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First published online May 11, 2006
doi: 10.1242/10.1242/dev.02345

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dILA neurons in the dorsal spinal cord are the product of terminal and non-terminal asymmetric progenitor cell divisions, and require Mash1 for their development

¹ Max-Delbrück-Centrum for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin-Buch, Germany.
² Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
³ National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

View larger version (81K): [in a new window]   Fig. 1. Temporal and spatial changes in the specification of neurons in the dorsal spinal cord. (A-C) Immunohistological analysis of dorsal spinal cords of wild type mice at stage E10.5-E12.5 using antibodies directed against Mash1 (blue), Lbx1 (red) and Tlx3 (green). The d4 and d5 (early) and dL (late) progenitor domains are indicated, as well as the dI3-5, dILA and dILB neuronal subtypes generated. Immunohistological analysis of spinal cords of wild-type animals at stage E12.5 using antibodies directed against (D,D') Tlx3 (red) and Pax2 (green), (E) Gsh1/2 (green) and Lbx1 (blue), (F) Mash1 (green), Olig3 (red) and Lbx1 (blue), (G,G') Mash1 (red) and Gsh1/2 (green), (H) Ptf1a (green) and Lbx1 (blue), and (H') Ptf1a (green), Lhx1/5 (blue) and Lmx1b (red). (I) Schematic display of the expression of transcription factors in progenitors of the alar plate and in dorsal neurons at E12.5. The yellow and orange bars indicate progenitor domains that express Ngn1 and Math1, respectively. Scale bars: 100 µm in A-H; 20 µm in D',G',H'.

View larger version (88K): [in a new window]   Fig. 2. Mash1 expressing progenitors give rise to dILA and dILB neurons. (A) Strategy used to replace the Mash1-coding sequence by GFP. The wild type Mash1 locus, the targeting vector, and the recombined allele before (Mash1GFPneo) and after deletion of the neo resistance cassette (Mash1GFP) are depicted. In the targeting vector, the GFP sequence (green) was fused to the initiation codon of Mash1, and the coding sequence of Mash1 (blue) was deleted. No additional polyA was introduced, and the exon-intron structure of the Mash1GFP allele is identical to the one of wild-type Mash1. (B,C) In situ hybridization of consecutive sections from a E12.5 Mash1GFP/+ spinal cord using probes directed against Mash1-coding sequence (B) or GFP (C). The expression patterns are identical. (D-F) Immunohistochemical analysis of spinal cords of Mash1GFP/+ mice at E12.5 with antibodies directed against (D) Lbx1 (red), GFP (green) and Mash1 (blue), (E) Lmx1b (red) and GFP (green), and (F) Lhx1/5 (red) and GFP (green). The spinal cord is shown at low (D) and high (E,F) magnification. All Lmx1b+ (dILB) neurons are also GFP+ in E; all Lhx1/5 (dILA) neurons are also GFP+ in F. Scale bars: 100 µm in B-D; 10 µm in E,F.

View larger version (92K): [in a new window]   Fig. 3. dILA neurons are the product of asymmetric cell divisions. (A) Summary of all possible cell divisions that could generate dILA or dILB neurons. Yes/no indicates if a particular type of progenitor cell division was observed by lineage tracing experiments using retroviral infection. (B) Quantification of the different types of independent two-cell clones observed after retroviral infection. (C-G) Two-cell clones residing in the dorsal spinal cord were identified after retroviral infection by the expression of ß-galactosidase, as assessed by immunohistochemisty (green). The molecular identity of cells was assessed by the use of antibodies directed against Lbx1 (red) and Lhx1/5 (blue). dILA neurons were defined as Lbx1+/Lhx1/5+ cells, dILB neurons as Lbx1+/Lhx1/5– cells, and progenitors as Lbx1-negative cells located close to the ventricle. Only those clones that contained at least one Lbx1+ neuron were assessed. Spinal cords are shown at low (C) and high (D-G) magnification. (E) High magnification of a two-cell clone containing one dILA (white) and one dILB (yellow) neuron. (G) High magnification of a two-cell clone containing two dILB neurons (yellow). Scale bar: 100 µm in A;10 µm in B-E.

View larger version (78K): [in a new window]   Fig. 4. Effect of the Mash1 mutation on the generation of dILA neurons. Dorsal neurons in control, Mash1–/– and Mash1Ngn2/Mash1Ngn2 embryos were analyzed at E12.5 using immunohistochemistry, with antibodies directed against (A-F) Lhx1/5 (green) and Tlx3 (red), (H-J) Pax2 (green) and Lmx1b (red). dILA neurons express Lhx1/5 and Pax2, and dILB neurons express Tlx3 and Lmx1b. (G,K) Numbers of BrdU+ dILA (dark-grey columns) and BrdU+ dILB (light grey columns) neurons in control (c), Mash1–/– (–/–) and Mash1Ngn2/Mash1Ngn2 (N2/N2) mutant mice, as assessed 24 hours after BrdU injection. The number of dILA neurons is reduced in Mash1–/– embryos. (L-N) Analysis of Ptf1a (green) and Mash1 (red) expression in the dorsal spinal cord of control (L), Mash1–/– (M) and Mash1Ngn2/Mash1Ngn2 (N) embryos. (O) Numbers of Ptf1a+ cells (black columns) in control (c), Mash1–/– (–/–) and Mash1Ngn2/Mash1Ngn2 (N2/N2) mutant mice. The error bars represent the standard deviation. (P-R) In situ hybridization of sections from E12.5 control, Mash1–/– and Mash1Ngn2/Mash1Ngn2 spinal cords using a probe directed against Hes5 mRNA. Scale bars: 100 µm in A-C,P-R; 50 µm in D-F,H-J,L-N.

