The Na+/PO4 cotransporter SLC20A1 gene labels distinct restricted subdomains of the developing pronephros in Xenopus and zebrafish embryos

The embryonic pronephric kidneys of Xenopus and zebrafish serve as models to study vertebrate nephrogenesis. Recently, multiple subdomains within the Xenopus pronephros have been defined based on the expression of several transport proteins. In contrast, very few studies on the expression of renal transporters have been conducted in zebrafish. We have recently shown that the anterior and posterior segments of the zebrafish pronephric duct may correspond to the proximal tubule and distal tubule/duct compartments of the Xenopus and higher vertebrate pronephros, respectively. Here, we report the embryonic expression pattern of the Na(+)/PO(4) cotransporter SLC20A1 (PiT1/Glvr-1) gene encoding a type III sodium-dependent phosphate cotransporter in Xenopus and zebrafish. In Xenopus, SLC20A1 mRNA is expressed in the somitic mesoderm and lower level of expression is detected in the neural tube, eye, and neural crest cells. From stage 25, SLC20A1 is also detectable in the developing pronephros where expression is restricted to the late portion of the distal pronephric tubules. In zebrafish, SLC20A1 is transcribed from mid-somitogenesis in the anterior part of the pronephros where its expression corresponds to the rostral portion of the expression of other proximal tubule-specific markers. Outside the pronephros, lower level of SLC20A1 expression is also observed in the posterior cardinal and caudal veins. Based on the SLC20A1 expression domain and that of other transporters, four segments have been defined within the zebrafish pronephros. Together, our data reveal that the zebrafish and Xenopus pronephros have non-identical proximo-distal organizations.     

Essential function of Wnt-4 for tubulogenesis in the Xenopus pronephric kidney

In the vertebrate embryo, development of the excretory system is characterized by the successive formation of three distinct kidneys: the pronephros, mesonephros, and metanephros. While tubulogenesis in the metanephric kidney is critically dependent on the signaling molecule Wnt-4, it is unknown whether Wnt signaling is equally required for the formation of renal epithelia in the other embryonic kidney forms. We therefore investigated the expression of Wnt genes during the pronephric kidney development in Xenopus. Wnt4 was found to be associated with developing pronephric tubules, but was absent from the pronephric duct. Onset of pronephric Wnt-4 expression coincided with mesenchyme-to-epithelium transformation. To investigate Wnt-4 gene function, we performed gain- and loss-of-function experiments. Misexpression of Wnt4 in the intermediate and lateral mesoderm caused abnormal morphogenesis of the pronephric tubules, but was not sufficient to initiate ectopic tubule formation. We used a morpholino antisense oligonucleotide-based gene knockdown strategy to disrupt Wnt-4 gene function. Xenopus embryos injected with antisense Wnt-4 morpholinos developed normally, but marker gene and morphological analysis revealed a complete absence of pronephric tubules. Pronephric duct development was largely unaffected, indicating that ductogenesis may occur normally in the absence of pronephric tubules. Our results show that, as in the metanephric kidney, Wnt-4 is critically required for tubulogenesis in the pronephric kidney, indicating that a common, evolutionary conserved gene regulatory network may control tubulogenesis in different vertebrate excretory organs.     

Expression profiling of the solute carrier gene family in chicken intestine from the late embryonic to early post-hatch stages

Intestinal development during late embryogenesis and early post-hatch has a long-term influence on digestive and absorptive capacity in chickens. The objective of this research was to obtain a global view of intestinal solute carrier (SLC) gene family member expression from late embryogenesis until 2 weeks post-hatch with a focus on SLC genes involved in uptake of sugars and amino acids. Small intestine samples from male chicks were collected on embryonic days 18 (E18) and 20 (E20), day of hatch and days 1, 3, 7 and 14 post-hatch. The expression profiles of 162 SLC genes belonging to 41 SLC families were determined using Affymetrix chicken genome microarrays. The majority of SLC genes showed little or no difference in level of expression during E18–D14. A number of well-known intestinal transporters were upregulated between E18 and D14 including the amino acid transporters rBAT , y ⁺ LAT-2 and EAAT3 , the peptide transporter PepT1 and the sugar transporters SGLT1 , GLUT2 and GLUT5 . The amino acid transporters CAT-1 and CAT-2 were downregulated. In addition, several glucose and amino acid transporters that are novel to our understanding of nutrient absorption in the chicken intestine were discovered through the arrays ( SGLT6 , SNAT1 , SNAT2 and AST ). These results represent a comprehensive characterization of the expression profiles of the SLC family of genes at different stages of development in the chicken intestine and lay the ground work for future nutritional studies.     

