Secretogranin-II plays a critical role in zebrafish neurovascular modeling

Secretogranin-II plays a critical role in zebrafish neurovascular modeling Abstract Secretoneurin (SN) is a neuropeptide derived from specific proteolytic processing of the precursor secretogranin II (SgII). In zebrafish and other teleosts, there are two paralogs named sgIIa and sgIIb. Our results showed that neurons expressing sgIIb were aligned with central arteries in the hindbrain, demonstrating a close neurovascular association. Both sgIIb−/− and sgIIa−/−/sgIIb−/− mutant embryos were defective in hindbrain central artery development due to impairment of migration and proliferation of central artery cells. Further study revealed that sgIIb is non-cell autonomous and required for central artery development. Hindbrain arterial and venous network identities were not affected in sgIIb−/− mutant embryos, and the mRNA levels of Notch and VEGF pathway-related genes were not altered. However, the activation of MAPK and PI3K/AKT pathways was inhibited in sgIIb−/− mutant embryos. Reactivation of MAPK or PI3K/AKT in endothelial cells could partially rescue the central artery developmental defects in the sgIIb mutants. This study provides the first in vivo evidence that sgIIb plays a critical role in neurovascular modeling of the hindbrain. Targeting the SgII system may, therefore, represent a new avenue for the treatment of vascular defects in the central nervous system. Secretogranin-II, secretoneurin, TALENs, neurovascular, zebrafish Introduction The development of the vascular system occurs by two processes: vasculogenesis and angiogenesis. Vasculogenesis is the de novo assembly of the first blood vessels, whereas angiogenesis is the coordinated growth of endothelial cells (ECs) from the pre-existing vasculature (Risau, 1997). Both processes are essential for the maintenance of tissue growth and organ function in development (Carmeliet, 2003a). Numerous congenital or acquired diseases are associated with pathological vasculogenesis or angiogenesis (Folkman, 1995; Carmeliet and Jain, 2000; Psaltis and Simari, 2015). Among them, brain tumors, ischemic stroke, and neurodegenerative diseases including Alzheimer’s disease are associated with abnormal brain angiogenesis (Krupinski et al., 1994; Zlokovic, 2005; Kim and Lee, 2009). Therapeutic is possible for the treatment of such diseases (Jain, 2001; Chu and Wang, 2012; Wong et al., 2016). However, some of the larger clinical trials using single angiogenic factors have not corroborated the exciting early results (Krichavsky and Losordo, 2011), but combinations of multiple angiogenic factors may be a more promising approach. It is therefore necessary to uncover new pro-angiogenic and anti-angiogenic factors and to have a comprehensive understanding of the mechanism of angiogenesis. Many ligands and their receptors are involved in the regulation of angiogenesis. These include Notch/Delta-like pathway (Siekmann and Lawson, 2007; Wang et al., 2014), VEGF pathway (Olsson et al., 2006), Wnt pathway (Dejana, 2010), shh pathway (Pola et al., 2001), angiopoietin (Suri et al., 1996), and netrins (Wilson et al., 2006) have been shown to be important. It is noteworthy that blood vessels are often aligned with nerves and display similar branching patterns. Some of these pathways or ligand–receptor complexes have been shown to act in parallel on both vascular and neural cells, demonstrating the interdependence and functional connection of the two systems sharing similar regulatory mechanisms (Larrivée et al., 2009). It may accelerate the discovery of mechanistic insights to realize that vascular system and nervous system use some common genetic pathways (Carmeliet, 2003b). Secretogranin-II (SgII) is mainly distributed in dense-core vesicles of many neurons and endocrine cells, and is overexpressed in some neuroendocrine tumors (Mahata et al., 1991). The neuropeptide secretoneurin (SN) is a short conserved peptide (31−43 amino acids) derived from the larger SgII precursor protein (~600 amino acids) by prohormone convertase-mediated processing (Fischer-Colbrie et al., 1995; Trudeau et al., 2012). While several potential peptides may arise from SgII processing, SN is the only highly abundant neuropeptide with known biological activities (Fischer-Colbrie et al., 1995). Various physiological roles have been assigned to SN (Trudeau et al., 2012), including those related to reproduction (Zhao et al., 2009), neuroinflammation (You et al., 1996), and neurotransmitter release (Reinisch et al., 1993). In the rodent brain, SN is found predominantly in the phylogenetically older parts, overlapping partly but not completely with established neurotransmitter or peptidergic systems (Fischer-Colbrie et al., 1995; Trudeau et al., 2012). Supporting this, in the goldfish, SN exhibits a more restricted central nervous system (CNS) distribution mainly to the preoptic magnocellular neurons co-expressing nonapetides in the oxytocin/vasopressin family, certain hypothalamic nuclei, and posterior projections to hindbrain structures (Canosa et al., 2011). Best described