View larger version (70K): [in a new window]   Fig. 5. Essential neurogenic function of Mash1 at E11.5 and subsequent stages in the dorsal spinal cord. (A-D) Spinal cords of Mash1–/– and control embryos were analyzed by immunohistochemistry at E12.5. (A,B) Differentiated neurons were visualized using the TuJ1 antibody (green), and YO-PRO1 (red) was used to stain nuclei. The width of the progenitor domain is indicated. (C,D) In control embryos, Lbx1+ cells (green) are rarely found in the progenitor domain, marked by the expression of Pax7 (red). In Mash1–/– embryos, many Lbx1+ cells can be found in the progenitor domain co-expressing Lbx1 and Pax7 (yellow). (E) Numbers of Pax7+ cells in the dorsal progenitor domain were determined in Mash1–/– (white columns) and control (black columns) embryos at the indicated time points. The number of progenitor cells is increased in Mash1–/– at E11.5 and at subsequent stages. (F) Quantification of newborn neurons at various developmental stages; to achieve this, BrdU was injected at various time points, and the number of dorsal BrdU+/NeuN+ neurons in Mash–/– and control embryos was determined 24 hours later. There is a decrease in the number of newborn neurons at E11.5 and at subsequent stages in Mash1–/– embryos. (G) The proliferation index of progenitor cells (number of dorsal BrdU+ progenitor cells/number of Pax7+ progenitor cells after a 2-hour BrdU pulse) was determined in Mash1–/– and control embryos at the indicated time points. The proliferation index of progenitor cells is not altered in Mash1–/– embryos. (H) The differentiation index (number of BrdU+NeuN+ dorsal neurons/total number of BrdU+ dorsal cells after a 24 hours BrdU pulse) was determined in Mash1–/– (white columns), Mash1Ngn2/Mash1Ngn2 (grey columns) and control (black columns) embryos at E12.5. The thickness of the optical section shown in A and B is 10 µm; the thickness of the optical sections shown in C and D is 1.2 µm. The error bars in E-H represent s.d. Scale bars: 100 µm in A,B; 50 µm in C,D.

View larger version (115K): [in a new window]   Fig. 6. Ectopic expression of Mash1 is not sufficient to induce dILA neurons. Chick neural tubes were electroporated at HH26 with constructs expressing GFP (A-C), Mash1 and GFP (D-F), or Ptf1a and GFP (G-I), and the effects on neuronal specification were assessed at HH29-30 using antibodies against GFP (red), Pax2 (green in A,D,G) and Tlx3 (green in B,E,H). The proportion of GFP+/Pax2+ (black columns) and GFP+/Tlx3+ cells (light-grey columns) after electroporation of the indicated constructs were determined in the alar plate. The error bars represent s.d. Scale bar: 100 µm.

View larger version (124K): [in a new window]   Fig. 7. Mash1 but not Ptf1a is expressed during all stages of the cell cycle. To determine at which stages of the cell cycle Mash1 or Ptf1a are expressed, BrdU was injected at the indicated time points before analysis of E12.5 embryos. (A-D) Analysis of dorsal spinal cords with antibodies directed against BrdU (green) and Mash1 (red). (E-H) Analysis of dorsal spinal cords with antibodies directed against BrdU (green) and Ptf1a (red). (I-L) Analysis of dorsal spinal cords with antibodies directed against BrdU (green) and phosphorylated Histone 3 (red), a marker for M-Phase cells. (M) Schematic display of the expression of the transcription factors Mash1 and Ptf1a during cell cycle at E12.5 (PD cell located in progenitor domain; MZ cell located in mantle zone). Scale bars: 100 µm and 10 µm.

View larger version (16K): [in a new window]   Fig. 8. Asymmetry of Mash1 function in the development of dILA neurons. Schematic diagram of progenitor cell divisions that generate a dILA daughter, and a model of the Mash1 function in their development. (A) dILA neurons are generated from progenitor cells by asymmetric cell divisions in control mice (left). These divisions are either non-terminal (top) and generate one dILA neuron and one progenitor, or are terminal (bottom) and generate one dILA and one dILB neuron. In Mash1–/– mice, aberrant progenitor cells (P*) are formed at the expense of dILA neurons (right). Supernumerary dorsal progenitors of Mash1–/– mice can incorporate BrdU and do thus replicate, but many are subsequently eliminated by apoptosis. (B) A model of Mash1 function in the development of dILA neurons that arise by asymmetric terminal divisions. Mash1 is expressed in the progenitor cell that gives rise to a dILA and a dILB neuron. In the dILA daughter, Mash1 exerts essential functions for neurogenesis and lineage specification. Mash1 allows a dILA progenitor (P1) to differentiate (P1N), and to express Ptf1a. By contrast, Mash1 is dispensable for the development of the dILB lineage.