QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 18 May 2005
doi: 10.1242/dev.01859

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Helms, A. W. Articles by Johnson, J. E. Search for Related Content PubMed PubMed Citation Articles by Helms, A. W. Articles by Johnson, J. E.

Sequential roles for Mash1 and Ngn2 in the generation of dorsal spinal cord interneurons

¹ Center for Basic Neuroscience, UT Southwestern Medical Center, Dallas, TX 75390, USA
² Division of Molecular Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

View larger version (70K): [in a new window]   Fig. 1. Colocalization of Mash1 and Ngn2 in the ventricular zone of the dorsal neural tube. E10.5 embryo transverse sections were processed for mRNA in situ hybridization (A) or immunofluorescence (B-F). (A) Pseudocolored overlay of adjacent sections hybridized with antisense Mash1 (pink) or Ngn2 (purple) probes to illustrate expression in the dorsoventral axis and possible overlap. (B) Double-label immunofluorescence shows Mash1 (red) and Ngn2 (green) in ventricular zone cells in the dorsal neural tube (region shown by bracket in A; ventricular zone is at the left in each panel). Arrows indicate Mash1 and Ngn2 co-expressing cells (yellow). (C-F) Co-labeling with BrdU incorporation was performed to detect cells in S phase. (C,E) Mash1 (red) cells incorporate BrdU (green) (detected as yellow, arrows). (D,F) Ngn2 (red) cells rarely score positive for BrdU incorporation (green). (E,F) Higher magnification images of the regions boxed in C,D. Scale bars: 90 µm in A; 25 µm in B-D; 15 µm in E,F.

View larger version (59K): [in a new window]   Fig. 2. Mash1 is required for dI3 and dI5 neuron formation, while Ngn2 represses formation of these populations. Immunofluorescence on transverse sections of neural tubes in wild-type (A,E,I), Mash1–/– (B,F,J), Ngn2–/– (C,G,K) and Mash1–/–;Ngn2–/– (D,H,L) E10.5 mouse embryos. (A-D) Co-labeling with Brn3a and Lhx1/5 (yellow) marks dI2 neurons. There is a significant increase in dI2 neurons in the Mash1–/– and Mash1–/–;Ngn2–/– embryos, but not Ngn2–/– relative to wild type. (E-H) Isl1 identifies dI3 neurons (green). In the Mash1–/– and Mash1–/–;Ngn2–/– mutant embryos, there is a dramatic decrease in dI3 neurons relative to wild type. By contrast, an increase in dI3 neurons in Ngn2–/– is detected. (I-L) Pax2 marks dI4 and dI6 neurons (green), and Lmx1b labels dI5 neurons (red). In the Mash1–/– and Mash1–/–;Ngn2–/– embryos, there is a complete loss of dI5. This is the opposite in Ngn2–/– embryos, where there is an increase in dI5 relative to wild type. dI4/6 neurons increase in the Mash1–/–, are unchanged in the Ngn2–/–, and are dramatically reduced in the Mash1–/–;Ngn2–/– relative to wild type embryos. (M) Individual populations in each mutant were counted on at least three sections from at least three embryos and cell counts are shown in the table. Because dI4 and dI6 neuronal populations cannot be distinguished in the Mash1 null, all Pax2 cells dorsal to the boundary where the first ventral Pax2 cell was found within the ventricular zone were counted, and as such they are labeled as dI4/6. Scale bar: 50 µm.

View larger version (37K): [in a new window]   Fig. 3. Excess Mash1 promotes dI3 and dI5 populations, while excess Ngn2 represses them in the chick neural tube. Immunofluorescence on transverse sections of E4 (HH23-24) chick neural tubes electroporated with pMiWIII-Mash1 (A,C,E,G) or pMiWIII-Ngn2 (B,D,F,H) at E3 (HH13-14). The arrow above each row of panels indicates the electroporated side of the neural tube. Neuronal population dI2 was labeled by Brn3a;Lhx1/5 (yellow) (A,B), dI3 by Isl1 (C,D), dI4 by Pax2 (E,F) and dI5 by Lmx1b (G,H). Each panel is representative of the phenotype seen in at least three sections from at least three electroporated embryos. (I) Relative induction or repression is expressed as the number of cells on the injected side divided by the number of cells on the control side and plotted in the graph on a logarithmic scale. ** P<0.001. Scale bar: 25 µm.

