Cast AE , Gao C , Amack JD , Ware SM .

HL088639 NHLBI NIH HHS , R01 HL088639-05 NHLBI NIH HHS , R01 HL088639 NHLBI NIH HHS

             convergent extension
             left/right pattern formation

Xla Wt + zic3 MO (fig.1.d, e)
Xla Wt + zic3 MO (fig.2.b)
Xla Wt + zic3 MO (fig.2.e)
Xla Wt + zic3 MO (fig.2.h)
Xla Wt + zic3 MO (fig.2.k)
Xla Wt + zic3 MO (fig.5.b)
Xla Wt + zic3 MO (fig.5.c)
Xla Wt + zic3 MO (fig.5.f)
Xla Wt + zic3 MO (fig.5.g)
Xla Wt + zic3 MO (fig.5.h)
Xla Wt + zic3 MO (fig.5.i)
Xla Wt + zic3 MO (fig.7.a, b)
Xla Wt + zic3 MO (fig.8.a)
Xla Wt + zic3 MO (fig.8.b)
Xla Wt + zic3 MO (fig.S1.a)
Xla Wt + zic3 MO (fig.S1.b)

	Fig. 1. Knockdown of Zic3 disrupts axial development in Xenopus. (A) Schematic of zic3 exon–intron structure showing translational-blocking (TB) and splice site blocking (SS) morpholino target sites. (B) RT-PCR analysis of zic3 in control (CTRL) and SS MO injected embryos. Asterisk denotes product that lacks exon 2. The top band is the full-length zic3 product. ODC was used as a loading control. (C) In vitro transcription–translation using Xenopus zic3 (Xzic3) or human HA-ZIC3 expression plasmids in the presence or absence of Zic3 TB MO. (D) Scaling system used to score Xenopus Zic3 morphant phenotypes that ranged from normal (1) to severe dorsal flexion with neural tube defect (5). (E) Average scores of control (CTRL), control morpholino (CTRL MO), Zic3 translational-blocking MO (TB MO), and Zic3 splice site morpholino (SS MO) injected embryos. Number of embryos (n) scored is shown above each injection group.
	Fig. 2. Xenopus Zic3 morphants exhibit defective gastrulation and convergent extension morphogenesis. (A) Normal control (CTRL) MO embryos at gastrula stage. (B) Sibling Zic3 morphants at stage 12.25 with delayed and abnormal blastopore closure. (C) The percentage of control morpholino and Zic3 morphant embryos that showed blastopore closure defects. (D–E, G–H) Whole-mount in situ hybridization (WISH) of brachyury at stage 13. (D) The blastopore area is outlined by the dashed line in control morpholino (D) and Zic3 morphant (E) embryos. (F) Average blastopore area in control and Zic3 morphant embryos. (G) Notochord length (L) and width (W) are designated by white lines in control morpholino embryos (G) and Zic3 morphants (H), which exhibited a wider, shorter notochord. (I) Average notochord length-to-width ratio in control morpholino and Zic3 morphant embryos. (J) Anterior–posterior (A–P) axis measurement (white line) of control morphant (J) and Zic3 morphant (K) embryos at the tailbud stage. (L) Average A–P axis lengths in control and Zic3 morphants. Error bars represent SEM. *p < 0.05; **p < 0.01 by t-test.
	Fig. 5. Reduction of Zic3 expression results in heart and gut abnormalities. (A–C) Ventral view of troponin WISH staining of the heart in Xenopus stage 46 embryos. (A) Normal heart looping with outflow tract from the right side. (B) Mirror phenotype, with outflow tract from the left side. (C) Abnormal heart looping, with no clear laterality of the outflow tract position. (D) Quantification of heart looping phenotypes in controls and Zic3 morphants. (E–I) Ventral view of Xenopus gut coiling in control and Zic3 morphant stage 46 embryos. (E) Normal gut coiling, right origin (RO) and counter-clockwise coil (CCW) direction. (F) Mirror gut coiling phenotype, left origin (LO) and clockwise coil (CW) direction. (G) Normal gut origin with reversed coil direction, clockwise. (H) Mirror gut origin, left side, with normal coil direction, counter-clockwise. (I) Abnormal gut coiling phenotype, no clear origin or coil direction. (J) Quantification of gut coiling phenotypes in control and Zic3 morphants. **p < 0.01 by Fisher's exact test; statistical significance was determined through comparison of control morphants with Zic3 morphants.
	Fig. 7. Abnormal L–R molecular marker expression in Xenopus and zebrafish Zic3 morphants. (A) Pitx2 expression in Xenopus Zic3 morphant embryos at stage 30. Embryos were photographed from both left and right sides. Red arrows highlight pitx2 expression. (B) Quantitative results of pitx2 expression in non-injected control (CTRL), control morpholino (CTRL MO) and Zic3 splice site morpholino (SS MO). (C) Southpaw (spaw) expression in zebrafish Zic3 morphant embryos. Red arrows highlight gene expression as left, right, bilateral or absent. (D) Quantitative results of spaw expression in control and Zic3 morphants.
	Fig. 8. Correlation between C-E morphogenesis and L–R patterning. (A) Percentage of control morphant (CTRL MO) and Zic3 morphant embryos with defective C-E phenotypes. (B) Percentage of control and Zic3 morphants with L–R patterning abnormalities. (C) Comparison of control and Zic3 morphants with defective C-E (score based on scale represented in Fig. 1D) and abnormal L–R patterning (based on pitx2 expression in the lateral plate mesoderm). C-E defects are categorized as normal to mild (C-E score of 1–3) and severe (C-E score of 4–5). **p < 0.01 by Fisher's exact test; statistical significance was determined through comparison of each experimental group with the CTRL MO (normal–mild CE) group.
	Supplemental Fig. 1. Xenopus Zic3 morpholino specificity. To demonstrate specificity, Zic3 morpholinos were injected singly at 2.65 ng, a dose not producing a phenotype. When injected together at a dose of 2.65 ng each, the morpholinos recapitulate the convergent extension and L phenotypes. (A) Percentage of embryos with normal convergent extension. (B) Percentage of embryos with normal L as assayed by pitx2 expression. **p < 0.0001 by Fisher's exact test; statistical significance was determined through comparison of each singly injected morpholino group with combined morpholino group.

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