A comparative study of cellular diversity between the Xenopus pronephric and mouse metanephric nephron.

The kidney is an essential organ that ensures bodily fluid homeostasis and removes soluble waste products from the organism. Nephrons, the functional units of the kidney, comprise a blood filter, the glomerulus or glomus, and an epithelial tubule that processes the filtrate from the blood or coelom and selectively reabsorbs solutes, such as sugars, proteins, ions, and water, leaving waste products to be eliminated in the urine. Genes coding for transporters are segmentally expressed, enabling the nephron to sequentially process the filtrate. The Xenopus embryonic kidney, the pronephros, which consists of a single large nephron, has served as a valuable model to identify genes involved in nephron formation and patterning. Therefore, the developmental patterning program that generates these segments is of great interest. Prior work has defined the gene expression profiles of Xenopus nephron segments via in situ hybridization strategies, but a comprehensive understanding of the cellular makeup of the pronephric kidney remains incomplete. Here, we carried out single-cell mRNA sequencing of the functional Xenopus pronephric nephron and evaluated its cellular composition through comparative analyses with previous Xenopus studies and single-cell mRNA sequencing of the adult mouse kidney. This study reconstructs the cellular makeup of the pronephric kidney and identifies conserved cells, segments, and associated gene expression profiles. Thus, our data highlight significant conservation in podocytes, proximal and distal tubule cells, and divergence in cellular composition underlying the capacity of each nephron to remove wastes in the form of urine, while emphasizing the Xenopus pronephros as a model for physiology and disease.

Tissue-Targeted CRISPR-Cas9-Mediated Genome Editing of Multiple Homeologs in F0-Generation Xenopus laevis Embryos.

Xenopus laevis frogs are a powerful developmental model that enables studies combining classical embryology and molecular manipulation. Because of the large embryo size, ease of microinjection, and ability to target tissues through established fate maps, X. laevis has become the predominant amphibian research model. Given that their allotetraploid genome has complicated the generation of gene knockouts, strategies need to be established for efficient mutagenesis of multiple homeologs to evaluate gene function. Here we describe a protocol to use CRISPR-Cas9-mediated genome editing to target either single alleles or multiple alloalleles in F0 X. laevis embryos. A single-guide RNA (sgRNA) is designed to target a specific DNA sequence encoding a critical protein domain. To mutagenize a gene with two alloalleles, the sgRNA is designed against a sequence that is common to both homeologs. This sgRNA, along with the Cas9 protein, is microinjected into the zygote to disrupt the genomic sequences in the whole embryo or into a specific blastomere for tissue-targeted effects. Error-prone repair of CRISPR-Cas9-generated DNA double-strand breaks leads to insertions and deletions creating mosaic gene lesions within the embryos. The genomic DNA isolated from each mosaic F0 embryo is sequenced, and software is applied to assess the nature of the mutations generated and degree of mosaicism. This protocol enables the knockout of genes within the whole embryo or in specific tissues in F0 X. laevis embryos to facilitate the evaluation of resulting phenotypes.

The Wnt/PCP formin Daam1 drives cell-cell adhesion during nephron development.

E-cadherin junctions facilitate assembly and disassembly of cell contacts that drive development and homeostasis of epithelial tissues. In this study, using Xenopus embryonic kidney and Madin-Darby canine kidney (MDCK) cells, we investigate the role of the Wnt/planar cell polarity (PCP) formin Daam1 (Dishevelled-associated activator of morphogenesis 1) in regulating E-cadherin-based intercellular adhesion. Using live imaging, we show that Daam1 localizes to newly formed cell contacts in the developing nephron. Furthermore, analyses of junctional filamentous actin (F-actin) upon Daam1 depletion indicate decreased microfilament localization and slowed turnover. We also show that Daam1 is necessary for efficient and timely localization of junctional E-cadherin, mediated by Daam1's formin homology domain 2 (FH2). Finally, we establish that Daam1 signaling promotes organized movement of renal cells. This study demonstrates that Daam1 formin junctional activity is critical for epithelial tissue organization.

Aquatic models of human ciliary diseases.

Cilia are microtubule-based structures that either transmit information into the cell or move fluid outside of the cell. There are many human diseases that arise from malfunctioning cilia. Although mammalian models provide vital insights into the underlying pathology of these diseases, aquatic organisms such as Xenopus and zebrafish provide valuable tools to help screen and dissect out the underlying causes of these diseases. In this review we focus on recent studies that identify or describe different types of human ciliopathies and outline how aquatic organisms have aided our understanding of these diseases.

