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Fig. 1. Spatial expression of Xenopus laevis hace1 in early development. Expression of hace1 during Xenopus laevis development was analyzed by whole-mount in situ hybridization using an antisense probe (upper panels) and sense probe as a control (lower panels). a, b At stage 4 (a) and 11 (b), hace1 was expressed throughout the presumptive ectoderm. c At stage 15, hace1 expression was weakly detected at the dorsal ectoderm. d At stage 23, hace1 was expressed in the neural tissue and also showed spotty expression in the epidermis. The rightmost panel shows a magnification of the red boxed region. e
hace1 was expressed in the brain, eye, neural tube, branchial arch, ear vesicle, and kidney at stage 31. The right panel shows a magnified view of the embryo. Animal is to the top in lateral views (a, b). Dorsal is to the top in lateral or anterior views (c, d, e). Anterior is to the left in dorsal or lateral views (c, d, e)
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Fig. 2. Dorsal depletion of hace1 leads to multiple developmental defects. a Immunoblot analysis shows that HACE1 MO inhibited translation of hace1-myc mRNA but not that of myc-hace1 mRNA used for rescue experiments. Xenopus laevis embryos were injected with the indicated sets of MOs (30 ng) and mRNAs (1.2 ng). α-tubulin was a loading control. b Embryos were injected with 30 ng of HACE1 MO alone (middle panel) or 30 ng of HACE1 MO plus 1.2 ng of myc-hace1 mRNA (right panel) into the animal region of two dorsal blastomeres at the four-cell stage. c, d Embryos were classified into severe, mild, and normal groups on the basis of the extent of each defect. *P < 0.05 (with Bonferroni correction), Mann–Whitney U-test. c
N, number of total right and left sides of embryos we evaluated. The right side and left side of each embryo were separately evaluated. d
N, number of total eyes we evaluated. The right eye and left eye of each embryo were separately evaluated. e, f The body length of embryos at stage 40-41 was quantified (Uninjected, n = 59; HACE1 MO, n = 53; HACE1 MO + myc-hace1 mRNA, n = 55). Values are expressed as the mean ± s.d. ***P < 0.001 (with Bonferroni correction), unpaired t-test
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Fig. 3.
hace1 knockdown inhibits convergent extension. a Keller sandwich explants were prepared from embryos injected with 30 ng of control MO or HACE1 MO into dorsal blastomeres at the four-cell stage. HACE1 MO (middle panel, n = 18), but not control MO (left panel, n = 18), caused a defect in elongation of explants. The defect in elongation caused by HACE1 MO was partly rescued by coinjection of 1.2 ng of myc-hace1 mRNA (right panel, n = 18). b The length-to-width ratio of explants was quantified. Values are expressed as the mean ± s.d. *P < 0.05, unpaired t-test
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Fig. 4. Knockdown of hace1 and overexpression of rac1-V12 cause a delay in neural tube closure. a Embryos were injected with the indicated MOs (30 ng) into the animal region of two dorsal blastomeres at the four-cell stage and the images were collected at stage 20. Anterior views with dorsal to the top. Representative results from two independent experiments are shown. b HACE1 MO (15 ng) was injected into the animal region of the right dorsal blastomere, and control MO (15 ng) was subsequently injected into that of the left dorsal blastomere. rac1-V12 (constitutively active Rac1) mRNA (10 pg) and gfp mRNA (90 pg) were injected into the animal region of the right dorsal blastomere, and then gfp mRNA alone was injected into the animal region of the left dorsal blastomere. Embryos at stage 13 were placed on a culture dish, and images were collected every 5 min until embryos completed neural tube closure. Still frames from a time-lapse movie show embryos from stage 14 (0 min) to stage 18 (120 min). Dorsal views with anterior to the top. Dotted lines indicate the midline. Transient posterior protrusion (e.g., middle left) was sometimes observed because embryos were firmly embedded in the agarose groove and thus were constantly subjected to the lateral-to-medial force. c A magnified view of embryos in b (60 min). Black dotted lines indicate the inner edges of neural folds. d The maximum distance between the dorsal midline and the left (i) or right (ii) inner edge of the neural fold was measured (shown as white double-headed arrows in c). The differences ((ii) minus (i)) were then calculated and plotted (Uninjected, n = 17; HACE1 MO, n = 20; Rac1-V12, n=11). Long central bar marks mean and upper and lower bars mark s.d. *P < 0.05, **P<0.01, Mann–Whitney U-test
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Fig. 5. Knockdown of hace1 by HACE1 MO2 leads to a phenotype similar to that caused by HACE1 MO treatment. a Embryos were injected with 50 ng of Control MO (left panel) or 50 ng of HACE1 MO2 (right panel) into dorsal blastomeres at the four-cell stage and fixed at stage 38/39. Representative results from two independent experiments are shown (Control MO, n = 39; HACE1 MO2, n = 40) (b, c) Embryos were classified into severe, mild, and normal groups on the basis of the extent of each defect. b
N, number of total right and left sides of embryos we evaluated. The right side and left side of each embryo were separately evaluated. c
N, number of total eyes we evaluated. The right eye and left eye of each embryo were separately evaluated. d, e The body length of embryos at stage 38/39 was quantified (Control MO, n = 39; HACE1 MO2, n = 40). Values are expressed as the mean ± s.d. ***P < 0.001, unpaired t-test. f Embryos were injected with 50 ng of Control MO (left panel) or 50 ng of HACE1 MO2 (right panel) into dorsal blastomeres at the four-cell stage and the images were collected at stage 20 (Control MO, n = 40; HACE1 MO2, n = 40). Anterior views with dorsal to the top
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Fig. 6. Defects in hace1 morphants are partly rescued by coinjection of Rac1 MO. a Temporal expression patterns of Xenopus laevis rac1 and rac2. The expression levels of rac1 and rac2 were analyzed by qRT-PCR. Expression levels were normalized to those of Xenopus laevis ornithine decarboxylase 1 (odc1). b Immunoblot analysis shows that Rac1 MO inhibited translation of endogenous Rac1. Xenopus laevis embryos were injected with the indicated MOs (70 ng in total) and gfp mRNA (0.2 ng). α-tubulin was a loading control. c Coinjection of Rac1 MO (50 ng) and HACE1 MO (30 ng) led to milder developmental defects than did coinjection of control MO (50 ng) and HACE1 MO (30 ng). Anterior is to the left, and dorsal is to the top. d, e Embryos were classified into severe, mild, and normal groups on the basis of the extent of each defect. f, g The body length of embryos at stage 40-41 was quantified. Dorsal views of embryos are shown with anterior to the left. Values are expressed as the mean ± s.d. ***P < 0.001, unpaired t-test h Embryos injected with 30 pg of Rac1-V12 (constitutively active Rac1) mRNA and 170 pg of gfp mRNA displayed phenotypes similar to those of embryos injected with HACE1 MO (n = 38). Anterior is to the left, and dorsal is to the top
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Fig. 7.
hace1 has a role in regulating differentiation into neural ectoderm. a–c HACE1 MO (15 ng) was injected into the animal region of the right dorsal blastomere, and control MO (15 ng) was injected into that of the left dorsal blastomere at the 4-cell stage. Embryos were fixed at the stages indicated, and expression of epidermal keratin, an epidermal ectoderm marker (a), sox2, a neural ectoderm marker (b), and Xbra, a mesodermal marker (c), were analyzed by in situ hybridization. Representative images from two independent experiments are shown (lower panels)
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hace1 (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 3, lateral view, animal up.
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hace1 (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 23, anterior view, dorsal up.
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hace1 (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
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