Ilya Skovorodkin

Novel perspectives for investigating congenital anomalies of the kidney and urinary tract (CAKUT)

Congenital anomalies of the kidney and urinary tract (CAKUT) are the commonest cause of chronic kidney disease in children. Structural anomalies within the CAKUT spectrum include renal agenesis, kidney hypo-/dysplasia, multicystic kidney dysplasia, duplex collecting system, posterior urethral valves and ureter abnormalities. While most CAKUT cases are sporadic, familial clustering of CAKUT is common, emphasizing a strong genetic contribution to CAKUT origin. Animal experiments demonstrate that alterations in genes crucial for kidney development can cause experimental CAKUT, while expression studies implicate mislocalization and/or aberrant levels of the encoded proteins in human CAKUT. Further insight into the pathogenesis of CAKUT will improve strategies for early diagnosis, follow-up and treatment. Here, we outline a collaborative approach to identify and characterize novel factors underlying human CAKUT. This European consortium will share the largest collection of CAKUT patients available worldwide and undertake multidisciplinary research into molecular and genetic pathogenesis, with extension into translational studies to improve long-term patient outcomes.

Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development.

The Wnt4 gene encodes a secreted signaling molecule controlling the development of several organs, such as the kidney, adrenal gland, ovary, mammary gland and pituitary gland. It is thought to act in the embryonic kidney as an auto-inducer of nephrogenesis controlling mesenchyme-to-epithelium transition, and Wnt4-deficient mice die soon after birth, probably of kidney failure. Given the requirement for Wnt4 signaling in the control of organogenesis, the targeting of Cre recombinase under the control of the Wnt4 promoter would provide a valuable tool for fate mapping and functional genomics. We report here on the generation and characterization of a Wnt4(EGFPCre) knock-in allele where the EGFPCre fusion cDNA and Neo selection cassette were targeted into the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates of the E14.5 embryonic kidney that normally express Wnt4 mRNA. Characterization of the pattern of recombination of the floxed Rosa26(LacZ) reporter with the Wnt4(EGFPCre) allele revealed that in addition to the embryonic kidney, reporter-derived staining was observed in the embryonic gonad, spinal cord, lung and adrenal gland, i.e. the sites of Wnt4 gene expression. Time-lapse fate mapping of the Wnt4(EGFPCre)-activated yellow fluorescent protein (YFP) from the Rosa26 locus in organ culture revealed that the cells that had expressed the Wnt4 gene contributed to the nephrons, some of the cells around the stalk of the developing ureter and also certain presumptive medullary stromal cells. Moreover, the time-lapse movies suggested that the first few pretubular cell aggregates may not mature into nephrons but instead appear to disintegrate. In association with this, Rosa26(YFP)-positive stromal cells emerge around these disintegrating structures. Such cells may be transient, since their derivatives are neither detected later in the more mature kidney nor is there an overlap of the Wnt4(EGFPCre); Rosa26(LacZ)-marked cells with those of the endothelial cells, the smooth muscle cells or the macrophages. The Wnt4(EGFPCre) allele provides a useful new tool for conditional mutagenesis and provides the first time-lapse-based map of the fate of nephron precursor cells.

A Secreted BMP Antagonist, Cer1, Fine Tunes the Spatial Organization of the Ureteric Bud Tree during Mouse Kidney Development

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.

Actin-Based Mechanism of Holospora obtusa Trafficking in Paramecium caudatum

Holospora obtusa, an alpha-proteobacterium, is an obligate endonuclear pathogen of the ciliate Paramecium caudatum. It is engulfed by the host cell in the course of phagocytosis but soon escapes from the phagosome and is transported across the host cell cytoplasm to the paramecium macronucleus. Electron microscopy reveals a comet-like tail resembling that of Listeria trailing after H. obtusa in the host cytoplasm. In this study we investigated the role of the host cell actin and Arp3 in the process of infection with Holospora. Cytochalasin D treatment significantly reduced the rate of nuclear infection. Using immunocytochemistry and experimental infection of GFP-actin-transfected paramecia we demonstrated that the Paramecium actin1-1 took part in the bacterial escape from the phagosome, its trafficking in the cytoplasm and entry into the host macronucleus. Rapid assembly/disassembly of actin filaments in P. caudatum led to quick loss of actin1-1 from the trails left by H. obtusa. Immunocytochemistry using anti-bovine Arp3 antibodies demonstrated the presence of Arp3 in these trails. Our data indicate that details of H. obtusa infection are rather similar to those of Listeria and Rickettsia.

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.

Cajal bodies and interchromatin granule clusters in cricket oocytes: Composition, dynamics and interactions

The organization and molecular composition of complicated Cajal bodies (CBs) and interchromatin granule clusters (IGCs) in oocytes of the house cricket, Acheta domesticus, were studied using immunofluorescent/confocal and Immunogold labeling/electron microscopy. In A. domesticus oocytes, the CB consists of the fibrillar matrix and a central cavity containing a predominantly granular body with insertions of tightly packed fibrillar material. The latter structure was identified as an “internal” IGC, since it is enriched with the SC35 protein, a marker for IGCs. The IGCs located outside the CB were also identified. Microinjections of the fluorescein‐tagged U7 snRNA into the ooplasm showed the targeting of the U7 to the matrix of the CB. Some other essential CB components (coilin, snRNPs, fibrillarin) were found to be colocalized in the matrix of the CB. Neither confocal nor Immunogold microscopy revealed significant amounts of RNA polymerase II (pol II) in the CB of A. domesticus oocytes. The splicing factor SC35 was detected in the matrix of the CB. In oocytes treated with DRB, the amount of IGCs in the nucleoplasm increased significantly, granular and fibrillar components of IGCs were segregated, and the fibrillar areas accumulated pol II. Additionally, IG‐like granules were shown to display on the surface of the CB probably due to a shifting from the internal IGC. We believe that in A. domesticus oocytes, CBs are involved in nuclear distribution of splicing factors, but their role in pol II transport is less significant. We also suggest that the formation of complicated CBs is a result of interconnection between two different nuclear domains, CBs and IGCs.

Wnt4 coordinates directional cell migration and extension of the Müllerian duct essential for ontogenesis of the female reproductive tract

The Müllerian duct (MD) is the anlage of the oviduct, uterus and upper part of the vagina, the main parts of the female reproductive tract. Several wingless-type mouse mammary tumor virus (MMTV) integration site family member (Wnt) genes, including Wnt4, Wnt5a and Wnt7a, are involved in the development of MD and its derivatives, with Wnt4 particularly critical, since the MD fails to develop in its absence. We use, here, Wnt4EGFPCre-based fate mapping to demonstrate that the MD tip cells and the subsequent MD cells are derived from Wnt4+ lineage cells. Moreover, Wnt4 is required for the initiation of MD-forming cell migration. Application of anti-Wnt4 function-blocking antibodies after the initiation of MD elongation indicated that Wnt4 is necessary for the elongation as well, and consistent with this, cell culture wound-healing assays with NIH3T3 cells overexpressing Wnt4 promoted cell migration by comparison with controls. In contrast to the Wnt4 null embryos, some Wnt4monomeric cherry/monomeric cherry (Wnt4mCh/mCh) hypomorphic mice survived to adulthood and formed MD in ∼45% of cases. Nevertheless, the MD of the Wnt4mCh/mCh females had altered cell polarization and basement membrane deposition relative to the controls. Examination of the reproductive tract of the Wnt4mCh/mCh females indicated a poorly coiled oviduct, absence of the endometrial glands and an undifferentiated myometrium, and these mice were prone to develop a hydro-uterus. In conclusion, the results suggest that the Wnt4 gene encodes signals that are important for various aspects of female reproductive tract development.

