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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02601

Development 133, 4367-4379 (2006)
Published by The Company of Biologists 2006
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Generation of GABAergic and dopaminergic interneurons from endogenous embryonic olfactory bulb precursor cells

Eva Vergaño-Vera1,2, María J. Yusta-Boyo2, Fernando de Castro3, Antonio Bernad4, Flora de Pablo2 and Carlos Vicario-Abejón1,2,*

1 Instituto Cajal, Consejo Superior de Investigaciones Científicas (CSIC), E-28002 Madrid, Spain.
2 Growth Factors in Vertebrate Development Group, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
3 Instituto de Neurociencias de Castilla y León, Universidad de Salamanca, Salamanca, Spain.
4 Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Madrid, Spain.


Figure 1
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  		Fig. 1. Cell marker expression in embryonic GE and OB. E12.5 and 13.5 mouse coronal or sagittal sections containing the GE or the OB were immunostained with specific antibodies. Gsh2 and Dlx2 were expressed strongly in the MGE and LGE (A-H), whereas no expression was observed in E13.5 OB (I,J). Most GABA+, Th+ and Tbr1+ cells were located in the MZ (K-N; a high magnification image is shown in N). LV, lateral ventricule; CTEX, cortex; NEZ, neuroepithelial zone; MZ, mantle zone. Similar expression patterns were observed in sections from two to five different mice. Scale bars: in N, 70 µm for A,C,G,I,J; 40 µm for B,D,E,F,H,N; 150 µm for K; 65 µm for L,M.

 

Figure 2
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  		Fig. 2. Gsh2 and Dlx2 expression and interneuron generation in short-term dissociated cultures of E13.5 OB and E13.5 GE. Cell suspensions of E13.5 OB and GE were cultured for 6 days. Representative fields of Gsh2/GAD-positive cells (OB, A-C; GE, G-I) and Dlx2/GAD-positive cells (OB, D-F; GE, J-L) in cultures. Arrows indicate double-labeled cells; arrowheads show GAD+ neurons that do not express Gsh2 or Dlx2. (M) Percentages of GAD+ neurons over total TuJ1+ neurons. (N) Percentages of Gsh2- or Dlx2-expressing GAD+ cells relative to total GAD+ cells. (O) Percentages of Gsh2- or Dlx2-expressing GAD+ cells relative to total Gsh2+ or total Dlx2+ cells. No (0%) Gsh2+ cell coexpressed GAD in OB cultures; Student's t test could thus not be calculated. *P<0.05 (OB versus GE). Results are the mean±s.e.m. of data from six cultures in three experiments. Scale bar in L: 25 µm.

 

Figure 3
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  		Fig. 3. Pax6 expression, BrdU labeling and interneuron generation in the OB. (A-C) E13.5 coronal sections immunostained with a Pax6-specific antibody showed abundant Pax6 expression in the NEZ and in cells of the MZ. (D-F) 52.0±1.1% (n=3) of GAD+ cells in culture expressed Pax6 (arrows). Arrowheads show GAD+ neurons that do not express Pax6. (G-I) Immunostaining of BrdU-labeled E13.5 mouse sections shows BrdU+/GABA+ cells (arrows) in the MZ. (J-L) Of cells in culture, 95.4±0.9% (n=3) of GABA+ were BrdU+ (arrows), indicating that the majority of interneurons are derived from dividing precursors. Similar expression patterns were observed in sections from two to four different mice. Scale bar in C: 60 µm for A; 45 µm for B; 40 µm for C; 35 µm for D-F; 25 µm in J-L; 15 µm in G-I.

 

Figure 4
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  		Fig. 4. Transplant of GFP-expressing E13.5 OB precursor cells in E13.5 wild-type OB slices. E13.5 OB cell suspensions were transplanted into E13.5 OB slices prepared from wild-type embryos; slices were then cultured for 2 days. (A-C) Representative images of GFP/GABA-positive cells. (D-F) Representative images of GFP/Th-positive cells. (G) Of the total GFP+ cells in the slices, 31.7% (n=15) expressed GABA and 12.1% (n=2) expressed Th. Scale bar in C: 25 µm for A-C; 20 µm for D-F.

 

Figure 5
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  		Fig. 5. Differentiation and maturation of GABAergic, dopaminergic and mitral/tufted neurons in short- and long-term dissociated cultures of E13.5 OB. Cell suspensions were cultured for 6 or 17 days, then single-(A,B) or double-immunostained (C-H). (A,B) Representative images of GABA- and Th-positive cells at 6 days in culture. (C-H) Representative images of MAP2ab/GABA, MAP2ab/Th, and MAP2ab/Tbr1 at 17 days in culture, revealing the morphological maturation of neurons. Images are representative of four to six cultures in three experiments. Scale bar in B: 20 µm for A,B; 30 µm for C-H.

 

