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A cardinal feature of the auditory pathway is frequency selectivity, represented in a tonotopic map from the cochlea to the cortex. The molecular determinants of the auditory frequency map are unknown. Here, we discovered that the transcription factor ISL1 regulates the molecular and cellular features of auditory neurons, including the formation of the spiral ganglion and peripheral and central processes that shape the tonotopic representation of the auditory map. We selectively knocked out Isl1 in auditory neurons using Neurod1Cre strategies. In the absence of Isl1, spiral ganglion neurons migrate into the central cochlea and beyond, and the cochlear wiring is profoundly reduced and disrupted. The central axons of Isl1 mutants lose their topographic projections and segregation at the cochlear nucleus. Transcriptome analysis of spiral ganglion neurons shows that Isl1 regulates neurogenesis, axonogenesis, migration, neurotransmission-related machinery, and synaptic communication patterns. We show that peripheral disorganization in the cochlea affects the physiological properties of hearing in the midbrain and auditory behavior. Surprisingly, auditory processing features are preserved despite the significant hearing impairment, revealing central auditory pathway resilience and plasticity in Isl1 mutant mice. Mutant mice have a reduced acoustic startle reflex, altered prepulse inhibition, and characteristics of compensatory neural hyperactivity centrally. Our findings show that ISL1 is one of the obligatory factors required to sculpt auditory structural and functional tonotopic maps. Still, upon Isl1 deletion, the ensuing central plasticity of the auditory pathway does not suffice to overcome developmentally induced peripheral dysfunction of the cochlea.
Assuntos
Vias Auditivas , Núcleo Coclear , Células Ciliadas Auditivas , Proteínas com Homeodomínio LIM , Neurogênese , Gânglio Espiral da Cóclea , Fatores de Transcrição , Animais , Vias Auditivas/embriologia , Cóclea/embriologia , Cóclea/inervação , Núcleo Coclear/embriologia , Células Ciliadas Auditivas/fisiologia , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/fisiologia , Camundongos , Neurogênese/genética , Gânglio Espiral da Cóclea/enzimologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
BACKGROUND: An altered sympathetic nervous system is implicated in many cardiac pathologies, ranging from sudden infant death syndrome to common diseases of adulthood such as hypertension, myocardial ischemia, cardiac arrhythmias, myocardial infarction, and heart failure. Although the mechanisms responsible for disruption of this well-organized system are the subject of intensive investigations, the exact processes controlling the cardiac sympathetic nervous system are still not fully understood. A conditional knockout of the Hif1a gene was reported to affect the development of sympathetic ganglia and sympathetic innervation of the heart. This study characterized how the combination of HIF-1α deficiency and streptozotocin (STZ)-induced diabetes affects the cardiac sympathetic nervous system and heart function of adult animals. METHODS: Molecular characteristics of Hif1a deficient sympathetic neurons were identified by RNA sequencing. Diabetes was induced in Hif1a knockout and control mice by low doses of STZ treatment. Heart function was assessed by echocardiography. Mechanisms involved in adverse structural remodeling of the myocardium, i.e. advanced glycation end products, fibrosis, cell death, and inflammation, was assessed by immunohistological analyses. RESULTS: We demonstrated that the deletion of Hif1a alters the transcriptome of sympathetic neurons, and that diabetic mice with the Hif1a-deficient sympathetic system have significant systolic dysfunction, worsened cardiac sympathetic innervation, and structural remodeling of the myocardium. CONCLUSIONS: We provide evidence that the combination of diabetes and the Hif1a deficient sympathetic nervous system results in compromised cardiac performance and accelerated adverse myocardial remodeling, associated with the progression of diabetic cardiomyopathy.
