XB-ART-55567
Nat Commun
2018 Apr 03;91:1319. doi: 10.1038/s41467-018-03715-w.
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Gon4l regulates notochord boundary formation and cell polarity underlying axis extension by repressing adhesion genes.
Williams MLK
,
Sawada A
,
Budine T
,
Yin C
,
Gontarz P
,
Solnica-Krezel L
.
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Anteroposterior (AP) axis extension during gastrulation requires embryonic patterning and morphogenesis to be spatiotemporally coordinated, but the underlying genetic mechanisms remain poorly understood. Here we define a role for the conserved chromatin factor Gon4l, encoded by ugly duckling (udu), in coordinating tissue patterning and axis extension during zebrafish gastrulation through direct positive and negative regulation of gene expression. Although identified as a recessive enhancer of impaired axis extension in planar cell polarity (PCP) mutants, udu functions in a genetically independent, partially overlapping fashion with PCP signaling to regulate mediolateral cell polarity underlying axis extension in part by promoting notochord boundary formation. Gon4l limits expression of the cell-cell and cell-matrix adhesion molecules EpCAM and Integrinα3b, excesses of which perturb the notochord boundary via tension-dependent and -independent mechanisms, respectively. By promoting formation of this AP-aligned boundary and associated cell polarity, Gon4l cooperates with PCP signaling to coordinate morphogenesis along the AP embryonic axis.
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Species referenced: Xenopus
Genes referenced: cldn5 dlx3 epcam gon4l itga3 myod1 prickle1 tbx6 wnt11
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Fig. 1. A forward genetic screen identifies ugly duckling(udu)/gon4l as a regulator of axis extension in zebrafish embryos. a Schematic of a synthetic screen to identify enhancers of the short axis phenotype in knym818/m818 zebrafish mutants. b–e Phenotypes at 24 hpf: wild type (WT) (b), knym818/m818 (c), vu66/vu66 (d), knym818/m818; vu66/vu66 compound mutants (e). Images are representative of phenotypes observed at Mendelian ratios in multiple independent clutches. f Diagram of mapping vu66 mutation to Chromosome 16. Bold numbers below specify the number of recombination events between vu66 and the indicated loci. g Diagram of the Gon4l protein encoded by the udu locus. Arrowheads indicate residues mutated in vu66 and other described udu alleles. h–k Live WT (h, j) and maternal zygotic (MZ)udu (i, k) embryos at yolk plug closure (YPC) (h–i) and 20 somite stage (j–k). 100 percent of MZudu mutants from more than 15 germline-replaced females exhibited the pictured phenotypes. l–m Whole mount in situ hybridization (WISH) for dlx3 (purple) and hgg1 (red) in WT (l) and MZudu−/− (m) embryos at two-somite stage. n–o WISH for myoD in WT (n) and MZudu−/− (o) embryos at eight-somite stage. Anterior is to the left in b–e, j–k; anterior is up in h–i, l–o. Fractions indicate the number of embryos with the pictured phenotype over the number of embryos examined. Scale bar is 500 μm in b–e and 300 μm in h–o | |
Fig. 2. MZudu mutant gastrulae exhibit irregular notochord boundaries. a–b Still images from live Nomarski time-lapse series of the dorsal mesoderm in WT (a) and MZudu−/− embryos (b) at the time points indicated. Images are representative of over 40 MZudu−/− gastrulae. Arrowheads indicate notochord boundaries. c–d Live confocal microscope images of representative WT (c, N = 23) and MZudu−/− (d, N = 43) embryos expressing membrane Cherry. Yellow lines mark notochord boundaries. e Quantification of notochord boundary straightness in live WT and MZudu−/− gastrulae throughout gastrulation. Symbols are means with SEM (two-way ANOVA, ****p < 0.0001). f–g Confocal microscope images of immunofluorescent staining for pan-Laminin in WT (f) and MZudu−/− (g) embryos at two-somite stage. N indicates the number of embryos analyzed. Scale bars are 50 μm. Anterior is up in all images | |
