Munoz WA et al. (2012), Plakophilin-3 is required for late embryonic am...

XB-ART-45120

PLoS One 2012 Jan 01;74:e34342. doi: 10.1371/journal.pone.0034342.

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Plakophilin-3 is required for late embryonic amphibian development, exhibiting roles in ectodermal and neural tissues.

Munoz WA , Kloc M , Cho K , Lee M , Hofmann I , Sater A , Vleminckx K , McCrea PD .

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The p120-catenin family has undergone a significant expansion during the evolution of vertebrates, resulting in varied functions that have yet to be discerned or fully characterized. Likewise, members of the plakophilins, a related catenin subfamily, are found throughout the cell with little known about their functions outside the desmosomal plaque. While the plakophilin-3 (Pkp3) knockout mouse resulted in skin defects, we find larger, including lethal effects following its depletion in Xenopus. Pkp3, unlike some other characterized catenins in amphibians, does not have significant maternal deposits of mRNA. However, during embryogenesis, two Pkp3 protein products whose temporal expression is partially complimentary become expressed. Only the smaller of these products is found in adult Xenopus tissues, with an expression pattern exhibiting distinctions as well as overlaps with those observed in mammalian studies. We determined that Xenopus Pkp3 depletion causes a skin fragility phenotype in keeping with the mouse knockout, but more novel, Xenopus tailbud embryos are hyposensitive to touch even in embryos lacking outward discernable phenotypes, and we additionally resolved disruptions in certain peripheral neural structures, altered establishment and migration of neural crest, and defects in ectodermal multiciliated cells. The use of two distinct morpholinos, as well as rescue approaches, indicated the specificity of these effects. Our results point to the requirement of Pkp3 in amphibian embryogenesis, with functional roles in a number of tissue types.

