Development
Abstract
Low-calorie sweetener (LCS) consumption in children has increased dramatically due to widespread presence in the food environment and efforts to mitigate obesity through sugar replacement. However, mechanistic studies on the long-term impact of early-life LCS consumption on cognitive function and physiological processes are lacking. Here, we developed a rodent model to evaluate the effects of daily LCS consumption (acesulfame potassium, saccharin, or stevia) during adolescence on adult metabolic, behavioral, gut microbiome, and brain transcriptomic outcomes. Results reveal that habitual early-life LCS consumption impacts normal post-oral glucose handling and impairs hippocampal-dependent memory in the absence of weight gain. Furthermore, adolescent LCS consumption yielded long-term reductions in lingual sweet taste receptor expression and alterations in sugar-motivated appetitive and consummatory responses. While early life LCS consumption did not produce robust changes in the gut microbiome, brain region-specific RNA sequencing analyses reveal LCS-induced changes in collagen- and synaptic signaling-related gene pathways in the hippocampus and nucleus accumbens, respectively, in a sex-dependent manner. Collectively, these results reveal that habitual early-life LCS consumption has long lasting implications for glucoregulation, sugar-motivated behavior, and hippocampal-dependent memory in rats, which may be based in part on changes in nutrient transporter, sweet taste receptor, and central gene pathway expression.
Authors
Linda Tsan, Sandrine Chometton, Anna M.R. Hayes, Molly E. Klug, Yanning Zuo, Shan Sun, Lana Bridi, Rae Lan, Anthony A. Fodor, Emily E. Noble, Xia Yang, Scott E. Kanoski, Lindsey A. Schier
Abstract
Lymphangiectasia, an anomalous dilation of lymphatic vessels first described in the 17th century, is frequently associated with chylous effusion, respiratory failure, and high mortality in young patients, yet the underlying molecular pathogenesis and effective treatments remain elusive. Here, we identify an unexpected causal link between MAPK activation and defective development of the lymphatic basement membrane that drives lymphangiectasia. Human pathological tissue samples from patients diagnosed with lymphangiectasia revealed sustained MAPK activation within lymphatic endothelial cells. Endothelial KRASG12D–mediated sustained MAPK activation in newborn mice caused severe pulmonary and intercostal lymphangiectasia, accumulation of chyle in the pleural space, and complete lethality. Pathological activation of MAPK in murine vasculature inhibited the Nfatc1-dependent genetic program required for laminin interactions, collagen crosslinking, and anchoring fibril formation, driving defective development of the lymphatic basement membrane. Treatment with ravoxertinib, a pharmacological inhibitor of MAPK, reverses nuclear-to-cytoplasmic localization of Nfatc1, basement membrane development defects, lymphangiectasia, and chyle accumulation, ultimately improving survival of endothelial KRAS mutant neonatal mice. These results reveal defective lymphatic basement membrane assembly and composition as major causes of thoracic lymphangiectasia and provide a potential treatment.
Authors
Harish P. Janardhan, Karen Dresser, Lloyd Hutchinson, Chinmay M. Trivedi
Abstract
Adaptation to increased insulin demand is mediated by β-cell proliferation and neogenesis among other mechanisms. Although it is known that pancreatic β-cells can arise from ductal progenitors, these observations have been limited mostly to the neonatal period. We have recently reported that the duct is a source of insulin secreting cells in adult insulin resistant states. To further explore the signaling pathways underlying the dynamic β-cell reserve during insulin resistance we undertook human islet and duct transplantations under the kidney capsule of immunodeficient NOD SCID gamma (NSG) mouse models that were either pregnant, insulin resistant or had insulin resistance superimposed upon pregnancy (pregnancy+insulin resistance), followed by single-nucleus RNA-sequencing (snRNA-seq) on snap-frozen graft samples. We observed an upregulation of proliferation markers (e.g., NEAT1), expression of islet endocrine cell markers (e.g., GCG and PPY) as well as mature β-cell markers (e.g., INS), in transplanted human duct grafts in response to high insulin demand. We also noted downregulation of ductal cell identity genes (e.g., KRT19 and ONECUT2) coupled with upregulation of β-cell development and insulin signaling pathways. These results indicate that subsets of ductal cells are able to gain β-cell identity and reflect a form of compensation during the adaptation to insulin resistance in both physiological and pathological states.