are the pro-angiogenic effects of SN. Synthetic SN can promote capillary tube formation in human umbilical vein ECs (HUVECs) in vitro (Kirchmair et al., 2004b) and induce neovascularization in the mouse cornea in vivo (Kirchmair et al., 2004b). The mechanism for the promotion of angiogenesis by SN may be not the same in different blood vessels. In vitro results show that the activation of mitogen-activated protein kinases (MAPK) by SN is dependent on vascular endothelial growth factor (VEGF) in human coronary artery ECs (HCAECs) (Albrecht-Schgoer et al., 2012), while in HUVECs, the angiogenic effects by SN are VEGF-independent (Kirchmair et al., 2004b). To date, most of the studies on angiogenesis have been carried out using exogenously applied SN. However, little is known about the effect of the endogenous SgII precursor protein or the SN neuropeptide on blood vessel formation as there are currently no sgII knockout animal models, and human mutations have not yet been identified. The aim of our study, therefore, was to investigate the role of sgII during early developmental stages. In zebrafish and other teleost fishes, there exist two paralogous genes, sgIIa and sgIIb that generate SNa and SNb peptides, respectively (Zhao et al., 2010). In this study, we have generated sgIIa−/−, sgIIb−/−, and sgIIa−/−/sgIIb−/− mutant zebrafish lines using transcription activator-like effector nucleases (TALENs), and found that sgIIb−/− and sgIIa−/−/sgIIb−/− mutants have specific defects in the development of hindbrain central arteries (CtAs). SgIIb plays a critical role in zebrafish neurovascular modeling that is mediated by MARK and PI3K/AKT signaling in vivo. Results sgIIb is expressed in the central nervous system of zebrafish embryos and sgIIb-expressing neurons and central arteries are aligned in the hindbrain We have established the expression pattern of sgIIb in wild-type (WT) zebrafish embryos by semi-quantitative reverse-transcriptase PCR (RT-PCR) and whole-mount in situ hybridization (WISH) technique. sgIIb mRNA levels were low at 10 h post-fertilization (hpf), then increased over the 14−24 hpf period, and stabilized after 36 hpf (Figure 1A). The WISH results revealed that sgIIb was mainly expressed in the CNS at 24 and 36 hpf, and concentrated in the brain by 45 hpf (Figure 1B). Then we examined the relationship between sgIIb-positive cells and Calcium/calmodulin-dependent protein kinase II delta 2 (Camk2d2)-expressing cells in the hindbrain. Camk2d2 belongs to the CaMKII family that functions in neuronal growth cone guidance and synaptic plasticity, among other functions (Mayford et al., 1995; Wen et al., 2004). Double-fluorescence in situ hybridization revealed colocalization of sgIIb and camk2d2 in the hindbrain (Figure 1C). To investigate the positional relationship between sgIIb-expressing cells and the vascular system in the hindbrain, double-fluorescence in situ hybridization with probes for sgIIb and the vascular marker kdrl (kinase insert domain receptor-like) was performed in 36−45 hpf embryos. Our results showed that sgIIb-expressing cells were aligned with the growing CtAs at 36−39 hpf (Figure 1D−I, P−R). At 42−45 hpf, sgIIb was widely distributed around CtAs in the hindbrain (Figure 1J−O, S−U). The expression of sgIIa was different from that of sgIIb. Firstly, sgIIa was detectable by 10 hpf (Supplementary Figure S1A), ~4 h earlier than sgIIb (Figure 1A). Thereafter, sgIIa expression increased gradually until it stabilized at ~24 hpf. sgIIa was highly expressed in the forebrain, midbrain, and ventral part of the neural tube but barely expressed in the hindbrain (Supplementary Figure S1B), which contrasts significantly with the abundance of sgIIb transcripts in this region (Figure 1B). Double-fluorescence WISH revealed that sgIIa-positive cells co-expressed the GABAergic neuron marker tal2 (basic helix-loop-helix transcription factor) and gad67, the mRNA encoding the GABA-synthesizing enzyme glutamic acid decarboxylase 67 (Supplementary Figure S1C). Figure 1 View largeDownload slide The developmental expression pattern of sgIIb in the CNS of zebrafish. (A) RT-PCR analysis for temporal expression of sgIIb mRNA during embryogenesis and early larval developmental stages. M, molecular size marker; C, no template control. (B) WISH of sgIIb in 24, 36, and 45 hpf zebrafish embryos. (C−U) Mapping expression of sgIIb in relation to camk2d2 and kdrl in the zebrafish hindbrain with double-fluorescence in situ hybridization. (C) Confocal imaging for sgIIb and camk2d2 mRNA at 36 hpf. camk2d2-positive cells co-express sgIIb (white arrowheads). (D−O) Maximal intensity projection of a confocal z-stack for sgIIb and kdrl mRNA. kdrl-positive cells are aligned with sgIIb-expressing cells (white arrowheads). (P−U) Single confocal planes showing sgIIb and kdrl mRNA. Embryos were examined at 36 hpf (D−F, P−R), 39 hpf (G−I), 42 hpf (J−L, S−U), and 45 hpf (M−O). Scale bar, 100 μm. Figure 1 View largeDownload slide The developmental expression pattern of sgIIb in the CNS of zebrafish. (A) RT-PCR analysis for temporal expression of sgIIb mRNA during embryogenesis