Figure 6
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  		Fig. 6. Transplant of E13.5 OB and E13.5 LGE precursor cells expressing GFP in P5-P7 wild-type OB. E13.5 OB (A-E,K,L) and LGE (F-K,L) cell suspensions were transplanted into the OB SEZ of P5 and P7 mouse pups. Grafted animals were analyzed 1 to 4 weeks after surgery. (A) One week post-transplant, GFP-expressing OB cells with a migratory morphology were observed in host tissue. (B,C) By 2 and 4 weeks, transplanted GFP-positive cells migrated away from the injection site to the GCL, the IPL, the boundary between the IPL and the ML, and the GL. (C,K) Most grafted OB cells reached the GCL and the limits established by the IPL and the EPL (ML + PL). (F-K) LGE-derived GFP+ cells migrated and differentiated within the OB; they migrated preferentially to the GCL and GL, with fewer cells reaching the ML + PL (G,H,K). The numbers of cells located in the different cell layers were counted and expressed relative to total transplanted GFP+ OB or GFP+ LGE cells found in the postnatal OB 2-4 weeks post-transplant (K). Results are the average±s.e.m. of data from mice receiving OB cells (n=4 mice) or LGE cells (n=4 mice). *P<0.05 (OB vs LGE; Student's t test). (D-J,L). Morphological analysis was performed on 50 neurons derived from GFP+ OB precursor cells (n=3 mice), and on 50 neurons from GFP+ LGE precursors (n=3 mice). Percentages of neurons with one apical dendrite, two apical dendrites or more than two dendrites are given. Note that the great majority of neurons found in mice transplanted with OB cells had one or two apical dendrites (D,E,L). Neurons with one apical dendrite were also abundant in mice transplanted with LGE cells (I,L). In these mice, however, the percentage of neurons possessing two apical dendrites, and of neurons that were multidendritic (J) was 2.3 times fewer and 4.1 times greater, respectively, than in mice grafted with OB cells *P<0.05 (OB vs LGE; Student's t test). m, more than two dendrites; o.a.d., one apical dentrite; t.a.d., two apical dendrites; GCL, granule cell layer; IPL, internal plexiform layer; ML, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. Scale bar in J: 90 µm for A-C,F,H; 50 µm for D-I; 20 µm for G; 15 µm for J.

 

Figure 7
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  		Fig. 7. Differentiation and maturation of E13.5 OB precursor cells expressing GFP in P5-P7 wild-type OB. E13.5 OB cell suspensions were transplanted and mice were analyzed 4 to 10 weeks after surgery. (A) Many granule neurons had a single, large apical dendrite that crossed the ML and extended dendritic branches with spines in the EPL. The granule neurons at the IPL-ML boundary (arrow in A) had two major apical dendrites that also extended branches with spines into the EPL (A,C,D, D inset). Staining with an anti-synaptophysin antibody (E,F) revealed close proximity of dendritic spines to synaptic boutons. (B) As host periglomerular neurons (inset in B shows a periglomerular/glomerular area stained for Th), some transplanted cells oriented their cell bodies horizontally. (G-O) Sections were stained with anti-GAD or anti-GABA antibodies. In some neurons, GAD or GABA and GFP colocalization was best observed in specific areas of the cell body. (P-R) The Th+ cell body surrounds the nuclear GFP+ signal (arrows). Neuron migration and differentiation was observed in all transplanted mice (n=6). Scale bar in R: 20 µm for A-C; 15 µm for D,G-I; 4 µm for D inset; 10 µm for E,F,J-R.

 

Figure 8
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  		Fig. 8. OBSC differentiation into GABAergic neurons. (A-G) E13.5- and 14.5-derived OBSC cultures were grown for 15-17 days in the absence of mitogens. Bdnf (20 ng/ml) was added to some cultures. Cells were double-immunostained with anti-GAD and anti-Dlx2 (A,B) and anti-MAP2ab and anti-GABA antibodies (C-F). (A,B) GAD+/Dlx2+ cells (arrows) and GAD+/Dlx2- cells (arrowheads) in the differentiating cultures. (E,F). Some Bdnf-treated cells showed a more complex neuronal morphology than controls. (G) Average percentages of GABA+ neurons relative to total MAP2ab+ neurons. Results are the mean±s.e.m. of data from four cultures in two experiments (passages 3-5). (H) Single cells were cultured to produce clonal neurospheres (I) which, after differentiation (J), generated GABA+ neurons (K,L). Scale bars in F and L: 40 µm for A-F; 9 µm for H; 30 µm for I,J; 20 µm for K,L.

 

Figure 9
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  		Fig. 9. Differentiation of OBSC into dopaminergic and mitral/tufted neurons. (A-D) Representative images of MAP2ab+ and Th+ cells in differentiating E13.5 OBSC cultures. (E) Average percentages of Th+ neurons relative to total MAP2ab+ neurons. Results are the mean±s.e.m. of data from four cultures in two experiments. Scale bar: in D, 40 µm. (F) Average percentages of Tbr1+ vs total MAP2ab+ neurons. Bdnf-treated cultures had 26% more Tbr1-labeled cells than controls (not statistically significant). Results are the mean±s.e.m. of data from four to six cultures in three experiments. (G-J) Representative images of MAP2ab+ and Tbr1+ cells. Arrows show Tbr1+ nuclei in the neurons. Scale bar in J: 50 µm.

 

Figure 10
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  		Fig. 10. TrkB expression in OB, and Bdnf action on OBSC-derived neurons. (A-E) Coronal sections from P6 and P21 mice were stained with an antibody to TrkB (A, ML), (B, GL), or double-stained with anti-TrkB and anti-SV2 (C-E), showing that in addition to expression in neuronal cell bodies, TrkB is highly expressed in synaptic terminals at the glomeruli. (F-Q) E13.5-derived OBSC cultures were grown for 15-17 days. Representative fields of TuJ1+ neurons (F,G) and synapsin I+ neurons (H,I) in control (F,H) and Bdnf-treated (G,I) cultures. Representative fields of GABA+/SV2+ (J-M) and Th+/SV2+ neurons (N-Q) in control (J,K,N,O) and Bdnf-treated (L,M,P,Q) cultures. Note that Bdnf-treated neurons show more synapsin I+ and SV2+ boutons than controls. Similar results were obtained in three experiments. Scale bar in Q: 35 µm for A,B; 25 µm for C-E; 30 µm for F-I; 15 µm for J-Q.

 

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© The Company of Biologists Ltd 2006