Slater PG et al. (2019), Characterization of Xenopus laevis guanine deam...

XB-ART-55652

Dev Dyn 2019 Apr 01;2484:296-305. doi: 10.1002/dvdy.14.

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Characterization of Xenopus laevis guanine deaminase reveals new insights for its expression and function in the embryonic kidney.

Slater PG , Cammarata GM , Monahan C , Bowers JT , Yan O , Lee S , Lowery LA .

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BACKGROUND: The mammalian guanine deaminase (GDA), called cypin, is important for proper neural development, by regulating dendritic arborization through modulation of microtubule (MT) dynamics. Additionally, cypin can promote MT assembly in vitro. However, it has never been tested whether cypin (or other GDA orthologs) binds to MTs or modulates MT dynamics. Here, we address these questions and characterize Xenopus laevis GDA (Gda) for the first time during embryonic development. RESULTS: We find that exogenously expressed human cypin and Gda both display a cytosolic distribution in primary embryonic cells. Furthermore, while expression of human cypin can promote MT polymerization, Xenopus Gda has no effect. Additionally, we find that the tubulin-binding collapsin response mediator protein (CRMP) homology domain is only partially conserved between cypin and Gda. This likely explains the divergence in function, as we discovered that the cypin region containing the CRMP homology and PDZ-binding domain is necessary for regulating MT dynamics. Finally, we observed that gda is strongly expressed in the kidneys during late embryonic development, although it does not appear to be critical for kidney development. CONCLUSIONS: Together, these results suggest that GDA has diverged in function between mammals and amphibians, and that mammalian GDA plays an indirect role in regulating MT dynamics. Developmental Dynamics 248:296-305, 2019. © 2019 Wiley Periodicals, Inc.

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Species referenced: Xenopus laevis
Genes referenced: gda odc1 slc5a9
GO keywords: microtubule [+]
???displayArticle.antibodies??? Kidney Ab1 Kidney Ab2
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	Figure 1. The CRMP homology domain is not conserved between human and XenopusGDA. Sequence alignment of X. laevishomeologs; X.tropicalis; Danio rerio; Gallus gallus; Homo sapiens; Rattus norvegicus; and Mus musculus GDA. The GDA N-terminal, including theZn2+ binding domain (yellow square), is well conserved among all species, while the CRMP homology (red square) and the PDZ-binding domains (green square) are less conserved.
	Figure 2. GDAs do not localize to the MT plus-end and only cypin promotes MT polymerization. A-B. Maximum intensity montage of 31 frames from a one-minute time-lapse image series, of Gda and cypin (green), and mKate2-MACF43 tracks (magenta), in cultured neuronal growth cones (A) and neural-derived embryonic mesenchymal cells (B), obtained from Xenopus embryos at stage 20-24. Scale bars 8 Î¼m (A) and 5 Î¼m (B). C. Schematic representation of the GFP-tagged constructs used, showing the critical domains. D-F.Quantification of mean values of MT growth velocity (D), growth lifetime (E) and growth length (F) in neuronal growth cones upon GFP, Gda, cypin and cypin chimera over-expression. (See Supplementary Movie 1 for a representative movie of mKate2-MACF43.) Bars on dot plots show mean and SEM. P < 0.01; * P < 0.001; **** P < 0.0001; n.s., not significant. A total of 138 control; 107 Gda; 154 cypin and 83 cypin chimera growth cones, from three independent experiments, were analyzed
	Figure 3. Xenopus GDA is expressed in the developing kidney.A. RT-PCR showing gdaexpression in the different developmental stages: 2 cell, blastula, neurula, 3 dpf, 6 dpf, and adult. ODC1 was used as an internal RT-PCR reference. B. Whole-mount in situ hybridization using an antisense probe specific to gda in different developmental stages. Scale bar: 0.5 mmC. Whole-mount in situ hybridization using an antisense probe specific to GDA or the kidney marker xSGLT-1K in 4.5 dpf embryos. Both gda and xSGLT-1K can be seen in a similar embryonic location. Scale bar: 0.5 mm and 0.25 mm for magnifications.
	Figure 4. Xenopus GDA knockdown results in edema formation but kidney morphology appears to be normal.A. Schematic representation of the MO effect on gdaRNA splicing (above) RT-PCR showing gdaknockdown (KD) when injecting increasing concentrations of MO (below). B. Representative images at 4 dpf control and Gda KD tadpoles. Scale bar: 1 mm.C. Schematic representation of a tadpole showing the measurements for edema identification. D.Quantification of the ratio: length face-to-chest (a) over length of cement gland-to-eye (b). E.Quantification of the percentage of tadpoles showing edema. F. Representative images of immunofluorescence using the kidney marker3G8 in addition to 4A6 at 4 dpf control and gdaKD tadpoles. F. Quantification of the ratio: average tubule diameter of the gdaMO-injected side over that of the uninjected side of the same tadpole. Bars on dot plots show mean and SEM. ns: not significant. **** P < 0.0001.

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Akhmanova, Tracking the ends: a dynamic protein network controls the fate of microtubule tips. 2008, Pubmed