Authors
Ercument Dirice, Giorgio Basile, Sevim Kahraman, Danielle Diegisser, Jiang Hu, Rohit N. Kulkarni
Abstract
Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease.
Authors
Dale Bryant, Valda Pauzuolyte, Neil J. Ingham, Aara Patel, Waheeda Pagarkar, Lucy A. Anderson, Katie E. Smith, Dale A. Moulding, Yeh C. Leong, Daniyal J. Jafree, David A. Long, Amina Al-Yassin, Karen P. Steel, Daniel J. Jagger, Andrew Forge, Wolfgang Berger, Jane C. Sowden, Maria Bitner-Glindzicz
Abstract
Infants born prematurely worldwide have up to a 50% chance of developing Bronchopulmonary Dysplasia (BPD), a clinical morbidity characterized by dysregulated lung alveolarization and microvascular development. It is known that Platelet-Derived Growth Factor Receptor Alpha positive (PDGFRA+) fibroblasts are critical for alveolarization, and that PDGFRA+ fibroblasts are reduced in BPD. A better understanding of fibroblast heterogeneity and functional activation status during pathogenesis is required to develop mesenchymal-targeted therapies for BPD. In this study, we utilized a neonatal hyperoxia mouse model (90% O2 PN0-PN7) and performed studies on sorted PDGFRA+ cells during injury and room air recovery. After hyperoxia injury, PDGFRA+ matrix and myofibroblasts decrease and PDGFRA+ lipofibroblasts increase by transcriptional signature and population size. PDGFRA+ matrix and myofibroblast recover during repair (PN10). After 7 days of in vivo hyperoxia, PDGFRA+ sorted fibroblasts have reduced contractility in vitro, reflecting loss of myofibroblast commitment. Organoids made with PN7 PDGFRA+ fibroblasts from hyperoxia mice exhibit reduced alveolar type 1 cell differentiation, suggesting reduced alveolar niche-supporting PDGFRA+ matrix fibroblast function. Pathway analysis predicted reduced WNT signaling in hyperoxia fibroblasts. In alveolar organoids from hyperoxia exposed fibroblasts WNT activation by CHIR increased size and number of alveolar organoids and enhanced alveolar type 2 cell differentiation.
Authors
Matthew R. Riccetti, Mereena George Ushakumary, Marion Waltamath, Jenna Green, John Snowball, Sydney E. Dautel, Mehari Endale, Bonny Lami, Jason Woods, Shawn K. Ahlfeld, Anne-Karina T. Perl
Abstract
The molecular mechanisms that drive the acquisition of distinct neural crest cell (NCC) fates is still poorly understood. Here, we identify Prdm6 as an epigenetic modifier that temporally and spatially regulates the expression of NCC specifiers and determines the fate of a subset of migrating Cardiac NCCs (CNCCs). Using transcriptomic analysis, genetic and fate mapping approaches in transgenic mice, we show that disruption of Prdm6 is associated with impaired CNCC differentiation, delamination, and migration, and leads to patent ductus arteriosus (DA)and ventricular noncompaction. Bulk and single-cell RNA-seq analyses of DA and CNCC identify Prdm6 as a regulator of a network of CNCC specification genes including Wnt1, Tfap2b, and Sox9. Loss of Prdm6 in CNCCs diminishes its expression in pre-EMT cluster, resulting in the retention of NCC in the dorsal neural tube. This defect is associated with diminished H4K20 mono-methylation and G1-S progression and augmented Wnt1 transcript levels in pre-EMT and neural tube clusters, which we show is the major driver of the impaired CNCC migration. Altogether, these findings reveal Prdm6 as a key regulator of CNCC differentiation and migration and identify Prdm6 and its regulated network as potential targets for the treatment of congenital heart diseases.
Authors
Lingjuan Hong, Na Li, Victor Gasque, Sameet Mehta, Lupeng Ye, Yinyu Wu, Jinyu Li, Andreas Gewies, Jürgen Ruland, Karen K. Hirschi, Anne Eichmann, Caroline Hendry, David van Dijk, Arya Mani
Abstract
Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole genome sequencing analysis on 146 non-syndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this dataset to a European cohort identified both overlapping and novel gene loci, and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene co-expression modules along cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations.
