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Cardiovascular Research Advance Access first published online on November 3, 2008
This version [Corrected Proof] published online on November 23, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn294
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org.

A crucial role of caldesmon in vascular development in vivo

Ping-Pin Zheng1, Lies-Anne Severijnen2, Marcel van der Weiden1, Rob Willemsen2,{dagger} and Johan M. Kros1,*,{dagger}

1 Department of Pathology, Erasmus Medical Center, JNI Room 230-c, Dr Molewaterplein 50, PO Box 1738, 3000 DR Rotterdam, The Netherlands
2 Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands

* Corresponding author. Tel: +31 10 7043905; fax: +31 10 7043905. E-mail address: j.m.kros{at}erasmusmc.nl

Received 24 July 2008; revised 28 October 2008; accepted 29 October 2008

Time for primary review: 13 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results and discussion
 References
 
Aims: We explored the in vivo effects of knockdown of caldesmon on vascular development in zebrafish.

Methods and results: We investigated the effects of caldesmon knockdown on the vascular development in a zebrafish model with special attention for the trunk and head vessels including the aortic arches. We examined the developing fishes at various time points. The vascular abnormalities observed in the caldesmon morphants were morphologically and functionally characterized in detail in fixed and living embryos. The knockdown of caldesmon caused serious defects in vasculogenesis and angiogenesis in zebrafish morphants, and the vascular integrity and blood circulation were concomitantly impaired.

Conclusion: The data provide the first functional assessment of the role of caldesmon in vascular development in vivo, indicating that this molecule plays a crucial role in vasculogenesis and angiogenesis in vivo. Interfering with caldesmon opens new therapeutic avenues for anti-angiogenesis in cancer and ischaemic cardiovascular disease.

KEYWORDS Caldesmon; Vascular development; Zebrafish model; Vasculogenesis; Angiogenesis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results and discussion
 References
 
Caldesmon (CaD) is evolutionally conserved among vertebrates.1 The zebrafish homologue is similar to mammalian low-molecular-weight caldesmon (l-CaD). Previously, we reported the specific upregulation of this protein (l-CaD) in glioma neovasculature and its association with migration and proliferation of endothelial cells (ECs) and endothelial progenitor cells (EPCs) in human tumours.2–7 The findings triggered us to explore the effects of this protein on the development of blood vessels in vivo for the design of new therapeutic strategies. Here we explored the effects of knockdown of CaD on the vascular development in zebrafish embryos. Zebrafish embryos can survive several days without a functioning circulatory system, allowing detailed analysis of the animals with severe cardiovascular defects.

The development of the vascular system in vertebrates occurs by two distinct processes: vasculogenesis and angiogenesis. The same primary vasculogenic vessels that establish the initial circulatory circuits in other vertebrate embryos are also present in the zebrafish. These vessels include the dorsal aorta (DA) and posterior cardinal vein (PCV) in the trunk and the internal carotid artery, the primordial hindbrain channel, the anterior cardinal vein, and the basilar artery in the head.8 The formation of most of the subsequent vessels in the embryo occurs by sprouting from pre-existing vessels in a process known as angiogenesis. Many (presumably angiogenic) blood vessels that subsequently develop in the zebrafish have orthologues in other vertebrates and among these are the central cranial arteries and the massive network of microvessels in the head, the dorsal longitudinal anastomotic vessels (DLAVs), the intersegmental vessels (ISVs), the subintestinal veins (SIVs), the caudal vessel plexus (CVP), the parachordal vessels, the vertebral vessels in the trunk, and more.9 The embryology of the aortic arch (AA) system in zebrafish is very similar to that of birds and mammals.10 Six pairs of vessels, connecting the ventral aorta to the lateral DA, emerge in a cranial-to-caudal sequence, each of which is embedded in its respective pharyngeal arch which is collectively known as the branchial AAs.10 The AA primordia arise by vasculogenesis and extend via angiogenesis.10 At the molecular level, several important genes, including VEGF, Flk-1/KDR, Fli-1, Flt-1, Tie-1, and Tie-2, have been cloned in zebrafish and show expression patterns similar to those in mammals.11,12 The striking conservation of vascular anatomy and the expression pattern of the associated genes across the vertebrate phyla indicate similar vasculogenic and angiogenic signalling pathways for blood vessel formation and patterning.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results and discussion
 References
 
