Delta-Notch signaling regulates oligodendrocyte specification

Hae-Chul Park and Bruce Appel*

Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA

* Author for correspondence (e-mail: b.appel{at}vanderbilt.edu)

Accepted 1 May 2003


    SUMMARY
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oligodendrocytes, the myelinating cell type of the central nervous system, arise from a ventral population of precursors that also produces motoneurons. Although the mechanisms that specify motoneuron development are well described, the mechanisms that generate oligodendrocytes from the same precursor population are largely unknown. By analysing mutant zebrafish embryos, we found that Delta-Notch signaling is required for spinal cord oligodendrocyte specification. Using a transgenic, conditional expression system, we also learned that constitutive Notch activity could promote formation of excess oligodendrocyte progenitor cells (OPCs). However, excess OPCs are induced only in ventral spinal cord at the time that OPCs normally develop. Our data provide evidence that Notch signaling maintains subsets of ventral spinal cord precursors during neuronal birth and, acting with other temporally and spatially restricted factors, specifies them for oligodendrocyte fate.

Key words: Delta, Notch, Oligodendrocytes, Motoneurons, Neural precursors, CNS, Spinal cord, Zebrafish


    INTRODUCTION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Delta-Notch signaling pathway regulates crucial aspects of neural cell fate specification and differentiation in both invertebrate and vertebrate embryos. For example, in Drosophila melanogaster, mutations that impair function of the Delta ligand, Notch receptor or other components of the signaling pathway resulted in the formation of excess neurons at the expense of epidermis (Campos-Ortega, 1995Go). Reduction of Notch signaling in vertebrate embryos also produced excess neurons at early stages of central nervous system (CNS) development, which correlated with loss of proliferative neural precursor cell populations and absence of some late-forming neurons (Chitnis et al., 1995Go; Henrique et al., 1997Go; Appel et al., 2001Go). Conversely, forced expression of Delta or constitutively active, intracellular forms of Notch (Notchac) blocked neuronal differentiation (Coffman et al., 1993Go; Chitnis et al., 1995Go; Henrique et al., 1997Go). These observations helped form the view that modulation of Delta-Notch signaling regulates the transition of proliferative neural precursors to post-mitotic differentiated cells. Thus, downregulation of Notch activity in subsets of precursors at different times might allow them to respond to other, temporally regulated instructive factors that specify them for different neuronal and glial fates (Bertrand et al., 2002Go). In this model, Notch signaling plays a permissive, rather than instructive, role in neural cell fate specification.

Several lines of evidence have now raised the possibility that Notch signaling directly promotes development of some types of glial cells. First, formation of Müller glia in the retina was promoted by forced expression of Notchac and by overexpression of Hes1 and Hes5, which are downstream components of the Notch signaling pathway (Furukawa et al., 2000Go; Hojo et al., 2000Go; Scheer et al., 2001Go). Second, in utero retroviral infection of the telencephelon of mouse embryos showed that Notchac could promote the formation of radial glia, which gave rise, postnatally, to astrocytes (Gaiano et al., 2000Go; Chambers et al., 2001Go). Third, in cells of the peripheral nervous system, activation of Notch signaling induced the formation of Schwann cells from purified neural crest stem cells (Morrison et al., 2000Go) and permitted glial development while blocking neurogenesis, in cultured quail neural crest cells (Wakamatsu et al., 2000Go). The manner in which Notch promotes glial development is unknown, although, in dorsal root ganglia, the asymmetric distribution of Numb proteins might influence Delta-Notch mediated decisions for neuronal or glial fate (Wakamatsu et al., 2000Go).

Until recently, most work investigating the role of Notch signaling in development of oligodendrocytes, the myelinating glial cell type of the CNS, focused on control of oligodendrocyte differentiation. When immature oligodendrocyte progenitor cells (OPCs) purified from rat optic nerve were exposed to Notch ligands, they did not differentiate, in contrast to control experiments (Wang et al., 1998Go). Consistent with this observation, selective inactivation of Notch1 in OPCs in vivo caused their premature differentiation (Genoud et al., 2002Go) and oligodendrocytes became myelinated too soon in mice heterozygous for a Notch1 mutation (Givogri et al., 2002Go). Additionally, astrocytes in demyelinating lesions of human multiple sclerosis patients upregulated Jagged1, a Notch ligand, and Jagged1 blocked maturation of purified human OPCs (John et al., 2002Go). These observations provide strong evidence that Notch signaling inhibits differentiation of cells once they enter the oligodendrocyte developmental pathway. Less clear is whether Notch signaling also promotes specification of OPCs from neural precursors. Neurospheres from delta-like 1 mutant mice produced a deficit of OPCs and, conversely, addition of a soluble Notch ligand to wild-type neurospheres enhanced their formation (Grandbarbe et al., 2003Go). However, multipotent adult hippocampus-derived progenitors produced fewer oligodendrocytes when they were transfected with Notchac (Tanigaki et al., 2001Go), although the markers used in this study did not distinguish between the possibilities that Notch activity blocked specification or subsequent differentiation. When electroporated into chick spinal cords, Notchac could promote formation of OPCs only when co-expressed with Olig2, a basic helix-loop-helix (bHLH) transcription factor necessary for oligodendrocyte development (Zhou et al., 2001Go).