Identification of Multicolor Fluorescent Probes for Heterogeneous Aß Deposits in Alzheimer's Disease.

Accumulation of amyloid-beta (Aß) into amyloid plaques and hyperphosphorylated tau into neurofibrillary tangles (NFTs) are pathological hallmarks of Alzheimer's disease (AD). There is a significant intra- and inter-individual variability in the morphology and conformation of Aß aggregates, which may account in part for the extensive clinical and pathophysiological heterogeneity observed in AD. In this study, we sought to identify an array of fluorescent dyes to specifically probe Aß aggregates, in an effort to address their diversity. We screened a small library of fluorescent probes and identified three benzothiazole-coumarin derivatives that stained both vascular and parenchymal Aß deposits in AD brain sections. The set of these three dyes allowed the visualization of Aß deposits in three different colors (blue, green and far-red). Importantly, two of these dyes specifically stained Aß deposits with no apparent staining of hyperphosphorylated tau or α-synuclein deposits. Furthermore, this set of dyes demonstrated differential interactions with distinct types of Aß deposits present in the same subject. Aß aggregate-specific dyes identified in this study have the potential to be further developed into Aß imaging probes for the diagnosis of AD. In addition, the far-red dye we identified in this study may serve as an imaging probe for small animal imaging of Aß pathology. Finally, these dyes in combination may help us advance our understanding of the relation between the various Aß deposits and the clinical diversity observed in AD.

Correction: DYRK1A-related intellectual disability: a syndrome associated with congenital anomalies of the kidney and urinary tract.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Divergent roles of the Wnt/PCP Formin Daam1 in renal ciliogenesis.

Kidneys are composed of numerous ciliated epithelial tubules called nephrons. Each nephron functions to reabsorb nutrients and concentrate waste products into urine. Defects in primary cilia are associated with abnormal formation of nephrons and cyst formation in a wide range of kidney disorders. Previous work in Xenopus laevis and zebrafish embryos established that loss of components that make up the Wnt/PCP pathway, Daam1 and ArhGEF19 (wGEF) perturb kidney tubulogenesis. Dishevelled, which activates both the canonical and non-canonical Wnt/PCP pathway, affect cilia formation in multiciliated cells. In this study, we investigated the role of the noncanoncial Wnt/PCP components Daam1 and ArhGEF19 (wGEF) in renal ciliogenesis utilizing polarized mammalian kidney epithelia cells (MDCKII and IMCD3) and Xenopus laevis embryonic kidney. We demonstrate that knockdown of Daam1 and ArhGEF19 in MDCKII and IMCD3 cells leads to loss of cilia, and Daam1's effect on ciliogenesis is mediated by the formin-activity of Daam1. Moreover, Daam1 co-localizes with the ciliary transport protein Ift88 and is present in cilia. Interestingly, knocking down Daam1 in Xenopus kidney does not lead to loss of cilia. These data suggests a new role for Daam1 in the formation of primary cilia.

DYRK1A-related intellectual disability: a syndrome associated with congenital anomalies of the kidney and urinary tract.

PURPOSE: Haploinsufficiency of DYRK1A causes a recognizable clinical syndrome. The goal of this paper is to investigate congenital anomalies of the kidney and urinary tract (CAKUT) and genital defects (GD) in patients with DYRK1A variants. METHODS: A large database of clinical exome sequencing (ES) was queried for de novo DYRK1A variants and CAKUT/GD phenotypes were characterized. Xenopus laevis (frog) was chosen as a model organism to assess Dyrk1a's role in renal development. RESULTS: Phenotypic details and variants of 19 patients were compiled after an initial observation that one patient with a de novo pathogenic variant in DYRK1A had GD. CAKUT/GD data were available from 15 patients, 11 of whom presented with CAKUT/GD. Studies in Xenopus embryos demonstrated that knockdown of Dyrk1a, which is expressed in forming nephrons, disrupts the development of segments of embryonic nephrons, which ultimately give rise to the entire genitourinary (GU) tract. These defects could be rescued by coinjecting wild-type human DYRK1A RNA, but not with DYRK1AR205* or DYRK1AL245R RNA. CONCLUSION: Evidence supports routine GU screening of all individuals with de novo DYRK1A pathogenic variants to ensure optimized clinical management. Collectively, the reported clinical data and loss-of-function studies in Xenopus substantiate a novel role for DYRK1A in GU development.

Transgenic Xenopus laevis Line for In Vivo Labeling of Nephrons within the Kidney.