Identification of six putative human transporters with structural similarity to the drug transporter SLC22 family

The solute carrier family 22 (SLC22) is a large family of organic cation and anion transporters. These are transmembrane proteins expressed predominantly in kidneys and liver and mediate the uptake and excretion of environmental toxins, endogenous substances, and drugs from the body. Through a comprehensive database search we identified six human proteins not yet cloned or annotated in the reference sequence databases. Five of these belong to the SLC22 family, SLC22A20, SLC22A23, SLC22A24, SLC22A25, and SPNS3, and the sixth gene, SVOPL, is a paralog to the synaptic vesicle protein SVOP. We identified the orthologs for these genes in mouse and rat and additional homologous proteins and performed the first phylogenetic analysis on the entire SLC22 family in human, mouse, and rat. In addition, we performed a phylogenetic analysis which showed that SVOP and SV2A-C are, in a comparison with all vertebrate proteins, most similar to the SLC22 family. Finally, we performed a tissue localization study on 15 genes on a panel of 30 rat tissues using quantitative real-time polymerase chain reaction.     

Comparative genomic and expression analysis of the conserved NTPDase gene family in Xenopus

The purines, ATP and adenosine, are important signaling molecules in the nervous system. ATP is sequentially degraded to adenosine by the ectonucleotidase proteins. The NTPDase (or CD39) family is a subfamily of these enzymes, which consists of nine members in mammals. In Xenopus embryos, we have shown that ATP, and its antagonist adenosine, regulate the rundown of swimming and we therefore proposed that ectonucleotidase proteins are key regulators of locomotor activity. Here, we report the cloning of all nine members of the NTPDase family in Xenopus laevis and Xenopus tropicalis. Our phylogenetic analysis shows that this family is highly conserved between the frog species and also during vertebrate evolution. In the adult frog, NTPDase genes are broadly expressed. During development, all NTPDase genes, except for NTPDase8, are expressed and display a distinct specific expression pattern, suggesting potentially different functions of these proteins during embryogenesis of X. laevis.     

Select nutrients in the ovine uterine lumen. ii. glucose transporters in the uterus and peri-implantation conceptuses

Total glucose in ovine uterine lumenal fluid increases 6-fold between Days 10 and 15 of gestation, but not the estrous cycle; however, mechanisms for glucose transport into the uterine lumen and uptake by conceptuses (embryo/fetus and associated membranes) are not established. This study determined the effects of the estrous cycle, pregnancy, progesterone (P4), and interferon tau (IFNT) on expression of both facilitative (SLC2A1, SLC2A3, and SLC2A4) and sodium-dependent (SLC5A1 and SLC5A11) glucose transporters in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy, as well as in conceptuses from Days 10 to 20 of pregnancy. The SLC2A1 and SLC5A1 mRNAs and proteins were most abundant in uterine luminal epithelia and superficial glandular epithelia (LE/sGE), whereas SLC2A4 was present in stromal cells and glandular epithelia (GE). SLC5A11 mRNA was most abundant in endometrial GE, whereas SLC2A3 mRNA was not detectable in endometria. SLC2A1, SLC2A3, SLC2A4, SLC5A1, and SLC5A11 were expressed in the trophectoderm and endoderm of conceptuses. Steady-state levels of SLC2A1, SLC5A1, and SLC5A11 mRNAs, but not SLC2A4 mRNA, were greater in endometria from pregnant than from cyclic ewes. Progesterone increased SLC2A1, SLC5A11, and SLC2A4 mRNAs in the LE/sGE and SLC5A1 in the GE of ovariectomized ewes. Expression of SLC5A1 was inhibited by ZK136,317 (progesterone receptor antagonist), and the combination of ZK136,317 and IFNT further decreased expression in GE. In constrast, P4 induced and IFNT stimulated expression of SLC2A1 and SLC5A11, and these effects were blocked by ZK136,317. Results of this study indicate differential expression of facilitative and sodium-dependent glucose transporters in ovine uteri and conceptuses for transport and uptake of glucose, and that P4 or P4 and IFNT regulate their expression during the peri-implantation period of pregnancy.     

