Transcriptional Regulation of AQP-8, a Caenorhabditis elegans Aquaporin Exclusively Expressed in the Excretory System, by the POU Homeobox Transcription Factor CEH-6

Due to the ever changing environmental conditions in soil, regulation of osmotic homeostasis in the soil-dwelling nematode Caenorhabditis elegans is critical. AQP-8 is a C. elegans aquaporin that is expressed in the excretory cell, a renal equivalent tissue, where the protein participates in maintaining water balance. To better understand the regulation of AQP-8, we undertook a promoter analysis to identify the aqp-8 cis-regulatory elements. Using progressive 5′ deletions of upstream sequence, we have mapped an essential regulatory region to roughly 300 bp upstream of the translational start site of aqp-8. Analysis of this region revealed a sequence corresponding to a known DNA functional element (octamer motif), which interacts with POU homeobox transcription factors. Phylogenetic footprinting showed that this site is perfectly conserved in four nematode species. The octamer site's function was further confirmed by deletion analyses, mutagenesis, functional studies, and electrophoretic mobility shift assays. Of the three POU homeobox proteins encoded in the C. elegans genome, CEH-6 is the only member that is expressed in the excretory cell. We show that expression of AQP-8 is regulated by CEH-6 by performing RNA interference experiments. CEH-6's mammalian ortholog, Brn1, is expressed both in the kidney and the central nervous system and binds to the same octamer consensus binding site to drive gene expression. These parallels in transcriptional control between Brn1 and CEH-6 suggest that C. elegans may well be an appropriate model for determining gene-regulatory networks in the developing vertebrate kidney. Due to the ever changing environmental conditions in soil, regulation of osmotic homeostasis in the soil-dwelling nematode Caenorhabditis elegans is critical. AQP-8 is a C. elegans aquaporin that is expressed in the excretory cell, a renal equivalent tissue, where the protein participates in maintaining water balance. To better understand the regulation of AQP-8, we undertook a promoter analysis to identify the aqp-8 cis-regulatory elements. Using progressive 5′ deletions of upstream sequence, we have mapped an essential regulatory region to roughly 300 bp upstream of the translational start site of aqp-8. Analysis of this region revealed a sequence corresponding to a known DNA functional element (octamer motif), which interacts with POU homeobox transcription factors. Phylogenetic footprinting showed that this site is perfectly conserved in four nematode species. The octamer site's function was further confirmed by deletion analyses, mutagenesis, functional studies, and electrophoretic mobility shift assays. Of the three POU homeobox proteins encoded in the C. elegans genome, CEH-6 is the only member that is expressed in the excretory cell. We show that expression of AQP-8 is regulated by CEH-6 by performing RNA interference experiments. CEH-6's mammalian ortholog, Brn1, is expressed both in the kidney and the central nervous system and binds to the same octamer consensus binding site to drive gene expression. These parallels in transcriptional control between Brn1 and CEH-6 suggest that C. elegans may well be an appropriate model for determining gene-regulatory networks in the developing vertebrate kidney. The Caenorhabditis elegans excretory system maintains osmotic homeostasis by expelling ionic and metabolic waste from the organism (1Nelson F.K. Albert P.S. Riddle D.L. J. Ultrastruct. Res. 1983; 82: 156-171Crossref PubMed Scopus (136) Google Scholar). The excretory system is comprised of four cells: the excretory duct cell, the binucleate excretory gland cell, the excretory pore cell, and the excretory (canal) cell. The excretory cell is a large fluid-filled cell consisting of a cell body, which is located ventral to the isthmus worm pharynx. From the cell body, two long canals extend posteriorly and two shorter canals extend anteriorly; the four canals are joined at the cell body forming an H-shape. The surface areas of the four canals are greatly increased by a system of canaliculi, which also provides exposure to the extracellular fluid-filled pseudocoelomic cavity (2Buechner M. Hall D.H. Bhatt H. Hedgecock E.M. Dev. Biol. 1999; 214: 227-241Crossref PubMed Scopus (107) Google Scholar). Developmental outgrowth of the excretory cell has been shown to be modulated by many of the same mechanisms that dictate neuronal guidance (3Buechner M. Trends Cell Biol. 2002; 12: 479-484Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Both of these tissues extend long cellular processes between their cell membranes and the epidermal basal lamina during development. Indeed, mutations in many genes, including lin-17, unc-5, unc-6, unc-34, unc-53, and unc-73, affect both neuronal and excretory cell development either by disrupting circumferential growth or by causing premature migrational termination of cellular processes (4Hedgecock E.M. Culotti J.G. Hall D.H. Stern B.D. Development. 1987; 100: 365-382Crossref PubMed Google Scholar). In addition, excretory cell tubulogenesis occurs in vitro using conditions intended for neuronal cell culturing (2Buechner M. Hall D.H. Bhatt H. Hedgecock E.M. Dev. Biol. 1999; 214: 227-241Crossref PubMed Scopus (107) Google Scholar). Consistent with the excretory cell's role in osmoregulation, previous experiments have shown that the excretory duct cell pumping rates in dauer larvae (a nematode diapause state induced by adverse environmental pressures) were inversely proportional to environmental osmotic pressures F.K. Riddle D.L. J. PubMed Scopus Google Scholar). of the excretory cell, duct cell, or pore cell to the worm F.K. Riddle D.L. J. PubMed Scopus Google Scholar). These two that the nematode excretory system is an and to the vertebrate kidney (1Nelson F.K. Albert P.S. Riddle D.L. J. Ultrastruct. Res. 1983; 82: 156-171Crossref PubMed Scopus (136) Google Scholar). The processes for a and the of the excretory cell an for transcriptional regulation and to both renal and neuronal development. 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