Abstract Background NR5A1 encodes a transcription factor essential for adrenal and gonadal development. Gene variants are a known cause of heterogeneous 46,XY disorders of sex development (DSD), but the mechanisms underlying the phenotypic variability remain unclear. We investigated how different NR5A1 variants affect downstream gene regulation and contribute to DSD pathogenesis. Methods We analyzed four naturally occurring NR5A1 variants identified in patients with 46,XY DSD—two novel (p.Cys65Ser, p.His310Arg) and two previously reported (p.Cys30Ser, p.Gln329*). We performed protein and transcriptomic analyses to characterize variant effects and identify dysregulated and candidate target genes, validated by qPCR and luciferase assays. Transcriptomic and CUT&Tag analyses focused on the p.Gln329* truncating variant. Results All four variants occurred at conserved residues and resulted in reduced NR5A1 protein expression and impaired nuclear localization upon transfection in HEK293T cells. Transcriptomic analysis using the p.Gln329* variant revealed broad downregulation of genes involved in steroidogenesis, including CYP11A1 , STAR , and CYP17A1 . Notably, AMHR2 and STARD8 were significantly downregulated and showed reduced CUT&Tag signal in variant-transfected cells. Promoter assays confirmed that all variants diminished CYP11A1 and AMHR2 promoter activity. Only the p.Gln329* variant affected STARD8 promoter activity. Conclusions These findings indicate that NR5A1 variants impair protein expression and localization, leading to transcriptional dysregulation of genes involved in steroid hormone biosynthesis and sexual development. Based on analysis of the p.Gln329* truncating variant, AMHR2 and STARD8 are strong candidate novel downstream targets of NR5A1, offering further insight into the mechanisms driving 46,XY DSD.

Claustrum orchestrates brain functions via its connections with numerous brain regions, but its molecular and cellular organization remains unresolved. Single-nucleus RNA sequencing of 227,750 macaque claustral cells identified 48 transcriptome-defined cell types, with most glutamatergic neurons similar to deep-layer insular neurons. Comparison of macaque, marmoset, and mouse transcriptomes revealed macaque-specific cell types. Retrograde tracer injections at 67 cortical and 7 subcortical regions defined four distinct distribution zones of retrogradely labeled claustral neurons. Joint analysis of whole-brain connectivity and single-cell spatial transcriptome showed that these four zones containing distinct compositions of glutamatergic (but not GABAergic) cell types preferentially connected to specific brain regions with a strong ipsilateral bias. Several macaque-specific glutamatergic cell types in ventral vs. dorsal claustral zones selectively co-projected to two functionally related areas-entorhinal cortex and hippocampus vs. motor cortex and putamen, respectively. These data provide the basis for elucidating the neuronal organization underlying diverse claustral functions.

Adventitial stem cells (ASCs), identified by Gli1 expression, have been proposed as key contributors to the vascular smooth muscle cell (SMC) population during atherosclerosis development. However, their precise role remains a subject of debate. To clarify this, a Gli1-CreER lineage tracing tool to track these cells in an atherosclerosis model is initially used. This fate-mapping studies revealed that Gli1+ cells contribute to a small subset of SMCs. To definitively differentiate between the true lineage transformation and potential ectopic labeling by Gli1-CreER in SMCs, a dual recombinase-mediated genetic strategy to label Gli1+ ASCs while eliminating ectopic labeling in SMCs is developed. The results from this dual lineage tracing approach demonstrated that Gli1+ ASCs do not contribute to the SMC population in atherosclerotic plaques. Instead, Gli1+ ASCs differentiate into a significant portion of SMCs after vascular anastomosis injury, suggesting their role is context-dependent. These findings challenge current paradigms and highlight the need to reconsider cellular targets for therapeutic interventions in atherosclerosis.