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Diabetes Mellitus Experimental , Cardiomiopatias Diabéticas , Animais , Camundongos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/complicações , Cardiomiopatias Diabéticas/genética , Coração/inervação , Miocárdio/metabolismo , Sistema Nervoso Simpático/metabolismoRESUMO
The molecular mechanisms regulating sympathetic innervation of the heart during embryogenesis and its importance for cardiac development and function remain to be fully elucidated. We generated mice in which conditional knockout (CKO) of the Hif1a gene encoding the transcription factor hypoxia-inducible factor 1α (HIF-1α) is mediated by an Islet1-Cre transgene expressed in the cardiac outflow tract, right ventricle and atrium, pharyngeal mesoderm, peripheral neurons, and hindlimbs. These Hif1aCKO mice demonstrate significantly decreased perinatal survival and impaired left ventricular function. The absence of HIF-1α impaired the survival and proliferation of preganglionic and postganglionic neurons of the sympathetic system, respectively. These defects resulted in hypoplasia of the sympathetic ganglion chain and decreased sympathetic innervation of the Hif1aCKO heart, which was associated with decreased cardiac contractility. The number of chromaffin cells in the adrenal medulla was also decreased, indicating a broad dependence on HIF-1α for development of the sympathetic nervous system.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Sistema Nervoso Simpático/crescimento & desenvolvimento , Medula Suprarrenal/embriologia , Medula Suprarrenal/inervação , Animais , Células Cromafins , Anomalias dos Vasos Coronários/embriologia , Vasos Coronários/embriologia , Feminino , Gânglios Simpáticos/embriologia , Gânglios Simpáticos/crescimento & desenvolvimento , Coração/embriologia , Coração/inervação , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Sistema Nervoso Simpático/enzimologiaRESUMO
SOX2 is essential for maintaining neurosensory stem cell properties, although its involvement in the early neurosensory development of cranial placodes remains unclear. To address this, we used Foxg1-Cre to conditionally delete Sox2 during eye, ear, and olfactory placode development. Foxg1-Cre mediated early deletion of Sox2 eradicates all olfactory placode development, and disrupts retinal development and invagination of the lens placode. In contrast to the lens and olfactory placodes, the ear placode invaginates and delaminates NEUROD1 positive neurons. Furthermore, we show that SOX2 is not necessary for early ear neurogenesis, since the early inner ear ganglion is formed with near normal central projections to the hindbrain and peripheral projections to the undifferentiated sensory epithelia of E11.5-12.5 ears. However, later stages of ear neurosensory development, in particular, the late forming auditory system, critically depend on the presence of SOX2. Our data establish distinct differences for SOX2 requirements among placodal sensory organs with similarities between olfactory and lens but not ear placode development, consistent with the unique neurosensory development and molecular properties of the ear.
Assuntos
Orelha Interna/embriologia , Neurogênese , Fatores de Transcrição SOXB1/metabolismo , Animais , Apoptose , Orelha Interna/citologia , Orelha Interna/metabolismo , Cristalino/embriologia , Cristalino/metabolismo , Camundongos , Camundongos Knockout , Mucosa Nasal/embriologia , Mucosa Nasal/metabolismo , Fatores de Transcrição SOXB1/genéticaRESUMO
Diabetes is a metabolic disease that involves the death or dysfunction of the insulin-secreting ß cells in the pancreas. Consequently, most diabetes research is aimed at understanding the molecular and cellular bases of pancreatic development, islet formation, ß-cell survival, and insulin secretion. Complex interactions of signaling pathways and transcription factor networks regulate the specification, growth, and differentiation of cell types in the developing pancreas. Many of the same regulators continue to modulate gene expression and cell fate of the adult pancreas. The transcription factor NEUROD1 is essential for the maturation of ß cells and the expansion of the pancreatic islet cell mass. Mutations of the Neurod1 gene cause diabetes in humans and mice. However, the different aspects of the requirement of NEUROD1 for pancreas development are not fully understood. In this study, we investigated the role of NEUROD1 during the primary and secondary transitions of mouse pancreas development. We determined that the elimination of Neurod1 impairs the expression of key transcription factors for α- and ß-cell differentiation, ß-cell proliferation, insulin production, and islets of Langerhans formation. These findings demonstrate that the Neurod1 deletion altered the properties of α and ß endocrine cells, resulting in severe neonatal diabetes, and thus, NEUROD1 is required for proper activation of the transcriptional network and differentiation