RESUMEN
Although the regulation of branching morphogenesis by spatially distributed cues is well established, the role of intracellular signaling in determining the branching pattern remains poorly understood. In this study, we investigated the regulation and function of phospholipase C gamma (PLCγ) in Fibroblast Growth Factor (FGF) signaling in lacrimal gland development. We showed that deletion of PLCγ1 in the lacrimal gland epithelium leads to ectopic branching and acinar hyperplasia, which was phenocopied by either mutating the PLCγ1 binding site on Fgfr2 or disabling any of its SH2 domains. PLCγ1 inactivation did not change the level of Fgfr2 or affect MAPK signaling, but instead led to sustained AKT phosphorylation due to increased PIP3 production. Consistent with this, PLCγ1 mutant phenotype can be reproduced by elevation of PI3K signaling in Pten knockout and attenuated by blocking AKT signaling. This study demonstrated that PLCγ modulates PI3K signaling by shifting phosphoinositide metabolism, revealing an important role of signaling dynamics in conjunction with spatial cues in shaping branching morphogenesis.
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BACKGROUND: Recent experimental studies of neuroinflammation in glaucoma pointed to cFLIP as a molecular switch for cell fate decisions, mainly regulating cell type-specific caspase-8 functions in cell death and inflammation. This study aimed to determine the importance of cFLIP for regulating astroglia-driven neuroinflammation in experimental glaucoma by analyzing the outcomes of astroglia-targeted transgenic deletion of cFLIP or cFLIPL. METHODS: Glaucoma was modeled by anterior chamber microbead injections to induce ocular hypertension in mouse lines with or without conditional deletion of cFLIP or cFLIPL in astroglia. Morphological analysis of astroglia responses assessed quantitative parameters in retinal whole mounts immunolabeled for GFAP and inflammatory molecules or assayed for TUNEL. The molecular analysis included 36-plexed immunoassays of the retina and optic nerve cytokines and chemokines, NanoString-based profiling of inflammation-related gene expression, and Western blot analysis of selected proteins in freshly isolated samples of astroglia. RESULTS: Immunoassays and immunolabeling of retina and optic nerve tissues presented reduced production of various proinflammatory cytokines, including TNFα, in GFAP/cFLIP and GFAP/cFLIPL relative to controls at 12 weeks of ocular hypertension with no detectable alteration in TUNEL. Besides presenting a similar trend of the proinflammatory versus anti-inflammatory molecules displayed by immunoassays, NanoString-based molecular profiling detected downregulated NF-κB/RelA and upregulated RelB expression of astroglia in ocular hypertensive samples of GFAP/cFLIP compared to ocular hypertensive controls. Analysis of protein expression also revealed decreased phospho-RelA and increased phospho-RelB in parallel with an increase in caspase-8 cleavage products. CONCLUSIONS: A prominent response limiting neuroinflammation in ocular hypertensive eyes with cFLIP-deletion in astroglia values the role of cFLIP in the molecular regulation of glia-driven neuroinflammation during glaucomatous neurodegeneration. The molecular responses accompanying the lessening of neurodegenerative inflammation also seem to maintain astroglia survival despite increased caspase-8 cleavage with cFLIP deletion. A transcriptional autoregulatory response, dampening RelA but boosting RelB for selective expression of NF-κB target genes, might reinforce cell survival in cFLIP-deleted astroglia.
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Astrocitos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Glaucoma , Enfermedades Neuroinflamatorias , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Ratones , Astrocitos/metabolismo , Astrocitos/patología , Glaucoma/metabolismo , Glaucoma/patología , Glaucoma/genética , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Ratones Transgénicos , Modelos Animales de Enfermedad , Citocinas/metabolismo , Retina/metabolismo , Retina/patología , Ratones Endogámicos C57BL , Nervio Óptico/patología , Nervio Óptico/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismoRESUMEN
In the prevailing model, Lgr5+ cells are the only intestinal stem cells (ISCs) that sustain homeostatic epithelial regeneration by upward migration of progeny through elusive upper crypt transit-amplifying (TA) intermediates. Here, we identify a proliferative upper crypt population marked by Fgfbp1, in the location of putative TA cells, that is transcriptionally distinct from Lgr5+ cells. Using a kinetic reporter for time-resolved fate mapping and Fgfbp1-CreERT2 lineage tracing, we establish that Fgfbp1+ cells are multi-potent and give rise to Lgr5+ cells, consistent with their ISC function. Fgfbp1+ cells also sustain epithelial regeneration following Lgr5+ cell depletion. We demonstrate that FGFBP1, produced by the upper crypt cells, is an essential factor for crypt proliferation and epithelial homeostasis. Our findings support a model in which tissue regeneration originates from upper crypt Fgfbp1+ cells that generate progeny propagating bi-directionally along the crypt-villus axis and serve as a source of Lgr5+ cells in the crypt base.
