Hirschsprung disease (HSCR) is the most frequent developmental anomaly of the enteric nervous system with an incidence of 1/5000 live births. Chronic intestinal pseudo-obstruction (CIPO) is less frequent and classified as neurogenic or myogenic. Isolated HSCR has an oligogenic inheritance with RET as the major disease-causing gene, while CIPO is genetically heterogeneous, caused by mutations in smooth muscle-specific genes. Here, we describe a series of patients with developmental disorders including gastrointestinal dysmotility, and investigate the underlying molecular bases. Trio-exome sequencing led to the identification of biallelic variants in ERBB3 and ERBB2 in eight individuals variably associating HSCR, CIPO, peripheral neuropathy and arthrogryposis. Thorough gut histology revealed aganglionosis, hypoganglionosis and intestinal smooth muscle abnormalities. The cell-type-specific ErbB3 and ErbB2 function was further analysed in mouse single-cell RNA sequencing data and in a conditional ErbB3-deficient mouse model, revealing a primary role for ERBB3 in enteric progenitors. The consequences of the identified variants were evaluated using RT-qPCR on patient-derived fibroblasts or immunoblot assays on Neuro-2a cells overexpressing either wild-type or mutant proteins, revealing either decreased expression or altered phosphorylation of the mutant receptors. Our results demonstrate that dysregulation of ERBB3 or ERBB2 leads to a broad spectrum of developmental anomalies including intestinal dysmotility.
Thuy-Linh Le, Louise Galmiche, Jonathan Levy, Pim Suwannarat, Debby M.E.I. Hellebrekers, Khomgrit Morarach, Franck Boismoreau, Tom E.J. Theunissen, Mathilde Lefebvre, Anna Pelet, Jelena Martinovic, Antoinette Gelot, Fabien Guimiot, Amanda Calleroz, Cyril Gitiaux, Marie Hully, Olivier Goulet, Christophe Chardot, Severine Drunat, Yline Capri, Christine Bole-Feysot, Patrick Nitschke, Sandra Whalen, Linda Mouthon, Holly E. Babcock, Robert Hofstra, Irenaeus F.M. de Coo, Anne-Claude Tabet, Thierry J. Molina, Boris Keren, Alice S. Brooks, Hubert J.M. Smeets, Ulrika Marklund, Christopher T. Gordon, Stanislas Lyonnet, Jeanne Amiel, Nadège Bondurand
LMNA mutations in patients are responsible for a dilated cardiomyopathy. Molecular mechanisms underlying the origin and development of the pathology are unknown. Herein, using mouse pluripotent embryonic stem cells (ESCs) and a mouse model both harboring the p.H222P Lmna mutation, we found early defects in cardiac differentiation of mutated ESCs and dilatation of mutated embryonic hearts at E13.5, pointing to a developmental origin of the disease. Using mouse ESCs, we demonstrated that cardiac differentiation of LmnaH222P/+ was impaired at the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene involved in epithelial-to-mesenchymal transition of epiblast cells, as well as Snai1 and Twist expression, was decreased in LmnaH222P/+ cells compared with WT cells in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. ChIP assay of H3K4me1 in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of Mesp1 and Twist in LmnaH222P/+ cells. Downregulation or inhibition of LSD1 that specifically demethylated H3K4me1 rescued the epigenetic landscape of mesodermal LmnaH222P/+ cells and in turn contraction of cardiomyocytes. Inhibition of LSD1 in pregnant mice or neonatal mice prevented cardiomyopathy in E13.5 LmnaH222P/H222P offspring and adults, respectively. Thus, LSD1 appeared to be a therapeutic target to prevent or cure dilated cardiomyopathy associated with a laminopathy.
Anne-Claire Guénantin, Imen Jebeniani, Julia Leschik, Erwan Watrin, Gisèle Bonne, Nicolas Vignier, Michel Pucéat
Postnatal failure of oligodendrocyte maturation has been proposed as a cellular mechanism of diffuse white matter injury (WMI) in premature infants. However, the molecular mechanisms for oligodendrocyte maturational failure remain unclear. In neonatal mice and cultured differentiating oligodendrocytes, sublethal intermittent hypoxic (IH) stress activated cyclophilin D–dependent mitochondrial proton leak and uncoupled mitochondrial respiration, leading to transient bioenergetic stress. This was associated with development of diffuse WMI: poor oligodendrocyte maturation, diffuse axonal hypomyelination, and permanent sensorimotor deficit. In normoxic mice and oligodendrocytes, exposure to a mitochondrial uncoupler recapitulated the phenotype of WMI, supporting the detrimental role of mitochondrial uncoupling in the pathogenesis of WMI. Compared with WT mice, cyclophilin D–knockout littermates did not develop bioenergetic stress in response to IH challenge and fully preserved oligodendrocyte maturation, axonal myelination, and neurofunction. Our study identified the cyclophilin D–dependent mitochondrial proton leak and uncoupling as a potentially novel subcellular mechanism for the maturational failure of oligodendrocytes and offers a potential therapeutic target for prevention of diffuse WMI in premature infants experiencing chronic IH stress.
