Let It Flow: Morpholino Knockdown in Zebrafish Embryos Reveals a Pro-Angiogenic Effect of the Metalloprotease Meprin α2

Background Meprin metalloproteases are thought to be involved in basic physiological functions such as cell proliferation and tissue differentiation. However, the specific functions of these enzymes are still ambiguous, although a variety of growth factors and structural proteins have been identified as meprin substrates. The discovery of meprins α1, α2 and β in teleost fish provided the basis for uncovering their physiological functions by gene silencing in vivo. Methodology/Principal Findings A Morpholino knockdown in zebrafish embryos targeting meprin α1 and β mRNA caused defects in general tissue differentiation. But meprin α2 morphants were affected more specifically and showed severe failures in the formation of the vascular system provoking the hypothesis of a pro-angiogenic effect. The blood circulation was largely diminished resulting in erythrocyte accumulation. These phenotypes mimic a previously described VEGF-A morphant, revealing a possible role of meprin α in VEGF-A activation. Indeed, human recombinant meprin α processed the vascular endothelial growth factor-A (VEGF-A) specifically, revealing the same cleavage products detectable for VEGF from zebrafish whole lysate. Conclusions/Significance Our results demonstrate that meprin metalloproteases are important for cell differentiation and proliferation already during embryogenesis, predominantly by the activation of growth factors. Thus, we conclude that meprins play a significant role in VEGF-A processing, subsequently regulating angiogenesis. Therefore, meprin α might be a new therapeutic target in cardiovascular diseases or in tumor growth inhibition.


Introduction
Meprin a and b exhibit unique features within the astacin family of zinc endopeptidases and the metzincin superfamily [1,2]. So far they could only be identified in vertebrates like fish, platypus, rodents and humans [3,4,5,6,7]. Striking are the molecular properties, revealing meprin a as the largest secreted protease known so far, due to oligomerisation up to 6 megadalton units [8,9]. Moreover, meprin b is the only astacin that stays predominantly membrane bound [10].
Originally, meprin expression has been observed on the apical side of epithelial brush borders in kidney proximal tubules and small intestine [5,6]. In the meantime, various other tissues have been found to express meprins differentially [11,12]. Meprins are secreted as zymogens, which are activated by proteolytic removal of amino terminal propeptides. Several ways for activation have been unraveled, depending on the expressing tissue. In the gut, both human enzymes are converted to their mature forms by trypsin [8]. Outside the intestine, there is selective activation of meprin a by plasmin [13], and of meprin b by tissue kallikreinrelated peptidase 4 (KLK4) [11], respectively. Upon secretion into the extracellular matrix (ECM), meprins are able to cleave a number of ECM proteins like laminin, fibronectin, collagen IV and nidogen, peptide hormones like bradykinin [10,14], as well as cytokines and growth factors like TGF-a, interleukin-1b and interleukin-18 [12,14,15,16].
The observed activation of interleukins by meprins and their expression in leukocytes of the intestinal lamina propria indicate a function in the immune response. This is supported by reports on a role for meprins in inflammatory bowel disease or Crohn's disease [17,18,19]. Furthermore, meprin a is expressed in certain tumors such as colorectal cancer and, hence, might play a role in tumor cell migration and invasion, and cancer progression [13,20]. Other sites of expression are human keratinocytes, where meprin a and b are found in the stratum basale and in the stratum granulosum, respectively [11].
In zebrafish, three homologous meprins, two meprin a variants and one b, are expressed in a broad array of tissues. Besides intestine and skin, the proteases could be found in kidney, head kidney, brain, gills, heart and liver [3]. Thus, the zebrafish appeared as a well suited model for studying the physiological function of meprins in vivo, which is made feasible by using morpholinos for knocking down meprin genes in zebrafish embryos.

