Sulf2b is expressed within Shh-producing cells whereas Sulf2a is restricted to neural progenitors. Here and in all subsequent panels, side views of embryos (a,c,e,g,k,l) are at the level of the trunk spinal cord, anterior to the left and dorsal to the top, and spinal cord transverse sections (b,d,f,h,i,j) are shown dorsal to the top. Developmental stages are indicated in each panel. (a–j) Detection of sulf2b (a–d) and sulf2a (e–j) mRNAs by whole-mount in situ hybridisation. Images are representative examples of at least five embryos. Grey outlines (b,d,f,h,i,j) label the spinal cord edge. Brackets in a,c,e,g indicate the position of the spinal cord. Dotted lines in b,d,f,h) delineate Shh-producing cells, i.e. the medial floor plate at 24 hpf and the medial floor plate + lateral floor plat at 36 hpf. Note that sulf2a mRNA is detected in neural progenitors but not in medial and lateral floor plate cells. (k-l) Double detection of sulf2a (red) and shh (green) mRNAs by fluorescent in situ hybridisation, showing that sulf2a expression does not overlap the shh-expressing domain (white brackets).

V3 interneuron production is increased upon Sulf2a depletion. Side views of 48 hpf embryos. (a–f) Detection (a–e) and quantification (f) of sim1a-expressing neurons in embryos injected with ctrlMO (a, n = 34), sulf2aMO^ATG (b, n = 20) or sulf2aMO^splice (c, n = 34) and in wild-type (d, n = 10) or sulf2a−/− (e, n = 12) embryos from two independent experiments in each case. Datasets were compared with Mann Whitney’s test (two-tailed). Data are presented as mean number of cells per embryo ± s.d (**p < 0.01, ****p < 0.0001).

Sulf2a depletion impairs OPC development. Side views of 48 hpf embryos. (a-f) Detection (a–e) and quantification (f) of OPCs by immunodetection of Sox10 (green in a–c, red in d,e) in Tg(olig2:DsRed) embryos (red in a–c) injected with ctrlMO (a, n = 73), sulf2aMO^ATG (b, n = 31) or sulf2aMO^splice (c, n = 47) and wild-type (d, n = 10) or sulf2a−/− (e, n = 48) Tg(olig2:GFP) embryos (green in d,e) from 9 and 6 independent experiments, respectively. Datasets were compared with Mann Whitney’s test (two-tailed). Data are presented as mean number of cells per embryo ± s.d (*p < 0.05, ****p < 0.0001).

The Nkx2.2a/p3 domain is dorsally expanded at the expense of the Olig2/pMN domain in 24 hpf sulf2a-depleted embryos. Side views of 24 hpf embryos. (a–c”) Double detection of olig2 mRNA and Sox2 in Tg(nkx2.2a:mEGFP) embryos injected with ctrlMO (a–a”), sulf2aMO^ATG (b–b”) or sulf2aMO^splice (c–c”). Vertical sets present successively the nkx2.2a:mEGFP signal (green in a”–c”), the olig2 mRNA staining (red in a”–c”) and the merged image together with Sox2 staining (blue in a”–c”). Note the dorsal expansion of the nkx2.2a:mEGFP signal associated with a strong decrease in the olig2 signal in sulf2a morphant embryos. (d, e) Quantification of nkx2.2a:mEGFP + /Sox2 + (d) and olig2 + /Sox2 + (e) progenitors in embryos injected with ctrlMO (n = 27), sulf2aMO^ATG (n = 14) and sulf2aMO^splice (n = 23) from two independent experiments. Datasets were compared with Mann Whitney’s test (two-tailed). Data are presented as a mean number of cells along the dorso-ventral axis per embryo ± s.d (****p < 0.0001).

