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The transcription factor Hypermethylated in Cancer 1 (HIC1) is associated with both tumorigenesis and the complex human developmental disorder Miller-Dieker Syndrome. While many studies have characterized HIC1 as a tumor suppressor, HIC1 function in development is less understood. Loss-of-function mouse alleles show embryonic lethality accompanied with developmental defects, including craniofacial abnormalities that are reminiscent of human Miller-Dieker Syndrome patients. However, the tissue origin of the defects has not been reported. In this study, we use the power of the Xenopus laevis model system to explore Hic1 function in early development. We show that hic1 mRNA is expressed throughout early Xenopus development and has a spatial distribution within the neural plate border and in migrating neural crest cells in branchial arches. Targeted manipulation of hic1 levels in the dorsal ectoderm that gives rise to neural and neural crest tissues reveals that both overexpression and knockdown of hic1 result in craniofacial defects with malformations of the craniofacial cartilages. Neural crest specification is not affected by altered hic1 levels, but migration of the cranial neural crest is impaired both in vivo and in tissue explants. Mechanistically, we find that Hic1 regulates cadherin expression profiles and canonical Wnt signaling. Taken together, these results identify Hic1 as a novel regulator of the canonical Wnt pathway during neural crest migration.
Fig. 1. Expression of hic1 RNA throughout early Xenopus laevis development. Representative image of RT-PCR using cDNA generated from whole embryos at indicated stages (Nieuwkoop-Faber, NF) across early developmental timepoints (A). In situ hybridization with hic1 anti-sense and sense control probes identifies mRNA expression pattern over early developmental timepoints (B). (i) Blastula NF stage 9 animal view and (ii) vegetal view, (iii) gastrula NF stage 10.5 animal view and (iv) vegetal view, (v) neurula NF stage 19 anterior view and (vi) dorsal view, (vii) tailbud NF stage 24 side view, (viii) tailbud NF stage 32 side view, (ix) tadpole NF stage 40 side view. Arrows indicate hic1 expression within the placodal ectoderm (v), neural plate border (vi) and branchial arches (vii, viii). In situ hybridization with hic1 anti-sense probe identifies mRNA expression pattern in Xenopus tropicalis embryos (C). (i) neurula NF stage 17 anterior view and (ii) dorsal view (iii) tailbud NF stage 27 side view and (iv) tadpole NF stage 35 side view. Arrows indicate hic1 expression within the neural plate border (i, ii) and branchial arches (iii).
Fig. 2. Disruption of normal hic1 expression results in head defects. Injection of either hic1 mRNA (500 pg total) or hic1 MO (50 ng total) in both dorsal animal cells of 8-cell stage embryos results in head defects including altered head shape and small eyes (A). Knockdown of hic1 expression by MO injection is rescued by co-injection with hic1 mRNA (50 pg) that lacks the MO binding site (B). Experiments were performed in biological and technical quadruplicate and phenotypes were quantified as percent of total embryos. Total number of embryos for each group is indicated above bars within the graphs (C). Immunofluorescence staining of transverse tissue sections shows Hic1 protein expression after injection of hic1-HA mRNA (250 pg) (D). Co-injection of hic1 MO (25 ng) in one side of embryo results in loss of Hic1 protein only when the MO binding site is present (hic1 5′UTR-HA, 250 pg) in injected hic1 mRNA. Dashed lines indicate region of co-injection as assessed by fluorescein dextran.
Fig. 3. Disruption of normal hic1 expression levels results in defects of the craniofacial cartilages. Unilateral injection of either hic1 mRNA (250 pg) or hic1 MO (25 ng) in one DA cell results in misshapen craniofacial regions and malformations of the craniofacial cartilages as shown by Alcian blue staining (A). Cartilage phenotypes were categorized as mild or severe depending on the extent of cartilages involved. Both dorsal and ventral views of individual heads are shown. Arrows indicate regions of disrupted cartilage development. CH: ceratohyal cartilage M: Meckel’s cartilage BR: branchial cartilage. Experiments were performed in biological and technical quadruplicate. Phenotypes were quantified and shown as percent of total embryos, with total embryo numbers indicated above bars within the graph (B).
