Technique to Target Microinjection to the Developing <em>Xenopus</em> Kidney

Abstract

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.

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

“…11 Microinjections were targeted to the V2 blastomere at the eight-cell stage, which provides major contributions to the development of the pronephros. [12][13][14] Ten nL of injection mix (described below) was injected into embryos. Ten ng of Dyrk1a morpholino 5'-TGCATCGTCC-TCTTTCAAGTCTCAT-3′ 15 or Standard morpholino 5′-CCTCTTACCTCAGTTACAATTTATA-3′ was co-injected with 50 pg RNA (either control βgalactosidase, human DYRK1A, or patient mutant DYRK1A R205* ) along with 1 ng membrane-RFP RNA 16 as a lineage tracer to verify that the correct blastomere was injected.…”

“…Xenopus produces large clutch sizes with hundreds of embryos that develop externally, and unilateral embryo injections allow for tissue-targeted knockdowns that are specific to organs such as the kidney. 12 Their embryonic kidney, the pronephros, can easily be visualized and imaged through a transparent epidermis, and they develop a fully functional kidney in ~56 hours. 23 Xenopus was chosen because it is an established model of nephron development, and gene expression studies demonstrate that the developing Xenopus nephron is anatomically and functionally similar to the mammalian nephron.…”

Dynamin binding protein is required forXenopus laeviskidney development

“…Cas9 protein (CP01; PNA Bio) and sgRNA were incubated together at room temperature for at least 5 min prior to microinjection . MOs, RNA constructs and Cas9/sgRNAs were co-injected with either membrane-RFP RNA, Alexa Fluor 488 fluorescent dextran or rhodamine dextran as a tracer (Davidson et al, 2006;DeLay et al, 2018;DeLay et al, 2016).…”

“…It is also possible to easily modulate gene expression in Xenopus embryos through overexpression, knockdown and knockout experiments via microinjection of RNA constructs, antisense morpholino oligonucleotides (MOs) and CRISPR constructs DeLay et al, 2018;Miller et al, 2011). The established cell fate maps of the early Xenopus embryo facilitate tissue-targeted modulation of gene expression by microinjection into the appropriate blastomere DeLay et al, 2016;Moody, 1987a;Moody, 1987b). Taken together, Xenopus is a powerful model for studying essential nephrogenesis genes.…”

Xenopus: leaping forward in kidney organogenesis

“…The previously characterized Xenopus cell-fate map allows for blastomere-specific microinjection and tissue-targeted disruption of genes [1–4]. Microinjection techniques can be used in combination with fluorescent tracers (chemical dyes and fluorescent constructs) as a method for generating a more detailed fate map and identifying stem and tissue progenitor cells.…”