Xenopus laevis embryos are an established model for studying kidney development. The nephron structure and genetic pathways that regulate nephrogenesis are conserved between Xenopus and humans, allowing for the study of human disease-causing genes. Xenopus embryos are also amenable to large-scale screening, but studies of kidney disease-related genes have been impeded because assessment of kidney development has largely been limited to examining fixed embryos. To overcome this problem, we have generated a transgenic line that labels the kidney. We characterize this cdh17:eGFP line, showing green fluorescent protein (GFP) expression in the pronephric and mesonephric kidneys and colocalization with known kidney markers. We also demonstrate the feasibility of live imaging of embryonic kidney development and the use of cdh17:eGFP as a kidney marker for secretion assays. Additionally, we develop a new methodology to isolate and identify kidney cells for primary culture. We also use morpholino knockdown of essential kidney development genes to establish that GFP expression enables observation of phenotypes, previously only described in fixed embryos. Taken together, this transgenic line will enable primary kidney cell culture and live imaging of pronephric and mesonephric kidney development. It will also provide a simple means for high-throughput screening of putative human kidney disease-causing genes.

Zinc transporters belonging to the Cation Diffusion Facilitator (CDF) family have complementary roles in transporting zinc out of the cytosol.

Zinc is an essential trace element that is required for the function of a large number of proteins. As these zinc-binding proteins are found within the cytosol and organelles, all eukaryotes require mechanisms to ensure that zinc is delivered to organelles, even under conditions of zinc deficiency. Although many zinc transporters belonging to the Cation Diffusion Facilitator (CDF) families have well characterized roles in transporting zinc into the lumens of intracellular compartments, relatively little is known about the mechanisms that maintain organelle zinc homeostasis. The fission yeast Schizosaccharomyces pombe is a useful model system to study organelle zinc homeostasis as it expresses three CDF family members that transport zinc out of the cytosol into intracellular compartments: Zhf1, Cis4, and Zrg17. Zhf1 transports zinc into the endoplasmic reticulum, and Cis4 and Zrg17 form a heterodimeric complex that transports zinc into the cis-Golgi. Here we have used the high and low affinity ZapCY zinc-responsive FRET sensors to examine cytosolic zinc levels in yeast mutants that lack each of these CDF proteins. We find that deletion of cis4 or zrg17 leads to higher levels of zinc accumulating in the cytosol under conditions of zinc deficiency, whereas deletion of zhf1 results in zinc accumulating in the cytosol when zinc is not limiting. We also show that the expression of cis4, zrg17, and zhf1 is independent of cellular zinc status. Taken together our results suggest that the Cis4/Zrg17 complex is necessary for zinc transport out of the cytosol under conditions of zinc-deficiency, while Zhf1 plays the dominant role in removing zinc from the cytosol when labile zinc is present. We propose that the properties and/or activities of individual CDF family members are fine-tuned to enable cells to control the flux of zinc out of the cytosol over a broad range of environmental zinc stress.

Tissue-Specific Gene Inactivation in Xenopus laevis: Knockout of lhx1 in the Kidney with CRISPR/Cas9.

Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, Xenopus laevis, is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in Xenopus The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the Xenopus kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of lhx1 results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in Xenopus by editing a gene (slc45a2) that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in Xenopus, providing a cost-effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques.

The gluconate shunt is an alternative route for directing glucose into the pentose phosphate pathway in fission yeast.

Glycolysis and the pentose phosphate pathway both play a central role in the degradation of glucose in all domains of life. Another metabolic route that can facilitate glucose breakdown is the gluconate shunt. In this shunt glucose dehydrogenase and gluconate kinase catalyze the two-step conversion of glucose into the pentose phosphate pathway intermediate 6-phosphogluconate. Despite the presence of these enzymes in many organisms, their only established role is in the production of 6-phosphogluconate for the Entner-Doudoroff pathway. In this report we performed metabolic profiling on a strain of Schizosaccharomyces pombe lacking the zinc-responsive transcriptional repressor Loz1 with the goal of identifying metabolic pathways that were altered by cellular zinc status. This profiling revealed that loz1Δ cells accumulate higher levels of gluconate. We show that the altered gluconate levels in loz1Δ cells result from increased expression of gcd1 By analyzing the activity of recombinant Gcd1 in vitro and by measuring gluconate levels in strains lacking enzymes of the gluconate shunt we demonstrate that Gcd1 encodes a novel NADP+-dependent glucose dehydrogenase that acts in a pathway with the Idn1 gluconate kinase. We also find that cells lacking gcd1 and zwf1, which encode the first enzyme in the pentose phosphate pathway, have a more severe growth phenotype than cells lacking zwf1 We propose that in S. pombe Gcd1 and Idn1 act together to shunt glucose into the pentose phosphate pathway, creating an alternative route for directing glucose into the pentose phosphate pathway that bypasses hexokinase and the rate-limiting enzyme glucose-6-phosphate dehydrogenase.

