Almeida AD , Wise HM , Hindley CJ , Slevin MK , Hartley RS , Philpott A .

G0500101 Medical Research Council , G0700758 Medical Research Council , R01CA095898 NCI NIH HHS , Wellcome Trust , R01 CA095898 NCI NIH HHS , G0800784 Medical Research Council , MRC_G0800784 Medical Research Council

	Figure 1. xCdc4α and xCdc4β protein are expressed throughout development, and transcripts accumulate in the neural crest and neural crest derived tissues. Expression of (A) xCdc4α and (B) xCdc4β in staged embryo lysates was determined by immunoblotting (IB) using anti-Cdc4 antibodies. In vitro translated (IVT) protein was used as a control. Equal loading was verified by IB for tubulin, with the equivalent of one embryo per lane loaded. (C-K) Developmental expression of xCdc4 was determined by whole mount ISH. At stage 15 (C), xCdc4 expression is detected broadly at the preplacodal ectoderm (ppe), in particular in the presumptive optic placode (pop) as well as in the prospective trunk neural crest (TNc). At late neurula stages (D,E), while xCdc4 transcripts continue accumulating in the trunk neural crest and optic placode (Opt P), xCdc4 is also detected in the cranial neural crest, particularly within the branchial aggregates (b). At stage 21 (F), xCdc4 is broadly detected in migrating cranial neural crest (Cc) and placodal regions. At stage 23 (G,H), xCdc4 is expressed in cranial neural crest that populates the branchial arches (Ba) and additionally in the myotome (My). At stage 26 (I), xCdc4 continues to be expressed in the myotome and branchial arches, as well as in several anterior placodes (arrowheads). xCdc4 is additionally expressed in the posterior fin mesenchyme (Fin). From stage 28 to 32 (J,K), xCdc4 expression is downregulated in the branchial arches, while persisting in the anterior placodal region (arrowheads), dorsal somites (s) and dorsal and ventral fin mesenchyme (Fin). Whole mount ISH at the indicated stages using xCdc4β sense probe confirms the specificity of the probe (L-N). Views in (C-N) are indicated bottom right after the stage (St): a, anterior view; d, dorsal view; l, lateral view.
	Figure 2. xCdc4 expression overlaps with expression of markers of neural crest and placodes. (A-R) Comparison of xCdc4 developmental expression (A,G,M) with the expression of neural crest and placodal gene markers in stage 15/16 (B-F), 18/19 (H-L) and 21/22 (N-R) embryos, as labeled. Asterisk indicates premigratory cranial crest; b, branchial crest; Cc, cranial neural crest; di, diencephalons; dlp, dorsolateral placodal area; Hg, hatching gland; h, hyoid crest; m, mandibular crest; me, mesencephalon; Nc, neural crest; Olf P, olfactory placode; Opt P, optic placode; plp, presumptive lens placode; pOlf P, presumptive olfactory placode; ppe, preplacodal ectoderm; pop, presumptive optic placode; s, somites; tel, telencephalon; TNc, trunk neural crest; yellow arrowhead, neural plate border region of the prospective rhombencephalon. Anterior view, dorsal up, stages as indicated.
	Figure 3. xCdc4 expression overlaps with expression of neural crest and placodal markers. (A-L). Comparison of xCdc4 developmental expression (A,G) with the expression of neural crest and placodal gene markers in the mid-tailbud stages 26/27 (A-F) and 32/33 (H-L). Arrowheads indicate the trigeminal placode; white asterisks indicate branchial arches; red asterisks indicate pharyngeal pouches. Ba1-2, branchial arches 1 and 2; Cg, cranial ganglia; Di/Me, diencephalon and midbrain boundary; E, eye; Ev, ear vesicle; F, forebrain; Ha, hyoid arch; Hg, hatching gland; M, mandibular arch; M*, mandibular crest surrounding the eye; Pr, profundal placode; Tel, telencephalon. Lateral view, anterior left, stages as indicated.
	Figure 4. xCdc4 is found in ectodermal and mesodermal derivatives. (A-L) xCdc4 ISH on transverse sections of stage 15, 18 and 22 embryos (A,B,G-I), including sense control (E,F,J-L) and comparative Snail2 expression on stage 15 (C) and stage 18 (D) embryo sections. xCdc4 expression is detected in the neural crest (NC, black arrows), preplacodal ectoderm (ppe) and neural plate border (NPB) as well as in the neural plate (np). In addition to the expression in both superficial and deep layers of the ectoderm, there is also expression of xCdc4 in the notochord (n) and in somitogenic mesoderm (sm).
	Figure 5. xCdc4α and xCdc4β block Cyclin E-mediated apoptosis, and this requires an intact F-box domain. Cyclin E mRNA (1 ng) was co-injected with 1 ng of other mRNAs as indicated into one cell of two-cell-stage embryos. (A) Embryos were scored for enlarged cells at stage 9 and apoptotic cells at stage 11 (n = 75-196). (B-M) Representative embryos from each injection are shown; enlarged cells are highlighted with white arrows (B,E-G,K) and shown enlarged in insets. Black arrows highlight apoptotic cells (H,L,M).
	Figure 6. Phosphorylated Cyclin E protein is degraded by xCdc4. (A) Two-cell-stage embryos were injected with 1 ng of amino-terminally FLAG-tagged Cyclin E mRNA (lanes 2 to 5) and either 1 ng of GFP mRNA (lane 2) or 1 ng of xCdc4 mRNA (lanes 3 to 5). The ability of xCdc4δFbox to block Cyclin E degradation was examined by co-injecting 1.5 ng of xCdc4δFbox mRNA (lane 5), with 1.5 ng of GFP mRNA or 1.5 ng of Skp2δFbox mRNA as controls (lanes 3 and 4). Embryos were allowed to develop to stage 10.5 and immunoblotting was performed for the FLAG epitope, while immunoblotting of actin was performed as a loading control. IR-Dye™-conjugated secondary antibodies were used, enabling accurate quantification of Cyclin E and actin expression. (B) The level of phospho-Cyclin E was normalized to the level of actin, then compared to the level of phospho-Cyclin E in GFP-injected embryos, showing mean ± standard error of the mean from four independent experiments.
