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First published online 30 May 2007
doi: 10.1242/dev.005520

Development 134, 2435-2447 (2007)
Published by The Company of Biologists 2007
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Physiological Notch signaling promotes gliogenesis in the developing peripheral and central nervous systems

Merritt K. Taylor*, Kelly Yeager and Sean J. Morrison{dagger}

Howard Hughes Medical Institute, Life Sciences Institute, Department of Internal Medicine, and Center for Stem Cell Biology, University of Michigan, Ann Arbor, MI 48109-2216, USA.


Figure 1
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  		Fig. 1. The numbers of NCSCs, neurons and glia are reduced in the gut after Rbpsuh deletion. (A,B) Transverse sections of the guts from E13.5 control (A) or Wnt1-Cre+ Rbpsuhfl/fl (B) mouse embryos were stained for neurons (TuJ1) and glia (BFABP). Scalebar: 10 µm. (C) The numbers of p75+ neural crest cells that initially colonized the gut did not differ between E10.5 control and Wnt1-Cre+ Rbpsuhfl/fl embryos (the number of p75+ cells per section; three sections per gut region per mouse, three mice per genotype). (D-F) At representative levels of the developing gut (foregut, midgut and hindgut), the numbers of neurons (TuJ1+) and glia (BFABP+) per section were similar to wild type in Wnt1-Cre+ Rbpsuhfl/fl embryos at E10.5 (D) but significantly (*, P<0.05) lower at E14.5 (E) and E18.5 (F). (G) There was no difference in the rate of proliferation of p75+ cells at E14.5 between control and mutant embryos (BrdU was administered for 30 minutes; three to seven sections per gut region per mouse, three to four mice per genotype). Additionally there was no significant difference in the rate of cell death between control and mutant mice at any stage of development (E10.5, E14.5 and E18.5) or at any level of the gut (foregut, midgut and hindgut) based on staining for activated caspase 3 (data not shown). (H) However, a significantly (*, P<0.05) lower percentage of cells from E13.5 Wnt1-Cre+ Rbpsuhfl/fl guts gave rise to multilineage NCSC colonies in clonal-density cultures in both standard medium and medium supplemented with neuregulin (+Nrg) (three to seven independent experiments). All error bars indicate s.d.

 

Figure 2
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  		Fig. 2. Rbpsuh is required for gliogenesis in sensory ganglia. (A-D) Transverse sections of dorsal root ganglia from control and Wnt1-Cre+ Rbpsuhfl/fl mouse embryos at E14.5 (A,B) and E18.5 (C,D) were stained for neurons (TuJ1+, green) and glia (BFABP+, red). Scale bar: 50 µm. (E) There was no difference between control and Wnt1-Cre+ Rbpsuhfl/fl embryos at E10.5 in terms of the number of p75+ neural crest progenitors that initially migrated into the ganglion (three sections per mouse and three mice per genotype). (F) Although no difference in neurogenesis was observed at E10.5 (glia could not be detected at this stage), neurogenesis was significantly (*, P<0.05) reduced in Wnt1-Cre+ Rbpsuhfl/fl sensory ganglia at E14.5 and E18.5 (four to nine sections per mouse, seven to eight mice per genotype). Gliogenesis was even more profoundly reduced, as virtually no glia could be detected in Wnt1-Cre+ Rbpsuhfl/fl sensory ganglia. (G,H) We did not detect any difference in the rate of proliferation (G, the percentage of cells that incorporated a 30-minute pulse of BrdU in vivo at E14.5), or cell death (H, the percentage of cells expressing activated caspase 3). Error bars indicate s.d.

 

Figure 3
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  		Fig. 3. Neural crest progenitors with glial potential persist in Wnt1-Cre+ Rbpsuhfl/fl sensory ganglia despite the lack of gliogenesis in vivo, but require stimulation by a gliogenic factor to undergo gliogenesis in culture. (A-N) We dissociated and cultured dorsal root ganglion cells from E13.5 control and Wnt1-Cre+ Rbpsuhfl/fl mouse embryos at clonal density (500 cells per 35 mm dish) under adherent conditions. (A,B) Multilineage colonies (N+G+M) contained neurons (N, peripherin+), glia (G, Gfap+) and myofibroblasts [M, Sma+ (Acta2/Actg1 - Mouse Genome Informatics)]. Other colonies were also counted. The data are expressed as the percentage of cells added to culture that formed each type of colony. Wnt1-Cre+ Rbpsuhfl/fl cells formed significantly fewer multilineage and glia-containing (G-containing) colonies (which included all multilineage, glia-only, and other colonies that contained glia) as compared with control cells (A; *, P<0.05). Wnt1-Cre+ Rbpsuhfl/fl cells did not differ from control cells in their ability to form neuron-containing or myofibroblast-containing colonies (A). Addition of the gliogenic factor neuregulin (Nrg) to sister cultures allowed Wnt1-Cre+ Rbpsuhfl/fl cells to form normal numbers of multilineage, glia-containing and other types of colonies as compared with control cells (B). (C-N) Representative photos of single fields of view from within multilineage colonies cultured from control cells in standard medium (C-E), Wnt1-Cre+ Rbpsuhfl/fl cells in standard medium (F-H), control cells in the presence of Nrg (I-K) and Wnt1-Cre+ Rbpsuhfl/fl cells in Nrg (L-N) show that Rbpsuh-deficient NCSCs rarely formed glia (H, Gfap, red), except in the presence of Nrg (N) (three to six independent experiments).

