Bright-field microscopy shows no macroscopic anomalies in rprmlmorphants/mutants. (A-C) Lateral view of live embryos by bright field microscopy at 48hpf. (A) wild type, (B) CRISPR-Cas9-rprml and (C) rprml MO-injected embryos reveals grossly normal morphology. (Bottom panel) Normal vascular development in the brain vessels of wild type and rprml morphant embryos

Similar phenotypes are obtained by disruption of rprml, either by CRISPR-Cas9 mutation or injection of antisense MOs. (A-E) lateral views of (A-C) live transgenic Tg(fli1:GFP) or (D-E) anti-RPRM IHC stained embryos at 48 hpf visualized by fluorescence and confocal microscopy, respectively. (A-A’) Normal vascular morphology is observed in control-MO-injected embryos. (B-C) Embryos injected with MOs or CRISPR-Cas9 targeting rprml exhibit reduced caudal hematopoietic tissue (CHT) territory (yellow double arrows). The yellow line represents the dorsal aorta (DA). (D-E) Rprm/Rprml protein expression is effectively blocked by rprml-MO. A human anti-RPRM/RPRML antibody labeled the Rohon-Beard neurons (RB, white arrows), the inter-somitic spaces (ISS, yellow arrows) and the pronephric tubule (pn, red brackets). A drastic reduction in immunoreactivity is observed in rprml MO-injected embryos compared to control MO-injected embryos. (F) Left panel: rprml CRISPR-Cas9 embryo genotyped by T7 endonuclease assay shows INDEL mutations of the expected sizes (500bp, 350bp). Right panel: in vitro DNA cleavage activity assay showing cropped areas containing Cas9-cleaved DNA bands of the expected sizes for rprml. A 1 kb DNA ladder was used as a molecular weight marker. (G) qPCR showing p53 relative expression in wild type and rprml MO specimens. Data shown as ΔCq ± SEM, (CI 95% = -8.41 ± 0.135 for wild type controls; CI 95% = -9.36 ± 0,65 for rprml morphants).

Early vascular development in rprml morphants. (A-D) Lateral view of embryonic zebrafish at 22 and 26 hpf analyzed by WISH for lmo2 (A-B) and fli1a (C-D). (A’- D’) Magnification of the trunk vessels.

CD41 expression is reduced in the CHT of rprml morphants. (A-D) Lateral view of the trunk with anterior to the left. (A-B) Whole mount immunohistochemistry/immunofluorescence showing Tg(CD41:GFP) expression pattern at 54 hpf. Staining with anti-GFP indicates expression of HSPC (yellow arrows) in the CHT (green bracket). (C-D) topro-3 staining (blue) shows the localization of the nuclei.

rprml deficiency by MOs results in reduce mpx+- and CD41+- cells during hematopoiesis. Fluorescent microscopy images for: (A-C) mpx+- and (D-F) CD41+ cell populations in transgenics Tg(mpx:GFP) and Tg(CD41:GFP) respectively . (B, E) rprml-MO injections cause reduction in mpx+- and CD41+ blood cells. (C, F) Co-injection rprml-MO and rprml mRNA significantly rescued the rprml morphant phenotype. (Bottom panels) Statistical significance was determined using two-tailed unpaired Student’s t-test. *** P ≤ 0.001, **** P ≤ 0.0001.

lack of rprml hinders normal activation of the Notch signaling pathway. (AD) Lateral views of 24 hpf analyzed by WISH for (A-B) arterial endothelial cells (kdr1) and (C-D) vascular arterial progenitor cells (dll4). (E-L) Fluorescent microscopy images of transgenics: (E-F) Tg(dll4:GFP), (G-I) Tg(flt0.8:RFP) and (J-L) Tg(Tp1:nRFP). Brackets indicated the positioning of the dorsal aorta (DA). (K-L) Asterisks indicated reduced Notch activity in CRISPR-Cas9-rprml and/or rprml-MO injected embryos (DA). The number of embryos with the phenotype shown as a fraction of the total number of embryos examined is indicated in the top right corner in J-L.

Knockdown of rprml impairs normal CHT niche morphogenesis. (A-B) Lateral view of double transgenic Tg(flk:mcherry;fli1:eGFP) embryos microinjected at 1-2 cell-stages with (A) control and (B) rprml morpholinos (MOs). The visualization at 48 hpf was done by live longitudinal confocal microscopy. Double arrows show the girth of the caudal hematopoietic tissue (CHT). Asterisks represent intervascular spaces. The number of embryos with the phenotype is shown as a fraction of the total number of examined embryos, indicated in the top right corner in A-B. Scale bars 50 µm.

