv2020

Prenatal psychosocial stress-induced behavioral and neuroendocrine abnormalities are associated with sex-specific alterations in synaptic transmission and differentially modulated by maternal environment

Prenatal stress (PS) is associated with increased vulnerability to affective disorders. Transplacental glucocorticoid passage and stress-induced maternal environment alterations are recognized as potential routes of transmission that can fundamentally alter neurodevelopment. However, molecular mechanisms underlying aberrant emotional outcomes or the individual contributions intrauterine stress versus maternal environment play in shaping these mechanisms remain unknown. Here, we report anxiogenic behaviors, anhedonia, and female hypothalamic-pituitary-adrenal axis hyperactivity as a consequence of psychosocial PS in mice. Sex-specific placental responses to stress and evidence of fetal amygdala programming precede these abnormalities. In adult offspring, we observe amygdalar transcriptional changes demonstrating sex-specific dysfunction in synaptic transmission and neurotransmitter systems. We find these abnormalities are primarily driven by in-utero stress exposure. Importantly, maternal care changes postnatally reverse anxiety-related behaviors and partially rescue gene alterations associated with neurotransmission. Our data demonstrate the influence maternal environment exerts in shaping offspring emotional development despite deleterious effects of intrauterine stress.

neuroscience↗

bioRxiv2018

Shared Nucleotide Flanks Confer Transcriptional Competency to bZip Core Motifs

Sequence-specific DNA binding recruits transcription factors (TFs) to the genome to regulate gene expression. Here, we perform high resolution mapping of CEBP proteins to determine how sequence dictates genomic occupancy. We demonstrate a fundamental difference between the sequence repertoire utilized by CEBPs in vivo versus the palindromic sequence preference reported by classical in vitro models, by identifying a palindromic motif at less than 1% of the genomic binding sites. On the native genome, CEBPs bind a diversity of related 10 bp sequences resulting from the fusion of degenerate and canonical half-sites. Altered DNA specificity of CEBPs in cells occurs through heterodimerization with other bZip TFs, and approximately 40% of CEBP-binding sites in primary human cells harbor motifs characteristic of CEBP heterodimers. In addition, we uncover an important role for sequence bias at core-motif-flanking bases for CEBPs and demonstrate that flanking bases regulate motif function across mammalian bZip TFs. Favorable flanking bases confer efficient TF occupancy and transcriptional activity, and DNA shape may explain how the flanks alter TF binding. Importantly, motif optimization within the 10-mer is strongly correlated with cell-type-independent recruitment of CEBP{beta}, providing key insight into how sequence sub-optimization affects genomic occupancy of widely expressed CEBPs across cell types.

genomics↗