Here, we provide in vivo, genetic evidence that Delta-Notch signaling is required for spinal cord oligodendrocyte specification. Spinal cord precursors of mutant zebrafish embryos that had reduced Notch signaling activity stopped dividing prematurely and developed as early-born neurons at the expense of later-born oligodendrocytes. By contrast, transgenic embryos in which Notchac was driven by a conditional expression system had a deficit of neurons and concomitant increase of OPCs. However, formation of OPCs in response to Notchac was restricted to their normal time and place of development. We also show that, subsequent to OPC specification, Notchac blocked oligodendrocyte differentiation, consistent with previous in vitro data. Our data indicate that Notch signaling is important throughout development of oligodendrocytes by promoting specification of OPCs from neural precursors and regulating their subsequent differentiation into mature oligodendrocytes.


    MATERIALS AND METHODS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Wild-type, mutant and transgenic zebrafish
Embryos were produced by pair matings of fish raised in the Vanderbilt University Zebrafish Facility. Except for heat-shock experiments, embryos were raised at 28.5°C and staged according to hours post-fertilization (hpf) and morphological criteria. We used the following mutant alleles: dladx2 (Appel et al., 1999Go); dld/aeiAR33 (Holley et al., 2000Go); and mibta52b (Jiang et al., 1996Go). For some dla;dld double mutant experiments, we used a new hypomorphic dld allele that we will describe elsewhere. We produced double mutant embryos by intercrossing dla+/-;dld+/- adults. Double mutant embryos were identified by phenotype, which included a somite defect characteristic of dld-/- embryos, an irregularly shaped brain, an abnormally curved body and absence of trunk pigmentation. The double mutant combination was fully penetrant, producing phenotype in approximately 1/16 of progeny from matings of doubly heterozygous adults. TG[hsp:GAL4] and TG[UAS:Notch1aac] transgenic lines were described previously (Scheer, 1999). To produce TG[hsp:GAL4];TG[UAS:Notch1aac] embryos, we crossed carriers that were either homozygous or heterozygous for each transgene.

Labeling methods and photomicroscopy
Manually dechorionated embryos were labeled with BrdU by incubating them for 20 minutes on ice in a solution of 10 mM BrdU and 15% DMSO in embryo medium (15 mM NaCl, 0.5 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 0.15 mM KH2PO4, 0.05 mM NH2PO4, 0.7 mM NaHCO3). The BrdU solution was replaced with embryo medium and embryos were incubated for 20 minutes at 28.5°C. The embryos were then anesthetized using 3-aminobenzoic acid ethyl ester, fixed in 4% paraformaldehyde, embedded in 1.5% agar/5% sucrose and frozen in 2-methyl-butane chilled by immersion in liquid nitrogen. Sections (10 µm) were obtained by using a cryostat microtome. Sections were treated with 2 N HCl for 1 hout, washed with PBS, blocked in PBS plus 2% sheep serum and 2 mg ml-1 bovine serum albumen (BSA) and then incubated with anti-BrdU antibody.

Previously described RNA probes included sox10 (Dutton et al., 2001Go), ngn1 (Blader et al., 1997Go), tlxa (Andermann and Weinberg, 2001Go), olig2 and plp1/dm20 (Park et al., 2002Go). In situ RNA hybridization was performed as described previously (Hauptmann and Gerster, 2000Go). Embryos for sectioning were treated as described above. Flat mounted embryos were dissected from the yolk and mounted in 75% glycerol.

For immunohistochemistry, we used the following primary antibodies: mouse anti-BrdU [G3G4, 1:1000, Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa, USA], mouse anti-HuC/D (1:20, Molecular Probes, Eugene, Oregon, USA), rabbit anti-phospho-histone-H3 (1:1000, Upstate Biotechnology, Charlottesville, Virginia, USA). For fluorescent detection of antibody labeling, we used Alexa Fluor 568 goat anti-mouse conjugate (1:200, Molecular Probes) and Alexa Fluor 488 goat anti-rabbit conjugate (1:200, Molecular Probes). Hoechst labeling was performed by incubating sections in Hoechst for 10 minutes following immunolabeling.

In situ hybridization and Hoechst/immunofluorescence images were obtained using a Spot digital camera mounted on a compound microscope. All other fluorescence images were obtained using a Zeiss LSM510 Meta laser scanning confocal microscope. All images were imported into Adobe Photoshop. Image manipulation was limited to levels, curves, hue and saturation adjustments.

Heat shock experiments
Heat shock was applied to transgenic and control embryos in embryo medium for 30 minutes at 40°C to induce Notch1aac expression. Following heat shock, embryos were incubated at 28.5°C. Heat-shocked, transgenic embryos were identified by phenotype, which consisted of touch insensitivity, abnormal brain morphology and indistinct somites. We confirmed our identification by labeling a subset of embryos with anti-Myc antibody, which detects the Myc epitope fused to Notch1aac (Scheer et al., 2001Go). To analyse the number of spinal cord olig2+ and sox10+ cells, we counted cells in 20 transverse sections from each of five nontransgenic and five transgenic embryos. Statistical significance was determined using Student's t test.


