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Koehler A
,
Schlupf J
,
Schneider M
,
Kraft B
,
Winter C
,
Kashef J
.
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Xenopus cadherin-11 (Xcadherin-11) is an exceptional cadherin family member, which is predominantly expressed in cranial neural crest cells (NCCs). Apart from mediating cell-cell adhesion it promotes cranial NCC migration by initiating filopodia and lamellipodia formation. Here, we demonstrate an unexpected function of Xcadherin-11 in NCC specification by interfering with canonical Wnt/β-catenin signaling. Loss-of-function experiments, using a specific antisense morpholino oligonucleotide against Xcadherin-11, display a nuclear β-catenin localization in cranial NCCs and a broader expression domain of the proto-oncogene cyclin D1 which proceeds c-myc up-regulation. Additionally, we observe an enhanced NCC proliferation and an expansion of specific NCC genes like AP2 and Sox10. Thereby, we could allocate NCC proliferation and specification to different gene functions. To clarify which domain in Xcadherin-11 is required for early NCC development we tested different deletion mutants for their rescue ability in Xcadherin-11 morphants. We identified the cytoplasmic tail, specifically the β-catenin binding domain, to be necessary for proper NCC development. We propose that Xcadherin-11 is necessary for controlled NCC proliferation and early NCC specification in tuning the expression of the canonical Wnt/β-catenin target genes cyclin D1 and c-myc by regulating the concentration of the nuclear pool of β-catenin.
Fig. 1. Depletion of Xcadherin-11 increases c-myc and cyclin D1. (A) Whole mount ISH results of Xcad-11-MO (8 ng) injected (*) in one dorsal animal blastomere of eight-cell-stage embryos analyzed at stage 16 and 19 for cyclin D1 and c-myc expression (dorsal view, anterior to top). Expression area of cyclin D1 is increased in both stages, whereas c-myc expression seems to be first unaffected at stage 16 and subsequently broadend at stage 19. (B) Statistical analyses of whole mount ISH results for cyclin D1 and c-myc. (C) qRT-PCR comparing cyclin D1 and c-myc expression between uninjected and Xcad-11-MO injected embryos at stage 16, 19 and 25. The expression of cyclin D1 and c-myc is increased in stage 19 and even more in stage 25. n=number of embryos (*p≤0.1; **p≤0.05).
Fig. 2. Expanded c-myc and cyclin D1 expression domains result from increased proliferation. Xenopus embryos were injected with Xcad-11-MO in one dorsal animal blastomere of eight-cell-stage embryo and incubated in DMSO only or in HUA/DMSO from stage 12–19. (A) Depletion of Xcadherin-11 results in expanded expression domains of cyclin D1 and c-myc. (B) Statistical analysis of cyclin D1 and c-myc expression in Xcad-11 depleted embros tretated with DMSO or DMSO/HUA. Treatment with HUA was able to rescue the expansion significantly. (*p≤0.05; **p≤0.01; ***p≤0.005) Injected side is marked with asterisk (*).
Fig. 3. Xcadherin-11 knockdown increases cranial NCC marker gene expression. (A) Whole mount ISH. Embryos were injected (*) with Xcad-11-MO (8 ng) in one dorsal animal blastomere of eight-cell-stage and analyzed at stage 16 and 19 (dorsal view, anterior to top) as well as stage 25 (lateral view) for AP2 and sox10 expression. At all stages investigated Xcadherin-11 morphants showed an increased AP2 expression. In comparison, almost all embryos revealed a reduced sox10 expression at stage 16 and 19 whereas at stage 25 most of the Xcad-11-MO injected embryos exhibited a broader expression domain of sox10. (B) Statistical analyses of whole mount ISH results for AP2 and sox10. (C) qRT-PCR analysis of AP2 and sox10 expression in stage 16, 19 and 25. We observed an increased expression of AP2 in stage 16 and 19 while sox10 was reduced in stage 19 and became increased in the later stage. n=number of embryos (*p≤0.1; **p≤0.05).
