Genetics and Genome Sciences

Most oligodendrocytes of the spinal cord originate from ventral progenitor cells of the pMN domain, characterized by expression of the transcription factor Olig2. A minority of oligodendrocytes is also recognized to emerge from dorsal progenitors during fetal development. The prevailing view is that generation of ventral oligodendrocytes depends on Sonic hedgehog (Shh) while dorsal oligodendrocytes develop under the influence of Fibroblast Growth Factors (FGFs). Using the well-established model of the chicken embryo, we evidence that ventral spinal progenitor cells activate FGF signaling at the onset of oligodendrocyte precursor cell (OPC) generation, as do they dorsal counterpart. Inhibition of FGF receptors at that time appears sufficient to prevent generation of ventral OPCs, highlighting that, in addition to Shh, FGF signaling is required also for generation of ventral OPCs. We further reveal an unsuspected interplay between Shh and FGF signaling by showing that FGFs serve dual ...

Background and Aims: As the major energy source for mammalian cells, mitochondria have been the subject of numerous studies. However, the isolation and purification of healthy mitochondria, especially from fresh tissue, remains challenging. The most popular methods and kits involve various centrifugation steps which require substantial time and equipment but do not consistently provide pure preparations of functional mitochondria. The aim of this study was to determine whether methods could be devised to improve the purity and yield of functional mitochondria from fresh tissue. Methods: Fresh mouse liver was homogenized, and cells lysed. Particle size analysis, quantitation of mitochondrial DNA, mitochondrial oxygen consumption, and purity of mitochondria (by electron microscopy) were measured in samples after various purification steps and significant differences determined. Results: A two-step procedure consisting of centrifugation followed by filtration through 1.2m and 0.8m filters resulted in uniform mitochondrial preparations with diameters between 520-540 nm, and approximately 5-times more pure samples. The mitochondria thus obtained had oxygen consumption and sensitivities to mitochondrial inhibitors that were indistinguishable from those purified by centrifugation alone. Electron microscopy confirmed the presence of more uniform and 4-5 times greater concentrations of mitochondria compared to centrifugation alone. Conclusions: A two-step procedure consisting of sequential centrifugation followed by filtration is a rapid method for the production of highly purified, uniform and functional mitochondria.

Background: Most oligodendrocytes of the spinal cord originate from ventral progenitor cells of the pMN domain, characterized by expression of the transcription factor Olig2. A minority of oligodendrocytes is also recognized to emerge from dorsal progenitors during fetal development. The prevailing view is that generation of ventral oligodendrocytes depends on Sonic hedgehog (Shh) while dorsal oligodendrocytes develop under the influence of Fibroblast Growth Factors (FGFs). Results: Using the well-established model of the chicken embryo, we show that ventral spinal progenitor cells activate FGF signaling at the onset of oligodendrocyte precursor cell (OPC) generation. Inhibition of FGF receptors at that time appears sufficient to prevent generation of ventral OPCs, highlighting that, in addition to Shh, FGF signaling is required also for generation of ventral OPCs. We further reveal an unsuspected interplay between Shh and FGF signaling by showing that FGFs serve dual essential functions in ventral OPC specification. FGFs are responsible for timely induction of a secondary Shh signaling center, the lateral floor plate, a crucial step to create the burst of Shh required for OPC specification. At the same time, FGFs prevent down-regulation of Olig2 in pMN progenitor cells as these cells receive higher threshold of the Shh signal. Finally, we bring arguments favoring a key role of newly differentiated neurons acting as providers of the FGF signal required to trigger OPC generation in the ventral spinal cord. Conclusion: Altogether our data reveal that the FGF signaling pathway is activated and required for OPC commitment in the ventral spinal cord. More generally, our data may prove important in defining strategies to produce large populations of determined oligodendrocyte precursor cells from undetermined neural progenitors, including stem cells. In the long run, these new data could be useful in attempts to stimulate the oligodendrocyte fate in residing neural stem cells.

In the ventral spinal cord, generation of neuronal and glial cell subtypes is controlled by Sonic hedgehog (Shh). This morphogen contributes to cell diversity by regulating spatial and temporal sequences of gene expression during development. Here, we report that establishing Shh source cells is not sufficient to induce the highthreshold response required to specify sequential generation of ventral interneurons and oligodendroglial cells at the right time and place in zebrafish. Instead, we show that Shh-producing cells must repeatedly upregulate the secreted enzyme Sulfatase1 (Sulf1) at two critical time points of development to reach their full inductive capacity. We provide evidence that Sulf1 triggers Shh signaling activity to establish and, later on, modify the spatial arrangement of gene expression in ventral neural progenitors. We further present arguments in favor of Sulf1 controlling Shh temporal activity by stimulating production of active forms of Shh from its source. Our work, by pointing out the key role of Sulf1 in regulating Shhdependent neural cell diversity, highlights a novel level of regulation, which involves temporal evolution of Shh source properties.

An imropved method for Lightsheet imaging sample preparation. We use this protocol for embryonic brain tissue (the embryonic cerebellum in particular), it could also be optimized for other types of tissues with few modifications to incubation periods. First, we fix the tissue for two hours on ice, then we pereamibilize using a mixture of Triton and Tween. An added ingenious step An added ingenious step is steaming the tissue in Citric acid to retrieve epitopes after crosslinking and bleach the autofluorescence of tissue and blood vessels. That simple step saves a lot of time avoiding H 2 O 2 bleaching steps in other staining protocols and improves the quality of antibody staining. After staining, the tissue clearing is based in concept on RTF method (Yu et al. 2018) with modifications and added incubation steps in increasing glycerol concentrations. The final imaging solution of 80% glycerol should already have a refractive index of 1.45 matching that of Zeiss lightsheet Z1 X5 lens, thus, it can be used to fill the lightsheet chamber for imaging and for sample storage.