of functional α and ß cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ilhotas Pancreáticas/citologia , Pâncreas/citologia , Pâncreas/embriologia , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Diabetes Mellitus/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/ultraestrutura , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Hearing depends on extracting frequency, intensity, and temporal properties from sound to generate an auditory map for acoustical signal processing. How physiology intersects with molecular specification to fine tune the developing properties of the auditory system that enable these aspects remains unclear. We made a novel conditional deletion model that eliminates the transcription factor NEUROD1 exclusively in the ear. These mice (both sexes) develop a truncated frequency range with no neuroanatomically recognizable mapping of spiral ganglion neurons onto distinct locations in the cochlea nor a cochleotopic map presenting topographically discrete projections to the cochlear nuclei. The disorganized primary cochleotopic map alters tuning properties of the inferior colliculus units, which display abnormal frequency, intensity, and temporal sound coding. At the behavioral level, animals show alterations in the acoustic startle response, consistent with altered neuroanatomical and physiological properties. We demonstrate that absence of the primary afferent topology during embryonic development leads to dysfunctional tonotopy of the auditory system. Such effects have never been investigated in other sensory systems because of the lack of comparable single gene mutation models.SIGNIFICANCE STATEMENT All sensory systems form a topographical map of neuronal projections from peripheral sensory organs to the brain. Neuronal projections in the auditory pathway are cochleotopically organized, providing a tonotopic map of sound frequencies. Primary sensory maps typically arise by molecular cues, requiring physiological refinements. Past work has demonstrated physiologic plasticity in many senses without ever molecularly undoing the specific mapping of an entire primary sensory projection. We genetically manipulated primary auditory neurons to generate a scrambled cochleotopic projection. Eliminating tonotopic representation to auditory nuclei demonstrates the inability of physiological processes to restore a tonotopic presentation of sound in the midbrain. Our data provide the first insights into the limits of physiology-mediated brainstem plasticity during the development of the auditory system.
Assuntos
Percepção Auditiva/genética , Percepção Auditiva/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Mesencéfalo/fisiologia , Percepção da Altura Sonora/fisiologia , Animais , Comportamento Animal/fisiologia , Mapeamento Encefálico , Núcleo Coclear/anatomia & histologia , Núcleo Coclear/fisiologia , Feminino , Audição/fisiologia , Colículos Inferiores/anatomia & histologia , Colículos Inferiores/fisiologia , Masculino , Mesencéfalo/embriologia , Camundongos , Camundongos Knockout , Gravidez , Reflexo de Sobressalto/genética , Reflexo de Sobressalto/fisiologia , Gânglio Espiral da Cóclea/citologia , Gânglio Espiral da Cóclea/fisiologia , Vestíbulo do Labirinto/anatomia & histologia , Vestíbulo do Labirinto/fisiologiaRESUMO
BACKGROUND: Epidemiological studies show that maternal diabetes predisposes offspring to cardiovascular and metabolic disorders. However, the precise mechanisms for the underlying penetrance and disease predisposition remain poorly understood. We examined whether hypoxia-inducible factor 1 alpha, in combination with exposure to a diabetic intrauterine environment, influences the function and molecular structure of the adult offspring heart. METHODS AND RESULTS: In a mouse model, we demonstrated that haploinsufficient (Hif1a+/-) offspring from a diabetic pregnancy developed left ventricle dysfunction at 12 weeks of age, as manifested by decreased fractional shortening and structural remodeling of the myocardium. Transcriptional profiling by RNA-seq revealed significant transcriptome changes in the left ventricle of diabetes-exposed Hif1a+/- offspring associated with development, metabolism, apoptosis, and blood vessel physiology. In contrast, both wild type and Hif1a+/- offspring from diabetic pregnancies showed changes in immune system processes and inflammatory responses. Immunohistochemical analyses demonstrated that the combination of haploinsufficiency of Hif1a and exposure to maternal diabetes resulted in impaired macrophage infiltration, increased levels of advanced glycation end products, and changes in vascular homeostasis in the adult offspring heart. CONCLUSIONS: Together our findings provide evidence that a global reduction in Hif1a gene dosage increases predisposition of the offspring exposed to maternal diabetes to cardiac dysfunction, and also underscore Hif1a as a critical factor in the fetal programming of adult cardiovascular disease.
