Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The major cause of limited life span in CF patients is progressive lung disease. CF models have been generated in 4 species (mice, rats, ferrets, and pigs) to enhance our understanding of the CF pathogenesis. Sheep may be a particularly relevant animal to model CF in humans due to the similarities in lung anatomy and development in the two species. Here, we describe the generation of a sheep model for CF using CRISPR/Cas9 genome editing and somatic cell nuclear transfer (SCNT) techniques. We generated cells with CFTR gene disruption and used them for production of CFTR–/– and CFTR+/– lambs. The newborn CFTR–/– sheep developed severe disease consistent with CF pathology in humans. Of particular relevance were pancreatic fibrosis, intestinal obstruction, and absence of the vas deferens. Also, substantial liver and gallbladder disease may reflect CF liver disease that is evident in humans. The phenotype of CFTR–/– sheep suggests this large animal model will be a useful resource to advance the development of new CF therapeutics. Moreover, the generation of specific human CF disease–associated mutations in sheep may advance personalized medicine for this common genetic disorder.
Zhiqiang Fan, Iuri Viotti Perisse, Calvin U. Cotton, Misha Regouski, Qinggang Meng, Chaim Domb, Arnaud J. Van Wettere, Zhongde Wang, Ann Harris, Kenneth L. White, Irina A. Polejaeva
Different nephron tubule segments perform distinct physiological functions, collectively acting as a blood filtration unit. Dysfunction of the proximal tubule segment can lead to Fanconi renotubular syndrome (FRTS), with major symptoms such as excess excretion of water, glucose, and phosphate in the urine. It has been shown that a mutation in HNF4A is associated with FRTS in humans and that Hnf4a is expressed specifically in proximal tubules in adult rat nephrons. However, little is known about the role of Hnf4a in nephrogenesis. Here, we found that Hnf4a is expressed in both presumptive and differentiated proximal tubules in the developing mouse kidney. We show that Hnf4a is required for the formation of differentiated proximal tubules but is dispensable for the formation of presumptive proximal tubules. Furthermore, we show that loss of Hnf4a decreased the expression of proximal tubule–specific genes. Adult Hnf4a mutant mice presented with FRTS-like symptoms, including polyuria, polydipsia, glycosuria, and phosphaturia. Analysis of the adult Hnf4a mutant kidney also showed proximal tubule dysgenesis and nephrocalcinosis. Our results demonstrate the critical role of Hnf4a in proximal tubule development and provide mechanistic insight into the etiology of FRTS.
Sierra S. Marable, Eunah Chung, Mike Adam, S. Steven Potter, Joo-Seop Park
Hemodynamic shear force has been implicated as modulating Notch signaling–mediated cardiac trabeculation. Whether the spatiotemporal variations in wall shear stress (WSS) coordinate the initiation of trabeculation to influence ventricular contractile function remains unknown. Using light-sheet fluorescent microscopy, we reconstructed the 4D moving domain and applied computational fluid dynamics to quantify 4D WSS along the trabecular ridges and in the groves. In WT zebrafish, pulsatile shear stress developed along the trabecular ridges, with prominent endocardial Notch activity at 3 days after fertilization (dpf), and oscillatory shear stress developed in the trabecular grooves, with epicardial Notch activity at 4 dpf. Genetic manipulations were performed to reduce hematopoiesis and inhibit atrial contraction to lower WSS in synchrony with attenuation of oscillatory shear index (OSI) during ventricular development. γ-Secretase inhibitor of Notch intracellular domain (NICD) abrogated endocardial and epicardial Notch activity. Rescue with NICD mRNA restored Notch activity sequentially from the endocardium to trabecular grooves, which was corroborated by observed Notch-mediated cardiomyocyte proliferations on WT zebrafish trabeculae. We also demonstrated in vitro that a high OSI value correlated with upregulated endothelial Notch-related mRNA expression. In silico computation of energy dissipation further supports the role of trabeculation to preserve ventricular structure and contractile function. Thus, spatiotemporal variations in WSS coordinate trabecular organization for ventricular contractile function.
Juhyun Lee, Vijay Vedula, Kyung In Baek, Junjie Chen, Jeffrey J. Hsu, Yichen Ding, Chih-Chiang Chang, Hanul Kang, Adam Small, Peng Fei, Cheng-ming Chuong, Rongsong Li, Linda Demer, René R. Sevag Packard, Alison L. Marsden, Tzung K. Hsiai
Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product–induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.
