Lichtig H , Artamonov A , Polevoy H , Reid CD , Bielas SL , Frank D .

R01 NS101597 NINDS NIH HHS

Xla + asxl3 MO (Fig. 1 B r1c2)
Xla + asxl3 MO (Fig. 1B r2c2)
Xla + asxl3 MO (Fig. 1 C)
Xla + asxl3 MO (Fig. 2 D 2rc2)
Xla + asxl3 MO (Fig. 3 B lanes 4,5)
Xla + asxl3 MO (Fig. 4 C lane 3)
Xla + asxl3 MO (Fig. 5. A r1/2 c3/4)
Xla Wt + asxl3 MO (Fig. 5. B)
Xla Wt + asxl3 MO (Fig. 5. C)
Xla Wt + asxl3 MO + animal cap amputation (Fig. 4 A lane 5)
Xla Wt + asxl3 MO + animal cap amputation (Fig. 4 B lane 6)
Xla Wt + meis3 (Fig. 3 A lane 4)
Xla Wt + meis3 + asxl3 MO (Fig. 3 A lane 6)

	FIGURE 1. ASXL3 knockdown causes neural tube defects and inhibits expression of posterior neural markers. (A) Neurula stage embryos were examined for neural tube defect phenotypes. Control embryos (CE – left panel) were injected with 15ng ASXL3-MO at the one cell stage (two right panels) The ASXL3 morphant embryos are squat, less elongated, with poor neural folding and open neural plates. In the CE group 87% of the embryos had normal neural folds (n = 158) versus the ASXL3-MO groups in which 86% had an open neural plate phenotype (n = 603). This was repeated in six independent experiments. The star marks the neural folds regions in the embryos. (B) Control embryos (CE, left panel) were injected at the one-cell stage with ASXL3-MO (15 ng, right panel). In situ hybridization was carried out on late neurula (st.18) embryos to the markers: n-tub (primary neuron), krox20 (hindbrain and lateral neural crest stripes) and cdx4 (spinal cord). Ntub was weakly expressed in 68% (n = 19) of the embryos; krox20 was weakly expressed in 85% (n = 20) of the embryos and cdx4 expression was normal in 81% (n = 21) of the ASXL3-MO injected embryos. (C) Control embryos (lane 2) were injected at the one-cell stage with ASXL3-MO (15 ng, lane 3). sqRT-PCR was carried out on total RNA isolated from pools of eight neurula (st.16) embryos from each group to the genes: krox20 (hindbrain) ntub (primary neuron), foxd3 (neural crest) and cdx1/4 (spinal cord). -RT was performed (lane 1) to total RNA isolated from the control uninjected embryos. The housekeeping gene ODC serves as positive control for RNA levels.
	FIGURE 2. Full-length and truncated/mutant human ASXL3 proteins rescue the ASXL3-MO Knockdown phenotype. (A) A schematic illustration of the main functional units of the full-length human and truncated/mutant ASXL3 proteins that were used in this study (Srivastava et al., 2016). (B) Control embryos (lane 2) were injected at the one-cell stage with ASXL3-MO (12.5 ng, lane 3) and increasing concentrations of the full-length mRNA (250–750 pg) encoding the human ASXL3 protein (lanes 4–6). sqRT-PCR was carried out on total RNA isolated from pools of seven neurula (st.16) embryos to the genes: krox20, ntub, and foxd3. -RT was performed (lane 1) to RNA isolated from the uninjected control embryos. The housekeeping gene Ef1α serves as positive control for RNA levels. (C) Control embryos (lane 2) were injected at the one-cell stage with ASXL3-MO (12.5 ng, lane 3) and the mRNAs (500 pg) encoding the full-length (FL – 2248 amino acids) or truncated/mutant (PT – 484 amino acids) human ASXL3 proteins (lanes 4–5). sqRT-PCR was carried out on total RNA isolated from pools of seven neurula (st.17–18) embryos to the genes: ntub, krox20, foxd3, cdx1, and cdx2. -RT was performed (lane 1) to RNA isolated from the uninjected control embryos. The housekeeping gene ODC serves as positive control for RNA levels. (D) Control embryos (CE, top panels) were injected at the one-cell stage with ASXL3-MO (middle panels) and mRNA (300 pg) encoding the full-length (FL) human ASXL3 protein (bottom panels). In situ hybridization was carried out in late neurula stage embryos to the markers: ntub (primary neuron) and twist (neural crest). Ntub was weakly expressed in 81% (n = 21) of the ASXL3-MO embryos versus controls (compare top/middle panels, left side); rescued expression was observed in 68% (n = 19) of the ASXL3-MO embryos (compare bottom/middle panels, left side). Twist was weakly expressed in 90% (n = 21) of the ASXL3-MO embryos versus controls (compare top/middle panels, right side); rescued expression was observed in 46% (n = 24) of the ASXL3-MO embryos (compare bottom/middle panels, right side).
	FIGURE 3. ASXL3 is necessary to induce posterior neural cell fates via Meis3 but not via retinoic acid. (A) One-cell stage embryos (lane 2) were injected at the one-cell stage with meis3 encoding RNA (750 pg, lane 4), ASXL3-MO (17.5 ng, lane 5) or both (lane 6). AC explants were removed from control (lane 3) and injected embryos (lanes 4–6) at blastula stage 9, and cultured to neurula stage 16. sqRT-PCR was carried out on total RNA isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3–6) to the genes: krox20, hoxd1 (hindbrain), cdx1 (spinal cord). -RT was performed for RNA levels. The housekeeping gene Ef1α serves as positive control for RNA levels. (B) Control embryos (lane 2) were injected at the one-cell stage with ASXL3-MO (7 and 9 ng, lanes 5 and 6, respectively). Upon reaching blastula stage 10 control embryos and the injected groups were placed in retinoic acid at 10–6 μg/ml (lanes 3, 6, 7). The Embryos were cultured to neurula stage 16. sqRT-PCR was carried out on total RNA isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3–8) to the genes: hoxa2, hoxb1, hoxb3, hoxb4, hoxd1, rar2.2α, krox20 (hindbrain), hoxb5 (spinal cord), and ntub (primary neuron). -RT was performed for RNA levels. The housekeeping gene ODC serves as positive control for RNA levels. (C) Control embryos (lane 2) were injected at the one-cell stage with ASXL3-MO 7 and 9 ng, (lanes 5 and 6, respectively). AC explants were removed from control (lane 3) and injected embryos (lanes 3–8) at blastula stage 9, and upon reaching blastula stage 10 AC controls and the injected groups were placed in retinoic acid at 10–6 μg/ml (lanes 4, 7, 8). The ACs were cultured to neurula stage 16. sqRT-PCR was carried out on total RNA isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3–8) to the genes: hoxa2, hoxb1, hoxb3, hoxb4, hoxd1, rar2.2α, krox20 (hindbrain), hoxb5 (spinal cord), and ntub (primary neuron). -RT was performed for RNA levels. The housekeeping gene ODC serves as positive control for RNA levels.
	FIGURE 4. Inhibition of ASXL3 differentially modifies noggin neuralizing activity. (A) Control embryos (lane 2) were injected at the one-cell stage with noggin encoding RNA (25 and 50 pg, lanes 4 and 5), ASXL3-MO (12 ng, lane 6), or both (lanes 7 and 8). AC explants were removed from control (lane 3) and injected embryos (lanes 4–8) at blastula stage 9, and cultured to neurula stage 16. sqRT-PCR was carried out on total RNA isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3–8) to the genes: nrp1, ncam (panneural), xanf1, foxg1, foxd1, otx2 (forebrain), and xag (cement gland). -RT was performed for RNA levels. The housekeeping gene ODC serves as positive control for RNA levels. (B) Control embryos (lane 2) were injected at the one-cell stage with ASXL3-MO (12 ng, lane 7) and mRNA (15, 25, 50 pg) encoding foxd1 (lanes 4–6), or both (lanes 8–10). AC explants were removed from control (lane 3) and injected embryos (lanes 4–10) at blastula stage, and cultured to neurula stage 16. sqRT-PCR was carried out on total RNA isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3–10). sqRT-PCR was carried out on total RNA isolated from pools of seven neurula (st.16) embryos to the genes: ncam (panneural), ntub (primary neurons), and otx2 (forebrain). -RT was performed (lane 1) to RNA isolated from the uninjected control embryos. The housekeeping gene Ef1α serves as positive control for RNA levels. (C) Control embryos (lane 2) were injected at the one-cell stage with ASXL3-MO (8, 12 ng; lanes 3–4). sqRT-PCR was carried out on total RNA isolated from pools of five neurula (st.16) embryos from each group to the genes: ncam, nrp1, xag, xanf, otx2, foxd1, foxg1, krox20, hoxb3, cdx1, cdx2, cdx4, ntub, snail2, and foxd3. -RT was performed (lane 1) to total RNA isolated from the control uninjected embryos. The housekeeping gene ODC serves as positive control for RNA levels. (D) Control embryos (lane 2) were injected at the one-cell stage with ASXL3-MO (8, 12 ng; lanes 3–4). sqRT-PCR was carried out on total RNA isolated from pools of six gastrula (st.11.5) embryos from each group to the genes: goosecoid (gsc), xnot, chordin (chd), myod, osr1, osr2, vent2, and brachyury (xbra). -RT was performed (lane 1) to total RNA isolated from uninjected control embryos. The housekeeping gene ODC serves as positive control for RNA levels. (E) Sibling embryos from (D) were taken for sqRT-PCR. Total RNA was isolated from pools of six neurula (st.16) embryos to the genes: engrailed2 (en2), krox20, cdx2/4, sox2/3, ncam, ntub, and foxd3. -RT (lane 1) to RNA isolated from the control embryos. The housekeeping gene ODC serves as positive control for RNA levels.
	FIGURE 5. ASXL3 knockdown disrupts head formation. (A) Stage 37 tadpole stage embryos were examined for axial defect phenotypes. Control embryos (CE – four left panels) were injected with 12 ng ASXL3-MO at the one-cell stage (four right panels) The ASXL3 morphant embryos are squat, less elongated, with poor trunk and head development. In the CE group 100% of the embryos developed normally development (n = 41) versus the ASXL3-MO group in which 100% of the embryos had a disrupted axial phenotype (n = 41). Four representative embryos are shown for each group. (B) Embryos were microinjected with ASXL3-MO (3 or 6 ng) into one-blastomere at the two-cell stage, so only half the embryo is perturbed. At tailbud stage 22, in situ hybridization was performed to the twist gene; twist is a marker for craniofacial neural crest cells (top panel control embryo/CE, 100% normal; n = 30). In middle and bottom panel, left side is injected and right side is normal. Twist expression is inhibited and (asymmetric, 60%; n = 40) in the left panel (ASXL3-MO), but its expression is normal on the uninjected side of the embryo in the right panel. (C) Embryos were injected with ASXL3-MO (12 ng) at the one-cell stage. Embryos were stained for cartilage formation at tadpole stages. Control embryos (90%, n = 27; panel) normal head cartilage staining (upper panel, left: head shot; right: dissected head cartilage). All morphant embryos (100%, n = 14) had reduced and abnormal head cartilage staining (lower panel, left: head shot; right: dissected head cartilage). (D,E)

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