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Protocadherins (Pcdhs), a major subfamily of cadherins, play an important role in specific intercellular interactions in development. These molecules are characterized by their unique extracellular domain (EC) with more than 5 cadherin-like repeats, a transmembrane domain (TM) and a variable cytoplasmic domain. PCNS (Protocadherin in Neural crest and Somites), a novel Pcdh in Xenopus, is initially expressed in the mesoderm during gastrulation, followed by expression in the cranial neural crest (CNC) and somites. PCNS has 65% amino acid identity to Xenopus paraxial protocadherin (PAPC) and 42-49% amino acid identity to Pcdh 8 in human, mouse, and zebrafish genomes. Overexpression of PCNS resulted in gastrulation failure but conferred little if any specific adhesion on ectodermal cells. Loss of function accomplished independently with two non-overlapping antisense morpholino oligonucleotides resulted in failure of CNC migration, leading to severe defects in the craniofacial skeleton. Somites and axial muscles also failed to undergo normal morphogenesis in these embryos. Thus, PCNS has essential functions in these two important developmental processes in Xenopus.
Fig. 2. PCNS expression in Xenopus embryos. (A) Developmental northern showing the temporal expression of PCNS. Xenopus embryo stages (Nieuwkoop and Faber, 1994) are indicated above the lanes. Strong expression is observed from late gastrula (stage 12) until late tailbud (stage 27) after which PCNS mRNA declines to background level. The gel was stained with ethidium bromide to assure equal loading of lanes. (B) Sagittal section of a mid-gastrulaembryo (stage 11), showing expression in the deep mesodermal cells. Animal pole at the top, dorsal on the right. The edges of the blastopore are indicated with arrowheads. (C) Expression in the CNC (black arrowheads) and somites (red arrowheads) in an early neurula stageembryo (stage 14). (D) Individual domains of CNC expression corresponding to prospective pharyngeal arches (black arrowheads) and a more posterior set of somites (red arrowheads) in late neurula stageembryo (stage 17). (E) Transverse section of embryo in panel D (plane indicated by dashed line) showing expression in the somite (red arrowhead), trunkNC (black arrowhead) and lateral plate mesoderm (blue arrowhead). Note that the color differences are an artifact of BM Purple staining. (F) Tailbudembryo (stage 25) expression in pharyngeal arches (black arrowheads) and posterior band of somites (red arrowhead). Anterior is to the right. (G) Stage 31embryo; pharyngeal arch expression has begun to fade as somite bands proceed further to the posterior. (H) Early tadpole (stage 34) somite expression exclusively at the most posterior tip of the tail (red arrowhead). PCNS RNA now detected in otic vesicle (blue arrowhead) and heart anlage (green arrowhead).
Fig. 3. PCNS overexpression phenotypearly neurula (stage 14). Injection of 1 ng RNA encoding PCNS resulted in gastrulation failure (Panel B; 96%, n = 28). Uninjected embryos were normal (panel A; 100%, n = 28).
Fig. 4. Dissociation and reaggregation assay. Dissociated inner ectodermal cells from embryos injected with 15 ng of RDx were mixed with inner ectodermal cells from embryos injected with RNAs encoding eGFP (panel A) or eGFP and PAPC (panel B) or eGFP and PCNS (panel C). Reaggregated cells were cultured until sibling embryos reached stage 28. PCNS expression yielded essentially the same degree of segregation as eGFP alone, while PAPC-expressing cells (green) largely separated from non-expressing cells (red). Each sample replicated in quadruplicate with similar results.
Fig. 5. PCNS loss of function. Whole-mount in situ hybridizations with NC markers to stage 19 (A), stage 22 (H) or stage 31 (L) embryos injected into one cell at the two-cell stage with 25 ng of M1 (B, D, G, K, O) or Co (A, C, I, M), or into one cell at the eight-cell stage with 6 ng of M1 (F) or Co (E), along with lacZ RNA as a lineage tracer. In situ probes are indicated on the individual panels at lower left. In each case, M1 resulted in severe inhibition of CNC cell migration into pharyngeal arches, and Co had no effect. In panels E and F, the lineage tracer is visible as red staining on the left side of the embryos. M1 injection inhibited migration of NC from rhombomere 5 (G; black arrowhead) without affecting the neural tube expression (red arrowhead). Panels A are dorsal views with the injected side on the left. In some cases, images were flipped horizontally to maintain this orientation. In panels H, the uninjected sides are shown on the left (H, J, L, N) and injected sides on the right (I, K, M, O). Images have been flipped horizontally to facilitate comparison of injected and uninjected sides. M1 strongly inhibited migration into posterior pharyngeal arches at stage 22 but had less effect on the mandibular arch migration (K; red arrowhead). Injection of Co had no effect (I). By stage 31 (L), Sox 9 expression in pharyngeal arches (N; red arrowheads) was essentially undetectable on the M1-injected side (O). Expression of Sox 9 in the otic vesicle was also strongly reduced (O; blue arrowhead). Injection of Co had no effect (M). Result statistics are summarized in Table 1.
Fig. 6. Craniofacial phenotype in tadpoles (stage 44). Embryos injected with 25 ng Co (A, B) or M1 (C, D) into one blastomere at the two-cell stage. The injected side is on the left in each panel. (A) Embryos injected with Co were normal overall (100%, n = 17) and had normal cranial cartilage as visualized with Alcian blue staining (panel B). (D) M1 resulted in cranial hypoplasia on the injected side in addition to a curved tail (87%, n = 15) and virtually complete loss of cranial cartilage, especially more posterior elements and gills. Some Meckel's cartilage was retained (arrowhead, panel C).
Fig. 8. Impaired somitogenesis in M1-injected embryos. (A) Whole-mount in situ hybridizations with MyoD (A), X-Delta-2 (D) or Thylacine 1 (G) to stage 20 (A) or stage 21 (D) embryos injected with 25 ng of Co or M1 into one cell at the two-cell stage. Injection of M1 inhibited somite segmentation for MyoD (panel B; 100%, n = 15) X-Delta-2 (panel E; 54%, n = 28) and Thylacine 1 (panel H; 53%, n = 15). Magnified images of somites in M1 embryos are shown in separate panels for MyoD (C), X-Delta-2 (F) and Thylacine 1 (I). Injection of Co had no effect (100% for MyoD, n = 29; 100%, n = 25 for X-Delta-2; 100%, n = 15 for Thylacine 1; panels A, D and G, respectively). All panels show dorsal view of embryos with anterior to the top and the injected side to the left.
Fig. 9. Parasagittal sections of a stage 24 embryo injected in one cell at two-cell stage with 25 ng M1. Immunostaining with 12/101, a muscle-specific antibody to section of (A) uninjected and (B) M1-injected side shows disturbed somite morphology. DAPI staining of (C) uninjected and (D) injected side sections shows that cell orientation is also disrupted.
