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Saharinen P
,
Helotera H
,
Miettinen J
,
Norrmen C
,
D'Amico G
,
Jeltsch M
,
Langenberg T
,
Vandevelde W
,
Ny A
,
Dewerchin M
,
Carmeliet P
,
Alitalo K
.
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The Claudin-like protein of 24 kDa (CLP24) is a hypoxia-regulated transmembrane protein of unknown function. We show here that clp24 knockdown in Danio rerio and Xenopus laevis results in defective lymphatic development. Targeted disruption of Clp24 in mice led to enlarged lymphatic vessels having an abnormal smooth muscle cell coating. We also show that the Clp24(-/-) phenotype was further aggravated in the Vegfr2(+/LacZ) or Vegfr3(+/LacZ) backgrounds and that CLP24 interacts with vascular endothelial growth factor receptor-2 (VEGFR-2) and VEGFR-3 and attenuates the transcription factor CREB phosphorylation via these receptors. Our results indicate that CLP24 is a novel regulator of VEGFR-2 and VEGFR-3 signaling pathways and of normal lymphatic vessel structure.
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-somitetail 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.
Supplemental Figure 4. D. rerio and X. laevis clp24 sequences and blood vascular effects in clp24 morpholino treated X. laevis embryos. A) D. rerio clp24 mRNA sequence. Black, bold text indicates a morpholino (MO) targeted against the 5untranslated region (clp24-UTR), blue underlined text indicates a MO (clp24-ATG)targeted against the translational start site (bold underlined) of clp24 mRNA. X. laevis partial mRNA sequence. Underlined is a MO targeted against the 5untranslated region, and in bold a MO targeted against the translational start site (bold underlined). B) Results from the experiments using the D. rerio clp24-ATG MO confirm the results obtained using the clp24-UTR MO, presented in Fig. 2. Toxic off-target effects of the second Clp24 MO assumably related to induction of a p53-dependent cell death pathway were attenuated by co-injection with a p53 morpholino as described (Robu et al. 2007). C-F) Control (C, D) and clp24 morpholino oligonucleotides (40 ng) (E, F) were injected into X. laevis embryos and the embryos were analyzed at st 33/34 by in situ hybridization with a msr probe staining the vasculature. At stage 32 msr was expressed in the heart and developing primary blood vessels. The clp24 morphants show less staining in the dorsal aorta (DA) and in the posterior cardinal vein (PCV), and have less intersomitic vessel (ISV) sprouts as compared to the control (3.67 0.39 (n=21) vs 4.1 0.33 (n=20), respectively), although this decrease did not reach statistical significance.
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