Assuntos
Doenças Cardiovasculares/genética , Diabetes Mellitus Experimental/complicações , Diabetes Gestacional , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mutação , Efeitos Tardios da Exposição Pré-Natal , Animais , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/fisiopatologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Gestacional/metabolismo , Diabetes Gestacional/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Interação Gene-Ambiente , Haploinsuficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Gravidez , Fatores de Risco , Função Ventricular Esquerda , Remodelação VentricularRESUMO
BACKGROUND: Hypoxia inducible factor 1 (HIF-1) activates protective pathways to counteract hypoxia and prevent tissue damage in conjunction with renal injury. The aim of this study was to evaluate a role of HIF-1 in diabetes-induced kidney damage. METHODS: We used a streptozotocin-induced diabetes mouse model and compared biochemical, histological and molecular parameters associated with kidney damage in Hif1α deficient (Hif1α +/- ) and wild-type mice. RESULTS: We showed that Hif1α deficiency accelerated pathological changes in the early stage of DN. Six weeks after diabetes-induction, Hif1α deficient mice showed more prominent changes in biochemical serum parameters associated with glomerular injury, increased expression of podocyte damage markers, and loss of podocytes compared to wild-type mice. These results indicate that Hif1α deficiency specifically affects podocyte survival in the early phase of DN, resulting in diabetic glomerular injury. In contrast, renal fibrosis was not affected by the global reduction of Hif1α, at least not in the early phase of diabetic exposure. CONCLUSIONS: Together our data reveal that HIF-1 has an essential role in the early response to prevent diabetes-induced tissue damage and that impaired HIF-1 signaling results in a faster progression of DN. Although the modulation of HIF-1 activity is a high-priority target for clinical treatments, further study is required to investigate HIF-1 as a potential therapeutic target for the treatment of DN.
Assuntos
Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/etiologia , Modelos Animais de Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Animais , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , PrognósticoRESUMO
BACKGROUND: Diabetic cardiomyopathy is associated with a number of functional and structural pathological changes such as left ventricular dysfunction, cardiac remodeling, and apoptosis. The primary cause of diabetic cardiomyopathy is hyperglycemia, the metabolic hallmark of diabetes. Recent studies have shown that a diabetic environment suppresses hypoxia-inducible factor (HIF)-1α protein stability and function. The aim of this study was to analyze the functional role of HIF-1α in the development of diabetic cardiomyopathy. We have hypothesized that the partial deficiency of HIF-1α may compromise cardiac responses under diabetic conditions and increase susceptibility to diabetic cardiomyopathy. METHODS: Diabetes was induced by streptozotocin in wild type (Wt) and heterozygous Hif1a knock-out (Hif1a+/-) mice. Echocardiographic evaluations of left ventricular functional parameters, expression analyses by qPCR and Western blot, and cardiac histopathology assessments were performed in age-matched groups, diabetic, and non-diabetic Wt and Hif1a+/- mice. RESULTS: Five weeks after diabetes was established, a significant decrease in left ventricle fractional shortening was detected in diabetic Hif1a+/- but not in diabetic Wt mice. The combination effects of the partial deficiency of Hif1a and diabetes affected the gene expression profile of the heart, including reduced vascular endothelial growth factor A (Vegfa) expression. Adverse cardiac remodeling in the diabetic Hif1a+/- heart was shown by molecular changes in the expression of structural molecules and components of the extracellular matrix. CONCLUSIONS: We have shown a correlation between heterozygosity for Hif1α and adverse functional, molecular, and cellular changes associated with diabetic cardiomyopathy. Our results provide evidence that HIF-1α regulates early cardiac responses to diabetes, and that HIF-1α deregulation may influence the increased risk for diabetic cardiomyopathy.
