Sulf2a controls Shh-dependent neural fate specification in the developing spinal cord.

Sulf2a belongs to the Sulf family of extracellular sulfatases which selectively remove 6-O-sulfate groups from heparan sulfates, a critical regulation level for their role in modulating the activity of signalling molecules. Data presented here define Sulf2a as a novel player in the control of Sonic Hedgehog (Shh)-mediated cell type specification during spinal cord development. We show that Sulf2a depletion in zebrafish results in overproduction of V3 interneurons at the expense of motor neurons and also impedes generation of oligodendrocyte precursor cells (OPCs), three cell types that depend on Shh for their generation. We provide evidence that Sulf2a, expressed in a spatially restricted progenitor domain, acts by maintaining the correct patterning and specification of ventral progenitors. More specifically, Sulf2a prevents Olig2 progenitors to activate high-threshold Shh response and, thereby, to adopt a V3 interneuron fate, thus ensuring proper production of motor neurons and OPCs. We propose a model in which Sulf2a reduces Shh signalling levels in responding cells by decreasing their sensitivity to the morphogen factor. More generally, our work, revealing that, in contrast to its paralog Sulf1, Sulf2a regulates neural fate specification in Shh target cells, provides direct evidence of non-redundant functions of Sulfs in the developing spinal cord.

Sulfatase 2 promotes generation of a spinal cord astrocyte subtype that stands out through the expression of Olig2.

Generation of glial cell diversity in the developing spinal cord is known to depend on spatio-temporal patterning programs. In particular, expression of the transcription factor Olig2 in neural progenitors of the pMN domain is recognized as critical to their fate choice decision to form oligodendrocyte precursor cells (OPCs) instead of astrocyte precursors (APs). However, generating some confusion, lineage-tracing studies of Olig2 progenitors in the spinal cord provided evidence that these progenitors also generate some astrocytes. Here, we addressed the role of the heparan sulfate-editing enzyme Sulf2 in the control of gliogenesis and found an unanticipated function for this enzyme. At initiation of gliogenesis in mouse, Sulf2 is expressed in ventral neural progenitors of the embryonic spinal cord, including in Olig2-expressing cells of the pMN domain. We found that sulf2 deletion, while not affecting OPC production, impairs generation of a previously unknown Olig2-expressing pMN-derived cell subtype that, in contrast to OPCs, does not upregulate Sox10, PDGFRα or Olig1. Instead, these cells activate expression of AP identity genes, including aldh1L1 and fgfr3 and, of note, retain Olig2 expression as they populate the spinal parenchyma at embryonic stages but also as they differentiate into mature astrocytes at postnatal stages. Thus, our study, by revealing the existence of Olig2-expressing APs that segregate early from pMN cells under the influence of Sulf2, supports the existence of a common source of APs and OPCs in the ventral spinal cord and highlights divergent regulatory mechanism for the development of pMN-derived OPCs and APs.

Dynamics of sonic hedgehog signaling in the ventral spinal cord are controlled by intrinsic changes in source cells requiring sulfatase 1.

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 high-threshold 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 Shh-dependent neural cell diversity, highlights a novel level of regulation, which involves temporal evolution of Shh source properties.

DSulfatase-1 fine-tunes Hedgehog patterning activity through a novel regulatory feedback loop.

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 stabilise expression domains of Hh target genes during wing disc development.

Robustness of positional specification by the Hedgehog morphogen gradient.

Spatial gradients of Hedgehog signalling play a central role in many patterning events during animal development, regulating cell fate determination and tissue growth in a variety of tissues and developmental stages. Experimental evidence suggests that many of the proteins responsible for regulating Hedgehog signalling and transport are themselves targets of Hedgehog signalling, leading to multiple levels of feedback within the system. We use mathematical modelling to analyse how these overlapping feedbacks combine to regulate patterning and potentially enhance robustness in the Drosophila wing imaginal disc. Our results predict that the regulation of Hedgehog transport and stability by glypicans, as well as multiple overlapping feedbacks in the Hedgehog response network, can combine to enhance the robustness of positional specification against variability in Hedgehog levels. We also discuss potential trade-offs between robustness and additional features of the Hedgehog gradient, such as signalling range and size regulation.