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Mucosa Intestinal , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Animales , Ratones , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citología , Células Madre/metabolismo , Células Madre/citología , Linaje de la Célula , Regeneración , Proliferación Celular , Células Epiteliales/metabolismo , Células Epiteliales/citología , Ratones Endogámicos C57BL , HomeostasisRESUMEN
Retinitis pigmentosa (RP) is one of the most common forms of hereditary neurodegeneration. It is caused by one or more of at least 3,100 mutations in over 80 genes that are primarily expressed in rod photoreceptors. In RP, the primary rod-death phase is followed by cone death, regardless of the underlying gene mutation that drove the initial rod degeneration. Dampening the oxidation of glycolytic end products in rod mitochondria enhances cone survival in divergent etiological disease models independent of the underlying rod-specific gene mutations. Therapeutic editing of the prolyl hydroxylase domain-containing protein gene (PHD2, also known as Egln1) in rod photoreceptors led to the sustained survival of both diseased rods and cones in both preclinical autosomal-recessive and dominant RP models. Adeno-associated virus-mediated CRISPR-based therapeutic reprogramming of the aerobic glycolysis node may serve as a gene-agnostic treatment for patients with various forms of RP.
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Células Fotorreceptoras Retinianas Bastones , Retinitis Pigmentosa , Animales , Humanos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/terapia , Células Fotorreceptoras Retinianas Conos/metabolismo , Modelos Animales de EnfermedadRESUMEN
Homology-directed repair (HDR) of double-strand DNA breaks (DSBs) is dependent on enzymatic resection of DNA ends by the Mre11/Rad50/Nbs1 complex. DNA resection is triggered by the CtIP/Sae2 protein, which allosterically promotes Mre11-mediated endonuclease DNA cleavage at a position internal to the DSB. Although the mechanics of resection, including the initial endonucleolytic step, are largely conserved in eucaryotes, CtIP and its functional counterpart in Saccharomyces cerevisiae (Sae2) share only a modest stretch of amino acid homology. Nonetheless, this stretch contains two highly conserved phosphorylation sites for cyclin-dependent kinases (T843 in mouse) and the damage-induced ATM/ATR kinases (T855 in mouse), both of which are required for DNA resection. To explore the function of ATM/ATR phosphorylation at Ctip-T855, we generated and analyzed mice expressing the Ctip-T855A mutant. Surprisingly, unlike Ctip-null mice and Ctip-T843A-expressing mice, both of which undergo embryonic lethality, homozygous CtipT855A/T855A mice develop normally. Nonetheless, they are hypersensitive to ionizing radiation, and CtipT855A/T855A mouse embryo fibroblasts from these mice display marked defects in DNA resection, chromosomal stability, and HDR-mediated repair of DSBs. Thus, although ATM/ATR phosphorylation of CtIP-T855 is not required for normal animal development, it enhances CtIP-mediated DNA resection in response to acute stress, such as genotoxin exposure.
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Proteínas Portadoras , Proteínas de Ciclo Celular , Mutágenos , Animales , Ratones , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Fosforilación , Saccharomyces cerevisiae/metabolismoRESUMEN
Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.