Fig. 3. Mediolateral cell polarity and cell intercalations are reduced in the axial mesoderm of MZudu−/− gastrulae. a–b, e–f Still images from live time-lapse confocal movies of the axial mesoderm in WT (a, b) and MZudu−/− (e, f) gastrulae at the time points indicated. Cell outlines are colored according to a cell’s position with respect to the notochord boundary. c, g Quantification of axial mesoderm cell orientation at 80% epiboly (left side) and +90 min (right side) time points. Each dot represents the orientation of the major axis of a single cell with respect to the embryonic ML axis and is colored according to that cell’s position with respect to the notochord boundary (as in images to the left). Bars indicate median values. Asterisks indicate significant differences between WT and MZudu−/− (Kolmogorov–Smirnov test, ***p < 0.001, ****p < 0.0001). d, h Quantification of axial mesoderm cell elongation at 80% epiboly (left side) and +90 min (right side) time points. Each dot represents the aspect ratio of a single cell and is color-coded as in c. Bars indicate mean values. Asterisks indicate significant differences between WT and MZudu−/− (Mann–Whitney test, ****p < 0.0001). i–j Cell intercalations detected in the axial mesoderm of WT (i) and MZudu−/− gastrulae (j). Cells gaining contacts with neighbors are green, cells losing contacts are magenta, and cells that both gain and lose contacts are yellow. k Quantification of cell intercalation events (T1 exchanges) in WT (blue bars) and MZudu−/− gastrulae (green bars) over 90 min. N indicates the number of embryos analyzed. Bars are means with SEM (T-test, *p < 0.05). l–m 200 μm confocal Z projections of immunofluorescent staining for phosphorylated Histone H3 (pH3) in WT (l) and MZudu−/− gastrulae (m) at 80% epiboly. Images are representative of eight independent trials. n Quantification of pH3+ cells/embryo at the stages indicated. Each dot represents a single embryo, dark lines are means with SEM (T-test, *p < 0.05, ***p < 0.001, ****p < 0.0001). Scale bars are 50 μm. Anterior/animal pole is up in all images | |
Fig. 4. Gon4l regulates axis extension independent of PCP signaling. a–d Live embryos at 24 hpf resulting from a cross between a germline-replaced udu−/−;kny fr6/+ female and an udu+/-;knyfr6/+ male. Genotypes are indicated in the upper right corner, fractions indicate the number of embryos in the clutch with the pictured phenotype. e Mosaically expressed Prickle (Pk)-GFP in WT and MZudu−/− gastrulae. Arrowheads indicate anteriorly localized Pk-GFP puncta. Membrane Cherry marks cell membranes, nuclear-RFP marks cells injected with pk-gfp RNA. Images are representative of three independent experiments. f Quantification of Pk-GFP localization shown in e (chi-square, p = 0.07). g Quantification of notochord boundary straightness in WT and kny−/− gastrulae. Symbols are means with SEM (two-way ANOVA, ****p < 0.0001). h, i, l, m Still images from live time-lapse confocal movies of the axial mesoderm in kny−/− (h–i) and kny−/−;udu−/− (l–m) gastrulae at the time points indicated. Cell outlines are colored as in Fig. 3. j, n Quantification of axial mesoderm cell orientation as in Fig. 3, bars are median values (Kolmogorov–Smirnov test, ***p = 0.0003). k, o Quantification of axial mesoderm cell elongation as in Fig. 3, bars are mean values. N indicates number of embryos analyzed (and number of cells in f). Scale bar is 500 μm in a–d, 10 μm in e, 50 μm in h–m | |
Fig. 5. Loss of Gon4l results in large-scale gene expression changes. a Plot of gene expression changes in MZudu mutants compared to WT at tailbud stage as assessed by RNA sequencing. Red dots indicate genes expressed at significantly different levels than WT (p ≤ 0.05, at least twofold change in transcript level). b Protein classes encoded by differentially expressed genes with decreased (top graph) or increased (bottom graph) expression in MZudu mutants. Percentages indicate the number of genes within a given class/total number of genes with increased or decreased expression, respectively. c–d P-values of the top ten most enriched gene ontology (GO) terms among genes with decreased (c) or increased (d) expression in MZudu−/− gastrulae compared to WT. e–f Heat maps of differentially expressed genes annotated as encoding signaling (e) or adhesion molecules (f). The four columns represent two biological and two technical replicates for each WT and MZudu−/− | |
