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Seasonal daylength, or circadian photoperiod, is a pervasive environmental signal that profoundly influences physiology and behavior. In mammals, the central circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus where it receives retinal input and synchronizes, or entrains, organismal physiology and behavior to the prevailing light cycle. The process of entrainment induces sustained plasticity in the SCN, but the molecular mechanisms underlying SCN plasticity are incompletely understood. Entrainment to different photoperiods persistently alters the timing, waveform, period, and light resetting properties of the SCN clock and its driven rhythms. To elucidate novel candidate genes for molecular mechanisms of photoperiod plasticity, we performed RNA sequencing on whole SCN dissected from mice raised in long (light:dark [LD] 16:8) and short (LD 8:16) photoperiods. Fewer rhythmic genes were detected in mice subjected to long photoperiod, and in general, the timing of gene expression rhythms was advanced 4-6 h. However, a few genes showed significant delays, including Gem. There were significant changes in the expression of the clock-associated gene Timeless and in SCN genes related to light responses, neuropeptides, gamma aminobutyric acid (GABA), ion channels, and serotonin. Particularly striking were differences in the expression of the neuropeptide signaling genes Prokr2 and Cck, as well as convergent regulation of the expression of 3 SCN light response genes, Dusp4, Rasd1, and Gem. Transcriptional modulation of Dusp4 and Rasd1 and phase regulation of Gem are compelling candidate molecular mechanisms for plasticity in the SCN light response through their modulation of the critical NMDAR-MAPK/ERK-CREB/CRE light signaling pathway in SCN neurons. Modulation of Prokr2 and Cck may critically support SCN neural network reconfiguration during photoperiodic entrainment. Our findings identify the SCN light response and neuropeptide signaling gene sets as rich substrates for elucidating novel mechanisms of photoperiod plasticity. Data are also available at http://circadianphotoperiodseq.com/, where users can view the expression and rhythmic properties of genes across these photoperiod conditions.
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Background: Microglial cell iron load and inflammatory activation are significant hallmarks of late-stage Alzheimer's disease (AD). In vitro, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and excess iron can augment cellular inflammation, suggesting a feed-forward loop between iron import mechanisms and inflammatory signaling. However, it is not understood whether microglial iron import mechanisms directly contribute to inflammatory signaling and chronic disease in vivo. These studies determined the effects of microglial-specific knockdown of Slc11a2 on AD-related cognitive decline and microglial transcriptional phenotype. Methods: In vitro experiments and RT-qPCR were used to assess a role for DMT1 in amyloid-ß-associated inflammation. To determine the effects of microglial Slc11a2 knockdown on AD-related phenotypes in vivo, triple-transgenic Cx3cr1 Cre - ERT2 ;Slc11a2 flfl;APP/PS1 + or - mice were generated and administered corn oil or tamoxifen to induce knockdown at 5-6 months of age. Both sexes underwent behavioral analyses to assess cognition and memory (12-15 months of age). Hippocampal CD11b + microglia were magnetically isolated from female mice (15-17 months) and bulk RNA-sequencing analysis was conducted. Results: DMT1 inhibition in vitro robustly decreased Aß-induced inflammatory gene expression and cellular iron levels in conditions of excess iron. In vivo, Slc11a2 KD APP/PS1 female, but not male, mice displayed a significant worsening of memory function in Morris water maze and a fear conditioning assay, along with significant hyperactivity compared to control WT and APP/PS1 mice. Hippocampal microglia from Slc11a2 KD APP/PS1 females displayed significant increases in Enpp2, Ttr, and the iron-export gene, Slc40a1, compared to control APP/PS1 cells. Slc11a2 KD cells from APP/PS1 females also exhibited decreased expression of markers associated with disease-associated microglia (DAMs), such as Apoe, Ctsb, Csf1, and Hif1α. Conclusions: This work suggests a sex-specific role for microglial iron import gene Slc11a2 in propagating behavioral and cognitive phenotypes in the APP/PS1 model of AD. These data also highlight an association between loss of a DAM-like phenotype in microglia and cognitive deficits in Slc11a2 KD APP/PS1 female mice. Overall, this work illuminates an iron-related pathway in microglia that may serve a protective role during disease and offers insight into mechanisms behind disease-related sex differences.