Fig. 4. in situ hybridization shows changes in CNC gene expression patterns upon hic1 manipulation. (A–D) Embryos were unilaterally injected with hic1 mRNA (250 pg) or hic1 MO (25 ng) and β-galactosidase mRNA (200 pg) as a tracer (red color) in one DA cell and ISH was performed at various stages throughout CNC development. NF stage 14 embryos were assessed for markers of neural ectoderm (sox2), non-neural ectoderm (xk70), neural plate border (ap2α, msx1) and neural crest (snail, slug) (A). NF stage 16 embryos were probed for the neural crest-expressed gene slug (B). NF stage 19–20 embryos were assessed for the expression of the neural crest-expressed genes slug, snail and sox9 (C). NF stage 24 embryos were assessed for expression of the neural crest-expressed gene sox10 (D). (E) Embryos were unilaterally injected with hic1 MO (25ng), hic1∗ mRNA (50 pg) and β-galactosidase as a tracer. NF stage 19–20 embryos were assessed for the neural crest marker slug. All experiments were performed in at least biological and technical triplicate and number of phenotypic embryos observed out of the total number of embryos assayed is indicated.
Fig. 5. hic1 knockdown leads to defects in migration of cranial neural crest transplants. Embryos were injected unilaterally with hic1 MO (25 ng) or control MO (25 ng) plus GFP-CAAX mRNA (200pg) in one DA cell. At NF stage 16, GFP + CNC were transplanted into a naïve stage-matched embryo and embryos were imaged at NF stage 29 for CNC migration by GFP fluorescence (A). Migration was categorized as normal (into the three branchial arches), aberrant (altered pattern of migration) or no migration (CNC did not leave the site of explant) and quantified as percent of total embryos. Total embryo numbers are indicated above bars within graph (B).
Fig. 6. hic1 knockdown leads to defects in migration of cranial neural crest explants. Embryos were injected unilaterally with hic1 MO (25 ng) or control MO (25 ng) plus GFP-CAAX mRNA (200pg) in one DA cell. (A–C) At NF stage 16, GFP + CNC were removed and explanted onto a fibronectin-coated dish. Explants were imaged 14–16 h later to assess for migration (A). Migrating cells from control MO embryos are GFP+, while migrating cells from hic1 MO embryos are GFP- (B). Migration was categorized as extensive (cells in a complete migrating monolayer), some (partial migration with some cells remaining clumped in the center of the explant) or no migration (all cells remain clumped in the explant) and quantified as percent of total explants. Total explant numbers are indicated above bars within graph (C). (D–E) At NF stage 16, GFP + CNC were removed, dissociated, and plated onto a fibronectin coated dish as single cells. Cells were imaged for 150 min and individual cell movements were tracked. Still images from start (time = 0) and end (time = 150) of imaging show individual cell tracks (D). Cell tracks were normalized to a zero-point origin and plotted to show individual cell movements within the population. N = 65 control MO, 63 hic1 MO taken over three separate experiments.
Fig. 7. hic1 knockdown disrupts Wnt signaling dynamics within migrating cranial neural crest cells. Embryos were injected unilaterally with hic1 MO (25 ng) or control MO (25 ng) plus GFP-CAAX mRNA (200pg) in one DA cell. At NF stage 16, GFP + CNC were explanted onto fibronectin-coated glass coverslips and allowed to develop for 30 min or 7 h. Explants were fixed and immunostaining was performed for β-catenin and filamentous actin (phalloidin) (A). β-catenin was quantified as fluorescence pixel intensity from immunostained explants and plotted against position along cell diameter. Regression analysis was performed to identify the overall pattern of β-catenin localization (B). Total RNA was collected from explant cells and qRT-PCR was performed to assess level of axin2 gene expression. Fold change in gene expression was calculated relative to control explants at the pre-migration stage. ∗p < 0.05, unpaired t-test.
Fig. 8. Wnt inhibition in hic1 morphant explants rescues neural crest spreading and cadherin expression. Embryos were injected unilaterally with hic1 MO (25 ng) or control MO (25 ng) plus GFP-CAAX mRNA (200pg) in one DA cell. At NF stage 16, GFP + CNC were explanted onto fibronectin-coated dishes and imaged after 30 min. Explants were treated with 5μM concentration of Tankyrase inhibitor XAV939, or DMSO control, and imaged after 15 h (A). Area of neural crest spreading was determined and presented as fold change in area as compared to pre-migratory explant (B). Experiment was performed in biological and technical triplicate. Statistical analysis was performed using the unpaired t-test. Total RNA was collected from explant cells and qRT-PCR was performed to assess cadherin gene expression levels (C). Experiment was performed in biological and technical quintuplicate. Gene expression was compared within each individual experiment relative to control pre-migratory explant cells and statistical analysis was performed using the paired t-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
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