Developments
Abstract
In vertebrate species, fertility is controlled by gonadotropin-releasing hormone (GnRH) neurons. GnRH cells arise outside the central nervous system, in the developing olfactory pit, and migrate along olfactory/vomeronasal/terminal nerve axons into the forebrain during embryonic development. Congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS) are rare genetic disorders characterized by infertility and they are associated with defects in GnRH neuron migration and/or altered GnRH secretion and signaling. Here, we documented the expression of Jagged 1/Notch signaling pathway in GnRH neurons and along the GnRH neuron migratory route both in zebrafish embryos and in human fetuses. Genetic knock-down of the zebrafish ortholog of JAG1 (jag1b) resulted in altered GnRH migration and olfactory axonal projections to the olfactory bulbs. Next-generation sequencing was performed in 467 CHH unrelated probands leading to the identification of heterozygous rare variants in JAG1. Functional in vitro validation of JAG1 mutants revealed that 7 out of the 9 studied variants exhibit reduced protein levels and altered subcellular localization. Altogether our data provide compelling evidence that Jag1/Notch signaling plays a prominent role in the development of GnRH neurons and we propose that JAG1 insufficiency may contribute to the pathogenesis of CHH in humans.
Authors
Ludovica Cotellessa, Federica Marelli, Paolo Duminuco, Michela Adamo, Georgios E. Papadakis, Lucia Bartoloni, Naoko Sato, Mariarosaria Lang-Muritano, Amineh Troendle, Waljit S. Dhillo, Annamaria Morelli, Giulia Guarnieri, Nelly Pitteloud, Luca Persani, Marco Bonomi, Paolo Giacobini, Valeria Vezzoli
Abstract
Ethanol (EtOH) is a commonly encountered teratogen that can disrupt organ development and lead to fetal alcohol spectrum disorders (FASDs); many mechanisms of developmental toxicity are unknown. Here, we used transcriptomic analysis in an established zebrafish model of embryonic alcohol exposure (EAE) to identify the ubiquitin-proteasome system (UPS) as a critical target of EtOH during development. Surprisingly, EAE alters 20S, 19S, and 11S proteasome gene expression and increases ubiquitylated protein load. EtOH and its metabolite acetaldehyde decrease proteasomal peptidase activity in a cell type–specific manner. Proteasome 20S subunit β 1 (psmb1hi2939Tg) and proteasome 26S subunit, ATPase 6 (psmc6hi3593Tg), genetic KOs define the developmental impact of decreased proteasome function. Importantly, loss of psmb1 or psmc6 results in widespread developmental abnormalities resembling EAE phenotypes, including growth restriction, abnormal craniofacial structure, neurodevelopmental defects, and failed hepatopancreas maturation. Furthermore, pharmacologic inhibition of chymotrypsin-like proteasome activity potentiates the teratogenic effects of EAE on craniofacial structure, the nervous system, and the endoderm. Our studies identify the proteasome as a target of EtOH exposure and signify that UPS disruptions contribute to craniofacial, neurological, and endodermal phenotypes in FASDs.
Authors
Olivia Weeks, Bess M. Miller, Brian J. Pepe-Mooney, Isaac M. Oderberg, Scott H. Freeburg, Colton J. Smith, Trista E. North, Wolfram Goessling
Abstract
FOXD1+ derived stromal cells give rise to pericytes and fibroblasts that support the kidney vasculature and interstitium but are also major precursors of myofibroblasts. ZEB2 is a SMAD-interacting transcription factor that is expressed in developing kidney stromal progenitors. Here we show that Zeb2 is essential for normal FOXD1+ stromal progenitor development. Specific deletion of mouse Zeb2 in FOXD1+ stromal progenitors (Zeb2 cKO) leads to abnormal interstitial stromal cell development, differentiation, and kidney fibrosis. Immunofluorescent staining analyses revealed abnormal expression of interstitial stromal cell markers MEIS1/2/3, CDKN1C, and CSPG4 (NG2) in newborn and 3-week-old Zeb2 cKO mouse kidneys. Zeb2 deficient FOXD1+ stromal progenitors also took on a myofibroblast fate that led to kidney fibrosis and kidney failure. Cell marker studies further confirmed that these myofibroblasts expressed pericyte and resident fibroblast markers including PDGFRβ, CSPG4, Desmin, GLI1, and NT5E. Notably, increased interstitial collagen deposition associated with loss of Zeb2 in FOXD1+ stromal progenitors was accompanied by increased expression of activated SMAD1/5/8, SMAD2/3, SMAD4, and AXIN2. Thus, our study identifies a key role of ZEB2 in maintaining the cell fate of FOXD1+ stromal progenitors during kidney development whereas loss of ZEB2 leads to differentiation of FOXD1+ stromal progenitors into myofibroblasts and kidney fibrosis.