2.1 Morpholino injections and verification of the specificity
The caldesmon antisense morpholino oligonucleotides (MOs) and 5-base mismatch controls were purchased from Gene Tools (Philomath, OR, USA). The caldesmon antisense MO1 5-AGTAAAGTCTCTTATTCTTCAACGC-3 and MO2 5-TAAGAGTTCATCCTGTAGAGTGATG-3 were designed to inhibit translation of the caldesmon RNA (gene: ENSDARG00000032052; transcript: ENSDART00000067366; translation: ENSDARP00000067365) and a 5-base mismatch control 5- AGAAAACTCTCTTATTGTTGAAGGC-3 was used. First, in a titration experiment, the MOs were injected into the yolk sac of zebrafish embryos between one- and two-cell stages at different concentrations (2, 4, and 8 ng/embryo), and the embryos were raised at 28.5°C until analysis under standard laboratory conditions. The concentration (4 ng/embryo) was used in all the subsequent experiments, because the survival of the embryos was satisfactory (>86%). The use of zebrafish embryos was approved by the Institutional Review Board for experimental animals.

There are several options to determine whether a phenotype is the result of knocking down a gene-of-interest by blocking translation with morpholinos (MO): (a) quantification of the target protein by an antibody (check if the translation is blocked); (b) RNA rescue experiments; (c) a control morpholino with 5 bp mismatch (discussed earlier); and (d) application of a second MO with similar blocking effects (discussed earlier). We verified the specificity of the MO phenotype in our experiments by combining the above-mentioned methods.

2.2 Quantification of the homologue protein of caldesmon
Quantification of the homologue protein was performed by vision assay (see whole-mount staining and section immunohistochemistry), dot blot, and whole-mount enzyme-linked immunosorbent assay (ELISA) by using the antibody EP050852 (Eurogentec, Belgium) against the protein. Western blotting was impossible because the antibody was not working in a reducing status.

In whole-mount ELISA, zebrafish embryos were fixed in 4% paraformaldehyde (PFA) at least 3 h, washed and permeabilized by TBST (0.05% Triton X-100 in TBS), and treated with alkaline phosphatase (AP) suppressor (0.5 M EDTA) to inhibit endogenous AP for 1 h. Next, the embryos were routinely blocked and incubated with EP050852 (1:50) for two to three overnights at 4°C, washed with TBST, and incubated with AP-conjugated secondary antibody (1:100) for two overnights at 4°C. After washing, the embryos were placed in a 96-well microplate, one embryo per well. p-nitrophenyl phosphate substrate (Sigma-Aldrich) was used as an enzyme substrate. The protein was quantified by measuring the optical density (OD) of the enzymatic end-product at 405 nm using a microplate reader (Thermo Multiskan Ascent). The embryos without primary antibody incubation were processed to estimate non-specific background, the mean value of which was subtracted from each 405 measurement. The mean value of the normal embryos was as the ODcontrol in the below formula. The MO inhibition effect of the protein was calculated by the following formula: %inhibition=[(ODcontrol–ODMO)/ODcontrolx100%].

