Heat-shock mediated overexpression of HNF1β mutations has differential effects on gene expression in the Xenopus pronephric kidney.

The transcription factor HNF1B, encoded by the TCF2 gene, plays an important role in the organogenesis of vertebrates. In humans, heterozygous mutations of HNF1B are associated with several diseases, such as pancreatic β-cell dysfunction leading to maturity-onset diabetes of the young (MODY5), defective kidney development, disturbed liver function, pancreas atrophy, and malformations of the genital tract. The African claw frog Xenopus laevis is an excellent model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a series of methods. In the present study, we overexpressed HNF1β mutants in the developing Xenopus embryo to assess their roles during organogenesis, particularly in the developing pronephric kidney. Towards this goal, we developed a heat-shock inducible binary Cre/loxP system with activator and effector strains. Heat-shock activation of the mutant HNF1B variants P328L329del and A263insGG resulted in malformations of various organs and the affected larvae developed large edemas. Defects in the pronephros were primarily confined to malformed proximal tubules. Furthermore, the expression of the proximal tubule marker genes tmem27 and slc3a1, both involved in amino acid transport, was affected. Both P328L329del and A263insGG downregulated expression of slc3a1. In addition, P328L329del reduced tmem27 expression while A263insGG overexpression decreased expression of the chloride channel clcnk and the transcription factor pax2. Overexpression of two mutant HNF1B derivatives resulted in distinct phenotypes reflected by either a reduction or an enlargement of pronephros size. The expression of selected pronephric marker genes was differentially affected upon overexpression of HNF1B mutations. Based on our findings, we postulate that HNF1B mutations influence gene regulation upon overexpression in specific and distinct manners. Furthermore, our study demonstrates that the newly established Cre/loxP system for Xenopus embryos is an attractive alternative to examine the gene regulatory potential of transcription factors in developing pronephric kidney as exemplified here for HNF1B

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

Gene expression mapping reveals 8 functionally distinct domains in the Xenopus pronephros. Interestingly, no structure equivalent to the mammalian collecting duct is identified

Prospects for the Xenopus Embryo Model in Therapeutics Technologies

Access to suitable animal models is essential in the field of therapeutics technologies. Recently, lower vertebrates have emerged as attractive low-cost animal models which offer new exciting applications in functional genomics and therapeutics technologies. Amphibian embryos of the genus Xenopus have long served as important models for the study of vertebrate development. Xenopus is evolutionary significantly less distant to humans than fish models, which suggests that experimental findings gained with Xenopus will more accurately predict human biology. Numerous experimental advantages, including external development, large size, identifiable blastomeres, and their ability to withstand extensive surgical intervention and culture in vitro, have favored the use of the Xenopus model in the past. More recently, the introduction of a simple efficient method to disrupt gene functions and the rapid development of genomic resources has further increased the attractiveness of this low-cost, high-throughput model for the analysis of vertebrate gene functions. Using the Xenopus embryo as the primary animal model, our research in the field of therapeutics technologies has focused on the identification and validation of novel drug targets by employing genomic and transcriptomic information in the analysis of the molecular and cellular processes underlying kidney organogenesis and vascular development. Furthermore, our research on signaling pathways controlling cellular differentiation of embryonic tissues provides important insights that may ultimately lead to the development of novel cell-based therapies in regenerative medicine. Finally, we are exploring the possibility of employing the Xenopus embryos in chemical library screens to identify novel chemical modulators of organogenesis

A Role for All-Trans-Retinoic Acid in the Early Steps of Lymphatic Vasculature Development

The molecular mechanisms that regulate the earliest steps of lymphatic vascular system development are unknown. To identify regulators of lymphatic competence and commitment, we used an in vitro vascular assay with mouse embryonic stem cell-derived embryoid bodies (EBs). We found that incubation with retinoic acid (RA) and, more potently, with RA in combination with cAMP, induced the expression of the lymphatic competence marker LYVE-1 in the vascular structures of the EBs. This effect was dependent on RA receptor (RAR)-α and protein kinase A signaling. RA-cAMP incubation also promoted the development of CD31+/LYVE-1+/Prox1+ cell clusters. In situ studies revealed that RAR-α is expressed by endothelial cells of the cardinal vein in ED 9.5–11.5 mouse embryos. Timed exposure of mouse and Xenopus embryos to excess of RA upregulated LYVE-1 and VEGFR-3 on embryonic veins and increased formation of Prox1-positive lymphatic progenitors. These findings indicate that RA signaling mediates the earliest steps of lymphatic vasculature development

The prepattern transcription factor Irx3 directs nephron segment identity

The nephron, the basic structural and functional unit of the vertebrate kidney, is organized into discrete segments, which are composed of distinct renal epithelial cell types. Each cell type carries out highly specific physiological functions to regulate fluid balance, osmolarity, and metabolic waste excretion. To date, the genetic basis of regionalization of the nephron has remained largely unknown. Here we show that Irx3, a member of the Iroquois (Irx) gene family, acts as a master regulator of intermediate tubule fate. Comparative studies in Xenopus and mouse have identified Irx1, Irx2, and Irx3 as an evolutionary conserved subset of Irx genes, whose expression represents the earliest manifestation of intermediate compartment patterning in the developing vertebrate nephron discovered to date. Intermediate tubule progenitors will give rise to epithelia of Henle’s loop in mammals. Loss-of-function studies indicate that irx1 and irx2 are dispensable, whereas irx3 is necessary for intermediate tubule formation in Xenopus. Furthermore, we demonstrate that misexpression of irx3 is sufficient to direct ectopic development of intermediate tubules in the Xenopus mesoderm. Taken together, irx3 is the first gene known to be necessary and sufficient to specify nephron segment fate in vivo

Gene expression analysis defines the proximal tubule as the compartment for endocytic receptor-mediated uptake in the Xenopus pronephric kidney

ISSN:0031-6768ISSN:1432-201

Label-free determination of hemodynamic parameters in the microcirculaton with third harmonic generation microscopy.

Determination of blood flow velocity and related hemodynamic parameters is an important aspect of physiological studies which in many settings requires fluorescent labeling. Here we show that Third Harmonic Generation (THG) microscopy is a suitable tool for label-free intravital investigations of the microcirculation in widely-used physiological model systems. THG microscopy is a non-fluorescent multi-photon scanning technique combining the advantages of label-free imaging with restriction of signal generation to a focal spot. Blood flow was visualized and its velocity was measured in adult mouse cremaster muscle vessels, non-invasively in mouse ear vessels and in Xenopus tadpoles. In arterioles, THG line scanning allowed determination of the flow pulse velocity curve and hence the heart rate. By relocating the scan line we obtained velocity profiles through vessel diameters, allowing shear rate calculations. The cell free layer containing the glycocalyx was also visualized. Comparison of the current microscopic resolution with theoretical, diffraction limited resolution let us conclude that an about sixty-fold THG signal intensity increase may be possible with future improved optics, optimized for 1200-1300 nm excitation. THG microscopy is compatible with simultaneous two-photon excited fluorescence detection. It thus also provides the opportunity to determine important hemodynamic parameters in parallel to common fluorescent observations without additional label

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs

Functional Genetic Targeting of Embryonic Kidney Progenitor Cells Ex Vivo

The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor-treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting