Figure 6

MHB expression of pax2a, eng2a and krox20 mRNA is normal, but patterning of pax2a (+) spinal interneurons and pronephric cells is disrupted, in brd2b morphants. Panels (A?L): Brightfield images of representative 24 hpf prim5 control and morphant embryos assayed by In situ hybridization for expression of patterning genes at the MHB region (A?I) and in spinal interneurons (J?L). Uninjected embryos (wt; A,D,G,J), embryos injected with control brd2b-5-base mismatch morpholino (mis5MO; B,E,H,K), and embryos injected with brd2bMO (brd2bMO1; C,F,I,L) were assayed for eng2a (A,B,C), krox20 (D,E,F,), and pax2a (G,H,I; J,K,L) expression. No differences in patterning gene expression at the MHB region are detected. Using pax2a(+) as a marker for spinal interneurons reveals mispairing on either side of the midline in morphants (J,K vs. L, gaps between lines). (M) Immunofluorescence with anti-Pax2a antibodies to visualize interneurons on either side of spinal cord, dorsal views of maximum projection confocal images. Representative control uninjected (wt) and brd2bMO1-injected (2bMO) embryos are shown with symmetrically paired interneurons linked by white lines; unpaired interneurons appear solo on one side of the central spinal cord. (N) The number of paired ?P? vs. unpaired ?U? interneurons in six embryos per treatment group (uninjected controls = none; brd2bMO-injected = 2bMO) was analyzed by chi-square contingency and is shown as a box plot (p < 0.0001). While, on average, only 3% of interneurons are unpaired in wild type, about 65% are unpaired in brd2bMO morphants. Panels (O?R): Analysis of Pax2a(+) pronephric precursor cells in the developing duct in brd2bMO and brd2aMO morphants by quantitative immunofluorescence. (O) Diagram of pronephric duct segments: G, glomerulus; N, neck; PCT, proximal convoluted tube; PST, proximal straight tube; DE, distal early tube; DL, distal late tube; C, cloaca. (P) Pax2a(+) immunofluorescence in uninjected (wt) and brd2bMO morphants (brd2bMO1). (Q) Pax2a(+) immunofluorescence in uninjected (wt) and brd2a morphants (brd2aMO1). Paralogs show opposite effects on distribution of Pax2a(+) pronephric cells (arrows). brd2b morphants show fewer cells in the more distal parts of the tube (PST, DE, DL) but more cells at the very end cloaca (C), compared to wild type, while brd2a morphants show excess cells along the tube, and a normal number of cells at the cloaca. (R) Quantitation of distribution of Pax2a(+) cells in uninjected (none) and brd2bMO1-injected embryos (2bMO), by pronephric segment. Boxplot of ANOVA and paired t-tests shows significant differences in the predicted direction between these two treatment groups in segments PST/DE/DL (p < 0.0001,) and in C (p < 0.0001), but not in G/PCT (p = 0.3971).