Figure 1.
Stage specific inhibition of Bmp in K. marmoratus.
Embryos were exposed to 100 µM dorsomorphin at the 32-cell (B), late blastula (C) and 80% epiboly (D) stages of development. Photographs of the embryos were taken 3 days post-fertilization. A1–3: Control (n = 20/20). B1–3: splitbody (phenotype variation details in Figure 3), this phenotype is characterised by absence of a distinct tail region (B1 arrowhead), morphologically undifferentiated head region (B2 arrowhead) and split body axis (B2 arrow), and cell islands in the posterior region (B3 arrowhead). C1–3: Curled tail (n = 8/10), this phenotype resembles snailhouse seen in zebrafish and is characterised by its curled tail (C3 arrowhead). D1–3: Bent tail (n = 12/12, this phenotype primarily displayed a bent tail (D3 arrowhead). Overview images are lateral views and head/tail images are dorsal views of the embryos. Scale bars: 250 µm.
Figure 2.
Bmp inhibition delays epiboly progression in K. marmoratus.
Epiboly coverage was recorded at day 1 (A) and day 2 (B) post-fertilization (dpf) in embryos exposed to 100 µM dorsomorphin (DM) at the 32-cell stage. Progression of the yolk syncytial layer (YSL) during gastrulation was assessed via staining of yolk syncytial nuclei (YSN) using Sytox Green. The green fluorescent YSN were observed 1 dpf (C). A1, 2: As control embryos reach c. 70% epiboly (A1 arrowhead, n = 10/10), DM treated embryos are delayed with epiboly covering c. 30% of the yolk (A2 arrowhead, n = 10/10). B1, 2: Controls reach the otic vesicle formation stage (B1, n = 10/10) whilst exposed embryos are lagging behind around 90% epiboly (B2 arrowhead, n = 10/10). C1, 2: Shortly after epiboly closure, control embryos enter the eye formation stage (C1, n = 10/10) (embryo and the eye are outlined) and YSN are spread all over the yolk. On the other hand DM exposed embryos are still mid-epiboly and fluorescent YSN are observed near the blastoderm margin (C2 arrowhead, n = 10/10), demonstrating that YSN are also delayed by inhibition of Bmp signalling. All images are lateral views of the embryos. Scale bars: 250 µm.
Figure 3.
The neural tube is separated in embryos of the splitbody phenotype.
K. marmoratus embryos were exposed to 100 µM dorsomorphin (DM) at the 32-cell stage (B, C), and 200 µM DM at the late blastula stage (D, E) of development. These embryos were then fixed 3 days post-fertilization and used for in situ hybridization using a sox3 probe (stains all neural tissue) (A–E2, A–E4) and Hoechst staining (a blue fluorescent DNA stain) (A–E1, A–E3) in order to examine body contour and split neural tube (A–E1, 2), and the nature of the posterior isolated cell lumps or cell islands (A–E3, 4). A1–4: Control embryo (n = 20/20). B, D: Splitbody individual with an opened end of the body axis and neural tube split (B1, 2 arrowheads n = 19/20, and D1, 2 arrowheads n = 12/20). Splitbody individuals with a closed end, as both strands of the body axis and neural tube join in their most posterior region (C1, 2 arrowheads n = 1/20, and E1, 2 arrowheads n = 8/20). All DM embryos presented here generated cell islands (B-E3 arrowheads) with distinct sox3 positive staining (B–E4 arrowheads). All images are dorsal views of the embryos. Scale bar: 250 µm.
Figure 4.
Somites and the notochord are divided in the splitbody phenotype.
K. marmoratus embryos were exposed to 100 µM dorsomorphin at the 32-cell stage and fixed 1 and 4 days post-fertilization in order to stain the notochord by in situ hybridization using a medaka ntl probe (A, B, C), and somites using the myosin antibody MF-20 (D, E). In control embryos at late gastrula, ntl stained axial mesoderm in the dorsal axis (A1 arrowhead, n = 10/10), whereas in DM treated embryos these cells appeared to have stayed in the lateral domains (A2 arrowhead, n = 10). At day 4, ntl stained the notochord in the tip of the tail for control embryos (B2 arrowhead, n = 10/10), whereas splitbody embryos had the tips of both body axes stained with ntl (C2 arrowheads, n = 10/10). For control embryos, somites are formed as pairs arranged either side of the neural axis (D1–3 arrowheads, n = 10/10). In the splitbody phenotype, somites were unpaired and separated in the two body axes (E1 Hoechst staining showing the body split; E2, 3 arrowheads, somites are present in both axes, n = 10/10). Photographs were taken at late gastrula for A, and 4 days post-fertilization for B–E. Images in A are lateral views and for B–E dorsal views of the embryos. Scale bars: 250 µm.
Figure 5.
Dorsomorphin inhibits phosphorylation of Smad1/5.
Bmp signalling activity was quantified by measuring phosphorylation of Smad 1/5 at the late gastrula stage. Embryos were exposed to 100 µM from 32-cell as well as 100 and 200 µM DM from late blastula. These were then frozen at the late gastrula stage and used for Western Blotting. A: Quantification of densitometry results obtained from 3 independent experiments (Mean ± SE), normalised to total Smad and indicated as fold increase over the resting control condition. All 3 treatments were significantly different from the control as indicated by asterisks (P<0.001), but no significant differences were observed between the treatments. B: Representative Western Blot of 3 independent experiments showing the levels of total Smad1/5/8 and phospho-Smad1/5 at late gastrula from the dose and stage specific treatments. These data demonstrate that all 3 treatments equally suppress phospho-Smad1/5 by late gastrula.