Results and Discussion
Peptide antibodies were generated against each of the three meprin variants and used to examine cryosections of 16 weeks old zebrafish by immunofluorescence microscopy, which revealed the intestine as the main expression site for all meprins, and the epidermis for meprin a 1 and b. Within the gut, meprin a 1 as well as b could be observed only in the brush border cells of the intestinal epithelia ( Figure 1A, E), whereas meprin a 2 signals were detected in the lamina propria mucosae ( Figure 1C). The distribution of fluorescence signals implies that meprin a 2 expression could occur in close proximity to endothelial cells ( Figure 1D). The distinct expression pattern of all three proteases indicates different functions in vivo. Recently it was shown that meprins are involved in certain intestinal pathologies like inflammatory bowel disease (IBD) or ulcerative colitis [21]. Meprin b 2/2 mice exhibit a diminished activation of pro-interleukin-18 (IL-18), which plays a key role in IBD [16]. Similar to IL-18, also IL-1b is processed by meprin b to its mature form [15].
Expression of meprin a 1 and b in zebrafish epidermis ( Figure 1B, F) correlates to the situation in human skin. We could show that meprin a and b are expressed in separate cell layers of human epidermis [11]. This is reflected by diverse effects of the recombinant enzymes on cultured keratinocytes (HaCaT). Here, meprin b induced a dramatic change in cell morphology and reduced the cell number, whereas meprin a seem to play a role in basal keratinocyte proliferation.
All three meprins (a 1 , a 2 and b) were identified by RT-PCR in developing zebrafish embryos starting at 4 hours post fertilization (hpf; data not shown). Thus, they are expressed at the end of the blastula stage, which is typical for zygotic genes activated during midblastula transition [22,23]. This suggests important roles for meprins in tissue formation and assembly in early embryonic development. To elucidate the functions of the three zebrafish meprins in vivo, we created knockdown embryos using morpholinos targeting each protease specifically [24]. As controls, ''standard morpholino oligomers'' were injected, not targeting any gene in zebrafish ( Figure 2A). Consequently, 98% of the injected control larvae did not show any morphant phenotype and the remaining 2% correspond to natural mortality, likewise observed for untreated embryos ( Figure 2B, F). To prove the successful knockdown we compared the intensity of fluorescence signals for meprin a 2 in cryosections of injected and wild type animals (see Figure 1G, H).
Obviously, the expression of meprin a 2 in the lamina propria of zebrafish intestine was significantly decreased in morphant animals ( Figure 1H). Meprin a 1 knockdown animals showed relatively mild, but clearly distinct alterations in comparison to wild type animals ( Figure 2C, A respectively). 44% of the injected embryos revealed a dilated pericardium or a distorted trunk and tail tissue, probably due to disorders in cell differentiation ( Figure 2F). By contrast, meprin b knockdown animals exhibited strikingly abnormal disorders of the whole trunk and tail in early development ( Figure 2D). Overall, the tissues seemed to be unstructured lacking any normal cell differentiation. The morphant embryos are viable in the beginning, but die within 24 hours post injection. This phenotype reveals very distinct and fundamental functions for meprin b in the differentiation of cells during embryonic development.
In the case of meprin a 2 knockdowns the epidermal cell layers seem to be widely disorganized in the trunk and especially in the tail region ( Figure 2E). But the most informative phenotype became visible in meprin a 2 morphants beyond the age of 48 hpf. These embryos exhibited a dramatically degenerated vascular system and the blood circulation was largely diminished or even completely interrupted ( Figure 3B). Consequently, red blood cells accumulated ventrally in the caudal region of a considerable number of phenotypes (see Figure 3C). To visualize the blood vessels in living embryos, tetramethyl rhodamine isothiocyanate- Dextran (TRITC-Dextran) was injected at the age of 48 hpf for microangiography [25]. This method uncovered the almost complete absence of intersegmental vessels (ISV) ( Figure 3B), which normally begin to sprout at the 26-somite stage (21 hpf) [26] in wild type embryos ( Figure 3A). The only prominent vessel was the large dorsal aorta (DA) extending ventrally from the heart to the tail vessels ( Figure 3B; supporting movie file Video S1). These phenotypes mimic even in detail previously described VEGF-A (vascular endothelial growth factor A) morphants, regarding the reduced vascular system and erythrocyte accumulation [27]. In corresponding morphants, the loss of the VEGF receptor flk-1 (VEGFR-2) resulted in the absence of angiogenic sprouting of all blood vessels as a consequence of disorganized endothelia [28]. Based on the morphant phenotypes observed here, we argued that  meprin a 2 might be involved in angiogenic blood vessel formation by processing VEGF-A. To test this hypothesis, we incubated recombinant human meprin a and b with recombinant human VEGF-A 165 , which is the predominant isoform in humans. By western blot analysis using a specific VEGF-A antibody, we were able to demonstrate that both human meprin a and b cleaved VEGF-A by limited proteolysis. This yielded in two distinct fragments of 19 kDa (meprin a) and 20 kDa (meprin b), derived from the 24 kDa unprocessed VEGF-A 165 monomer ( Figure 4A). Moreover, by western blotting we detected zebrafish VEGF in cell lysates of wildtype fish, displaying the same cleavage pattern in accordance to the cleavage of human VEGF-A by meprin a ( Figure 4A). By N-terminal sequencing, we were able to identify the cleavage site in VEGF-A 165 incubated with meprin a, between Ala4 and Glu5 ( Figure 4B). Since this cleavage is not decisive for the different molecular weights of the emerging fragments, we propose, that VEGF-A is cleaved additionally within the Cterminal region. However, no distinctive bands could be observed corresponding to C-terminal fragments, probably due to multiple processing events (data not shown).
Hence, meprin metalloproteases might trigger angiogenesis in two possible ways. On the one hand, unique N-terminal processing of the growth factor could increase its potential to enhance endothelial cell proliferation. On the other hand, removal of the inhibitory C-terminal region from the anti-angiogenic factor VEGF-A xxx b would likewise cause a pro-angiogenic effect [29].
The disturbed organization of the epidermal cells and the deformation of tail and trunk as seen in meprin a and b morphants could be due to the proteolytic activity of meprins on cytokines like VEGF. This assumption is supported by the co-localization of meprins and VEGF in human keratinocytes. Here, VEGF plays an important role in permeability barrier homeostasis and dermal angiogenesis [30]. It has been shown previously that meprins cleave various other cytokines, growth factors and peptides, which take part in different situations like cell migration or tissue formation. For instance, TGF-a and IL-8 are processed and thereby activated by meprin a during inflammatory disease in human lung [12].
In summary, we could demonstrate by in vivo knockdown studies that meprins have fundamental physiological effects in the early embryonic development of zebrafish. The data shows that meprin metalloproteases are involved in general tissue differentiation. Moreover, we conclude that meprin a 2 is required to process VEGF-A, thereby triggering angiogenesis in the zebrafish.