Zoya Niatsetskaya, Sergey Sosunov, Anna Stepanova, James Goldman, Alexander Galkin, Maria Neginskaya, Evgeny Pavlov, Vadim Ten
The maternal perinatal environment modulates brain formation, and altered maternal nutrition has been linked to the development of metabolic and psychiatric disorders in the offspring. Here, we showed that maternal high-fat diet (HFD) feeding during lactation in mice elicits long-lasting changes in gene expression in the offspring’s dopaminergic circuitry. This translated into silencing of dopaminergic midbrain neurons, reduced connectivity to their downstream targets, and reduced stimulus-evoked dopamine (DA) release in the striatum. Despite the attenuated activity of DA midbrain neurons, offspring from mothers exposed to HFD feeding exhibited a sexually dimorphic expression of DA-related phenotypes, i.e., hyperlocomotion in males and increased intake of palatable food and sucrose in females. These phenotypes arose from concomitantly increased spontaneous activity of D1 medium spiny neurons (MSNs) and profoundly decreased D2 MSN projections. Overall, we have unraveled a fundamental restructuring of dopaminergic circuitries upon time-restricted altered maternal nutrition to induce persistent behavioral changes in the offspring.
R.N. Lippert, S. Hess, P. Klemm, L.M. Burgeno, T. Jahans-Price, M.E. Walton, P. Kloppenburg, J.C. Brüning
The transcription factor ISL1 is expressed in pituitary gland stem cells and the thyrotrope and gonadotrope lineages. Pituitary-specific Isl1 deletion causes hypopituitarism with increased stem cell apoptosis, reduced differentiation of thyrotropes and gonadotropes, and reduced body size. Conditional Isl1 deletion causes development of multiple Rathke’s cleft-like cysts, with 100% penetrance. Foxa1 and Foxj1 are abnormally expressed in the pituitary gland and associated with a ciliogenic gene expression program in the cysts. We confirmed expression of FOXA1, FOXJ1 and stem cell markers in human Rathke's cleft cyst tissue, but not craniopharyngiomas, which suggests these transcription factors are useful, pathological markers for diagnosis of Rathke's cleft cysts. These studies support a model whereby expression of ISL1 in pituitary progenitors drives differentiation into thyrotropes and gonadotropes, and without it, activation of FOXA1 and FOXJ1 permits development of an oral epithelial cell fate with mucinous cysts. This pituitary specific Isl1 mouse knockout sheds light on the etiology of Rathke's cleft cysts and the role of ISL1 in normal pituitary development.
Michelle L. Brinkmeier, Hironori Bando, Adriana C. Camarano, Shingo Fujio, Koji Yoshimoto, Flávio S. J. de Souza, Sally A. Camper
Esophageal atresia (EA/TEF) are common congenital abnormalities of the gastrointestinal tract. The etiology of EA/TEF is not well understood. We hypothesized that EA/TEF may be the direct consequence of abnormal expression of Noggin (NOG) signaling cascade. Here we showed that, in neonates with EA/TEF, NOG was missing from the atretic esophagus, resulting in immature esophagus that contains respiratory glands, and cilia. When using mouse esophageal organoid units (EOUs) or tracheal organoid units (TOU) as a model of foregut development in vitro, NOG determined the fate of foregut progenitors by allowing expression of esophageal epithelium proteins. When NOG was present in the culture of mTOU, it altered the cell morphology of the organoid unit epithelium, allowing expression of squamous cell proteins normally found in esophagus. On the other hand, when NOG was inhibited in mEOU, the organoid epithelium began to express respiratory markers mimicking the phenotype seen in pathology samples of human EA/TEF. Moreover, human EOU derived from EA/TEF patients were small, fibrotic and lack esophageal epithelium, but when NOG was added, the EOU grew larger, healthier and express esophageal proteins. These results indicate that Noggin is a critical regulator of cell fate decisions between esophageal and pulmonary morphogenesis.
Carolina Pinzon-Guzman, Sreedhara Sangadala, Katherine M. Riera, Evgenya Y. Popova, Elizabeth Manning, Won Jae Huh, Matthew S. Alexander, Julia S. Shelton, Scott D. Boden, James R. Goldenring
Fowler syndrome is a rare autosomal recessive brain vascular disorder caused by mutation in FLVCR2 in humans. The disease occurs during a critical period of brain vascular development, is characterized by glomeruloid vasculopathy and hydrocephalus, and is almost invariably prenatally fatal. Here, we sought to gain insights into the process of brain vascularization and the pathogenesis of Fowler Syndrome by inactivating Flvcr2 in mice. We show that Flvcr2 is necessary for angiogenic sprouting in the brain, but surprisingly dispensable for maintaining the blood brain barrier. Endothelial cells lacking Flvcr2 have altered expression of angiogenic factors, fail to adopt tip-cell properties and display reduced sprouting leading to vascular malformations similar to those seen in humans with Fowler Syndrome. Brain hypo-vascularization is associated with hypoxia and tissue infarction, ultimately causing hydrocephalus and death of mutant animals. Strikingly, despite severe vascular anomalies and brain tissue infarction, the blood-brain barrier is maintained in Flvcr2 mutant mice. Our new Fowler syndrome models therefore define the pathobiology of this disease, and provide new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.
