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. 2012 Feb;19(2):209-19.
doi: 10.1038/cdd.2011.84. Epub 2011 Jul 1.

Jagged2 controls the generation of motor neuron and oligodendrocyte progenitors in the ventral spinal cord

M A Rabadán  1 , J Cayuso, G Le Dréau, C Cruz, M Barzi, S Pons, J Briscoe, E Martí
Affiliations

Jagged2 controls the generation of motor neuron and oligodendrocyte progenitors in the ventral spinal cord

M A Rabadán et al. Cell Death Differ. 2012 Feb.

Abstract

In the developing spinal cord, motor neurons (MNs) and oligodendrocytes arise sequentially from a common pool of progenitors. However, the genetic network responsible for this neurogenesis to gliogenesis switch is largely unknown. A transcriptome analysis identified the Notch ligand Jagged2 (JAG2) as a Sonic hedgehog-regulated factor transiently expressed in MN progenitors (pMNs). In vivo loss- and gain-of-function experiments show that JAG2 schedules the differentiation of the pMN progenitors. At early developmental stages, Olig2 expressing pMN progenitors that enter the differentiation pathway exclusively generate MNs. At these times, the activation of the Notch pathway by JAG2 maintains selected pMN progenitors in an undifferentiated state by two mechanisms; first it inhibits MN generation by reducing Olig2 proteins levels, and second it directly inhibits the premature generation of oligodendrocyte progenitors (OLPs) by maintaining high levels of Hes5. Later, extinction of JAG2 from the pMN results in the loss of Hes5 expression, heralding the gliogenic phase of pMN progenitors. Strikingly, downregulation of JAG2 in pMN progenitors is sufficient to promote the precocious generation of OLPs. Together these data provide evidence that JAG2 is a key regulator of the timely and ordered generation of two of the defining cell types in the spinal cord, MNs and OLPs.