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Células Endoteliales , Células Madre Pluripotentes , Ratones , Humanos , Animales , Adulto , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , Pulmón , Blastocisto/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismoRESUMEN
BACKGROUND: Cones are essential for color recognition, high resolution, and central vision; therefore cone death causes blindness. Understanding the pathophysiology of each cell type in the retina is key to developing therapies for retinal diseases. However, studying the biology of cone cells in the rod-dominant mammalian retina is particularly challenging. In this study, we used a bacterial artificial chromosome (BAC) recombineering method to knock in the "CreERT2" sequence into the Gnat2 and Arr3 genes, respectively and generated three novel inducible CreERT2 mice with different cone cell specificities. RESULTS: These models (Gnat2CreERT2, Arr3T2ACreERT2, and Arr3P2ACreERT2) express temporally controllable Cre recombinase that achieves conditional alleles in cone photoreceptors. Cre-LoxP recombination can be induced as early as postnatal day (PD) two upon tamoxifen injection at varying efficiencies, ranging from 10 to 15% in Gnat2CreERT2, 40% in Arr3T2ACreERT2, and 100% in Arr3P2ACreERT2. Notably, knocking in the P2A-CreERT2 cassette does not affect cone cell morphology and functionality. Most cone-phototransduction enzymes, including Opsins, CNGA3, etc. are not altered except for a reduction in the Arr3 transcript. CONCLUSIONS: The Arr3P2ACreERT2 mouse, an inducible cone-specific Cre driver, is a valuable line in studying cone cell biology, function, as well as its relationship with rod and other retinal cells. Moreover, the Cre activity can be induced by delivering tamoxifen intragastrically as early as PD2, which will be useful for studying retinal development or in rapid degenerative mouse models.
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The various functions of the skeleton are influenced by extracellular cues, hormones, and neurotransmitters. One type of neuronal regulation favors bone mass accrual by inhibiting sympathetic nervous system (SNS) activity. This observation raises questions about the transcriptional mechanisms regulating catecholamine synthesis. Using a combination of genetic and pharmacological studies, we found that the histone deacetylase sirtuin 1 (SIRT1) is a transcriptional modulator of the neuronal control of bone mass. Neuronal SIRT1 reduced bone mass by increasing SNS signaling. SIRT1 did so by increasing expression of monoamine oxidase A (MAO-A), a SIRT1 target that reduces brain serotonin levels by inducing its catabolism and by suppressing tryptophan hydroxylase 2 (Tph2) expression and serotonin synthesis in the brain stem. SIRT1 upregulated brain catecholamine synthesis indirectly through serotonin, but did not directly affect dopamine ß hydroxylase (Dbh) expression in the locus coeruleus. These results help us to understand skeletal changes associated with selective serotonin reuptake inhibitors (SSRIs) and may have implications for treating skeletal and metabolic diseases.
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Envejecimiento , Serotonina , Sirtuina 1 , Animales , Ratones , Envejecimiento/genética , Catecolaminas , Monoaminooxidasa/genética , Monoaminooxidasa/metabolismo , Serotonina/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
The current strategy to detect acute injury of kidney tubular cells relies on changes in serum levels of creatinine. Yet serum creatinine (sCr) is a marker of both functional and pathological processes and does not adequately assay tubular injury. In addition, sCr may require days to reach diagnostic thresholds, yet tubular cells respond with programs of damage and repair within minutes or hours. To detect acute responses to clinically relevant stimuli, we created mice expressing Rosa26-floxed-stop uracil phosphoribosyltransferase (Uprt) and inoculated 4-thiouracil (4-TU) to tag nascent RNA at selected time points. Cre-driven 4-TU-tagged RNA was isolated from intact kidneys and demonstrated that volume depletion and ischemia induced different genetic programs in collecting ducts and intercalated cells. Even lineage-related cell types expressed different genes in response to the 2 stressors. TU tagging also demonstrated the transient nature of the responses. Because we placed Uprt in the ubiquitously active Rosa26 locus, nascent RNAs from many cell types can be tagged in vivo and their roles interrogated under various conditions. In short, 4-TU labeling identifies stimulus-specific, cell-specific, and time-dependent acute responses that are otherwise difficult to detect with other technologies and are entirely obscured when sCr is the sole metric of kidney damage.