For the dot blot assay, a nitrocellulose membrane (NM) was used for spotting the samples. The extracts were made by identical numbers of control embryos, wild type (WT), and caldesmon morphants (CaD-MOs) at an identical developmental stage, which were homogenized by an identical volume of lysis buffer. The protein concentration was adjusted at 2 µg/µL. The extract (2 µL) from controls, WT, and CaD-MOs was spotted on the NM, respectively. The NM was air-dried and blocked in blocking solution (10% non-fat dry milk) for 1 h at room temperature (RT). The NM was incubated with EP050852 (1:100) for 2 h at RT, washed in PBS (3 x 10 min), followed by incubating with horseradish peroxidase-conjugated secondary antibody (1:1000) for 2 h at RT and washed (3 x 10 min). The target protein spots were visualized by enhanced chemiluminescence (Amersham Biosciences Corp., Piscataway, NJ, USA). The films were scanned for analysis and imaging.

2.3 Morpholino rescue experiment
The cDNA of human CaD served as a template containing a T7 RNA polymerase promoter. RNA was in vitro synthesized using the mMESSAGE mMACHINE kit (Ambion, Austin, TX, USA) according to the manufacturer's protocol and co-injected with the morpholinos.

2.4 Immunostaining of whole mount and sections
Briefly, embryos were fixed by 4% PFA at RT for minimal 3 h following standard procedures. Embryos were treated with 1 M NH4Cl at RT for 3 h to quench autofluorescence, permeabilized, blocked by 5% goat serum in PBST for 1 h at RT, and incubated with the selected primary antibodies: VEGFR2/Flk1 (Lab Vision), VEGFR1/Flt1 (Lab Vision), endothelial nitric oxide synthase (eNOS) (Lab Vision), CD105 (Lab Vision), Glut-1 (Dako), occludin (Zymed), ZO-1 (Zymed), and Tie-2 (R&D System) at dilution 1:50 to 100 for two to three overnights at 4°C. After post-incubation washing, embryos were incubated with FITC- or rhodamine-conjugated goat-anti-rabbit or goat-anti-mouse (Jackson ImmunoResearch Laboratories, Inc.) at a dilution of 1:100 for two overnights at 4°C. After thoroughly washing, fluorescence images were recorded by a fluorescence microscope and/or confocal laser scanning microscopy (CLSM). Embryos at identical developmental stages, processed without primary antibody, were used as controls for each experiment. 4',6-diamidino-2-phenylindole (DAPI) was used for nuclei counterstaining.

Immunohistochemical analysis of sections was performed by standard methods. Briefly, embryos were fixed, dehydrated, embedded in paraffin, and sectioned (5 µm). Sections were deparaffinized, blocked, antigen-retrieved, incubated with the selected primary antibodies: EP050852 (Eurogentec) at 1:100 and Glut-1 (Dako) at 1:150, washed, and stained by AP-conjugated secondary antibody. AP-based substrate was used for visualization.

The cross-species reactivity of the antibodies used was confirmed by immunohistochemistry, western blotting, and dot blot, unless already tested by others,13–15 or the antibody was specifically raised in zebrafish (Tie-2).

2.5 Whole-mount endogenous alkaline phosphatase (EAP) staining
EAP staining was applied following fixation and permeabilization of the embryos at a defined stage in 4% PFA and 0.05% Triton X-100 in PBS (PBST). The staining procedures were as described previously.16

2.6 Whole-mount lectin staining
Briefly, embryos were fixed with 4% PFA, permeabilized with PBST, and washed by PBS, followed by incubation at 4°C overnight with FITC-conjugated BSI-B4 (Sigma, 10 µg/mL) in PBS. After subsequent washes, fluorescent images were recorded by fluorescence microscopy and/or CLSM.

2.7 Parameters for assessment of the cardiovascular system and the circulation
The parameters for assessing the cardiovascular system included: (a) DA and PCV; (b) ISV; (c) DLAV; (d) SIV; (e) CVP; (f) cranial vessels (CVs); and (g) AA. The parameters for monitoring the circulation included: (a) relative number of circulating red blood cells (RBCs); (b) axial circulation; (c) ISV and DLAV circulation; and (d) circulation shunting.