	Figure 7. xCdc4δFbox does not affect the cell cycle. xCdc4δFbox or xCdc4 mRNA (1 ng) was injected into one cell of two-cell-stage embryos. GFP mRNA (1 ng) was injected as a control, and β-gal mRNA was injected as a lineage tracer (light blue unilateral staining). Whole mount antibody staining against phH3 was performed to detect mitotic cells. The average percentage difference in the number of mitotic cells on the injected side, compared to the uninjected side, was calculated as indicated in Materials and methods. Representative embryos are shown (anterior view, dorsal up, injected side right).
	Figure 8. xCdc4δFbox inhibits neural crest development. xCdc4δFbox or xCdc4 mRNA (1 ng) was injected into one cell of two-cell-stage embryos. GFP mRNA (1 ng) was injected as a control, and β-gal mRNA was injected as a lineage tracer (light blue or red unilateral staining). (A-U) Whole mount ISH was performed for Snail2 (A-C), Snail (D-F), c-Myc (G-I), Pax3 (J-L), Opl (M-O) and Msx1 (P-R), and the early neuronal marker Sox2 (S-U). Representative embryos are shown (anterior view, dorsal up, injected side right).
	Figure 9. Loss of neural crest induced by xCdc4δFbox is rescued by co-injection of xCdc4. xCdc4δFbox mRNA (1 ng) was co-injected with either 1 ng of xCdc4 or 1 ng of GFP mRNA into one cell of two-cell-stage embryos. As controls, 1 ng of xCdc4 mRNA was co-injected with 1 ng of GFP mRNA, as well as injection of 2 ng of GFP mRNA as a separate control. β-gal mRNA was injected as a lineage tracer. (A-H) Embryos were allowed to develop to stages 18/19 and whole mount ISH was performed for the neural crest markers Snail2 (A-D) and Snail (E-H). Representative embryos are shown (anterior view, dorsal up, injected side right). Arrows indicate loss of expression of each marker on the injected side of xCdc4δFbox expressing embryos. The reduction in expression of each marker on the injected side of xCdc4δFbox expressing embryos is rescued by co-injection of xCdc4 mRNA (arrowheads).
	Figure 10. xCdc4δFbox inhibits neural crest specification. xCdc4δFbox or xCdc4 mRNA (1 ng) was injected into one cell of two-cell-stage embryos. GFP mRNA (1 ng) was injected as a control, and β-gal mRNA was injected as a lineage tracer (light blue unilateral staining). (A-F) Embryos were allowed to develop to stage 13/14 and the expression of Snail2 (A-C) and Snail (D-F) in the prospective neural crest region was examined by whole mount ISH. Representative embryos are shown (anterior view, dorsal up, injected side right). Arrows indicate loss of expression of each marker on the injected side of xCdc4δFbox expressing embryos.
	Figure 11. xCdc4δFbox impairs melanocyte development. xCdc4δFbox, xCdc4 or control GFP mRNA (0.5 ng) were injected into each of the two dorsal cells of four-cell-stage embryos. (A-C)Embryos were allowed to develop to stage 43 and subsequently the distribution and number of melanocytes were compared: xCdc4δFbox (A), xCdc4 (B) and GFP (C). (A-C) Representative embryos are shown (lateral views, anterior side right). (D)The average number of melanocytes for each condition was calculated and the mean ± standard error of the mean from two independent experiments are shown (n = 31 to 57). There was a significant decrease in the number of melanocytes following injection with xCdc4δFbox when compared to GFP or xCdc4 (***P << 0.0001), and a smaller decrease following injection with xCdc4 when compared to injection with GFP (*P < 0.01).
	Figure 12. xCdc4δFbox does not reduce neural crest by inducing apoptosis. (A,B) xCdc4δFbox or xCdc4 mRNA (1 ng) was injected into one cell of two-cell-stage embryos. (C) GFP mRNA (1 ng) was injected as a control, and β-gal mRNA was injected as a lineage tracer (light blue unilateral staining). Apoptosis was assessed using TUNEL staining. Arrow indicates TUNEL-positive cells. Representative embryos are shown (anterior view, dorsal up, injected side right). (D) The area of Snail2 staining on the injected side was expressed as a ratio of the area on the uninjected side, and for each experiment an average ratio was obtained (see Materials and methods). Mean ± standard error of the mean ratio from two independent experiments are shown (n = 57-82). (E-J) xCdc4δFbox, xCdc4 or GFP mRNA (1 ng) was injected into one cell of two-cell-stage embryos, along with 1 ng of hBclXL or 1 ng of GFP mRNA as indicated, with β-gal mRNA as a lineage tracer (light blue unilateral staining). Whole mount ISH for Snail2 was performed on stage 18 embryos. The reduction in expression of Snail2 on the injected side of xCdc4δFbox expressing embryos is not rescued by co-injection of hBclXL (arrowheads). Representative embryos from the indicated injections are shown (anterior view, dorsal up, injected side right).
	fbxw7 ( F-box and WD repeat domain containing 7, E3 ubiquitin protein ligase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	fbxw7 ( F-box and WD repeat domain containing 7, E3 ubiquitin protein ligase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior right, dorsal up.
	fbxw7 ( F-box and WD repeat domain containing 7, E3 ubiquitin protein ligase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.

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