Charitharth Vivek Lal, Nelida Olave, Colm Travers, Gabriel Rezonzew, Kalsang Dolma, Alexandra Simpson, Brian Halloran, Zubair Aghai, Pragnya Das, Nirmal Sharma, Xin Xu, Kristopher Genschmer, Derek Russell, Tomasz Szul, Nengjun Yi, J. Edwin Blalock, Amit Gaggar, Vineet Bhandari, Namasivayam Ambalavanan
The inverse relationship between gestational age at birth and postviral respiratory morbidity suggests that infants born preterm (PT) may miss a critical developmental window of T cell maturation. Despite a continued increase in younger PT survivors with respiratory complications, we have limited understanding of normal human fetal T cell maturation, how ex utero development in premature infants may interrupt normal T cell development, and whether T cell development has an effect on infant outcomes. In our longitudinal cohort of 157 infants born between 23 and 42 weeks of gestation, we identified differences in T cells present at birth that were dependent on gestational age and differences in postnatal T cell development that predicted respiratory outcome at 1 year of age. We show that naive CD4+ T cells shift from a CD31–TNF-α+ bias in mid gestation to a CD31+IL-8+ predominance by term gestation. Former PT infants discharged with CD31+IL8+CD4+ T cells below a range similar to that of full-term born infants were at an over 3.5-fold higher risk for respiratory complications after NICU discharge. This study is the first to our knowledge to identify a pattern of normal functional T cell development in later gestation and to associate abnormal T cell development with health outcomes in infants.
Kristin M. Scheible, Jason Emo, Nathan Laniewski, Andrea M. Baran, Derick R. Peterson, Jeanne Holden-Wiltse, Sanjukta Bandyopadhyay, Andrew G. Straw, Heidie Huyck, John M. Ashton, Kelly Schooping Tripi, Karan Arul, Elizabeth Werner, Tanya Scalise, Deanna Maffett, Mary Caserta, Rita M. Ryan, Anne Marie Reynolds, Clement L. Ren, David J. Topham, Thomas J. Mariani, Gloria S. Pryhuber
CHD7, an ATP-dependent chromatin remodeler, is disrupted in CHARGE syndrome, an autosomal dominant disorder characterized by variably penetrant abnormalities in craniofacial, cardiac, and nervous system tissues. The inner ear is uniquely sensitive to CHD7 levels and is the most commonly affected organ in individuals with CHARGE. Interestingly, upregulation or downregulation of retinoic acid (RA) signaling during embryogenesis also leads to developmental defects similar to those in CHARGE syndrome, suggesting that CHD7 and RA may have common target genes or signaling pathways. Here, we tested three separate potential mechanisms for CHD7 and RA interaction: (a) direct binding of CHD7 with RA receptors, (b) regulation of CHD7 levels by RA, and (c) CHD7 binding and regulation of RA-related genes. We show that CHD7 directly regulates expression of Aldh1a3, the gene encoding the RA synthetic enzyme ALDH1A3 and that loss of Aldh1a3 partially rescues Chd7 mutant mouse inner ear defects. Together, these studies indicate that ALDH1A3 acts with CHD7 in a common genetic pathway to regulate inner ear development, providing insights into how CHD7 and RA regulate gene expression and morphogenesis in the developing embryo.
Hui Yao, Sophie F. Hill, Jennifer M. Skidmore, Ethan D. Sperry, Donald L. Swiderski, Gilson J. Sanchez, Cynthia F. Bartels, Yehoash Raphael, Peter C. Scacheri, Shigeki Iwase, Donna M. Martin
The adrenal cortex undergoes remodeling during fetal and postnatal life. How zona reticularis emerges in the postnatal gland to support adrenarche, a process whereby higher primates increase prepubertal androgen secretion, is unknown. Using cell-fate mapping and gene deletion studies in mice, we show that activation of PKA has no effect on the fetal cortex, while it accelerates regeneration of the adult cortex, triggers zona fasciculata differentiation that is subsequently converted into a functional reticularis-like zone, and drives hypersecretion syndromes. Remarkably, PKA effects are influenced by sex. Indeed, testicular androgens increase WNT signaling that antagonizes PKA, leading to slower adrenocortical cell turnover and delayed phenotype whereas gonadectomy sensitizes males to hypercorticism and reticularis-like formation. Thus, reticularis results from ultimate centripetal conversion of adult cortex under the combined effects of PKA and cell turnover that dictate organ size. We show that PKA-induced progenitor recruitment is sexually dimorphic and may provide a paradigm for overrepresentation of women in adrenal diseases.