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Lesión Renal Aguda , ARN , Animales , Perfilación de la Expresión Génica , Ratones , ARN/metabolismoRESUMEN
Prelamin A is a farnesylated precursor of lamin A, a nuclear lamina protein. Accumulation of the farnesylated prelamin A variant progerin, with an internal deletion including its processing site, causes Hutchinson-Gilford progeria syndrome. Loss-of-function mutations in ZMPSTE24, which encodes the prelamin A processing enzyme, lead to accumulation of full-length farnesylated prelamin A and cause related progeroid disorders. Some data suggest that prelamin A also accumulates with physiological aging. Zmpste24-/- mice die young, at â¼20 wk. Because ZMPSTE24 has functions in addition to prelamin A processing, we generated a mouse model to examine effects solely due to the presence of permanently farnesylated prelamin A. These mice have an L648R amino acid substitution in prelamin A that blocks ZMPSTE24-catalyzed processing to lamin A. The LmnaL648R/L648R mice express only prelamin and no mature protein. Notably, nearly all survive to 65 to 70 wk, with â¼40% of male and 75% of female LmnaL648R/L648R mice having near-normal lifespans of 90 wk (almost 2 y). Starting at â¼10 wk of age, LmnaL648R/L648R mice of both sexes have lower body masses than controls. By â¼20 to 30 wk of age, they exhibit detectable cranial, mandibular, and dental defects similar to those observed in Zmpste24-/- mice and have decreased vertebral bone density compared to age- and sex-matched controls. Cultured embryonic fibroblasts from LmnaL648R/L648R mice have aberrant nuclear morphology that is reversible by treatment with a protein farnesyltransferase inhibitor. These novel mice provide a model to study the effects of farnesylated prelamin A during physiological aging.
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Lamina Tipo A/metabolismo , Longevidad , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Progeria/genética , Animales , Sitios de Unión , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Ratones , Mutación , Fenotipo , PrenilaciónRESUMEN
Retinitis pigmentosa (RP) is caused by one of many possible gene mutations. The National Institutes of Health recommends high daily doses of vitamin A palmitate for RP patients. There is a critical knowledge gap surrounding the therapeutic applicability of vitamin A to patients with the different subtypes of the disease. Here, we present a case report of a patient with RP caused by a p.D190N mutation in Rhodopsin (RHO) associated with abnormally high quantitative autofluorescence values after long-term vitamin A supplementation. We investigated the effects of vitamin A treatment strategy on RP caused by the p.D190N mutation in RHO by exposing Rhodopsin p.D190N (RhoD190N/+) and wild-type (WT) mice to experimental vitamin A-supplemented and standard control diets. The patient's case suggests that the vitamin A treatment strategy should be further studied to determine its effect on RP caused by p.D190N mutation in RHO and other mutations. Our mouse experiments revealed that RhoD190N/+ mice on the vitamin A diet exhibited higher levels of autofluorescence and lipofuscin metabolites compared to WT mice on the same diet and isogenic controls on the standard control diet. Vitamin A supplementation diminished photoreceptor function in RhoD190N/+ mice while preserving cone response in WT mice. Our findings highlight the importance of more investigations into the efficacy of clinical treatments like vitamin A for patients with certain genetic subtypes of disease and of genotyping in the precision care of inherited retinal degenerations.
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Degeneración Retiniana , Retinitis Pigmentosa , Animales , Suplementos Dietéticos , Ratones , Mutación , Degeneración Retiniana/genética , Retinitis Pigmentosa/tratamiento farmacológico , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Vitamina ARESUMEN
Pparg, a nuclear receptor, is downregulated in basal subtype bladder cancers that tend to be muscle invasive and amplified in luminal subtype bladder cancers that tend to be non-muscle invasive. Bladder cancers derive from the urothelium, one of the most quiescent epithelia in the body, which is composed of basal, intermediate, and superficial cells. We find that expression of an activated form of Pparg (VP16;Pparg) in basal progenitors induces formation of superficial cells in situ, that exit the cell cycle, and do not form tumors. Expression in basal progenitors that have been activated by mild injury however, results in luminal tumor formation. We find that these tumors are immune deserted, which may be linked to down-regulation of Nf-kb, a Pparg target. Interestingly, some luminal tumors begin to shift to basal subtype tumors with time, down-regulating Pparg and other luminal markers. Our findings have important implications for treatment and diagnosis of bladder cancer.
