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First published online 7 March 2007
doi: 10.1242/dev.001255

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Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord

Michiya Sugimori^1,2, Motoshi Nagao¹, Nicolas Bertrand^3,*, Carlos M. Parras^3,, François Guillemot³ and Masato Nakafuku^1,4,5,

¹ Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
² Department of Neurobiology, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
³ Division of Molecular Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
⁴ Departments of Pediatrics and Neurosurgery, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267, USA.
⁵ Solution Oriented Research for Science and Technology (SORST), Japan Science and Technology Agency, 3-4-15, Nihonbashi, Chuo-ku, Tokyo 103-0027, Japan.

View larger version (123K): [in this window] [in a new window]   Fig. 1. Expression of Mash1 in relation to oligodendrocyte specification in the ventral spinal cord. (A-E) All panels show transverse sections of the rat embryo spinal cord (the dorsal side is top). Molecules stained in red and green are shown on the top, and the stage and axial level of the sections are shown on the left. Horizontal lines indicate the boundaries between the Pax6+ (p2), Olig2+ (p*; arrows) and Nkx2.2+ (p3) progenitor domains. Insets in Aa-Ad, Ba and Ca-Cd are higher magnification views of the areas indicated by thick arrows. Circles in Aa and Ba indicate Olig2+ and Nkx2.2+ cells migrating in the MZ. Insets show co-expression (Ac,Ad,Cc,Cd) or segregation (Aa,Ab,Ba,Ca,Cb) of two markers in single cells. Small arrowheads indicate double-positive cells. Db is a higher magnification view of the area boxed in Da. Scale bars: in Ad,Ba,Cd, 100 µm for Aa-Ad,Ba,Ca-Cd; in Bc,Df,Ee,Eg, 50 µm for Bb,Bc,Db-Df,Ea-Eg; in Da, 200 µm.

View larger version (125K): [in this window] [in a new window]   Fig. 2. Expression of inhibitory HLH factors in differentiating astrocytes. (Aa-Ag) Expression of Id1 and Hes1 in S100ß+ astrocyte at E16.5. Arrowheads in Aa-Ac indicate the loss of expression of Pax6, Olig2, and Nkx2.2 in some Id1+ cells in the VZ (higher magnification views in insets), whereas those in Ad-Ag indicate the co-expression of Id1 and Hes1 with Pax7 and S100ß. (Ba-Be) Segregation of GFAP+ cells with Pax6+, Olig2+, and Nkx2.2+ cells at E18.5. Arrowheads indicate no overlap of GFAP+ cells with Pax6+, Olig2+ and Nkx2.2+ cells. (Bf-Bj) Expression of Id1 and Hes1 in GFAP+ and S100ß+ astrocytes at E18.5. Arrowheads in Bf-Bj show co-expression of GFAP and S100ß with Id1 and Hes1. Bc,Be,Bg,Bi are higher magnification views of the areas boxed in Bb,Bd,Bf,Bh, respectively. (Ca-Db) Downregulation of Id1, Olig2 and Nkx2.2 in Sox2+ VZ cells between E18.5 and 22.5. Dashed lines indicate the border of the VZ. Ca and Cb show the staining for Id1 and Sox2, respectively, in the same section. Arrowheads show the lack of Id1 expression in some Sox2+ cells. FP, floor plate. Scale bars: in Ac, 50 µm for Aa-Ac,Ad,Af,Ag,Bc,Be,Bg,Bi,Bj,Cc,Cd; in Ae,Ba,Bb,Bd, 200 µm; in Bf,Bh, 100 µm; and in Cb,Db, 25 µm for Ca,Cb,Da,Db. (E) Schematic representation of the expression patterns of patterning and proneural/inhibitory HLH factors during the course of neuro/gliogenesis. Colored boxes indicate the expression of Pax6 (green), Olig2 (red) and Nkx2.2 (blue) in progenitor domains. Paler colors between E16.5 and E20.5 indicate the gradual loss of their expression in the VZ. The expression of proneural and inhibitory HLH factors is indicated by black lines. The periods of generation of neurons (N), oligodendrocytes (O) and astrocytes (A) are indicated by red, blue and green lines, respectively.

View larger version (60K): [in this window] [in a new window]   Fig. 3. Cell-fate specification activities of patterning and proneural/inhibitory HLH factors. (A-H) Neurospheres subjected to the clonal (A-D) and population (E-H) assays. The top and bottom panels in A show the fluorescence and brightfield pictures, respectively, of GFP+ (left) and GFP- (right) neurospheres. Arrowheads show a virus-infected GFP+ neurosphere colony and dissociated cells. (Ia,Ib) Effects of overexpression of patterning and HLH factors and their combinations in the clonal assay. Viruses used for infection, either alone (Ia) or in combination (Ib), are shown in the top row. The percentages of various progenitor subtypes are shown in the next 11 rows of each table. The ranges and averages of the total cell number (clone size) and numbers of neurons and oligodendrocytes per clone (mean±s.d.) are shown in the bottom six rows of each table. `N,' `O' and `A' stand for neurons, oligodendrocytes and astrocytes, respectively. The data shown in red and blue are values larger and smaller, respectively, compared with the control culture. All data are the summary of three to four independent experiments. *, P<0.05; **, P<0.01 compared with the control virus; %, P<0.01 compared with Ngn2 or Ngn3 virus; $, P<0.01, compared to Mash1 virus; &, P<0.01, compared with Id1 virus; and #, P<0.01, compared with Hes1 virus. Scale bars: in D, 100 µm for A-D; in H, 50 µm for E-H.