Fig. 4. The cytoplasmic domain of Xcadherin-11 mediates proper c-myc expression and proliferation. (A) Scheme of the Xcadherin-11 mutants used for rescue experiments. (B) Statistical analysis of whole mount ISH results for c-myc. Full length Xcad-11 and Xcad-11∆e, but not Xcad-11∆c co-injection was able to rescue c-myc expression. (C) Whole mount ISH. Embryos were injected with Xcad-11-MO (8 ng) alone or together with 75 pg full length Xcad-11, 75 pg Xcad-11Δc or 50 pg Xcad-11Δe, respectively, and analyzed for c-myc expression at stage 19. Dorsal view, anterior to top, asterisk marks the injected side. (D) Statistical analyses of whole mount ISH results. The area of c-myc stained tissue was compared in horizontally sliced embryos. The area of NCC positive tissue is increased by Xcad-11-MO, and this effect cannot be reverted by co-injection of Xcad-11 ∆c. On the other hand co-injection of Xcad-11 ∆e resulted in an area of a comparable size (1.2fold increase) as in uninjected tissue (set as 1). (E) Statistical analysis of pH3 positive nuclei at stage 19 in NCC positive tissue. PH3 positive nuclei on the uninjected side were set as 1. The increase of pH3 positive nuclei after Xcadherin-11 depletion could be significantly rescued after Xcad-11Δe co-injection. Only 1.05fold of pH3 positive nuclei could be detected by immunohistochemistry. Co-injection of Xcad-11Δc was not able to restore the increase of pH3 positive nuclei and resulted in an increase in mitotic nuclei by 1.52fold. n=number of sections (*p≤0.05).
Fig. 5. The β-catenin binding site is essential for Xcadherin-11 function. (A) Whole mount ISH. Embryos were injected with Xcad-11ΔeΔβcat (50 pg) either with or without Xcad-11-MO in one dorsal animal blastomere of eight-cell-stage and analyzed at stage 19 for c-myc and AP2 expression. While overexpression of Xcad-11ΔeΔβcat did not result in a distinct phenotype the expansion of c-myc and AP2 staining by Xcad-11-MO could not be rescued by Xcad-11ΔeΔβcat. Dorsal view, anterior to top, asterisk marks the injected side. (B) Statistical analysis of whole mount ISH results. 67% of the analyzed embryos with Xcad-11-MO and Xcad-11ΔeΔβcat showed an expanded c-myc expression domain. The overexpression of Xcad-11ΔeΔβcat did not show any significant phenotype (80% normal expression). Similar results were obtained for AP2 (53% increased expression in embryos co-injected with Xcad-11-MO and Xcad-11 ∆e∆β-cat). n=number of embryos.
Fig. 6. β-catenin is translocated in the nucleus after Xcadherin-11 depletion. Transversal sections of Xcad-11-MO injected (one dorsal animal blastomere at eight-cell-stage) embryos analyzed at stage 19. (A–C) Co-injection with mbGFP (200 pg) was used as lineage tracer and for membrane staining. (D–F) Immunostaining for β-catenin. Note nuclear β-catenin staining at the injected side (red arrows in F). (G–I) Nuclei were stained with DAPI. (J–L) Merged images, squares indicate areas of higher magnification. Crop1: uninjected side, crop2: injected side. Scale bar 50 µm.
Fig. 7. Depletion of Xcad-11 results in an activation of the Wnt-signaling pathway. (A) Scheme of reporter gene experiment. Embryos were injected with the displayed constructs in one dorsal animal blastomere of eight-cell-stage embryo. At neurula stages the NCC area was explanted and underwent luciferase assay. (B,C) Injection of Xcad-11-MO resulted in strong activation of the siamois (B) and cyclin D1 (C) promoters. While the siamois promoter was activated in a similar manner by constitutively active β-catenin as positive control and Xcad-11-MO, the increase in cyclin D1 expression was even more prominent in Xcad-11-MO injected embryos (*p≤0.05; **p≤0.01; ***p≤0.005). (D) Enhanced expression of c-myc and AP2 by Xcad-11-MO can be prevented by co-injection of β-catenin-MO. (E,F) Statistical analysis of ISH. In Xcad-11-MO injected embryos 63% of the embryos show an increased expression domain of AP2 (E) and 44% of c-myc (F). When β-catenin-MO was co-injected, only 29% of the embryos resulted in an increased AP2 and only 24% in an increased c-myc expression area. (**p≤0.01) β-catenin-MO injection alone led to decreased NCC formation.