 

Figure 4
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  		Fig. 4. Rbpsuh deletion leads to profound defects in gliogenesis despite normal neurogenesis in sympathetic ganglia. (A,B) Transverse sections of the sympathetic chain from control or Wnt1-Cre+ Rbpsuhfl/fl mouse embryos at E13.5 (A) or E18.5 (B) were stained for neurons (TuJ1+, green) and glia (BFABP+, red). Scale bar: 20 µm. (C) The number of migratory neural crest cells (p75+) in the sympathetic ganglion of the control and Wnt1-Cre+ Rbpsuhfl/fl embryos did not differ at E10.5. (D) The numbers of neurons (TuJ1+) and glia (BFABP+) per section through the E10.5 (three mice per genotype), E14.5 (four to seven mice per genotype) and E18.5 (four to seven mice per genotype) sympathetic chain. Numbers of BFABP+ glia in the Wnt1-Cre+ Rbpsuhfl/fl sympathetic chain were significantly reduced relative to control (P<0.05). (E,F) We did not detect any difference in the rates of proliferation (E, the percentage of cells that incorporated a 30-minute pulse of BrdU in vivo at E14.5) or cell death (F, the percentage of cells expressing activated caspase 3) (three mice per genotype). For all developmental stages, between five and twelve sections were counted per mouse at upper and lower thoracic levels. Error bars represent s.d.; *, P<0.05.

 

Figure 5
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  		Fig. 5. Undifferentiated neural crest progenitors persist in the sympathetic chain in the absence of Rbpsuh and can undergo gliogenesis in culture in the presence of neuregulin (Nrg). (A,B) E13.5 sympathetic ganglia from control and Wnt1-Cre+ Rbpsuhfl/fl mouse embryos were dissociated and plated at clonal density (500 cells per 35 mm dish) under adherent conditions in the absence (A, standard medium; nine independent experiments) and presence (B, four independent experiments) of 5 nM Nrg. In standard medium (A), Wnt1-Cre+ Rbpsuhfl/fl cells formed significantly (*, P<0.05) fewer multilineage colonies (N+G+M) and all glia-containing colonies (G-containing). In the presence of Nrg (B), Wnt1-Cre+ Rbpsuhfl/fl cells and controls cells formed similar numbers of all colony types, including multilineage and glia-containing colonies. (C-R) Typical photos of a single field of view from within a multilineage colony formed by control cells under standard conditions (C-F), an N+M colony formed by Rbpsuh-deficient cells under standard conditions (G-J), a multilineage colony formed by control cells in the presence of Nrg (K-N) and a multilineage colony formed by Rbpsuh-deficient cells in the presence of Nrg (O-R).

 

Figure 6
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  		Fig. 6. Deletion of Rbpsuh reduces the generation of astrocytes and increases the generation of oligodendrocytes without affecting the numbers of neurons in the E14.5 spinal cord. (A-F) Sections from the thoracic neural tube of E14.5 Nestin-Cre+ Rbpsuhfl/fl mice contained fewer BFABP+ radial glia (A,B) and more Olig2+ oligodendrocyte progenitors (C,D) than sections from control littermates, although levels of neurogenesis appeared similar (E,F). The differences in gliogenesis were particularly pronounced in the center of the neural tube, which was marked by a disorganized or absent central canal in Nestin-Cre+ Rbpsuhfl/fl mice (compare A to B, and H to I). (G) Nestin-Cre+ Rbpsuhfl/fl mice had significantly fewer BFABP+ cells per section and significantly more Olig2+ cells per section (three to seven sections per mouse, three mice per genotype). (H-O) Higher magnification images showed that the center of the Nestin-Cre+ Rbpsuhfl/fl neural tube contained mainly Olig2+ oligodendrocyte progenitors (I), in contrast to control mice that contained mainly BFABP+ radial glia (H). The small numbers of Olig2+ cells present in control mice did not co-label with BrdU (N, arrow), whereas at least some of the Olig2+ cells from Nestin-Cre+ Rbpsuhfl/fl mice did (O, arrowheads). We did not observe any difference between Nestin-Cre+ Rbpsuhfl/fl mice and littermate controls in the frequency of cells undergoing cell death (activated caspase-3+ cells, data not shown). Panels A-F,H,I are montages of multiple non-overlapping images of the same section. Scale bars: 50 µm in A,B for A-F; 50 µm in H,I; 20 µm in J,K for J-O.