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Figures

Figure 1
Figure 1
The Notch ligand Jagged2 is transiently expressed in the ventral spinal cord. (a) A transcriptional profiling strategy for Shh identified patterning determinants and components of the Notch pathway. Chick embryos were electroporated with activator or inhibitors of the Shh pathway (see Materials and Methods), transfected GFP-expressing cells were FACS sorted, and extracted RNA was used to hybridise full genome genechips. In response to activators of the Shh pathway (Gli3Act), genes expressed in the ventral NT were upregulated, whereas dorsal genes appeared downregulated. In addition, expression of the Notch ligand Serrate2/Jagged2 appeared upregulated. (b–e) Expression of Jagged2 revealed by in situ hybridisation in sections of chick embryos at the indicated stages. (c) Shows double labelling with the intermediate neuronal subtypes (Pax2). (f, g) Expression of Jagged2 revealed by fluorescent in situ (red), double labelled with the pMN marker Olig2 (f) or the MN marker islet1 (g). (h) Double immnunohistochemistry for Olig2/Nkx2.2 shows the generation of OLP. (i) Summary of the expression of Jagged2 in relation to the generation of MNs and OLPs from the pMN domain
Figure 2
Figure 2
Jagged2 activity controls the timely differentiation of MNs. (a) Embryos electroporated (EP) at HH14, with the indicated DNAs, were analysed at 72 h post EP with the early MN marker MNR2 (red). EP side is shown to the right, GFP (green) shows transfected cells. Quantification of motor neurons and their spatial distribution was performed analysing confocal images. (b) Histograms represent the number of MNR2-positive cells depending on the distance to the lumen; TZ, transition zone; MZ, mantle zone. (c) Schematic representation of the MN birth dating experiments following Jagged2 manipulations. Embryos EP at HH14 were labelled with a single BrdU pulse 24 h PE, and analysed at 48 h PE for BrdU/MN markers. (d) Schematic representation of motor neurons birth dates in the brachial spinal cord of the chick embryo, data adapted from Hollyday and Hamburguer. (e) Example sections showing double staining for the MN marker Islet1 (green) and BrdU (red). EP side is shown to the right, GFP (blue) shows transfected cells. (f) Quantitative data showing the proportion of born MNs, plots correspond to the ratio of EP versus non-EP side in each experimental condition (control, black dots; GOF-Jag2, red dots; LOF-Jag2, green dots). Time is represented as hours of incubation and HH stages. First dot corresponds to the EP time, second to the BrdU−/Islet1+ cells (MNs born at the time of the BrdU pulse), third dot represents the total Islet1+ cells (MNs terminally differentiated 48 h PE). Bars correspond to the standard error (S.E.M.)
Figure 3
Figure 3
Overexpression of Jagged2 retains Olig2+ cells in a progenitor stage. (a) Model representing the fate of pMN progenitors along three cell cycles, in control situation in which 50% divisions are asymmetric, giving rise to one MN (green) and one progenitor cell (red). Percentages of asymmetric divisions are adapted from data provided by BrdU incorporation experiments in the presence or the absence of Jagged2. Drawing shows the resulting proportion of neurons (N) and progenitors (P). Plots represent the ratio of theoretical MNs generated in each condition, dots correspond to each cell cycle. Plots represent the ratio of theoretical progenitors that remain in each condition, dots correspond to each cell cycle. (b) Quantitative data showing the ratio of Olig2+ cells, plots correspond to the ratio of EP versus non-EP side in each experimental condition (control, black dots; GOF-Jag2, red dots; LOF-Jag2, green dots). Time is represented as hours of incubation and HH stages. Dots correspond to 12 h 24 h, 48 h and 72 h PE. Bars correspond to the standard error (S.E.M.). (c) Sample sections, EP with indicated DNAs, and immunostained with Olig2 (red). EP sides are shown to the right, GFP (green) shows transfected cells
Figure 4
Figure 4
Jagged2 reduces Olig2 protein levels and inhibits the Olig2 capacity to differentiate MNs. (a) Western blots from NT total lysates were immunobloted with anti-Olig2, anti-Myc and anti-RFP antibodies. Embryos co-EP at HH14 with Jagged2-Myc, Olig2 and pCAGGS-ires-RFP were dissected at 12 h PE. Note that Olig2 protein is distributed within two separate bands that were both shifted but maintained in the presence of calf intestine alkaline phosphatase (CIAP), showing that both forms are phosphoproteins. These two bands might reflect alternate translation initiation sites. Anti-Myc reveals the presence or absence of Jagged2, anti-RFP as EP control. (b) Quantitative analysis of upper and lower Olig2 bands, in the presence or the absence of Jagged2, normalised to RFP levels. Olig2 protein levels are significantly reduced in NT co-transfected with Jagged2. (c) The Olig2 capacity to differentiate MNs is prevented by Jagged2 activity. Embryos EP at HH14 with the indicated DNAs were analysed 12 h PE for the MN progenitor marker Olig2 (red) and the MN definitive marker MNR2 (green). GFP is shown in blue. EP of Olig2 is sufficient for the generation of ectopic MNR2+ MNs. Quantification of ratio of MNR2+ cells in EP versus control non-EP side revealed a ratio of 0.85±0.07 in control EP embryos, whereas in Olig2-EP increased to 2.02±0.03. EP of Jag2 prevents the generation of MNR2+ cells to 0.58±0.02, whereas co-EP of Olig2 and Jag2 also reduced the appearance of MNR2+ cells to 0.51±0.05 in the normal ventral domain, but also prevented the generation of ectopic dorsal MNs. (d) Summary model of Jagged2 activity in the pMN domain, Jagged2 activity splits Olig2+ progenitors into two different identities
Figure 5
Figure 5
Depletion of Jagged2 activity at the time of MN generation results in premature OLP generation. (a–h) Embryos electroporated at HH14 with the indicated DNAs were analysed at 48 h PE with OLP markers. EP side is shown to the right, GFP (green) shows transfected cells. (a and b) Olig2 is expressed at pMN in control NT, whereas GOF-JAG2 EP shows migratory Olig2+ cells. (c) Co-EP with human Jagged2 (GOF-JAG2) restores Olig2+ cells to pMN. (d and e) Sox9 is expressed throughout the VZ in control NT, whereas LOF-JAG2 electroporated NT shows depletion of Sox9+ cells at pMN and migratory Sox9+ cells. (f) Co-EP with GOF-JAG2 restores Sox9 expression in pMN. (g) Olig2+ migratory cells (red) co-expressed the OLP marker O4 (green). (h) Quantitative analysis of Olig2+ and Sox9+ migratory cells at 48 h PE after electroporation of the indicated DNAs. Numbers are shown as migratory marker expressing cell in each experimental condition. Bars correspond to the standard error (S.E.M.). (i–n) At 72 h PE of LOF-JAG2, Sox9 is extinguished from the VZ, and premature expression of the OLP markers Sox10 and PLPDM 20 are detected by in situ hybridisation. In control electroporated spinal cords, Sox10 and PLPDM20 are only expressed in the ventral roots. (o) Premature OLP generation as revealed by the real-time–PCR detection of a significant increase in the expression of PDGF receptor α and in the myelin-specific gene PLP at 48 h PE of LOF-JAG2. (p) Premature OLP generation as revealed by the increased activity of the MBP-Luc reporter 24 h PE of LOF-JAG2
Figure 6
Figure 6
Persistent overexpression of Jagged2 inhibits OLP generation. Embryos EP at HH18 with Jagged2 (GOF-JAG2), or control empty vector (Control), were analysed at 4, 5 days after EP for oligodendrocyte generation. (a and c) Oligodendrocytes are identified as migratory cells departing from the pMN expressing Olig2 (red) and Nkx2.2 (green). Electroporated side is shown to the right, GFP (blue) shows transfected cells. Controls show numerous migratory OLPs. GOF-JAG inhibits OLP migration. (b and d) Oligodendrocytes are identified as migratory expressing Olig2 (red) and O4 (green). Electroporated side is shown to the right, GFP (blue) shows transfected cells. Controls show numerous migratory OLPs. GOF-JAG inhibits OLP migration. (e) Quantitative analysis of Olig2+ migratory cells at 72 h and 108 h after electroporation of the indicated DNAs. Numbers are shown as migratory marker expressing cell in each experimental condition. Bars correspond to the standard error (S.E.M.)
Figure 7
Figure 7
Hes5 acts downstream of Jagged2 to prevent OLP generation. (a–c) In situ hybridisation of wild-type embryos shows that Hes5 is transiently expressed in pMN at the time of MN generation and extinguished after MN generation. (d) Control EP show transient expression of Jagged2 at the pMN. (e) Overexpression of Jagged2 (GOF-JAG2 48 h PE at HH14) maintains Hes5 expression at pMN. (f) Depletion of Jagged2 (LOF-JAG2 48 h PE of shJagged2 at HH14) inhibits Hes5 expression. (g–j) Hes5 overexpression retains Olig2+ and Sox9+ cells at pMN, even in the absence of Jagged2 function. Electroporated side is shown to the right, GFP (green) shows transfected cells. (k) Quantitative analysis of migratory Olig2+ and Sox9+ cells. Numbers are shown as total migratory cells on the electroporated side in each experimental condition. Bars correspond to the standard error (S.E.M.). (l) Summary of the genetic network operating for OLP generation
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