Fish Maintenance
Zebrafish (Danio rerio) were bred and kept under constant conditions at a temperature of 28uC and a schedule of 14 h light and 10 h darkness. From embryonic stadium, fish were fed daily with dry food and weekly with living food (Artemia salina). Embryos were staged according to morphological criteria [23].

Morpholino Sequences
Antisense-morpholino phosphorodiamidate oligonucleotides were designed against following sequences (GeneTools, Philomath, USA). Each oligo was tagged with 39 fluorescein to visualize the distribution in the injected cells. As control, a standard morpholino oligo targeted against a mutation in the human beta-globin pre-mRNA was used (59-CCT CTT ACC TCA GTT ACA ATT TAT A-39).

VEGF-A Substrate Assay
500 ng of recombinant human VEGF-A 165 (Immunotools, Friesoythe, Germany) was incubated with 85 nM meprin a or meprin b, respectively, for different times (1, 5, 15 and 30 minutes) at 37uC. By western blotting, the resulting fragments were identified using an anti-human VEGF-A antibody (1:200, VEGF(C-1) sc7269, Santa Cruz Biotechnology, Santa Cruz, USA). Recombinant human meprin a and b were expressed, purified and activated as described before [8,11]. For N-terminal sequencing, proteins were blotted onto PVDF membranes, stained with Coomassie Brilliant Blue and sequenced at the protein microsequencing center of the Institut Fédératif de Recherche (IFR) 128 (Lyon, France). In zebrafish whole lysate (from adult fish), VEGF was detected by western blotting using a monoclonal anti-zebrafish VEGF antibody (R&D Systems, Wiesbaden, Germany).

Immunofluorescence Analysis
Cryosections of unfixed 4 weeks old zebrafish were generated with the cryostat HM 560 (Microm, Walldorf, Germany) and incubated with 5% goat serum in phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 9.2 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) to block non-specific binding. Afterwards the samples were incubated for 2 h at 4uC with polyclonal antizebrafish meprin antibodies (1:200 in 0.5% goat serum/PBS). The polyclonal antisera from rabbit and guinea pig were generated against the following peptides. IgG fractions were purified by a sepharose-6B-column (Pineda, Berlin, Germany): meprin a 1 : NH 2 -CTLDPSDGFWRGPSK-CONH 2 meprin a 2 : NH 2 -CHDAKVQSERFYNSEGYAY-CONH 2 meprin b: NH 2 -CVREYTAENPKGDLRL-CONH 2 After removal of unbound primary antibody by washing with PBS, the samples were incubated with Alexa 568 goat anti-rabbit IgG or Alexa 488 goat anti-guinea pig IgG fluorescent antibody, respectively (1:400 in 0.5% goat serum/PBS; Invitrogen, Karlsruhe, Germany) for 90 min. Moreover, 4.6-Diamidino-2-phenylindol (DAPI) was added to label the nuclei. Immunofluorescence detection was carried out using a DM IRBE microscope (Leica, Wetzlar, Germany) with fluorescence facility. The fluorescence intensity was compared on cryosections from intestine of wild type zebrafish and meprin a 2 morphants. The software ImageJ (U. S. National Institutes of Health, Bethesda, USA) was used to measure and visualize the grade of intensity.

Supporting Information
Video S1 The movie shows the phenotype of the living meprin a 2 knockdown zebrafish. First scene displays the embryo in bright field microscopy, with the visible heart beating. This is followed by fluorescence microscopy, revealing a degenerated vascular system compared to the wildtype fish, visualized by microangiography. Found at: doi:10.1371/journal.pone.0008835.s001 (3.15 MB MOV)