rab11 genes are highly conserved in vertebrates
We first examined Rab11 protein phylogeny in zebrafish [Rab-11A (NP_001007360); Rab-11BA (NP_999935); Rab-11BB (NP_001002555)] and other representative organisms including worm [Caenorhabditis elegans, RAB-11.1 (NP_490675); RAB-11.2 (NP_001251691)], fruit fly [Drosophila melanogaster, Rab11 isoform A (NP_599137); Rab11 isoform B (NP_477170)], medaka [Oryzias latipes, Rab-11A (XP_004086096); Rab-11B (XP_004068250); Rab-11B-like (XP_004068248)], frog [Xenopus tropicalis, Rab-11A (NP_001016481); Rab-11B.1 (NP_001120303)], chicken [Gallus gallus, Rab-11A (NP_001005827); Rab-11B (NP_001012569)], mouse [Mus musculus, Rab-11A (NP_059078); Rab-11B (NP_033023)], rat [Rattus norvegicus, Rab-11A (NP_112414); Rab-11B (NP_116006)] and human [Homo sapiens, Rab-11A isoform 1 (NP_004654); Rab-11B (NP_004209)].
The zebrafish Rab11a, Rab11ba and Rab11bb proteins are highly conserved during evolution, especially the GTPase domain, suggesting their important functions. While these paralogues differ in the C-terminal part adjacent to the GTPase domain (Fig.1A). Zebrafish rab11a was clustered in the rab11a group, and rab11ba and rab11bb in the Rab11b group (Fig.1B) in the phylogenic tree constructed according to amino acid sequences of all the Rab11 proteins above.
Expression of rab11 genes analyzed by RT-PCR
We examined rab11a expression using RT-PCR. High expression level of maternal rab11a was detected in both 1-cell stage fertilized eggs and cleavage stages (Fig.2). Rab11a exhibited steady expression through 96 hpf with a slight lower level found at 12 hpf. In adult zebrafish, rab11a was found highly expressed in the eye, fin, brain, eggs and muscle. Its expression in the nervous system is much restricted to the brain, being very weak in the spinal cord. With much similarity to rab11a, rab11ba is steadily expressed from 1-cell stage through 96 hpf, except for a slightly low expression period around 12 hpf. Like rab11a, rab11ba is maternally expressed, as detected at 1-cell stage and in the eggs. Rab11ba is also highly expressed in the brain, eggs and muscle and is very weak in the spinal cord. As a paralogue, rab11bb was expressed differently. Rab11bb only showed extremely weak maternal mRNA expression. High zygotic rab11bb expression was not observed until 24 hpf and remained through 96 hpf. Rab11bb was detected at high level in kidney, eye, fin and brain, and weakly expressed in muscle. Unlike rab11a and rab11ba, rab11bb expression level in the spinal cord is high.
Rab11a expression during zebrafish embryonic development
We then performed in situ hybridization to further study the spatial-temporal expression pattern of rab11a as well as rab11ba and rab11bb (Fig.3-5). High level of maternal rab11a mRNA was detected at 2 hpf (64-cell) (Fig.3A). At 12 hpf, zygotic rab11a mRNA was found in the whole organism without tissue specificity (Fig.3B). As seen at 20 hpf to 24 hpf, rab11a expression began to decrease in the trunk and was mainly restricted to the brain and head structures, and also in the caudal spinal cord, caudal vein, pronephric duct and the proctodeum (Fig.3C-D’). The mRNA expression in the brain remained strong until 36 hpf and decreased in other tissues (Fig.3E). From 48 hpf to 72 hpf, rab11a mRNA was expressed mainly in the brain, with expression in the posterior hindbrain becoming weaker (Fig.3F, G). At 48 hpf, rab11a expression was also confirmed to express in the brain especially the cerebral cortex by histological analysis of sectioned embryos (Fig.3I-I’’). At 4 dpf, rab11a was strongly expressed in the brain and also in the pharyngeal arches and the digestive duct (Fig.3 H-H’’).
Rab11ba expression during zebrafish embryonic development
Similar to rab11a, maternal rab11ba mRNA was detected at 3 hpf (1k-cell) (Fig.4A). At 16 hpf, rab11ba was expressed in the whole organism and especially in the neural tube (Fig.4B-B’). At 20 hpf, rab11ba expression in the brain remained strong, but its mRNA was not detected in the spinal cord (Fig.4C, C’). Like that of rab11a, rab11ba mRNA was seen in the pronephric duct and proctodeum (Fig.4C). At 24 hpf, rab11ba was maintained in the brain (Fig.4D). At this stage, rab11ba started its expression in the pectoral fins. Rab11ba expression in the spinal cord and the caudal vein was significantly decreased at 36 hpf and again increased at 48 hpf (Fig.4E-F). At this stage, rab11ba was expressed at proctodeum at 48 hpf. Transverse section analysis demonstrated almost the same expression patterns of Rab11ba as that of Rab11a where it was expressed in the brain at 48 hpf as well (Fig.4I-I’’). From 72 hpf to 96 hpf, rab11ba was expressed in the brain, retina, olfactory placode and pectoral fins (Fig.4G-H’). Rab11ba was also found highly expressed in cranio-facial neuromasts at 72 hpf and 96 hpf, which was unique compared to rab11a and rab11bb (Fig.4G-H’).
Rab11bb expression during zebrafish embryonic development
No maternal message was detected of rab11bb at 2 hpf (64-cell) (Fig.5A). Rab11bb expression was seen in the brain, retina, and spinal cord except for the caudal part and the proctodeum from 20 hpf to 24 hpf (Fig.5B, C). Its expression in the spinal cord turned weaker at 36 hpf (Fig.5D, D’). At 48 hpf and 72 hpf, rab11bb expression was restricted to the brain, with expression in the retina and ear weakly detected and no detectable expression in other tissues (Fig.5E-F’). Similar to rab11a and rab11ba, rab11bb was also observed to express in the brain at 48 hpf but with a seemingly lower level by comparing the hybridization signals (Fig.5H-H’’). At 4 dpf, its expression in the brain remained strong, while its mRNA was also detected in the retina, ear and pharyngeal arches (Fig.5G, G’).