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Species referenced: Xenopus laevis
Genes referenced: ctnnd1 foxd3 myc pkp2 pkp3 tuba4b twist1
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	Figure 1. Pkp3 domain structure and amino acid sequence alignment.(A) Diagram of Pkp3 protein characteristics. Potential alternative translation initiation start sites are indicated with arrows, while a conserved, potential caspase cleavage site is labeled with an asterisk. (B) Sequence alignment of Xenopus laevis, human and two isoforms of mouse Pkp3. Identical and similar residues are indicated by grey highlighting. The Armadillo domain is underlined. Black boxes indicate methionines conserved with mouse that might serve as alternative translation initiation sites. Potential caspase cleavage sites are labeled with asterisks.
	Figure 2. Xenopus Pkp3 temporal expression profiles.(A) Semi-quantitative real-time RT-PCR analyses indicating Pkp3 transcripts are deposited maternally at a relatively low level and then levels increase following gastrulation. (B) The 75 kDa Pkp3 protein isoform increases following gastrulation, while the 110 kDa isoform exhibits an inverse pattern. The polyclonal antibody generated against Xenopus Pkp3 amino acids 1–350 (N-terminal domain) recognizes Pkp3 protein products migrating at approximately 110 kDa and 75 kDa. Further, in early development, a fainter and more slowly migrating band is detected at approximately 120 kDa. Immuno-blot detection of GAPDH serves as a loading control.
	Figure 3. Xenopus Pkp3 spatial expression profiles.(A) Whole-mount in situ RNA hybridization reveals Pkp3 mRNA signals in the anterior and dorsal neural fold regions of neurula embryos (subpanel a). At elongation stages (stage 22; subpanel b), staining of the skin remains apparent, as does a concentration in dorsal structures. At tadpole stages, neural derived tissues including the brain, branchial arches and the spinal cord are stained, as are somites (subpanels c–d). As a basis for comparison (negative controls), we undertook sense-probe hybridizations in parallel (subpanels E–H). (B) Immuno-blotting on adult Xenopus tissue extracts. We used an affinity purified rabbit polyclonal antibody directed against the N-terminal domain of Pkp3, which clearly resolved the Pkp3 protein isoform migrating at approximately 75 kDa. This product was strongly expressed in heart, lung, muscle and skin. Weak, reproducible expression was detected in brain and kidney.
	Figure 4. Pkp3 subcellular localization.Following earlier injection of exogenous Myc-tagged Pkp3 mRNA (0.5 ng injected into one-cell stage embryo), Pkp3 was visualized in the naïve ectoderm (animal cap) cells of blastula embryos at the plasma membrane, in the cytoplasm along what appear to be fibrous structures (possibly the intermediate filament network), and in the nuclear compartment (subpanel a). At late neurula stage the exogenous Pkp3 was again detected at the plasma membrane (arrowhead), in the nuclear compartment (though much weaker), and as punctate apically disposed spots (conceivably basal bodies or other cilia associated structures, arrow), in multiciliated cells intercalating upwards from the deeper sensoral layer (subpanel a″).
	Figure 5. Depletion of endogenous Pkp3 results in skin fragility.(A) Diagram of morpholino-based depletion strategy. MO 3 targets the Pkp3 mRNA translational start site and MO 1 targets a non-overlapping upstream sequence in the 5′ UTR. Both morpholinos were designed to block translation initiation. (B) Immuno-blotting of hatching embryo extracts (stage 27) confirmed reductions of both Pkp3 protein isoforms following morpholino injection, but no reductions of Pkp2 protein (40 ng of each morpholino when injected individually or 20 ng of each morpholino when injected together into one-cell stage embryos). GAPDH serves as a loading control. (C) Injection of MOs 1 or 3 (40 ng into one-cell stage embryos) results in skin fragility (subpanels b and c, respectively), contributing to what can become lethal ectodermal damage sustained as the embryo hatches from the Vitelline membrane (higher magnification in subpanels b′, b″, and c′). Negative control embryos injected with standard control morpholino (40 ng) did not exhibit significant phenotypes (subpanels a-a′). (D) Quantification of the skin fragility effects are indicated in subpanel “d”, where P-values indicate statistical significance. As indicated, either 40 or 20 ng of each morpholino was injected into one-cell stage embryos. (E) Skin of tailbud stage embryos injected at one-cell stage with 40 ng standard control morpholino (subpanel a) compared to 40 ng MO 1 or 3 injected embryos (subpanels b and c, respectively). Pkp3 depleted ectoderm contains fewer basally disposed desmosomes (black arrow heads), while in apical regions desmosomes appear equally abundant. Note also that in skin of Pkp3 knockdown embryos, mitochondria have become more apically disposed (white arrowheads), and secretory vesicles have become much larger along the apical surface (black arrows).
	Figure 6. Pkp3 depletion results in tactile hyposensitivity and neural defects.(A) MO 1 or 3 injected embryos, that survive hatching have permanently kinked axes (subpanels b and c, respectively) (40 ng at one-cell stage). This is quantified in subpanel d, where the P-values indicate statistical significance. (B) Further observation revealed that Pkp3 depleted embryos were non-responsive to gentle tactile stimuli as observed by light shaking of their incubation dish, while controls attempted to escape from such stimuli. Upon more pronounced stimulation, it became evident that the embryos were able to swim normally, indicating retained muscle function (etc.) (data not shown). (C) Through the use of an established neural marker (acetylated alpha tubulin), examination of motoneurites arranged at somite boundaries revealed no large differences between controls and Pkp3 depleted embryos. (D) Neural processes present in the posterior region (tailbud) of control tailbud stage embryos (subpanel a), as well as a tadpole stage lateral neural tract of unknown identity (subpanels b), were reproducibly found to be less apparent and/or foreshortened following Pkp3 depletion (subpanels a′ and b′, respectively). Morpholinos (40 ng each) were injected at the one-cell stage.
	Figure 7. Neural crest induction and migration is disrupted upon Pkp3 depletion.(A) Injection of MO 1 or 3 resulted in loss of pigmentation and reduced eye size. 40 ng of either morpholino was injected at the one-cell stage. (B) In situ hybridization of the neural crest marker, Twist, following Pkp3 depletion (20 ng total MO into a single dorsal blastomere at the four-cell stage). At neurula stage 16, early (inductive) neural crest expression of Twist is reduced or lost (subpanel b). At neurula stage 21, later (migratory) neural crest expression likewise appears affected, as the tracts in Pkp3 depleted embryos do not extend (subpanel d–e) in the normal pattern (subpanel c). (C) Expression of the neural crest markers, Twist and FoxD3, was analyzed by in situ hybridization following knockdown of Pkp3. Pkp3 morphants showed reductions of both markers in the neural crest domain, that could be partially rescued upon co-injection of exogenous Pkp3 lacking the morpholino targeted sequence(s). 20 ng of each morpholino and 100 pg of either Myc-tagged beta-galactosidase or Myc-tagged Pkp3 mRNA was injected in a single dorsal blastomere. Quantification of these results is represented in the accompanying bar graph, where P-values indicate statistical significance.

References [+] :

Achtstätter, Cytokeratin filaments and desmosomes in the epithelioid cells of the perineurial and arachnoidal sheaths of some vertebrate species. 1989, Pubmed, Xenbase