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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), hans6 mutant embryos (C, G), osr1 morphant (osr1 MO) embryos (D, H) and hans6 mutant embryos injected with osr1 morpholino (hans6 + 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 hans6 mutants (C, n=11), absent (48%) or reduced (48%) in osr1 morphants (D, n=25), and relatively normal in hans6 + 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 hans6 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 hans6+ 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 ?m.