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Inhibitory interneurons regulate cortical circuit activity, and their dysfunction has been implicated in autism spectrum disorder (ASD). 16p11.2 microdeletions are genetically linked to 1% of ASD cases. However, few studies investigate the effects of this microdeletion on interneuron development. Using ventral telencephalic organoids derived from human induced pluripotent stem cells, we have investigated the effect of this microdeletion on organoid size, progenitor proliferation and organisation into neural rosettes, ganglionic eminence marker expression at early developmental timepoints, and expression of the neuronal marker NEUN at later stages. At early stages, deletion organoids exhibited greater variations in size with concomitant increases in relative neural rosette area and the expression of the ventral telencephalic marker COUPTFII, with increased variability in these properties. Cell cycle analysis revealed an increase in total cell cycle length caused primarily by an elongated G1 phase, the duration of which also varied more than normal. At later stages, deletion organoids increased their NEUN expression. We propose that 16p11.2 microdeletions increase developmental variability and may contribute to ASD aetiology by lengthening the cell cycle of ventral progenitors, promoting premature differentiation into interneurons.
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Transtorno do Espectro Autista , Células-Tronco Pluripotentes Induzidas , Humanos , Transtorno do Espectro Autista/metabolismo , Telencéfalo , Neurônios/metabolismo , Interneurônios/metabolismo , OrganoidesRESUMO
BACKGROUND: Autism spectrum condition or 'autism' is associated with numerous genetic risk factors including the polygenic 16p11.2 microdeletion. The balance between excitatory and inhibitory neurons in the cerebral cortex is hypothesised to be critical for the aetiology of autism making improved understanding of how risk factors impact on the development of these cells an important area of research. In the current study we aim to combine bioinformatics analysis of human foetal cerebral cortex gene expression data with anatomical and electrophysiological analysis of a 16p11.2+/- rat model to investigate how genetic risk factors impact on inhibitory neuron development. METHODS: We performed bioinformatics analysis of single cell transcriptomes from gestational week (GW) 8-26 human foetal prefrontal cortex and anatomical and electrophysiological analysis of 16p11.2+/- rat cerebral cortex and hippocampus at post-natal day (P) 21. RESULTS: We identified a subset of human interneurons (INs) first appearing at GW23 with enriched expression of a large fraction of risk factor transcripts including those expressed from the 16p11.2 locus. This suggests the hypothesis that these foetal INs are vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2+/- rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells. LIMITATIONS: The human foetal gene expression data was acquired from cerebral cortex between gestational week (GW) 8 to 26. We cannot draw inferences about potential vulnerabilities to genetic autism risk factors for cells not present in the developing cerebral cortex at these stages. The analysis 16p11.2+/- rat phenotypes reported in the current study was restricted to 3-week old (P21) animals around the time of weaning and to a single interneuron cell-type while in human 16p11.2 microdeletion carriers symptoms likely involve multiple cell types and manifest in the first few years of life and on into adulthood. CONCLUSIONS: We have identified developing interneurons in human foetal cerebral cortex as potentially vulnerable to monogenic autism risk factors and the 16p11.2 microdeletion and report interneuron phenotypes in post-natal 16p11.2+/- rats.