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The gain-of-function mutation in the TALK-1 K+ channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of ß-cell electrical activity and glucose-stimulated insulin secretion. The KCNK16 gene encoding TALK-1 is the most abundant and ß-cell-restricted K+ channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the CD-1 (ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell ß-cell K+ currents resulting in blunted glucose-stimulated Ca2+ entry and loss of glucose-induced Ca2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impairs glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet insulin secretion during development. These data suggest that TALK-1 is an islet-restricted target for the treatment for diabetes.
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Diabetes Mellitus Tipo 2 , Glucagón , Glucosa , Secreción de Insulina , Ratones Endogámicos C57BL , Animales , Masculino , Ratones , Animales Recién Nacidos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Glucagón/metabolismo , Glucosa/metabolismo , Homeostasis , Insulina/metabolismo , Secreción de Insulina/efectos de los fármacos , Secreción de Insulina/genética , Islotes Pancreáticos/metabolismo , Mutación , Canales de Potasio/metabolismo , Canales de Potasio/genéticaRESUMEN
Seasonal daylength, or circadian photoperiod, is a pervasive environmental signal that profoundly influences physiology and behavior. In mammals, the central circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus where it receives retinal input and synchronizes, or entrains, organismal physiology and behavior to the prevailing light cycle. The process of entrainment induces sustained plasticity in the SCN, but the molecular mechanisms underlying SCN plasticity are incompletely understood. Entrainment to different photoperiods persistently alters the timing, waveform, period, and light resetting properties of the SCN clock and its driven rhythms. To elucidate novel molecular mechanisms of photoperiod plasticity, we performed RNAseq on whole SCN dissected from mice raised in Long (LD 16:8) and Short (LD 8:16) photoperiods. Fewer rhythmic genes were detected in Long photoperiod and in general the timing of gene expression rhythms was advanced 4-6 hours. However, a few genes showed significant delays, including Gem . There were significant changes in the expression clock-associated gene Timeless and in SCN genes related to light responses, neuropeptides, GABA, ion channels, and serotonin. Particularly striking were differences in the expression of the neuropeptide signaling genes Prokr2 and Cck , as well as convergent regulation of the expression of three SCN light response genes, Dusp4 , Rasd1 , and Gem . Transcriptional modulation of Dusp4 and Rasd1, and phase regulation of Gem, are compelling candidate molecular mechanisms for plasticity in the SCN light response through their modulation of the critical NMDAR-MAPK/ERK-CREB/CRE light signaling pathway in SCN neurons. Modulation of Prokr2 and Cck may critically support SCN neural network reconfiguration during photoperiodic entrainment. Our findings identify the SCN light response and neuropeptide signaling gene sets as rich substrates for elucidating novel mechanisms of photoperiod plasticity.
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ABSTRACT: Escherichia coli and Staphylococcus aureus are two of the most common bacterial species responsible for sepsis. While it is observed that they have disparate clinical phenotypes, the signaling differences elicited by each bacteria that drive this variance remain unclear. Therefore, we used human whole blood exposed to heat-killed E. coli or S. aureus and measured the transcriptomic signatures. Relative to unstimulated control blood, heat-killed bacteria exposure led to significant dysregulation (upregulated and downregulated) of >5,000 genes for each experimental condition, with a slight increase in gene alterations by S. aureus. While there was significant overlap regarding proinflammatory pathways, Gene Ontology overrepresentation analysis of the most altered genes suggested biological processes like macrophage differentiation and ubiquinone biosynthesis were more unique to heat-killed S. aureus, compared with heat-killed E. coli exposure. Using Ingenuity Pathway Analysis, it was demonstrated that nuclear factor erythroid 2-related factor 2 signaling, a main transcription factor in antioxidant responses, was predominately upregulated in S. aureus exposed blood relative to E. coli. Furthermore, the use of pharmacologics that preferentially targeted the nuclear factor erythroid 2-related factor 2 pathway led to differential cytokine profiles depending on the type of bacterial exposure. These findings reveal significant inflammatory dysregulation between E. coli and S. aureus and provide insight into the targeting of unique pathways to curb bacteria-specific responses.
