Corkins ME et al. (2018), Transgenic Xenopus laevis Line for In Vivo Labe...

XB-ART-54809

Genes (Basel) 2018 Apr 06;94:. doi: 10.3390/genes9040197.

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

Corkins ME , Hanania HL , Krneta-Stankic V , DeLay BD , Pearl EJ , Lee M , Ji H , Davidson AJ , Horb ME , Miller RK .

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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.

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Species referenced: Xenopus laevis
Genes referenced: cdh17 daam1

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	Figure 1. Schematic of cdh17:eGFP transgenic line creation. (A) cdh17:eGFP construct flanked by I-SceI sites was co-injected with I-SceI meganuclease into one-cell embryos. (B) Eight female and 11 male Xenopus laevis adults were grown from these F0 embryos. (C) Upon mating of an F0 transgenic male to a wild type female, 14 F1 females were generated.
	Figure 2. cdh17:eGFP expression co-localizes with the kidney markers 3G8 and 4A6. Stage 42 cdh17:eGFP transgenic X. laevis embryos were co-immunostained with antibodies for green fluorescent protein (GFP) and the kidney markers 3G8 and 4A6. (A) GFP immunostained cdh17:eGFP embryo. (B) 3G8/4A6 immunostained embryo. (C) Merged differential interference contrast (DIC) and fluorescent image of cdh17:eGFP embryo. Embryo is surrounded in a bubble of 1:2 volume benzyl alcohol to benzyl benzoate (BABB).
	Figure 3. Subcellular GFP expression in the cdh17:eGFP line is largely cytoplasmic and nuclear. Fixed stage 42 cdh17:eGFP embryos were stained with (A,E) anti-GFP, (B,F) 3G8/4A6, and (C,G) DAPI (40 ,6-diamidino-2-phenylindole). (E–H) Magnified image of proximal tubules.
	Figure 4. cdh17:eGFP labels the kidney throughout pronephric development. Stage 32 through stage 44, cdh17:eGFP transgenic X. laevis embryos were immunostained with antibodies for GFP and the kidney markers 3G8 and 4A6. GFP expression is present throughout pronephric kidney development. (Left) GFP expression, (right) GFP (Green) and 3G8/4A6 (red) kidney marker expression.
	Figure 5. Live imaging of kidney development using cdh17:eGFP reporter line. Kidney development within a transgenic embryo was visualized by GFP expression from stages 30 to 44.
	Figure 6. cdh17:eGFP transgenic line labels the kidney for tracking of rhodamine secretion. Rhodamine-dextran was injected into the coelomic cavity of stage 42 X. laevis embryos and tracked using GFP as a marker for the kidney.
	Figure 7. X. laevis cdh17:eGFP transgenic line is useful for primary culture of kidney tubules. (A) Diagram showing region of embryo that was explanted. (B) Schematic of methodology used to culture cells. Media used for each step is as indicated DFA (Danilchik’s for Amy) and CMFM (Calcium Magnesium Free Media). (C) Image of GFP-positive cells cultured for 24 h in DFA. Cells are outlined using dashed lines.
	Figure 8. X. laevis adult mesonephric tubules express cdh17:eGFP. Adult kidneys were dissected, then imaged from an F1 generation cdh17:eGFP animal. (A) Brightfield image of X. laevis cdh17:eGFP kidney. (B,C) GFP fluorescence imaging of cdh17:eGFP kidney. Dashed boxes indicate regions taken for fluorescence imaging.
	Figure 9. Disruption of kidney development using Daam1 morpholino (MO) can be tracked by cdh17:eGFP. cdh17:eGFP transgenic embryos were injected with Standard or Daam1 morpholino, then stage 42 embryos were assayed for kidney abnormalities. (A) Representative images of kidneys under indicated conditions. (B) Percentage of abnormal kidneys under indicated conditions. (C) Daam1 morpholino was injected into both V2 blastomeres, resulting in the development of edema, which indicates kidney functional defects. Arrow indicates site of edema.

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