    RESULTS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Delta-Notch signaling is required for spinal cord oligodendrocyte formation
We showed previously that zebrafish embryos mutant for deltaA (dla), which encodes one of four known zebrafish Delta-like ligands, had reduced numbers of proliferative spinal cord cells and excess neurons (Appel et al., 2001Go). We extend those data here to show that, at 24 hpf, spinal cords of double mutant embryos lacking function of dla and the related deltaD (dld) gene (dla-/-;dld-/- embryos) had few S-phase cells as revealed by BrdU incorporation (Fig. 1B). Similarly, spinal cord cells of embryos homozygous mutant for mind bomb (mib), which encodes a ubiquitin ligase necessary for efficient Notch signaling (Itoh et al., 2003Go), did not incorporate BrdU (Fig. 1C). Immunocytochemical labeling of phosphorylated histone H3, which marks M-phase cells, and Hu proteins, which reveals mostly post-mitotic neurons and some neural cells in S-phase (Marusich et al., 1994), showed that 24 hpf wild-type embryos had a small number of M-phase cells located near the ventricle and neurons distributed along the pial surface of the spinal cord (Fig. 1D). By contrast, dla-/-;dld-/- and mib-/- embryos had few, if any, M-phase cells and large excesses of neurons (Fig. 1E,F). In mutant embryos, neurons appeared in positions normally occupied by proliferative cells. In fact, nearly every spinal cord cell was Hu+. Thus, Delta-Notch signaling limits formation of spinal cord neurons and maintains a population of proliferative neural precursors.



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Fig. 1. Delta-Notch is required to maintain the spinal cord precursor pool and for OPC specification. All images show transverse sections, dorsal to the top. (A-C) 1 dpf embryos treated with BrdU and anti-BrdU antibody (pink) to mark S-phase cells and Hoechst (blue) to reveal nuclei. dla-/-;dld-/- (B) and mib-/- (C) embryos had fewer S-phase spinal cord cells than wild type (A). Spinal cords are outlined. (D-F) 1 dpf embryos labeled with anti-phospho-histone-H3 antibody to mark M-phase cells (green) and pan neuronal anti-Hu antibody (red). dla-/-;dld-/- (E) and mib-/- (C) embryos had excess neurons and a deficit of M-phase cells relative to wild type (D). (G-I) 2 dpf embryos probed for sox10 expression, which marks OPCs, by in situ RNA hybridization. Spinal cord cells of dla-/-;dld-/- (H) and mib-/- (I) embryos did not express sox10. (J-L) 3 dpf embryos probed for plp1/dm20 RNA expression, which marks premyelinating oligodendrocytes. dla-/-;dld-/- (K) and mib-/- (L) embryos did not express plp1/dm20. Scale bars: 20 µm.

 
A ventral spinal cord precursor domain, called pMN, produces first motoneurons and then oligodendrocytes (Richardson et al., 2000Go; Zhou and Anderson, 2002Go). Previous work showed that reduction of Notch activity in zebrafish embryos resulted in formation of excess primary motoneurons (Dornseifer et al., 1997Go; Appel and Eisen, 1998Go; Haddon et al., 1998Go; Appel et al., 2001Go). As production of excess neurons occurred at the expense of proliferative precursors in dla-/-;dld-/- and mib-/- embryos, we predicted that these embryos would lack oligodendrocytes. In zebrafish, the earliest gene expression apparently specific to oligodendrocyte lineage cells is that of sox10, which, at 48 hpf, marks OPCs (Park et al., 2002Go) (Fig. 1G). Neither dla-/-;dld-/- nor mib-/- embryos expressed sox10 in spinal cord (Fig. 1H,I). To confirm the absence of OPCs from mutant embryos, we tested expression of plp1/dm20, which marks premyelinating oligodendrocytes (Timsit et al., 1995Go; Park et al., 2002Go). By contrast to 3 days post-fertilization (dpf) wild-type embryos (Fig. 1J), neither dla-/-;dld-/- nor mib-/- embryos expressed plp1/dm20 (Fig. 1K,L). These observations are consistent with the possibility that, within the pMN domain, Delta-Notch signaling limits formation of early-born primary motoneurons and promotes formation of later-born OPCs at least in part by maintaining a subset of proliferative precursors.