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PPAR gamma/metabolismo , Transducción de Señal , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/patología , Animales , Biomarcadores de Tumor/metabolismo , Carcinogénesis , Carcinógenos/toxicidad , Diferenciación Celular , Proliferación Celular , Proteína Vmw65 de Virus del Herpes Simple/genética , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Humanos , Ratones , Ratones Transgénicos , PPAR gamma/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/metabolismo , Urotelio/efectos de los fármacos , Urotelio/inmunología , Urotelio/patologíaRESUMEN
Achromatopsia is characterized by amblyopia, photophobia, nystagmus, and color blindness. Previous animal models of achromatopsia have shown promising results using gene augmentation to restore cone function. However, the optimal therapeutic window to elicit recovery remains unknown. Here, we attempted two rounds of gene augmentation to generate recoverable mouse models of achromatopsia including a Cnga3 model with a knock-in stop cassette in intron 5 using Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) and targeted embryonic stem (ES) cells. This model demonstrated that only 20% of CNGA3 levels in homozygotes derived from target ES cells remained, as compared to normal CNGA3 levels. Despite the low percentage of remaining protein, the knock-in mouse model continued to generate normal cone phototransduction. Our results showed that a small amount of normal CNGA3 protein is sufficient to form "functional" CNG channels and achieve physiological demand for proper cone phototransduction. Thus, it can be concluded that mutating the Cnga3 locus to disrupt the functional tetrameric CNG channels may ultimately require more potent STOP cassettes to generate a reversible achromatopsia mouse model. Our data also possess implications for future CNGA3-associated achromatopsia clinical trials, whereby restoration of only 20% functional CNGA3 protein may be sufficient to form functional CNG channels and thus rescue cone response.
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Defectos de la Visión Cromática/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Modelos Animales de Enfermedad , Edición Génica , Mutación , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Defectos de la Visión Cromática/metabolismo , Técnicas de Sustitución del Gen , Ratones , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/fisiologíaRESUMEN
Quiescent neural stem cells (NSCs) in the adult mouse ventricular-subventricular zone (V-SVZ) undergo activation to generate neurons and some glia. Here we show that platelet-derived growth factor receptor beta (PDGFRß) is expressed by adult V-SVZ NSCs that generate olfactory bulb interneurons and glia. Selective deletion of PDGFRß in adult V-SVZ NSCs leads to their release from quiescence, uncovering gliogenic domains for different glial cell types. These domains are also recruited upon injury. We identify an intraventricular oligodendrocyte progenitor derived from NSCs inside the brain ventricles that contacts supraependymal axons. Together, our findings reveal that the adult V-SVZ contains spatial domains for gliogenesis, in addition to those for neurogenesis. These gliogenic NSC domains tend to be quiescent under homeostasis and may contribute to brain plasticity.
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Células Madre Adultas/fisiología , Ventrículos Cerebrales/fisiología , Ventrículos Laterales/fisiología , Células-Madre Neurales/fisiología , Neuroglía/fisiología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Astrocitos/citología , Astrocitos/fisiología , Axones/fisiología , Diferenciación Celular , División Celular , Ventrículos Cerebrales/citología , Epéndimo/citología , Epéndimo/fisiología , Femenino , Perfilación de la Expresión Génica , Homeostasis , Ventrículos Laterales/citología , Masculino , Ratones , Neurogénesis , Bulbo Olfatorio/citología , Bulbo Olfatorio/fisiología , Oligodendroglía/citología , Oligodendroglía/fisiología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
Lactate metabolism has been shown to have increasingly important implications in cellular functions as well as in the development and pathophysiology of disease. The various roles as a signaling molecule and metabolite have led to interest in establishing a new method to detect lactate changes in live cells. Here we report our development of a genetically encoded metabolic indicator specifically for probing lactate (GEM-IL) based on superfolder fluorescent proteins and mutagenesis. With improvements in its design, specificity, and sensitivity, GEM-IL allows new applications compared with the previous lactate indicators, Laconic and Green Lindoblum. We demonstrate the functionality of GEM-IL to detect differences in lactate changes in human oncogenic neural progenitor cells and mouse primary ventricular myocytes. The development and application of GEM-IL show promise for enhancing our understanding of lactate dynamics and roles.
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Ácido Láctico , Células-Madre Neurales , Humanos , Animales , Ratones , Ácido Láctico/metabolismo , Células-Madre Neurales/metabolismo , Miocitos Cardíacos/metabolismo , Transducción de SeñalRESUMEN
Optogenetic genome engineering tools enable spatiotemporal control of gene expression and provide new insight into biological function. Here, we report the new version of genetically encoded photoactivatable (PA) Cre recombinase, PA-Cre 3.0. To improve PA-Cre technology, we compare light-dimerization tools and optimize for mammalian expression using a CAG promoter, Magnets, and 2A self-cleaving peptide. To prevent background recombination caused by the high sequence similarity in the dimerization domains, we modify the codons for mouse gene targeting and viral production. Overall, these modifications significantly reduce dark leak activity and improve blue-light induction developing our new version, PA-Cre 3.0. As a resource, we have generated and validated AAV-PA-Cre 3.0 as well as two mouse lines that can conditionally express PA-Cre 3.0. Together these new tools will facilitate further biological and biomedical research.
