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.