2.8 Staining of circulating red blood cells
Non-fixed embryos were stained by o-dianisidine for 15 min in the dark, as described previously.17

2.9 General strategy of labelling of the developing vasculature
Quantification of mRNA does not necessarily provide information about the amount of active proteins in a cell because of nonsense-mediated mRNA decay and microRNA-mediated mRNA silencing.18 These mechanisms are conserved in vertebrates.19 Therefore, in this study, we preferentially used protein-based approaches for fine characterization of the effects on the various blood vessels in the CaD-MOs.

For the overall screening of the structural defects of the developing vasculature, we used whole-mount EAP and BS-l isolectin B4 staining. RBC expression of globulin was used to examine the functional integrity of the circulatory system. In vivo imaging was used for monitoring the dynamics of the circulation. At the molecular level, we used the molecular markers VEGFR2/Flk1, VEGFR1/Flt1, eNOS, CD105, occludin, Glut-1, ZO-1, and Tie-2 to specifically label the angiogenic ECs/EPCs and their interaction as well as the regionally specialized vessels according to their labelling peculiarity. VEGFR2/Flk1 and VEGFR1/Flt1, encoding receptor-type protein tyrosine kinases, are specific markers for angiogenic EC/EPC.12,20 The expression of VEGFR2/Flk1 diminishes in the late developmental stages. For example, by 26hpf, the expression of VEGFR2/Flk1 can be seen in the trunk in the domains, where the ISVs will form and the expression is still detected in newly forming ISVs after the onset of the blood circulation.21 By 36hpf, the VEGF2 expression is weak or not detectable in newly formed vessels.21 In contrast, VEGFR1/Flt1 retains its expression until later stages.22 Clearly, the expression of the various markers is dependent on the specific developmental stage. eNOS and CD105 are well-characterized endothelial-lineage markers, predominantly expressed in relatively differentiated EPCs and mature ECs.23,24 Occludin and ZO-1 are major components of tight junctions (TJ) of endothelium,15 and the molecules serve well as a monitor of vascular integrity. Glut-1, a marker for the integrity of the brain–blood barrier (BBB), is expressed in the ECs of microvessels with a barrier property.25,26 Tie-2 is an endothelium-specific receptor tyrosine kinase.27

2.10 Sequence identification and alignment of the zebrafish homologue to human CALD1
Based on a reciprocal BLAST analysis of the Ensembl zebrafish genomic sequence database, the following gene (Gene ID: ENSDARG00000032052; peptide ID: ENSDARP00000067365) was identified as a homologue (a putative orthologue) to human CALD1 in the Ensembl Gene Report. The human caldesmon (CALD1) sequences were acquired from public databases (NCBI). We aligned the homologue against the peptide sequences (each isoform) of the human CALD1. The sequence comparisons resulted in a nearly equal identity and similarity for each isoform against the homologue (Table 1). According to domain mapping of the human CALD1, there are two major functional domains: N-terminal myosin/calmodulin-binding domains and C-terminal calmodulin/actin-binding domains, which are conserved in each isoform.28–30 The functional domains were further aligned with the homologue, respectively (Table 1).


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  		Table 1 Alignment of human CALD1 isoforms to the zebrafish homologue

 

   3. Results and discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results and discussion
References
 
The evolutionarily conserved similarities of the amino acid sequences at the two major functional domains between the zebrafish homologue and the human CaD reach 67.5 and 55.6% (Table 1), respectively. Therefore, the homologue may be regarded as an orthologue of the human caldesmon gene (CALD1). The expression pattern of CaD in 2dpf, 3dpf, and 4dpf is similar. It is predominantly expressed in the AA (Figure 1A1, C1 and E1); the jaw primordium (Figure 1A1); the subintestinal vessels (Figure 1B1); DA; PCV; ISVs; and other regions such as somites (Figure 1D1 and E2). At earlier stages, CaD is predominantly expressed in DA and PCV (Figure 1F).


