Rab32 and Rab38 genes in chordate pigmentation: an evolutionary perspective - PubMed (original) (raw)

Rab32 and Rab38 genes in chordate pigmentation: an evolutionary perspective

Ugo Coppola et al. BMC Evol Biol. 2016.

Abstract

Background: The regulation of cellular membrane trafficking in all eukaryotes is a very complex mechanism, mostly regulated by the Rab family proteins. Among all membrane-enclosed organelles, melanosomes are the cellular site for synthesis, storage and transport of melanin granules, making them an excellent model for studies on organelle biogenesis and motility. Specific Rab proteins, as Rab32 and Rab38, have been shown to play a key role in melanosome biogenesis. We analysed the Rab32 and Rab38 genes in the teleost zebrafish and in the cephalochordate amphioxus, gaining insight on their evolutionary history following gene and genome duplications.

Results: We studied the molecular evolution of Rab supergroup III in deuterostomes by phylogenetic reconstruction, intron and synteny conservation. We discovered a novel amino acid stretch, named FALK, shared by three related classes belonging to Rab supergroup III: Rab7L1, Rab32LO and Rab32/Rab38. Among these, we demonstrated that the Rab32LO class, already present in the last common eukaryotic ancestor, was lost in urochordates and vertebrates. Synteny shows that one zebrafish gene, Rab38a, which is expressed in pigmented cells, retained the linkage with tyrosinase, a protein essential for pigmentation. Moreover, the chromosomal linkage of Rab32 or Rab38 with a member of the glutamate receptor metabotropic (Grm) family has been retained in all analysed gnathostomes, suggesting a conserved microsynteny in the vertebrate ancestor. Expression patterns of Rab32 and Rab38 genes in zebrafish, and Rab32/38 in amphioxus, indicate their involvement in development of pigmented cells and notochord.

Conclusions: Phylogenetic, intron conservation and synteny analyses point towards an evolutionary scenario based on a duplication of a single invertebrate Rab32/38 gene giving rise to vertebrate Rab32 and Rab38. The expression patterns of Rab38 paralogues highlight sub-functionalization event. Finally, the discovery of a chromosomal linkage between the Rab32 or Rab38 gene with a Grm opens new perspectives on possible conserved bystander gene regulation across the vertebrate evolution.

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Figures

Fig. 1

Fig. 1

Evolution of the Rab32 and Rab38 subfamily in deuterostomes. Numbers at the branches indicate replicates obtained using the Maximum Likelihood estimation method. 247 sites were used for tree inference. Colored boxes highlight three classes of proteins: Rab7L1 present in invertebrates and vertebrates (blue box), Rab32LO present in protostomes, echinoderms and cephalochordates (yellow box), the Rab32 and Rab38 cluster present in all the deuterostomes (red box). Black arrow indicates the acquisition of FALK amino acid stretch at the stem of Rab7L1, Rab32LO, Rab32 and Rab38 evolution. Triangles indicate monophyletic clades belonging to Rab supergroup III (Rab7, Rab9, Rab23)

Fig. 2

Fig. 2

Alignment of Rab supergroup III domains. The alignment shows the amino acid conservation of three Rab domains: the P-loop, involved in trafficking (green), the Switch I (turquoise) and the Switch II (magenta) important for supporting the binding with other molecules. Downstream of Switch I, four ultra-conserved amino acids are highlighted, named FALK (yellow). The white background indicates changes in amino acid composition during evolution. At the bottom, human representatives of other Rab supergroups are reported to show the absence of FALK

Fig. 3

Fig. 3

Rab32 and Rab38 synteny conservation in gnathostomes. The Rab32 (a) and Rab38 (b) loci harbour several genes (grey boxes) conserved across evolution; in blue boxes Rab genes we studied, in red boxes Rab38 genes that are physically linked to tyrosinase (green). During the gnathostome evolution, the Rab32 is always linked to Grm1, while Rab38 is linked to Grm5. The scheme 3b ’ shows the functional relationship between Rab38 and Tyr during mammalian melanosome biogenesis (adapted from [55]). The position of the genes above or below the chromosome (horizontal line) indicates their transcriptional orientation on positive or negative strand, respectively

Fig. 4

Fig. 4

Rab32/38 expression pattern during amphioxus embryogenesis. Rab32/38 has been observed in notochord presumptive territories at the gastrula stage (a, black arrow), while later in development at the neurula stage it is expressed in notochord, mainly in the rostral part (b-c, black arrow). d is a vibratome section (15 μm) of the neurula specimen showed in b-c, at the level of the vertical dashed line. At the pre-mouth larval stage, Rab32/38 expression in the notochord turns off while a positive signal appears in the pharynx region (e, arrowhead)

Fig. 5

Fig. 5

Rab32a expression pattern during zebrafish embryogenesis. Rab32a has been observed in the presumptive posterior axial mesoderm (arrowhead) at 6 hpf (a) and in the developing notochord (white arrow) and Kupffer’s vesicle (arrowhead) at 8 hpf (b). At 24 hpf, it is present in RPE (white arrowhead), notochord and migrating neural crest cells (black arrows) (c-f). At 48 hpf the signal disappears in neural crest cells, but persists in RPE and notochord (white arrows) (g-h) and appears in the swim bladder (g, white asterisk). The expression in the swim bladder persists at 72 hpf larvae (i-j, white asterisk)

Fig. 6

Fig. 6

Rab38s expression pattern during zebrafish embryogenesis. Rab38a is expressed across the pharyngula embryonic period (24–48 hpf) (a-d): at 24 hpf there is a faint signal in RPE (arrowhead) and a strong one in migrating melanoblasts (a, b, white arrowhead) and a low level of expression is detectable in the mid-ventral region of brain (a); at 48 hpf the expression is visible only in the RPE (black arrowhead) (c, d). Rab38b is expressed only at late developmental stages (e-h): at 48 hpf in a small region of the pharyngeal arches (white arrow) and in the developing swim bladder (e, f, white asterisks), while at 72 hpf only in swim bladder (g, h, white asterisks). The gene Rab38c gene is strongly expressed in the head region (i and j for 24 hpf larvae). Lateral view in all images (anterior is on the left) except d and f that are ventral (anterior on the top)

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