Fig 1.
Evolutionary history of the reprimo gene family in gnathostome vertebrates.
Reprimo genes (RPRM, RPRML and RPRM3) diversified as a product of the two rounds of whole genome duplication occurred in the last common ancestor of vertebrates. After that, they were differentially retained among the different group of vertebrates. Teleost fish (e.g. zebrafish) underwent an extra round of whole genome duplication that further expanded this group of genes, however, not all reprimo genes were retained in all species. Thus, teleost species retained duplicated copies only in the RPRM gene lineage, while in the other two (RPRML and RPRM3), only one of them was retained. Diffuse lines represent gene lineages that are not retained in actual species. Divergence time estimates were obtained from the TimeTree public database [38]. The 615 mya estimates correspond to the last common ancestor of vertebrates.
Fig 2.
RPRM protein family multiple sequence alignment.
The N-terminus of the proteins shows most of the sequence variance and insertions, while the C-terminus is greatly conserved. Some amino acid substitutions can be observed which mark differences between the two proteins. RPRM- and RPRML-specific amino acids are highlighted in blue and red, respectively. An * (asterisk) indicates positions which have a single, fully conserved residue. A: (colon) indicates conservation between groups of strongly similar properties—scoring > 0.5 in the Gonnet PAM 250 matrix. A. (period) indicates conservation between groups of weakly similar properties—scoring ≤ 0.5 in the Gonnet PAM 250 matrix. Potential transmembrane domain predicted using Geneius software.
Fig 3.
rprm expression patterns during early embryogenesis.
(A-I) The expression patterns of rprma, rprmb and rprml were visualized by whole-mount in situ hybridization (WISH) during zebrafish embryonic development. Developmental stages are expressed as hours post-fertilization (hpf). (A, C, D, F, G, I) Lateral (anterior to the left) and (B, E, H) dorsal views are shown. Blue arrows in D-E indicate the expression in the somites (ss), while (C, F, I) the red inset magnifications indicate the expression in the notochord.
Fig 4.
RPRM expression patterns are conserved between zebrafish and human brain.
(A-D) RPRM expression patterns were examined using whole-mount in situ hybridization in wild-type embryos at 28 and (E-H) 72 hours post-fertilization [hpf]. (A-D) Frontal views of the embryo heads. (E-H) Dorsal views of the embryo heads. (I-L) Retina cross-sections. (A-C) At 28 hpf rprma, rprmb and rprml transcripts are expressed in neuronal populations such as dorsal thalamus (DT), preoptic region (Po) and ventral thalamus, respectively (black arrows). (D) rprm3 is ubiquitously expressed throughout the brain. (E-G) At 72hpf rprma and rprml are expressed in the DT and the VT, respectively (white arrows), while rprmb is not expressed in those regions (asterisks). (H) At the same developmental stage, rprm3 mRNA is expressed in the Po and the optic chiasma (Oc). (I-L) Cross sections of the retina. (I, J, L) rprma, rprmb and rprm3 expression are restricted in the retina to the retinal ganglion cell layer (RGL, black arrow). (K) In contrast, rprml transcript expression is absent in the RGL (asterisk). (M, M’, N, N’) IHC staining for RPRM of white and grey matter sections from adult human samples (400x; inset magnifications 600x). (M-N) RPRM protein is expressed in the cytoplasm and axons of neurons (black arrowheads). (O-O’) RPRM protein is expressed in the nuclei of astrocytes.
Fig 5.
RPRM is expressed during zebrafish angiogenesis and in adult human vasculature.
(A-C). Whole-mount in situ hybridization at 72hpf. Lateral view of the head vasculature. rprma/b and l are expressed in the mesencephalic vein (MsV), the dorsal longitudinal vein (DLV) and the primordial hindbrain channel (PHBC). (D-F) Lateral view of the trunk vessels. rprma/b and l are expressed at low levels in the dorsal aorta (DA, red bracket) the posterior cardinal vein (PCV, blue bracket) and the intersegmental vessels (ISV, red arrow). (D’-F’) Inset magnifications (red brackets) and (D”-F”) transverse histological cross-sections of the posterior trunk region. (G-H) IHC staining for RPRM in tissue sections showing small blood vessels from adult human samples (400x; inset magnification 600x). (G, G’) RPRM protein is expressed in the nuclei of endothelial cells (EC, black arrows) from a small artery. (H-H’) RPRM protein is expressed in the nuclei of EC and vascular smooth muscle cells (vSMCs, black arrow) of a muscular artery.
Fig 6.
Expression of rprm genes during zebrafish gut development and human intestinal tissue.
(A-F) Lateral views of whole-mount in situ hybridization (WISH) at 72hpf. (A-B) WISH for two well-known gut markers (A) platelet-derived growth factor receptor (pdgfrβ), (B) smooth muscle actin (sm22, also known as transgelin1), and the rprm genes (C) rprm3, (D) rprma (E) rprmb and (F) rprml. At 3dpf, rprma/-b and rprml are expressed in the embryonic intestinal tube. At the same developmental stage, there is a lack of expression of rprm3 in the intestinal tissue (C, asterisk). (G-I) Lateral cross sections of the zebrafish gut, anterior to the left. (J-J’) RPRM IHC staining (400x; inset magnification 600x) showing nuclear positive expression in enterocytes at the base of the intestinal crypts. (K-K’) RPRM IHC, (400x; inset magnification 600x) showing nuclear positive expression in smooth muscle cells from the muscularis propria of the small intestine.