and early larval developmental stages. M, molecular size marker; C, no template control. (B) WISH of sgIIb in 24, 36, and 45 hpf zebrafish embryos. (C−U) Mapping expression of sgIIb in relation to camk2d2 and kdrl in the zebrafish hindbrain with double-fluorescence in situ hybridization. (C) Confocal imaging for sgIIb and camk2d2 mRNA at 36 hpf. camk2d2-positive cells co-express sgIIb (white arrowheads). (D−O) Maximal intensity projection of a confocal z-stack for sgIIb and kdrl mRNA. kdrl-positive cells are aligned with sgIIb-expressing cells (white arrowheads). (P−U) Single confocal planes showing sgIIb and kdrl mRNA. Embryos were examined at 36 hpf (D−F, P−R), 39 hpf (G−I), 42 hpf (J−L, S−U), and 45 hpf (M−O). Scale bar, 100 μm. Establishment of sgII mutant zebrafish lines with TALENs To investigate the role of sgII during neurovascular development in vivo, two pairs of TALENs were designed for the zebrafish sgIIa and sgIIb genes. The TALEN target sites of sgIIa and sgIIb were both chosen following the ATG start site and in front of SNa and SNb domains (Figure 2A and C). The sgIIa heterozygote with 7-bp deletion and 2-bp insertion (−7,+2 bp) or just 7-bp deletion (−7 bp) and the sgIIb heterozygote with a 7-bp insertion and 5-bp (−5,+7 bp) deletion or just 10-bp deletion (−10 bp) were screened out and further used to establish the sgIIa−/− and sgIIb−/− homozygous mutant line (Figure 2A and C). All these mutant lines resulted in open reading frame-shift mutants of sgIIa or sgIIb gene, and thus generating truncated proteins with no SN peptides (Figure 2B and D). The sgIIa−/−/sgIIb−/− homozygous mutant line (sgIIa−/−−7,+2 bp;sgIIb−/− −5,+7 bp) was obtained by crossing the sgIIa−/− homozygote (−7,+2 bp) with the sgIIb−/− homozygote (−5,+7 bp). All crossings were performed using in vitro fertilization. Both the real-time quantitative PCR (RT-qPCR) and WISH results revealed that mRNA levels of sgIIa and sgIIb were significantly decreased in sgIIa (Figure 2E and F) and sgIIb (Figure 2E and G) mutant embryos, respectively, compared to WT, indicating a mechanism of nonsense-mediated mRNA decay (Chang et al., 2007). Since the pituitary is a major production site of SgIIa protein and SN peptide, we monitored SN immunoreactivity in adults to determine the effects of mutations on protein production using a well-characterized polyclonal antibody (Zhao et al., 2006b) that recognizes zebrafish SNa but not SNb (Supplementary Figure S2). Two bands corresponding to the full-length precursor (~62.2 kDa) and an intermediate fragment (~59 kDa) were observed in WT zebrafish pituitaries. Our results revealed that SgIIa precursor protein or proteolitically processed SNa-immunoreactive fragments were not detectable in the adult pituitary gland of sgIIa−/− mutant and sgIIa−/−/sgIIb−/− double mutant fish. The ~30 kDa SNa-immunoreactive fragment of SgIIa was dramatically increased in adult sgIIb−/− mutant fish (Figure 2H), suggesting a compensatory expression of sgIIa in adult sgIIb−/− mutant fish. Figure 2 View largeDownload slide Mutation of sgIIb causes defects of CtA development in vivo. (A and C) The location of the TALEN-binding sites (underlined) on zebrafish sgIIa or sgIIb gene and two mutant lines of TALEN-targeted sgIIa alleles (A) or sgIIb alleles (C). Deletions and insertions are indicated by dashes and red letters, respectively. (B and D) Schematic representation of the putative WT SgIIa or SgIIb protein and two mutated SgIIa proteins (B) or SgIIb proteins (D). (E) WISH of WT, sgIIa−/− mutant, and sgIIb−/− mutant embryos. Antisense probes against sgIIa and sgIIb were visualized at 36 hpf. Scale bar, 100 μm. (F and G) Relative mRNA level of sgIIa (F) or sgIIb (G) in 36 hpf sgIIa−/− mutant or sgIIb−/− mutant embryos, respectively, as measured by RT-qPCR. Data shown are mean ± SEM of three independent experiments. Statistical significance was assessed using the two-tailed Student’s t-test. (H) Western blotting analysis of the pituitary samples from WT, sgIIa−/− mutant, sgIIb−/− mutant, and sgIIa−/−/sgIIb−/− mutant adults at 120 dpf. (I) Schematic vascular modeling in the hindbrain of 36 hpf and 45 hpf WT zebrafish embryos. PHBCs are in blue, BA is in red, and CtAs are in green. (J) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP), sgIIb−/−;Tg(kdrl:EGFP) (TG2), and sgIIa−/−/sgIIb−/−;Tg(kdrl:EGFP) (TG3) zebrafish hindbrain. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. Embryos were examined at 36, 39, 42, 45, and 48 hpf. Scale bar, 50 μm. (K−N) Quantitative analysis of CtAs in Tg(kdrl:EGFP), TG2, and TG3 zebrafish hindbrains. Numbers of total CtAs (K), normal CtAs (L), and disorganized CtAs (M) and total length of CtAs (N) were determined. Embryos were 45 and 48 hpf. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. Figure 2 View largeDownload slide Mutation of sgIIb causes defects of CtA development in vivo. (A and C) The location of the TALEN-binding sites (underlined) on zebrafish sgIIa or sgIIb gene and two mutant lines of TALEN-targeted sgIIa alleles (A) or sgIIb alleles (C). Deletions and insertions are indicated by dashes and red letters, respectively. (B and D) Schematic representation of the putative WT SgIIa or SgIIb protein and two mutated SgIIa proteins (B) or SgIIb proteins (D). (E) WISH of WT, sgIIa−/− mutant, and sgIIb−/− mutant embryos. Antisense probes against sgIIa and sgIIb were visualized at 36 hpf. Scale bar, 100 μm. (F and G) Relative mRNA level of sgIIa (F) or sgIIb (G) in 36 hpf sgIIa−/− mutant or sgIIb−/− mutant embryos, respectively, as measured by RT-qPCR. Data shown are mean ± SEM of three independent experiments. Statistical significance was assessed using the two-tailed Student’s t-test. (H) Western blotting analysis of the pituitary samples from WT, sgIIa−/− mutant, sgIIb−/− mutant, and sgIIa−/−/sgIIb−/− mutant adults at 120 dpf. (I) Schematic vascular modeling in the hindbrain of 36 hpf and 45 hpf WT zebrafish embryos. PHBCs are in blue, BA is in red, and CtAs are in green. (J) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP), sgIIb−/−;Tg(kdrl:EGFP) (TG2), and sgIIa−/−/sgIIb−/−;Tg(kdrl:EGFP) (TG3) zebrafish hindbrain. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. Embryos were examined at 36, 39, 42, 45, and 48 hpf. Scale bar, 50 μm. (K−N) Quantitative analysis of CtAs in Tg(kdrl:EGFP), TG2, and TG3 zebrafish hindbrains. Numbers of total CtAs (K), normal CtAs (L), and disorganized CtAs (M) and total length of CtAs (N) were determined. Embryos were 45 and 48 hpf. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. Mutation of sgIIb causes defects of CtA development in vivo The sgIIa−/−, sgIIb−/−, and sgIIa−/−/sgIIb−/− mutant zebrafish lines were crossed with the Tg(kdrl:EGFP) line that expresses green fluorescent protein (GFP) in the vascular system. Their progeny was raised to adulthood and intercrossed to establish three homozygous mutant lines expressing GFP in vascular system, i.e. TG1: sgIIa−/−;Tg(kdrl:EGFP), TG2: sgIIb−/−;Tg(kdrl:EGFP), and TG3: sgIIa−/−/sgIIb−/−;Tg(kdrl:EGFP). CtAs are a set of vessels penetrating the hindbrain; they grow from the primordial hindbrain channels (PHBCs) to the basilar artery (BA) and interconnect the PHBCs and BA (Figure 2I). The percentage of embryos with CtA defects was significantly increased in TG2 embryos (Supplementary Figure S3A). In vivo confocal imaging results showed specific defects of hindbrain CtA development in TG2 and TG3 embryos at 36, 39, 42, 45, and 48 hpf (Figure 2J). Normal CtAs are those extend to BA along an inverted V pattern, as shown previously (Fujita et al., 2011), while disorganized CtAs are those do not migrate along an inverted V pattern. Compared to the Tg(kdrl:EGFP) line, we found that the number of normal CtAs, the number of total CtAs, and the total length of CtAs were all significantly decreased (Figure 2K, L, and N), whereas the number of disorganized CtAs increased (Figure 2M) in TG2 and TG3 embryos at 45 and 48 hpf, respectively. The appearance of the CtA defects in TG2 and TG3 embryos was highly similar (Figure 2J−N). In addition, the overall morphology and survival rate were not affected in sgIIb−/− mutant embryos (Supplementary Figure S4A and B). Other blood vessels outside the hindbrain remained normal in TG2 embryos (Figure 3A−J). Midbrain vasculature was statistically analyzed following methods in a previous report (Chen et al., 2012). Total length and segment number of midbrain vasculature were not affected in TG2 embryos (Figure 3I and J). These data demonstrate that sgIIb is critical for neurovascular modeling specifically in the hindbrain. sgIIb and camk2d2 co-expressing neuronal cells were not affected in the hindbrain of sgIIb−/− mutant embryos (Figure 3L). Brain morphology and the expression of the neuronal markers HuC, neurod4, and robo3 in rhombomeres or rhombomere boundaries (Park et al., 2000; Challa et al., 2001; Wang et al., 2003) also remained normal (Figure 3K, M−O), demonstrating that CtA defects in sgIIb mutants are not an indirect consequence of changes in brain growth or patterning. To examine the key roles of sgIIb in CtA development more precisely, we carried out time-lapse imaging from 36 to 45 hpf using Tg(kdrl:EGFP) and TG2. Our results showed that some CtAs failed to sprout or connect to the BA in TG2 embryos that lack SgIIb (Supplementary Video S1) compared with Tg(kdrl:EGFP) (Supplementary Video S2). By 52 hpf in TG2 and TG3 embryos, the number of disorganized CtAs still remained somewhat high (Supplementary Figure S5C), but some CtAs that did not extend to BA at 48 hpf recovered to their normal appearance (Supplementary Videos S3 and S4, Figure S5A and B). Defects were also visualized using WISH for cadherin 5 (cdh5) in the hindbrain vasculature of 45 hpf sgIIb−/− mutant embryos (Supplementary Figure S3B), an observation consistent with the results from 45 hpf TG2 or TG3 embryos. Importantly, sgIIb knockdown by morpholino injections caused similar defects of CtA development (Supplementary Figures S3C, S6A and B) as noted with the sgIIb knockout experiments. In contrast, CtA development was not affected in TG1 embryos (Supplementary Figure S7A). Since sgIIa is weakly expressed in the hindbrain of zebrafish embryos (Supplementary Figure S1B), and the sgIIa mRNA level remained unchanged in sgIIb mutant embryos (Supplementary Figure S7B and C), sgIIa is unlikely to participate in the development of hindbrain CtAs. Figure 3 View largeDownload slide sgIIb is critical for neurovascular modeling specifically in the hindbrain. (A−H) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP) and TG2 embryos at 45 hpf. (A−D) Lateral views of Tg(kdrl:EGFP) and TG2 vasculature in the head (A and B) and trunk (C and D). (E and G) Dorsal views of Tg(kdrl:EGFP) and TG2 vasculature in the anterior head region. (F and H) Lateral views of magnified Tg(kdrl:EGFP) and TG2 vasculature in the trunk region. (I and J) Quantitative analysis of midbrain vasculature in Tg(kdrl:EGFP) and TG2 zebrafish lines. (K) Brain morphology of WT and sgIIb−/− mutant embryos at 45 hpf (dorsal view). (L−O) Maximal intensity projection of a confocal z-stack for sgIIb mRNA together with camk2d2 (L), HuC (M), neurod4 (N), and robo3 (O) mRNA at 36 hpf WT and sgIIb−/− mutant embryos using double-fluorescence in situ hybridization (lateral view). DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; h, hindbrain; ISV, intersegmental vessel; m, midbrain; MCeV, mid-cerebral vein; mhb, midbrain−hindbrain boundary; MsV, mesencephalic vein; MtA, metencephalic artery; PCV, posterior cardinal vein. Scale bar, 50 μm. Figure 3 View largeDownload slide sgIIb is critical for neurovascular modeling specifically in the hindbrain. (A−H) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP) and TG2 embryos at 45 hpf. (A−D) Lateral views of Tg(kdrl:EGFP) and TG2 vasculature in the head (A and B) and trunk (C and D). (E and G) Dorsal views of Tg(kdrl:EGFP) and TG2 vasculature in the anterior head region. (F and H) Lateral views of magnified Tg(kdrl:EGFP) and TG2 vasculature in the trunk region. (I and J) Quantitative analysis of midbrain vasculature in Tg(kdrl:EGFP) and TG2 zebrafish lines. (K) Brain morphology of WT and sgIIb−/− mutant embryos at 45 hpf (dorsal view). (L−O) Maximal intensity projection of a confocal z-stack for sgIIb mRNA together with camk2d2 (L), HuC (M), neurod4 (N), and robo3 (O) mRNA at 36 hpf WT and sgIIb−/− mutant embryos using double-fluorescence in situ hybridization (lateral view). DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; h, hindbrain; ISV, intersegmental vessel; m, midbrain; MCeV, mid-cerebral vein; mhb, midbrain−hindbrain boundary; MsV, mesencephalic vein; MtA, metencephalic artery; PCV, posterior cardinal vein. Scale bar, 50 μm. sgIIb is non-cell autonomous and is required for migration and proliferation of central artery ECs in vivo To date, the only known bioactive peptide generated from the SgII precursor is SN. When we injected SNb mRNA into one-cell stage embryos of the TG2 line, the defects of CtA development observed in TG2 embryos could be partly rescued (Figure 4A−C). No ectopic sprouting of blood vessel was observed in Tg(kdrl:EGFP) following SNb mRNA injection (Supplementary Figure S8A−D). Cell transplantation experiments were carried out to investigate whether sgIIb is non-cell autonomous in CtA development. Donor Tg(kdrl:EGFP) or TG2 embryos (donor-derived ECs were marked with GFP) were injected with Tritc-dextran (to mark all donor-derived cells) at the one-cell stage. WT and sgIIb−/− embryos were used as hosts. Cell transplantation results showed that the percentage of embryos with EGFP-positive CtAs was higher in WT hosts, irrespective of which donor embryos were used (Figure 4D and F). More specifically, percentages of embryos with EGFP-positive CtAs were 70% (14/20) in WT–WT group and 61 % (11/18) in the sgIIb−/−–WT group (Figure 4F), indicating that transplanted sgIIb−/− cells could develop to CtAs at a similar degree with WT cells in WT embryo hosts. Importantly, sgIIb expression was detected in camk2d2-positive neurons but not the CtAs (Figure 1C and P−U), indicating that the source of SgIIb/SNb is not the ECs, and thus any effect should be non-cell autonomous. Only 13% (3/23) of the chimeras had EGFP-positive CtAs in WT-sgIIb−/− group (Figure 4F), suggesting that CtA development from WT cells was rather poor, when surrounded by sgIIb−/− cells in the recipient embryos. Moreover, when SNb mRNA-injected TG2 cells were transplanted into areas adjacent to CtAs in TG2 embryos, CtA defects in the sgIIb mutants could be partially rescued (Figure 4E and G). Transplanted cells were not EGFP-positive, demonstrating that transplanted cells were not CtA ECs, while CtA defects could be rescued by these non-ECs. Thus, it can be confirmed that the role of SgIIb/SNb in CtA development was non-cell autonomous. Furthermore, the duration of CtA migration from PHBC to BA was significantly longer in sgIIb mutant fish (Figure 4H), indicating that CtA migration speed was affected by deletion of sgIIb. Anti-phosphorylated histone H3 (PH3) antibody was used to detect the cells in M-phase of the cell cycle. Whole-mount immunohistochemistry results indicated that the percentage of PH3-positive ECs within CtAs was significantly decreased in sgIIb−/− mutant embryos (Figure 4I and N), while the percentage of PH3-positive non-ECs in the hindbrain was not affected (Figure 4J and N). The percentage of PH3-positive cells within PHBC, trunk, forebrain, and midbrain was also not affected in sgIIb−/− mutant embryos (Figure 4K−M and O−Q). Therefore, sgIIb was required for EC proliferation in zebrafish CtAs specifically. These findings demonstrate that sgIIb is non-cell autonomous and required for migration and proliferation of CtA ECs in vivo. Figure 4 View largeDownload slide sgIIb is non-cell autonomously required for migration and proliferation of CtA ECs in vivo. (A) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP) and TG2 embryos injected with SNb or RFP mRNA. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. (B and C) Quantitative analysis of CtAs in the hindbrain of Tg(kdrl:EGFP)+RFP mRNA, TG2+RFP mRNA, and TG2+SNb mRNA-injected zebrafish. Numbers of normal CtAs (B) and disorganized CtAs (C) were counted in 45 hpf zebrafish hindbrain. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. (D) Maximal intensity projection of a confocal z-stack of chimeras at 45 hpf. White arrowheads indicate CtAs. (E) Maximal intensity projection of a confocal z-stack of chimeras at 45 hpf. (F) Percentage of chimeras with EGFP-positive CtAs. Numbers in the bracket of each group represent the number of chimeras with EGFP-positive CtAs/the number of chimeras with EGFP-positive ECs in the hindbrain. (G) Quantitative analysis of normal CtAs in 45 hpf TG2SNb mRNA > TG2 and TG2RFP mRNA > TG2 embryos. (H) Quantitative analysis of CtA migration duration in Tg(kdrl:EGFP) and TG2 embryos. (I−Q) Percentage of PH3-positive ECs and non-ECs in 42 hpf Tg(kdrl:EGFP) and TG2 embryos. The percentage of PH3-positive ECs in CtA (I and N), PHBC (K and O), forebrain and midbrain (L and P), and trunk (M and Q). The percentage of PH3-positive non-ECs in the hindbrain (J). Data shown are mean ± SEM (n = the number of embryos analyzed). DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel; MCeV, mid-cerebral vein; MsV, mesencephalic vein. Scale bar, 50 μm. Figure 4 View largeDownload slide sgIIb is non-cell autonomously required for migration and proliferation of CtA ECs in vivo. (A) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP) and TG2 embryos injected with SNb or RFP mRNA. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. (B and C) Quantitative analysis of CtAs in the hindbrain of Tg(kdrl:EGFP)+RFP mRNA, TG2+RFP mRNA, and TG2+SNb mRNA-injected zebrafish. Numbers of normal CtAs (B) and disorganized CtAs (C) were counted in 45 hpf zebrafish hindbrain. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. (D) Maximal intensity projection of a confocal z-stack of chimeras at 45 hpf. White arrowheads indicate CtAs. (E) Maximal intensity projection of a confocal z-stack of chimeras at 45 hpf. (F) Percentage of chimeras with EGFP-positive CtAs. Numbers in the bracket of each group represent the number of chimeras with EGFP-positive CtAs/the number of chimeras with EGFP-positive ECs in the hindbrain. (G) Quantitative analysis of normal CtAs in 45 hpf TG2SNb mRNA > TG2 and TG2RFP mRNA > TG2 embryos. (H) Quantitative analysis of CtA migration duration in Tg(kdrl:EGFP) and TG2 embryos. (I−Q) Percentage of PH3-positive ECs and non-ECs in 42 hpf Tg(kdrl:EGFP) and TG2 embryos. The percentage of PH3-positive ECs in CtA (I and N), PHBC (K and O), forebrain and midbrain (L and P), and trunk (M and Q). The percentage of PH3-positive non-ECs in the hindbrain (J). Data shown are mean ± SEM (n = the number of embryos analyzed). DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel; MCeV, mid-cerebral vein; MsV, mesencephalic vein. Scale bar, 50 μm. Mutation of sgIIb does not affect arterial−venous identity or Notch and VEGF pathways in the hindbrain The establishment of arterial–venous identity is essential in the development of blood vessels (Lawson and Weinstein, 2002). We therefore examined artery- and vein-specific markers in the hindbrain of 36 hpf embryos by WISH. The expression of vein-specific markers (dab2 and flt4) and artery-specific markers (hey2, flt1, and dll4) was not affected in sgIIb mutant compared to WT embryos (Figure 5A and B). Double-fluorescence in situ hybridization revealed that the artery-specific marker flt1 was still expressed in CtAs of sgIIb mutant embryos (Figure 5C). Thus, CtA defects in sgIIb mutants are not associated with changes to arterial–venous identity in the hindbrain. It has been reported that Notch and VEGF pathways are critical for angiogenesis (Jakobsson et al., 2009; Blanco and Gerhardt, 2013). Fluorescence-activated cell sorting was used to obtain ECs from Tg(kdrl:EGFP) and TG2 embryonic heads. The mRNA level of Notch and VEGF pathway-related genes was determined using RT-qPCR. As shown in Supplementary Figures S9 and S10A, the mRNA levels of Notch pathway-related genes (including notch1a, notch1b, notch2, dll4, hey2, hey1, hey6, dlc, dld) and VEGF pathway-related genes (including vegfaa, vegfab, vegfb, vegfc, vegfd, flt1, kdrl, flt4, nrp1a, nrp1b) in ECs were not affected by mutation of sgIIb. VEGFA protein