Figure 1
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  		Figure 1 Expression of the caldesmon (CaD) homologue in zebrafish embryos and its knockdown in the caldesmon morphants (CaD-MOs). (A1) CaD expression in aortic arch (AA) (square) and JP (arrows) in a 3dpf wild-type (WT). (A2) A reduction of CaD expression in this region was seen in the corresponding CaD-MOs (square). (B1) CaD are expressed in the subintestinal vessel in a 3dpf WT (arrow). (B2) Loss of this protein from this region is seen in the corresponding CaD-MO (arrow). (C1) CaD is expressed in AA (square) in a 4dpf control. (C2) Massive loss of expression in this region is seen in the corresponding CaD-MO. (D1) CaD is expressed in dorsal aorta (DA), posterior cardinal vein (PCV) (arrowheads), intersegmental vessels, and other regions such as somites in 4dpf control. (D2) Reduced expression of this protein in these regions is seen in the corresponding CaD-MO. (E1) CaD expression in AA (arrow) in a 2dpf WT. (E2) Expression pattern of CaD in the trunk of a 2dpf WT. The expression pattern is similar to that in 3dpf and 4dpf. (F) CaD is expressed in DA and PCV (arrows) in 1.5dpf±WT. (G) An overall reduction of the protein expression in the CaD-MOs was assessed by the dot blot assay. (H) The mismatch-morpholino (MM)-injected embryos did not show a reduction in the expression. (A1) to (B2): section staining; (C1) to (E2): whole-mount staining, lateral views, anterior towards the left, and dorsal is up; (F): whole-mount staining, whole embryo view. Scale bar, 50 µm.

 
We compared the ATG- and the control-MO-injected embryos at various developmental time points. ATG MOs knockdown of CaD resulted in severe and reproducible phenotypes. Morphological changes were apparent by visual inspection at 2dpf and became even more pronounced at 3dpf through later time points. The phenotypes observed in early stages were still present in 5dpf CaD-MOs. The morpholino phenotype was highly penetrant as representatively demonstrated by 2dpf (88%; n = 96), 3dpf (86%; n = 96), and 4.5/5dpf (92%; n = 126) vs. the control-injected embryos 2dpf (6%; n = 98) and 3dpf (5%; n = 86). All paired comparisons were highly significant (P < 0.001). The specificity of the morpholino phenotype was verified by the following approaches. Reduced or absent expression of this protein was observed in the CaD-MOs by immunohistochemistry (Figure 1A2, B2, C2, and D2). Confirmation of the reduction was obtained by whole-mount ELISA and dot blot assay (Figure 1G). The protein was inhibited ~86% in CaD-MOs as measured by whole-mount ELISA. These results are evidence that the targeted translation was successfully blocked and proof the specificity of the knockdown. Further support for the specificity of the knockdown was the similar effects of a second morpholino when compared with the first one and the insignificant penetrance (~6%) of the 5-base mismatch control morpholino. The specificity was further confirmed by the RNA rescue experiments. Co-injection of the ATG MOs with the RNA resulted in a significant rescue (89%, n = 66, P < 0.001).

The vasculogenic axial vessels (DA and PCV) and the angiogenic trunk vessels (ISV, DLAV, SIV, and CVP) in the CaD-MOs were either completely or partially missing or abnormally patterned (Figure 2). Concomitant impairment of the circulation (Table 2 and Figure 2) and vascular integrity (Figure 2) such as decreased RBCs in the circulation system, disrupted TJs, and haemorrhages were recorded in the morphants. Functionally, in live imaging, the control embryos showed vigorously circulating blood cells throughout the length of the body, including the axial vessels, ISV, and DLAV. In the CaD-MOs, we found a significantly reduced number of circulating blood cells moving sluggishly through the axial vessels with absent or reduced circulating blood cells in the ISV and DLAV (Table 2). The circulatory dysfunction and vascular immaturity are the sequel of abnormal vessels, rather than a defect in RBC development, as normal looking RBCs were still seen in the lumina of the vessels of the CaD-MOs. Other major circulatory defects in the CaD-MOs included misconnection of arteries and veins and shunting (Figure 2 and Table 2).


