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.

In the developing spinal cord, oligodendrocyte progenitors (OLPs) originate from the ventral neuroepithelium and the specification of this lineage depends on the inductive activity of Sonic hedgehog (Shh) produced by ventral midline cells. On the other hand, it has been shown that OLP identity is acquired by the coexpression of the transcription factors olig2 and nkx2.2. Although initially expressed in adjacent nonoverlapping domains of the ventral neuroepithelium, these transcription factors become coexpressed in the pMN domain at the time of OLP specification through dorsal extension of the Nkx2.2 domain. Here we show that Shh is sufficient to promote the coexpression of Olig2 and Nkx2.2 in neuroepithelial cells. In addition, Shh activity is necessary for this coexpression since blocking Shh signalling totally abolishes Olig2 expression and impedes dorsal extension of Nkx2.2. Although Shh at these stages affects neuroepithelial cell proliferation, the dorsal extension of the Nkx2.2 domain is not due to progenitor proliferation but to repatterning of the ventral neuroepithelium. Finally, Shh not only stimulates OLP specification but also simultaneously restricts the ventral extension of the astrocyte progenitor (AP) domain and reduces astrocyte development. We propose that specification of distinct glial lineages is the result of a choice that depends on Shh signalling.

Recent evidence indicates that oligodendrocytes originate initially from the ventral neural tube. We have documented in chick embryos the effect of early ventralization of the dorsal neural tube on oligodendrocyte differentiation. Notochord or floor plate grafted at stage 10 in dorsal position induced the development of oligodendrocyte precursors in the dorsal spinal cord. In vitro, oligodendrocytes differentiated from medial but not intermediate neural plate explants, suggesting that the ventral restriction of oligodendrogenesis is established early. Furthermore, quail fibroblasts overexpressing the ventralizing signal Sonic Hedgehog induced oligodendrocyte differentiation in both the intermediate neural plate and the E4 dorsal spinal cord. These results strongly suggest that the emergence of the oligodendrocyte lineage is related to the establishment of the dorso-ventral polarity of the neural tube.

Although glial cells missing (gcm) genes are known as glial determinants in the fly embryo, the role of vertebrate orthologs in the central nervous system is still under debate. Here we show for the first time that the chicken ortholog of fly gcm (herein referred to as c-Gcm1), is expressed in early neuronal lineages of the developing spinal cord and is required for neural progenitors to differentiate as neurons. Moreover, c-Gcm1 overexpression is sufficient to trigger cell cycle exit and neuronal differentiation in neural progenitors. Thus, c-Gcm1 expression constitutes a crucial step in the developmental cascade that prompts progenitors to generate neurons: c-Gcm1 acts downstream of proneural (neurogenin) and progenitor (Sox1-3) factors and upstream of NeuroM neuronal differentiation factor. Strikingly, this neurogenic role is not specific to the vertebrate gene, as fly gcm and gcm2 are also sufficient to induce the expression of neuronal markers. Interestingly, the neurogenic role is restricted to post-embryonic stages and we identify two novel brain neuronal lineages expressing and requiring gcm genes. Finally, we show that fly gcm and the chick and mouse orthologs induce expression of neural markers in HeLa cells. These data, which demonstrate a conserved neurogenic role for Gcm transcription factors, call for a re-evaluation of the mode of action of these genes during evolution.

Summary Sulfs are secreted sulfatases that catalyse removal of sulfate from Heparan Sulfate Proteoglycans (HSPGs) in the extracellular space. These enzymes are well known to regulate a number of crucial signalling pathways during development. In this study, we report that DSulfatase-1 (DSulf1), the unique Drosophila Sulf protein, is a regulator of Hedgehog (Hh) signalling during wing development. DSulf1 activity is required in both Hh source and Hh receiving cells for proper positioning of Hh target gene expression boundaries. As assessed by loss- and gain-of-function experiments in specific compartments, DSulf1 displays dual functions with respect to Hh signalling, acting as a positive regulator in Hh producing cells and a negative regulator in Hh receiving cells. In either domain, DSulf1 modulates Hh distribution by locally lowering the concentration of the morphogen at the apical pole of wing disc cells. Thus, we propose that DSulf1, by its desulfation catalytic activity, lowers Hh/HSPG interaction in both Hh source and target fields, thereby enhancing Hh release from its source of production and reducing Hh signalling activity in responding cells. Finally, we show that Dsulf1 pattern of expression is temporally regulated and depends on EGFR signalling, a Hh-dependent secondary signal in this tissue. Our data reveal a novel Hh regulatory feedback loop, involving DSulf1, which contributes to maintain and stabilize expression domains of Hh target genes during wing disc development.

A substantial amount of data has highlighted the crucial influence of Shh signalling on the generation of diverse classes of neurons and glial cells throughout the developing central nervous system. A critical step leading to this diversity is the establishment of distinct neural progenitor cell domains during the process of pattern formation. The forming spinal cord, in particular, has served as an excellent model to unravel how progenitor cells respond to Shh to produce the appropriate pattern. In recent years, considerable advances have been made in our understanding of important parameters that control the temporal and spatial interpretation of the morphogen signal at the level of Shh-receiving progenitor cells. Although less studied, the identity and position of Shh source cells also undergo significant changes over time, raising the question of how moving the Shh source contributes to cell diversification in response to the morphogen. Here, we focus on the dynamics of Shh-producing cells and discuss specific roles for these time-variant Shh sources with regard to the temporal events occurring in the receiving field.