Figure 7

Co-knockdown of Brd2a and Brd2b suppresses morphant defects in both brain and pronephros of morphant embryos at 24 hpf. Double knockdown was used to test for genetic interaction between paralogs. Panels (A?D): Brightfield images of head and trunk of representative 24 hpf prim5 embryos from indicated treatment groups assessed for morphology: (A) uninjected, (B) brd2bMO-injected, (C) brd2aMO-injected, and (D) brd2bMO + brd2aMO co-injected. Panels (E?H): Darkfield images of representative embryos from the same treatment groups assessed by TUNEL assay for apoptotic nuclei: (E) uninjected, (F) brd2bMO-injected, (G) brd2aMO-injected, and (H) brd2bMO + brd2aMO co-injected. Co-injection of MOs from each paralog suppresses morphant defects in both morphology and cell death in the brain and trunk, indicating genetic interaction with functional antagonism between paralogs. Black arrows indicate reduced brain, ill-formed MHB region, disorganized PBI common to both morphants (B,C), and plug of excess cells at the cloaca unique to brd2bMO morphants (upper arrow in B, trunk). White arrows show increased apoptosis in brain and PBI common to both morphants (F,G, head; G, trunk), while lack of apoptosis is seen at the cloaca only in brd2bMO morphants (F, trunk). See Table 1 for population morphology data and Figure 5 for quantitative TUNEL data. Panels (I?L): Darkfield images of pronephric duct in trunks of representative 24 hpf prim 5 embryos from the indicated treatment groups assessed for Pax2a(+) cells by immunofluorescence: (I) uninjected, (J) brd2bMO-injected, (K) brd2aMO-injected, and (L) brd2bMO + brd2aMO co-injected. Cloacal opening is indicated by white arrow in left panels. Distribution along the ductal tube of Pax2a(+) pronephric precursor cells is highlighted between white arrows in right panels. Co-knockdown suppresses morphant defects of each paralog, bringing distribution of cells along the duct and at the cloaca closer to wildtype. (see also Figure 6O,P,Q). Panels (M?P) Darkfield images of spinal interneurons along dorsal trunks of representative 24 hpf embryos from the indicated treatments groups assessed for Pax2a(+) cells by immunofluorescence: (M) uninjected, (N) brd2bMO-injected, (O) brd2aMO-injected, and (P) brd2bMO + brd2aMO co-injected. Co-knockdown suppresses the morphant mis-pairing defect of each paralog, and restores interneuron number and pairing to near wildtype levels. Lines connect symmetrically paired neurons; unpaired neurons appear solo on one or the other side of the central nerve cord.