Identification and Characterization of Pkhd1, the Mouse Orthologue of the Human ARPKD Gene

Abstract

Abstract. PKHD1, the gene mutated in human autosomal recessive polycystic kidney disease has recently been identified. Its translation products are predicted to belong to a superfamily of proteins involved in the regulation of cellular adhesion and repulsion. One notable aspect of the gene is its unusually complex pattern of splicing. This study shows that mouse Pkhd1 and its translation products have very similar properties to its human orthologue. Mouse Pkhd1 extends over approximately 500 kb of genomic DNA, includes a minimum of 68 nonoverlapping exons, and exhibits a complex pattern of splicing. The longest ORF encodes a protein of 4059aa predicted to have an N-terminal signal peptide, multiple IPTs and PbH1 repeats, a single transmembrane span (TM), and a short cytoplasmic C-terminus. Although the protein sequence is generally well conserved (approximately 73% average identity), the C-termini share only 55% identity. The pattern of Pkhd1 expression by in situ hybridization was also examined in developing and adult mouse tissues over a range of ages (E12.5 to 3 mo postnatal). High levels of expression were present in renal and biliary tubular structures at all time points examined. Prominent Pkhd1 signals were also found in a number of other organs and tissues. Tissue-specific differences in transcript expression were revealed through the use of single exon probes. These data show that key features of human PKHD1 are highly conserved in the mouse and suggest that the complicated pattern of splicing is likely to be functionally important.

A mouse model for cystic biliary dysgenesis in autosomal recessive polycystic kidney disease (ARPKD)†

“…We previously characterized the murine Pkhd1 gene and performed detailed expression analyses in embryonic and adult tissues. 9 This study also provided evidence that differentially spliced Pkhd1 mRNAs are expressed in a tissuespecific manner. Because analyses by multiple tissue Northern blots and in situ hybridization indicated that our probe for exon 40 (homologous to exon 41 of the human gene) hybridized with the largest, most abundant transcripts in liver and kidney and also with differentially spliced smaller transcripts in testis, we decided to disrupt exon 40 by insertion of a LacZ-PGK-neo selection cassette (Fig.…”

“…We previously observed the highest levels of Pkhd1 mRNA expression in kidney and lower levels in liver. 9 Northern blot hybridization using exon 40 as a probe and RT-PCR amplification using an exon 40 primer pair failed to detect Pkhd1 mRNA expression in kidney and liver of homozygous mutants (Fig. 1C,E).…”

“…We chose to mutate exon 40, because it is part of the most abundant mRNA expressed in liver and kidney. 9 The Pkhd1 transcript expressed in kidney and liver of Pkhd1ex40 mice is only slightly modified, encoding a protein lacking amino acid residues 2160-2223 due to skipping exon 40. Analyses by RT-PCR and LacZ staining indicated that the entire exon 40 including the targeting cassette is removed by splicing from the mutated transcript.…”

“…8 We recently identified the murine Pkhd1 orthologue and showed that the overall protein structure and the complex pattern of splicing is highly conserved. 9 We then aimed to investigate the role of polyductin/fibrocystin during mouse development and therefore constructed mice with a targeted Pkhd1 germline mutation.…”

Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function

“…These findings are consistent with our previous report 31 and indicate that FPC may modulate the morphogenesis and maintenance of tubular/ductal architectures in organs generated from the primary duct system. 50 Several recent studies indicated that FPC is involved in notch-like processing and that its extremely large extracellular domain is released from the primary cilia of renal epithelial cells. 51 That GFP-positive signals were detected at the microvilli of tubular epithelia in our Pkhd1 Ϫ/Ϫ mice agrees with the report that FPC may be released into the tubular/ductal lumen.…”