The appearance of neurofilaments (NFs) and vimentin (Vim) in the nervous system of the mouse embryo was documented using immunohistochemical techniques.The three NF protein subunits appear early and simultaneously in central and peripheral neurons at 9 to 10 days of gestation. The onset of NF expression is concomitant with axon elongation and correlates extremely well with neurofibrillar differentiation and, in the case of autonomic ganglia, with the expression of adrenergic neurotransmitter properties. In the central and peripheral nervous system, NF expression is preceded by that of Vim, and both types of intermediate filaments coexist within the same cell for a short period of time.

In the embryonic chick ventral spinal cord, the initial emergence of oligodendrocytes is a relatively late event that depends on prolonged Sonic hedgehog (Shh) signaling. In this report, we show that specification of oligodendrocyte precursors (OLPs) from ventral Nkx2.2-expressing neural progenitors occurs precisely when these progenitors stop generating neurons, indicating that the mechanism of the neuronal/oligodendroglial switch is a common feature of ventral OLP specification. We further show that an experimental early increase in the concentration of Shh is sufficient to induce premature specification of OLPs at the expense of neuronal genesis indicating that the relative doses of Shh received by ventral progenitors determine whether they become neurons or glia. Accordingly, we observe that the Shh protein accumulates at the apical surface of Nkx2.2-expressing cells just before OLP specification, providing direct evidence that these cells are subjected to a higher concentration of the morphogen when they switch to an oligodendroglial fate. Finally, we show that this abrupt change in Shh distribution is most likely attributable to the timely activity of Sulfatase 1 (Sulf1), a secreted enzym that modulates the sulfation state of heparan sulfate proteoglycans. Sulf1 is expressed in the ventral neuroepithelium just before OLP specification, and we show that its experimental overexpression leads to apical concentration of Shh on neuroepithelial cells, a decisive event for the switch of ventral neural progenitors toward an oligodendroglial fate.

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.

In the developing ventral spinal cord, motor neurons (MNs) and oligodendrocyte precursor cells (OPCs) are sequentially generated from a common pool of neural progenitors included in the so-called pMN domain characterized by Olig2 expression. Here, we establish that the secreted Sulfatase 1 (Sulf1) is a major component of the mechanism that causes these progenitors to stop producing MNs and change their fate to generate OPCs. We show that specification of OPCs is severely affected in sulf1-deficient mouse embryos. This defect does not rely on abnormal patterning of the spinal cord or failure in maintenance of pMN progenitors at the onset of OPC specification. Instead, the efficiency of OPC induction is reduced, only few Olig2 progenitors are recruited to generate OPCs, meanwhile they continue to produce MNs beyond the normal timing of the neuroglial switch. Using the chicken embryo, we show that Sulf1 activity is required precisely at the stage of the MN-to-OPC fate switch. Finally, we bring arguments supporting the view that Sulf1 controls the level of Sonic Hedgehog (Shh) signaling activity, behaving as an enhancer rather than an obligatory component in the Shh pathway. Our study provides additional insights into the temporal control of Olig2 progenitor cell fate change by the identification of Sulf1 as an extracellular timing signal in the ventral spinal cord.