level in sgIIb mutant embryos was also not changed (Supplementary Figure S10C and D). vegfaa dominantly functions in VEGFa signaling and is also expressed in neural cells in addition to ECs (Wild et al., 2017). The expression of vegfaa was not affected in the sgIIb mutant hindbrain, as determined using WISH (Supplementary Figure S10B). These results indicate that Notch and VEGF pathways are not affected in the hindbrain of sgIIb mutant fish. Figure 5 View largeDownload slide Mutation of sgIIb inhibits the activation of MAPK and PI3K/AKT pathways. (A and B) WISH was performed in 36 hpf WT and sgIIb mutant zebrafish embryos with venous markers dab2 and flt4 (A) and arterial markers hey2, flt1, and dll4 (B). Black arrowheads indicate the expression of venous markers and red arrowheads indicate the expression of venous markers in the hindbrain. (C) Confocal imaging for kdrl and flt1 mRNA in WT and sgIIb mutant zebrafish embryos at 36 hpf. White arrowheads point to sprouting CtAs (kdrl-positive) that co-express flt1. (D) Whole-mount immunohistochemistry for p-ERK1/2 expression in 36 hpf WT and sgIIb−/− mutant embryos. The frequency of embryos with the indicated phenotypes is shown in the bracket of each group. Scale bar, 100 μm. (E) Western blots for phospho-AKT (p-AKT), total AKT, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2 expression in 36 hpf WT and sgIIb−/− mutant embryos. Actin was used as a loading control. (F) Single confocal planes of EGFP and p-ERK1/2 in 42 hpf Tg(kdrl:EGFP) and TG2 embryonic hindbrain (lateral view). Scale bar, 50 μm. (G) Magnified views of single confocal planes of EGFP-positive CtAs and p-ERK1/2 from dashed box in F. Dashed areas delineate CtAs that co-express EGFP and p-ERK1/2. Scale bar, 25 μm. (H and I) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (J) Relative density of p-ERK signals at CtAs in 42 hpf Tg(kdrl:EGFP) and TG2 embryos. Data shown are mean ± SEM (n = the number of CtAs analyzed). Figure 5 View largeDownload slide Mutation of sgIIb inhibits the activation of MAPK and PI3K/AKT pathways. (A and B) WISH was performed in 36 hpf WT and sgIIb mutant zebrafish embryos with venous markers dab2 and flt4 (A) and arterial markers hey2, flt1, and dll4 (B). Black arrowheads indicate the expression of venous markers and red arrowheads indicate the expression of venous markers in the hindbrain. (C) Confocal imaging for kdrl and flt1 mRNA in WT and sgIIb mutant zebrafish embryos at 36 hpf. White arrowheads point to sprouting CtAs (kdrl-positive) that co-express flt1. (D) Whole-mount immunohistochemistry for p-ERK1/2 expression in 36 hpf WT and sgIIb−/− mutant embryos. The frequency of embryos with the indicated phenotypes is shown in the bracket of each group. Scale bar, 100 μm. (E) Western blots for phospho-AKT (p-AKT), total AKT, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2 expression in 36 hpf WT and sgIIb−/− mutant embryos. Actin was used as a loading control. (F) Single confocal planes of EGFP and p-ERK1/2 in 42 hpf Tg(kdrl:EGFP) and TG2 embryonic hindbrain (lateral view). Scale bar, 50 μm. (G) Magnified views of single confocal planes of EGFP-positive CtAs and p-ERK1/2 from dashed box in F. Dashed areas delineate CtAs that co-express EGFP and p-ERK1/2. Scale bar, 25 μm. (H and I) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (J) Relative density of p-ERK signals at CtAs in 42 hpf Tg(kdrl:EGFP) and TG2 embryos. Data shown are mean ± SEM (n = the number of CtAs analyzed). The role of sgIIb in neurovascular modeling is mediated by MAPK and PI3K/AKT signaling in vivo Previous studies have demonstrated that MAPK and PI3K/AKT pathways participate in the proliferation and migration of ECs (Mavria et al., 2006; Karar and Maity, 2011). We examined the MAPK and PI3K/AKT pathways in sgIIb mutant embryos with defects of CtA development. The expression levels of p-ERK1/2 and p-AKT in sgIIb mutant embryos were much lower than that in WT, while the levels of total ERK1/2 and total AKT were unchanged (Figure 5D, E, H, and I). Whole-mount immunohistochemistry and confocal imaging results showed that p-ERK1/2 was expressed in CtAs (Figure 5F and G; Supplementary Figure S11). Furthermore, the relative density of p-ERK signal was significantly decreased in CtAs of TG2 embryos (Figure 5F, G, and J). These results suggest that the activation of MAPK and PI3K/AKT was inhibited in the CtAs of sgIIb mutants. Accordingly, when we injected SNb mRNA into one-cell stage sgIIb mutant embryos, the expression levels of p-ERK1/2 and p-AKT were significantly increased at 36 hpf (Figure 6A−C), while the levels of total ERK1/2 and total AKT remained unchanged. N-arachidonoyl-L-serine (ARA) is a known activator of MAPK and PI3K/AKT pathways (Milman et al., 2006). Delivery of ARA (50 μM) increased p-ERK1/2 and p-AKT levels in sgIIb mutant embryos (Figure 6E−G). Furthermore, CtA developmental defects in TG2 fish could be partially rescued by ARA treatment (Figure 6D, H, and I). The constitutively active forms of the protein Mek1(S218E, S222D) (Mansour et al., 1994) or myristoylated Akt1 (Kubota et al., 2005) were specifically