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Graphical abstract
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Fig. 1. Loss of mink1 Induces Abnormal Heart Development. A) Schematic of Xenopus tropicalis Mink1 protein domains, site of kinase-dead (KD) mutation, and Crispr target sites (yellow). Protein homology between human and X. tropicalis Mink1 is 76%. B) Representative images of Xenopus tropicalis normal and abnormal cardiac outflow tracts (ventral views with anterior at top) at stage 45. OFT: outflow tract, V: ventricle. C) Quantification of abnormal cardiac outflow tract looping in mink1 crispants. 5, 3 and 12 biological replicates included for CRISPRs 1, 2 and 3, respectively. D) Rescue of mink1 depletion cardiac looping phenotype with human MINK1 mRNA. 5 biological replicates. E) Quantification of abnormal cardiac OFT looping in embryos with overexpression of wild-type and kinase-dead human MINK1 RNA. Lower dose of MINK1 RNA used for rescue than for overexpression (see Methods). 7 and 4 biological replicates included. P < 0.0001 = ∗∗∗∗, p < 0.005 = ∗∗, p < 0.05 = ∗ by two-tailed T-test.
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Fig. 2. Mink1 is Required for LRO Patterning. A) Representative images of pitx2c expression at stage 19. Pitx2c is normally expressed on the left lateral plate mesoderm (arrow, top row). Pitx2c expression is impaired in mink1 crispants (typically bilateral, arrows lower row). B) Quantification of abnormal pitx2c in mink1 crispants. 6 biological replicates included. C) Representative images of dand5 expression at stage 19 and stage 14. Anterior is to the top and posterior is to the bottom; ventral view. Dand5 expression is absent in the left-right Organizer of mink1 crispants after initiation of flow, stage 19 (top row), and before the initiation of flow, stage 14 (bottom row). D) Quantification of abnormal dand5 expression in mink1 crispants at stages 14 and 19.4 and 3 biological replicates included for stage 19 and 14, respectively. E) Representative images of nodal1 expression at stage 14. Anterior is to the top and posterior is to the bottom; ventral view. Nodal1 expression is also absent in the left-right Organizer of mink1 crispants before the initiation of flow, stage 14. F) Quantification of abnormal nodal1 expression in mink1 crispants. 4 biological replicates included. P < 0.0005 = ∗∗∗, p < 0.05 = ∗ by two-tailed T-test.
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Fig. 3. Mink1 Regulates Canonical Wnt Signaling. A) Representative images of foxj1 expression in stage 10.5 uninjected embryos and mink1 crispants along with quantification. Pink lines represent angle from center measurements made in ImageJ. 3 biological replicates B) Representative images of goosecoid expression in stage 10.5 uninjected embryos and mink1 crispants along with quantification. 9 biological replicates. C) Representative images of nodal3 expression in stage 10.5 uninjected embryos and mink1 crispants along with quantification. 3 biological replicates. D) Injection of β-catenin RNA rescues loss of Spemann Organizer gene expression in mink1 crispants. Representative images of goosecoid expression along with quantification. 4 biological replicates. E) Levels of total β-catenin protein are reduced in mink1 crispants at stage 10 as assayed by Western blot. F) Injection of β-catenin RNA also rescues abnormal pitx2c expression in mink1 crispants. 3 biological replicates. P < 0.0001 = ∗∗∗∗, p < 0.005 = ∗∗, p < 0.05 = ∗ by two-tailed T-test.
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Fig. 4. Global Phosphoproteomic Analysis Reveals HMGA2 As a Potential Downstream MINK1 Effector. A) Schematic of experimental pathway for phosphoenriched mass spectrometry experiment. B) Hmga2 exhibited loss of protein and phosphorylation at S18 residue due to depletion of mink1. Graphic representation of global phospoproteome due to mink1 depletion, X-axis represents protein log2 fold change, Y-axis represents phosphorylation log2 fold change. Red represents stage 10, green represents stage 11, blue represents stage 12. Yellow circles represent Hmga2 protein level changes. C) Quantification of abnormal outflow tract looping in hmga2 crispants. 3 biological replicates. D) Hmga2 is required for Spemann Organizer gene expression. Representative images and quantification of reduced goosecoid expression in hmga2 crispants. 3 biological replicates. P < 0.0001 = ∗∗∗∗, p < 0.005 = ∗∗, p < 0.05 = ∗ by two-tailed T-test.