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Transtorno Autístico , Interneurônios , Humanos , Ratos , Animais , Transtorno Autístico/genética , Neurônios , Córtex Cerebral , Fatores de RiscoRESUMO
BACKGROUND: While Pace of Life Syndrome predicts behavioural differences between individuals with differential growth and survival, testing these predictions in nature is challenging due to difficulties with measuring individual behaviour in the field. However, recent advances in acoustic telemetry technology have facilitated measurements of individual behaviour at scales not previously possible in aquatic ecosystems. METHODS: Using a Walleye (Sander vitreus) population inhabiting Black Bay, Lake Superior, we examine whether life history characteristics differ between more and less mobile individuals as predicted by Pace of Life Syndrome. We tracked the movement of 192 individuals from 2016 to 2019 using an acoustic telemetry study, relating patterns in annual migratory behaviour to individual growth, and seasonal changes in optimal thermal-optical habitat. RESULTS: We observed two consistent movement patterns in our study population-migratory individuals left Black Bay during late summer to early fall before returning to the bay, whereas residents remained within the bay year-round. The average maximum length of migrant Walleye was 5.5 cm longer than residents, and the sex ratios of Walleye caught during fall surveys was increasingly female-biased towards the mouth of Black Bay, suggesting that a majority of migrants were females. Further, Walleye occupancy outside of Black Bay was positively associated with increasing thermal-optical habitat. CONCLUSIONS: Walleye in Black Bay appear to conform to Pace of Life Syndrome, with migrant individuals gaining increased fitness through increased maximum size, which, given size-dependent fecundity in this species, likely results in greater reproductive success (via greater egg deposition vs. non-migrants). Further, apparent environmental (thermal) controls on migration suggest that migratory Walleye (more so than residents) may be more sensitive to changing environmental conditions (e.g., warming climate) than residents.
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Pax6 is a well-known regulator of early neuroepithelial progenitor development. Its constitutive loss has a particularly strong effect on the developing prethalamus, causing it to become extremely hypoplastic. To overcome this difficulty in studying the long-term consequences of Pax6 loss for prethalamic development, we used conditional mutagenesis to delete Pax6 at the onset of neurogenesis and studied the developmental potential of the mutant prethalamic neurons in vitro. We found that Pax6 loss affected their rates of neurite elongation, the location and length of their axon initial segments, and their electrophysiological properties. Our results broaden our understanding of the long-term consequences of Pax6 deletion in the developing mouse forebrain, suggesting that it can have cell-autonomous effects on the structural and functional development of some neurons.
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Proteínas de Homeodomínio , Fatores de Transcrição Box Pareados , Animais , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Camundongos , Neurônios/metabolismo , Fator de Transcrição PAX6/genética , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismoRESUMO
OBJECTIVE: The inclusion of peer-delivered services in organizations providing behavioral health care has significantly increased in recent years, and substantial resources are being directed toward implementing recovery-oriented mental health services using peer-provided programs. Previous research found that participants in such programs have improved recovery outcomes. While there are demonstrated positive associations between recovery outcomes and peer-provided services, there is limited research on the effectiveness of specific peer-provided interventions. Veteran X is a peer-led program developed in the Department of Veterans Affairs in which participants serve as a recovery team for a fictitious Veteran who faces numerous social and mental health issues. This study compared the effectiveness of the Veteran X program with treatment as usual on measures of recovery wellbeing, symptoms and functioning, and risk and protective factors for substance use disorders. METHODS: Participants were recruited (N = 80) over a period of ten months, and had self-selected into treatment as usual (TAU, N = 37), or treatment as usual plus Veteran X (N = 43). RESULTS: No baseline differences were found on the pretest measures. Both groups improved on all measures after 60 days of participation, however Veteran X participants improved significantly more than TAU participants on the measures of recovery wellbeing and symptoms and functioning. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: The results of this study appear to support the positive contribution of the Veteran X program in improving recovery wellbeing and symptoms and functioning among participating veterans. (PsycInfo Database Record (c) 2021 APA, all rights reserved).
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Recuperação da Saúde Mental , Serviços de Saúde Mental , Transtornos Relacionados ao Uso de Substâncias , Veteranos , Humanos , Grupo AssociadoRESUMO
The interaction between cell surface heparan sulphate and diffusible ligands such as FGFs is of vital importance for downstream signaling, however, there are few techniques that can be used to investigate this binding event. The ligand and carbohydrate engagement (LACE) assay is a powerful tool which can be used to probe the molecular interaction between heparan sulphate and diffusible ligands and can detect changes in binding that may occur following genetic or pharmacological intervention. In this protocol we describe an FGF17:FGFR1 LACE assay performed on embryonic mouse brain tissue. We also describe the method we have used to quantify changes in fluorescent LACE signal in response to altered HS sulphation.
