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.

Recent studies using single-cell RNA-sequencing have revealed cellular heterogeneity in the developing mammalian cerebellum, yet the regulatory logic underlying this cellular diversity remains to be elucidated. Using integrated single-cell RNA and ATAC analyses, we resolved developmental trajectories of cerebellar progenitors and identified putative trans- and cis-elements that control cell state transition. We reverse engineered gene regulatory networks (GRNs) of each cerebellar cell type. Through in silico simulations and in vivo experiments, we validated the efficacy of GRN analyses and uncovered the molecular control of a posterior transitory zone (PTZ), a distinct progenitor zone residing immediately anterior to the morphologically defined rhombic lip (RL). We showed that perturbing cell fate specification in the PTZ and RL causes posterior cerebellar vermis hypoplasia, the most common cerebellar birth defect in humans. Our study provides a foundation for comprehensive studies of developmental programs of the mammalian cerebellum.

BackgroundMost 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).ResultsUsing 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.ConclusionAltogether 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.