IGF signalling is critical for cardiac valve development in zebrafish. (A,C) Heart region of IGF-2a or IGF- 2b morphant embryos at 72 hpf. Hearts are enlarged and incompletely looped (white outline) with blood reflux between the two chambers. (B,D) Co-injection of igf-2a or igf-2b RNA with the corresponding morpholino rescues the IGF-2 morphant phenotype. Hearts in rescued embryos are identical to hearts in uninjected embryos. (E,J) Expression of bmp4 and notch1b in embryos injected with control morpholinos. Note the restriction of expression at the atrioventricular boundary (black arrowheads). The white outline indicates the shape of the heart. (F,K) Bmp4 and notch1b are restricted to the atrioventricular boundary in IGF- 2a morphant embryos. (G,H,L,M) IGF-2b and IGF-2(a+b) morphant embryos show a loss of bmp4 and notch1b expression at the atrioventricular boundary (black arrowheads). The heart is also incompletely looped at this stage (white outline). (I,N) DN-IGF-1R injected embryos show a loss of bmp4 and notch1b at the atrioventricular boundary. (O) Expression of eln2 at the outflow tract of the heart in embryos injected with control morpholinos (white arrowhead). (P,Q,R) IGF-2a, IGF-2b and IGF-2(a+b) morphant embryos show a decrease of eln2 expression at the outflow tract. (S) DN-IGF-1R injected embryos show a loss of eln2 at the outflow tract. Frequency of embryos displaying this expression pattern: F, 30/30; G, 20/40; H, 15/27; I, 29/ 44; K, 33/33; L, 14/32; M, 12/30; N, 49/56; P, 5/24; Q, 31/36; R, 24/26; S, 31/39. (T) Normal cardiac looping in control injected embryos at 72 hpf as shown by the expression of cmlc2 (heart loops to the right in this view). Variety of abnormal cardiac looping phenotypes in IGF-2(a+b) morphant embryos. (The frequencies of the irregular looping phenotypes are given in Table 2.) A-D and O-S are lateral views, E-N and T-X are ventral views.
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