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.