Amino acid cotransporter SLC3A2 is selectively expressed in the early proximal segment of Xenopus pronephric kidney nephrons

The transport of amino acids across membranes is critical to all cells. As amino acids freely pass through the glomerular filtration barrier of the kidney, they must be efficiently resorbed to avoid depletion of circulating amino acid reserves. Not only do defects in amino acid resorption lead to costly wastage, they also cause congenital aminoacidurias. A clone encoding Xenopus SLC3A2 was identified and shown to be expressed at high levels in the early segment of the pronephric proximal tubules in developing tadpoles. The type II membrane glycoprotein encoded by this gene can associate with a wide variety of protein partners and participates in a broad spectrum of biological processes. In this report, the first whole-mount analysis of SLC3A2 during early embryonic development is presented. The expression pattern of SLC3A2 in the early proximal segment of the Xenopus pronephros is analogous to that of a previously described SLC7A8/XAA2 amino acid transporter. In mammals, SLC3A2 and SLC7A8/XAA2 associate to form a functional neutral amino acid transporter complex and coexpression of these two genes in a small domain within the pronephric tubules indicates that this is also the situation in the developing Xenopus kidney.     

Sequential gene expression during pronephric tubule formation in vitro in Xenopus ectoderm

The kidney has been used as a model organ to analyze organogenesis. In in vitro experiments using Xenopus blastula ectoderm, the development of pronephric tubules (the prototype of the kidney) may be induced by treatment with activin A and retinoic acid (RA). The present study examined whether pronephric tubules induced in ectodermal explants exhibited similar characteristics to those of normal embryos at the molecular level. The experimental conditions required for high frequency induction (100%) of pronephric tubule formation from presumptive ectoderm without the development of muscle and notochord were determined. The developmental expression of the pronephros marker genes Xlim-1 and Xlcaax-1 was examined in induced pronephric tubules. After treatment with 10 ng/mL activin A and 10⁻⁴ mol/L RA, only pronephric tubules were induced at a high frequency. Induced pronephric tubules showed the same timing and patterns of expression for the marker genes Xlim-1 and Xlcaax-1 as normal embryos. These results suggest that the in vitro development of pronephric tubules induced in the presumptive ectoderm by activin A and RA parallels normal development at the molecular level.     

Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric kidney tubules

The embryonic kidneys of larval aquatic vertebrates such as fish and frogs serve as excellent model systems for exploring the early development of nephric organs. These experimental systems can easily be manipulated by microsurgery, microinjection, genetics, or combinations of these approaches. However, little is known about how physiologically similar these simple kidneys are to the more complex mammalian adult kidneys. In addition, almost nothing is known about proximo-distal patterning of nephrons in any organism. In order begin to explore the physiological specialization of the pronephric tubules along the proximo-distal axis, a combination of uptake assays using fluorescently tagged proteins, LDL particles and dextrans, and an informatics-targeted in situ screen for transport proteins have been performed on embryos of the frog, Xenopus laevis. Genes identified to be expressed within unique subdomains of the pronephric tubules include an ABC transporter, two amino acid cotransporters, two sodium bicarbonate cotransporters, a novel sodium glucose cotransporter, a sodium potassium chloride cotransporter (NKCC2), a sodium chloride organic solute cotransporter (ROSIT), and a zinc transporter. A novel combination of colorimetric and fluorescent whole-mount in situ hybridization (FCIS) was used to precisely map the expression domain of each gene within the pronephros. These data indicate specialized physiological function and define multiple novel segments of the pronephric tubules, which contain at least six distinct transport domains. Uptake studies identified functional transport domains and also demonstrated that early glomeral leakage can allow visualization of protein movement into the pronephric tubules and thus establish a system for investigating experimentally induced proteinuria and glomerulonephritis.     

Functional characterisation of human SGLT-5 as a novel kidney-specific sodium-dependent sugar transporter

Sodium glucose cotransporters (SGLT) actively catalyse carbohydrate transport across cellular membranes. Six of the 12 known SGLT family members have the capacity to bind and/or transport monosaccharides (SGLT-1 to 6); of these, all but SGLT-5 have been characterised. Here we demonstrate that human SGLT-5 is exclusively expressed in the kidney. Four splice variants were detected and the most abundant SGLT-5-mRNA was functionally characterised. SGLT-5 mediates sodium-dependent [(14)C]-α-methyl-D-glucose (AMG) transport that can be inhibited by mannose, fructose, glucose, and galactose. Uptake studies using demonstrated high capacity transport for mannose and fructose and, to a lesser extent, glucose, AMG, and galactose. SGLT-5 mediated mannose, fructose and AMG transport was weakly (μM potency) inhibited by SGLT-2 inhibitors. In summary, we have characterised SGLT-5 as a kidney mannose transporter. Further studies are warranted to explore the physiological role of SGLT-5.     