Delta-Notch signaling regulates olig2 and ngn1 expression differently
A combinatorial code of bHLH proteins might specify pMN precursors for motoneuron or oligodendrocyte fates, whereby cells that express Olig2 and Ngns develop as motoneurons and those that express only Olig2 develop as oligodendrocytes (Zhou and Anderson, 2002Go). Thus, we examined expression of zebrafish olig2 and ngn1 to gain further insight into the basis of the mutant phenotypes described above. In 24 hpf wild-type embryos, a discrete group of ventral spinal cord cells uniformly expressed olig2 (Fig. 2A) (Park et al., 2002Go). By contrast, ngn1 was expressed in a mosaic throughout the dorsoventral spinal cord axis (Fig. 2B). Double RNA in situ hybridization showed that a subset of olig2+ cells expressed ngn1 (Fig. 2C). These expression patterns are consistent with immunolocalization of Olig2 and Ngn2 proteins in bird and rodent embryos (Mizuguchi et al., 2001Go; Novitch et al., 2001Go; Zhou et al., 2001Go; Zhou and Anderson, 2002Go) and consistent with the idea that motoneurons develop from olig2+, ngn1+ cells, whereas oligodendrocytes arise from olig2+, ngn1- cells.



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Fig. 2. Delta-Notch signaling maintains olig2 expression and inhibits ngn1 expression. (A-C) Transverse sections, dorsal up, of 24 hpf embryos. Whereas a discrete group of ventral spinal cord cells expressed olig2 (A), cells that expressed ngn1 were scattered throughout the spinal cord (B). In situ double RNA hybridization revealed that a subset of olig2+ cells (C, red) expressed ngn1 (blue, arrows). (D-G) Dorsal views, anterior left, of 12 hpf embryos. (D,E) Embryos hybridized with probes for olig2 and tlxa. Brackets indicate olig2 expression. tlxa expression marks prospective Rohon-Beard (RB) sensory neurons. In wild-type embryos, olig2 expression was maintained throughout the length of the trunk neural keel (D, arrow). In dla-;dld- mutant embryos, identified by the excess Rohon-Beard phenotype, anterior neural keel cells did not maintain olig2 expression (E). (F,G) Embryos hybridized with probe to reveal ngn1 expression. Brackets indicate region of neural keel that expressed olig2. Excess cells in dla-;dld- mutant embryos expressed ngn1 at high level (G) compared to wild type (F). Scale bar: 20 µm (A-C); 50 µm (D-G).

 
At 24 hpf, spinal cord cells of dla-/-;dld-/- and mib-/- embryos did not express olig2 or ngn1 (H.-C. Park, unpublished). One explanation for this observation is that prematurely differentiating neurons of mutant embryos did not maintain olig2 and ngn1 expression. Thus, we examined mutant embryos at early stages of neurogenesis. At 10.5 hpf, medial neural plate cells of wild-type embryos, which occupy ventral spinal cord upon completion of neurulation, expressed olig2 (Park et al., 2002Go). All embryos produced by intercrosses of dla+/-;dld+/- adults appeared to express olig2 RNA similarly at 10.5 hpf (data not shown). At a comparable stage, the excess primary motoneuron phenotype of dla-/- and mib-/- embryos was evident (Appel et al., 2001Go). However, by the seven-somite stage (12 hpf), anterior ventromedial neural keel cells of double mutant embryos lacked detectable levels of olig2 transcripts (Fig. 2E), whereas wild-type cells maintained olig2 expression along the length of the neural keel (Fig. 2D). mib-/- embryos expressed olig2 similarly to dla-/-;dld-/- embryos (H.-C. Park, unpublished).

Excess neural plate cells of Notch signaling deficient zebrafish embryos expressed ngn1, consistent with the excess primary neuron phenotype of the same embryos (Appel et al., 2001Go). Similarly, at 12 hpf, when olig2 expression was absent from the anterior neural keel, the density of ngn1+ cells was greater in dla-/-;dld-/- embryos than in wild-type (Fig. 2F,G). Taken together, these data indicate that Delta-Notch signaling creates a combinatorial bHLH protein code by inhibiting ngn1 expression and maintaining olig2 expression.

Notch signaling promotes OPC specification and inhibits oligodendrocyte differentiation
One interpretation of our data is that the requirement for Notch signaling in formation of spinal cord oligodendrocytes indirectly arises from the well-known role of Notch signaling in maintaining neural precursor populations. In this view, Notch signaling prevents neuronal differentiation of a subset of precursors, which then might be specified for oligodendrocyte development by another, instructive signal. An alternative, but not mutually exclusive, possibility is that Notch signaling actively promotes specification of spinal cord oligodendrocytes. To test this possibility, we used a transgenic GAL4/UAS system controlled by a heat-shock promoter to drive ubiquitous expression of a constitutively active form of zebrafish Notch1a (Notch1aac). Previous studies showed that Notch1aac expressed in this way persisted for at least 17 hours, effectively blocked neurogenesis and promoted formation of Müller glia in the retina (Scheer et al., 2001Go; Scheer et al., 2002Go).