Sulf2a depletion does not affect formation of the Nkx2.2a/Olig2-co-expressing p* domain but reduces the number of progenitors that only express Olig2 at the onset of gliogenesis. Side views of 36 hpf embryos. (a–c”’): Double detection of olig2 mRNA and Sox2 in Tg(nkx2.2a:mEGFP) embryos injected with ctrlMO (a–a”’), sulf2aMO^ATG (b–b”’) or sulf2aMO^splice (c–c”’). Vertical sets present successively the nkx2.2a:mEGFP signal (green in a”–c”, a”’–c”’), the olig2 mRNA staining (red in a”–c”,a”’–c”’), the merged image of nkx2.2a:mEGFP and olig2 signals and the merged image together with Sox2 staining (blue in a”’–c”’). Dotted line represents the dorsal limit of nkx2.2a:mEGFP-expressing domain. (d-g) Cell quantification performed in embryos injected with ctrlMO (n = 16), sulf2aMO^ATG (n = 11) and sulf2aMO^splice (n = 15) from three independent experiments. Cell counts in (d,e) correspond to the population of Sox2 + progenitors expressing either nkx2.2a:mEGFP (d) or olig2(e). Quantifications in (f,g) correspond to Sox2 + progenitors that either co-express olig2 and nkx2.2a:mEGFP (f) or express olig2 mRNA but not nkx2.2a:mEGFP (g). Except in e where datasets were compared with Student’s t-test (unpaired two-tailed), datasets were compared with Mann Whitney’s test (two-tailed). Results are presented as a mean number of cells along the dorso-ventral axis per embryo ± s.d (*p < 0,05, ** p < 0,005, ***p < 0.001,****p < 0.0001).

Sulf2a depletion causes a preferential deficit of non-myelinating OPCs. Side views of 48 hpf embryos. (a–d”) Immunodetection of Sox10 in Tg(nkx2.2a:mEGFP; olig2:DsRed2) embryos injected with ctrlMO (a–a”) or sulf2aMO^splice (b–b”) and wild-type (c–c”) or mutant for sulf2a (d–d”). Horizontal sets present successively the Sox10 staining (red) in combination with the olig2:DsRed2 signal (blue, a–d), with the nkx2.2a:mEGFP signal (green, a’–d’) and the merged image of the three signals (a”–d”). White arrows point to non-myelinating OPCs (Nkx2.2a:mEGFP-/olig2:DsRed2 + /Sox10 + cells) while open arrowheads indicate myelinating OPCs (Nkx2.2a:mEGFP + /olig2:DsRed2 + /Sox10 + cells). (e–g) Quantification of OPCs in embryos injected with ctrlMO (n = 70) or sulf2aMO^splice (n = 59) and in wild-type (n = 23) or sulf2a−/− (n = 24) embryos from five and three independent experiments, respectively. Cell counts in (e) correspond to the total number of OPCs co-expressing Sox10 and olig2:DsRed2. Quantifications of nkx2.2a:mEGFP + /myelinating OPCs (f) and nkx2.2a:mEGFP-/non-myelinating OPCs (g) were presented separately. Datasets were compared with Mann Whitney’s test (two-tailed). Data are presented as mean number of cells per embryo ± s.d (*p < 0.05, **p < 0.01, *** p < 0.0005, ****p < 0.0001).

Sulf2a is excluded from Nkx2.2a-expressing progenitors but expressed, at gliogenic stage, in a subset of Olig2 progenitors. Side views (a,c,e) and transverse sections (b,d,f) of 24 hpf (a,b) and 36 hpf (c–f) embryos. (a–f) Detection of sulf2a mRNA (red) in Tg(nkx2.2a:mEGFP) embryos (green, a–d) or in Tg(olig2:GFP) embryos (green, e,f). Images show representative examples of at least three embryos. Note that sulf2a expression dorsally abuts the nkx2.2a:mEGFP-expressing domain both at 24 and 36 hpf (a–d) and is heterogeneously expressed within the population of Olig2 progenitors at 36 hpf (e,f).

