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Protocadherins represent the biggest subgroup within the cadherin superfamily of transmembrane glycoproteins. In contrast to classical type I cadherins, protocadherins in general exhibit only moderate adhesive activity. During embryogenesis, they are involved in cell signaling and regulate diverse morphogenetic processes, including morphogenetic movements during gastrulation and neural crest migration. The two protocadherins paraxial protocadherin (PAPC) and axial protocadherin (AXPC) are indispensable for proper gastrulation movements in Xenopus and zebrafish. The closest relative PCNS instead, is required for neural crest and somite formation. Here, we show that cranial neural crest (CNC) cells in addition to PCNS express PAPC, but not AXPC. Overexpression of PAPC resulted in comparable migration defects as knockdown of PCNS. Moreover, reconstitution experiments revealed that PAPC is able to replace PCNS in CNC cells, indicating that both protocadherins can regulate CNC migration.
Figure 1. PAPC is expressed in cranial neural crest (CNC) cells. (a) In situ hybridization (ISH) for PAPC and PCNS in tailbud embryos (stage 23). PCNS is expressed in the pharyngeal arches. PAPC transcripts are detected in the otic vesicle, but not in neural crest cells. (b) Quantitative real-time PCR exhibited PAPC expression in CNC explants. Explants were dissected at the indicated stage. All values are normalized to ODC expression. The expression of the CNC-specific marker genes twist, snail, and slug are shown for comparison. The bars indicate average and standard deviation of at least three independent experiments.
Figure 2. Overexpressed PAPC disturbs CNC migration. (a) Overexpression of PAPC resulted in a concentration-dependent migration defect as shown by the mislocalization of twist expression. One dorsal blastomere of Xenopus eight-cell stage embryos was injected with the indicated amount of PAPC mRNA. The asterisks mark the injected side. (b) Quantification of the migration defects in "n" embryos. **P < 0.005 according to Student's t test. Error bars indicate the standard error.
Figure 3. PAPC knockdown does not affect CNC migration. (a) Fluorescently labeled CNC cells grafted into wild-type embryos were able to migrate into the pharyngeal pouches as did CNC cells coinjected with PAPC-specific antisense morpholino (PAPC MO). In contrast, CNC cells coinjected with PCNS-specific antisense morpholino oligonucleotide (PCNS MO) show impaired CNC migration. Anterior is to the left and dorsal to the top. Shown are merged brightfield and FITC images of representative embryos. Data indicate the penetrance of the represented phenotype. (b) Injection of a PCNS-specific antisense morpholino oligonucleotide (PCNS MO), but not of a PAPC-specific morpholino (PAPC MO) inhibited CNC migration. The asterisks mark the injected side. (c) Quantification of the migration defects in �n� embryos with high (2 pmol) and low (0.4 pmol) PCNS MO. Error bars indicate the standard error.
Figure 4. PAPC compensates for the loss of PCNS. (a) Injection of a PCNS-specific antisense morpholino oligonucleotide (PCNS MO) caused severe migration defects, which were rescued by coinjection of 500 pg PAPC mRNA. The asterisks mark the injected side. Lower concentrations of PAPC mRNA were less effective. (b) Quantification of the migration defects in �n� embryos. **P < 0.005 according to Student's t test. Error bars indicate the standard error.