Typhanie Dumontet, Isabelle Sahut-Barnola, Amandine Septier, Nathanaëlle Montanier, Ingrid Plotton, Florence Roucher-Boulez, Véronique Ducros, Anne-Marie Lefrançois-Martinez, Jean-Christophe Pointud, Mohamad Zubair, Ken-Ichirou Morohashi, David T. Breault, Pierre Val, Antoine Martinez
Incomplete penetrance of congenital heart defects (CHDs) was observed in a mouse model. We hypothesized that the contribution of a major genetic locus modulates the manifestation of the CHDs. After genome-wide linkage mapping, fine mapping, and high-throughput targeted sequencing, a recessive frameshift mutation of the heterogeneous nuclear ribonucleoprotein A1 (Hnrnpa1) gene was confirmed (Hnrnpa1ct). Hnrnpa1 was expressed in both the first heart field (FHF) and second heart field (SHF) at the cardiac crescent stage but was only maintained in SHF progenitors after heart tube formation. Hnrnpa1ct/ct homozygous mutants displayed complete CHD penetrance, including truncated and incomplete looped heart tube at E9.5, ventricular septal defect (VSD) and persistent truncus arteriosus (PTA) at E13.5, and VSD and double outlet right ventricle at P0. Impaired development of the dorsal mesocardium and sinoatrial node progenitors was also observed. Loss of Hnrnpa1 expression leads to dysregulation of cardiac transcription networks and multiple signaling pathways, including BMP, FGF, and Notch in the SHF. Finally, two rare heterozygous mutations of HNRNPA1 were detected in human CHDs. These findings suggest a role of Hnrnpa1 in embryonic heart development in mice and humans.
Zhe Yu, Paul L.F. Tang, Jing Wang, Suying Bao, Joseph T. Shieh, Alan W.L. Leung, Zhao Zhang, Fei Gao, Sandra Y.Y. Wong, Andy L.C. Hui, Yuan Gao, Nelson Dung, Zhi-Gang Zhang, Yanhui Fan, Xueya Zhou, Yalun Zhang, Dana S.M. Wong, Pak C. Sham, Abid Azhar, Pui-Yan Kwok, Patrick P.L. Tam, Qizhou Lian, Kathryn S.E. Cheah, Binbin Wang, You-Qiang Song
Vertebrates possess 2 proteins with vitamin K oxidoreductase (VKOR) activity: VKORC1, whose vitamin K reduction supports vitamin K–dependent (VKD) protein carboxylation, and VKORC1-like 1 (VKORC1L1), whose function is unknown. VKD proteins include liver-derived coagulation factors, and hemorrhaging and lethality were previously observed in mice lacking either VKORC1 or the γ-glutamyl carboxylase (GGCX) that modifies VKD proteins. Vkorc1–/– mice survived longer (1 week) than Ggcx–/– mice (midembryogenesis or birth), and we assessed whether VKORC1L1 could account for this difference. We found that Vkorc1–/–;Vkorc1l1–/– mice died at birth with severe hemorrhaging, indicating that VKORC1L1 supports carboxylation during the pre- and perinatal periods. Additional studies showed that only VKORC1 sustains hemostasis beyond P7. VKORC1 expression and VKOR activity increased during late embryogenesis and following birth, while VKORC1L1 expression was unchanged. At P0, most (>99%) VKOR activity was due to VKORC1. Prothrombin mRNA, protein, and carboxylation also increased during this period, as did mRNA levels of coagulation factors encoding genes F7, F9, and F10. VKORC1L1 levels in Vkorc1–/– mouse liver may therefore be insufficient for supporting carboxylation beyond day 7. In support of this conclusion, VKORC1L1 overexpression in liver rescued carboxylation and hemostasis in adult Vkorc1–/– mice. These findings establish that VKORC1L1 supports VKD protein carboxylation in vivo.
Julie Lacombe, Mark A. Rishavy, Kathleen L. Berkner, Mathieu Ferron
Kit receptor tyrosine kinase is highly expressed in the developing mammalian brain, yet little is known about its contribution to neural cell development and function. Here we introduced a brain-specific conditional Kit loss-of-function mutation in mice and observed severe hypoplasia of the central nervous system. This was accompanied by an increase in apoptotic cell death in the early embryonic brain and the gradual loss of the self-renewal capacity of neuronal stem/precursor cells. A single copy of the brain-specific conditional Kit loss-of-function allele resulted in the observed phenotype, including impaired in vitro differentiation of neural cells from Kit-haploinsufficient embryonic stem (ES) cells. Our findings demonstrate that Kit signaling is required for the early development of neural cells. This potentially novel Kit-haploinsufficient lethal phenotype may represent an embryonic lethal phenomenon previously unobserved because of its dominantly acting nature.
Hitomi Aoki, Akira Hara, Takahiro Kunisada
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