Figure 2. POU-IV is postmitotically expressed in hair cells of tentacular ectoderm at metamorphosis in the sea anemone.

Confocal sections of Nematostella vectensis at metamorphosis, labeled with antibodies against POU-IV, and/or tyrosinated ∂-tubulin (‘tyrTub’). Filamentous actin is labeled with phalloidin (Pha), and nuclei are labeled with DAPI (dapi). Proliferative cells are labeled by the thymidine analogue EdU. A shows a section through the presumptive tentacle primordia with the blastopore/mouth facing up. E, I, M-P show sections through developing oral tentacles with the distal end of the tentacle facing up; M–P are tangential sections of tentacles at the level of the surface ectoderm and parts of the endoderm (en). B–D, F–H, and J–L are magnified views of the boxed regions in A, E, and I, respectively, with the apical epithelial surface facing up. A-D: late planula. E–H: tentacle-bud. I–P: primary polyp. At the late planula stage prior to hair cell differentiation, POU-IV-positive nuclei are primarily localized at the basal and middle layers of the ectoderm of presumptive tentacle primordia (arrowheads in B–D); few POU-IV-positive nuclei are detectable at the superficial stratum. At the tentacle-bud stage, hair cells with pronounced stereovilli (st) and POU-IV-positive nuclei begin to develop in the superficial stratum of the ectodermal epithelium in tentacle primordia (arrowheads in F–H). POU-IV-positive nuclei in the superficial layer specifically occur in hair cells (white arrowheads in J–L) and not in adjacent support cells (purple arrowheads in J–L). The inset in J shows a magnified view of stereovilli (st) of a POU-IV-positive hair cell; note the presence of stereovillar rootlets (rl). In addition to hair cells, cnidocytes express POU-IV in the tentacular ectoderm (Figure 2—figure supplement 2; Tournière et al., 2020). POU-IV-positive cells are EdU-negative (arrowheads in I–L), evidencing their postmitotic cell-cycle status. Abbreviations: ec, ectoderm; en, endoderm. Scale bar: 10 µm (A–P); 2 µm (inset in J).

Figure 2—figure supplement 1. The pattern of immunoreactivity with an anti-POU-IV antibody recapitulates that of pou-iv mRNA expression.

Confocal sections of oral tentacles of Nematostella vectensis at the primary polyp stage, labeled with an anti-POU-IV antibody (‘POU-IV protein’; A−D), or an antisense riboprobe against pou-iv mRNA (‘pou-iv mRNA’; E−H). Microtubules are labeled by antibodies against tyrosinated ∂-tubulin (‘tyrTub’; A, B) or acetylated ∂-tubulin (‘acTub’; E, F). Nuclei are labeled with DAPI (dapi). All panels show sections through oral tentacles with the distal end of the tentacle facing up. Anti-POU-IV immunoreactivity occurs in a subset of apical nuclei (white arrowhead in A−D) and the majority of basal nuclei (gray arrowhead in A−D) within the pseudostratified ectodermal epithelium (ec), suggesting nuclear localization of POU-IV proteins in these cells. Consistent with the pattern of anti-POU-IV immunoreactivity, in situ hybridization signals of pou-iv mRNA occur in a subset of ectodermal epithelial cells whose cell bodies are located apically (white arrowhead in E−H), and in those ectodermal cells whose cell bodies are localized basally (bracketed in E−H). Anti-POU-IV immunoreactivity is absent in nuclei of tentacular endodermal cells (en in A−D), and in situ hybridization signals for pou-iv mRNA are not detectable in these cells (en in E−H), indicating the lack of pou-iv expression in tentacular endoderm. Scale bar: 10 μm.

Figure 2—figure supplement 2. POU-IV localizes to the nuclei of cnidocytes in tentacular ectoderm of the sea anemone.

Confocal sections of an oral tentacle of Nematostella vectensis at the primary polyp stage, labeled with antibodies against POU-IV and/or minicollagen 3 (‘Ncol3’; Zenkert et al., 2011). Nuclei are labeled with DAPI (dapi). All panels show longitudinal sections through the center of the tentacle; the distal end of the tentacle is to the left. Arrowheads show POU-IV-positive nuclei of Ncol3-positive cnidocytes that reside in the tentacular ectoderm. Scale bar: 10 µm.

Figure 2—figure supplement 3. Immunostaining with a preadsorbed anti-POU-IV antibody.

Confocal sections of oral tentacles of Nematostella vectensis at the primary polyp stage, labeled with an anti-POU-IV antibody (‘anti-POU-IV’; A-C) and an anti-POU-IV antibody preadsorbed with the POU-IV antigen (CQPTVSESQFDKPFETPSPINamide) used to generate the antibody (‘preadsorbed anti-POU-IV’; D–F). Nuclei are labeled with DAPI (dapi). In all panels, the distal end of the tentacle is to the top, and sections are at the level of surface ectoderm. Arrowheads indicate nuclear immunoreactivity that is abolished when the preadsorbed antibody is used, indicating that the anti-POU-IV reacts with nuclear POU-IV. Scale bar: 10 μm.