Development
Abstract
Previous studies have demonstrated the presence of microbial DNA in the fetal environment. However, it remains unclear whether this DNA represents viable bacteria and how it relates to the maternal microbiota across different body sites. We studied the microbiota of human and mouse dyads to understand these relationships, localize bacteria in the fetus, and demonstrate bacterial viability. In human preterm and full-term mother-infant dyads at the time of Cesarean delivery, the oral cavity and meconium of newborn infants born as early as 24 weeks of gestation contained a microbiota that was predicted to originate from in utero sources including the placenta. Using operative deliveries of pregnant mice under highly controlled, sterile conditions in the laboratory, composition, visualization, and viability of bacteria in the in utero compartment and fetal intestine were demonstrated by 16S rRNA gene sequencing, fluorescence in situ hybridization, and bacterial culture. The composition and predicted source of the fetal gut microbiota shifted between mid- and late gestation. Cultivatable bacteria in the fetal intestine were found during mid-gestation but not late gestation. Our results demonstrate a dynamic, viable mammalian fetal microbiota during in utero development.
Authors
Noelle Younge, Jessica R. McCann, Julie Ballard, Catherine Plunkett, Suhail Akhtar, Félix Araújo-Pérez, Amy Murtha, Debra Brandon, Patrick C. Seed
Abstract
Left ventricular noncompaction (LVNC) is one of the most common forms of genetic cardiomyopathy characterized by excessive trabeculation and impaired myocardial compaction during fetal development. Patients with LVNC are at higher risk of developing left/right ventricular failure or both. Although the key regulators for cardiac chamber development are well studied, the role of semaphorin (Sema)/plexin signaling in this process remains poorly understood. In this article, we demonstrate that genetic deletion of Plxnd1, a class-3 Sema receptor in endothelial cells, leads to severe cardiac chamber defects. They were characterized by excessive trabeculation and noncompaction similar to patients with LVNC. Loss of Plxnd1 results in decreased expression of extracellular matrix proteolytic genes, leading to excessive deposition of cardiac jelly. We demonstrate that Plxnd1 deficiency is associated with an increase in Notch1 expression and its downstream target genes. In addition, inhibition of the Notch signaling pathway partially rescues the excessive trabeculation and noncompaction phenotype present in Plxnd1 mutants. Furthermore, we demonstrate that Semaphorin 3E (Sema3E), one of PlexinD1’s known ligands, is expressed in the developing heart and is required for myocardial compaction. Collectively, our study uncovers what we believe to be a previously undescribed role of the Sema3E/PlexinD1 signaling pathway in myocardial trabeculation and the compaction process.
Authors
Reddemma Sandireddy, Dasan Mary Cibi, Priyanka Gupta, Anamika Singh, Nicole Tee, Akiyoshi Uemura, Jonathan A. Epstein, Manvendra K. Singh
Abstract
The ang1-Tie2 pathway is required for normal vascular development, but its molecular effectors are not well-defined during cardiac ontogeny. Here we show that endocardial specific attenuation of Tie2 results in mid-gestation lethality due to heart defects associated with a hyperplastic but simplified trabecular meshwork (fewer but thicker trabeculae). Reduced proliferation and production of endocardial cells (ECs) following endocardial loss of Tie2 results in decreased endocardial sprouting required for trabecular assembly and extension. The hyperplastic trabeculae result from enhanced proliferation of trabecular cardiomyocyte (CMs), which is associated with upregulation of Bmp10, increased retinoic acid (RA) signaling, and Erk1/2 hyperphosphorylation in the myocardium. Intriguingly, myocardial phenotypes in Tie2-cko hearts could be partially rescued by inhibiting in utero RA signaling with pan-retinoic acid receptor antagonist BMS493. These findings reveal two complimentary functions of endocardial Tie2 during ventricular chamber formation: ensuring normal trabeculation by supporting EC proliferation and sprouting, and preventing hypertrabeculation via suppression of RA signaling in trabecular CMs.
Authors
Xianghu Qu, Cristina Harmelink, H. Scott Baldwin
Abstract
Children with trisomy 21 (Down syndrome [DS]) have a 130-fold increased incidence of Hirschsprung Disease (HSCR), a developmental defect where the enteric nervous system (ENS) is missing from distal bowel (i.e., distal bowel is aganglionic). Treatment for HSCR is surgical resection of aganglionic bowel, but many children have bowel problems after surgery. Post-surgical problems like enterocolitis and soiling are especially common in children with DS. To determine how trisomy 21 affects ENS development, we evaluated the ENS in two DS mouse models, Ts65Dn and Tc1. These mice are trisomic for many chromosome 21 homologous genes, including Dscam and Dyrk1a, which are hypothesized to contribute to HSCR risk. Ts65Dn and Tc1 mice have normal ENS precursor migration at E12.5 and almost normal myenteric plexus structure as adults. However, Ts65Dn and Tc1 mice have markedly reduced submucosal plexus neuron density throughout the bowel. Surprisingly, the submucosal neuron defect in Ts65Dn mice is not due to excess Dscam or Dyrk1a, since normalizing copy number for these genes does not rescue the defect. These findings suggest the possibility that the high frequency of bowel problems in children with DS and HSCR may occur because of additional unrecognized problems with ENS structure.