BACKGROUND:Antibody-drug conjugate (ADC), combining monoclonal antibodies with cytotoxic payloads through covalent linkers, is leading a new era of targeted cancer therapy. Despite its therapeutic success, cardiotoxicity has been a recognized concern in early approved ADCs such as trastuzumab, yet the cardiac safety profiles and mechanisms of recently approved ADCs remain poorly characterized. Assessing the nature of their cardiac events using the FDA Adverse Event Reporting System (FAERS) database is vital for risk assessment in surgical oncology. This study aimed to analyze cardiac adverse events (cAEs) related to ADCs in the FAERS database to detect cardiac risk signals, characterize clinical patterns, and investigate mechanistic pathways, ultimately informing risk mitigation strategies in clinical practice. MATERIALS AND METHODS:This systematic study analyzed FAERS data (Jan 2019-Sep 2023) for ADC-related adverse events (AEs). AEs were standardized using MedDRA terminology. Disproportionality analysis using the reporting odds ratio (ROR) method was performed to identify ADC-related cAEs. Potential risk factors were evaluated through logistic regression analyses. To understand the molecular basis, TCGA transcriptome data were analyzed to explore mechanisms of ADC-related cAEs. RESULTS:cAEs comprised 11.77% (range: 8.63-23.50%) of all ADC-related reports from 2019 to 2023. A total of 49 cAE categories were identified. The mean age of reports with ADC-related cAEs was 60 (±13) years with 8.67% of reports having a fatal outcome. Breast cancer indications dominated. Combining ADCs with dexamethasone significantly reduced the risk of cardiac failure (ROR decreased from 3.18 to 0.85). ADC-related cAEs correlated with dysregulated HSP70 binding ( R = 0.82, P = 4.66e-4) and heat shock protein pathways. CONCLUSIONS:This study underscores the importance of recognizing and managing the cAEs associated with ADC therapy. Our findings provide a nuanced understanding of the burden, risk factors, and potential biological mechanisms of ADC-related cAEs, which can inform clinical decision-making and guide the development of safer and more effective ADC therapies.

In the brain, a precise excitation-inhibition balance underpins every neural operation. Strikingly, across the vertebrate CNS, excitatory and inhibitory neurons often emerge from common progenitors (termed EX-IN lineage), yet how such dichotomous fates diverge has remained enigmatic. Here, exploiting spinal v2a (excitatory)–v2b (inhibitory) and retinal bipolar (excitatory) - amacrine (inhibitory) lineages as EX-IN lineage paradigm, we report a single, conserved genetic program: excitatory neural fate is the default state when sibling-cell communication is silenced; progenitor- and excitatory neuron-expressed homeobox genes actively repress the inhibitory program, while Notch signaling releases this repression to unleash inhibitory identity. Thus, we define a unifying principle governing EX-IN lineage divergence that generate the fundamental dichotomy of neural circuits. ### Competing Interest Statement The authors have declared no competing interest. Creative Research Groups of the National Natural Science Foundation of China, 32321003 STI2030-Major Projects, 2021ZD0204500 National Natural Science Foundation of China, 32471029, 31871035, 32500853 Shanghai Natural Science Foundation, 25ZR1402526 China Postdoctoral Science Foundation, 2021M703308 Shanghai Post-doctoral Excellence Program, 2021400

This study aims to investigate the role of RNF149 and tetraspanin CD63 in lipopolysaccharide/Toll-like receptor 4 (LPS/TLR4) signal transduction. TNF-α was assessed using enzyme-linked immunosorbent assay. The distribution of TLR4 was examined through flow cytometry after CD63 knockdown. Real-time polymerase chain reaction was used to analyze the expression of the target genes RNF149 and CD63 under different conditions. Western blotting was employed to detect gene expression, while immunoprecipitation and confocal microscopy were used to evaluate protein interactions. Transcriptome array data from stimulated monocytes (GSE7547) was obtained from GEO and subjected to bioinformatic analysis. It is suggested that CD63 may serve as a substrate of RNF149, with RNF149 capable of directly interacting with CD63. RNF149 degrades CD63 through covalent modification of CD63 at lysine 29 of the ubiquitin monomer, leading to the formation of a multiubiquitin chain. Both RNF149 and CD63 interact with TLR4, with CD63 promoting LPS/TLR4 signaling and RNF149 inhibits it. CD63 does not impact the distribution of TLR4 on the cell surface and does not directly interact with TIRAP, IRAK4, or TRAF6, but does interact with Myd88.RNF149 plays a negative regulatory role in LPS/TLR4 signal transduction by mediating ubiquitination-induced CD63 degradation.