We first established that heat induction of Notch1aac expression inhibits the formation of spinal cord neurons during embryonic periods relevant to OPC specification. When we induced Notch1aac expression repeatedly at 10, 24 and 36 hpf, to ensure a high level of Notch1aac expression, we found that transgenic embryos lacked most spinal cord neurons, marked by Hu immunofluorescence, at 48 hpf (Fig. 3). Labeling to detect the Myc epitope fused to Notch1aac confirmed that cells strongly expressed Notch1aac at 48 hpf (H.-C. Park, unpublished). Those neurons present in heat-shocked embryos probably were born before Notch1aac accumulated to high level. Consistent with this, when we induced Notch1aac at mid-gastrulation (8 hpf), no spinal cord neurons were present at 24 hpf (H.-C. Park, unpublished). Thus, prolonged Notch1aac expression effectively inhibits spinal cord neurogenesis.



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Fig. 3. Notch1aac expression blocks neurogenesis. Transverse sections, dorsal to top, labeled by immunocytochemistry to reveal Hu proteins. (A) Wild-type control embryo revealing normal distribution of neurons at 48 hpf. (B) 48 hpf transgenic embryo, in which Notch1aac expression was induced at 10, 24 and 36 hpf, had a deficit of neurons. Scale bar: 20 µm.

 
We next examined olig2 expression in transgenic embryos. Heat induction of Notch1aac expression at 8 and 24 hpf did not alter spinal cord olig2 expression at 26 hpf, although olig2 expression was induced in somite cells (Fig. 4B,E). However, at 36 hpf, transgenic embryos that had been heat shocked at 8 and 24 hpf had a 2.2-times increase in the number of olig2+ ventral spinal cord cells, although olig2 transcripts were no longer evident in somites (Fig. 4D,E). At 48 hpf, the number of olig2+ spinal cord cells in transgenic embryos that had been heat shocked at 8, 24 and 36 hpf did not appear to differ significantly from control embryos (H.-C. Park, unpublished). These data raise several possibilities. First, the ectopic somite expression of olig2 indicates that olig2 might be a regulatory target of the Notch signaling pathway. However, Notch activity alone was not sufficient to promote olig2 expression in spinal cord. Second, the increase in the number of spinal cord cells that expressed olig2 in transgenic embryos, compared with the wild type, occurred between 24 and 36 hpf. This is the period when secondary motoneurons, which arise from olig2+ precursors, differentiate. As Notch1aac expression blocked the formation of neurons, we interpret our data to mean that Notch activity maintains a population of proliferative olig2+ precursors during the time of secondary motoneuron formation. Finally, the apparent downregulation of olig2 by 48 hpf in transgenic embryos corresponds to the time that olig2 is downregulated and OPCs are specified in normal embryos (Park et al., 2002Go). One possible explanation for this observation is that Notch1aac drives olig2+ precursors into the OPC developmental pathway at 48 hpf.



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Fig. 4. Notch1aac increases the number of ventral spinal cord cells that express olig2. Transverse sections, dorsal to top, labeled by olig2 RNA hybridization. (A) Wild-type control embryo revealing normal distribution of olig2+ cells at 26 hpf. (B) 26 hpf transgenic embryo in which Notch1aac was induced at 8 and 24 hpf expressed olig2 similarly to wild type in spinal cord (sc) but expressed olig2 ectopically in somites (so). (C) Wild-type control embryo revealing normal distribution of olig2+ cells at 36 hpf. (D) Excess spinal cord cells expressed olig2 at 36 hpf in transgenic embryo that was heat shocked at 8 and 24 hpf. (E) Quantification of olig2+ cells at 24 and 36 hpf in wild-type (wt) and Notch1aac-expressing embryos. Bars indicate the average number of olig2+ cells per transverse section. Data were obtained from 20 sections from each of five control and five heat-shocked transgenic embryos for each time point. P<0.00000001 by Student's t test. Scale bar: 20 µm.

 
To test whether Notch activity can promote formation of OPCs, we examined sox10 expression. Although somites of 26 hpf embryos that had been heat shocked at 8 and 24 hpf strongly expressed sox10, spinal cord cells did not (Fig. 5B), indicating that Notch1aac is not sufficient to promote premature formation of OPCs. 52 hpf transgenic embryos that were heat shocked at 8, 24 and 36 hpf had a 1.9-times excess of spinal cord sox10+ cells compared with control embryos (Fig. 5D,E). The excess sox10+ spinal cord cells occupied positions at which motoneurons normally reside. Because the increase in the number of sox10+ spinal cord cells is similar to the increase in olig2+ cells at 36 hpf, these data support the possibility that continuous Notch signaling directs ventral spinal cord cells that normally develop as neurons into the oligodendrocyte pathway. In contrast to embryos assayed at 26 hpf, somites of Notch1aac-expressing embryos of 52 hpf embryos did not express sox10, indicating that Notch activity cannot continuously promote sox10 expression in mesodermal cells. However, these embryos had many sox10+ cells adjacent to the spinal cord and notochord (Fig. 5D). In wild-type embryos, sox10+ cells that occupy similar positions originate in neural crest (Dutton et al., 2001Go) (Fig. 5C). Thus, Notch activity might promote formation of neural crest or particular subsets of neural crest, consistent with previous data showing that Notch signaling is required for neural crest formation in zebrafish (Cornell and Eisen, 2000Go) and that Notch activity can promote formation of Schwann cells from cultured neural crest stem cells (Morrison et al., 2000Go). Taken together, our data show that Notch signaling can promote sox10 expression and oligodendrocyte specification in the spinal cord, but that these functions are limited to the normal time and place of oligodendrocyte development.



