Fig. 8

hand2 and osr1 act in opposing, parallel pathways to regulate pronephron development.

(A–A'') Fluorescent in situ hybridization depicts overlap (A) of hand2 (A') and osr1 (A'') expression in wild-type embryos; dorsal views, anterior to the left, of three-dimensional reconstructions at the 6 somite stage. (B–I) In situ hybridization depicts pax2a (B–E) and atp1a1a.4 (F–I) expression at 24 hpf in wild-type embryos (B, F), han^s6 mutant embryos (C, G), osr1 morphant (osr1 MO) embryos (D, H) and han^s6 mutant embryos injected with osr1 morpholino (han^s6 + osr1 MO) (E, I); dorsal views, anterior to the left. (B–E) Compared to wild-type (B), pax2a expression in the glomerular and neck precursors (arrow) was expanded in 100% of han^s6 mutants (C, n=11), absent (48%) or reduced (48%) in osr1 morphants (D, n=25), and relatively normal in han^s6 + osr1 MO embryos (E, n=11). While the extent of marker expression was generally comparable to wild-type in the double loss-of-function embryos, the stereotypic patterning of this population was often somewhat disrupted. Expression in overlying spinal neurons (asterisk) was unaffected. (F–I) Compared to wild-type (F), atp1a1a.4 expression in the pronephric tubules was wide in 85% of han^s6 mutants (G, n=13), while many osr1 morphants (H, n=133) had tubules with shortened anterior expression (18%) or tubules with segmental losses (35%), and 47% of osr1 morphants had a wild-type appearance. 85% of han^s6+ osr1 MO embryos (I, n=46) resembled wild-type, whereas 11% had a shortened anterior tubule and 4% had segmental losses in the tubules. Scale bars represent 100 μm.