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Introduction: Maternal diabetes is a recognized risk factor for both short-term and long-term complications in offspring. Beyond the direct teratogenicity of maternal diabetes, the intrauterine environment can influence the offspring's cardiovascular health. Abnormalities in the cardiac sympathetic system are implicated in conditions such as sudden infant death syndrome, cardiac arrhythmic death, heart failure, and certain congenital heart defects in children from diabetic pregnancies. However, the mechanisms by which maternal diabetes affects the development of the cardiac sympathetic system and, consequently, heightens health risks and predisposes to cardiovascular disease remain poorly understood. Methods and results: In the mouse model, we performed a comprehensive analysis of the combined impact of a Hif1a-deficient sympathetic system and the maternal diabetes environment on both heart development and the formation of the cardiac sympathetic system. The synergic negative effect of exposure to maternal diabetes and Hif1a deficiency resulted in the most pronounced deficit in cardiac sympathetic innervation and the development of the adrenal medulla. Abnormalities in the cardiac sympathetic system were accompanied by a smaller heart, reduced ventricular wall thickness, and dilated subepicardial veins and coronary arteries in the myocardium, along with anomalies in the branching and connections of the main coronary arteries. Transcriptional profiling by RNA sequencing (RNA-seq) revealed significant transcriptome changes in Hif1a-deficient sympathetic neurons, primarily associated with cell cycle regulation, proliferation, and mitosis, explaining the shrinkage of the sympathetic neuron population. Discussion: Our data demonstrate that a failure to adequately activate the HIF-1α regulatory pathway, particularly in the context of maternal diabetes, may contribute to abnormalities in the cardiac sympathetic system. In conclusion, our findings indicate that the interplay between deficiencies in the cardiac sympathetic system and subtle structural alternations in the vasculature, microvasculature, and myocardium during heart development not only increases the risk of cardiovascular disease but also diminishes the adaptability to the stress associated with the transition to extrauterine life, thus increasing the risk of neonatal death.
Assuntos
Doenças Cardiovasculares , Diabetes Gestacional , Insuficiência Cardíaca , Animais , Criança , Feminino , Humanos , Recém-Nascido , Camundongos , Gravidez , Doenças Cardiovasculares/metabolismo , Diabetes Gestacional/metabolismo , Coração , Miocárdio/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
AIM: The transcriptional factor HIF-1α is recognized for its contribution to cardioprotection against acute ischemia/reperfusion injury. Adaptation to chronic hypoxia (CH) is known to stabilize HIF-1α and increase myocardial ischemic tolerance. However, the precise role of HIF-1α in mediating the protective effect remains incompletely understood. METHODS: Male wild-type (WT) mice and mice with partial Hif1a deficiency (hif1a +/-) were exposed to CH for 4 weeks, while their respective controls were kept under normoxic conditions. Subsequently, their isolated perfused hearts were subjected to ischemia/reperfusion to determine infarct size, while RNA-sequencing of isolated cardiomyocytes was performed. Mitochondrial respiration was measured to evaluate mitochondrial function, and western blots were performed to assess mitophagy. RESULTS: We demonstrated enhanced ischemic tolerance in WT mice induced by adaptation to CH compared with their normoxic controls and chronically hypoxic hif1a +/- mice. Through cardiomyocyte bulk mRNA sequencing analysis, we unveiled significant reprogramming of cardiomyocytes induced by CH emphasizing mitochondrial processes. CH reduced mitochondrial content and respiration and altered mitochondrial ultrastructure. Notably, the reduced mitochondrial content correlated with enhanced autophagosome formation exclusively in chronically hypoxic WT mice, supported by an increase in the LC3-II/LC3-I ratio, expression of PINK1, and degradation of SQSTM1/p62. Furthermore, pretreatment with the mitochondrial division inhibitor (mdivi-1) abolished the infarct size-limiting effect of CH in WT mice, highlighting the key role of mitophagy in CH-induced cardioprotection. CONCLUSION: These findings provide new insights into the contribution of HIF-1α to cardiomyocyte survival during acute ischemia/reperfusion injury by activating the selective autophagy pathway.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia , Hipóxia , Mitofagia , Infarto do Miocárdio , Animais , Mitofagia/fisiologia , Camundongos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/genética , Hipóxia/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Adaptação Fisiológica/fisiologia , Camundongos Endogâmicos C57BLRESUMO
Cardiovascular malformations are the most common manifestation of diabetic embryopathy. The molecular mechanisms underlying the teratogenic effect of maternal diabetes have not been fully elucidated. Using genome-wide expression profiling, we previously demonstrated that exposure to maternal diabetes resulted in dysregulation of the hypoxia-inducible factor 1 (HIF-1) pathway in the developing embryo. We thus considered a possible link between HIF-1-regulated pathways and the development of congenital malformations. HIF-1α heterozygous-null (Hif1a(+/-)) and wild type (Wt) littermate embryos were exposed to the intrauterine environment of a diabetic mother to analyze the frequency and morphology of congenital defects, and assess gene expression changes in Wt and Hif1a(+/-) embryos. We observed a decreased number of embryos per litter and an increased incidence of heart malformations, including atrioventricular septal defects and reduced myocardial mass, in diabetes-exposed Hif1a(+/-) embryos as compared to Wt embryos. We also detected significant differences in the expression of key cardiac transcription factors, including Nkx2.5, Tbx5, and Mef2C, in diabetes-exposed Hif1a(+/-) embryonic hearts compared to Wt littermates. Thus, partial global HIF-1α deficiency alters gene expression in the developing heart and increases susceptibility to congenital defects in a mouse model of diabetic pregnancy.
