Uso di cellule staminali embrionali per lo studio in vitro dei meccanismi molecolari di regionalizzazione della corteccia cerebrale

Durante lo sviluppo della corteccia gradienti di morfogeni attraversano il neuroepitelio suddividendolo in distinti domini funzionali. L'informazione contenuta in tali gradienti è successivamente tradotta nell'attivazione di specifiche costellazioni di geni nella progenie postmitotica. Una classe di morfogeni particolarmente importante nel patterning corticale è quella dei fattori FGF. Infatti l'espressione di FGF8 in posizione mediale anteriore nel telencefalo di topo in via di sviluppo è necessaria per la corretta specificazione delle strutture corticali più rostrali. Anche COUP-TF1, un gene essenziale per la specificazione dell'identità corticale caudale, è espresso nel neuroepitelio neocorticale con un gradiente caudo-rostrale complementare a quello di FGF8. Inoltre l'espressione di questi due fattori è mutualmente regolata; pertanto diminuendo l'espressione dell'uno, l'altro risponde espandendo il proprio dominio d'espressione. \\In questo lavoro abbiamo messo appunto un modello per lo studio in vitro del patterning corticale mediante utilizzo di neuroni ottenuti dal differenziamento di cellule staminali embrionali di topo. Abbiamo quindi utilizzato tale modello per caratterizzare il meccanismo di regolazione di COUP-TF1 da parte di FGF8. Abbiamo dimostrato come il trattamento tardivo con FGF8 determini una riduzione nell'espressione di COUP-TF1 senza alterare l'identità corticale dei neuroni generati. Inoltre tale repressione sembra essere mediata -almeno in parte- a livello post-trascrizionale, poiché l'overespressione della 3'UTR di COUP-TF1 scherma il trascritto endogeno dalla repressione traduzionale mediata da FGF8. Infine abbiamo identificato alcuni microRNA upregolati dal trattamento con FGF8 e possibili candidati come repressori traduzionali di COUP-TF1

The microRNA miR-21 Is a Mediator of FGF8 Action on Cortical COUP-TFI Translation

The morphogen FGF8 plays a pivotal role in neocortical area patterning through its inhibitory effect on COUP-TFI/Nr2f1 anterior expression, but its mechanism of action is poorly understood. We established an in vitro model of mouse embryonic stem cell corticogenesis in which COUP-TFI protein expression is inhibited by the activation of FGF8 in a time window corresponding to cortical area patterning. Interestingly, overexpression of the COUP-TFI 3′UTR reduces the inhibitory effect of FGF8 on COUP-TFI translation. FGF8 induces the expression of few miRNAs targeting COUP-TFI 3′UTR in silico. We found that the functional inhibition of miR-21 can effectively counteract the inhibitory effect of FGF8 in vitro and regulate COUP-TFI protein levels in vivo. Accordingly, miR-21 expression is complementary to COUP-TFI expression during corticogenesis. These data support a translational control of COUP-TFI gradient expression by FGF8 via miR-21 and contribute to our understanding of how regionalized expression is established during neocortical area mapping

Effect Of Priming On Seed Vigor Of Wheat (Triticum Aestivum L.)

Priming is a process that controls the process of hydration of seeds for the ongoing metabolic processes before germination. Research on priming was conducted at ICERI seed laboratory from May to September 2009 to evaluate the effect of different priming methods on wheat seed vigor. Physical properties and chemical composition of seed were evaluated before seeds were treated. The priming treatment were conducted by soaking 250 g of seed in 500 mL of solution for hydropriming and halopriming. Two seed lots of Nias and Dewata variety were subjected to heated and unheated distilled water for 12hours and subjected to KCl and CaCl2 at 10, 20, and 30 ppm and unprimed seed. The experiment were arranged in completely randomized design, replicated thrice. Vigor evaluation by observed seed germination, simultaneity growth, germination rate, seedling dry weight, electric conductivity of seed leakage and length of primary root. The results showed that highest germination, simultaneity growth, seedling dry weight, and length of primary root, were priming treatment with KCl 30 ppm and CaCl2 20 and 30 ppm. Priming with distilled water for 12 hours gave higher germination percentage and simultaneity growth

A eutherian-specific microRNA controls the translation of Satb2 in a model of cortical differentiation