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Infecciones por Escherichia coli , Infecciones Estafilocócicas , Humanos , Escherichia coli , Staphylococcus aureus , Factor 2 Relacionado con NF-E2/genética , Regulación de la Expresión GénicaRESUMEN
The gain-of-function mutation in the TALK-1 K + channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of ß-cell electrical activity and glucose-stimulated insulin secretion (GSIS). The KCNK16 gene encoding TALK-1, is the most abundant and ß-cell-restricted K + channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the mixed C57BL/6J:CD-1(ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell ß-cell K + currents resulting in blunted glucose-stimulated Ca 2+ entry and loss of glucose-induced Ca 2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impaired glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet hormone secretion during development. These data strongly suggest that TALK-1 is an islet-restricted target for the treatment of diabetes.
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Biomedical sciences PhDs pursue a wide range of careers inside and outside academia. However, there is little data regarding how career interests of PhD students relate to the decision to pursue postdoctoral training or to their eventual career outcomes. Here, we present the career goals and career outcomes of 1452 biomedical sciences PhDs who graduated from Vanderbilt University between 1997 and 2021. We categorized careers using an expanded three-tiered taxonomy and flags that delineate key career milestones. We also analyzed career goal changes between matriculation and doctoral defense, and the reasons why students became more- or less-interested in research-intensive faculty careers. We linked students' career goal at doctoral defense to whether they did a postdoc, the duration of time between doctoral defense and the first non-training position, the career area of the first non-training position, and the career area of the job at 10 years after graduation. Finally, we followed individual careers for 10 years after graduation to characterize movement between different career areas over time. We found that most students changed their career goal during graduate school, declining numbers of alumni pursued postdoctoral training, many alumni entered first non-training positions in a different career area than their goal at doctoral defense, and the career area of the first non-training position was a good indicator of the job that alumni held 10 years after graduation. Our findings emphasize that students need a wide range of career development opportunities and career mentoring during graduate school to prepare them for futures in research and research-related professions.
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Caloric restriction (CR) extends organismal lifespan and health span by improving glucose homeostasis mechanisms. How CR affects organellar structure and function of pancreatic beta cells over the lifetime of the animal remains unknown. Here, we used single nucleus transcriptomics to show that CR increases the expression of genes for beta cell identity, protein processing, and organelle homeostasis. Gene regulatory network analysis link this transcriptional phenotype to transcription factors involved in beta cell identity (Mafa) and homeostasis (Atf6). Imaging metabolomics further demonstrates that CR beta cells are more energetically competent. In fact, high-resolution light and electron microscopy indicates that CR reduces beta cell mitophagy and increases mitochondria mass, increasing mitochondrial ATP generation. Finally, we show that long-term CR delays the onset of beta cell aging and senescence to promote longevity by reducing beta cell turnover. Therefore, CR could be a feasible approach to preserve compromised beta cells during aging and diabetes.
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OBJECTIVE: ASCL1, a pioneer transcription factor, is essential for neural cell differentiation and function. Previous studies have shown that Ascl1 expression is increased in pancreatic ß-cells lacking functional KATP channels or after feeding of a high fat diet (HFD) suggesting that it may contribute to the metabolic stress response of ß-cells. METHODS: We generated ß-cell-specific Ascl1 knockout mice (Ascl1ßKO) and assessed their glucose homeostasis, islet morphology and gene expression after feeding either a normal diet or HFD for 12 weeks, or in combination with a genetic disruption of Abcc8, an essential KATP channel component. RESULTS: Ascl1 expression is increased in response to both a HFD and membrane depolarization and requires CREB-dependent Ca2+ signaling. No differences in glucose homeostasis or islet morphology were observed in Ascl1ßKO mice fed a normal diet or in the absence of KATP channels. However, male Ascl1ßKO mice fed a HFD exhibited decreased blood glucose levels, improved glucose tolerance, and increased ß-cell proliferation. Bulk RNA-seq analysis of islets from Ascl1ßKO mice from three studied conditions showed alterations in genes associated with the secretory function. HFD-fed Ascl1ßKO mice showed the most extensive changes with increased expression of genes necessary for glucose sensing, insulin secretion and ß-cell proliferation, and a decrease in genes associated with ß-cell dysfunction, inflammation and dedifferentiation. HFD-fed Ascl1ßKO mice also displayed increased expression of parasympathetic neural markers and cholinergic receptors that was accompanied by increased insulin secretion in response to acetylcholine and an increase in islet innervation. CONCLUSIONS: Ascl1 expression is induced by stimuli that cause Ca2+-signaling to the nucleus and contributes in a multifactorial manner to the loss of ß-cell function by promoting the expression of genes associated with cellular dedifferentiation, attenuating ß-cells proliferation, suppressing acetylcholine sensitivity, and repressing parasympathetic innervation of islets. Thus, the removal of Ascl1 from ß-cells improves their function in response to metabolic stress.
