Fig. 4

Fig. 4

Reintroduction of endodermal cells into the endodermless embryo rescues defective migration of the head kidney and the PCV.

(A–N) The inhibition of osr1 expression led to a rescue of renal, interrenal and PCV phenotypes in the sox32 morphant. Dorsal views of Tg(wt1b:GFP) (A–D,A'–D'), Tg(sox17:EGFP)^s870(E–H) and Tg(fli1:EGFP)^y1(I–L,I'–L',I”–L”) embryos injected with sox32MO, osr1MO, sox32/osr1 double-MOs or STD-MO, respectively, and harvested at either 34 hpf (A–D,A'–D',E–H) or 36 hpf (I–L,I'–L',I”–L”) for labelling steroidogenic IR (orange arrows) by 3β-Hsd activity staining. PG and pronephric tubules (pt) were detected by GFP expression in Tg(wt1b:GFP), while the gut tube was detected by GFP in Tg(sox17:EGFP)^s870. The embryos are oriented with anterior to the top. Distances between bilateral IRs and the outer edges of the PCVs at the level of the IR as described in (I–L,I'–L') are quantified in (M,N). When the osr1 MO was co-injected with the sox32 MO, the convergence defects of bilateral IRs and PCVs were significantly alleviated. Red brackets mark lateral boundaries of the PCV branches at the level of the IR. (O–U) Transplantation of sox32-expressing cells into the sox32 morphant rescued the defective migration of bilateral PCVs and IRs. (O) Schematic of the transplantation approach using Sox32 to target donor cells to the endoderm. (P) The lateral view of a representative recipient (n = 11/12) shows that grafted sox32RNA⁺Dextran⁺ cells were associated with the PCV endothelium. The sox32RNA⁺Dextran⁺ cells near the PCV are marked by white arrowheads. The embryo is oriented with anterior to the left. Dorsal views of the IR and the PCV in the control, the sox32 morphant and the recipient are shown in (Q–S), and the distance between bilateral IRs and PCVs are quantified in (T,U) respectively. *P < 0.05; **P < 0.005; ***P < 0.001 (Student's t-test). Scale bar, 50 μm.