Authors
Sudhir Kumar, Xueping Fan, Hila Milo Rasouly, Richa Sharma, David J. Salant, Weining Lu
Abstract
Preeclampsia is a serious pregnancy disorder that lacks effective treatments other than delivery. Improper sensing of oxygen changes during placentation by prolyl hydroxylases (PHD), specifically PHD2, causes placental Hypoxia-Inducible Factor-1 (HIF1) buildup and abnormal downstream signaling in early-onset preeclampsia; yet therapeutic targeting of HIF1 has never been attempted. Here we generated a conditional (placenta-specific) knockout of Phd2 in mice (Phd2-/- cKO) to reproduce HIF1 excess and to assess anti-HIF therapy. Conditional deletion of Phd2 in the junctional zone (JZ) during pregnancy increased placental HIF1 content, resulting in abnormal placentation, impaired remodeling of the uterine spiral arteries, and fetal growth restriction. Pregnant dams developed new-onset hypertension at mid-gestation (E9.5) in addition to proteinuria and renal and cardiac pathology, hallmarks of severe preeclampsia in humans. Daily injection of acriflavine, a small-molecule inhibitor of HIF1, to pregnant Phd2-/- cKO mice from E7.5 (prior to hypertension) or E10.5 (after hypertension has been established) to E14.5 corrected placental dysmorphologies and improved fetal growth. Moreover, it reduced maternal blood pressure and reverted renal and myocardial pathology. Thus, therapeutic targeting of the HIF pathway may improve placental development and function, as well as maternal and fetal health, in preeclampsia.
Authors
Julien Sallais, Chanho Park, Sruthi Alahari, Tyler Porter, Ruizhe Liu, Merve Kurt, Abby Farrell, Martin Post, Isabella Caniggia
Abstract
Maternal hyperthyroidism is associated with an increased incidence of congenital abnormalities at birth, but it is not clear which of those defects arise from a transient developmental excess of thyroid hormone, and which depend on pregnancy stage, antithyroid drug choice, or unwanted subsequent fetal hypothyroidism. To address this issue we studied a mouse model of comprehensive developmental thyrotoxicosis secondary to a lack of type 3 deiodinase (DIO3). Dio3-/- mice exhibit reduced neonatal viability on most genetic backgrounds and perinatal lethality on a C57BL/6 background. Dio3-/- mice exhibit severe growth retardation during the neonatal period and cartilage loss. Mice surviving after birth manifest brain and cranial dysmorphisms, severe hydrocephalus, choanal atresia, and cleft palate. These abnormalities are noticeable in C57BL/6J Dio3-/- mice at fetal stages, in addition to a thyrotoxic heart with septal defects and thin ventricular walls. Our findings stress the protecting role of DIO3 during development and support the hypothesis that human congenital abnormalities associated with hyperthyroidism during pregnancy are caused by transient thyrotoxicosis before clinical intervention. Our results also suggest thyroid hormone involvement in the etiology of idiopathic pathologies including cleft palate, choanal atresia, Chiari malformations, Kaschin-Beck disease, and Temple and other cranio-encephalic and heart syndromes.
Authors
M. Elena Martinez, Ilka Pinz, Marilena Preda, Christine R. Norton, Thomas Gridley, Arturo Hernandez
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
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