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Fig. 5. Notch1aac promotes OPC specification. Transverse sections, dorsal to top, labeled by sox10 RNA hybridization. (A) 26 hpf wild-type embryo. Spinal cord (sc), somite (so) and notochord (nc) cells did not express sox10. (B) Transgenic embryo in which Notch1aac was induced at 8 and 24 hpf. Somite cells expressed sox10 but spinal cord and notochord cells did not. (C) 52 hpf wild-type embryo hybridized with sox10 probe. sox10+ cells had mostly dispersed from ventral spinal cord (bracket). Neural crest-derived cells on the medial migratory pathway also expressed sox10 (arrow). (D) Transgenic embryo in which Notch1aac was induced at 8, 24 and 36 hpf. Excess ventral spinal cord cells expressed sox10 (bracket) as did presumptive neural crest cells (arrows). (E) Quantification of sox10+ cells at 52 hpf in wild-type (wt) and Notch1aac-expressing embryos. Bars indicate the average number of sox10+ cells per transverse section. Data were obtained from 20 sections from each of 5 control and 5 heat-shocked transgenic embryos. P<0.00000001 by Student's t test. Scale bar: 20 µm.

 
Because experiments using cultured cells showed that Notch activity can block OPC differentiation (Wang et al., 1998Go; John et al., 2002Go), we induced Notch1aac expression at 60 hpf, after OPC specification, and assayed expression of dm20/plp1, which marks premyelinating oligodendrocytes, at 3.5 dpf. Compared with control embryos, few cells in experimental embryos expressed dm20/plp1 (Fig. 6A,B). Thus, by driving ubiquitous Notch1aac expression after OPC specification, we prevented the progression of OPCs to a more mature developmental stage. Taken together, our data provide in vivo evidence that Notch signaling first promotes OPC specification and then inhibits their differentiation into mature oligodendrocytes.



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Fig. 6. Notch1aac inhibits oligodendrocyte differentiation. Side views of 3.5 dpf embryos, dorsal to top. (A) Wild-type embryo showing normal distribution of plp1/dm20+ cells in spinal cord (sc). (B) Transgenic embryo in which Notch1aac was induced at 60 hpf. The number of plp1/dm20+ cells was greatly reduced. Arrows indicate plp1/dm20+ cells in ventral spinal cord. Notochord indicated by nc. Scale bar: 20 µm.

 

    DISCUSSION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Here, we presented two tests of a hypothesis that Delta-Notch signaling regulates specification of a common population of neural precursors for neuronal and oligodendrocyte fates. First, we found that mutant zebrafish embryos that had reduced Notch signaling activity failed to maintain the proliferative neural precursor population, had excess neurons and lacked oligodendrocytes. Second, we found that transgenic, conditional expression of a constitutively active form of Notch blocked neuronal development and promoted formation of excess OPCs in ventral spinal cord. Our data support a model in which Delta-Notch signaling prevents ventral spinal cord precursors from developing as neurons and then, acting with other temporally and spatially limited factors, specifies them for oligodendrocyte fate.

Delta-Notch signaling is required for OPC specification
Because mouse embryos that are homozygous for null mutations of Delta or Notch genes die at early stages of neural development, there are few data that address the requirement of Notch signaling for vertebrate CNS glial specification. Recently, this limitation was circumvented through analysis of mice in which Notch1 was conditionally inactivated in the cerebellum. These mice prematurely expressed neuronal markers and had reduced number of mutant cerebellar cells that expressed the glial marker GFAP (Lutolf et al., 2002Go). In an alternative approach, neurospheres were derived from Delta-like 1 mutant mice. After culturing, mutant neurospheres produced excess neurons and a deficit of oligodendrocytes and astrocytes compared with controls (Grandbarbe et al., 2003Go). Additionally, retinas of mice that were homozygous for a mutation of Hes5, which encodes a downstream effector of Notch signaling, had fewer Müller glia than the wild type (Hojo et al., 2000Go). These observations are consistent with the idea that Delta-Notch signaling regulates neuronal-glial fate decisions.

Several lines of evidence point toward a role for Delta-Notch signaling in regulating specification of motoneuron and oligodendrocyte fates in zebrafish. First, prospective primary motoneurons were usually replaced when they were removed at the 11-somite stage (Appel et al., 2001Go) but not at the 13-somite stage (Eisen et al., 1989Go). This is similar to observations that ablated neuroblasts were replaced by neighboring cells in grasshoppers (Doe and Goodman, 1985Go) and raised the possibility that primary motoneurons, like grasshopper neuroblasts, inhibit neighboring precursors from adopting the same fate. Second, prospective primary motoneurons expressed higher levels of dla and dld than neighboring cells (Appel and Eisen, 1998Go; Haddon et al., 1998Go), indicating that Notch ligands are present at the right time and place to regulate specification of cells that arise in close proximity to primary motoneurons. Third, mutant zebrafish that had reduced levels of Notch signaling had excess primary motoneurons and a concomitant deficit of later-born secondary motoneurons, showing that Delta-Notch signaling regulates specification of neural precursors for different neuronal fates (Appel et al., 2001Go). Finally, medial neural plate cells, which occupy ventral spinal cord upon completion of neurulation, gave rise to primary motoneurons and oligodendrocytes (Park et al., 2002Go). Thus, Delta proteins expressed by primary motoneurons could regulate specification of nearby cells for oligodendrocyte fate.