View larger version (43K): [in this window] [in a new window]   Fig. 4. Cell-fate specification activities of patterning and proneural/inhibitory HLH factors in the population assay. (A-L) The percentages of neuronal and glial marker-positive cells among total virus-infected cells are shown (mean±s.d. from four independent experiments). Viruses used for infection are shown on the x axis. Dashed horizontal lines indicate the values in the control culture. *, P<0.05; **, P<0.01 compared with the control virus; %, P<0.01 compared with Ngn1, Ngn2 or Ngn3 virus; $, P<0.05; $$, P<0.01 compared with Mash1 virus; and &, P<0.01 compared with Id1 virus.

View larger version (90K): [in this window] [in a new window]   Fig. 5. Deficiency in oligodendrogenesis in Mash1 and Ngn2 mutants. (Aa-De) Early generation of OLPs in various mutant mice at E12.5. Arrowheads indicate marker-positive cells. Insets show higher magnification views of the p* domain and adjacent areas. Intense tubular staining in Ad-Dd and Ae-De derived from blood vessels. (E,F) Decrease in the numbers of Olig1+ and NG2+ cells in Mash1-/- mice between E12.5 and E16.5 compared with wild type. The number of marker-positive cells per transverse section of the lumbar spinal cord, including both the VZ and MZ, was quantified. Data are mean±s.d. obtained from staining of six to ten sections derived from two to three embryos for each genotype (*, P<0.01). (G-M) Comparison of the numbers of cells expressing OLP markers in various mutant mice at E12.5 (G-L) and E13.5 (M). In G and H, Olig2+ and Nkx2.2+ cells only in the MZ were counted, whereas in I-M, both the VZ and MZ were subjected to quantification (indicated in boxes). Percentages are values relative to the wild type. Data are mean±s.d. obtained from staining of eight to ten sections derived from two to three embryos for each genotype. *, P<0.01 compared to the wild type; $, P<0.05; and $$, P<0.01 compared to Mash1-/- mice. Scale bars: in Dd, 100 µm for Aa-Dd; in De, 50 µm for Ae-De.

View larger version (80K): [in this window] [in a new window]   Fig. 6. Deficiency in neurogenesis in Mash1 and Ngn2 mutants. (Aa-Dc) Generation of motoneurons and Islet1+/Olig2+ neurons at E12.5. Circles in Aa-Da indicate the cluster of motoneurons in the ventral MZ. Arrowheads in Ca and Cc indicate ectopic HB9+ cells in and adjacent to the VZ in Mash1KI Ngn2/+ mice. Thick arrows in Aa-Da indicate the locations of the areas magnified in Ab-Db. (E,F) The numbers of HB9+ and Islet1+ cells per section in the motoneuron cluster in the ventral MZ (E) and in the area close to the p* VZ (F) in the wild-type and various mutant mice. (G) The number of Islet1+/Olig2+ cells in the lateral MZ. (H) Schematic diagrams summarizing the defects in neurogenesis and oligodendrogenesis in the Olig2+ domain in various mutants. Ngn2+ cells and neurons are shown in red, whereas Mash1+ cells and oligodendrocytes are in blue. Cross marks and thick circles indicate a decrease or absence, and increase, respectively, of specific cell populations in the mutants. Scale bars: in Da, 100 µm for Aa-Da; in Dc, 50 µm for Ab-Db,Ac-Dc.

View larger version (61K): [in this window] [in a new window]   Fig. 7. Defects in gliogenesis in Pax6-/- mutants. (A-E) Ectopic and precocious oligodendrogenesis in Pax6-/- mice. The left and right sides of each panel show the staining of heterozygote (+/-) and homozygote (-/-) mice, respectively, at E13.5. Three horizontal lines indicate the dorsal limit of the Nkx6.1+ domain (upper), the ventral limit of the Irx3+ domain (middle), and the dorsal limit of the Nkx2.2+ domain (lower) in the heterozygote. The location of the normal Pax6+ domain is indicated by brackets. The reduced Olig2+ domain in the homozygous mutant is indicated by an arrow in B. Arrowheads indicate ectopic OLPs. (F,G) The numbers of ectopic Olig2+, Olig1+, and Nkx2.2+ cells in the MZ (F) and in the dorsal (normally Pax6+) VZ (G) in the Pax6+/- and Pax6-/- mice. Percentages are values relative to Pax6+/- mice. Data are mean±s.d. obtained from staining of six to eight sections from two to three embryos for each genotype. **, P<0.01. (H,I) Premature astrogenesis in Pax6-/- mice. In H, early astrocytes were detected as GS+ cells (arrowheads) at E15.5. In I, the number of GS+ cells detected per transverse section of the lumbar spinal cord is compared between Pax6+/- (black bars) and Pax6-/- (red bars) mice. (J) Schematic diagrams summarizing the defects in gliogenesis in Pax6+/- mice. Notice that the generation of V0-2 interneurons is also defective in the mutant (Scardigli et al., 2001). Scale bars: 100 µm in E,H.

View larger version (27K): [in this window] [in a new window]   Fig. 8. Combinatorial actions of patterning and proneural/inhibitory HLH factors in specifying neuronal and glial cell lineages. The activities of individual transcription factors (patterning factors, left; proneural/inhibitory HLH factors, top) and their combinations are summarized. Arrows and blunt-ended lines indicate stimulation and inhibition, respectively, of differentiation into specific cell types. The thickness of each arrow arbitrarily reflects the strength of the activity. Olig, oligodendrocyte.