Authors
Clara Sze Man Tang, Mimmi Mononen, Wai-Yee Lam, Sheng Chih Jin, Xuehan Zhuang, Maria-Mercè Garcia-Barcelo, Qiongfen Lin, Yujia Yang, Makoto Sahara, Elif Eroglu, Kenneth R. Chien, Haifa Hong, Paul K.H. Tam, Peter J. Gruber
Abstract
The genetic bases for the Congenital Disorders of Glycosylation (CDG) continue to expand but an understanding of how glycosylation defects cause patient phenotypes remains largely unknown. Here we combined developmental phenotyping and biochemical studies in a new zebrafish model (pmm2sa10150) of PMM2-CDG to uncover a protease-mediated pathogenic mechanism relevant to craniofacial and motility phenotypes exhibted by mutant embryos. Mutant embryos have reduced phosphomannomutase activity and modest decreases in N-glycan occupancy as detected by MALDI MS imaging. Cellular analyses of cartilage defects in pmm2 sa10150 embryos revealed a block in chondrogenesis that is associated with defective proteolytic processing, but seemingly normal N-glycosylation, of the cell adhesion molecule N-cadherin. The activities of the proconvertases and matrix metalloproteinases responsible for N-cadherin maturation were significantly altered in pmm2sa10150 mutant embryos. Importantly, pharmacologic and genetic manipulation of proconvertase activity restored matrix metalloproteinase activity, N-cadherin processing and cartilage pathology in pmm2 sa10150 embryos. Collectively, these studies demonstrate in CDG that targeted alterations in protease activity create a pathogenic cascade that impacts the maturation of cell adhesion proteins critical for tissue development.
Authors
Elsenoor J. Klaver, Lynn Dukes-Rimsky, Brijesh Kumar, Zhi-Jie Xia, Tammie Dang, Mark A. Lehrman, Peggi Angel, Richard R. Drake, Hudson H. Freeze, Richard Steet, Heather Flanagan-Steet
Abstract
We recently described a previously unknown trans-tentorial venous system (TTVS) connecting venous drainage throughout the brain in humans. Prior to this finding, it was believed that the embryologic tentorial plexus regresses, resulting in a largely avascular tentorium. Our finding contradicted this understanding and necessitated further investigation into the development of the newly described TTVS. Herein we sought to investigate mice as a model to study the development of this system. First, using vascular casting and ex vivo micro-computed tomography (micro-CT), we demonstrate that this TTVS is conserved in adult mice. Next, using high-resolution magnetic resonance imaging (MRI), we found the primitive tentorial venous plexus in murine embryo at day 14.5. We also found that, at this embryologic stage, the tentorial plexus drains the choroid plexus. Finally, using vascular casting and micro-CT, we found that the TTVS is the dominant venous drainage in the early postnatal period (P8). Herein, we demonstrate that the TTVS is conserved between mice and humans and present a longitudinal study of its development. In addition, our findings establish mice as a translational model for further study of this newly described system and its relationship to intracranial physiology.
Authors
Pashayar P. Lookian, Vikram Chandrashekhar, Anthony Cappadona, Jean-Paul Bryant, Vibhu Chandrashekhar, Jessa M. Tunacao, Danielle R. Donahue, Jeeva P. Munasinghe, James G. Smirniotopoulos, John D. Heiss, Zhengping Zhuang, Jared S. Rosenblum
Abstract
Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex ‘organization’ of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter, in mice, to study expression of its ligand, ephrinB2, and analysed developmental phenotypes following conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells, and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization, and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.
Authors
Oliver Lyons, James Walker, Christopher Seet, Mohammed Ikram, Adam Kuchta, Andrew Arnold, Magda Hernández-Vásquez, Maike Frye, Gema Vizcay-Barrena, Roland A. Fleck, Ashish S. Patel, Soundrie Padayachee, Peter Mortimer, Steve Jeffery, Siren Berland, Sahar Mansour, Pia Ostergaard, Taija Makinen, Bijan Modarai, Prakash Saha, Alberto Smith
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