Lung injury activates epithelial stem or progenitor cells for alveolar repair and regeneration. Unraveling the origin and fate of injury-induced progenitors is crucial for elucidating lung repair mechanisms. Here, we report that p63-expressing progenitors emerge upon bleomycin-induced mouse lung injury. Single-cell RNA sequencing and clonal analysis reveal that these p63+ progenitors proliferate rapidly and differentiate into alveolar type 1 and type 2 cells through different trajectories. Dual recombinase-mediated sequential genetic-lineage tracing demonstrates that p63+ progenitors originate from airway secretory cells and subsequently generate alveolar cells. Functionally, p63 activation is essential for efficient alveolar regeneration from secretory cells post injury. Our study identifies secretory-cell-derived p63+ progenitors as contributors to alveolar repair, suggesting a potential therapeutic avenue for lung regeneration following injury.

Epithelial cells (ECs) have been proposed to contribute to myofibroblasts or fibroblasts through epithelial-mesenchymal transition (EMT) during renal fibrosis. However, since EMT may occur dynamically, transiently, and reversibly during kidney fibrosis, conventional lineage tracing based on Cre-loxP recombination in renal ECs could hardly capture the transient EMT activity, yielding inconsistent results. Moreover, previous EMT research has primarily focused on renal proximal tubule ECs, with few reports of distal tubules and collecting ducts. Here, we generated dual recombinases-mediated genetic lineage tracing systems for continuous monitoring of transient mesenchymal gene expression in E-cadherin+ and EpCAM+ ECs of distal tubules and collecting ducts during renal fibrosis. Activation of key EMT-inducing transcription factor (EMT-TF) Zeb1 and mesenchymal markers αSMA, vimentin, and N-cadherin, were investigated following unilateral ureteral obstruction (UUO). Our data revealed that E-cadherin+ and EpCAM+ ECs did not transdifferentiate into myofibroblasts, nor transiently expressed these mesenchymal genes during renal fibrosis. In contrast, in vitro a large amount of cultured renal ECs upregulated mesenchymal genes in response to TGF-β, a major inducer of EMT.

A genetic system, ProTracer, has been recently developed to record cell proliferation in vivo. However, the ProTracer is initiated by an infrequently used recombinase Dre, which limits its broad application for functional studies employing floxed gene alleles. Here we generated Cre-activated functional ProTracer (fProTracer) mice, which enable simultaneous recording of cell proliferation and tissue-specific gene deletion, facilitating broad functional analysis of cell proliferation by any Cre driver.

Grain size is an important agronomic trait, but our knowledge about grain size determination in crops is still limited. Endoplasmic reticulum (ER)-associated degradation (ERAD) is a special ubiquitin proteasome system that is involved in degrading misfolded or incompletely folded proteins in the ER. Here, we report that SMALL GRAIN 3 (SMG3) and DECREASED GRAIN SIZE 1 (DGS1), an ERAD-related E2-E3 enzyme pair, regulate grain size and weight through the brassinosteroid (BR) signaling pathway in rice (Oryza sativa). SMG3 encodes a homolog of Arabidopsis (Arabidopsis thaliana) UBIQUITIN CONJUGATING ENZYME 32, which is a conserved ERAD-associated E2 ubiquitin conjugating enzyme. SMG3 interacts with another grain size regulator, DGS1. Loss of function of SMG3 or DGS1 results in small grains, while overexpression of SMG3 or DGS1 leads to long grains. Further analyses showed that DGS1 is an active E3 ubiquitin ligase and colocates with SMG3 in the ER. SMG3 and DGS1 are involved in BR signaling. DGS1 ubiquitinates the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) and affects its accumulation. Genetic analysis suggests that SMG3, DGS1, and BRI1 act together to regulate grain size and weight. In summary, our findings identify an ERAD-related E2-E3 pair that regulates grain size and weight, which gives insight into the function of ERAD in grain size control and BR signaling.

