Rspo1 is required for HSC specification. The HSC markers runx1 (A,B,D,E) and cmyb (G,H,J,K), which identify HSCs emerging from the dorsal aorta, as well as the T lymphocyte marker rag1 (M,N,P,Q), found in thymic T cells, were examined at 24 hpf, 36 hpf and 96 hpf as indicated, by whole-mount in situ hybridization (WISH) in wild-type (WT; A,G,M), Rspo1 MO-injected (B,H,N), Rspo1 wild-type and heterozygous siblings (D,J,P), or rspo1 dtt^y135 homozygous mutants (E,K,Q). Red arrowheads indicate HSC precursors in the dorsal aorta (A,B,D,E,G,J,K) or T lymphocytes in the thymus (M,P), when present. Numbers of embryos displaying the depicted phenotype are indicated. (C,F,I,L,O,R) Histograms illustrate percentages of phenotypes. Lateral mid-trunk or ventral head views at 160× magnification.

Rspo1 is required for normal wnt16 expression. Wild-type (WT; A,D,G) or Rspo1 MO-injected animals (B,E,H) were examined by WISH for expression of wnt16 at the time points indicated. Wnt16 is natively found in the dorsal anterior domain of each formed somite, overlapping the rspo1 expression domain, and is diminished in Rspo1 MO-injected animals. Black arrows indicate areas of lost wnt16 expression. Numbers of embryos displaying the depicted phenotype are indicated. (C,F,I) Histograms illustrate percentages of phenotypes. Lateral views of the mid trunk at 160× magnification. (J) qRT-PCR of wnt16 transcript at 18 hpf, 20 hpf, and 24 hpf, normalized to expression of ef1a, reveals decreased wnt16 in Rspo1 knockdown (Rspo1 MO) compared with wild-type (WT) (mean±s.e.m.). *P≤0.0189, determined by one-way ANOVA.

Notch pathway gene expression in Rspo1 morphants. Wild-type (A,D,G,J,M,P) or Rspo1 MO-injected animals (B,E,H,K,N,Q) were examined by WISH for expression of the Notch ligands dlc (A,B), dld (D,E), or Notch receptors notch1a (G,H), notch1b (J,K), notch2 (M,N) and notch3 (P,Q) at the time points indicated. Compared with wild-type (A,D), dlc and dld are specifically decreased (B,E) in the formed somites (below red bars), but not the pre-somitic mesoderm. Notch receptor expression is unaffected. Flat mount dorsal views of the trunk or lateral views at 160× magnification. Numbers of embryos displaying the depicted phenotype are indicated. (C,F,I,L,O,R) Histograms illustrate percentages of phenotypes. (S,T) Decreased dlc and dld mRNA in Rspo1 knockdown (Rspo1 MO) was confirmed by qRT-PCR, normalized to expression of ef1a, of transcript isolated from dissected embryo trunks (mean±s.e.m.). *P≤0.0006, determined by one-way ANOVA.

Rspo1 is required for normal vegfaa expression. Wild-type (A,D,G,J,M,P) or Rspo1 MO injected animals (B,E,H,K,N,Q) were examined by WISH for expression of vegfaa (A,B), shha (D,E), the Shh transcriptional target ptch2 (G,H), tgfb1a (J,K), tgfb1b (M,N), or tgfbr2 (P,Q) at the time points indicated. vegfaa, which is expressed in the medial somite of uninjected embryos, is diminished in Rspo1 MO-injected animals (A,B). shha in the hypochord and its target, ptch2, in the somite are unaffected (D,E,G,H). Tgfb1a and tgfb1b are also suppressed in Rspo1 knockdown embryos (J,K,M,N). Red arrowhead in J indicates dorsal aorta. Tgfbr2 in the somite is unaffected (P,Q). Numbers of embryos displaying the depicted phenotype are indicated. (C,F,I,L,O,R) Histograms illustrate percentages of phenotypes. Lateral views of the mid trunk at 160× magnification. (S) Diminished vegfaa expression was confirmed by qRT-PCR normalized to expression of ef1a at 20 hpf, 22 hpf, and 24 hpf (mean±s.e.m.). *P≤0.0008, determined by one-way ANOVA.

Dlc, dld and vegfaa-165 can rescue HSC specification in Rspo1 morphants. Expression of the HSC markers runx1 (A,D,G,J,M,P,S) and cmyb (B,E,H,K,N,Q,T), and the T lymphocyte marker rag1 (C,F,I,L,O,R,U) in the thymus were examined at 24 hpf, 36 hpf, and 96 hpf as indicated, by WISH in wild-type embryos (A-C), or embryos injected with 5 ng Rspo1 MO (D-F), or 5 ng Rspo1 MO and mRNAs encoding candidate rescue genes dlc (100 ng; G-I), dld (50 ng; J-L), dlc+dld (50 ng and 25 ng, respectively; M-O), vegfaa-165 (100 ng; P-R) or vegfaa-121 (100 ng; S-U). dlc and dld mRNA were independently able to partially rescue HSCs. Co-injection of dlc and dld caused a more complete rescue of HSCs. vegfaa-165, but not vegfaa-121, was able to rescue HSCs. Red arrowheads indicate runx1⁺ (A,D,G,J,M,P,S), and cmyb⁺ (B,E,H,K,N,Q,T) HSCs, or rag1⁺ T lymphocytes (C,I,L,O,R), when visible. Numbers of embryos displaying the depicted phenotype are indicated. Lateral views of the mid trunk at 160× magnification.