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The 593 kbp 16p11.2 copy number variation (CNV) affects the gene dosage of 29 protein coding genes, with heterozygous 16p11.2 microduplication or microdeletion implicated in about 1% of autism spectrum disorder (ASD) cases. The 16p11.2 CNV is frequently associated with macrocephaly or microcephaly indicating early defects of neurogenesis may contribute to subsequent ASD symptoms, but it is unknown which 16p11.2 transcripts are expressed in progenitors and whose levels are likely, therefore, to influence neurogenesis. Analysis of human fetal gene expression data revealed that KIF22, ALDOA, HIRIP3, PAGR1, and MAZ transcripts are expressed in neural progenitors with ALDOA and KIF22 significantly enriched compared to post-mitotic cells. To investigate the possible roles of ALDOA and KIF22 proteins in human cerebral cortex development we used immunohistochemical staining to describe their expression in late first and early second trimester human cerebral cortex. KIF22 protein is restricted to proliferating cells with its levels increasing during the cell cycle and peaking at mitosis. ALDOA protein is expressed in all cell types and does not vary with cell-cycle phase. Our expression analysis suggests the hypothesis that altered neurogenesis in the cerebral cortex contributes to ASD in 16p11.2 CNV patients.
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Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Córtex Cerebral/metabolismo , Regulação da Expressão Gênica/genética , Adulto , Ciclo Celular , Córtex Cerebral/crescimento & desenvolvimento , Variações do Número de Cópias de DNA , Feminino , Feto/metabolismo , Deleção de Genes , Duplicação Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Neurais/metabolismo , GravidezRESUMO
Barrier removal is a recognized solution for reversing river fragmentation, but restoring connectivity can have consequences for both desirable and undesirable species, resulting in a connectivity conundrum. Selectively passing desirable taxa while restricting the dispersal of undesirable taxa (selective connectivity) would solve many aspects of the connectivity conundrum. Selective connectivity is a technical challenge of sorting an assortment of things. Multiattribute sorting systems exist in other fields, although none have yet been devised for freely moving organisms within a river. We describe an approach to selective fish passage that integrates ecology and biology with engineering designs modeled after material recycling processes that mirror the stages of fish passage: approach, entry, passage, and fate. A key feature of this concept is the integration of multiple sorting processes each targeting a specific attribute. Leveraging concepts from other sectors to improve river ecosystem function may yield fast, reliable solutions to the connectivity conundrum.
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Organisms living in environments with oscillating temperatures may rely on plastic traits to sustain thermal tolerance during high temperature periods. Phenotypic plasticity in critical thermal maximum (CTmax) is a powerful thermoregulative strategy that enables organisms to adjust CTmax when ambient temperatures do not match thermal preference. Given that global temperatures are increasing at an unprecedented rate, identifying factors that affect the plastic response in CTmax can help predict how organisms are likely to respond to changes in their thermal landscape. Using an experimental thermal chamber in the field, we investigated the effect of short-term acclimation on the CTmax and thermal safety margin (TSM) of wild-caught redside dace, Clinostomus elongatus, (n = 197) in a northern population in Two Tree River, Ontario. Streamside CTmax trials were used to identify the maximum temperature at which redside dace maintain equilibrium, providing a powerful tool for understanding how thermal stress affects individual performance. CTmax and TSM of redside dace were sensitive to changes in temperature, regardless of season, suggesting that temperature pulses caused by climate change or urban activities can impose negative fitness consequences year round. Interestingly, an individual's recent thermal history was more influential to its thermal tolerance than the current ambient water temperature. While the CTmax of redside dace increased with body size, the effect of body size on TSM remains unclear based on our models. The results provide insight into the thermal performance of redside dace that, to date, has been difficult to assess due to the species' rarity and lack of suitable streamside protocols.
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Organotypic slice culture is a powerful technique for exploring the embryonic development of the mammalian brain. In this protocol we describe a basic slice culture technique we have used for two sets of experiments: axon guidance transplant assays and bead culture assays.
