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PLoS One
2017 Jul 10;127:e0180465. doi: 10.1371/journal.pone.0180465.
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A functional approach to understanding the role of NCKX5 in Xenopus pigmentation.
Williams RM
,
Winkfein RJ
,
Ginger RS
,
Green MR
,
Schnetkamp PP
,
Wheeler GN
.
???displayArticle.abstract??? NCKX5 is an ion exchanger expressed mostly in pigment cells; however, the functional role for this protein in melanogenesis is not clear. A variant allele of SLC24A5, the gene encoding NCKX5, has been shown to correlate with lighter skin pigmentation in humans, indicating a key role for SLC24A5 in determining human skin colour. SLC24A5 expression has been found to be elevated in melanoma. Knockdown analyses have shown SLC24A5 to be important for pigmentation, but to date the function of this ion exchanger in melanogenesis has not been fully established. Our data suggest NCKX5 may have an alternative activity that is key to its role in the regulation of pigmentation. Here Xenopus laevis is employed as an in vivo model system to further investigate the function of NCKX5 in pigmentation. SLC24A5 is expressed in the melanophores as they differentiate from the neural crest and develop in the RPE of the eye. Morpholino knockdown and rescue experiments were designed to elucidate key residues and regions of the NCKX5 protein. Unilateral morpholino injection at the 2 cell stage resulted in a reduction of pigmentation in the eye and epidermis of one lateral side of the tadpole. Xenopus and human SLC24A5 can rescue the morpholino effects. Further rescue experiments including the use of ion exchange inactive SLC24A5 constructs raise the possibility that full ion exchanger function of NCKX5 may not be required for rescue of pigmentation.
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Fig 1. Expression of SLC24A5 in X.laevis.
(A-K) Whole mount in situ hybridisation, (L) RT-PCR. (A-D) Expression of SLC24A5. (A) Expression initiates in the neural crest (arrow) and RPE at stage 25. (B) Expression continues in the melanophores as they mature from the neural crest as indicated by arrows. (C) Expression continues in the melanophores in the lateral pigmentation (LP) and increases in the RPE. (D) By stage 38 strong expression can be seen in melanophores of the lateral pigmentation, tail pigmentation (TP) and RPE. (E-H) Expression of the melanophore marker DCT in X.laevis. (E) Expression in the arising neural crest is indicated by arrow, (F) DCT is expressed in the melanophores as they mature from the neural crest as indicated by arrows. (G) and (H) Expression is maintained as melanophores migrate into lateral and tail pigmentation, expression continues in the RPE of the eye as indicated. (I-K) Histological analysis of SLC24A5 expression. (I) Section through the head reveals restricted expression to the RPE and dorsal head. (J) Section through the trunk shows expression in the flanks of the tadpoletrunk and head. (K) whole embryo before sectioning, red line indicate approximate area where above sections were taken. (L) RT PCR analysis of SLC24A5 from various stages of X.laevis development. DCT was used a melanophores marker comparison, H4, loading control. Cells used were X.laevis melanophores (kind gift from Vladimir Gelfand).
https://doi.org/10.1371/journal.pone.0180465.g001
Fig 2. Morpholino knockdown of SLC24A5.
(A) Scale of morpholino effect, a, ai, aii, scale 0 no effect; b, bi, bii, scale 1 subtle effect; c, ci, cii, scale 2 clear effect; d, di, dii, scale 3 obvious effect. a-d non injected control sides, ai-di injected sides showing effect, aii-dii dorsal view of embryos in a-d and ai-di highlighting relative effects in the eye. (B) Cryosectioning of morpholino treated embryos, a injected side; b non injected side; c and d sections of embryo in a and b, relative locations of sections are shown on a. (C) Dose response of morpholino, CMO control morpholino.
https://doi.org/10.1371/journal.pone.0180465.g002
Fig 3. Expression of DCT following morpholino knockdown of SLC24A5.
(A) Non injected side. (B) 100ng morpholino injected side. (C) Embryo in (A) following DCT in situ. (D) Embryo in (B) following DCT in situ. (E,F) Non injected controls following DCT in situ, n = 19.
https://doi.org/10.1371/journal.pone.0180465.g003
Fig 4. Rescuing the morpholino phenotype.
Embryos were injected with 100ng morpholino and 5ng cRNA of the various constructs. Embryos were allowed to develop to stage 38. The experiments were repeated in triplicate and the results were combined, n values shown at top of graph. (A) schematic of rescue constructs. (B) Embryos scored for pigmentation effect as scale in Fig 2. NI non injected, WT wild type, AT A111T, DN D383N, XL X.laevis, mo morpholino, CMO control morpholino. Kruskal Wallis test revealed significant difference between 100ng ATG MO and the WT, AT, DN and XL constructs, p = 0.001 for all, but not 4C p = 0.988.
https://doi.org/10.1371/journal.pone.0180465.g004
Fig 5. Rescuing with Xenopus mutants.
Embryos were injected with 100ng morpholino and 5ng cRNA of the mutant constructs. Embryos were allowed to develop to stage 38. The experiments were repeated in triplicate and the results were combined, n values shown at top of graph.
https://doi.org/10.1371/journal.pone.0180465.g005
Fig 6. Rescuing the morpholino with chimeric constructs.
5ng RNA of the constructs was co-injected with 100ng morpholino, the embryos were raised to stage 38. The experiments were repeated in triplicate and the results were combined, n values shown at top of graph. (A-D) shows schematics of chimera rescue contructs. (E) Embryos were scored according to the scale in Fig 2. Kruskal Wallis revealed a significant difference between 100ng ATG MO alone and the NCKX5 and NCKX5 ntl2 constructs, p = 0.01 and p = 0.022 respectively.
https://doi.org/10.1371/journal.pone.0180465.g006
slc24a5 (solute carrier family 24 member 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anteriorleft, dorsal up.
slc24a5 (solute carrier family 24 member 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anteriorleft, dorsal up. Arrows point to melanophores.
slc24a5 (solute carrier family 24 member 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 38, lateral view, anteriorleft, dorsal up.
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