Laterality defects in laf/alk8 mutant embryos. (A–C) Wild-type sibling (A) and laf/alk8 mutant (B) embryo at 30 hpf stained for cmlc2 expression to reveal positioning of the heart and quantification of the results (C). (D–G) Wild-type sibling (D) and laf/alk8 mutant (E, F) embryos at 50 hpf stained for foxa3 expression to reveal the positioning of the liver (l), pancreas (p) and gut (g) and quantification of the results (G). (H–K) Wild-type sibling (H) and laf/alk8 mutant (I, J) embryos at 22 somite stage stained for lefty1 expression in the dorsal diencephalon and quantification of the results (K). All pictures are of dorsal views with anterior to the top (M, middle; L, left; R, right; B, bilateral; A, absent).

Bmp regulates LR patterning at two distinct developmental time points. (A–E) Spw expression in the LPM and ntl expression in the midline in wild-type (A), Tg(hs::noggin3) (B, C) and Tg(hs::bmp2b) (D, E). Heat-shock induced noggin3 expression at tb stage results in bilateral spw expression (B) while noggin3 induction at the 16-somite stage has no effect on spw expression (C). Heat-shock induced bmp2b expression at the tb stage results in the absence of spw expression (D) while bmp2b induction at the 16-somite stage has no effect (E). (F–J) At the 22 somite stage pitx2 is asymmetrically expressed in the LPM (F, arrow). Left-sided pitx2 expression is absent in Tg(hs::noggin3) embryos heat-shocked at the tb stage (G). Normal left-sided pitx2 expression in Tg(hs::noggin3) embryos heat-shocked at the 16 somite stage (H). Bilateral pitx2 expression in the LPM of Tg(hs::bmp2b) embryos heat-shocked at the tb stage (I). Normal left-sided pitx2 expression in Tg(hs::bmp2b) embryos heat-shocked at the 16 somite stage (J). (K–O) Lefty1 expression in the midline and left cardiac field at the 22-somite stage of a wild-type embryo (K). Induction of noggin3 (L, M) or bmp2b (N, O) at tb or 16-somite stage, results in the down-regulation of lefty1 in the cardiac field. In the notochord, lefty1 expression is repressed by ectopic noggin3 (L, M) and induced by bmp2b (N, O). (P–T′) Lefty2 expression in the left cardiac field of a 25-somite stage wild-type embryo (P). Induction of noggin3 at the tb (Q) or 16-somite stage (R) or the induction of bmp2b at the tb stage (S) results in the down-regulation of lefty2 expression. Induction of bmp2b expression at the 16-somite stage results in ectopic lefty2 expression in the right cardiac field (T, arrowhead) and in the notochord (T2). (U–W) Foxa3 expression in the visceral organs of a wild-type embryo at 50 hpf (U). Induction of noggin3 at the tb stage results in visceral organ laterality defects, such as organ duplications and absence of gut looping (V) while ectopic noggin3 expression induced at the 16-somite stage has no effect on the laterality of the visceral organs (W). All embryos are shown as dorsal views with anterior to the top of the panel.

Ectopic noggin3 expression prior to the 22-somite stage affects normal cardiac jogging. (A–E) Schematic depiction of the bilateral cardiac fields during heart tube formation at the indicated stages corresponding to the stage when embryos were heat-shocked. (F–J) Induction of noggin3 in Tg(hs::noggin3) embryos by a heat-shock at the 10-somite stage (F), 15-somite stage (G), 18-somite stage (H), 22-somite stage (I) or 28-somite stage (J). All are dorsal views with anterior to the top of the panel showing cmlc2 expression at 26–30 hpf. L, left; R, right.

Bmp4 expression at early and late somite stages. Lateral view of a 10-somite stage embryo showing Bmp4 expression in an anterior and posterior expression domain (A). Boxed area shown in panel A is enlarged in panel B and shows bmp4 expression in and around Kupffer's vesicle (arrow). At the 20–22 somite stage (C), bmp4 expression is confined to the cardiac field and LPM where it is stronger on the left compared with the right side. Injection of a spw MO results in a down-regulation of bmp4 expression in the left LPM (D). Lateral views with anterior to the right (A, B) and dorsal views with anterior to the top (C, D). L, left; R, right.

