Jansen EJ , van Bakel NH , Coenen AJ , van Dooren SH , van Lith HA , Martens GJ .

	Fig. 1. Phylogenetic analysis and alignment of Ac45 [accessory subunit of vacuolar (H+)-ATPase] and Ac45-like protein (Ac45LP) sequences. a Sequence alignment. Underlining Transmembrane region, asterisks conserved cysteine residues. The alignment contains colors utilized by the ClustalX coloring algorithm in JalView (conservation grade colors and coloring of amino acid residues according to physicochemical criteria). b Phylogenetic tree: AaAedes aegypti, AcAnolis carolinensi, BfBranchiostoma floridae, CeCaenorhabditis elegans, CfCanis familiaris, DmDrosophila melanogaster, DrDanio rerio, GaGasteroteus aculeatus, GgGallus gallus, HsHomo sapiens, TgTaeniopygia guttata, MdMonodelphis domestica, MmMus musculus, OaOrnithorhynchus anatinus, RnRattus norvegicus, XlXenopus laevis, XtXenopustropicalis
	Fig. 2. The evolutionary history of the Ac45 and Ac45LP genes. a Schematic overview of genes surrounding the (presumptive) Ac45LP- and Ac45 genes. The Ac45LP gene was identified in chicken and Xenopus, but not in zebrafish. In human and mouse, only a remnant of the presumptive Ac45LP gene (exon 9) was found. The Ac45LP-neighboring genes were fully conserved. The Ac45 gene and its neighboring genes were fully conserved among species, with the exception of chicken. b Alignment of the partial human and mouse presumptive Ac45LP amino acid sequences (deduced from exon 9 Ac45LP sequences) with Ac45LP amino acid sequences from various species. Asterisk Stop codon preceding the exon 9 Ac45LP open reading frame. AcAnolis carolinensi, GgGallus gallus, HsHomo sapiens, TgTaeniopygia guttata, MmMus musculus, OaOrnithorhynchus anatinus, XlXenopus laevis, XtX.tropicalis. c Lineage-dependent evolutionary fate of the Ac45 and Ac45LP genes
	Fig. 3. Exogenous expression of Ac45 and Ac45LP in Xenopus embryos. Western blot analysis of lysates of embryos injected with synthetic Ac45 mRNA or Ac45LP mRNA. Ac45 and Ac45LP proteins were detected with an anti-Ac45-C antibody and visualized by chemoluminescence
	Fig. 4. Expression of Ac45 and Ac45LP mRNAs in developing Xenopus. Total RNA was extracted from Xenopus embryos (developmental stages are indicated). Reverse transcriptase-PCR analysis was performed as described in the Materials and Methods. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control for RNA integrity. -RT Absence of reverse transcriptase (negative control). Staging of Xenopus embryos was according to Nieuwkoop and Faber [30]
	Fig. 5. Whole-mount in situ hybridization for Ac45 and Ac45LP mRNAs in stage-30 Xenopus embryos. Hybridizations with Ac45-anti-sense- (a) and Ac45-sense (b) DIG-labeled probes. Ac45 mRNA expression was predominantly found in developing neural tissues (brain, spinal cord and eye). c No signals were observed with the Ac45LP-anti-sense probe. d Negative control using the Ac45LP-sense probe
	Fig. 6. Expression of Ac45 and Ac45LP mRNAs in neural tissue and neural crest cells of Xenopus embryos. Sections of developmental stage-30 Xenopus embryos hybridized with an Ac45-anti-sense probe showed Ac45 expression in the developing brain (a), in migrating neural crest cells between the pharyngeal arches and in the developing eye (b, arrows). No signals were detected with the Ac45-sense probe (c). Ac45LP mRNA expression was detected in the developing brain (d), in the migrating neural crest cells (e, f, arrows) and to a lower extent in the developing eye (e). Ac45LP mRNA-positive cells were also detected in the ventral mesoderm (g). No hybridization signal was found with the Ac45LP-sense probe (h). Asterisk Non-specific staining, e eye, Pros prosencephalon, Mes mesencephalon
	Fig. 7. Tissue distributions of Xenopus Ac45 and Ac45LP mRNAs. RNA was extracted from various tissues and RT-PCR analysis was performed as described in the Material and Methods. GAPDH served as an internal control for RNA integrity. Si Small intestine, Lu lung, Te testes, St stomach, Ov ovary, Li liver, Sp spleen, Ga gall bladder, Ki kidney, Br brain, Mu muscle, He heart, Oo oocytes, Rt absence of reverse transcriptase (negative control)
	atp6ap1.1 (ATPase, H+ transporting, lysosomal accessory protein 1, gene 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior right, dorsal up.

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