View larger version (55K): [in a new window]   Fig. 4. Mash1 cells primarily become dI3 and dI5 neurons. Immunofluorescence on transverse sections of Mash1+/– (A,C,E) or Mash1–/– (B,D,F) mouse embryos at E11.5 containing the transgene M1-GIC (see Fig. S1 in the supplementary material) and the Cre reporter allele R26R-YFP. (A-F) Anti-GFP antibodies detect both GFP from M1-GIC and YFP when R26R-YFP reports the Cre activity from M1-GIC. These green labeled cells reflect cells with Mash1 or progeny of Mash1+ cells. (A,B) Brn3a;Lhx1/5 (pink) label dI2. These cells would appear white if they co-labeled with antibodies to GFP/YFP. The absence of white cells indicate dI2 do not derive from Mash1+ precursors (A). (C,D) Brn3a;Isl1 (pink) label dI3. White cells indicate co-labeling with antibodies to GFP/YFP and demonstrate Mash1+ cells give rise to dI3 neurons (C, arrowheads) and these cells are absent in the null (D). (E,F) Lmx1b (red) labels dI5. The co-labeling with GFP/YFP (yellow) indicates dI5 are derived from Mash1+ cells (E, arrowheads) and these cells are absent in the null (F). Lhx1/5 (blue) labels dI4. Co-labeling with GFP/YFP (turquoise) indicates rare dI4 cells derived from Mash1+ cells (E, arrow), and these cells do not increase in Mash1–/– (F). The increase in dI2 and dI4 seen at E10.5 cannot be attributed to fate switching of dI3 and dI5 precursor cells. The color of each neuronal population is shown on the left without and with (black background) co-labeling with the GFP/YFP. The total number of cells for each neuronal subtype and the number of each that co-labels with GFP/YFP are shown in the table. (G) Individual populations in each mutant were counted on at least three sections from at least three embryos each. Scale bar: 25 µm.

View larger version (66K): [in a new window]   Fig. 5. Mash1 inhibits Ngn1 but not Ngn2 expression in the dorsal neural tube. Immunofluorescence (A,B,D,E) and in situ hybridization (G-J) on transverse sections of E10.5 mouse neural tubes. (A-C) There is no change in levels of Ngn2 between wild-type (A) and Mash1–/– (B) embryos. (D-F) There is no change in expression of Mash1 between wild-type (D) and Ngn2–/– (E) embryos. (G-J) The dorsal domain of Ngn1 is expanded in Mash1–/– (H, arrow) and Mash1–/–;Ngn2–/– (J, arrow) relative to wild-type (G) embryos. No change was detected in Ngn1 in Ngn2–/– (I) embryos. Each panel is representative of the phenotype seen in at least three sections from at least three embryos. Scale bar: 50 µm in A,B,D,E; 225 µm in G,H.

View larger version (78K): [in a new window]   Fig. 6. Distinct functions for Mash1 and Ngn2 in specification of dorsal neurons. Immunofluorescence on cross-sections of E11.5 mouse neural tubes where Mash1 and Ngn2 have been swapped into the Ngn2 and Mash1 locus, respectively: wild type (A,F,K), Mash1KI Ngn2/+ (B,G,L), Mash1KI Ngn2 (C,H,M), Ngn2KI Mash1/+ (D,I,N) and Ngn2KI Mash1(E,J,O). (A-E) Co-localization of Brn3a (red) and Lhx1/5 (green) detects dI2 neurons (yellow). (F-J) Isl1 (green) detects dI3 neurons. (K-O) Pax2 (green) and Lmx1b (red) detect dI4 and dI5 neurons, respectively. Cell counts for each marker on at least three sections from at least three embryos of each genotype are shown in the table. (P) Because dI4 and dI6 populations cannot be distinguished in the Mash1 null, all Pax2 counts were completed by drawing a boundary line at the first Pax2-expressing cell found nearest the ventricular zone, and as such, they are labeled as dI4/6. **P<0.001 and *P<0.01. Scale bar: 50 µm.