Nicolas Santander, Carlos Omar Lizama, Eman Meky, Gabriel L. McKinsey, Bongnam Jung, Dean Sheppard, Christer Betsholtz, Thomas D. Arnold
A majority (~95%) of the gas-exchange surface area is generated through septa formation during alveologenesis. Disruption of this process leads to alveolar simplification and bronchopulmonary dysplasia (BPD), a prevalent disorder in premature infants. Although several models have been proposed, the mechanism of septa formation remains under debate. Here we show that inactivation of myosin light chain kinase (MLCK), a key factor required for myofibroblast contraction, disrupted septa formation, supporting the myofibroblast contraction model of alveologenesis. The alveoli simplification phenotype was accompanied by decreased yes-associated protein (YAP), a key effector in the Hippo mechanotransduction pathway. Expression of activated YAP in Mlck-mutant lungs led to partial reversal of alveolar simplification. In the adult, although Mlck inactivation did not lead to simplification, it prevented reseptation during compensatory regrowth in the pneumonectomy model. These findings revealed that myofibroblast reactivation and contraction are requisite steps toward regenerating the gas-exchange surface in diseases such as BPD and chronic obstructive pulmonary disease (COPD).
Rongbo Li, Xiaoping Li, James Hagood, Min-Sheng Zhu, Xin Sun
The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide. Although gene-environment interactions have been implicated in the etiology of several disorders, the impact of paternal and/or maternal metabolic syndrome on the clinical phenotypes of offspring and the underlying genetic and epigenetic contributors of NAFLD have not been fully explored. To this end, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique nondietary model manifesting 3 hallmarks that confer high risk for the development of NAFLD: hyperglycemia, insulin resistance, and dyslipidemia. We report that parental metabolic syndrome epigenetically reprograms members of the TGF-β family, including neuronal regeneration–related protein (NREP) and growth differentiation factor 15 (GDF15). NREP and GDF15 modulate the expression of several genes involved in the regulation of hepatic lipid metabolism. In particular, NREP downregulation increases the protein abundance of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and ATP-citrate lyase (ACLY) in a TGF-β receptor/PI3K/protein kinase B–dependent manner, to regulate hepatic acetyl-CoA and cholesterol synthesis. Reduced hepatic expression of NREP in patients with NAFLD and substantial correlations between low serum NREP levels and the presence of steatosis and nonalcoholic steatohepatitis highlight the clinical translational relevance of our findings in the context of recent preclinical trials implicating ACLY in NAFLD progression.
Dario F. De Jesus, Kazuki Orime, Dorota Kaminska, Tomohiko Kimura, Giorgio Basile, Chih-Hao Wang, Larissa Haertle, Renzo Riemens, Natalie K. Brown, Jiang Hu, Ville Männistö, Amélia M. Silva, Ercument Dirice, Yu-Hua Tseng, Thomas Haaf, Jussi Pihlajamäki, Rohit N. Kulkarni
Prenatal alcohol exposure (PAE) affects at least 10% of newborns globally and leads to the development of fetal alcohol spectrum disorders (FASDs). Despite its high incidence, there is no consensus on the implications of PAE on metabolic disease risk in adults. Here, we describe a cohort of adults with FASDs that had an increased incidence of metabolic abnormalities, including type 2 diabetes, low HDL, high triglycerides, and female-specific overweight and obesity. Using a zebrafish model for PAE, we performed population studies to elucidate the metabolic disease seen in the clinical cohort. Embryonic alcohol exposure (EAE) in male zebrafish increased the propensity for diet-induced obesity and fasting hyperglycemia in adulthood. We identified several consequences of EAE that may contribute to these phenotypes, including a reduction in adult locomotor activity, alterations in visceral adipose tissue and hepatic development, and persistent diet-responsive transcriptional changes. Taken together, our findings define metabolic vulnerabilities due to EAE and provide evidence that behavioral changes and primary organ dysfunction contribute to resultant metabolic abnormalities.
Olivia Weeks, Gabriel D. Bossé, Isaac M. Oderberg, Sebastian Akle, Yariv Houvras, Paul J. Wrighton, Kyle LaBella, Isabelle Iversen, Sahar Tavakoli, Isaac Adatto, Arkadi Schwartz, Daan Kloosterman, Allison Tsomides, Michael E. Charness, Randall T. Peterson, Matthew L. Steinhauser, Pouneh K. Fazeli, Wolfram Goessling
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