 

Figure 7
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  		Fig. 7. Rbpsuh deletion reduces the generation of astrocytes and increases the generation of oligodendrocytes in the E19.5 mouse spinal cord without depleting progenitors that could form multilineage colonies in culture. (A-F) The differences observed at E14.5 persisted in the E19.5 spinal cord, including decreased numbers of Gfap+ astrocytes (A,B) and increased numbers of Mbp+ oligodendrocytes (C,D) in the Nestin-Cre+ Rbpsuhfl/fl spinal cord as compared with littermate controls. We also observed more Sox10+ Pdgfr{alpha}+ oligodendrocyte lineage cells in the Nestin-Cre+ Rbpsuhfl/fl spinal cord (E,F, arrows). C,D represent montages of multiple non-overlapping images of the same section. (G) Quantification revealed a significant (*, P<0.05) reduction in the number of BFABP+ astrocytes and significant increases in the numbers of Olig2+, Sox10+ and Mbp+ oligodendrocyte lineage cells in the Nestin-Cre+ Rbpsuhfl/fl spinal cord (three to seven sections per mouse, three to five mice per genotype). Because glia are difficult to count in sections based on filamentous Gfap staining, we acutely dissociated cells from E19.5 spinal cord and plated them in culture at low density for 6 hours then stained for Gfap expression. (H) We observed a significantly lower percentage of Nestin-Cre+ Rbpsuhfl/fl cells in culture that were Gfap+ (*, P<0.05; 1000 cells counted per mouse, three mice per genotype). There was no significant difference in the number of cells undergoing cell death (activated caspase 3+) in control and Nestin-Cre+ Rbpsuhfl/fl mice (data not shown). (I-P) Dissociated E19.5 thoracic spinal cord cells were cultured at clonal density then stained for neurons (TuJ1+), astrocytes (Gfap+) and oligodendrocytes (O4+). Representative multilineage colonies from control (I-L) and Nestin-Cre+ Rbpsuhfl/fl (M-P) spinal cord cells show more Gfap+ cells and stronger Gfap staining in control colonies. (Q) The percentage of spinal cord cells that formed each type of colony. Nestin-Cre+ Rbpsuhfl/fl spinal cord cells were significantly more likely than control cells to form multilineage colonies (N+A+O) and oligodendrocyte-containing colonies (O-containing), but significantly less likely to form astrocyte-only colonies (A). P<0.05. The increase in neuron-containing colonies reflected the increase in multilineage colonies, not an increase in neuronal-restricted progenitors. Scale bars: 50 µm in A-F.

 

Figure 8
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  		Fig. 8. Rbpsuh is necessary for the maintenance of Sox9 expression after E12.5 in the spinal cord. (A-E) Transverse sections of the spinal cord from control and Nestin-Cre+ Rbpsuhfl/fl mouse embryos from E11.5 to E19.5 were stained for Sox9. A-E represent montages of multiple non-overlapping images of the same section. In control embryos, Sox9+ cells were in the ventricular zone early in development but gradually dispursed throughout the spinal cord by E19.5. (F) In Nestin-Cre+ Rbpsuhfl/fl embryos, Sox9+ cells were in the ventricular zone from E11.5 to E13.5, but became increasingly depleted relative to control embryos after E12.5 (three to six mice per genotype and time point). (G) Only rare Sox9+ cells appeared to undergo cell death at E13.5 and E14.5, but Sox9+ cells from Nestin-Cre+ Rbpsuhfl/fl embryos exhibited increased cell death at E19.5. (H) Nestin-Cre+ Rbpsuhfl/fl embryos exhibited reduced expression of Rbpsuh and Sox9 but not Scl by qRT-PCR at E14.5.

 

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© The Company of Biologists Ltd 2007