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First published online March 1, 2004
doi: 10.1242/10.1242/dev.01008

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Distinct domains within Mash1 and Math1 are required for function in neuronal differentiation versus neuronal cell-type specification

Center for Basic Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9111, USA

View larger version (49K): [in a new window]   Fig. 4. Essential structural component of Mash1 required for its neuronal differentiation function is identified. (A) Schematic of the Mash1 and MyoD chimeric bHLH domains used to identify amino acids required for the neuronal differentiation activity of Mash1. Mash1 sequences are shown in blue and MyoD sequences are in red. The first ten constructs are in the context of Mash1 with the domain replaced by MyoD sequences shown in parentheses. The next ten constructs are in the context of MyoD with the domain replaced by Mash1 sequences shown in parentheses. One MyoD/Math1 chimeric protein was tested; the Math1 HLH sequence is shown in green. Mash1NR-AQ, Mash1R-G, and Mash1E-G (black) are DNA binding mutants. The ability of each chimeric protein to drive neuronal differentiation was assayed by the fluorescence intensity of GFP found preferentially in the lateral half of the neural tube (+). (B-M) Representative sections from chick embryos expressing the chimeric proteins. B-G are in the context of full-length Mash1, and H-M are in the context of full-length MyoD. The white dots outline the electroporated side of the neural tube. Ventral is on the left in each panel. Expression of each construct was verified using immunofluorescence with myc antibody (data not shown). (N,O) Quantification of these data shown as a ratio of fluorescence intensity (FI) in the lateral half versus the medial half of the neural tube. Helix 1 of Mash1 is necessary (E) and sufficient (in the context of MyoD) (K) for the neuronal differentiation phenotype. **P<0.001.

View larger version (88K): [in a new window]   Fig. 1. Neural bHLH factors induce neuronal differentiation in the chick neural tube. (A-Q) HH14-16 chick neural tubes were electroporated in ovo with constructs expressing myc-tagged control vector (A,B), Mash1 (C,D), Math1 (E,F), Mash1NR-AQ (G,H), Mash1R-G (I,J), MyoD (K,L), MyoD(Mash1H1) (M,N), MyoD(Mash1HLH) (O), Mash1(MyoDH1) (P) and harvested 24 hours later. Cross sections through the neural tube are shown at 20x (A-P) and 40x (A'-P'). Immunofluorescence with anti-Mash1 or anti-myc antibodies indicates the expression of the transgene in the electroporated cells (A-P, red). Double labeling with the Tuj1 antibody was used to detect differentiating neurons (A,C,E,G,I,K,M,O,P, green), or with anti-BrdU to detect proliferating cells in the ventricular zone (B,D,F,H,J,L,N, green). (Q) The percentage of electroporated cells expressing Tuj1. (R) The percentage of electroporated cells incorporating BrdU. Expression of Mash1, Math1, MyoD(Mash1H1), and MyoD(Mash1HLH) caused cells to move laterally out of the ventricular zone, express the neuronal marker Tuj1, and exit the cell cycle. These activities require DNA binding since the DNA binding mutants Mash1NR-AQ and Mash1R-G lack these activities. Expression of the non-neural bHLH MyoD and Mash1(MyoDH1) also lacked these activities. The electroporated side is on the right in A-P. **P<0.001, *P<0.01.

View larger version (52K): [in a new window]   Fig. 2. Neuronal sub-type specification activity of the neural bHLH factors. (A-H) HH14-16 chick neural tubes were electroporated in ovo with expression constructs Mash1 (A,B), Mash1NR-AQ (C,D), Mash1R-G (E,F) and Math1 (G,H), and harvested at 24 hours. Immunofluorescence with antibodies to Lhx2/9, Islet1, Lhx1/5, or Pax2 as indicated illustrate dorsal interneuron populations dI1-dI3, dI4/6 (see diagram in M). dI2 cells are distinguished from dI4 and dI6 by their lack of Pax2 expression (B,D,F, red cells). In each case the electroporated side is on the right (arrowhead) and should be compared with the control side on the left. The white line indicates the midline. (I-L) Quantification of the data in A-H. Mash1 increases dI3 neurons (A) at the expense of dI1 (A) and dI2 (B) neurons. This activity of Mash1 requires Mash1 to bind DNA since the Mash1NR-AQ and Mash1R-G mutants, which lack DNA binding, do not have this phenotype (C-F). Math1 increases dI1 (G, H) at the expense of dI2 (H) and dI3 (G) neurons. (M) Diagram of different populations of progenitors (dP1-dP6 defined by bHLH expression) and interneurons (dI1-dI6 defined by LIM HD factors), and a summary of how the interneurons change in response to overexpression of the bHLH factors. **P<0.001.