Sulf2a restricts dorsal extent of the high-threshold Shh response without affecting expression of shh. Side views (a,c) and transverse sections (b,d,f,g) of 24 hpf embryos. a-d: Immunostaining of Sox2 in Tg(GBS-ptch2:EGFP) embryos injected with ctrlMO (a,b) or sulf2aMO^ATG (c,d). Dotted lines in b and d delineate the medial floor plate. Note the dorsal expansion of the GBS-ptch2:EGFP signal in sulf2a morphants. (e) Quantification of GBS-ptch2:EGFP + /Sox2 + cells in embryos injected with ctrlMO (n = 13), sulf2aMO^ATG (n = 12) or sulf2aMO^splice (n = 9) from two independent experiments. Datasets were compared with Mann Whitney’s test (two-tailed). Data are presented as mean number of cells along the dorso-ventral axis on each side of the lumen per embryo ± s.d (***p < 0.0005, ** p < 0.005). (f–h) Double detection of patched2 mRNA (red, f, g) and Sox2 (blue, f, g) in embryos injected with ctrlMO (f, n = 5) or sulf2aMO^ATG (g, n = 5). Patched2 relative fluorescence intensity in neural progenitors (white frame in f,g) was quantified along the dorso-ventral (DV) axis (grey line for ctrlMO and purple for sulf2aMO^ATG). Datasets were compared with two-way ANOVA test. Data are presented in arbitrary units of fluorescence (AU; mean values ± s.d) along the DV axis per section (*p < 0.005).

Model for Sulf2a function. Scheme showing Sulf2a and Shh-dependent gene expression in ventral neural progenitors during neuronal production (24 hpf, a,a’) and at the onset of gliogenesis (36 hpf, b,b’) in wild-type (a,b) and sulf2a depleted (a’,b’) contexts. In 24 hpf wild-type embryos (a), the ventral-most progenitors of the p3 domain (green), adjacent to the medial floor plate (MFP, brown), express the high-threshold Shh responsive gene Nkx2.2a and generate V3 interneurons. Sulf2a (turquoise blue frame), expressed in dorsally located Olig2/pMN progenitors (red), limits the dorsal extent of high-threshold Shh response to prevent activation of Nkx2.2a and thus allows maintenance of Olig2 expression (red) required to orient these cells toward the MN lineage. When sulf2a is depleted (a’), the range of high-threshold Shh response extends dorsally, leading to dorsal misexpression of Nkx2.2a which in turn represses Olig2. Subsequent changes in the sizes of the p3 and pMN domains cause overproduction of V3 interneurons at the expense of MNs. In 36hpf wild-type embryos (b), the Shh

source has expanded in the former Nkx2.2a/p3 domain to form the lateral floor plate (LFP, brown). Immediately adjacent progenitors, i.e. the ventral-most cells of the Olig2/pMN domain, because they activate high-threshold Shh response, upregulate Nkx2.2a. At that stage, Nkx2.2a no more represses Olig2, leading to formation of the p* domain (yellow), populated by progenitors co-expressing Olig2 and Nkx2.2a. Sulf2a expression (turquoise blue frame) is excluded from p* cells but maintained in dorsally located Olig2 progenitors (red) where the enzyme, again, prevents high-threshold Shh response. Then, the two distinct populations of Olig2 progenitors, expressing Nkx2.2a (yellow) or not (red) generate the myelinating (yellow) and non-myelinating (red) OPC, respectively. In 36 hpf sulf2a-depleted embryos (b’), LFP formation is not affected but, because of the dorsal expansion of the Nkx2.2a/p3 domain that occurred at earlier stage, the dorsally adjacent progenitors express Nkx2.2a but not Olig2 (green), in contrast to the wild-type situation. Nonetheless, Sulf2a depletion, again, allows activation of high-threshold Shh response that causes dorsal misexpression of Nkx2.2a. Subsequently, the p* domain (yellow), while positioned dorsally, forms properly but, the reduced size of the Olig2/pMN domain leads to a deficit in progenitors that only express Olig2 and, consequently to a strong reduction in the population of non-myelinating OPCs (red). It has to be noted that the population of myelinating OPC (yellow) is also mildly reduced possibly due to a secondary effect of aberrant neuronal populations (see discussion).