Assuntos
Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/embriologia , Coração/embriologia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Proteínas Musculares/biossíntese , Gravidez em Diabéticas/metabolismo , Animais , Embrião de Mamíferos/patologia , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Camundongos , Camundongos Mutantes , Proteínas Musculares/genética , Gravidez , Gravidez em Diabéticas/genética , Gravidez em Diabéticas/patologiaRESUMO
NEUROD1 is a transcription factor that helps maintain a mature phenotype of pancreatic ß cells. Disruption of Neurod1 during pancreatic development causes severe neonatal diabetes; however, the exact role of NEUROD1 in the differentiation programs of endocrine cells is unknown. Here, we report a crucial role of the NEUROD1 regulatory network in endocrine lineage commitment and differentiation. Mechanistically, transcriptome and chromatin landscape analyses demonstrate that Neurod1 inactivation triggers a downregulation of endocrine differentiation transcription factors and upregulation of non-endocrine genes within the Neurod1-deficient endocrine cell population, disturbing endocrine identity acquisition. Neurod1 deficiency altered the H3K27me3 histone modification pattern in promoter regions of differentially expressed genes, which resulted in gene regulatory network changes in the differentiation pathway of endocrine cells, compromising endocrine cell potential, differentiation, and functional properties.
Assuntos
Células Endócrinas , Células Secretoras de Insulina , Diferenciação Celular/genética , Fatores de Transcrição , Ativação TranscricionalRESUMO
BACKGROUND: Glucose homeostasis is dependent on functional pancreatic α and ß cells. The mechanisms underlying the generation and maturation of these endocrine cells remain unclear. RESULTS: We unravel the molecular mode of action of ISL1 in controlling α cell fate and the formation of functional ß cells in the pancreas. By combining transgenic mouse models, transcriptomic and epigenomic profiling, we uncover that elimination of Isl1 results in a diabetic phenotype with a complete loss of α cells, disrupted pancreatic islet architecture, downregulation of key ß-cell regulators and maturation markers of ß cells, and an enrichment in an intermediate endocrine progenitor transcriptomic profile. CONCLUSIONS: Mechanistically, apart from the altered transcriptome of pancreatic endocrine cells, Isl1 elimination results in altered silencing H3K27me3 histone modifications in the promoter regions of genes that are essential for endocrine cell differentiation. Our results thus show that ISL1 transcriptionally and epigenetically controls α cell fate competence, and ß cell maturation, suggesting that ISL1 is a critical component for generating functional α and ß cells.
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Neuronal development in the inner ear is initiated by expression of the proneural basic Helix-Loop-Helix (bHLH) transcription factor Neurogenin1 that specifies neuronal precursors in the otocyst. The initial specification of the neuroblasts within the otic epithelium is followed by the expression of an additional bHLH factor, Neurod1. Although NEUROD1 is essential for inner ear neuronal development, the different aspects of the temporal and spatial requirements of NEUROD1 for the inner ear and, mainly, for auditory neuron development are not fully understood. In this study, using Foxg1Cre for the early elimination of Neurod1 in the mouse otocyst, we showed that Neurod1 deletion results in a massive reduction of differentiating neurons in the otic ganglion at E10.5, and in the diminished vestibular and rudimental spiral ganglia at E13.5. Attenuated neuronal development was associated with reduced and disorganized sensory epithelia, formation of ectopic hair cells, and the shortened cochlea in the inner ear. Central projections of inner ear neurons with conditional Neurod1 deletion are reduced, unsegregated, disorganized, and interconnecting the vestibular and auditory systems. In line with decreased afferent input from auditory neurons, the volume of cochlear nuclei was reduced by 60% in Neurod1 mutant mice. Finally, our data demonstrate that early elimination of Neurod1 affects the neuronal lineage potential and alters the generation of inner ear neurons and cochlear afferents with a profound effect on the first auditory nuclei, the cochlear nuclei.
