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 have 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 utilize CRISPR-Cas9-mediated genome editing to target either single alleles or multiple alloalleles in F0 X. laevis embryos. A single guide (sg) RNA 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\u27s 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

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

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

Transgenic <i>Xenopus laevis</i> 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

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

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

<div><p>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 <i>Schizosaccharomyces pombe</i> 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 <i>cis4</i> or <i>zrg17</i> leads to higher levels of zinc accumulating in the cytosol under conditions of zinc deficiency, whereas deletion of <i>zhf1</i> results in zinc accumulating in the cytosol when zinc is not limiting. We also show that the expression of <i>cis4</i>, <i>zrg17</i>, and <i>zhf1</i> 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.</p></div

FRET responses of wild-type cells expressing ZapCY1 or ZapCY2 during zinc shock.

<p><b>(A)</b> Wild-type cells expressing ZapCY1 were grown overnight in ZL-EMM. At t = 0 cells were exposed to 0, 1, 10, or 100 μM. Cells were harvested at the indicated time points and total cellular zinc measured using Atomic Absorption Spectroscopy. The final concentration of zinc/cell was calculated by comparing values to a standard curve. <b>(B and C)</b> Wild-type cells expressing ZapCY1 were grown overnight in ZL-EMM. Cells were transferred to temperature-controlled cuvettes and were assayed for FRET by spectrofluorometry. At t = 0 cells were shocked with 0–1000 μM Zn²⁺ and the changes in FRET monitored over time. The FRET ratio was determined by dividing the FRET emission at 535 nm by the eCFP emission at 475 nm following excitation of samples at 434 nm. Panel B shows a representative experiment and panel C shows the average values from 3 independent experiments with error bars representing standard deviations. <b>(D)</b> Wild-type cells expressing ZapCY1 were grown overnight in ZL-EMM. At t = 0 cells were shocked with 100 μM Zn²⁺. At the indicated time point cells were harvested and crude protein extracts prepped for immunoblot analysis. Immunoblots were incubated with antibodies to GFP and loading control Act1. <b>(E and F)</b> Wild-type cells expressing ZapCY2 were grown overnight in ZL-EMM. At t = 0 cells were shocked with 0–1000 μM Zn²⁺ and the FRET response measured as described in panel B. Panel E shows a representative experiment and panel F shows the average values from 3 independent experiments with error bars representing standard deviations. <b>(G)</b> Wild-type cells expressing ZapCY2 were grown overnight in ZL-EMM. At t = 0 cells were shocked with 10 μM Zn²⁺. Crude protein extracts were then prepped for immunoblot analysis as described in panel D.</p

<i>zrt1</i>Δ cells accumulate lower levels of zinc in the cytosol.

<p><b>(A)</b><i>zrt1</i>Δ cells expressing ZapCY1 were grown overnight in ZL-EMM. At t = 0 cells were exposed to 0–1000 μM. Total cellular zinc was then determined by AAS as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007262#pgen.1007262.g003" target="_blank">Fig 3A</a>. <b>(B-F)</b> <i>zrt1</i>Δ cells expressing ZapCY1 or ZapCY2 were grown overnight in ZL-EMM. Cells were transferred to temperature-controlled cuvettes and at t = 0 shocked with 0–1000 μM Zn²⁺. Changes in the FRET ratio were measured as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007262#pgen.1007262.g003" target="_blank">Fig 3B</a>. Panels B and D show representative experiments and panels C and E show the average values from 3 independent experiments with error bars representing standard deviations. Panel F shows a comparison of a 1 μM zinc shock in wild-type and <i>zrt1</i>Δ cells expressing ZapCY1 <b>(G-I)</b> Wild-type and <i>zrt1</i>Δ cells expressing ZapCY1 or ZapCY2 were grown overnight in ZL-EMM. At t = 0 cells were shocked with 100 μM Zn²⁺. Cells were harvested at the indicated time points and crude protein extracts prepped for immunoblot analysis. Immunoblots were incubated with antibodies to GFP and loading control Act1.</p

<i>zhf1</i>Δ cells accumulate higher levels of zinc in the cytosol following a zinc shock.

<p><b>(A and B)</b><i>zhf1</i>Δ cells expressing ZapCY1 were grown and subject to zinc shock as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007262#pgen.1007262.g003" target="_blank">Fig 3B</a>. Panel A shows a representative experiment and panel B shows the average values from 3 independent experiments with error bars representing standard deviations. <b>(C)</b> <i>zhf1</i>x expressing ZapCY1 were grown overnight in ZL-EMM. At t = 0 cells were shocked with 100μM Zn²⁺. Cells were harvested at the indicated time points and crude protein extracts prepped for immunoblot analysis. Immunoblots were incubated with antibodies to GFP and loading control Act1. <b>(D)</b> Wild-type (1), <i>zhf1</i>Δ (2), <i>cis4</i>Δ (3), and <i>zrg17</i>Δ (4) cells expressing ZapCY1 were grown overnight in ZL-EMM. Cells were harvested for immunoblot analysis following the overnight growth (t = 0) or after a zinc shock with 100 μM Zn²⁺ for 90 min (t = 90). Immunoblots were performed as described in panel C. <b>(E)</b> <i>zhf1</i>Δ ZapCY1cells were grown overnight in ZL-EMM. Cells were transferred to temperature-controlled cuvettes and were treated with +/- 50 μM NaPT for 20 min followed by the addition of 0–1000 μM Zn²⁺. Changes in the FRET ratio were determined as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007262#pgen.1007262.g003" target="_blank">Fig 3B</a>. <b>(F)</b> <i>zhf1</i>Δ cells expressing ZapCY2 were grown overnight in ZL-EMM. Cells were shocked with 100 μM Zn²⁺ and cell harvested for immunoblot analysis at the indicated time points. Immunoblots were performed as described in panel C. Results show the average values from 3 independent experiments with error bars representing standard deviations. <b>(G-I)</b> <i>zhf1</i>Δ cells expressing ZapCY2 were grown and subject to zinc shock as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007262#pgen.1007262.g003" target="_blank">Fig 3B</a>. Panel F shows a representative experiment and panel G shows the average values from 3 independent experiments with error bars representing standard deviations. Panel I shows a comparison of a 1 μM zinc shock in wild-type and <i>zhf1</i>Δ cells expressing ZapCY2.</p