View larger version (51K): [in a new window]   Fig. 3. Math1 and Mash1 are transcriptional activators in their neurogenic and neuronal cell-type-specific activities. (A-L) HH14-16 chick neural tubes were electroporated in ovo with expression constructs VP16 (A,B), Math1bHLH-VP16 (C,D), Mash1bHLH-VP16 (E,F), EnR (G,H), Math1bHLH-EnR (I,J) and Mash1bHLH-EnR (K,L), harvested at 24 hours. (A,C,E,G,I,K) Immunofluorescence using anti-myc antibodies to assay for expression of the transgene demonstrates the movement of the cells to the lateral neural tube where differentiating neurons reside. White dots outline the injected side of the neural tube and arrows indicate the lateral (C,E) or medial (I,K) position of the electroporated cells within the neural tube. (M) Quantification of these data shown as a ratio of fluorescence intensity (FI) in the lateral half versus the medial half of the neural tube (M). (B,D,F,H,J,L) Interneuron populations dI1 and dI3 were detected using anti-Lhx2/9 (red) or Islet1 (green). (N) Quantification of these data shown as a ratio of the number of labeled cells on the electroporated side (right, indicated by arrowhead) versus the number of labeled cells on the control side (left). VP16 and EnR on their own have no effect in these assays. Math1bHLH-VP16 and Mash1bHLH-VP16 approximate the activity of full-length Math1 and Mash1 in both the neuronal differentiation and cell-type specification activities. In contrast, Math1bHLH-EnR and Mash1bHLH-EnR have the opposite effect. **P<0.001.

View larger version (51K): [in a new window]   Fig. 5. Essential structural components of Math1 and Mash1 required for their neuronal specification properties are identified. (A) Schematic of the Math1 and Mash1 chimeric bHLH domains used to identify amino acids required for their neuronal specification properties. Math1 sequences are shown in green and Mash1 sequences are in blue. The first six constructs are in the context of Math1 with the domain replaced by Mash1 sequences shown in parentheses. The next six constructs are in the context of Mash1 with the domain replaced by Math1 sequences shown in parentheses. Math1 HLH (green) and Mash1 HLH (blue) were also tested in the context of MyoD (red). The effect of each chimeric protein, in increasing (+) or decreasing (-) dI1 and dI3 interneuron populations as described in Fig. 2, is shown. (B) Quantification of Lhx2/9- and Islet1-expressing cells in the electroporated side versus the control side for each construct. **P<0.001. (C) Double immunofluorescence with Lhx2/9 (red) and Islet1 (green) showing the aberrant co-expression (yellow) of these HD factors in neural tubes electroporated with Mash1 with the helix 2 of Math1.

View larger version (26K): [in a new window]   Fig. 6. Modeling of Class II bHLH structures place subclass-specific amino acids residues on an external surface of the dimer. (A) Alignment of amino acid residues from the bHLH domain of neural bHLH factors and MyoD. A representative from mouse for each neural sub-class is shown; Mash1, the Achaete-scute family; Math1, the Atonal family; Ngn1, the Neurogenin family; Nscl1, the Nscl family; NeuroD, the NeuroD family. Residues in red highlight the residues conserved between all Class II bHLH factors. Residues in blue are conserved within members of a specific sub-family but are unique between the sub-families. (B) Mash1 bHLH monomer modeled onto the crystal structure of MyoD. The interfaces between the E-protein heterodimer partner and DNA are shown. Note the residues conserved between all Class II bHLH factors provide these interfaces (red). The residues unique to each sub-family (blue) provide surfaces for additional interactions.

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