Development
Abstract
Neonatal life is marked by rapid antigen exposure, necessitating establishment of peripheral immune tolerance via conversion of naïve CD4+ T cells into regulatory T cells (Tregs). Here, we demonstrated heightened capacity for FOXP3 expression and tolerogenic function among cord blood versus adult blood naive CD4+ T cells and showed that this is linked to their unique metabolic profile and elevated expression of the NADase, CD38. Early-life naïve CD4+ T cells demonstrated a metabolic preference for glycolysis, which directly facilitated their differentiation trajectory. We revealed an age-dependent gradient in CD38 levels on naïve CD4+ T cells and showed that high CD38 expression contributes to both the glycolytic state and tolerogenic potential of neonatal CD4+ T cells, effects that were mediated at least in part via the NAD-dependent deacetylase SIRT1. Thus, the early-life window for peripheral tolerance in humans is critically enabled by the immunometabolic state of the naïve CD4+ compartment.
Authors
Laura R. Dwyer, Andrea M. DeRogatis, Sean Clancy, Victoire Gouirand, Charles Chien, Elizabeth E. Rogers, Scott P. Oltman, Laura L. Jelliffe-Pawlowski, Theo van den Broek, Femke van Wijk, Susan V. Lynch, Rachel L. Rutishauser, Allon Wagner, Alexis J. Combes, Tiffany C. Scharschmidt
Abstract
Germline loss-of-function folliculin (FLCN) gene mutations cause Birt-Hogg-Dubé (BHD) syndrome, in which pulmonary cysts are present in up to 90% of the patients. The pathogenic mechanisms underlying lung cyst development in BHD are almost entirely unknown because of the limited availability of BHD patient lung samples and the lack of authentic BHD lung disease models. We generated lung mesenchyme–specific and lung epithelium–specific Flcn-knockout mice using a Cre/loxP approach. We found that deletion of Flcn in lung mesenchymal cells, but not in lung epithelial cells, resulted in alveolar enlargement starting from early postnatal life, with evidence of cyst formation in adult mice, resembling the pulmonary disease in human BHD. These changes were associated with increased mechanistic target of rapamycin complex 1 (mTORC1) activity in the lungs of both patients with BHD and Flcn-knockout mice. Attenuation of mTORC1 activity by knocking out Raptor gene (Rptor) or pharmacologic inhibition using rapamycin substantially rescued the pulmonary pathology caused by Flcn deletion in mice. Taken together, these human and mouse data support a model in which mTORC1 hyperactivation drives pulmonary cystic pathology in BHD.
Authors
Ke Cao, Hui Chen, Ling Chu, Hong-Jun Wang, Jianhua Zhang, Yongfeng Luo, Joanne Chiu, Damir Khabibullin, Nicola Alesi, Matthew E. Thornton, Brendan H. Grubbs, Ali Ataya, Nishant Gupta, Francis X. McCormack, Kathryn A. Wikenheiser-Brokamp, Elizabeth P. Henske, Wei Shi
Abstract
Muscle cell fusion is critical for the formation and maintenance of multinucleated myotubes during skeletal muscle development and regeneration. However, the molecular mechanisms directing cell-cell fusion are not fully understood. Here, we identified platelet-derived growth factor receptor β (PDGFRβ) signaling as a key modulator of myocyte function in adult muscle cells. Our findings demonstrated that genetic deletion of Pdgfrb enhanced muscle regeneration and increased myofiber size, whereas Pdgfrb activation impaired muscle repair. Inhibition of PDGFRβ activity promoted myonuclear accretion in both mouse and human myotubes, whereas PDGFRβ activation stalled myotube development by preventing cell spreading to limit fusion potential. Furthermore, PDGFRβ activity cooperated with TGF-β signaling to regulate myocyte size and fusion. Mechanistically, PDGFRβ signaling required STAT1 activation, and blocking STAT1 phosphorylation enhanced myofiber repair and size during regeneration. Collectively, PDGFRβ signaling acts as a regenerative checkpoint and represents a potential clinical target to improve skeletal muscle repair.
Authors
Siwen Xue, Abigail M. Benvie, Jamie E. Blum, Benjamin D. Cosgrove, Anna E. Thalacker-Mercer, Daniel C. Berry
Abstract
Alternative splicing-triggered nonsense-mediated mRNA decay (AS-NMD) critically regulates gene expression, but the extent to which neuronal genes are regulated by AS-NMD remains understudied. Here, we identified more than 3,000 developmentally regulated AS-NMD exons in mouse and human brains, and validated them in cultured neurons. AS-NMD suppresses synaptic genes during brain development and differentially regulates more than 200 causal genes for neurodevelopmental disorders (NDDs). We detected an AS-NMD exon in GRIA2 and identified splice-switching antisense oligonucleotides that suppressed GRIA2 NMD and increased its functional isoforms. In summary, this study uncovers genes repressed by AS-NMD in the brain and nominates amenable splice-switching targets for treating dominant NDDs such as autism spectrum disorders and developmental epileptic encephalopathy.
