Stephen LA, Johnson EJ, Davis GM, McTeir L, Pinkham J, Jaberi N, Davey MG.

BB/D526745/2 Biotechnology and Biological Sciences Research Council , BB/F024347/1 Biotechnology and Biological Sciences Research Council , BBS/E/R/00001614 Biotechnology and Biological Sciences Research Council , BB_BBS/E/R/00001614 Biotechnology and Biological Sciences Research Council , BBS/E/D/20320000 Biotechnology and Biological Sciences Research Council

	Figure 1. Organ asymmetry is abnormal in Talpid3−/− mouse, but normal in talpid3 chicken embryos. Wildtype mouse and chicken embryos exhibit dextral cardiac looping (a, c). This is reversed in the Talpid3−/− mouse (b) but not in the talpid3 chicken (d). Regression of the right gonad occurs in both the wildtype and talpid3 chicken by E10 (e, f). Abbreviations: GR, Right gonad; GL, Left gonad; C, Kidney cyst; K, Kidney. a–d magnification comparable, e, f magnification comparable.
	Figure 2. FOXJ1 expression is not sufficient to induce motile cilia formation in the chicken node. (a) FOXJ1 expression in the future choroid plexus of hindbrain, forebrain (outlined, arrows), and otic vesicle (asterisk) of stage 20HH embryos which precedes motile ciliogenesis, compared with stage 3HH and 4HH embryos, where no FOXJ1 can be observed (all embryos photographed underwent RNA in situ hybridization in the same tube). Red arrow indicates node, green arrow indicates primitive streak. (b) Expression of FOXJ1 by RT-PCR does show expression in stage 3HH and 4HH embryos compared to stage 20HH hindbrain (H) and forebrain (F) [negative control is stage 20HH limb bud (L)]. (c) qPCR, shows that FOXJ1 at 4HH is approximately 60% of that of the hindbrain (P = 0.000245). (d–f) Stage 4HH embryos underwent immunocytochemistry for acetylated tubulin (ciliary axoneme, green) and γ tubulin (basal body, red) to identify cilia (white arrows). Short cilia were identified in the mesoderm of the node (arrows e), while longer cilia were identified in greater abundance, in the epiblast (epi) and hypoblast (hypo) peripheral to the node (arrows f, arrowheads indicate telophase bridges which look similar to cilia but are not associated with a basal body). PG-Primitive groove; d-magnification 10×; e-magnification 50×; f-magnification 100×.
	Figure 3. Cilia are present and functional in the node of 4HH-8HH talpid3 chickens. (a–f). Stage 4HH wildtype and talpid3 embryos underwent section immunocytochemistry for acetylated tubulin (ciliary axoneme, green) and γ tubulin (basal body, red) to identify cilia (white arrows). Short cilia were identified in the mesoderm of the nodes of both genotypes (b, e, arrows), while longer cilia were identified in greater abundance, in the epiblast (epi) and hypoblast (hypo) peripheral to the node (c, f, arrows. Arrowheads indicate telophase bridges which look similar to cilia but are not associated with a basal body). (g–j). Immunocytochemistry was carried out on 8HH embryos for acetylated tubulin to identify cilia (red arrows; blue arrow denotes telophase bridge). Cilia were identified in both nodal (g, i) and nonnodal tissues (h, j) in both wildtype (g, h) and talpid3 (i, j) embryos at 8HH. (k) qPCR show no significant difference between expression of PTCH1 in wildtype and talpid3 embryos (P = 0.84). PG-Primitive groove. (a–f) Magnifications comparable: a, d-magnification 10×, b-magnification 50×, c, e, f-magnification 100×. Magnification of g–j the same (not comparable to a–f).
	Figure 4. The core PCP component VANGL2 is normally localized in talpid3 chickens at 4HH. (a, b) examples of wholemount labeled 4HH embryos, green = VANGL2 localization, red = actin, outlining the edges within the epiblast. Insets show magnification of cells lateral to the primitive streak and examples of cells outlined in white with a green asterisk highlighting VANGL2 localization. In wildtype embryos (a) and talpid3 embryos (b), VANGL2 protein within the cell was orientated toward the primitive streak in 72.4% of embryos (green, c), while 83.58% of talpid3 embryos were orientated toward the primitive streak (red, c). Abbreviations: N, Hensen's Node; dotted line—primitive streak.
	Figure 5. SHH expression is normal in the talpid3 chicken at 5HH. SHH is expressed bilaterally at the node, through the rostral notochord at 3–4HH (a, b) but is lateralized, becoming expressed on the left side of the node from 5HH (arrows, c–f) in both wildtype (c, e) and talpid3 (d, f) embryos. Magnification a–d comparable (1.6×), e–f (4×).
	Figure 6. Laterality markers are unperturbed in the talpid3 chicken. In situ hybridization in mouse embryos demonstrated expression of laterality markers Lefty1/2 in wildtype mouse embryos (a), where Lefty2 is expressed the left lateral plate mesoderm and cardiac folds (arrows) and in the Talpid3−/− mouse which exhibits expression bilaterally (arrows, b). LEFTY1 in wildtype chicken embryos at 8HH (c) and talpid3 chicken embryos (d) show the same expression throughout the notochord. PITX2 is expressed in the left lateral plate mesoderm (arrow e, f) in both the wildtype (e) and talpid3 chicken in both wildtype and talpid3 chickens.

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