Here, we showed that dla-/-;dld-/- and mib-/- embryos did not produce OPCs or premyelinating oligodendrocytes. Additionally, neural precursors prematurely exited the cell cycle and differentiated as neurons in these embryos. As secondary motoneurons and oligodendrocytes arise after primary motoneurons, one interpretation of our data is that Notch signaling prevents a subset of ventral spinal cord precursors from developing as primary motoneurons, enabling them to take later neuronal or oligodendrocyte fates. In this view, downregulation of delta gene expression during primary motoneuron differentiation would result in a decrease of Notch activity in neighboring precursors. A release from Notch-mediated inhibition soon after primary motoneuron specification might allow a cell to develop as a secondary motoneuron, whereas a later release might result in oligodendrocyte development (Fig. 7). Thus, temporal regulation of Notch signaling might underlie the temporal switch in production of primary motoneurons to secondary motoneurons to oligodendrocytes.



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Fig. 7. Model for Delta-Notch regulated specification of pMN precursors for motoneuron and oligodendrocyte fates. Primary motoneurons (pmn) are the first cells to differentiate and activate Notch in neighboring cells via Delta. Notch activation maintains these cells as precursors during the period of primary motoneuron specification. Downregulation of Delta expression in differentiating primary motoneurons results in loss of Notch activity in some precursors, which then adopt secondary motoneuron (smn) fate. These cells upregulate Delta expression, maintaining high Notch activity in remaining precursors, which are specified as OPCs. Maintenance of high Notch activity in OPCs inhibits their differentiation into premyelinated oligodendrocytes (pre oligo). In dla-/-;dld-/- embryos, which have reduced levels of Notch activity, excess pMN precursors are specified as primary motoneurons at the expense of secondary motoneurons and oligodendrocytes. This model does not account for possible lineage relationships between different cell types.

 
A switch between production of neurons and glial cells was proposed to be regulated by bHLH proteins (Vetter, 2001Go). In the ventral spinal cord, motoneuron and oligodendrocyte precursors expressed Olig bHLH proteins, which are structurally similar to proneural Ngns (Lu et al., 2000Go; Takebayashi et al., 2000Go; Zhou et al., 2000Go). During the period of motoneuron production, a subset of Olig+ cells expressed Ngns (Mizuguchi et al., 2001Go; Novitch et al., 2001Go; Zhou et al., 2001Go) (data shown here). Later, Ngn expression subsided, coincident with the time at which oligodendrocytes are thought to be specified (Zhou et al., 2001Go). These observations, coupled with various functional tests, led to the proposal that Ngn and Olig proteins create a simple bHLH protein code in which Ngn and Olig expression together specify motoneuron development and Olig alone, upon Ngn downregulation, specifies oligodendrocyte development (Zhou et al., 2001Go; Zhou and Anderson, 2002Go).

Our data provide evidence supporting the importance of a bHLH protein code to motoneuron and oligodendrocyte specification and show that Delta-Notch signaling is required to establish the code. We have shown that the failure to restrict ngn1 expression to a subset of medial neural plate cells in Notch signaling deficient zebrafish embryos correlated with formation of excess neurons, consistent with other observations that Notch signaling inhibits proneural genes expression and neuronal development in vertebrate and invertebrate embryos. Furthermore, dla-/-;dld-/- and mib-/- embryos failed to maintain a proliferative population of olig2+ cells. We interpret this to mean that, in the absence of Delta-Notch mediated inhibition, uniformly high levels of Ngns cause all olig2+ neural precursors to stop dividing and differentiate as neurons at the expense of oligodendrocytes. Thus, in normal embryos, high levels of Notch activity prevents ngn gene expression in a subset of olig2+ neural precursors, reserving them to produce other cell types, such as oligodendrocytes, at a later time. In this view, Delta-Notch signaling might play a purely permissive role in neural cell fate diversification, by regulating the ability of neural precursors to respond to other instructive signals.

