Fig. 2

Spaw expression. (A-D) Four to six somite stage. (A) Dig-spaw probe, dorsal close-up of tailbud. (B-D) Two-color in situ hybridization: spaw probe and a spadetail or ntl probe. (B) Dig-spaw (purple) + Fl-spt (red), dorsal view of tailbud. (C) Dig-spaw (purple) + Fl-ntl (red), lateral view of tailbud. n, prospective notochord. (D) Dig-spaw (purple) + Fl-ntl (red), dorsal view of tailbud. (E) Ten to 12 somite stage. Dig-spaw, dorsal view, onset of asymmetric left-sided expression (arrowhead). (F,G) Twelve to 13 somites, dorsal views, comparing spaw (F) and one-eyed pinhead (G) expression. Anterior is towards the top. (H,I) Seven to nine somite stage, lateral view, close-up of heads. Anterior is towards the right. (H) Dig-spaw probe. (I) Dig-oep probe. (J-M) Dorsal views, anterior towards the top. (J) Fifteen to 18 somites, DNP-spaw plus a DNP-netrin probe. Arrowhead indicates left-sided spaw domain. Arrow indicates central stripe of netrin in floor plate. (K,L) Twenty to 23 somite stage. (K) Dig-spaw (purple) showing left-sided spaw domain along most of the length of the embryo. White arrow indicates bilateral spaw flanking tailbud. (L) Left-sided spaw domain has expanded laterally and is fading posteriorly. (M) Twenty-two to 24 somite stage; Dig-spaw probe (inset provides a lateral view of the tailbud).

Fig. 3

Comparison of spaw with lefty2 and pitx2. (A,C,D) Dig-spaw (purple) + DNP-lefty2 (red) probes. Dorsal close-up; A is at a lower magnification than C and D; lefty2 marks the heart field. (A,C,D) The patterns seen in embryos from a pool of 20-23 somite embryos. (A) spaw is expressed in this embryo but lefty2 is not. The converse pattern (lefty2+,spaw–) was never seen. (B) Dig-spaw probe alone, 20-23 somite stage. Dorsal close-up near heart field. Comparison of B (spaw probe) with C (spaw + lefty2 probes) emphasizes the extensive overlap between the left-sided spaw and lefty2 expression domains in C. (D) Asymmetric spaw and lefty2 zones are present as largely non-overlapping domains. (E) Dorsal close-up. DNP-lefty2 probe (red) only, 20-23 somite embryo. (F) Twenty to 23 somite embryo. Dig-spaw (purple) + DNP-pitx2 (red) probes. Lateral view, anterior towards the left, comparing the anterior border of spaw (black arrowhead) in the lateral plate with the anterior position of left-sided pitx2 (arrow) in the diencephalon.

Fig. 4

Spaw expression in midline mutants. (A-E) Dorsal views, anterior is towards the top. Digspaw probe. (F) Coordinate axes: A, anterior; P, posterior; L, left; R, right. (A,B) Ten to 12 somite stage, ntlb160/ntlb160 (abbreviated ntl–/– in figure) Asterisk indicates tailbud; arrowheads indicate left and right lateral plate. (C) Seventeen to 19 somites. Wild-type sibling (sib) from a ntlb160/+ incross. Arrow indicates ectopic spaw. (D) Sixteen to 20 somite embryo, sqtcz35/sqtcz35 (abbreviated sqt–/– in figure) Arrowheads indicate bilateral spaw expression in lateral plate. Inset shows lateral view of tailbud. (E) Seventeen to 18 somites, bozm168/bozm168. Arrowhead indicates rightsided spaw domain. (G,H) Shield stage, looking down on blastoderm, dorsal towards the right, Dig-goosecoid probe. (G) Uninjected control. (H) Injected with 25 pg pCS2+ActHASpaw plasmid.

Fig. 5

Morphological effects of spaw knockdown. (A-D) Uninjected and Spaw- MO1-injected embryos (10 ng), 55-60 hours post-fertilization embryos. (A) Lateral view, anterior towards the left. Asterisks indicate posterior end of yolk tube extension. (B,C) Higher magnifications of embryos in A, focusing on somites and notochord. (D) Dorsal view, anterior towards the left. (E) Sixty to 70% epiboly, Dig-gsc probe, dorsal view. Uninjected embryo (left); embryo injected with 10 ng Spaw-MO1 (right). (F-H) Ventral anterior view of 30 hours post-fertilization embryos injected with 6 ng Spaw-MO1. Dig-nkx2.5 probe shows the heart tube (arrowheads). Hearts exhibit left jog (F), no jog (G) or right jog (H). (I,J) MF20 myosin antibody staining highlighting normal Dlooping (I) and reversed L-looping (J) of the heart in two embryos injected with 10 ng each of Spaw-MO1. Ventral views, 60 hours post-fertilization. The ventricle (arrowhead) stains more intensely. (K,L) Dorsal view of 48 hours postfertilization embryos (anterior towards the left), Dig-preproinsulin probe, right (K) and left (L) displacements of the pancreas (arrowheads) from the midline. R, right side; L, left side.