Cerebral cortical development is controlled by key transcription factors that specify the neuronal identities in the different layers. The mechanisms controlling their expression in distinct cells are only partially known. We investigated the expression and stability of Tbr1, Bcl11b, Fezf2, Satb2, and Cux1 mRNAs in single developing mouse cortical cells. We observe that Satb2 mRNA appears much earlier than its protein and in a set of cells broader than expected, suggesting an initial inhibition of its translation, subsequently released during development. Mechanistically, Satb2 30UTR modulates protein translation of GFP reporters during mouse corticogenesis. We select miR541, a eutherian-specific miRNA, and miR-92a/b as the best candidates responsible for SATB2 inhibition, being strongly expressed in early and reduced in late progenitor cells. Their inactivation triggers robust and premature SATB2 translation in both mouse and human cortical cells. Our findings indicate RNA interference as a major mechanism in timing cortical cell identities

A cross-species spatiotemporal proteomic analysis identifies UBE3A-dependent signaling pathways and targets

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A. Restoring UBE3A levels is a potential disease-modifying therapy for AS and has recently entered clinical trials. There is paucity of data regarding the molecular changes downstream of UBE3A hampering elucidation of disease therapeutics and biomarkers. Notably, UBE3A plays an important role in the nucleus but its targets have yet to be elucidated. Using proteomics, we assessed changes during postnatal cortical development in an AS mouse model. Pathway analysis revealed dysregulation of proteasomal and tRNA synthetase pathways at all postnatal brain developmental stages, while synaptic proteins were altered in adults. We confirmed pathway alterations in an adult AS rat model across multiple brain regions and highlighted region-specific differences. UBE3A reinstatement in AS model mice resulted in near complete and partial rescue of the proteome alterations in adolescence and adults, respectively, supporting the notion that restoration of UBE3A expression provides a promising therapeutic option. We show that the nuclear enriched transketolase (TKT), one of the most abundantly altered proteins, is a novel direct UBE3A substrate and is elevated in the neuronal nucleus of rat brains and human iPSC-derived neurons. Taken together, our study provides a comprehensive map of UBE3A-driven proteome remodeling in AS across development and species, and corroborates an early UBE3A reinstatement as a viable therapeutic option. To support future disease and biomarker research, we present an accessible large-scale multi-species proteomic resource for the AS community (https://www.angelman-proteome-project.org/)

An Eutherian-Specific microRNA Controls the Translation of Satb2 in a Model of Cortical Differentiation

Cerebral cortical development is controlled by key transcription factors that specify the neuronal identities in the different cortical layers. These transcription factors are crucial for the identity of the different neurons, but the mechanisms controlling their expression in distinct cells are only partially known. Here we investigate the expression and stability of the mRNAs of Tbr1, Bcl11b, Fezf2, Satb2 and Cux1 in single developing mouse cortical cells. We focus on Satb2 and find that its mRNA expression occurs much earlier than its protein synthesis and in a set of cells broader than expected, suggesting an initially tight control of its translation, which is subsequently de-repressed at late developmental stages. Mechanistically, Satb2 3\u2019UTR modulates protein translation of GFP reporters during mouse corticogenesis. By in vitro pull-down of Satb2 3\u2019UTR-associated miRNAs, we select putative miRNAs responsible for SATB2 inhibition, focusing on those strongly expressed in early progenitor cells and reduced in late cells. miR-541, an Eutherian-specific miRNA, and miR-92a/b are the best candidates and their inactivation triggers robust and premature SATB2 translation in both mouse and human cortical cells. Our findings indicate that RNA interference plays a major role in the timing of cortical cell identity and may be part of the toolkit involved in specifying supra-granular projection neurons

Functional imaging of brain organoids using high-density microelectrode arrays

Studies have provided evidence that human cerebral organoids (hCOs) recapitulate fundamental milestones of early brain development, but many important questions regarding their functionality and electrophysiological properties persist. Highdensity microelectrode arrays (HD-MEAs) represent an attractive analysis platform to perform functional studies of neuronal networks at the cellular and network scale. Here, we use HD-MEAs to derive large-scale electrophysiological recordings from sliced hCOs. We record the activity of hCO slices over several weeks and probe observed neuronal dynamics pharmacologically. Moreover, we present results on how the obtained recordings can be spike-sorted and subsequently studied across scales. For example, we show how to track single neurons across several days on the HD-MEA and how to infer axonal action potential velocities. We also infer putative functional connectivity from hCO recordings. The introduced methodology will contribute to a better understanding of developing neuronal networks in brain organoids and provide new means for their functional characterization.ISSN:0883-7694ISSN:1938-142