Development
Abstract
Ehlers-Danlos Syndrome, Classic-Like, 2 (clEDS2) is a rare genetic disorder caused by biallelic mutations in the AEBP1 gene, which encodes Aortic carboxypeptidase-like protein (ACLP). Patients with clEDS2 exhibit hallmark features such as loose connective tissues, osteoporosis, and scoliosis. Despite its clinical significance, the molecular mechanisms underlying AEBP1 mutations in skeletal development remain poorly understood, and effective therapeutic strategies are currently unavailable. Here, using OsxCre conditional knockout mice, we show that Aebp1 deletion in osteoprogenitors reduces body size and bone mass, recapitulating key skeletal features reported in clEDS2. In primary osteoblasts, both genetic deletion and siRNA-mediated knockdown of Aebp1 impair osteoblast differentiation. Mechanistically, Aebp1 loss attenuates Wnt/β-catenin signaling in bone. Restoration of Wnt/β-catenin signaling by injecting BIO, a small molecule inhibitor of GSK3, substantially rescued bone mass reduction in Aebp1 knockout mice. These findings support a model in which Aebp1 sustains baseline Wnt/β-catenin tone in osteoblast-lineage cells and suggest that Wnt-targeted approaches may help mitigate clEDS2-related skeletal defects.
Authors
Shuhao Feng, Zihang Feng, Zhonghao Deng, Yiran Wei, Ru Lian, Yangchen Jin, Shiqi Zhao, Yu Jin, Zhongmin Zhang, Liang Zhao
Abstract
SLC26A9 is an epithelial chloride channel that was identified as a genetic modifier of disease severity of cystic fibrosis (CF) and other chronic muco-obstructive lung diseases. However, data on the in vivo role of SLC26A9 function in lung health and disease remain limited. Here, we investigated the effect of genetic deletion of Slc26a9 (Slc26a9-/-) on the pulmonary phenotype of neonatal mice. We found that lack of Slc26a9 causes severe neonatal respiratory distress with high mortality. Histology, immunohistochemistry and micro-computed tomography imaging studies identified airway obstruction with MUC5B-positive mucus plugs in neonatal Slc26a9-/- mice. Bioelectric measurements demonstrated a reduced transepithelial potential difference indicative of reduced chloride secretion across tracheal explants of neonatal Slc26a9-/- compared to wild-type mice. In addition, neonatal Slc26a9-/- mice displayed hypoxic degeneration of airway epithelial cells associated with sterile neutrophilic airway inflammation. Collectively, our data show that SLC26A9-mediated chloride secretion is critical for proper mucociliary clearance, respiratory function and survival after birth, and identify a novel role of SLC26A9 in neonatal adaptation during the transition from fetal to neonatal life.
Authors
Pamela Millar-Büchner, Johanna J. Salomon, Julia Duerr, Stephan Spahn, Pinelopi Anagnostopoulou, Willi L. Wagner, Mark O. Wielpuetz, Hermann-Josef Gröne, Anita Balázs, Marcus A. Mall
Abstract
Secreted high mobility group box protein 1 (HMGB1) regulates the adaptive immune response and acts as a biosensor for cells undergoing necrosis, stress, and inflammatory stimulation. However, its role in B cells remains enigmatic. Here, we demonstrate that HMGB1 is critical for peripheral B cell homeostasis and humoral immunity. Conditional deletion of Hmgb1 in B cells led to expanded marginal zone B cells, reduced B1a cells, and impaired antigen-specific antibody responses. Mechanistically, HMGB1 deficiency enhanced proximal and distal B cell receptor (BCR) signaling, probably via increased CD21 expression, which lowered the BCR activation threshold. This phenotype was linked to reduced lymphoid enhancer-binding factor 1 (LEF1) levels, a Wnt-responsive transcription factor, as HMGB1 directly bound the Lef1 promoter to sustain its transcription, thereby repressing Cd21. Furthermore, HMGB1 constrained actin reorganization by suppressing the MST1/DOCK8/WASP axis, which feedback-modulated BCR clustering and signalosome recruitment. Collectively, HMGB1 ensures optimal BCR signaling by transcriptionally and cytoskeletally tuning activation thresholds, highlighting its dual role as a nuclear regulator and cytoskeletal modulator in B cell immunity.