Figure 2
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  		Figure 2 Defects of developing trunk vasculature and circulation in the caldesmon morphants (CaD-MOs). (A1) VEGFR2/Flk1 labelling in a 3dpf control. The intense-stained sites are located in the tip endothelial cells (ECs) (green arrowhead) and the budding regions (blue arrowheads). (A2) The tip cells are aligned on the bottom of the tissue section (square). (A3) The signal of VEGFR2/Flk1 is perturbed in the corresponding CaD-MO, as reflected by a significant loss of the tip and budding EC population instead of the dot-patchy patterns of dorsal aorta (DA) and posterior cardinal vein (PCV) (arrowheads). (B1) A normally patterned intersegmental vessel (ISV) is revealed by VEGFR1/Flt1 labelling in a 3dpf control. (B2) A complete or partial loss of ISVs with an interrupted DA and loss of dorsal longitudinal anastomotic vessel (DLAV) is seen in the corresponding CaD-MO. (B3) Bright red dots (the cell bodies of ECs) stained by VEGFR1/Flt1 are aligned in the midline without formation of ISVs in a 5dpf CaD-MO. (C1) Occludin is expressed in the trunk vasculature of a 2dpf control. (C2) The signal of occludin is absent in the corresponding regions of the CaD-MO. (D1) Tie-2 outlines the trunk vasculature in a 2dpf control. The expression pattern is similar to that of VEGFR1/Flt1 in (B1). (D2) The Tie-2 signal almost completely disappeared in the CaD-MO. (E1) The caudal vessel plexus (CVP) shows normal patterning as revealed by endothelial nitric oxide synthase (eNOS) staining in a 5dpf control. (E2) A less-developed CVP region with reduced endothelial sheets is seen in the corresponding CaD-MO. (F1) The endothelium of PCV labelled by eNOS is seen as a monolayer in a 5dpf control. (F2) A multilayered staining pattern is seen in the corresponding CaD-MO. (G1) A normal patterning of subintestinal vein (SIV) is shown in a 3dpf control. (G2) An abortive SIV is seen in the corresponding CaD-MO. (H1) Lectin staining outlines the trunk vasculature in a 5dpf control. (H2) Bright green dots (the cell bodies of ECs) labelled by lectin are chaotically distributed. Most dots are out of the normal vascular areas and some are aggregated in clusters. (I1) Red blood cells (RBCs) travel through the body length of a 5dpf control as revealed by RBC staining. (I2) In the corresponding CaD-MO, a remarkable reduction in the circulating RBCs is shown in the axial vessels (DA and PCV), ISV, and DLAV. Extravasation of RBCs from the heart (I2, circle) or haemorrhage (I3). (J1) The PCV is precisely anchored at the ventromost part of the midtrunk in a 5dpf control, as revealed by the RBC staining. The distance between DA and PCV (height) is precisely determined. (J2) The PCV in the corresponding region of the corresponding CaD-MO is detached from the ventromost part by dorsal shifting of the axial vein as revealed by RBC staining. The linear pattern of the blood flow is lost, while widening is noticed in the CaD-MO. In addition, the flow in the PCV is conducted erroneously into the ISV (arrow). (K1) The normal pattern of the caudal circulation is noticed by the continuous blood flow from DA into the caudal artery and its return via the caudal vein in a 5dpf control. (K2) The flow is interrupted in the CaD-MO. Instead, there is direct return into the PCV by shunting (arrows). All panels: whole-mount staining, lateral views, anterior towards the left, dorsal is up. Scale bar, 100 µm.