Expression of three <Emphasis Type="Italic">spalt </Emphasis>(<Emphasis Type="Italic">sal</Emphasis>) gene homologues in zebrafish embryos

Three homologues of the Drosophilaregion-specific homeotic gene spalt (sal) have been isolated in zebrafish, sall1a, sall1b and sall3. Phylogenetic analysis of these genes against known salDNA sequences showed zebrafish sall1aand sall1b to be orthologous to other vertebrate sal-1 genes and zebrafish sall3to be orthologous to other vertebrate sal-3 genes, except Xenopus sall3. Phylogenetic reconstruction suggests that zebrafish sall1a and sall1bresulted from a gene duplication event occurring prior to the divergence of the ray-finned and lobe-finned fish lineages. Analysis of the expression pattern of the zebrafish sal genes shows that sall1a and sall3 share expression domains with both orthologous and non-orthologous vertebrate sal genes. Both are expressed in various regions of the CNS, including in primary motor neurons. Outside of the CNS, sall1a expression is observed in the otic vesicle (ear), heart and in a discrete region of the pronephric ducts. These analyses indicate that orthologies between zebrafish sal genes and other vertebrate sal genes do not imply equivalence of expression pattern and, therefore, that biological functions are not entirely conserved. However we suggest that, like other vertebrate sal genes, zebrafish sal genes have a role in neural development. Also, expression of zebrafish sall1a in the otic vesicle, heart sac and the pronephric ducts of zebrafish embryos is possibly consistent with some of the abnormalities seen in Sall1-deficient mice and in Townes-Brocks Syndrome, a human disorder which is caused by mutations in the human spalt gene SALL1.     

Towards a molecular anatomy of the Xenopus pronephric kidney.

Kidney development is distinguished by the sequential formation of three structures of putatively equivalent function from the intermediate mesoderm, the pronephros, mesonephros, and metanephros. While these organs differ morphologically, their basic structural organization exhibits important similarities. The earliest form of the kidney, the pronephros, is the primary blood filtration and osmoregulatory organ of fish and amphibian larvae. Simple organization and rapid formation render the Xenopus pronephric kidney an ideal model for research on the molecular and cellular mechanisms dictating early kidney organogenesis. A prerequisite for this is the identification of genes critical for pronephric kidney development. This review describes the emerging framework of genes that act to establish the basic components of the pronephric kidney: the corpuscle, tubules, and the duct. Systematic analysis of marker gene expression, in temporal and spatial resolution, has begun to reveal the molecular anatomy underlying pronephric kidney development. Furthermore, the emerging evidence indicates extensive conservation of gene expression between pronephric and metanephric kidneys, underscoring the importance of the Xenopus pronephric kidney as a simple model for nephrogenesis. Given that Xenopus embryos allow for easy testing of gene function, the pathways that direct cell fate decisions in the intermediate mesoderm to make the diverse spectrum of cell types of the pronephric kidney may become unraveled in the future.     

Organization of the pronephric kidney revealed by large-scale gene expression mapping

Background

The pronephros, the simplest form of a vertebrate excretory organ, has recently become an important model of vertebrate kidney organogenesis. Here, we elucidated the nephron organization of the Xenopus pronephros and determined the similarities in segmentation with the metanephros, the adult kidney of mammals.

Results

We performed large-scale gene expression mapping of terminal differentiation markers to identify gene expression patterns that define distinct domains of the pronephric kidney. We analyzed the expression of over 240 genes, which included members of the solute carrier, claudin, and aquaporin gene families, as well as selected ion channels. The obtained expression patterns were deposited in the searchable European Renal Genome Project Xenopus Gene Expression Database. We found that 112 genes exhibited highly regionalized expression patterns that were adequate to define the segmental organization of the pronephric nephron. Eight functionally distinct domains were discovered that shared significant analogies in gene expression with the mammalian metanephric nephron. We therefore propose a new nomenclature, which is in line with the mammalian one. The Xenopus pronephric nephron is composed of four basic domains: proximal tubule, intermediate tubule, distal tubule, and connecting tubule. Each tubule may be further subdivided into distinct segments. Finally, we also provide compelling evidence that the expression of key genes underlying inherited renal diseases in humans has been evolutionarily conserved down to the level of the pronephric kidney.