rprml is dispensable for primitive hematopoiesis but required for HSPC formation. (A–F) Dorsal and lateral views of 10 somite-stage zebrafish embryos analyzed by WISH. White and black arrowheads represent the anterior- (ALM) and posterior-lateral plate mesoderm (PLM), respectively. (A,C,E,G,I) control MO-injected embryos; (B,D,F,H,J) rprml MO-injected embryos. (A–F) Expression patterns of typical hemangioblast markers: (A,B) lmo2, (C,D) fli1a, (E,F) tal1/scl1. (G,H) Lateral view of 24 hpf control MO and rprml MO-injected embryos stained by WISH for tal1/scl1. Black arrows indicate the intermediate cell mass (ICM) and red arrowheads normal expression pattern for tal1/scl1. (I,J) 28 hpf embryos stained by WISH for cmyb comparing controls versus rprml MO-injected embryos. Black arrowheads indicate normal expression pattern for cmyb. rprml morphant embryos retain retinal expression of cmyb (eye, e). The number of embryos with the phenotype shown as a fraction of the total number of embryos examined is indicated in the top right corner in (A–J). Scale bars, 100 μm (A–F) and 200 μm (G–J).

rprml is required for transient-definitive hematopoiesis. (A) Schematic diagram for zebrafish hematopoiesis at 28 hpf showing the intermediate cell mass (ICM) and the posterior blood island (PBI). (B) Schematic for the transient-definitive wave of hematopoiesis where EMP give rise to erythroid myeloid and granulocytic/neutrophils cell population at the PBI. (C,E,G,I) Lateral views of control MO and (D,F,H,J) rprml MO-injected embryos. (C–H) 28 hpf embryos analyzed by WISH against erythroid/myeloid progenitor and granulocyte markers: (C–D) tal1/scl1 for erythroid precursor cells, (E,F) pu.1 for myeloid precursor cell, (G,H) mpx for neutrophils. Inset magnifications from (C) to (H) show the posterior blood island (PBI). Black arrowheads in (D,F,H) show normal expression of tal1/scl1, pu.1 and mpx in the anterior primitive hematopoietic territories. (I,J) Representative images of 48 hpf transgenic Tg(mpx:GFP) embryos analyzed by fluorescent microscopy. (I,I′) control and (J,J′) rprml morphant embryos. Inset magnifications in (I′,J′) show positive fluorescence in neutrophils at the caudal hematopoietic tissue. Number of embryos with the phenotype shown as a fraction of the total number of embryos examined is indicated in the top right corner in (C–J). Scale bars, 200 μm (C–H) and 100 μm (I,J).

rprml is required for specification of hematopoietic precursor/stem cells (HSPCs) from the hemogenic endothelium at the ventral dorsal aorta. (A) Lateral view of zebrafish embryo with anterior to the left, showing overview of regions magnified in the fluorescent imaging (B–K) and WISH (L,M) panels. Red region denotes the left thymic lobe, yellow region the anterior gonad mesonephros/ventral dorsal aorta (AGM/VDA), green region the posterior caudal vein plexus/caudal hematopoietic tissue (PCVP/CHT) and white region the pronephric-tubule. (B–M) Lateral views of zebrafish embryos with anterior to the left at denoted developmental stages (48, 54 and 84 hpf). Loss-of function of rprml impairs normal formation of CD41-GFP^low+ HSPCs (yellow arrows in B–E) originating from the AGM/VDA (B–E, red bar graphs in N) and their migration to the CHT (F–I, green bar graphs in N) and thymus (J,K, yellow bar graphs in N). (D,E) Double transgenic Tg(flt0.8:mcherry;CD41:GFP) CRISPR-Cas9-rprml injected embryos show the same decrease in HSPCs originating from the AGM/VDA as rprml-morphant embryos. (L,M) WISH showing that rprml-morphant embryos show a remarkable absence of rag2 (lymphocyte marker) in the thymus. Statistical significance was determined using two-tailed unpaired Student’s t-test. ***P ≤ 0.001, ****P ≤ 0.0001. Scale bars, 50 μm (B–I) and 100 μm (J–M).

Lack of rprml increases cell apoptosis within the HSPC niche at the CHT. (A) Schematic diagram showing zebrafish vasculature at 48 hpf. In green: (1) aorta-gonad-mesonephros (AGM) where the HSPCs are born, and (2) the posterior caudal vein plexus/caudal hematopoietic tissue (PCVP/CHT), which constitutes the embryonic HSPC niche. Red and blue colors correspond to the artery and vein, respectively. (B–I) Confocal spinning-disk images of the CHT of Tg(kdrl:eGFP) at 48 hpf. (B,C) Note that the spaces are slightly larger in rprml morphants compared to control embryos. Right panel shows a schematic representation of the observed phenotypes from (B,C). Dotted areas indicate intervascular spaces within the CHT. (E,F,H,I) Anti-activated caspase-3 (Casp-3, red) labels apoptotic cells. (D,F) A control MO-injected embryo showing (D) normal morphology of the CHT and (E,F) standard levels of Casp-3 activity. (G–I) rprml-MO injected embryos show (H,I) increased Casp-3 immunoreactivity within the PCVP/CHT. Number of embryos with the phonotype shown as a fraction of the total number of embryos examined is indicated in the bottom left corner in (F,I). (J,K) Statistical significance was determined using two-tailed unpaired Student’s t-test. **P ≤ 0.01, ***P ≤ 0.001.