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Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, but OXPHOS also supports biosynthesis during proliferation. In contrast, the role of OXPHOS during quiescence, beyond ATP production, is not well understood. Using mouse models of inducible OXPHOS deficiency in all cell types or specifically in the vascular endothelium that negligibly relies on OXPHOS-derived ATP, we show that selectively during quiescence OXPHOS provides oxidative stress resistance by supporting macroautophagy/autophagy. Mechanistically, OXPHOS constitutively generates low levels of endogenous ROS that induce autophagy via attenuation of ATG4B activity, which provides protection from ROS insult. Physiologically, the OXPHOS-autophagy system (i) protects healthy tissue from toxicity of ROS-based anticancer therapy, and (ii) provides ROS resistance in the endothelium, ameliorating systemic LPS-induced inflammation as well as inflammatory bowel disease. Hence, cells acquired mitochondria during evolution to profit from oxidative metabolism, but also built in an autophagy-based ROS-induced protective mechanism to guard against oxidative stress associated with OXPHOS function during quiescence.Abbreviations: AMPK: AMP-activated protein kinase; AOX: alternative oxidase; Baf A: bafilomycin A1; CI, respiratory complexes I; DCF-DA: 2',7'-dichlordihydrofluorescein diacetate; DHE: dihydroethidium; DSS: dextran sodium sulfate; ΔΨmi: mitochondrial inner membrane potential; EdU: 5-ethynyl-2'-deoxyuridine; ETC: electron transport chain; FA: formaldehyde; HUVEC; human umbilical cord endothelial cells; IBD: inflammatory bowel disease; LC3B: microtubule associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; mtDNA: mitochondrial DNA; NAC: N-acetyl cysteine; OXPHOS: oxidative phosphorylation; PCs: proliferating cells; PE: phosphatidylethanolamine; PEITC: phenethyl isothiocyanate; QCs: quiescent cells; ROS: reactive oxygen species; PLA2: phospholipase A2, WB: western blot.
Assuntos
Autofagia , Doenças Inflamatórias Intestinais , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cisteína/metabolismo , DNA Mitocondrial/metabolismo , Dextranos/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Formaldeído/metabolismo , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Isotiocianatos , Lipopolissacarídeos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Fosfatidiletanolaminas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Respiração , SirolimoRESUMO
Diabetes is a chronic metabolic disorder characterized by hyperglycemia and associated with many health complications due to the long-term damage and dysfunction of various organs. A consequential complication of diabetes in men is reproductive dysfunction, reduced fertility, and poor reproductive outcomes. However, the molecular mechanisms responsible for diabetic environment-induced sperm damage and overall decreased reproductive outcomes are not fully established. We evaluated the effects of type 2 diabetes exposure on the reproductive system and the reproductive outcomes of males and their male offspring, using a mouse model. We demonstrate that paternal exposure to type 2 diabetes mediates intergenerational and transgenerational effects on the reproductive health of the offspring, especially on sperm quality, and on metabolic characteristics. Given the transgenerational impairment of reproductive and metabolic parameters through two generations, these changes likely take the form of inherited epigenetic marks through the germline. Our results emphasize the importance of improving metabolic health not only in women of reproductive age, but also in potential fathers, in order to reduce the negative impacts of diabetes on subsequent generations.
