Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo - PubMed (original) (raw)

Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo

Janna Krueger et al. Development. 2011 May.

Abstract

Endothelial tip cells guide angiogenic sprouts by exploring the local environment for guidance cues such as vascular endothelial growth factor (VegfA). Here we present Flt1 (Vegf receptor 1) loss- and gain-of-function data in zebrafish showing that Flt1 regulates tip cell formation and arterial branching morphogenesis. Zebrafish embryos expressed soluble Flt1 (sFlt1) and membrane-bound Flt1 (mFlt1). In Tg(flt1(BAC):yfp) × Tg(kdrl:ras-cherry)(s916) embryos, flt1:yfp was expressed in tip, stalk and base cells of segmental artery sprouts and overlapped with kdrl:cherry expression in these domains. flt1 morphants showed increased tip cell numbers, enhanced angiogenic behavior and hyperbranching of segmental artery sprouts. The additional arterial branches developed into functional vessels carrying blood flow. In support of a functional role for the extracellular VEGF-binding domain of Flt1, overexpression of sflt1 or mflt1 rescued aberrant branching in flt1 morphants, and overexpression of sflt1 or mflt1 in controls resulted in short arterial sprouts with reduced numbers of filopodia. flt1 morphants showed reduced expression of Notch receptors and of the Notch downstream target efnb2a, and ectopic expression of flt4 in arteries, consistent with loss of Notch signaling. Conditional overexpression of the notch1a intracellular cleaved domain in flt1 morphants restored segmental artery patterning. The developing nervous system of the trunk contributed to the distribution of Flt1, and the loss of flt1 affected neurons. Thus, Flt1 acts in a Notch-dependent manner as a negative regulator of tip cell differentiation and branching. Flt1 distribution may be fine-tuned, involving interactions with the developing nervous system.

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Figures

Fig. 1.

Fig. 1.

Expression of flt1 isoforms in zebrafish embryos. (A) Structural organization of membrane-bound flt1 (mflt1) and soluble flt1 (sflt1). sflt1 contains a unique mRNA sequence, not present in mflt1, that encodes the 5′ region of intron 10. (B) Taqman analysis shows higher expression of sflt1 than mflt1 in control embryos at 24 hpf, 30 hpf and 48 hpf. f.c., fold change relative to mflt1 expression at indicated time-points. Error bars indicate s.e.m. (200 embryos/group from four separate experiments). (C) Whole-mount in situ hybridization with antisense riboprobes directed against mflt1 or sflt1; embryos at 30 hpf. Note that mflt1 is expressed in segmental sprouts, aorta and cardinal vein; sflt1 is expressed in segmental sprouts and aorta. Ao, dorsal aorta; PCV, posterior cardinal vein; Se, segmental artery.

Fig. 2.

Fig. 2.

Flt1 regulates segmental vessel branching morphogenesis. (A,B) Reduced mFlt1 and sFlt1 protein levels (A) and excessive segmental vessel branching (B) in zebrafish flt1 morphants. The red arrowheads in B indicate the aberrant connection between adjacent segmental vessels. (C,D) Injection of mRNA encoding mflt1 or sflt1 rescued segmental branching defects in flt1 morphants. MO, flt1 ATG morpholino. _y_-axis shows percentage of examined embryos with aberant segmental vessel branching, as shown in B (lower middle panel; 120 embryos/group from four separate experiments). (E,F) Overexpression of mflt1 (top row) or sflt1 (bottom row) in control embryos results in short segmental artery sprouts (E, left) and reduced filopodia extensions (E, right). The boxed regions are shown at higher magnification in the right-hand panels. _y_-axis shows percentage of embryos with reduced segmental vessel sprouts, as shown in E. **, P<0.01; ***, P<0.001; Student's _t_-test. Error bars indicate s.e.m. (120 embryos/group from three separate experiments).

Fig. 3.

Fig. 3.

Flt1 regulates endothelial tip cell formation. (A,B) Time-lapse in vivo imaging shows increased tip cell numbers and proliferating endothelial tip cells in segmental vessels of zebrafish flt1 morphants. Note that in flt1 morphants the leading cell number 1 gives rise to two progeny cells termed 1.1 and 1.2. _y_-axis shows the percentage of examined flt1 morphants with proliferation in tip or stalk cells. (C) Quantification of endothelial cell numbers in four consecutive segmental vessel (ISV) – dorsal longitudinal anastomotic vessel (DLAV) loops reveals an increase in flt1 morphants. (D) Anti-phospho-histone H3 staining confirms endothelial proliferation in segmental vessels of flt1 morphants. **, P<0.01; Student's _t_-test. Error bars indicate s.e.m.