 


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  		Table 2 Frequencies (%) of circulation defects at 2, 3, and 4.5dpf caldesmon morphants

 
Despite the fact that ISVs are generated by sprouting angiogenesis, it seems more appropriate to describe this process as vasculogenesis type II. There is almost exclusive migration of angioblasts followed by tubular formation and little or no cell division,31,32 rather than traditional angiogenesis including cellular proliferation and migration.33 Thus, the development of the ISVs serves as an ideal model for studying EC migration during vascular development.21 The ISVs are generated by synchronously collective migration of ECs in a two-step process.32 The first step requires the sprouting and migration of ECs from the DA to form a primary network of the ISV segments, whereas the second step encompasses the sprouting of ECs from the PCV interface with this primary network to form the ISV network.32 Anatomically, the ISVs are typically along the vertical somite boundaries (myoseptal boundaries).34 A schematic illustration for the network of the ISVs as well as its relationship to DA, PCV, DLAV, and somites is shown (Figure 3). A well-formed DA and PCV are prerequisites for the formation of an intact primary and secondary network of the ISV segments. In the completed primary ISV network, ECs are located at the DLAV–primary segment junction (ECD), at the level of the parachordal vessels (ECp), and at the DA–primary segment junction (ECA)32 (Figure 3). The normal pattern of ISVs is outlined by Flt1 (Figure 2B1), occludin (Figure 2C1), Tie-2 (Figure 2D1), and lectin (Figure 2H1). The pattern is indicative of the proper alignment of the ECs. In the primary network of the ISVs, a defect in the formation of the DA results in an initially defective ECA with complete loss of the sprouts of the ISVs (Figure 2B3, C2, D2, and H2). The absence of ECp and ECD in the CaD-MOs most likely results from the failure of migration of the ECs, which are committed to become ECp and ECD. The sequel is partial loss of dorsal sprouts of ISVs (Figure 2B2). The ISV formation is completed before 3dpf during normal development.35 However, in 5dpf CaD-MOs, ECs are still aligned in the midline (Figure 2B3 and H2), additional evidence of impaired EC migration. These results proof a role for CaD in the regulation of adhesion-dependent signalling of cytoskeletal organization and cell migration, consistent with various in vitro studies.36–39


Figure 3
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  		Figure 3 Relationship of the trunk vasculature with somites. The scheme illustrates the anatomical relationship of dorsal aorta, posterior cardinal vein, intersegmental vessel, dorsal longitudinal anastomotic vessel, and somites.

 
The large CVs were replaced by disorganized vascular channels, and the cranial microvasculature was significantly reduced (Figure 4). The developing AAs were structurally and functionally damaged in the CaD-MOs (Figure 5). AA defects may associate with other defects in pharyngeal arch-derived structures. However, defects in aortic and pharyngeal arches may also occur separately,10 because the pharyngeal arch structure is highly heterogeneous and receives contributions from endoderm, mesoderm, ectoderm, and neural crest.10 Dissociated defect of aortic and pharyngeal arches is seen in the CaD-MOs (Figure 5G1–G3). In most vertebrates, TJs between adjacent ECs are major components of the BBB,15 including tetraspanning transmembrane proteins such as occludin and claudin or cytoplasmic-anchoring proteins such as ZO-1.40 The BBB in zebrafish is revealed by the expression of ZO-1 in the cerebral microvessels.15 The BBB in zebrafish is functionally and structurally similar to that of higher vertebrates such as humans,15 which is illustrated by overlapping expression of Glut-1 and ZO-1 (Figure 4C1 and C2). In the CaD-MOs, the development of the BBB was interrupted (Figure 4D2).