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Acetilcolina , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Insulina , Animales , Masculino , Ratones , Adenosina Trifosfato/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Glucosa , Insulina/metabolismo , Secreción de Insulina , Estrés FisiológicoRESUMEN
Caloric restriction (CR) extends organismal lifespan and health span by improving glucose homeostasis mechanisms. How CR affects organellar structure and function of pancreatic beta cells over the lifetime of the animal remains unknown. Here, we used single nucleus transcriptomics to show that CR increases the expression of genes for beta cell identity, protein processing, and organelle homeostasis. Gene regulatory network analysis link this transcriptional phenotype to transcription factors involved in beta cell identity (Mafa) and homeostasis (Atf6). Imaging metabolomics further demonstrates that CR beta cells are more energetically competent. In fact, high-resolution light and electron microscopy indicates that CR reduces beta cell mitophagy and increases mitochondria mass, increasing mitochondrial ATP generation. Finally, we show that long-term CR delays the onset of beta cell aging and senescence to promote longevity by reducing beta cell turnover. Therefore, CR could be a feasible approach to preserve compromised beta cells during aging and diabetes.
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Repressive KRAB domain-containing zinc-finger proteins (KRAB-ZFPs) are abundant in mammalian genomes and contribute both to the silencing of transposable elements (TEs) and to the regulation of developmental stage- and cell type-specific gene expression. Here we describe studies of zinc finger protein 92 (Zfp92), an X-linked KRAB-ZFP that is highly expressed in pancreatic islets of adult mice, by analyzing global Zfp92 knockout (KO) mice. Physiological, transcriptomic and genome-wide chromatin binding studies indicate that the principal function of ZFP92 in mice is to bind to and suppress the activity of B1/Alu type of SINE elements and modulate the activity of surrounding genomic entities. Deletion of Zfp92 leads to changes in expression of select LINE and LTR retroelements and genes located in the vicinity of ZFP92-bound chromatin. The absence of Zfp92 leads to altered expression of specific genes in islets, adipose and muscle that result in modest sex-specific alterations in blood glucose homeostasis, body mass and fat accumulation. In islets, Zfp92 influences blood glucose concentration in postnatal mice via transcriptional effects on Mafb, whereas in adipose and muscle, it regulates Acacb, a rate-limiting enzyme in fatty acid metabolism. In the absence of Zfp92, a novel TE-Capn11 fusion transcript is overexpressed in islets and several other tissues due to de-repression of an IAPez TE adjacent to ZFP92-bound SINE elements in intron 3 of the Capn11 gene. Together, these studies show that ZFP92 functions both to repress specific TEs and to regulate the transcription of specific genes in discrete tissues.
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Elementos Transponibles de ADN , Islotes Pancreáticos , Animales , Femenino , Masculino , Ratones , Glucemia , Cromatina , Islotes Pancreáticos/metabolismo , Mamíferos/genética , Proteínas Represoras/genética , Retroelementos/genética , Dedos de Zinc/genéticaRESUMEN
During early embryogenesis, the transcription factor SOX17 contributes to hepato-pancreato-biliary system formation and vascular-hematopoietic emergence. To better understand Sox17 function in the developing endoderm and endothelium, we developed a dual-color temporal lineage-tracing strategy in mice combined with single-cell RNA sequencing to analyze 6934 cells from Sox17-expressing lineages at embryonic days 9.0-9.5. Our analyses showed 19 distinct cellular clusters combined from all 3 germ layers. Differential gene expression, trajectory and RNA-velocity analyses of endothelial cells revealed a heterogenous population of uncommitted and specialized endothelial subtypes, including 2 hemogenic populations that arise from different origins. Similarly, analyses of posterior foregut endoderm revealed subsets of hepatic, pancreatic, and biliary progenitors with overlapping developmental potency. Calculated gene-regulatory networks predict gene regulons that are dominated by cell type-specific transcription factors unique to each lineage. Vastly different Sox17 regulons found in endoderm versus endothelial cells support the differential interactions of SOX17 with other regulatory factors thereby enabling lineage-specific regulatory actions.