Fig. 4.

Fig. 4.

Tip and stalk cell marker expression and Notch signaling in flt1 morphants. (A,B) Expression of kdrl, flt4 and dll4 in control and flt1 morphants. Expression of the tip cell marker kdrl in segmental sprouts in Tg(kdrl:hras-mcherry)s896 transgenic zebrafish embryos (A, left) and kdrl in toto in situ hybridization (A, overview and detail). Note the ectopic expression of flt4 in the dorsal aorta of flt1 morphants (A, fourth panel). Expression of the stalk cell markers nrarpa and nrarpb is reduced, but expression of dll4 is maintained in morphants (A, fifth panel; B). (C,D) In situ hybridization and Taqman analysis of the trunk region show reduced expression of the receptors notch1a, notch1b, notch2 and notch3 and of the Notch ligands jag1a, jag1b and jag2 in flt1 morphants. Reduced expression of efnb2a suggests loss of Notch signaling in flt1 morphants. (E) Conditional overexpression of notch1a intracellular cleaved domain (NICD) in flt1 morphants (right) rescues segmental arterial branching defects. (F) Segmental branching pattern in dll4 morphants (red arrowhead) or in embryos treated with the Notch γ-secretase inhibitor DAPT (red arrow) did not phenocopy the branching pattern of flt1 morphants (yellow arrowhead). The boxed regions are shown at higher magnification in the right-hand panels. DA, dorsal aorta; PCV, posterior cardinal vein. *, P<0.05; **, P<0.01; Student's _t_-test. Error bars indicate s.e.m. f.c., fold change relative to age-matched control embryos.

Fig. 5.

Fig. 5.

Distribution of Flt1 in vessels and nerves. (A) Comparison of flt1BAC:yfp and kdrl:cherry expression in segmental artery sprouts of 30 hpf Tg(kdrl:ras-cherry)s916 × Tg(flt1BAC:yfp) double-transgenic zebrafish embryos, in controls (top row) and flt1 morphants (bottom row). In controls, note the overlap of flt1BAC:yfp and kdrl:cherry expression in tip and stalk cells. At 30 hpf, expression of flt1BAC:yfp is restricted to the vascular compartment. After injection of flt1 ATG-blocking MO, expression of flt1BAC:yfp in sprouts appears less intense, but is not completely lost. (B) Injection of flt1 ATG-blocking MO in Tg(kdrl:ras-cherry)s916 × Tg(flt1BAC:yfp) double-transgenic embryos causes hyperbranching of segmental vessels and affects neurons. In controls at 48 hpf, flt1BAC:yfp marks segmental vessels and a subset of spinal cord neurons (arrowhead). Injection of the flt1 ATG-blocking MO induces aberrant vessel patterning and the neuronal flt1BAC:yfp expression domain is reduced. Injection of the flt1 ATG-blocking MO in Tg(kdrl:hras-mcherry)s896 × Tg(huC:egfp) double-transgenic embryos affects neurons. (C-E″) Flt1 immunostaining in Tg(huC:egfp) neuronal reporter embryos shows Flt1 throughout the neural tube. Overview of neural tube (C-C″) and different focal planes from dorsal to ventral (D-E″) are shown. (F) Flt1 immunostaining in Tg(flt1BAC:yfp) embryos shows co-localization of flt1BAC:yfp with Flt1 antibody staining in spinal cord neurons (boxed). (G) Immunostaining for Flt1 and zn-12 shows Flt1 staining of Rohon-Beard sensory neurons. (H) Overexpression of fli1ep:cherry-sflt1 in Tg(huC:egfp) embryos results in neuronal sFlt1-cherry expression throughout the neural tube. Arrowheads indicate co-localization of huC:egfp with sFlt1-cherry on neuronal cell bodies. (I) Overexpression of kdrl:egfp-sflt1 in Tg(kdrl:hras-mcherry)s896 embryos shows sFlt1-egfp expression in vessels and the neural tube (arrowheads). Nt, neural tube; Se, segmental artery.

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