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Integrasas/metabolismo , Recombinación Genética/genética , Animales , Codón/genética , Ingeniería Genética/métodos , Integrasas/genética , Luz , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Optogenética , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/efectos de la radiación , Recombinación Genética/efectos de la radiaciónRESUMEN
Millions of people worldwide with incurable end-stage lung disease die because of inadequate treatment options and limited availability of donor organs for lung transplantation1. Current bioengineering strategies to regenerate the lung have not been able to replicate its extraordinary cellular diversity and complex three-dimensional arrangement, which are indispensable for life-sustaining gas exchange2,3. Here we report the successful generation of functional lungs in mice through a conditional blastocyst complementation (CBC) approach that vacates a specific niche in chimeric hosts and allows for initiation of organogenesis by donor mouse pluripotent stem cells (PSCs). We show that wild-type donor PSCs rescued lung formation in genetically defective recipient mouse embryos unable to specify (due to Ctnnb1cnull mutation) or expand (due to Fgfr2cnull mutation) early respiratory endodermal progenitors. Rescued neonates survived into adulthood and had lungs functionally indistinguishable from those of wild-type littermates. Efficient chimera formation and lung complementation required newly developed culture conditions that maintained the developmental potential of the donor PSCs and were associated with global DNA hypomethylation and increased H4 histone acetylation. These results pave the way for the development of new strategies for generating lungs in large animals to enable modeling of human lung disease as well as cell-based therapeutic interventions4-6.
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Enfermedades Pulmonares/terapia , Pulmón/crecimiento & desarrollo , Células Madre Pluripotentes/metabolismo , Regeneración/genética , Acilación/genética , Animales , Blastocisto/metabolismo , Diferenciación Celular/genética , Metilación de ADN/genética , Modelos Animales de Enfermedad , Histonas/genética , Humanos , Pulmón/patología , Enfermedades Pulmonares/patología , Ratones , Organogénesis/genética , Células Madre Pluripotentes/trasplante , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , beta Catenina/genéticaRESUMEN
Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/ß-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.
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Proteínas de Homeodominio/metabolismo , Tejido Parenquimatoso/fisiología , Diente/citología , Diente/embriología , Adolescente , Animales , Femenino , Proteínas de Homeodominio/genética , Humanos , Incisivo/citología , Incisivo/embriología , Ratones Endogámicos , Tercer Molar/citología , Técnicas de Cultivo de Órganos , Tejido Parenquimatoso/citología , Embarazo , Regiones Promotoras Genéticas , Regeneración , Células del Estroma/fisiología , Porcinos , Factor A de Crecimiento Endotelial Vascular/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismoRESUMEN
D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light.
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Células Fotorreceptoras Retinianas Bastones/metabolismo , Retinitis Pigmentosa/patología , Rodopsina/genética , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Electrorretinografía , Técnicas de Sustitución del Gen , Glicosilación , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Estructura Terciaria de Proteína , Retina/metabolismo , Retina/patología , Retinitis Pigmentosa/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Alineación de SecuenciaRESUMEN
Genomic approaches to developing new diagnostic and therapeutic strategies in retinal dystrophies are among the most advanced applications of genetics (Tsang SH, Gouras P (1996) Molecular physiology and pathology of the retina. In: Duane TD, Tasman W, Jaeger AE (eds) Duane's clinical opthalmology. Lippincott-Raven, Philadelphia). The notion that "nothing can be done" for patients with retinal dystrophies is no longer true. Electrophysiological testing and autofluorescence imaging help to diagnose and predict the patient's course of disease. Better phenotyping can contribute to better-directed, cost-efficient genotyping. Combining fundoscopy, autofluorescent imaging, and electrophysiological testing is essential in approaching patients with retinal dystrophies. Emerging are new gene-based treatments for these devastating conditions.