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Figure 2. Clp24 is required for vascular and lymphatic development in D. rerio and X. laevis. (A) Control-injected (A,C) and clp24 MO- injected (B,D) embryos of a fluorescent zebrafish (Fli1:eGFPy1) line were screened at 6 dpf (A,B) and 48 hpf (C,D). Note extra branching of the ISVs (arrows in B) and absence of TD (arrowheads in B) at 6 dpf, and absence of parachordal lymphatic precursors (PL) (arrow in C) in clp24 morphants (arrowheads in D). (E,F) TD formation in clp24 morphants. (E) Percentage of affected embryos. (F) Average TD length over 10-somite tail segment at 6 dpf. (*) P < 0.05 versus control. (G,H) At stage 46, clp24 MO-treated (H) but not control-treated (G) X. laevis tadpoles had massive edema in heart, gut, and cloaca region (arrowheads). (I) Transgenic Tg(Flk1:eGFP) X. laevis embryos were injected with 40 ng of clp24 (J,L) or control MO (I,K). (I,J) Small capillaries, but not the main blood vessels, were to a large extent missing in clp24 morphants (arrowheads). (J) Both the VCLV and the DCLV failed to assemble into a compact lymph vessel, and the LECs appear dispersed and disorganized. (K,L) Lymphangiography for MO-treated tadpoles. Note the absence of dye uptake by the malformed VCLV in clp24 morphants (arrowhead, L) as compared with control (arrowheads, K). (M,N) Control (M) and clp24 (N) MO-treated X. laevis embryos were analyzed at stage 35/36 by prox1 ISH. The clp24 morphants show less staining in the PCV, heart (H), and rostral lymph sac (RLS), and in the area of the future lymph hearts (LH) and of the future VCLV. Note a reduced number of prox1-positive cells migrating across the tail from the VCLV to form the DCLV (arrowheads). Staining in liver (L) seems similar to control. (DA) Dorsal aorta; (DLAV) dorsal longitudinal anastomosing vessel. |