R-spondin-1 (rspo1) expression. (A-K) Embryos fixed at the times indicated and processed by WISH for expression of rspo1 transcript. Lateral (A,C,E,G,I), dorsal (B,J) views or transverse sections (D,F,H,K) are presented, at 70X (A,B) or 160X (C-K) magnification. Black dotted lines indicate the level of transverse sections (C,E,G,I). Red arrowheads identify regions of more abundant rspo1 expression. Rspo1 was expressed in the neural tube and in the trunk mesoderm of embryos 22 hpf – 36 hpf (A-H). Rspo1 expression was limited to the lateral line (I), a cranial ventricular region (J), and an area ventral to the dorsal aorta (K) in 51 hpf embryos. (L) RT-PCR for rspo1 message in mRNA from whole embryos at the stages indicated. No reverse transcriptase (RT) control lanes are indicated (-). (M) Rspo1 mRNA levels (mean±SEM) relative to ef1 control quantified by q-RT-PCR from uninjected and Rspo1 MO injected animals. * = statistically significant (p ≤ 0.007) determined by one-way ANOVA

Rspo1, runx1, wnt16 levels in Rspo1 MO or Wnt16 MO animals. (A) Image of WT embryo at 24 hpf showing area of images taken and labeled with the location of the neural tube (NT, gray), dorsal aorta (DA, red line), posterior cardinal vein (PCV, blue line), and yolk sac (YS). Representative wild type (WT; B,E,H), Rspo1 MO (C,F,I), and Wnt16 MO (D,G,J) embryos processed by WISH for rspo1 (B-D), runx1 (E-G), and wnt16 (H-J) mRNA expression at 22 hpf or 24 hpf as indicated in each image. Lateral views of the trunk at 160x magnification. Numbers of embryos displaying the depicted phenotype are indicated.

Rspo1 knockdown does not alter primary vascular specification. Wild-type (A,C,E,G) or Rspo1 MO injected animals (B,D,F,H) were examined by WISH for expression of endothelium (cdh5, A,B), arterial identity (dlc, C,D and efnb2a, E,F), or by fluorescence for vessel patterning (fli1a:EGFP, G,H) at the time points indicated, revealing normal patterning. (H) Rspo1 morphants have stunted intersegmental vessel growth at 26 hpf. Numbers of embryos displaying the depicted phenotype are indicated. Magnification 160X.

Rspo1 phenotype is specific. Wild-type (A,C,E,G) or Rspo1 MO injected animals (B,D,F,H) were examined by WISH for markers of primitive erythrocytes (gata1, A,B), myotome (myoda, C,D), neural crest (crestin, E,F), or thymic epithelium (foxn1, G,H) at the time points indicated, revealing normal patterning. Numbers of embryos displaying the depicted phenotype are indicated. Lateral trunk (A-F) or ventral head views (G,H) are presented. Magnification 160X.

Dlc and dld expression is decreased in Wnt16 morphants. (A) Lateral view image of wild-type 17 hpf embryo with box indicating the location of in situ images. The cartoon shows a flat mount section of the tail of the embryo with formed somites and the pre-somitic mesoderm indicated. Wild-type (B,D), and Wnt16 MO (C,E) embryos were examined by WISH for expression dlc (B,C) or dld (D,E). Numbers of embryos displaying the depicted phenotype are indicated. Magnification 160X.

Vegfa does not rescue dlc and dld expression in Rspo1 morphants. (A) Lateral view image of wild-type 17 hpf embryo with box indicating the location of in situ images. The cartoon shows a flat mount section of the tail of the embryo with formed somites and the pre-somitic mesoderm indicated. Wild-type (B,F), Rspo1 MO (C,G), Rspo1 MO + vegfaa-165 mRNA (D,H), or Rspo1 MO + vegfaa-121 mRNA (E,I) were examined by WISH for expression dlc (B-E) or dld (F-I). Numbers of embryos displaying the depicted phenotype are indicated. Magnification 160X. (J-K) dlc and dld mRNA (mean±SEM) levels relative to ef1 control quantified by qRT-PCR from uninjected (WT), Rspo1 MO Rspo1 MO + vegfaa-165 mRNA and Rspo1 MO + vegfaa-121 mRNA injected animals. * significantly different from WT (p ≤ 0.005) determined by one-way ANOVA.

Wnt16, Dlc, and Dld do not rescue vegfaa expression in Rspo1 morphants. (A) Diagram of the location of interest. Vegfaa expression is found in the medial somite of the trunk and tail at 24 hpf. In situ hybridization for vegfaa expression in wild-type (WT; B), Rspo1 morphants (Rspo1 MO; C), Rspo1 MO plus dlc mRNA (Rspo1 MO + dlc, D), Rspo1 MO plus dld mRNA (Rspo1 MO + dld, E), Rspo1 MO plus dlc and dld mRNA (Rspo1 MO + dlc + dld, F), and Rspo1 MO plus wnt16 mRNA (Rspo1 MO + wnt16, G). Numbers of embryos displaying the depicted phenotype are indicated. Magnification 160X.