Bmp4 morpholino knock down. (A) Genomic sequence of bmp4 showing exon1 (capitalized), intron1 (non-capitalized) and exon2 (capitalized). Location of the bmp4 splice MO is indicated and the primers used for RT-PCR are indicated by black arrows. (B–D) RT-PCR on mRNA from uninjected control embryos (lane 1) or embryos injected with 1 ng bmp4 splice MO (lane 2) and 0.5 ng bmp4 splice MO (lane 3) and control PCR (-RT) on control embryos (lane 4) or embryos injected with 1 ng bmp4 splice MO (lane 5) and 0.5 ng bmp4 splice MO (lane 6). PCR on the ef1a gene was used as a control for RNA isolation procedure and the RT reaction (B). PCR using the exon-intron primers 1 and 2 will amplify unspliced bmp4 RNA (C) which is induced by bmp4 splice MO injections. PCR using exon–exon primers 1 and 3 will results in the amplification of normal spliced bmp4 mRNA (lane 1) or alternatively spliced bmp4 mRNA after bmp4 splice MO injection (lanes 2 and 3). Band “A” in lane 1 indicates normal spliced bmp4 mRNA and band “B” indicates alternatively spliced bmp4 mRNA with 54 bp insertion (both determined by sequencing the fragments). The asterisk in lane 1 indicates non-specific amplification (determined by sequencing). Arrowheads indicate various alternatively spliced products induced by injection of 1 ng of bmp4 splice MO. (E, F) Injection of 1.6 pg of wt bmp4 mRNA results in a strong ventralization of the embryos (91%, n = 116) (E), while injection up to 40 pg of the bmp4 mRNA with the 54 bp insertion (bmp4-splice) has no effect (0% affected, n = 54) (F). (G–I) Injection of 0.8 ng of bmp4 splice MO results in the loss of ventral structures such as ventral tail fin (arrow) and blood islands (asterisks), both characteristics of mild C1 and C2 dorsalization (H) (in 49% of injected embryos, n = 138) which can be rescued by coinjection of bmp4 splice MO and 0.8 pg bmp4 RNA (I) (15% C1 and C2 dorsalization, n = 156).

Bmp4 is required for LR patterning. (A, B) Spw expression in the left LPM and ntl expression in the notochord in control MO-injected embryos (A) and bilateral spw expression in bmp4 splice MO-injected embryos (B) at the 18 somite stage. (C, D) pitx2 expression in the posterior LPM (arrow) of control MO-injected embryos at the 22 somite stage (C) is predominantly absent or reduced when embryos are injected with bmp4 splice MO (D). (E, F) Cyclops is broadly expressed but stronger in the left anterior LPM of wt or control MO-injected embryos at the 22 somite stage (arrow in panel E). Although still being expressed, the asymmetry in cyclops expression is lost when embryos have been injected with bmp4 splice MO (F). (G, H) Normal lefty1 expression in the notochord and left cardiac field of 22 somite stage embryos (G) is absent from embryos injected with bmp4 splice MO (H). (I, J) Normal cardiac jogging in control MO-injected embryos (I) and loss of cardiac jogging in embryos injected with bmp4 splice MO (J) revealed by cmlc2 expression. Dorsal view, anterior to the top of 30 hpf embryos. (L, M) Visceral organ laterality revealed by foxa3 expression in control MO-injected embryos (L) and embryos injected with bmp4 splice MO (M) resulting in organ duplications and failed gut looping.

Bmp4 in Kupffer's vesicle is required for LR patterning. All embryos shown here were injected at the 512 cell stage to target the bmp4 MOs to the dorsal forerunner cells (DFC) that will form Kupffer's vesicle. (A, B) Ntl expression in 10 somite stage embryos marks Kupffer's vesicle. No difference in vesicle morphology was observed in DFC^{ctr MO}-injected embryos (A) compared to DFC^{bmp4 MO}-injected embryos (B). Lateral views of tail bud, arrowhead points to KV. (C, D) LR dynein (dnah9) is expressed in Kupffer's vesicle. No difference was observed in DFC^{ctr MO}-injected embryos (C) compared to DFC^{bmp4 MO}-injected embryos (D). Ventral views of tail bud at 1 somite stage, arrowhead points to Kupffer's vesicle. (E–H) Ectopic spw expression in the right LPM upon DFC^{bmp4 MO} injection (F) compared with DFC^{ctr MO}-injected embryos (E). No effect on spw expression was observed after DFC^{bmp2b MO} (G) or DFC^{bmp7 MO} (H) injection. Dorsal views of 18 somite stage embryos. (I–L) Lefty1 is expressed in the head region and in the notochord (arrow) at the 10 somite stage (I, J) and in the left heart field and notochord at the 22 somite stage (K, L). Lefty1 expression is absent from the notochord but normally present in the head region in DFC^{bmp4 MO}-injected embryos (J) when compared to of DFC^{ctr MO}-injected embryos (I). At the 22 somite stage lefty1 expression is absent from the DFC^{bmp4 MO}-injected embryos (L) compared to DFC^{ctr MO}-injected embryos (K). (I, J) Lateral view of 10 somite stage embryos. Arrow points to the lefty1 expression in the posterior notochord. (K, L) Dorsal views of 22 somite stage embryos. (M, N) Expression of lefty2 in the LPM is not affected in the DFC^{bmp4 MO}-injected embryos (N) compared to DFC^{ctr MO}-injected embryos (M). Dorsal views of 25 somite stage embryos.

Comparable nkx2.5 expression in laf/alk8 siblings (A) and mutant (B) embryos at the 15-somite stage. Heart tube in laf/alk8 siblings jogs to the left (C) while it remains in the middle in mutant embryos (D). Embryos at 28 hpf double stained with vmhc (red color) and amhc (blue color). laf/alk8 sibling (E) and mutant (F) embryos show absence of cardiac looping in the mutant embryo. (A–D) Dorsal views with anterior to the bottom of the panel. (E, F) Ventral views.

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