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Fig. 5. Phosphomimetic HMGA2 rescues mink1 LOF phenotypes. A) Representative images goosecoid gene expression in mink1 crispants injected with WT, S18D, or S43D HMGA2 mRNA. 4 biological replicates B) Quantification of pitx2c expression in mink1 crispants injected with WT, S18D, or S43D HMGA2 mRNA. 4 biological replicates P < 0.0001 = ∗∗∗∗, p < 0.0005 = ∗∗∗, p < 0.005 = ∗∗, p < 0.05 = ∗ by two-tailed T-test.
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Fig. 6. HMGA2 Rescues Spemann Organizer Cell Fates in β-catenin Depleted Embryos. A) Overexpression of WT HMGA2 is sufficient to induce broad ectopic goosecoid expression in the animal pole of stage 10.5 embryos. Overexpression of S18D and S43D HMGA2 induces localized expression of goosecoid. 3 biological replicates. B) In β-catenin morphants, HMGA2 overexpression can rescue Spemann Organizer gene expression. Wild-type HMGA2 rescues goosecoid expression in a broad pattern on the animal pole, while S18D and S43D rescue in a more localized pattern 3 biological replicates. C) MINK1 overexpression cannot rescue Spemann Organizer gene expression in β-catenin morphants. 3 biological replicates.
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Supplementary Figure 1: mink1 CRISPR sgRNA Efficacy. A) Schematic representation of mink1 gene structure and crispr sgRNA target sites. B-D) Quantification of insertions and deletions generated by CRISPR. B) ICE results for mink1 CR 3 targeting exon 4 C) ICE results for mink1 CR 2 targeting exon 4 D) ICE results for mink1 CR 1 targeting exon 25. Histograms reflect the percentage of PCR products that have either frame shift indels (blue) or in frame indels (orange). E) Protein alignment between human MINK1 and Xenopus tropicalis Mink1. CR3 deletes 6 nucleotide removing two highly conserved amino acids 55 and 56 (valine and methionine highlighted in yellow) or 54 and 55 (lysine and valine highlighted in yellow). CR 1 induces a two nucleotide insertion (red) which results in an early stop codon (red asterisk).
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Supplementary Figure 2: MINK1 Kinase Dead Mutation Results in Loss of Function A) Quantification of abnormal OFT looping in mink1 CR3 and then an additional injection of sibling mink1 CR3 embryos with MINK1 kinase dead (KD) mRNA. Lower dose of MINK1 (KD) RNA used for rescue than for overexpression. 2 biological replicates.
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Supplementary Figure 3: Abnormal OFT looping and LR axis patterning correlate with gastrulation defects in mink1 crispants. A) Representative images of gastrulation defects seen in mink1 crispants at stage 19 and stage 45. Dorsal view, anterior to left- posterior to right. B) mink1 crispants had significant gastrulation defects. 3 biological replicates included. C) Gastrulation defects were highly correlated with abnormal cardiac outflow tract looping in mink1 crispants. 3 biological replicates. D) Gastrulation defects were highly correlated with abnormal pitx2c expression in mink1 crispants. 3 biological replicates. P < 0.0001= ****, p < 0.0005= ***, p < 0.005= ** by two-tailed T-test.
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Supplementary Figure 4: Global Phosphoproteomic Analysis of mink1 Crispants Uncovered Multiple Potential Downstream Effectors. A) Chaf1a, Hmga2, Nucks1 and Rpl30 all exhibited substantial loss of phosphorylation in mink1 crispants, as well as moderate changes in protein levels. B) Hmga2 crispants exhibited the most substantial loss of Spemann Organizer gene expression. C-D: Graphic representation of global phospoproteome due to mink1 depletion, X-axis represents protein log2 fold change, Y-axis represents phosphorylation log2 fold change. Red represents stage 10, green represents stage 11, blue represents stage 12. Yellow circles represent Hmga2 protein level changes. C) β-catenin exhibited a loss of protein and gain in phosphorylation at serine-552 in mink1 Crispants. D) GSK3 exhibited a substantial gain of protein in mink1 Crispants.
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Supplementary Figure 5: hmga2 CRISPR sgRNA Efficacy. A) ICE results for hmga2 CR 2. Blue bars represent frame shift indels, orange represents in frame indels.
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Supplementary Figure 6: Relations between hmga2 and β-catenin. A) Total β-catenin protein levels remain unchanged in stage 10.5 Hmga2 crispants. B) Hmga2 is expressed ubiquitously throughout the animal pole of stage 10.5 embryos and spreads into the Spemann Organizer (arrow). Hmga2 transcript expression does not change in β-catenin morphants. 3 biological replicates included.
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pitx2 (paired like homeodomain 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left and right, dorsal up.
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