Shifted, the Drosophila ortholog of Wnt inhibitory factor-1, controls the distribution and movement of Hedgehog.

We here identify and characterize an extracellular modulator of Hedgehog signaling in Drosophila, Shifted. Shifted is required for high levels of long-range signaling in the developing wing imaginal disc. Surprisingly, shifted encodes the only Drosophila ortholog of the secreted vertebrate protein Wnt Inhibitory Factor-1 (WIF-1), whose known role is to bind to extracellular Wnts and inhibit their activity. However, Shifted does not regulate Hedgehog signaling by affecting Wingless or Wnt signaling. We show instead that Shifted is a secreted protein that acts over a long distance and is required for the normal accumulation of Hh protein and its movement in the wing. Our data further indicate that Shf interacts with Hh and the heparan sulfate proteoglycans. Therefore, we propose that Shf stabilizes the interaction between Hh and the proteoglycans, an unexpected role for a member of the WIF-1 family.

Patterns in evolution: veins of the Drosophila wing.

The development of the Drosophila wing is a classical model for studying the genetic control of tissue size, shape and patterning. A detailed picture of how positional information is interpreted by cells in the imaginal disc and translated into the adult wing vein pattern has recently emerged. It highlights the central role of dose-dependent activation of distinct cell transcription programs in response to the Hedgehog (Hh) and Decapentaplegic (Dpp) morphogens, as well as an early role of Notch signalling, in connecting the positioning of vein primordia and vein differentiation proper. The biochemical basis of the cross-talk that operates between these different signalling pathways is less well understood. New strategies made possible by the genome sequencing of several insect models should provide an important complement to the knowledge obtained from >60 years of genetic studies.

Vein-positioning in the Drosophila wing in response to Hh; new roles of Notch signaling.

The Drosophila wing is a classical model for studying the generation of developmental patterns. Previous studies have suggested that vein primordia form at boundaries between discrete sectors of gene expression along the antero-posterior (A/P) axis in the larval wing imaginal disc. Observation that the vein marker rhomboid (rho) is expressed at the centre of wider vein-competent domains led to propose that narrow vein primordia form first, and produce secondary short-range signals activating provein genes in neighbouring cells (see Curr. Opin. Genet. Dev. 10 (2000) 393). Here, we examined how the central L3 and L4 veins are positioned relative to the limits of expression of Collier (Col), a dose-dependent Hedgehog (Hh) target activated in the wing A/P organiser. We found that rho expression is first activated in broad domains adjacent to Col-expressing cells and secondarily restricted to the centre of these domains. This restriction which depends upon Notch (N) signaling sets the L3 and L4 vein primordia off the boundaries of Col expression. N activity is also required to fix the anterior limit of Col expression by locally antagonising Hh activation, thus precisely positioning the L3 vein primordium relative to the A/P compartment boundary. Experiments using Nts mutants further indicated that these two activities of N could be temporally uncoupled. Together, these observations highlight new roles of N in topologically linking the position of veins to prepattern gene expression.

Connecting Hh, Dpp and EGF signalling in patterning of the Drosophila wing; the pivotal role of collier/knot in the AP organiser.

Hedgehog (Hh) signalling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely unknown. We have re-examined the role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary, the AP organiser in the imaginal wing disc. We found that col mutant wings are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. We link these phenotypes to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation, the EGFR ligand Vein and the intervein determination gene blistered. We further show that attenuation of Dpp signalling in the AP organiser is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signalling pathways in the wing AP organiser is presented.

Notch and Wingless modulate the response of cells to Hedgehog signalling in the Drosophila wing.

During Drosophila wing development, Hedgehog (Hh) signalling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signalling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. Here, we describe a functional hierarchy of these signalling pathways that highlights the importance of competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc revealed that collier/knot (col/kn), patched (ptc), and decapentaplegic (dpp) are repressed at the DV boundary by N signalling. Attenuation of Hh signalling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. We show that this down-regulation depends on activity of the canonical Wg signalling pathway. We propose that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. Our findings suggest a paradigm of repression of the Hh response by N and/or Wnt signalling that may be applicable to signal integration in vertebrate appendages.