Figure 4
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  		Figure 4 Defects of cranial vasculature in the caldesmon morphants (CaD-MOs). (A1) Functional cephalic vessels are revealed by actively passing through red blood cells (RBCs) in a 5dpf control. (A2) Normal patterned vessels are greatly reduced and replaced by dilated, aneurysmatic channels (arrows) in the corresponding CaD-MO. (B1) Well-developed cranial microvasculature is seen by CD105 staining in a 5dpf control. (B2) The microvasculature is greatly decreased in the corresponding CaD-MO. (C1) and (C2) On frozen sections of 3dpf wild-type, double labelling of ZO-1 and Glut1 was performed. The image shows brain area. Both antibodies stain microvessels or capillaries and the signals overlap well. (D1) Glut-1 is expressed in the blood–brain barrier capillaries of the normal brain in a 3dpf control. (D2) Loss of Glut-1 expression is observed in the corresponding CaD-MO. (C1), (C2), (D1), and (D2): section staining; all others: whole-mount staining; ventral view; anterior towards top [(A1) and (A2)]; and ventrolateral view and anterior towards the left [(B1) and (B2)]. Scale bar, 50 µm.

 


Figure 5
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  		Figure 5 Defects of aortic arches (AAs) in the caldesmon morphants (CaD-MOs). (A1) AA is seen in a 3dpf control by endogenous alkaline phosphatase (EAP) staining (circle). (A2) AA is mostly lost in the corresponding CaD-MO. (B1) Glut-1 is expressed in AA region in a 3dpf control. (B2) Glut-1 signal is largely lost in the corresponding CaD-MO. (C1) Glut-1 is expressed in the AA region in a tissue section of a 3dpf wild-type (arrow). (C2) The Glut-1 signal is completely lost in the corresponding CaD-MO (arrow). (D1) A more developed AA is seen in a 5dpf control by EAP staining (circle). (D2) AA is lost in the corresponding CaD-MO. (E1) Occludin is expressed in the AA region in the 5dpf control (circle). (E2) The occludin signal is completely lost in the corresponding CaD-MO. (F1) Functional AA is revealed by the red blood cell staining in a 5dpf control (square). (F2) Loss of normal functional AA is seen in the corresponding CaD-MO. [(G1) to (G3)] Glut-1 signal is lost in the CaD-MO (G1), but retains the pharyngeal arches as counterstained by DAPI (G2). (C1) and (C2): section staining; all others: whole-mount staining; ventrolateral view and anterior towards the left. Scale bar, 100 µm.

 
The structural integrity and functional maturity of the blood vessels are not only determined by the presence of normally functioning ECs, but also involve interendothelial junctions and mural cells (smooth muscle cells or pericytes), which are generally conserved among vertebrates. However, some interspecies differences exist. For instance, because there are no additional layers of elastic lamina or mural cells in the vascular walls of the early developing trunk vasculature of zebrafish embryos, vascular integrity is predominantly determined by intact endothelium and associated TJs.41,42 The junction-associated molecules may well serve as sensitive markers of normal or disturbed function of the developing vasculature (Figure 2C1). The haemorrhages (Figure 2I2 and I3), the regional and systemic arrest of blood circulation (Figure 2I2 and Table 2), and the shunting of blood (Figure 2J2 and K2 and Table 2) all are consequences of vascular dysfunction and/or immaturity.

The present study provides the first functional assessment of CaD in embryonic development in vivo by showing that knockdown of its expression severely interferes with vascular development in zebrafish. The data indicate that CaD is crucial for a proper vascular development in vivo, and this molecule deserves to be further explored as a therapeutic target for anti-angiogenesis or be used as a stimulator of neoangiogenesis in ischaemic diseases.


   Acknowledgements
 
The authors thank W.C. Hop (Department of Biostatistics, Erasmus Medical Center, Rotterdam, The Netherlands) for his assistance with the statistical analysis.

Conflict of interest: none declared.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results and discussion
 References
 

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P.-P. Zheng, L.-A. Severijnen, R. Willemsen, and J. M. Kros
Functional Cardiac Phenotypes in Zebrafish Caldesmon Morphants: A Digital Motion Analysis
Circulation, October 27, 2009; 120(17): e145 - e146.
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