Assuntos
Diabetes Mellitus Tipo 2/genética , Infertilidade/genética , Herança Paterna/genética , Fenótipo , Espermatozoides/fisiologia , Animais , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/induzido quimicamente , Dieta Hiperlipídica/efeitos adversos , Feminino , Infertilidade/sangue , Infertilidade/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Herança Paterna/efeitos dos fármacos , Gravidez , Espermatozoides/efeitos dos fármacos , Estreptozocina/toxicidadeRESUMO
Ear development requires the transcription factors ATOH1 for hair cell differentiation and NEUROD1 for sensory neuron development. In addition, NEUROD1 negatively regulates Atoh1 gene expression. As we previously showed that deletion of the Neurod1 gene in the cochlea results in axon guidance defects and excessive peripheral innervation of the sensory epithelium, we hypothesized that some of the innervation defects may be a result of abnormalities in NEUROD1 and ATOH1 interactions. To characterize the interdependency of ATOH1 and NEUROD1 in inner ear development, we generated a new Atoh1/Neurod1 double null conditional deletion mutant. Through careful comparison of the effects of single Atoh1 or Neurod1 gene deletion with combined double Atoh1 and Neurod1 deletion, we demonstrate that NEUROD1-ATOH1 interactions are not important for the Neurod1 null innervation phenotype. We report that neurons lacking Neurod1 can innervate the flat epithelium without any sensory hair cells or supporting cells left after Atoh1 deletion, indicating that neurons with Neurod1 deletion do not require the presence of hair cells for axon growth. Moreover, transcriptome analysis identified genes encoding axon guidance and neurite growth molecules that are dysregulated in the Neurod1 deletion mutant. Taken together, we demonstrate that much of the projections of NEUROD1-deprived inner ear sensory neurons are regulated cell-autonomously.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Ciliadas Auditivas/metabolismo , Fibras Nervosas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Apoptose/genética , Axônios/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Epitélio/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Ciliadas Auditivas/patologia , Células Ciliadas Auditivas/ultraestrutura , Camundongos Knockout , Modelos Biológicos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Órgão Espiral/patologia , Fatores de Transcrição SOXB1/metabolismo , Gânglio Espiral da Cóclea/metabolismoRESUMO
The programming of cell fate by transcription factors requires precise regulation of their time and level of expression. The LIM-homeodomain transcription factor Islet1 (Isl1) is involved in cell-fate specification of motor neurons, and it may play a similar role in the inner ear. In order to study its role in the regulation of vestibulo-motor development, we investigated a transgenic mouse expressing Isl1 under the Pax2 promoter control (Tg +/- ). The transgenic mice show altered level, time, and place of expression of Isl1 but are viable. However, Tg +/- mice exhibit hyperactivity, including circling behavior, and progressive age-related decline in hearing, which has been reported previously. Here, we describe the molecular and morphological changes in the cerebellum and vestibular system that may cause the hyperactivity of Tg +/- mice. The transgene altered the formation of folia in the cerebellum, the distribution of calretinin labeled unipolar brush cells, and reduced the size of the cerebellum, inferior colliculus, and saccule. Age-related progressive reduction of calbindin expression was detected in Purkinje cells in the transgenic cerebella. The hyperactivity of Tg +/- mice is reduced upon the administration of picrotoxin, a non-competitive channel blocker for the γ-aminobutyric acid (GABA) receptor chloride channels. This suggests that the overexpression of Isl1 significantly affects the functions of GABAergic neurons. We demonstrate that the overexpression of Isl1 affects the development and function of the cerebello-vestibular system, resulting in hyperactivity.
Assuntos
Cerebelo/metabolismo , Cerebelo/patologia , Hipercinese/metabolismo , Hipercinese/patologia , Proteínas com Homeodomínio LIM/biossíntese , Fator de Transcrição PAX2/biossíntese , Fatores de Transcrição/biossíntese , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Vestíbulo do Labirinto/metabolismo , Vestíbulo do Labirinto/patologiaRESUMO
Male infertility is a worldwide problem associated with genetic background, environmental factors, and diseases. One of the suspected contributing factors to male infertility is diabetes mellitus. We investigated the molecular and morphological changes in sperms and testicular tissue of diabetic males. The study was performed in streptozotocin-induced type 1 diabetes mouse model. Diabetes decreased sperm concentration and viability and increased sperm apoptosis. Changes in protamine 1/protamine 2 ratio indicated reduced sperm quality. The testicular tissue of diabetic males showed significant tissue damage, disruption of meiotic progression, and changes in the expression of genes encoding proteins important for spermiogenesis. Paternal diabetes altered sperm quality and expression pattern in the testes in offspring of two subsequent generations. Our study revealed that paternal diabetes increased susceptibility to infertility in offspring through gametic alternations. Our data also provide a mechanistic basis for transgenerational inheritance of diabetes-associated pathologies since protamines may be involved in epigenetic regulations.