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Heparan sulfate (HS) is a cell surface and extracellular matrix carbohydrate extensively modified by differential sulfation. HS interacts physically with canonical fibroblast growth factor (FGF) proteins that signal through the extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) pathway. At the embryonic mouse telencephalic midline, FGF/ERK signaling drives astroglial precursor somal translocation from the ventricular zone of the corticoseptal boundary (CSB) to the induseum griseum (IG), producing a focus of Slit2-expressing astroglial guidepost cells essential for interhemispheric corpus callosum (CC) axon navigation. Here, we investigated the cell and molecular function of a specific form of HS sulfation, 2-O HS sulfation catalyzed by the enzyme Hs2st, in midline astroglial development and in regulating FGF protein levels and interaction with HS. Hs2st-/- embryos of either sex exhibit a grossly enlarged IG due to precocious astroglial translocation and conditional Hs2st mutagenesis and ex vivo culture experiments show that Hs2st is not required cell autonomously by CC axons or by the IG astroglial cell lineage, but rather acts non-cell autonomously to suppress the transmission of translocation signals to astroglial precursors. Rescue of the Hs2st-/- astroglial translocation phenotype by pharmacologically inhibiting FGF signaling shows that the normal role of Hs2st is to suppress FGF-mediated astroglial translocation. We demonstrate a selective action of Hs2st on FGF protein by showing that Hs2st (but not Hs6st1) normally suppresses the levels of Fgf17 protein in the CSB region in vivo and use a biochemical assay to show that Hs2st (but not Hs6st1) facilitates a physical interaction between the Fgf17 protein and HS.SIGNIFICANCE STATEMENT We report a novel non-cell-autonomous mechanism regulating cell signaling in developing brain. Using the developing mouse telencephalic midline as an exemplar, we show that the specific sulfation modification of the cell surface and extracellular carbohydrate heparan sulfate (HS) performed by Hs2st suppresses the supply of translocation signals to astroglial precursors by a non-cell-autonomous mechanism. We further show that Hs2st modification selectively facilitates a physical interaction between Fgf17 and HS and suppresses Fgf17 protein levels in vivo, strongly suggesting that Hs2st acts selectively on Fgf17 signaling. HS interacts with many signaling proteins potentially encoding numerous selective interactions important in development and disease, so this class of mechanism may apply more broadly to other biological systems.
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Astrócitos/metabolismo , Heparitina Sulfato/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Células-Tronco Neurais/metabolismo , Prosencéfalo/enzimologia , Sulfatos/metabolismo , Sulfotransferases/fisiologia , Animais , Biomarcadores , Linhagem da Célula , Movimento Celular , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Homeodomínio/análise , Camundongos , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/fisiologia , Prosencéfalo/citologia , Prosencéfalo/embriologia , Sulfotransferases/deficiência , Fatores de Transcrição/análiseRESUMO
Our aim was to study the mechanisms that contribute to the development of discrete thalamic nuclei during mouse embryogenesis (both sexes included). We characterized the expression of the transcription factor coding gene Zic4 and the distribution of cells that expressed Zic4 in their lineage. We used genetic fate mapping to show that Zic4-lineage cells mainly contribute to a subset of thalamic nuclei, in particular the lateral geniculate nuclei (LGNs), which are crucial components of the visual pathway. We observed that almost all Zic4-lineage diencephalic progenitors express the transcription factor Pax6 at variable location-dependent levels. We used conditional mutagenesis to delete either one or both copies of Pax6 from Zic4-lineage cells. We found that Zic4-lineage cells carrying either homozygous or heterozygous loss of Pax6 contributed in abnormally high numbers to one or both of the main lateral geniculate nuclei (LGNs). This could not be attributed to a change in cell production and was likely due to altered sorting of thalamic cells. Our results indicate that positional information encoded by the levels of Pax6 in diencephalic progenitors is an important determinant of the eventual locations of their daughter cells.