Unraveling cell fate plasticity during tissue homeostasis and repair can reveal actionable insights for stem cell biology and regenerative medicine. In the pancreas, it remains controversial whether lineage transdifferentiation among the exocrine cells occur under pathophysiological conditions. Here, to address this question, we used a dual recombinase-mediated genetic system that enables simultaneous tracing of pancreatic acinar and ductal cells using two distinct genetic reporters, avoiding the "ectopic" labeling by Cre-loxP recombination system. We found that acinar-to-ductal transdifferentiation occurs after pancreatic duct ligation or during caerulein-induced pancreatitis, but not during homeostasis or after partial pancreatectomy. On the other hand, pancreatic ductal cells contribute to new acinar cells after significant acinar cell loss. By genetic tracing of cell proliferation, we also quantify the cell proliferation dynamics and deduce the turnover rate of pancreatic exocrine lineages during homeostasis. Together, these results suggest that the lineage transdifferentiation happens between acinar cells and ductal cells in the pancreatic exocrine glands under specific conditions.

Breakdown of self-incompatibility has frequently been attributed to loss-of-function mutations of alleles at the locus responsible for recognition of self-pollen (i.e. the S-locus). However, other potential causes have rarely been tested. Here, we show that self-compatibility of S1S1-homozygotes in selfing populations of the otherwise self-incompatible Arabidopsis lyrata is not due to S-locus mutation. Between-breeding-system cross-progeny are self-compatible if they combine S-1 from the self-compatible cross-partner with recessive S-1 from the self-incompatible cross-partner, but self-incompatible with dominant S-alleles. Because S1S1 homozygotes in outcrossing populations are self-incompatible, mutation of S-1 cannot explain self-compatibility in S1S1 cross-progeny. This supports the hypothesis that an S-1-specific modifier unlinked to the S-locus causes self-compatibility by functionally disrupting S-1. Self-compatibility in S19S19 homozygotes may also be caused by an S-19-specific modifier, but we cannot rule out a loss-of-function mutation of S-19. Taken together, our findings indicate that breakdown of self-incompatibility is possible without disruptive mutations at the S-locus.

After severe heart injury, fibroblasts are activated and proliferate excessively to form scarring, leading to decreased cardiac function and eventually heart failure. It is unknown, however, whether cardiac fibroblasts are heterogeneous with respect to their degree of activation, proliferation and function during cardiac fibrosis. Here, using dual recombinase-mediated genetic lineage tracing, we find that endocardium-derived fibroblasts preferentially proliferate and expand in response to pressure overload. Fibroblast-specific proliferation tracing revealed highly regional expansion of activated fibroblasts after injury, whose pattern mirrors that of endocardium-derived fibroblast distribution in the heart. Specific ablation of endocardium-derived fibroblasts alleviates cardiac fibrosis and reduces the decline of heart function after pressure overload injury. Mechanistically, Wnt signaling promotes activation and expansion of endocardium-derived fibroblasts during cardiac remodeling. Our study identifies endocardium-derived fibroblasts as a key fibroblast subpopulation accounting for severe cardiac fibrosis after pressure overload injury and as a potential therapeutic target against cardiac fibrosis.

Following severe liver injury, when hepatocyte-mediated regeneration is impaired, biliary epithelial cells (BECs) can transdifferentiate into functional hepatocytes. However, the subset of BECs with such facultative tissue stem cell potential, as well as the mechanisms enabling transdifferentiation, remains elusive. Here we identify a transitional liver progenitor cell (TLPC), which originates from BECs and differentiates into hepatocytes during regeneration from severe liver injury. By applying a dual genetic lineage tracing approach, we specifically labeled TLPCs and found that they are bipotent, as they either differentiate into hepatocytes or re-adopt BEC fate. Mechanistically, Notch and Wnt/β-catenin signaling orchestrate BEC-to-TLPC and TLPC-to-hepatocyte conversions, respectively. Together, our study provides functional and mechanistic insights into transdifferentiation-assisted liver regeneration.

