Skip to main content
. 2021 Dec 23;10:e74336. doi: 10.7554/eLife.74336

Figure 8. POU-IV activates the expression of polycystin 1 specifically in hair cells.

(A-F) Confocal sections of primary polyps labeled with an antisense riboprobe against polycystin 1 transcript (‘pkd1’) and antibodies against acetylated ∂-tubulin (‘acTub’) and minicollagen 3 (‘Ncol3”; Zenkert et al., 2011). Nuclei are labeled with DAPI (‘dapi’). A–C are side views of the animal with the oral opening facing up. Expression of polycystin 1 occurs exclusively in the ectoderm of the oral tentacles (te). (D–F) Side views of a polycystin 1-expressing epithelial cell (arrowhead) in the tentacular ectoderm (ec) with its apical surface facing up. Note that the cell body is localized apically and lacks minicollagen 3 expression. (G) A schematic of the polycystin 1 locus, showing the distribution of POU-IV ChIP DNA (‘POU-IV’) and input DNA from adult polyps. JGI gene models (‘Genes’) and the revised gene model based on RTPCR (‘Revised’) and the locations of the consensus POU-IV-binding motif – AT(A/T)ATT(A/T)AT – are numbered as I and II. X-axis shows the position along the genomic scaffold, and Y-axis shows the number of reads. * shows an exon whose sequence is missing in the publicly available Nematostella vectensis genome (v1.0; Putnam et al., 2007). (H–K) Confocal sections of an oral tentacle of a primary polyp injected with polycystin 1::kaede construct, labeled with an antibody against Kaede (‘pkd1::kaede’). Filamentous actin is labeled with phalloidin (Pha). The apical surface of the tentacular ectodermal epithelium is to the top. Note that the Kaede-positive cell (arrowhead) has an apical cilium (ci) and stereovilli (st), a central nucleus (nu), and basal neurites (ne), exhibiting morphological hallmarks of a hair cell. No other cell types were found to be Kaede-positive. L–S: Confocal sections of a homozygous pou-iv mutant (‘F2 pou-iv -/-’, P-S) and its sibling control (F2 pou-iv +/+ or pou-iv +/-, ‘F2 sibling ctrl’, L–O) at the primary polyp stage, labeled with an antisense riboprobe against polycystin 1 transcript (‘pkd1’) and antibodies against acetylated ∂-tubulin (‘acTub’) and minicollagen 3 (‘Ncol3’; Zenkert et al., 2011). Panels show side views of the animal with the oral opening facing up. Animals lacking mature cnidocysts based on Ncol3 staining were assumed to be pou-iv -/- mutants; animals with mature cnidocysts were assumed to be pou-iv +/+ or pou-iv +/-. O and S are magnified views of tentacles boxed in M and Q, respectively. Note that cell-type-specific expression of polycystin 1 in tentacular ectoderm (arrowhead in M; O) is absent in the POU-IV null mutant (arrowhead in Q; S), demonstrating that POU-IV is necessary for polycystin 1 expression. Abbreviations: en, endoderm; cn, cnidocyst; nu, nucleus; ms, muscle fiber. Scale bar: 50 µm (A–C, L–S); 10 µm (D–F, H–K).

Figure 8.

Figure 8—figure supplement 1. The predicted protein structure of Nematostella vectensis Polycystin 1-like.

Figure 8—figure supplement 1.

The predicted protein structure of N. vectensis Polycystin 1-like. Extracellular domains are shown in blue, the intracellular domains in orange, and the transmembrane domains in purple. Transmembrane-spanning regions were predicted by using Phyre2 (Kelley et al., 2015) and based on alignment with human and Fugu Polycystin 1 sequences. Non-transmembrane-spanning regions were predicted by using NCBI conserved domain search with default Blast search parameters. The PKD domain and the REJ module are specific to Polycystin 1 (PKD1) proteins. Abbreviations: WSC, cell wall integrity and stress response component; REJ, receptor for egg jelly; GPCR, G-protein-coupled receptor; TM, transmembrane domain; PLAT, polycystin-1, lipoxygenase, and alpha toxin; TOP, tetragonal opening for polycystins.
Figure 8—figure supplement 1—source data 1. Partial cDNA sequence of Nematostella vectensis polycystin 1-like, in which the start codon and stop codon are highlighted in green and red, respectively.
Figure 8—figure supplement 1—source data 2. Translated amino acid sequence of Nematostella vectensis polycystin 1-like, in which conserved domains are highlighted in light blue for transmembrane-spanning regions and in purple for other domains.
Figure 8—figure supplement 2. Nematostella vectensis polycystin 1-like belongs to the Polycystin 1/PKD1 group.

Figure 8—figure supplement 2.

Unrooted maximum likelihood phylogeny of Polycystin protein families based on the alignment of Polycystin 1 (PKD1) and Polycystin 2 (PKD2) protein sequences that span the conserved TOP and transmembrane domains over 323 amino acid sites. NCBI accession numbers are shown with the name of each sequence. Bootstrap support values are shown at each node except when lower than 50%. The unit of the branch length is the number of substitutions per site. Note the strong support for the placement of N. vectensis polycystin 1-like (N. vectensis PKD1, highlighted in purple) within the PKD1 group, and for that of N. vectensis PKD2-like within the PKD2 group. The JGI ID for N. vectensis PKD2 (polycystin 2)-like is 160849.
Figure 8—figure supplement 2—source data 1. Alignment of polycystin 1 and polycystin 2 amino acid sequences used to construct phylogeny shown in Figure 8—figure supplement 2.