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Regulação da Expressão Gênica no Desenvolvimento/genética , Heterozigoto , Proteínas de Homeodomínio/genética , Homozigoto , Fator de Transcrição PAX6/genética , Fatores de Transcrição/genética , Animais , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Núcleos Talâmicos , Fatores de Transcrição/metabolismo , Vias Visuais/fisiologiaRESUMO
GABAergic interneurons (GABA, γ-aminobutyric acid) regulate neural-circuit activity in the mammalian cerebral cortex. These cortical interneurons are structurally and functionally diverse. Here, we use single-cell transcriptomics to study the origins of this diversity in the mouse. We identify distinct types of progenitor cells and newborn neurons in the ganglionic eminences, the embryonic proliferative regions that give rise to cortical interneurons. These embryonic precursors show temporally and spatially restricted transcriptional patterns that lead to different classes of interneurons in the adult cerebral cortex. Our findings suggest that shortly after the interneurons become postmitotic, their diversity is already patent in their diverse transcriptional programs, which subsequently guide further differentiation in the developing cortex.
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Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Neurônios GABAérgicos/classificação , Interneurônios/classificação , Neurogênese/genética , Animais , Embrião de Mamíferos/citologia , Feminino , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Perfilação da Expressão Gênica , Interneurônios/citologia , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Mitose/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Análise de Célula Única , Transcrição Gênica , TranscriptomaRESUMO
Fibroblast growth factor (FGF) morphogen signalling through the evolutionarily ancient extracellular signalling-regulated kinase/mitogen activated protein kinase (ERK/MAPK) pathway recurs in many neural and non-neural developmental contexts, and understanding the mechanisms that regulate FGF/ERK function are correspondingly important. The glycosaminoglycan heparan sulphate (HS) binds to FGFs and exists in an enormous number of differentially sulphated forms produced by the action of HS modifying enzymes, and so has the potential to present an extremely large amount of information in FGF/ERK signalling. Although there have been many studies demonstrating that HS is an important regulator of FGF function, experimental evidence on the role of the different HS modifying enzymes on FGF gradient formation has been lacking until now. We challenged ex vivo developing mouse neural tissue, in which HS had either been enzymatically removed by heparanase treatment or lacking either the HS modifying enzymes Hs2st (Hs2st-/- tissue) or Hs6st1 (Hs6st1-/- tissue), with exogenous Fgf8 to gain insight on how HS and the function of these two HS modifying enzymes impacts on Fgf8 gradient formation from an exogenously supplied source of Fgf8 protein. We discover that two different HS modifying enzymes, Hs2st and Hs6st1, indeed differentially modulate the properties of emerging Fgf8 protein concentration gradients and the Erk signalling output in response to Fgf8 in living tissue in ex vivo cultures. Both Hs2st and Hs6st1 are required for stable Fgf8 gradients to form as rapidly as they do in wild-type tissue while only Hs6st1 has a significant effect on suppressing the levels of Fgf8 protein in the gradient compared to wild type. Next we show that Hs2st and Hs6st1 act to antagonise and agonise the Erk signalling in response to Fgf8 protein, respectively, in ex vivo cultures of living tissue. Examination of endogenous Fgf8 protein and Erk signalling outputs in Hs2st-/- and Hs6st1-/- embryos suggests that our ex vivo findings have physiological relevance in vivo Our discovery identifies a new class of mechanism to tune Fgf8 function by regulated expression of Hs2st and Hs6st1 that is likely to have broader application to the >200 other signalling proteins that interact with HS and their function in neural development and disease.