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Desarrollo Embrionario , Células Endoteliales , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Factores de Transcripción SOXF , Animales , Ratones , Diferenciación Celular , Linaje de la Célula/genética , Endodermo/metabolismo , Células Endoteliales/metabolismo , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Análisis de Secuencia de ARN , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Factores de Transcripción/metabolismo , Desarrollo Embrionario/genéticaRESUMEN
Brown adipose tissue (BAT) has in recent times been rediscovered in adult humans, and together with work from preclinical models, has shown to have the potential of providing a variety of positive metabolic benefits. These include lower plasma glucose, improved insulin sensitivity, and reduced susceptibility to obesity and its comorbidities. As such, its continued study could offer insights to therapeutically modulate this tissue to improve metabolic health. It has been reported that adipose-specific deletion of the gene for protein kinase D1 (Prkd1) in mice enhances mitochondrial respiration and improves whole-body glucose homeostasis. We sought to determine whether these effects were mediated specifically through brown adipocytes using a Prkd1 brown adipose tissue (BAT) Ucp1-Cre-specific knockout mouse model, Prkd1BKO . We unexpectedly observed that upon both cold exposure and ß3 -AR agonist administration, Prkd1 loss in BAT did not alter canonical thermogenic gene expression or adipocyte morphology. We took an unbiased approach to assess whether other signaling pathways were affected. RNA from cold-exposed mice was subjected to RNA-Seq analysis. These studies revealed that myogenic gene expression is altered in Prkd1BKO BAT after both acute and extended cold exposure. Given that brown adipocytes and skeletal myocytes share a common precursor cell lineage expressing myogenic factor 5 (Myf5), these data suggest that loss of Prkd1 in BAT may alter the biology of mature brown adipocytes and preadipocytes in this depot. The data presented herein clarify the role of Prkd1 in BAT thermogenesis and present new avenues for the further study of Prkd1 function in BAT.
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Tejido Adiposo Pardo , Transducción de Señal , Humanos , Ratones , Animales , Tejido Adiposo Pardo/metabolismo , Obesidad/metabolismo , Ratones Noqueados , Termogénesis/fisiología , Expresión Génica , Proteínas Quinasas/metabolismo , Ratones Endogámicos C57BLRESUMEN
The autonomic nervous system regulates pancreatic function. Islet capillaries are essential for the extension of axonal projections into islets, and both of these structures are important for appropriate islet hormone secretion. Because beta cells provide important paracrine cues for islet glucagon secretion and neurovascular development, we postulated that beta cell loss in type 1 diabetes (T1D) would lead to a decline in intraislet capillaries and reduction of islet innervation, possibly contributing to abnormal glucagon secretion. To define morphological characteristics of capillaries and nerve fibers in islets and acinar tissue compartments, we analyzed neurovascular assembly across the largest cohort of T1D and normal individuals studied thus far. Because innervation has been studied extensively in rodent models of T1D, we also compared the neurovascular architecture between mouse and human pancreas and assembled transcriptomic profiles of molecules guiding islet angiogenesis and neuronal development. We found striking interspecies differences in islet neurovascular assembly but relatively modest differences at transcriptome level, suggesting that posttranscriptional regulation may be involved in this process. To determine whether islet neurovascular arrangement is altered after beta cell loss in T1D, we compared pancreatic tissues from non-diabetic, recent-onset T1D (<10-yr duration), and