Identification and Expression Analysis of the Complete Family of Zebrafish pkd Genes

Polycystic kidney disease (PKD) proteins are trans-membrane proteins that have crucial roles in many aspects of vertebrate development and physiology, including the development of many organs as well as left–right patterning and taste. They can be divided into structurally-distinct PKD1-like and PKD2-like proteins and usually one PKD1-like protein forms a heteromeric polycystin complex with a PKD2-like protein. For example, PKD1 forms a complex with PKD2 and mutations in either of these proteins cause Autosomal Dominant Polycystic Kidney Disease (ADPKD), which is the most frequent potentially-lethal single-gene disorder in humans. Here, we identify the complete family of pkd genes in zebrafish and other teleosts. We describe the genomic locations and sequences of all seven genes: pkd1, pkd1b, pkd1l1, pkd1l2a, pkd1l2b, pkd2, and pkd2l1. pkd1l2a/pkd1l2b are likely to be ohnologs of pkd1l2, preserved from the whole genome duplication that occurred at the base of the teleosts. However, in contrast to mammals and cartilaginous and holostei fish, teleosts lack pkd2l2, and pkdrej genes, suggesting that these have been lost in the teleost lineage. In addition, teleost, and holostei fish have only a partial pkd1l3 sequence, suggesting that this gene may be in the process of being lost in the ray-finned fish lineage. We also provide the first comprehensive description of the expression of zebrafish pkd genes during development. In most structures we detect expression of one pkd1-like gene and one pkd2-like gene, consistent with these genes encoding a heteromeric protein complex. For example, we found that pkd2 and pkd1l1 are expressed in Kupffer's vesicle and pkd1 and pkd2 are expressed in the developing pronephros. In the spinal cord, we show that pkd1l2a and pkd2l1 are co-expressed in KA cells. We also identify potential co-expression of pkd1b and pkd2 in the floor-plate. Interestingly, and in contrast to mouse, we observe expression of all seven pkd genes in regions that may correspond to taste receptors. Taken together, these results provide a crucial catalog of pkd genes in an important model system for elucidating cell and developmental processes and modeling human diseases and the most comprehensive analysis of embryonic pkd gene expression in any vertebrate.

3). This figure shows the region of the polycystin-cation-channel domain that is present in all of the proteins and was used to construct the phylogenetic tree in Fig. 3B. Different families of PKD2-like proteins are color-coded with the same color. This same color-coding is used in Figure 3B. Numbers on either side of each sequence, indicate amino acid positions in the full-length sequences of each protein.  PCR primers and conditions used to amplify and sequence overlapping fragments of zebrafish pkd1, pkd1l2a and pkd1l2b transcripts (genes for which annotations in Ensembl are incomplete). Column 1 lists names given to each primer set. Columns 2 and 3 list PCR primers used to map transcript region indicated in column 6. Columns 4 and 5 list annealing temperatures and extension times of PCR protocols used in each case. The rest of the protocol is provided in materials and methods. *These primers were used on a circularized cDNA product following inverse PCR to generate 5' sequence. + These primers were used in a series of nested PCR reactions. Nested Set 1 primers were used on a circularized cDNA product following inverse PCR to generate 5' sequence. Nested Set 2 primers were used in reactions seeded with the Nested Set 1 PCR product. See materials and methods for details. § These reverse primers contain sequence for T3 RNA Polymerase promoter at their 5' end, since they were also used to generate in situ hybridization riboprobes (see Supplementary Table 3 for riboprobe primers). ^ Since some of our mapped sequence data is not present in the current Ensembl chromosome 1 genomic sequence (GRCz10), we cannot map all exon boundaries for pkd1 and so instead we show the base pair locations within the mapped mRNA transcript that were amplified and sequenced by these primers (see Figure 1 text for further information).

Supplementary Table 3. PCR primers for creating in situ hybridization riboprobes
Primer sequences used to generate riboprobes for in situ hybridization. Expected PCR product sizes (in base pairs) are indicated in column 4. * indicates primers published in Coxam et al., (2014). All other riboprobe primers were designed during this study. T3 RNA polymerase promoter sequence at 5' end of each reverse primer is bold and underlined. pkd1 -Set 1, pkd1l1 -Set 1 and pkd1l2a -Set 1 primers generated riboprobes that gave the strongest expression in our assays and so were used exclusively in this study. See materials and methods for further information. Shares synteny with teleost and mammalian pkd1 genes (data not shown). Phylogeny (Fig. 3A). Elephant shark pkd1 (two pkd1 genes in genome) SINCAMG00000009812 Scaffold_312(+): 34635-113581

Species Percentage Amino Acid Identity to Full-Length Mouse PKD1L3 Protein
Shares synteny with teleost and mammalian pkd1 genes (data not shown). Phylogeny (Fig. 3A).

2273648-2326286
Does not share synteny with teleost pkd1b genes (data not shown). Formerly called pkd1 but phylogeny suggests that this gene is pkd1b (Fig.  3A). All PKD1L3 genes examined in this study are syntenic with the DHODH gene. In the elephant shark, dhodh is present on Scaffold_12 and is also flanked by the genes znf821 and atxn11, which also surround the mammalian PKD1L3 genes. Tblastn analysis with either the polycystin-cation-channel domains of zebrafish PKD proteins or the full-length mouse PKD1L3 protein failed to detect any hits adjacent to these genes on Scaffold_12 in the elephant shark genome, although we cannot rule out the possibility that a pkd1l3 ortholog might exist elsewhere in the genome.   Shares synteny with green spotted pufferfish, stickleback and medaka pkd2 genes (data not shown). Phylogeny (Fig. 3B).
Supplementary Table 5. Characterization of pkd genes in the genomes of holostei and cartilaginous fish.
Column one indicates the species. Column 2 lists gene name used in current Ensembl genome assembly (see materials and methods). Column 3 lists Ensembl gene ID. Column 4 shows position in current version of appropriate genome. Column 5 lists data that support the annotations shown here (see results for more info). pkd genes were first identified by performing textual searches of the appropriate genome assembly and their synteny and phylogeny examined (data not shown, Fig. 3A-B). We then searched for additional pkd genes that are present in either mammalian or teleost genomes, but had not been identified by our previous searches, by performing Tblastn analyses with the polycystin-cation-channel domain of the zebrafish Pkd protein, or the full-length sequence of the mouse PKD protein in question.