Fig. 6

The effects of spaw inhibition on midline development are restricted to effects on late lefty1 expression. (A-G) Comparison of lefty1 expression in uninjected (u) and Spaw-MO1-injected (10 ng) embryos at various stages. Dig-lefty1 probe. (A,B) Shield stage. (C,D) One to 3 somite stage. (E,F) Seven to 8 somite stage. (G) nineteen to 22 somite stage. (A,B) View onto blastoderm, dorsal is downwards. (C-F) Lateral views, anterior towards the top. (G) Lateral views, anterior towards the upper right. Arrowheads indicate presence (left) or absence (right) of asymmetric lefty1 expression in diencephalon. Asterisks indicate presence (left) or absence (right) of lefty1 in posterior notochord. (H,I) Comparison of ntl expression in uninjected (u; left in H, right in I) and Spaw-MO1- injected (10 ng; right in H, left in I) embryos at shield and 6-7 somite stages. Dig-ntl probe. (H) Arrowheads indicate ntl expression in dorsal shield and forerunner cells. (I) Views of dorsal midline, anterior is towards top.

Fig. 7

spaw regulates asymmetric gene expression. (A-C) Seventeen to 18 somite stage, DNP-netrin + DNP-spaw probes. Arrows indicate netrin (net) in the CNS. Arrowheads indicate the presence (A,C) or absence (B) of spaw in the left lateral plate. Asterisk indicates spaw expression in the tailbud. (A) 10 ng NCMO; (B) 10 ng Spaw-MO1; (C) uninjected (u). (D) Twelve to 14 somite stage, Dig-oep probe. (Top) Uninjected; (Bottom) injected with 14 ng Spaw- MO1. (E) Twenty-two to 24 somite stage; Dig-pitx2 probe, dorsal view of embryos injected with 10 ng NCMO (left), 10 ng of Spaw-MO1 (middle) or not injected (right). Black arrows indicate position of left-sided pitx2c near gut. Black arrowheads indicate bilateral pitx2a/2c in putative Rohon-Beard neurons. (F) Twenty to 22 somite stage; Dig-lefty2 probe; dorsal view with anterior towards the top. (Left) 10 ng Spaw-MO1 treated; (Right) 10 ng NC-MO treated. Arrowheads indicate presence (right) or absence (left) of left-sided lefty2 domain. (G) Nineteen to 22 somite stage; Diglefty1 probe; dorsal view with anterior towards the top. (Right) Uninjected; (Left) injected with 10 ng Spaw- MO1. Arrowheads indicate presence (right) or absence (left) of left-sided lefty1 domain in heart. (H) Nineteen to 22 somites, lateral view, anterior towards the left. Dig-lefty1 probe. Embryos injected with 10 ng of Spaw- MO1 (left) or of NC-MO (right). Asterisks indicate site of lefty1 expression domain in posterior notochord. Arrowheads indicate location of lefty1 domain on left side of dorsal diencephalon. (I-K) Eighteen to 22 somites; DNP-cyclops probe; lateral view of head, anterior towards the left. (I) Embryo injected with 10 ng of NC-MO. (J) Embryo injected with 10 ng Spaw-MO1. (K) Uninjected embryo. Arrowheads indicate expression (I,K) or absence (J) of cyclops in the left part of the dorsal diencephalon. (L,M) Twelve to 14 somites, anterior towards the bottom; Dig-oep probe, dorsal view, focusing on the epiphysis. (L) Uninjected embryo. (M) Embryo injected with 14 ng Spaw-MO1. (N,O) Nineteen to 23 somites; Dig-flh probe, dorsal view, focusing on epiphysis. (N) Uninjected embryo. (O) Embryo injected with 10 ng Spaw-MO2. (P,Q) Twenty-one to 23 somite stage; DNP-pitx2 probe; lateral views (left side), anterior towards the left. Arrowheads indicate region of the dorsal diencephalon where pitx2c mRNA is normally restricted to the left side. (P) Embryo injected with 10 ng of Spaw-MO1. (Q) Uninjected embryo. (R,S) Twenty-one to 23 somite stage, DNP-pitx2 probe, frontal view. Arrowheads indicate region of the dorsal diencephalon where pitx2c mRNA is normally restricted to the left side. (R) Embryo injected with 10 ng of Spaw-MO1. (S) Embryo injected with 10 ng of NC-MO. (T-W) Sixteen to 18 somite stage; anterior is towards the top, dorsal view. DNP-spaw (–MATR) probe is specific for endogenous spaw transcripts. (T) Uninjected embryo. (U) Embryo injected with 9 ng of Spaw-MO1. (V,W) Two examples of ‘spaw-reversed’ embryos, achieved by co-injection of 9 ng Spaw-MO1 and 40 pg ActHASpaw RNA

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