C. elegans lifespan extension by osmotic stress requires FUdR, base excision repair, FOXO, and sirtuins.

Moderate stress can increase lifespan by hormesis, a beneficial low-level induction of stress response pathways. 5'-fluorodeoxyuridine (FUdR) is commonly used to sterilize Caenorhabditis elegans in aging experiments. However, FUdR alters lifespan in some genotypes and induces resistance to thermal and proteotoxic stress. We report that hypertonic stress in combination with FUdR treatment or inhibition of the FUdR target thymidylate synthase, TYMS-1, extends C. elegans lifespan by up to 30%. By contrast, in the absence of FUdR, hypertonic stress decreases lifespan. Adaptation to hypertonic stress requires diminished Notch signaling and loss of Notch co-ligands leads to lifespan extension only in combination with FUdR. Either FUdR treatment or TYMS-1 loss induced resistance to acute hypertonic stress, anoxia, and thermal stress. FUdR treatment increased expression of DAF-16 FOXO and the osmolyte biosynthesis enzyme GPDH-1. FUdR-induced hypertonic stress resistance was partially dependent on sirtuins and base excision repair (BER) pathways, while FUdR-induced lifespan extension under hypertonic stress conditions requires DAF-16, BER, and sirtuin function. Combined, these results demonstrate that FUdR, through inhibition of TYMS-1, activates stress response pathways in somatic tissues to confer hormetic resistance to acute and chronic stress. C. elegans lifespan studies using FUdR may need re-interpretation in light of this work.

The double zinc finger domain and adjacent accessory domain from the transcription factor loss of zinc sensing 1 (loz1) are necessary for DNA binding and zinc sensing.

The Loz1 transcription factor from Schizosaccharomyces pombe plays an essential role in zinc homeostasis by repressing target gene expression in zinc-replete cells. To determine how Loz1 function is regulated by zinc, we employed a genetic screen to isolate mutants with impaired zinc-dependent gene expression and analyzed Loz1 protein truncations to map a minimal zinc-responsive domain. In the screen, we isolated 36 new loz1 alleles. 27 of these alleles contained mutations resulting in the truncation of the Loz1 protein. The remaining nine alleles contained point mutations leading to an amino acid substitution within a C-terminal double zinc finger domain. Further analysis of two of these substitutions revealed that they disrupted Loz1 DNA activity in vitro. By analyzing Loz1 protein truncations, we found that the last 96 amino acids of Loz1 was the smallest region that was able to confer partial zinc-dependent repression in vivo. This 96-amino acid region contains the double zinc finger domain and an accessory domain that enhances DNA binding. These results were further supported by the findings that MtfA, a transcription factor from Aspergillus nidulans that contains a related double zinc finger, is unable to complement loz1Δ, whereas a chimera of MtfA containing the Loz1 accessory domain is able to complement loz1Δ. Together, our studies indicate that the double zinc finger domain and adjacent accessory domain preceding zinc finger 1 are necessary for DNA binding and zinc-dependent repression.

Zinc finger protein Loz1 is required for zinc-responsive regulation of gene expression in fission yeast.

In Schizosaccharomyces pombe, alcohol dehydrogenase 1 (Adh1) is an abundant zinc-requiring enzyme that catalyses the conversion of acetaldehyde to ethanol during fermentation. In a zinc-replete cell, adh1 is highly expressed. However, in zinc-limited cells, adh1 gene expression is repressed, and cells induce the expression of an alternative alcohol dehydrogenase encoded by the adh4 gene. In our studies examining this zinc-dependent switch in alcohol dehydrogenase gene expression, we isolated an adh1Δ strain containing a partial loss of function mutation that resulted in higher levels of adh4 transcripts in zinc-replete cells. This mutation also led to the aberrant expression of other genes that are typically regulated by zinc. Using linkage analysis, we have mapped the position of this mutation to a single gene called Loss Of Zinc sensing 1 (loz1). Loz1 is a 55-kDa protein that contains a double C2H2-type zinc finger domain. The mapped mutation that disrupts Loz1 function leads to an arginine to glycine substitution in the second zinc finger domain, suggesting that the double zinc finger domain is important for Loz1 function. We show that loz1Δ cells hyperaccumulate zinc and that Loz1 is required for gene repression in zinc-replete cells. We also have found that Loz1 negatively autoregulates its own expression. We propose that Loz1 is a unique metalloregulatory factor that plays a central role in zinc homeostasis in S. pombe.

