The embryonic kidney of Xenopus laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 – 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

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.

Somites give rise to the vertebral column and segmented musculature of adult vertebrates. The cell movements that position cells within somites along the anteroposterior and dorsoventral axes are not well understood. Using a fate mapping approach, we show that at the onset of Xenopus laevis gastrulation, mesoderm cells undergo distinct cell movements to form myotome fibers positioned in discrete locations within somites and along the anteroposterior axis. We show that the distribution of presomitic cells along the anteroposterior axis is influenced by convergent and extension movements of the notochord. Heterochronic and heterotopic transplantations between presomitic gastrula and early tailbud stages show that these cells are interchangeable and can form myotome fibers in locations determined by the host embryo. However, additional transplantation experiments revealed differences in the competency of presomitic cells to form myotome fibers, suggesting that maturation within the tailbud presomitic mesoderm is required for myotome fiber differentiation.

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.

While kidney donations stagnate, the number of people in need of kidney transplants continues to grow. Although transplanting culture-grown organs is years away, pursuing the engineering of the kidney de novo is a valid means of closing the gap between the supply and demand of kidneys for transplantation. The structural organization of a mouse kidney is similar to that of humans. Therefore, mice have traditionally served as the primary model system for the study of kidney development. The mouse is an ideal model organism for understanding the complexity of the human kidney. Nonetheless, the elaborate structure of the mammalian kidney makes the discovery of new therapies based on de novo engineered kidneys more challenging. In contrast to mammals, amphibians have a kidney that is anatomically less complex and develops faster. Given that analogous genetic networks regulate the development of mammalian and amphibian nephric organs, using embryonic kidneys of Xenopus laevis (African clawed frog) to analyze inductive cell signaling events and morphogenesis has many advantages. Pioneering work that led to the ability to generate kidney organoids from embryonic cells was carried out in Xenopus. In this review, we discuss how Xenopus can be utilized to compliment the work performed in mammalian systems to understand kidney development.

Highlights d Daam1 localizes to cell-cell contacts in developing nephron d Daam1 regulates the cytoskeletal membrane dynamics d Daam1 promotes E-cadherin localization d Formin homology domain 2 (FH2) of Daam1 mediates Ecadherin localization

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An image of an X. laevis tadpole showing rhodamine‐labeled transplanted presomitic mesoderm cells (red) in which a subset of cells have differentiated into myotome fibers (shown in orange). The differentiated myotome fibers are stained with the antibody 12/101 (green) and the notochord is stained with Tor 70 (blue). The image was digitally duplicated twice and fused at the anterior end. From Krneta‐Stankic et al., Developmental Dynamics 239:1162–1177, 2010.