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During vertebrate eye morphogenesis, a transient fissure forms at its inferior part, known as the optic fissure. This will gradually close, giving rise to a healthy, spherical optic cup. Failure of the optic fissure to close gives rise to an ocular disorder known as coloboma. During this developmental process, Foxg1 is expressed in the optic neuroepithelium, with highest levels of expression in the nasal optic stalk. Foxg1-/- mutant mice have microphthalmic eyes with a large ventral coloboma. We found Wnt8b expression upregulated in the Foxg1-/- optic stalk and hypothesized that, similar to what is observed in telencephalic development, Foxg1 directs development of the optic neuroepithelium through transcriptional suppression of Wnt8b To test this, we generated Foxg1-/-;Wnt8b-/- double mutants of either sex and found that the morphology of the optic cup and stalk and the closure of the optic fissure were substantially rescued in these embryos. This rescue correlates with restored Pax2 expression in the anterior tip of the optic fissure. In addition, although we do not find evidence implicating altered proliferation in the rescue, we observe a significant increase in apoptotic cell density in Foxg1-/-;Wnt8b-/- double mutants compared with the Foxg1-/- single mutant. Upregulation of Wnt/ß-catenin target molecules in the optic cup and stalk may underlie the molecular and morphological defects in the Foxg1-/- mutant. Our results show that proper optic fissure closure relies on Wnt8b suppression by Foxg1 in the nasal optic stalk to maintain balanced apoptosis and Pax2 expression in the nasal and temporal edges of the fissure.SIGNIFICANCE STATEMENT Coloboma is an ocular disorder that may result in a loss of visual acuity and accounts for â¼10% of childhood blindness. It results from errors in the sealing of the optic fissure (OF), a transient structure at the bottom of the eye. Here, we investigate the colobomatous phenotype of the Foxg1-/- mutant mouse. We identify upregulated expression of Wnt8b in the optic stalk of Foxg1-/- mutants before OF closure initiates. Foxg1-/-;Wnt8b-/- double mutants show a substantial rescue of the Foxg1-/- coloboma phenotype, which correlates with a rescue in molecular and cellular defects of Foxg1-/- mutants. Our results unravel a new role of Foxg1 in promoting OF closure providing additional knowledge about the molecules and cellular mechanisms underlying coloboma formation.
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Fatores de Transcrição Forkhead/deficiência , Proteínas do Tecido Nervoso/deficiência , Disco Óptico/embriologia , Disco Óptico/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/biossíntese , Animais , Feminino , Fatores de Transcrição Forkhead/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Gravidez , Fatores de Transcrição/deficiência , Fatores de Transcrição/efeitos dos fármacos , Proteínas Wnt/genéticaRESUMO
BACKGROUND: The tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/- hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3. RESULTS: Although TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/- hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/- hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex. CONCLUSIONS: Mosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing.
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Linhagem da Célula , Camundongos Transgênicos/genética , Mosaicismo , Transgenes/genética , Proteínas tau/genética , Animais , Expressão Gênica , CamundongosRESUMO
In this issue of Neuron, Rani et al. (2016) address important questions about the mechanisms of cerebral cortical evolution. They describe how a primate-specific long non-coding RNA titrates the levels of a microRNA that regulates an ancient signaling pathway controlling neuronal numbers.
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Córtex Cerebral , DNA Intergênico , Animais , Humanos , MicroRNAs/genética , Neurônios , RNA Longo não CodificanteRESUMO
Heparan sulfate (HS) is a linear carbohydrate composed of polymerized uronate-glucosamine disaccharide units that decorates cell surface and secreted glycoproteins in the extracellular matrix. In mammals HS is subjected to differential sulfation by fifteen different heparan sulfotransferase (HST) enzymes of which Hs2st uniquely catalyzes the sulfation of the 2-O position of the uronate in HS. HS sulfation is postulated to be important for regulation of signaling pathways by facilitating the interaction of HS with signaling proteins including those of the Fibroblast Growth Factor (Fgf) family which signal through phosphorylation of extracellular signal-regulated kinases Erk1/2. In the developing mouse telencephalon Fgf2 signaling regulates proliferation and neurogenesis. Loss of Hs2st function phenocopies the thinned cerebral cortex of mutant mice in which Fgf2 or Erk1/2 function are abrogated, suggesting the hypothesis that 2-O-sulfated HS structures play a specific role in Fgf2/Erk signaling pathway in this context in vivo. This study investigated the molecular role of 2-O sulfation in Fgf2/Erk signaling in the developing telencephalic midline midway through mouse embryogenesis at E12.5. We examined the expression of Hs2st, Fgf2, and Erk1/2 activity in wild-type and Hs2st-/- mice. We found that Hs2st is expressed at high levels at the midline correlating with high levels of Erk1/2 activation and Erk1/2 activation was drastically reduced in the Hs2st-/- mutant at the rostral telencephalic midline. We also found that 2-O sulfation is specifically required for the binding of Fgf2 protein to Fgfr1, its major cell-surface receptor at the rostral telencephalic midline. We conclude that 2-O sulfated HS structures generated by Hs2st are needed to form productive signaling complexes between HS, Fgf2 and Fgfr1 that activate Erk1/2 at the midline. Overall, our data suggest the interesting possibility that differential expression of Hs2st targets the rostral telencephalic midline for high levels of Erk signaling by increasing the sensitivity of cells to an Fgf2 signal that is rather more widespread.