Identification of cellular surface markers that distinguish tumorous from normal vasculature is important for the development of tumor vessel-targeted therapy. Here, we show that Apj, a G protein-coupled receptor, is highly enriched in tumor endothelial cells but absent from most endothelial cells of adult tissues in homeostasis. By genetic targeting using Apj-CreER and Apj-DTRGFP-Luciferase, we demonstrated that hypoxia-VEGF signaling drives expansion of Apj(+) tumor vessels and that targeting of these vessels, genetically and pharmacologically, remarkably inhibits tumor angiogenesis and restricts tumor growth. These in vivo findings implicate Apj(+) vessels as a key driver of pathological angiogenesis and identify Apj(+) endothelial cells as an important therapeutic target for the anti-angiogenic treatment of tumors.

Genetic technology using site-specific recombinases, such as the Cre-loxP system, has been widely employed for labeling specific cell populations and for studying their functions in vivo. To enhance the precision of cell lineage tracing and functional study, a similar site-specific recombinase system termed Dre-rox has been recently used in combination with Cre-loxP. To enable more specific cell lineage tracing and ablation through dual recombinase activity, we generated two mouse lines that render Dre- or Dre+Cre-mediated recombination to excise a stop codon sequence that prevents the expression of diphtheria toxin receptor (DTR) knocked into the ubiquitously expressed and safe Rosa26 locus. Using different Dre- and Cre-expressing mouse lines, we showed that the surrogate gene reporters tdTomato and DTR were simultaneously expressed in target cells and in their descendants, and we observed efficient ablation of tdTomato+ cells after diphtheria toxin administration. These mouse lines were used to simultaneously trace and deplete the target cells of interest through the inducible expression of a reporter and DTR using dual Cre and Dre recombinases, allowing a more precise and efficient study of the role of specific cell subsets within a heterogeneous population in pathophysiological conditions in vivo.

The tumor microenvironment (TME) represents a milieu enabling cancer cells to develop malignant properties, while concerted interactions between cancer and stromal cells frequently shape an “activated/reprogramed” niche to accelerate pathological progression. Here we report that a soluble factor epiregulin (EREG) is produced by senescent stromal cells, which non-cell-autonomously develop the senescence-associated secretory phenotype (SASP) upon DNA damage. Genotoxicity triggers EREG expression by engaging NF-κB and C/EBP, a process supported by elevated chromatin accessibility and increased histone acetylation. Stromal EREG reprograms the expression profile of recipient neoplastic cells in a paracrine manner, causing upregulation of MARCHF4, a membrane-bound E3 ubiquitin ligase involved in malignant progression, specifically drug resistance. A combinational strategy that empowers EREG-specific targeting in treatment-damaged TME significantly promotes cancer therapeutic efficacy in preclinical trials, achieving response indices superior to those of solely targeting cancer cells. In clinical oncology, EREG is expressed in tumor stroma and handily measurable in circulating blood of cancer patients post-chemotherapy. This study establishes EREG as both a targetable SASP factor and a new noninvasive biomarker of treatment-damaged TME, thus disclosing its substantial value in translational medicine.

Tissue-resident macrophages play essential functions in the maintenance of tissue homeostasis and repair. Recently, the endocardium has been reported as a de novo hemogenic site for the contribution of hematopoietic cells, including cardiac macrophages, during embryogenesis. These observations challenge the current consensus that hematopoiesis originates from the hemogenic endothelium within the yolk sac and dorsal aorta. Whether the developing endocardium has such a hemogenic potential requires further investigation. Here, we generated new genetic tools to trace endocardial cells and reassessed their potential contribution to hematopoietic cells in the developing heart. Fate-mapping analyses revealed that the endocardium contributed minimally to cardiac macrophages and circulating blood cells. Instead, cardiac macrophages were mainly derived from the endothelium during primitive/transient definitive (yolk sac) and definitive (dorsal aorta) hematopoiesis. Our findings refute the concept of endocardial hematopoiesis, suggesting that the developing endocardium gives rise minimally to hematopoietic cells, including cardiac macrophages.