Fig. 6. DamID-seq identifies putative direct targets of Gon4l during gastrulation. a Box plot of normalized uniquely aligned DamID reads at each of three categories of genomic regions. Center lines are medians, box limits are upper and lower quartiles, whiskers are highest and lowest values. P-values indicate significant differences between Gon4l-Dam samples and GFP-Dam controls (T-test). b Fold change (log2) Gon4l-Dam over GFP-Dam reads (RPKM) at each of five gene features. c–f Genome browser tracks of Gon4l-Dam and GFP-Dam association at the histh1 (c), wnt11r (d), cldn5 (e), and tbx6 (f) loci. Scale is number of reads. Each track represents one biological replicate at tailbud stage. g Venn diagram of genes with regions of significant Gon4l enrichment within gene bodies (blue) or promoters (pink), and genes differentially expressed in MZudu−/− gastrulae (gray). h Correlation of Gon4l enrichment levels across gene bodies (blue dots) and promoters (red dots) with relative transcript levels of genes differentially expressed in MZudu mutants. A positive correlation was detected between increased expression in WT relative to MZudu mutants and Gon4l enrichment in both gene bodies (Spearman correlation p = 0.0095) and promoters (p = 0.0098). Dotted lines are linear regressions of these correlations | |
Fig. 7. Gon4l regulates notochord boundary straightness by limiting itga3b expression. a–c Genome browser tracks of Gon4l-Dam and GFP-Dam association at the epcam (a) and itga3b (b) locus. An expanded view of the itga3b promoter is shown in c. d–e Quantitative RT-PCR for epcam (d) and itga3b (e) in WT and MZudu−/− embryos at tailbud stage. N indicates the number of independent clutches tested with technical triplicates of each, bars are means with SEM. f–g Quantification of notochord boundary straightness in WT epcam (f) or itga3b (g) overexpressing embryos (two-way ANOVA, ****p < 0.0001). h–i Notochord boundary straightness in MZudu−/− epcam (h) or itga3b (i) morphants and sibling controls (two-way ANOVA, ****p < 0.0001). j Quantification of notochord boundary straightness in WT itga3b-overexpressing embryos with or without lama5 MO (two-way ANOVA, ****p < 0.0001). N indicates the number of embryos analyzed, symbols are means with SEM. k–m Itgα3b-GFP localization in WT embryos in the tissues and at the time points indicated. Insets are enlarged from regions in white squares. Yellow lines and arrowheads mark notochord boundaries. Images are representative of three independent trials. Scale bar is 50 μm. n–o Live images of kny−/− embryos at 24 hpf injected with 200 pg GFP (n) or 200 pg itga3b (o) RNA. Images are representative of four independent experiments. p Quantification of axis length of injected kny−/− embryos at 24 hpf. Each dot represents one embryo, bars are means with SEM (T-tests, ***p < 0.001, ****p < 0.0001). N indicates number of embryos analyzed | |
Fig. 8. Loss of Gon4l and excess epcam reduce notochord boundary tension. a Diagram of laser ablation experiments to measure tension at axial mesoderm cell interfaces. b Still images from confocal time-lapse movies of each of the three types of cell interfaces (Edge, V junctions, and T junctions) before and after laser ablation. Arrowheads indicate cell vertices adjacent to the ablated interface. Images are representative of 36 independent experiments. c–e Quantification of cell vertex recoil distance immediately after laser ablation of Edge (c), V junction (d), and T junction (e) interfaces at the time points indicated in WT, MZudu−/−, WT epcam overexpressing, and WT itga3b-overexpressing gastrulae. Symbols are means with SEM. Asterisks are colored according to key and indicate significant differences compared to WT controls (two-way ANOVA, **** p < 0.0001, **p < 0.01). f–g Quantification of notochord boundary straightness in WT itga3b (f) or epcam (g) overexpressing embryos with or without Calyculin A (two-way ANOVA, ****p < 0.0001, ***p < 0.001). N indicates the number of embryos analyzed, symbols are means with SEM. h Graphical model of the roles of Gon4l and PCP signaling in regulating ML cell polarity of axial mesoderm cells and notochord boundary formation |
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