Authors
Ellen M. Schill, Christina M. Wright, Alisha Jamil, Jonathan M. LaCombe, Randall J. Roper, Robert O. Heuckeroth
Abstract
Biomechanical forces and endothelial-to-mesenchymal transition (EndoMT) are known to mediate valvulogenesis. However, the relative contributions of myocardial contractile and hemodynamic shear forces remain poorly understood. We integrated 4-D light-sheet imaging of transgenic zebrafish models with moving-domain computational fluid dynamics to determine effects of changes in contractile forces and fluid wall shear stress (WSS) on ventriculobulbar (VB) valve development. Augmentation of myocardial contractility with isoproterenol increased both WSS and Notch1b activity in the developing outflow tract (OFT) and resulted in VB valve hyperplasia. Increasing WSS in the OFT, achieved by increasing blood viscosity through EPO mRNA injection, also resulted in VB valve hyperplasia. Conversely, decreasing myocardial contractility by Tnnt2a morpholino oligonucleotide (MO) administration, 2,3-butanedione monoxime treatment, or Plcγ1 inhibition completely blocked VB valve formation, which could not be rescued by increasing WSS or activating Notch. Decreasing WSS in the OFT, achieved by slowing heart rate with metoprolol or reducing viscosity with Gata1a MO, did not affect VB valve formation. Immunofluorescent staining with the mesenchymal marker, DM-GRASP, revealed that biomechanical force-mediated Notch1b activity is implicated in EndoMT to modulate valve morphology. Altogether, increases in WSS result in Notch1b- EndoMT-mediated VB valve hyperplasia, whereas decreases in contractility result in reduced Notch1b activity, absence of EndoMT, and VB valve underdevelopment. Thus, we provide developmental mechanotransduction mechanisms underlying Notch1b-mediated EndoMT in the OFT.
Authors
Jeffrey J. Hsu, Vijay Vedula, Kyung In Baek, Cynthia Chen, Junjie Chen, Man In Chou, Jeffrey Lam, Shivani Subhedar, Jennifer Wang, Yichen Ding, Chih-Chiang Chang, Juhyun Lee, Linda L. Demer, Yin Tintut, Alison L. Marsden, Tzung K. Hsiai
Abstract
During endochondral bone formation, chondrocyte hypertrophy represents a crucial turning point from chondrocyte differentiation to bone formation. Both parathyroid hormone-related protein (PTHrP) and histone deacetylase 4 (HDAC4) inhibit chondrocyte hypertrophy. Using multiple mouse genetics models, we demonstrate in vivo that HDAC4 is required for the effects of PTHrP on chondrocyte differentiation. We further show in vivo that PTHrP leads to reduced HDAC4 phosphorylation at the 14-3-3–binding sites and subsequent HDAC4 nuclear translocation. The Hdac4-KO mouse shares a similar but milder phenotype with the Pthrp-KO mouse, indicating the possible existence of other mediators of PTHrP action. We identify HDAC5 as an additional mediator of PTHrP signaling. While the Hdac5-KO mouse has no growth plate phenotype at birth, the KO of Hdac5 in addition to the KO of Hdac4 is required to block fully PTHrP action on chondrocyte differentiation at birth in vivo. Finally, we show that PTHrP suppresses myocyte enhancer factor 2 (Mef2) action that allows runt-related transcription factor 2 (Runx2) mRNA expression needed for chondrocyte hypertrophy. Our results demonstrate that PTHrP inhibits chondrocyte hypertrophy and subsequent bone formation in vivo by allowing HDAC4 and HDAC5 to block the Mef2/Runx2 signaling cascade. These results explain the phenotypes of several genetic abnormalities in humans.
Authors
Shigeki Nishimori, Forest Lai, Mieno Shiraishi, Tatsuya Kobayashi, Elena Kozhemyakina, Tso-Pang Yao, Andrew B. Lassar, Henry M. Kronenberg
Abstract
Maternal malnutrition, which causes prenatal exposure to excessive glucocorticoid, induces adverse metabolic programming, leading to hypertension in offspring. In offspring of pregnant rats receiving a low-protein diet or dexamethasone, a synthetic glucocorticoid, mRNA expression of angiotensin receptor type 1a (Agtr1a) in the paraventricular nucleus (PVN) of the hypothalamus was upregulated, concurrent with reduced expression of DNA methyltransferase 3a (Dnmt3a), reduced binding of DNMT3a to the Agtr1a gene, and DNA demethylation. Salt loading increased BP in both types of offspring, suggesting that elevated hypothalamic Agtr1a expression is epigenetically modulated by excessive glucocorticoid and leads to adult-onset salt-sensitive hypertension. Consistent with this, dexamethasone treatment of PVN cells upregulated Agtr1a, while downregulating Dnmt3a, and decreased DNMT3a binding and DNA demethylation at the Agtr1a locus. In addition, Dnmt3a knockdown upregulated Agtr1a independently of dexamethasone. Hypothalamic neuron–specific Dnmt3a-deficient mice exhibited upregulation of Agtr1a in the PVN and salt-induced BP elevation without dexamethasone treatment. By contrast, dexamethasone-treated Agtr1a-deficient mice failed to show salt-induced BP elevation, despite reduced expression of Dnmt3a. Thus, epigenetic modulation of hypothalamic angiotensin signaling contributes to salt-sensitive hypertension induced by prenatal glucocorticoid excess in offspring of mothers that are malnourished during pregnancy.
Authors
Fumiko Kawakami-Mori, Mitsuhiro Nishimoto, Latapati Reheman, Wakako Kawarazaki, Nobuhiro Ayuzawa, Kohei Ueda, Daigoro Hirohama, Daisuke Kohno, Shigeyoshi Oba, Tatsuo Shimosawa, Takeshi Marumo, Toshiro Fujita
Abstract
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.
Authors
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
Abstract
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.
Authors
Sierra S. Marable, Eunah Chung, Mike Adam, S. Steven Potter, Joo-Seop Park
Abstract
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.
Authors
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
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