Zinc-dependent regulation of the Adh1 antisense transcript in fission yeast.

In yeast, Adh1 (alcohol dehydrogenase 1) is an abundant zinc-binding protein that is required for the conversion of acetaldehyde to ethanol. Through transcriptome profiling of the Schizosaccharomyces pombe genome, we identified a natural antisense transcript at the adh1 locus that is induced in response to zinc limitation. This antisense transcript (adh1AS) shows a reciprocal expression pattern to that of the adh1 mRNA partner. In this study, we show that increased expression of the adh1AS transcript in zinc-limited cells is necessary for the repression of adh1 gene expression and that the increased level of the adh1AS transcript in zinc-limited cells is a result of two mechanisms. At the transcriptional level, the adh1AS transcript is expressed at a high level in zinc-limited cells. In addition to this transcriptional control, adh1AS transcripts preferentially accumulate in zinc-limited cells when the adh1AS transcript is expressed from a constitutive promoter. This secondary mechanism requires the simultaneous expression of adh1. Our studies reveal how multiple mechanisms can synergistically control the ratio of sense to antisense transcripts and highlight a novel mechanism by which adh1 gene expression can be controlled by cellular zinc availability.

C. elegans Notch signaling regulates adult chemosensory response and larval molting quiescence.

BACKGROUND: The conserved DOS-motif proteins OSM-7 and OSM-11 function as coligands with canonical DSL (Delta, Serrate, and LAG-2) ligands to activate C. elegans Notch receptors during development. We report here that Notch ligands, coligands, and the receptors LIN-12 and GLP-1 regulate two C. elegans behaviors: chemosensory avoidance of octanol and quiescence during molting lethargus. RESULTS: C. elegans lacking osm-7 or osm-11 are defective in their response to octanol. We find that OSM-11 is secreted from hypodermal seam cells into the pseudocoelomic body cavity and acts non-cell autonomously as a diffusible factor. OSM-11 acts with the DSL ligand LAG-2 to activate LIN-12 and GLP-1 Notch receptors in the neurons of adult animals, thereby regulating octanol avoidance response. In adult animals, overexpression of osm-11 and consequent Notch receptor activation induces anachronistic sleep-like quiescence. Perturbation of Notch signaling alters basal activity in adults as well as arousal thresholds and quiescence during molting lethargus. Genetic epistasis studies reveal that Notch signaling regulates quiescence via previously identified circuits and genetic pathways including the egl-4 cGMP-dependent kinase. CONCLUSIONS: Our findings indicate that the conserved Notch pathway modulates behavior in adult C. elegans in response to environmental stress. Additionally, Notch signaling regulates sleep-like quiescence in C. elegans, suggesting that Notch may regulate sleep in other species.

OSM-11 facilitates LIN-12 Notch signaling during Caenorhabditis elegans vulval development.

Notch signaling is critical for cell fate decisions during development. Caenorhabditis elegans and vertebrate Notch ligands are more diverse than classical Drosophila Notch ligands, suggesting possible functional complexities. Here, we describe a developmental role in Notch signaling for OSM-11, which has been previously implicated in defecation and osmotic resistance in C. elegans. We find that complete loss of OSM-11 causes defects in vulval precursor cell (VPC) fate specification during vulval development consistent with decreased Notch signaling. OSM-11 is a secreted, diffusible protein that, like previously described C. elegans Delta, Serrate, and LAG-2 (DSL) ligands, can interact with the lineage defective-12 (LIN-12) Notch receptor extracellular domain. Additionally, OSM-11 and similar C. elegans proteins share a common motif with Notch ligands from other species in a sequence defined here as the Delta and OSM-11 (DOS) motif. osm-11 loss-of-function defects in vulval development are exacerbated by loss of other DOS-motif genes or by loss of the Notch ligand DSL-1, suggesting that DOS-motif and DSL proteins act together to activate Notch signaling in vivo. The mammalian DOS-motif protein Deltalike1 (DLK1) can substitute for OSM-11 in C. elegans development, suggesting that DOS-motif function is conserved across species. We hypothesize that C. elegans OSM-11 and homologous proteins act as coactivators for Notch receptors, allowing precise regulation of Notch receptor signaling in developmental programs in both vertebrates and invertebrates.