Authors
Qiuyue Chen, Ziyin Zhang, Nanshu Xiang, Li Luo, Xin Dai, Danqing Kang, Lu Yang, Yingzi Zhu, Jiang Chang, Yukai Jing, Na Li, Qianglin Chen, Panpan Jiang, Ju Liu, Yanmei Huang, Heather Miller, Xinyuan Zhou, Fang Zheng, Quan Gong, Chaohong Liu
Abstract
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein. Several therapeutic approaches boosting SMN are approved for human patients, delivering remarkable improvements in lifespan and symptoms. However, emerging phenotypes, including neurodevelopmental comorbidities, are being reported in some treated SMA patients, indicative of alterations in brain development. Here, using a mouse model of severe SMA, we revealed an underlying neurodevelopmental phenotype in SMA where prenatal SMN-dependent defects in translation drove disruptions in non-motile primary cilia across the central nervous system (CNS). Low levels of SMN caused widespread perturbations in translation at embryonic day (E) 14.5 targeting genes associated with primary cilia. The density of primary cilia in vivo, as well as cilial length in vitro, was significantly decreased in prenatal SMA mice. Proteomic analysis revealed downstream perturbations in primary cilia-regulated signalling pathways, including Wnt signalling. Cell proliferation was concomitantly reduced in the hippocampus of SMA mice. Prenatal transplacental therapeutic intervention with SMN-restoring risdiplam rescued primary cilia defects in SMA mouse embryos. Thus, SMN protein is required for normal cellular and molecular development of primary cilia in the CNS. Early, systemic treatment with SMN-restoring therapies can successfully target neurodevelopmental comorbidities in SMA.
Authors
Federica Genovese, Yu-Ting Huang, Anna A.L. Motyl, Martina Paganin, Gaurav Sharma, Ilaria Signoria, Deborah Donzel, Nicole C.H. Lai, Marie Pronot, Rachel A. Kline, Helena Chaytow, Kimberley J. Morris, Kiterie M.E. Faller, Thomas M. Wishart, Ewout J.N. Groen, Michael A. Cousin, Gabriella Viero, Thomas H. Gillingwater
Abstract
Recent findings suggest that the small intestine (SI) is a novel site for B cell lymphopoiesis during fetal and neonatal life. However, the unique and/or conserved features that enable B cell development at this site remain unclear. To investigate the molecular and cellular scaffolds for B cell lymphopoiesis in mouse and human fetal intestines we leveraged single-cell RNA sequencing, in situ immunofluorescence, spatial transcriptomics and high-dimensional spectral flow cytometry. We found that SI mesenchymal and stromal cells expressed higher levels of chemokines known to recruit common lymphoid progenitors. Importantly, local lymphatic endothelial cells expressed IL7 and TSLP in proximity to IL7R+ precursor B cells, likely promoting their differentiation in the SI. Notably, we found that fetal-derived lymphoid tissue inducer (LTi) cells were required for B cell development and localization in the SI, but not fetal liver. These findings identify a lymphoid tissue development-independent role for this immune cell in B cell development. Collectively, our data reveal a conserved intestinal B cell niche in mice and humans, challenging traditional models of lymphopoiesis. The identification of a requisite cellular/molecular scaffold for fetal B cell development allows future studies to test the importance of this de novo B cell lymphopoiesis to long-term immunity.
Authors
Kimberly A. Carroll, Weihong Gu, Long Phan, Eduardo Gonzalez Santiago, Wenjia Wang, George C. Tseng, Liza Konnikova, Shruti Sharma
Abstract
Bronchopulmonary dysplasia (BPD), a prevalent and chronic lung disease affecting premature newborns, results in vascular rarefaction and alveolar simplification. Although the vasculature has been recognized as a main player in this disease, the recently found capillary heterogeneity and cellular dynamics of endothelial subpopulations in BPD remain unclear. Here, we show Cap2 cells are damaged during neonatal hyperoxic injury, leading to their replacement by Cap1 cells which, in turn, significantly decline. Single cell RNA-seq identifies the activation of numerous p53 target genes in endothelial cells (ECs), including Cdkn1a (p21). While global deletion of p53 results in worsened vasculature, endothelial-specific deletion of p53 reverses the vascular phenotype and improves alveolar simplification during hyperoxia. This recovery is associated with the emergence of a transitional EC state, enriched for oxidative stress response genes and growth factors. Notably, this transitional EC gene signature is conserved in an aberrant capillary population identified in human BPD with pulmonary hypertension, underscoring the biological and clinical relevance of our findings. These results reveal a key role for p53 in maintaining endothelial lineage fidelity during pulmonary capillary repair following hyperoxic injury and highlight the critical contribution of the endothelium to BPD pathogenesis.
Authors
Lisandra Vila Ellis, Jonathan D. Bywaters, Amanda Ceas, Yun Liu, Jennifer M.S. Sucre, Jichao Chen
Abstract
Asthma is characterized by exacerbated response to triggers such as allergen. While pulmonary neuroendocrine cells (PNECs), a rare population of airway epithelial cells, are essential for amplifying allergen-induced asthma response, how PNECs are regulated to achieve this role remains poorly understood. Here we show that in the adult mouse airway, inactivation of achaete-scute-like protein 1 gene in PNECs led to loss of these cells. Intriguingly, exposure of these mutants to house dust mites (HDM), a common allergen, led to reappearance of PNECs. Similarly, exposure of wild-type mice to HDM led to PNEC hyperplasia, a result of proliferation of existing PNECs and transdifferentiation from club cells. Single-cell RNA-Seq experiments revealed PNEC heterogeneity, including the emergence of an allergen-induced PNEC subtype. Notch signaling was downregulated in HDM-treated airway, and treatment with Notch agonist prevented PNEC hyperplasia. These findings together suggest that HDM-induced PNEC hyperplasia may contribute to exacerbated asthma response.