Conclusion

The present study validates the Xenopus pronephros as a genuine model that may be used to elucidate the molecular basis of nephron segmentation and human renal disease.     

LPS/CD14 activation triggers SGLT-1-mediated glucose uptake and cell rescue in intestinal epithelial cells via early apoptotic signals upstream of caspase-3

Recent findings indicate that enhanced glucose uptake protects enterocytes from excessive apoptosis and barrier defects induced by LPS exposure. The aim of this study was to characterize the mechanisms responsible for increased sodium-dependent glucose cotransporter (SGLT)-1 activity in enterocytes challenged with LPS. SGLT-1-transfected Caco-2 cells were incubated with LPS in high glucose media. LPS increased SGLT-1 activity in dose- and time-dependent fashion, and is due to increased V(max) of the cotransporter. Elevated apical expression of SGLT-1 was also demonstrated. This LPS-induced effect was colchicine-inhibitable, suggesting microtubule-dependent translocation of SGLT-1 onto apical surface. Immunofluorescence staining showed expression of CD14 on the apical surface, but no TLR-4, on these cells. Neutralizing anti-CD14 decreased the LPS-induced upregulation of SGLT-1 activity, whereas anti-TLR-4 had no effect. Pharmacological studies indicated that signaling for LPS-mediated SGLT-1 glucose uptake depends on caspase-8 and -9 activation, but occurs independently of caspase-3. The findings describe a novel feedback mechanism within the apoptotic signaling pathway for SGLT-1-dependent cytoprotection. The observation suggests a new function for CD14 on enterocytes, involving the induction of the caspase-dependent SGLT-1 activity, which ultimately leads to cell rescue. The understanding of these signaling events may shed light on enterocytic cytoprotection and homeostasis mechanism upon pro-apoptotic challenges.     

Two novel Xenopus frizzled genes expressed in developing heart and brain.

A family of genes related to the Drosophila wingless receptor frizzled have been found in vertebrates. We have cloned full length cDNAs of two novel frizzled genes from embryonic Xenopus tissue. We are calling them Xfz7 and Xfz9 (for Xenopus frizzled) because their deduced peptide sequences show extensive similarity to other vertebrate frizzled molecules. Xfz7 is closely related to human, chick and mouse frz-7 and Xfz9 is most related to human FZD9 and mouse fzd9. Xfz7 is expressed in a broad, complex and dynamic pattern beginning at gastrulation. At later stages Xfz7 expression is found in neural crest, neural tube, eye, pronephric duct and the heart. Xfz9 expression in contrast is more restricted to the neuroectoderm and, at later stages of development, to the dorsal regions of the mid- and hindbrain.     

Mito-nuclear genetic comparison in a Wolbachia infected weevil: insights on reproductive mode, infection age and evolutionary forces shaping genetic variation

Background

The composition and expression of vertebrate gene families is shaped by species specific gene loss in combination with a number of gene and genome duplication events (R1, R2 in all vertebrates, R3 in teleosts) and depends on the ecological and evolutionary context. In this study we analyzed the evolutionary history of the solute carrier 1 (SLC1) gene family. These genes are supposed to be under strong selective pressure (purifying selection) due to their important role in the timely removal of glutamate at the synapse.

Results

In a genomic survey where we manually annotated and analyzing sequences from more than 300 SLC1 genes (from more than 40 vertebrate species), we found evidence for an interesting evolutionary history of this gene family. While human and mouse genomes contain 7 SLC1 genes, in prototheria, sauropsida, and amphibia genomes up to 9 and in actinopterygii up to 13 SLC1 genes are present. While some of the additional slc1 genes in ray-finned fishes originated from R3, the increased number of SLC1 genes in prototheria, sauropsida, and amphibia genomes originates from specific genes retained in these lineages. Phylogenetic comparison and microsynteny analyses of the SLC1 genes indicate, that theria genomes evidently lost several SLC1 genes still present in the other lineage. The genes lost in theria group into two new subfamilies of the slc1 gene family which we named slc1a8/eaat6 and slc1a9/eaat7.

Conclusions

The phylogeny of the SLC1/EAAT gene family demonstrates how multiple genome reorganization and duplication events can influence the number of active genes. Inactivation and preservation of specific SLC1 genes led to the complete loss of two subfamilies in extant theria, while other vertebrates have retained at least one member of two newly identified SLC1 subfamilies.