longstanding T1D (>10-yr duration) donors. Recent-onset T1D showed greater islet and acinar capillary density compared to non-diabetic and longstanding T1D donors. Both recent-onset and longstanding T1D had greater islet nerve fiber density compared to non-diabetic donors. We did not detect changes in sympathetic axons in either T1D cohort. Additionally, nerve fibers overlapped with extracellular matrix (ECM), supporting its role in the formation and function of axonal processes. These results indicate that pancreatic capillaries and nerve fibers persist in T1D despite beta cell loss, suggesting that alpha cell secretory changes may be decoupled from neurovascular components.NEW & NOTEWORTHY Defining the neurovascular architecture in the pancreas of individuals with type 1 diabetes (T1D) is crucial to understanding the mechanisms of dysregulated glucagon secretion. In the largest T1D cohort of biobanked tissues analyzed to date, we found that pancreatic capillaries and nerve fibers persist in human T1D despite beta cell loss, suggesting that alpha cell secretory changes may be decoupled from neurovascular components. Because innervation has been studied extensively in rodent T1D models, our studies also provide the first rigorous direct comparisons of neurovascular assembly in mouse and human, indicating dramatic interspecies differences.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Células Secretoras de Glucagón , Islotes Pancreáticos , Humanos , Ratones , Animales , Diabetes Mellitus Tipo 1/metabolismo , Islotes Pancreáticos/metabolismo , Glucagón/metabolismo , Capilares/metabolismo , Células Secretoras de Glucagón/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Fibras Nerviosas/metabolismoRESUMEN
Pancreatic islet beta cells are essential for maintaining glucose homeostasis. To understand the impact of aging on beta cells, we performed meta-analysis of single-cell RNA sequencing datasets, transcription factor (TF) regulon analysis, high-resolution confocal microscopy, and measured insulin secretion from nondiabetic donors spanning most of the human life span. This revealed the range of molecular and functional changes that occur during beta cell aging, including the transcriptional deregulation that associates with cellular immaturity and reorganization of beta cell TF networks, increased gene transcription rates, and reduced glucose-stimulated insulin release. These alterations associate with activation of endoplasmic reticulum (ER) stress and autophagy pathways. We propose that a chronic state of ER stress undermines old beta cell structure function to increase the risk of beta cell failure and type 2 diabetes onset as humans age.
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Leigh syndrome (LS) is a rare, inherited neurometabolic disorder that presents with bilateral brain lesions caused by defects in the mitochondrial respiratory chain and associated nuclear-encoded proteins. We generated human induced pluripotent stem cells (iPSCs) from three LS patient-derived fibroblast lines. Using whole-exome and mitochondrial sequencing, we identified unreported mutations in pyruvate dehydrogenase (GM0372, PDH; GM13411, MT-ATP6/PDH) and dihydrolipoyl dehydrogenase (GM01503, DLD). These LS patient-derived iPSC lines were viable and capable of differentiating into progenitor populations, but we identified several abnormalities in three-dimensional differentiation models of brain development. LS patient-derived cerebral organoids showed defects in neural epithelial bud generation, size and cortical architecture at 100â days. The double mutant MT-ATP6/PDH line produced organoid neural precursor cells with abnormal mitochondrial morphology, characterized by fragmentation and disorganization, and showed an increased generation of astrocytes. These studies aim to provide a comprehensive phenotypic characterization of available patient-derived cell lines that can be used to study Leigh syndrome.