Authors
Estelle Kim, Brian K. Wells, Hannah Indralingam, Yujuan Su, Jamie Verheyden, Xin Sun
Abstract
Mammalian skin is a vital barrier with the epidermis serving as its protective outer layer, continually undergoing renewal. Given that loss of the epidermis or its barrier function is lethal for mammals, multiple stem cell populations likely exist for the interfollicular epidermis (IFE), enhancing evolutionary survival. Here, we demonstrate that transcription factor KROX20 marks a heterogeneous stem cell population in the upper and middle mouse hair follicle (HF), partially overlapping with known HF stem cell markers in those regions. Lineage tracing in mice using different reporter lines shows that Krox20-lineage cells migrate from the HF to the IFE, contributing to both basal and suprabasal layers during adulthood. Spatial transcriptomics data corroborate our findings. Depletion of epithelial Krox20-expressing cells leads to epidermal hyperplasia and a disruption of stratification during morphogenesis and homeostasis. Our study highlights the contribution of hair follicle Krox20-lineage cells to the IFE and the regulation of epidermal homeostasis.
Authors
Elnaz Ghotbi, Edem Tchegnon, Ze Yu, Tracey Shipman, Zhiguo Chen, Yumeng Zhang, Renee M. McKay, Chao Xing, Chung-Ping Liao, Lu Q. Le
Abstract
Systemic arterial stenosis, including moyamoya disease (MMD) and middle aortic syndrome (MAS), is a rare condition of unclear etiology. MMD is a cerebral angiopathy, and MAS affects the abdominal and thoracic aorta. Although some genetic associations with MAS have been identified, the causes remain elusive. In this study, de novo heterozygous missense variants of RING finger protein 213 (RNF213) (p.His4058Pro and p.Thr4155Pro) in 2 unrelated families with MAS and MMD were studied by whole-exome sequencing. To elucidate the significance of these variants, we produced knockin mice carrying the Rnf213 p.His4058Pro variant. Homozygous knockin mice exhibited perinatal lethality because of respiratory failure and lung dysplasia, suggesting that this variant is pathogenic. Lung dysplasia in homozygous knockin mice was associated with upregulated innate immunity and inflammatory responses and downregulated cell proliferation. These findings suggested that in mice, the RNF213 p.His4058Pro variant plays critical roles in regulation of innate immunity and inflammation that affect lung development, revealing the complexity of RNF213 function in various tissues and species. In conclusion, this study provides insights into the genetic basis of MAS and MMD, highlights the potential involvement of RNF213 variants in systemic vasculopathy, and identifies unexpected associations with lung development and immune processes.
Authors
Ayako Kashimada, Tomoko Mizuno, Eriko Tanaka, Susumu Hosokawa, Tomohiro Udagawa, Yuichi Hiraoka, Keisuke Uchida, Tomohiro Morio, Kenjiro Kosaki, Masatoshi Takagi
Abstract
PDX1 mutations are associated with multiple forms of diabetes, including syndromic, neonatal, mature onset diabetes of the young (MODY), and type 2 diabetes. Two PDX1 missense mutations (Thr151Met and Asn196Thr) were identified in a pediatric female patient that cause permanent neonatal diabetes, pancreas hypoplasia, and a malformed gallbladder. We found that the mouse Pdx1 Asn197Thr variant (homologous to human PDX1 Asn196Thr), but not Pdx1 Thr152Met (homologous to human PDX1 Thr151Met), altered its nuclear localization and disrupted the PDX1-ONECUT1 interaction. Neither variant substantially affected PDX1 protein stability, but both reduced PDX1 binding to the Pdx1 gene promoter. Importantly, the Pdx1 Asn197Thr variant caused pancreas agenesis and reduced enteroendocrine cells in the duodenum in genetically engineered mice, due at least in part to reduced Pdx1 promoter binding and disrupted PDX1-ONECUT1 interaction.
Authors
Xiaodun Yang, Angela Zanfardino, Riccardo Schiaffini, Jeff Ishibashi, Bareket Daniel, Matthew W. Haemmerle, Novella Rapini, Alessia Piscopo, Emanuele Miraglia del Giudice, Maria Cristina Digilio, Raffaele Iorio, Mafalda Mucciolo, Stefano Cianfarani, Dario Iafusco, Fabrizio Barbetti, Doris A. Stoffers
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