Fig. 8. Model of Xcadherin-11 function in NCC specification. (A) Temporal course of Wnt and Xcadherin-11 protein levels during embryo development stages and the corresponding phases of NCC formation (color code see Steventon et al., 2005). Increased expression of Xcadherin-11 at neurula stages is correlated with an increased Wnt level. (B) Competition of Xcadherin-11 and Wnt for the cytoplasmic pool of β-catenin. Knockdown of Xcadherin-11 leads to increased levels of β-catenin in the cytoplasm and an enhanced nuclear localization resulting in an up-regulation of NCC specifiers c-myc and cyclin D1.
Supplement Fig. 1. Xcadherin-11 knockdown increases cell proliferation. Xenopus embryos were injected with Xcad-11-MO together with mbGFP (200 pg) as tracer in one dorsal animal blastomere of eight-cell-stage embryo. (A) At stage 16 and stage 19 embryos were fixed, ISH stained for AP2 (stage 16) or c-myc (stage 19), horizontally sectioned and analyzed for phospho H3 (pH3) staining. (B) Horizontal section of an embryo stained with NCC marker c-myc (bright field, left) and immunostained for pH3 (middle). Area of positive NCC marked tissue is surrounded by white lines in the merged image (right). (C) Statistical analysis of NCC stained area and pH3 positive nuclei at stage 16 and 19. The NCC positive area is significantly increased on the Xcadherin-11 injected side until up to 2fold in stage 19. PH3 positive nuclei on the uninjected side were set as 1. The number of pH3 positive nuclei in the analyzed sections was increased by 1.6fold at stage 16 and by 1.8fold at stage 19 in the NCC positive tissue (* p≤0.05; ** p≤0.005). (D) Treatment of embryos with HUA results in a strong reduction of pH3 positive nuclei in the embryo. * marks the injected side
Supplement Fig. 2. Xcadherin-11 is involved in gastrulation movements independent of cranial NCC specification. (A) Comparison of blastopore closure between wildtype and Xcad-11-MO (16 ng) injected embryos at stage 11 and stage 12. (B,C) Keller open-face explants of Xcad-11-MO (16 ng) injected embryos alone or together with 75 pg Xcad-11ï„c or 50 pg Xcad-11ï„e. Explants were scored for elongation (upper panel) and constriction (lower panel). # = number of explants (*p≤0.005 to wildtype; **p≤0.005 to Xcad-11-MO).
Supplement Fig. 3. Xcadherin-11 depletion results in decreased expression of neural markers. (A) ISH of Xcad-11-MO (8 ng) injected in one dorsal animal blastomere of eight-cell-stage embryos analyzed with neural markers sox2, krox20 and en2. In stage 16, 19 and 25 the expression domain of sox2 appears broader and more diffuse on the injected side (*). Krox20 and en2 signals are strongly decreased or lost on the injected side. (B) Statistical analysis of ISH. (C) qRT-PCR of sox2 expression confirms the ISH data. A significant decrease of sox2 expression is observed in stage 19 embryos. (D) Similar results are obtained for a second neural marker, N-CAM in qRT-PCR. A significant reduction in expression is observed in stages 19 and 25 (**p≤0.05; *p≤0.1).
Supplement Fig. 4. Xcadherin-11 depletion results in nuclear localization of β-catenin. Statistical analysis of nuclear β-catenin staining of three different embryos in regard to Fig. 6. On the Xcad-11-MO injected side a significantly higher number of cell nuclei (stained by DAPI) display a nuclear localization of β-catenin compared to the uninjected side. (**p≤0.01).