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Células Madre Pluripotentes Inducidas , Enfermedad de Leigh , Células-Madre Neurales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Leigh/genética , Enfermedad de Leigh/metabolismo , Mutación/genética , Células-Madre Neurales/metabolismo , Organoides/metabolismoRESUMEN
Clonal evolution in myelodysplastic syndrome (MDS) can result in clinical progression and secondary acute myeloid leukemia (sAML). To dissect changes in clonal architecture associated with this progression, we performed single-cell genotyping of paired MDS and sAML samples from 18 patients. Analysis of single-cell genotypes revealed patient-specific clonal evolution and enabled the assessment of single-cell mutational cooccurrence. We discovered that changes in clonal architecture proceed via distinct patterns, classified as static or dynamic, with dynamic clonal architectures having a more proliferative phenotype by blast count fold change. Proteogenomic analysis of a subset of patients confirmed that pathogenic mutations were primarily confined to primitive and mature myeloid cells, though we also identify rare but present mutations in lymphocyte subsets. Single-cell transcriptomic analysis of paired sample sets further identified gene sets and signaling pathways involved in two cases of progression. Together, these data define serial changes in the MDS clonal landscape with clinical and therapeutic implications. SIGNIFICANCE: Precise clonal trajectories in MDS progression are made possible by single-cell genomic sequencing. Here we use this technology to uncover the patterns of clonal architecture and clonal evolution that drive the transformation to secondary AML. We further define the phenotypic and transcriptional changes of disease progression at the single-cell level. See related article by Menssen et al., p. 330 (31). See related commentary by Romine and van Galen, p. 270. This article is highlighted in the In This Issue feature, p. 265.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Neoplasias Primarias Secundarias , Evolución Clonal/genética , Células Clonales/patología , Humanos , Leucemia Mieloide Aguda/genética , Mutación , Síndromes Mielodisplásicos/genéticaRESUMEN
Insm1, Neurod1, and Pax6 are essential for the formation and function of pancreatic endocrine cells. Here, we report comparative immunohistochemical, transcriptomic, functional enrichment, and RNA splicing analyses of these genes using gene knock-out mice. Quantitative immunohistochemical analysis confirmed that elimination of each of these three factors variably impairs the proliferation, survival, and differentiation of endocrine cells. Transcriptomic analysis revealed that each factor contributes uniquely to the transcriptome although their effects were overlapping. Functional enrichment analysis revealed that genes downregulated by the elimination of Insm1, Neurod1, and Pax6 are commonly involved in mRNA metabolism, chromatin organization, secretion, and cell cycle regulation, and upregulated genes are associated with protein degradation, autophagy, and apoptotic process. Elimination of Insm1, Neurod1, and Pax6 impaired expression of many RNA-binding proteins thereby altering RNA splicing events, including for Syt14 and Snap25, two genes required for insulin secretion. All three factors are necessary for normal splicing of Syt14, and both Insm1 and Pax6 are necessary for the processing of Snap25. Collectively, these data provide new insights into how Insm1, Neurod1, and Pax6 contribute to the formation of functional pancreatic endocrine cells.
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Células Endocrinas , Factores de Transcripción , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Células Endocrinas/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Ratones , Factor de Transcripción PAX6/genética , ARN , Empalme del ARN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genéticaRESUMEN
Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and ß cells, and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled more than 40,000 cells from normal human islets by single-cell RNA-Seq and stratified α and ß cells based on combinatorial TF expression. Subpopulations of islet cells coexpressing ARX/MAFB (α cells) and MAFA/MAFB (ß cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-Seq, MAFA/MAFB-coexpressing ß cells showed enhanced electrophysiological activity. Thus, these results indicate that combinatorial TF expression in islet α and ß cells predicts highly functional, mature subpopulations.
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Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Adulto , Fenómenos Electrofisiológicos , Expresión Génica , Células Secretoras de Glucagón/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Factor de Transcripción MafB/genética , Factor de Transcripción MafB/metabolismo , Persona de Mediana Edad , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transcriptoma , Adulto JovenRESUMEN
To gain a deeper understanding of pancreatic ß-cell development, we used iterative weighted gene correlation network analysis to calculate a gene co-expression network (GCN) from 11 temporally and genetically defined murine cell populations. The GCN, which contained 91 distinct modules, was then used to gain three new biological insights. First, we found that the clustered protocadherin genes are differentially expressed during pancreas development. Pcdhγ genes are preferentially expressed in pancreatic endoderm, Pcdhß genes in nascent islets, and Pcdhα genes in mature ß-cells. Second, after extracting sub-networks of transcriptional regulators for each developmental stage, we identified 81 zinc finger protein (ZFP) genes that are preferentially expressed during endocrine specification and ß-cell maturation. Third, we used the GCN to select three ZFPs for further analysis by CRISPR mutagenesis of mice. Zfp800 null mice exhibited early postnatal lethality, and at E18.5 their pancreata exhibited a reduced number of pancreatic endocrine cells, alterations in exocrine cell morphology, and marked changes in expression of genes involved in protein translation, hormone secretion and developmental pathways in the pancreas. Together, our results suggest that developmentally oriented GCNs have utility for gaining new insights into gene regulation during organogenesis.