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Embrião de Mamíferos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Sulfotransferases/fisiologia , Telencéfalo/metabolismo , Animais , Western Blotting , Embrião de Mamíferos/citologia , Feminino , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Transdução de Sinais , Telencéfalo/citologiaRESUMO
INTRODUCTION: There is limited understanding of the type and extent of maternal postures that midwives should encourage or support during labor. The aims of this study were to identify a set of postures and movements commonly seen during labor, to develop an activity monitoring system for use during labor, and to validate this system design. METHODS: Volunteer student midwives simulated maternal activity during labor in a laboratory setting. Participants (N = 15) wore monitors adhered to the left thigh and left shank, and adopted 13 common postures of laboring women for 3 minutes each. Simulated activities were recorded using a video camera. Postures and movements were coded from the video, and statistical analysis conducted of agreement between coded video data and outputs of the activity monitoring system. RESULTS: Excellent agreement between the 2 raters of the video recordings was found (Cohen's κ = 0.95). Both sensitivity and specificity of the activity monitoring system were greater than 80% for standing, lying, kneeling, and sitting (legs dangling). DISCUSSION: This validated system can be used to measure elected activity of laboring women and report on effects of postures on length of first stage, pain experience, birth satisfaction, and neonatal condition. This validated maternal posture-monitoring system is available as a reference-and for use by researchers who wish to develop research in this area.
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Trabalho de Parto , Tocologia/métodos , Monitorização Fisiológica , Movimento , Postura , Adolescente , Adulto , Feminino , Humanos , Dor do Parto , Pessoa de Meia-Idade , Monitorização Fisiológica/normas , Atividade Motora , Gravidez , Resultado da Gravidez , Reprodutibilidade dos Testes , Gravação em Vídeo , Adulto JovemRESUMO
The corpus callosum (CC) connects the left and right cerebral hemispheres in mammals and its development requires intercellular communication at the telencephalic midline mediated by signaling proteins. Heparan sulfate (HS) is a sulfated polysaccharide that decorates cell surface and extracellular matrix proteins and regulates the biological activity of numerous signaling proteins via sugar-protein interactions. HS is subject to regulated enzymatic sulfation and desulfation and an attractive, although not proven, hypothesis is that the biological activity of HS is regulated by a sugar sulfate code. Mutant mouse embryos lacking the heparan sulfotransferases Hs2st or Hs6st1 have severe CC phenotypes and form Probst bundles of noncrossing axons flanking large tangles of midline glial processes. Here, we identify a precocious accumulation of Sox9-expressing glial cells in the indusium griseum region and a corresponding depletion at the glial wedge associated with the formation of Probst bundles along the rostrocaudal axis in both mutants. Molecularly, we found a surprising hyperactivation of Erk signaling in Hs2st(-/-) (2-fold) and Hs6st1(-/-) (6-fold) embryonic telencephalon that was most striking at the midline, where Erk signaling is lowest in wild-types, and a 2-fold increase in Fgf8 protein levels in Hs6st1(-/-) embryos that could underpin Erk hyperactivation and excessive glial movement to the indusium griseum. The tightly linked Hs6st1(-/-) CC glial and axonal phenotypes can be rescued by genetic or pharmacological suppression of Fgf8/Erk axis components. Overall, our data fit a model in which Hs2st and Hs6st1 normally generate conditions conducive to CC development by generating an HS-containing environment that keeps Erk signaling in check.