Delta-Notch signaling promotes oligodendrocyte specification
Another possibility we sought to test is that Notch activity might specify neural precursors for oligodendrocyte fate. Although several reports already addressed this possibility, the data are ambiguous. For example, Notch activity blocked formation of oligodendrocytes from cultured adult hippocampus-derived progenitors (AHPs) (Tanigaki et al., 2001Go). However, this study assessed oligodendrocyte development using only markers that reveal late stages of oligodendrocyte differentiation. Because Notch activity inhibits oligodendrocyte differentiation (see below), OPCs might have been produced from AHPs but not detected in these experiments. In a different cell culture assay, Notch activity promoted formation of both OPCs and astrocytes, apparently from common precursors, within neurospheres (Grandbarbe et al., 2003Go). However, there are no data that support the presence of a common precursor of oligodendrocytes and astrocytes in vivo, leaving open the question of whether Notch signaling specifies OPCs during development. Finally, expression of constitutively active Notch, alone, by electroporation was not sufficient to promote OPC formation in chick spinal cords (Zhou et al., 2001Go).

Here, we used a transgenic, conditional expression system to drive high levels of Notch1aac in intact zebrafish embryos throughout the period of neurogenesis. These embryos did not prematurely nor ectopically express OPC markers. However, they had an approximately twofold excess of olig2+ and sox10+ ventral spinal cord cells at 36 and 52 hpf, respectively. By contrast, spinal cord cells of dla-/-;dld-/- and mib-/- embryos initiated but did not maintain olig2 expression and never expressed sox10. Because Notch1aac simultaneously blocked neuronal development, we interpret our results to mean that Notch diverts precursors that would otherwise develop as ventral spinal cord neurons toward an oligodendrocyte fate (Fig. 7). In contrast to canonical models of Notch signaling, which suggest that Notch activity must be downregulated for precursor cells to enter a developmental pathway, our work shows that continuous Notch activity causes ventral spinal cord precursors to develop as oligodendrocytes instead of neurons.

Our observation that Notch1aac expression promoted formation of excess OPCs only in ventral spinal cord at the normal time of OPC specification indicates that Notch must act with other spatially and temporally regulated factors. One candidate is Olig2, as co-electroporation of plasmids that encode Olig2 and constitutively active Notch promoted ectopic OPC development in chick embryos (Zhou et al., 2001Go). However, in zebrafish, OPCs do not appear to be specified for at least 36 hours after initiation of olig2 expression. The Notch pathway must be active in a subset of these cells throughout the period of olig2 expression to block motoneuron development. This raises the possibility that the factor that controls the timing of oligodendrocyte specification is downstream of olig2.

One important problem our data do not address is the nature of motoneuron and oligodendrocyte precursors. At one extreme, pMN precursors might be multipotent, having equivalent potential to develop as primary motoneurons, secondary motoneurons or oligodendrocytes. At the other extreme, pMN precursors might be restricted to a single fate. A third alternative is that some precursors produce primary and secondary motoneurons whereas others produce only oligodendrocytes. Lineage analysis performed in chick embryos showed that motoneurons and oligodendrocytes can share a precursor but left unresolved the question of when these cell types become separated in the lineage (Leber et al., 1990Go). A second problem that must be considered is whether pMN cells give rise only to motoneurons and oligodendrocytes or whether they also produce other cell types. Various kinds of ventral spinal cord interneurons in zebrafish embryos have been described by morphology but their origin is unknown (Bernhardt et al., 1990Go; Bernhardt et al., 1992Go; Hale et al., 2001Go). A complete understanding of the mechanism by which Delta-Notch signaling regulates motoneuron, oligodendrocyte and possibly interneuron specification will require careful cell lineage analysis combined with conditional manipulation of Notch signaling activity.

Delta-Notch signaling couples oligodendrocyte specification and differentiation
Significantly, various data indicate that Notch signaling inhibits oligodendrocyte differentiation. In vitro experiments showed that Notch activity could inhibit differentiation of purified OPCs (Wang et al., 1998Go), selective inactivation of Notch1a in OPCs in vivo caused their premature differentiation (Genoud et al., 2002Go) and oligodendrocytes became myelinated too soon in mice heterozygous for a Notch1 mutation (Givogri et al., 2002Go). Additionally, astrocytes in demyelinating lesions of human multiple sclerosis patients upregulated Jagged1, a Notch ligand, and Jagged1 blocked maturation of purified human OPCs (John et al., 2002Go). Our observation that Notch1aac inhibited expression of plp1/dm20 in vivo provides support for the idea that Notch signaling regulates maturation of oligodendrocytes. Taken together with our demonstration that Delta-Notch signaling promotes OPC formation, our work indicates that Notch signaling both promotes specification of neural precursors for oligodendrocyte fate and subsequently regulates their differentiation (Fig. 7). Thus, Notch activity couples the control of oligodendrocyte specification and differentiation, which might help to match the development of myelinating oligodendrocytes to their target axons.


    ACKNOWLEDGMENTS
 
We thank L. Solnica-Krezel, C. Chiang and S. Oh for comments on the manuscript, J. Campos-Ortega and his lab members for developing and distributing the GAL4/UAS transgenic lines, and D. Ward and L. McClung for technical assistance. The anti-BrdU antibody, developed by S. J. Kaufman, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Confocal microscopy was performed using equipment made available by the VUMC Cell Imaging Core Resource, supported by NIH grants 1S10RR15682-1, CA68485 and DK20593. Funds for this work were provided by the Whitehall Foundation and NIH grant HD38118.


    REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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