Plasmon-Resonant Gold Nanostars With Variable Size as Contrast Agents for Imaging Applications

Plasmon-resonant nanostars (NSts) have recently found applications in biophotonics due to their unique optical and chemical characteristics, showing comparable or superior properties than other anisotropic plasmon-resonant nanoparticles. In this paper, we synthesized gold NSts by the seed-mediated surfactant-free method. By varying the diameters and amount of spherical seeds we tuned the final NSts tip-to-tip sizes to 50, 82, 100, and 120 nm ensuring the plasmon-resonant peak location between 710 and 830 nm, and the scattering/absorption ratio at the plasmon-resonant wavelengths being 0.12, 0.25, 0.30, 0.35 correspondingly. We investigated the application of the NSts as contrast agents for imaging techniques operating at visible and infrared wavelengths: optical coherence tomography (OCT) and Doppler OCT with the spectrum centered at 930-nm wavelength, as well as for conventional confocal laser scanning microscopy (CLSM) working at 633 nm. The most intense OCT signal was registered from the largest NSts, in correspondence with spectroscopy measurements at 930-nm wavelength. For imaging of nanoparticles incubated with living cells, we applied CLSM in combined scattering and transmission modes, and observed localization of the NSts on the cell surface. Due to the highest scattering at the CLSM operating wavelength, the strongest signal was obtained from the 82-nm particles; the lowest intensity of the CLSM backscattered signal was detected from the cells labeled with the smallest NSts. Thus, by tuning the initial concentration of seeds, it is possible to adjust the size (and scattering properties) of the nanostars to the operating wavelength of the optical device to achieve the best performance.

Stable transfection of the hypotrichous ciliate Stylonychia lemnae with tagged α1 tubulin minichromosomes

Summary Macronuclei of Stylonychia lemnae contain millions of gene sized minichromosomes. These minichromosomes in general harbour only one gene. Their nontranscribed regions are usually less than 400 bp long and most likely carry the signals for DNA replication and transcription. Therefore, S. lemnae seems to be an ideal object to study these processes in a protist organism. By microinjecting defined linear minichromosomal DNA directly into macronuclei of vegetative cells, we have developed a suitable in vivo assay system for S. lemnae. Transfections were carried out with α1 tubulin minichromosomes whose gene was internally tagged by a 19 bp-long insertion. Although the endogenous ocl tubulin minichromosome is present in approximately 100,000 copies per cell, clonal cell lines were generated which stably contained tagged minichromosomes in up to a fivefold excess of endogenous copy numbers. As demonstrated by Southern blot analysis, the copy number of endogenous α1 tubulin minichromosomes was not reduced in these cell lines, and transfected minichromosomes were not noticeably rearranged by the cells. Moreover, tagged ocl tubulin genes were unequivocally expressed. Transcription of these genes was initiated correctly, and the amount of transcript present in steady-state RNA correlated with gene copy number.

Plasmon-Resonant Gold Nanoparticles with Variable Morphology as Optical Labels and Drug Carriers for Cytological Research

In this work, two types of nanocomposites, silica-coated nano-sea-urchins and silica-coated gold nanostars, were fabricated. CTAB-coated nano-sea-urchins with an average size of about 100 nm demonstrate an absorption peak near 600-700 nm and stability in aqueous suspension. CTAB was exchanged with m-PEG-SH by an intermediate PEG layer. A layer of silica was synthesized on the nano-sea-urchins surface with thickness of about 20 nm. Nanostars with an average size of about 60 nm with a number of thin sharp branches were fabricated and functionalized by PVP to improve their stability. PVP-coated nanostars were used in optical coherence tomography experiments to show their contrasting properties. After silica-coating, stable and monodispersed nanoparticles with silica shell thickness about 60 nm were obtained. Nontoxicity of the silica-coated nanostars at least until the concentration of nanoparticles about 400 μg/mL was showed by fluorescent cell viability assay using propidium iodide. Extinction coefficient of the gold nanostars and nanocomposites was estimated by a spectrophotometer system in collimated transmission regime.