Authors
Kaining Hu, Runwei Yang, Jiaming Qiu, Xinran Feng, Kayleigh J. LaPre, Jessica Tanouye, Yalan Yang, Xiaochang Zhang
Abstract
How β-Catenin (βCat) mediates tissue hyperplasia is poorly understood. To explore this, we employed the adrenal cortex as a model system given its stereotypical spatial organization and the important role βCat plays in homeostasis and disease. For example, excessive production of aldosterone by the adrenal cortex (primary aldosteronism, PA) constitutes a major cause of cardiovascular morbidity and is associated with βCat gain-of-function (βCat-GOF). Adherens junctions (AJs) connect the actin cytoskeletons of adjacent zona Glomerulosa (zG) cells via a cadherin-βCat-α-Catenin complex and mediate aldosterone production. Whether βCat-GOF drives zG hyperplasia, a key feature of PA, via AJs is unknown. Here, we showed that aldosterone secretagogues (K+, AngII) and βCat-GOF mediated AJ formation via Rho/ROCK/actomyosin signaling. In addition, Rho/ROCK inhibition led to altered zG rosette morphology and decreased aldosterone production. Mice with zG-specific βCat-GOF demonstrated increased AJ formation and zG hyperplasia, which was blunted by Rho/ROCK inhibition and deletion of α-Catenin. βCat also impacted AJ formation independently of its role as a transcription factor. Furthermore, analysis of human aldosterone-producing adenomas revealed high levels of βCat expression were associated with increased membranous expression of K-Cadherin. Together, our findings identified Rho/ROCK signaling and αCat as key mediators of AJ formation and βCat-driven hyperplasia.
Authors
Mesut Berber, Betul Haykir, Nick A. Guagliardo, Vasileios Chortis, Kleiton Silva Borges, Paula Q. Barrett, Felix Beuschlein, Diana L. Carlone, David T. Breault
Abstract
The lymphatic system maintains tissue fluid balance, and FOXC2 mutations cause lymphoedema-distichiasis syndrome, which is characterized by lymphatic valve defects. Although oscillatory shear stress regulates FOXC2 expression, other extracellular regulators remain unclear. In this study, we identified LPA4 and LPA6, two Gα12/Gα13-coupled receptors for the bioactive lipid lysophosphatidic acid (LPA), as key regulators of FOXC2 expression and lymphatic valve development. Lymphatic endothelial cell (LEC)-specific Lpa4;Lpa6-deficient mice exhibited impaired lymphatic valve formation and maintenance, which resembled phenotypes of LEC-specific Foxc2-deficient mice, including abnormal lymphatic vessel patterning. Mechanistically, lymphatic endothelial Lpa4/Lpa6 ablation reduced FOXC2 expression in vitro and in vivo. NF-κB was found essential for LPA-induced FOXC2 expression through the LPA4/LPA6-Gα12/Gα13-Rho kinase signaling axis. Accordingly, pharmacological inhibition of NF-κB and Rho kinase impaired lymphatic valve maintenance in mice. These results suggested that lymphatic endothelial LPA4 and LPA6 synergistically regulate FOXC2 expression through NF-κB activation and play an important role in lymphatic valve formation and maintenance. Our findings provide a molecular basis for lymphatic vessel development with a therapeutic potential for targeting lymphatic-associated diseases.
Authors
Daisuke Yasuda, Nana Sato, Keisuke Yanagida, Tomomi Hashidate-Yoshida, Tomohiro Shiiya, Hideo Shindou, Atsuki Taira, Takashi Ebihara, Takao Shimizu, Masanori Hirashima, Seiya Mizuno, Satoru Takahashi, Satoshi Ishii
Abstract
Lymphatics maintains fluid homeostasis, immune surveillance, and tissue integrity. Here, we identified the E26 transformation-specific (ETS) transcription factors Erg and Fli1 as essential, cooperative regulators of lymphatic integrity and function. Using inducible, lymphatic endothelial cell-specific deletion in mice, we demonstrated that combined loss of Erg and Fli1 in adults results in fatal lymphatic failure, including chylothorax, chylous ascites, and impaired lymphatic drainage. Single-cell transcriptomic analysis revealed that loss of Erg and Fli1 caused disrupted lymphatic heterogeneity and dysregulation of key lymphatic genes, including valve-specific gene profiles. Erg and Fli1 coordinated lymphatic-immune crosstalk by transcriptionally regulating C-C motif chemokine ligand 21 (Ccl21), which mediates dendritic cell trafficking. Their loss also induced pro-inflammatory and pro-thrombotic gene expression, further contributing to lymphatic dysfunction. During embryonic development, the co-deletion led to lymphatic mis-patterning and loss of valve-initiating lymphatic endothelial cell clusters. The impact of loss of Erg and Fli1 function on lymphatic development in mice is consistent with FOXC2 mutations in lymphedema-distichiasis syndrome or ERG gene variants underlying primary lymphoedema in humans. Moreover, Erg and Fli1 were required for regenerative lymphangiogenesis and lymphatic repair following injury in adults. Our findings establish Erg and Fli1 as core transcriptional regulators of lymphatic identity, integrity, and function.
Authors
Myung Jin Yang, Seok Kang, Seon Pyo Hong, Hokyung Jin, Jin-Hui Yoon, Cheolhwa Jin, Chae Min Yuk, Lidiya G Gebeyehu, Junho Jung, Sung-hwan Yoon, Hyuek jong Lee, Gou Young Koh
Abstract
The adaptor protein LNK/SH2B3 negatively regulates hematopoietic stem cell (HSC) homeostasis. Lnk-deficient mice show marked expansion of HSCs without premature exhaustion. Lnk deficiency largely restores HSC function in Fanconi Anemia (FA) mouse models and primary FA patient cells, albeit protective mechanisms remain enigmatic. Here, we uncover a novel role for LNK in regulating translesion synthesis (TLS) during HSC replication. Lnk deficiency reduced replication stress-associated DNA damage, particularly in the FA background. Lnk deficiency suppressed single-strand DNA breaks, while enhancing replication fork restart in FA-deficient HSCs. Diminished replication-associated damage in Lnk-deficient HSCs occurred commensurate with reduced ATR-p53 checkpoint activation that is linked to HSC attrition. Notably, Lnk deficiency ameliorated HSC attrition in FA mice without exacerbating carcinogenesis during ageing. Moreover, we demonstrated that enhanced HSC fitness from Lnk deficiency was associated with increased TLS activity via REV1 and, to a lesser extent, TLS polymerase eta. TLS polymerases are specialized to execute DNA replication in the presence of lesions or natural replication fork barriers that stall replicative polymerases. Our findings implicate elevated use of these specialized DNA polymerases as critical to the enhanced HSC function imparted by Lnk deficiency, which has important ramifications for stem cell therapy and regenerative medicine in general.
Authors
Brijendra Singh, Md Akram Hossain, Xiao Hua Liang, Jeremie Fages, Carlo Salas Salinas, Roger A. Greenberg, Wei Tong
Abstract
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 increased 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 tissues driving AOS pathogenesis, we employed conditional genetics to isolate the contribution of the vascular endothelium 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 reduced Notch1 signaling in the vasculature is a key driver of pathogenesis in this AOS mouse model.
Authors
Alyssa F. Solano, Kristina Preusse, Brittany Cain, Rebecca Hotz, Parthav Gavini, Zhenyu Yuan, Benjamin Bowen, Gabrielle Maco, Hope Neal, Ellen K. Gagliani, Christopher Ahn, Hee-Woong Lim, Laura Southgate, Rhett A. Kovall, Raphael Kopan, Brian Gebelein
Abstract
Tay-Sachs and Sandhoff disease are fatal neurodegenerative diseases without an effective therapy that are caused by mutations in the HEXA and HEXB genes, respectively. Together they encode the heterodimeric isozyme of hexosaminidase (HexA) that degrades GM2 ganglioside. This report describes a 5 year-long study using a bidirectional AAV9 vector (AAV9-Bic_HexA/HexB) encoding both HEXA and HEXB in the Tay-Sachs sheep model. Bidirectional AAV9 was delivered intravenously or through various cerebral spinal fluid (CSF) delivery routes: intracerebroventricular (ICV), cisterna magna (CM) and lumbar delivery (LIT). The longest survival and best distribution were achieved by multipoint CSF delivery (combined CM, ICV and LIT) with treated animals survived up to 5 years of age (untreated Tay-Sachs animals die ~9 months). Extension in survival was accompanied by lasting improvement in neurological examination and maze testing. Improvement in biomarkers of efficacy including MRI, MR spectroscopy, diffusion tensor imaging as well as CSF levels of GM2 ganglioside and hexosaminidase A (HexA) activity was evident. Post-mortem assessments showed broad HexA distribution, GM2 ganglioside clearance and vector genome distribution, especially in deep brain structures. Therapeutic efficacy documented in this study supports translation of bidirectional vector and multipoint CSF delivery to a clinical trial in Tay-Sachs and Sandhoff disease patients.
Authors
Toloo Taghian, Jillian Gallagher, Stephanie Bertrand, William C. Baker, Kalajan Lopez Mercado, Hector R. Benatti, Erin Hall, Yvette Lopez, Abigail McElroy, John T. McCarthy, Sanjana Pulaparthi, Deborah Fernau, Samuel Mather, Sophia Esteves, Elise Diffie, Amanda Gross, Hannah G. Lahey, Xuntian Jiang, Elizabeth Parsley, Rachael Gately, Rachel Prestigiacomo, Siauna Johnson, Amanda Taylor, Lindsey Bierfeldt, Susan Tuominen, Jennifer Koehler, Guangping Gao, Jun Xie, Qin Su, Robert King, Matthew J. Gounis, Vania Anagnostakou, Ajit Puri, Ana Rita Batista, Miguel Sena-Esteves, Douglas R. Martin, Heather Gray-Edwards
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Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)