expressed in ECs through injection of 50 pg Tol2-kdrl-Mek1-P2A-mCherry-Tol2 or Tol2-kdrl-Myr-Akt1-P2A-mCherry-Tol2. We found that upregulated MAPK or PI3K/AKT activity in ECs partially rescued CtA defects in sgIIb mutant embryos (Figure 6K and L). To detect whether upregulation of MAPK or PI3K/AKT signal can rescue proliferation defects in sgIIb mutant embryos, whole-mount immunohistochemistry with PH3 antibody was conducted after the injection of 75 pg Mek1(S218E, S222D) or myristoylated Akt1 mRNA. As shown in Figure 6M and N, the percentage of PH3-positive ECs within sgIIb mutant CtAs was significantly increased with Mek1(S218E, S222D) or myristoylated Akt1 mRNA injection. These findings demonstrate that the role of sgIIb in neurovascular modeling is mediated by MAPK and PI3K/AKT signaling in CtA ECs. Figure 6 View largeDownload slide The role of sgIIb in neurovascular modeling is mediated by MAPK and PI3K/AKT signaling in CtA ECs. (A) Western blots for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 in 36 hpf sgIIb−/− mutant and sgIIb−/− mutant embryos injected with SNb mRNA. Actin was used as a loading control. (B and C) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (D) Maximal intensity projection of a confocal z-stack of 45 hpf Tg(kdrl:EGFP) or TG2 embryos incubated with DMSO or ARA. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. Scale bar, 50 μm. (E) Western blots for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 in 36 hpf sgIIb−/− mutant embryos incubated with DMSO or ARA. Actin was used as a loading control. (F and G) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (H and I) Quantitative analysis of CtAs in Tg(kdrl:EGFP) + ARA, TG2 + DMSO, and TG2 + ARA zebrafish hindbrains. Numbers of normal CtAs (H) and disorganized CtAs (I) were counted in 45 hpf zebrafish hindbrains. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. (J) The Tol2 transposon cassettes of the control (Tol2-kdrl-mCherry-Tol2), myristoylated Akt1 (Tol2-kdrl-Myr-Akt1-P2A-mCherry-Tol2), and Mek1 (Tol2-kdrl-Mek1-P2A-mCherry-Tol2) expression plasmids. (K) Maximal intensity projection of a confocal z-stack of 45 hpf Tg(kdrl:EGFP) or TG2 embryos injected with control, myristoylated Akt1, or Mek1 expression vector. (L) Percentage of mCherry-positive normal CtAs. Numbers in the bracket of each group represent the number of mCherry-positive normal CtAs/the number of mCherry-positive total CtAs. (M and N) Percentage of PH3-positive ECs in 45 hpf Tg(kdrl:EGFP) or TG2 embryo CtAs injected with RFP mRNA, Myr-Akt1 mRNA, or Mek1 mRNA. Figure 6 View largeDownload slide The role of sgIIb in neurovascular modeling is mediated by MAPK and PI3K/AKT signaling in CtA ECs. (A) Western blots for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 in 36 hpf sgIIb−/− mutant and sgIIb−/− mutant embryos injected with SNb mRNA. Actin was used as a loading control. (B and C) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (D) Maximal intensity projection of a confocal z-stack of 45 hpf Tg(kdrl:EGFP) or TG2 embryos incubated with DMSO or ARA. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. Scale bar, 50 μm. (E) Western blots for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 in 36 hpf sgIIb−/− mutant embryos incubated with DMSO or ARA. Actin was used as a loading control. (F and G) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (H and I) Quantitative analysis of CtAs in Tg(kdrl:EGFP) + ARA, TG2 + DMSO, and TG2 + ARA zebrafish hindbrains. Numbers of normal CtAs (H) and disorganized CtAs (I) were counted in 45 hpf zebrafish hindbrains. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. (J) The Tol2 transposon cassettes of the control (Tol2-kdrl-mCherry-Tol2), myristoylated Akt1 (Tol2-kdrl-Myr-Akt1-P2A-mCherry-Tol2), and Mek1 (Tol2-kdrl-Mek1-P2A-mCherry-Tol2) expression plasmids. (K) Maximal intensity projection of a confocal z-stack of 45 hpf Tg(kdrl:EGFP) or TG2 embryos injected with control, myristoylated Akt1, or Mek1 expression vector. (L) Percentage of mCherry-positive normal CtAs. Numbers in the bracket of each group represent the number of mCherry-positive normal CtAs/the number of mCherry-positive total CtAs. (M and N) Percentage of PH3-positive ECs in 45 hpf Tg(kdrl:EGFP) or TG2 embryo CtAs injected with RFP mRNA, Myr-Akt1 mRNA, or Mek1 mRNA. Discussion The neuropeptide SN, processed from SgII, has multiple physiological functions (Zhao et al., 2009; Albrecht-Schgoer et al., 2012; Trudeau et al., 2012). It has been reported that synthetic SN can promote angiogenesis in vitro and in vivo (Kirchmair et al., 2004b). Therefore, the function of SN in angiogenesis has attracted considerable interest in recent years, yet the role of SN in CNS-specific angiogenesis has remained unknown until now. To our knowledge, no other early developmental roles for sgII have been reported previously. To investigate the role of sgII in CNS-specific angiogenesis, we generated sgII mutant zebrafish lines with GFP expressed in the vascular system. Our in vivo confocal imaging results showed that the number of normal CtAs was decreased while the number of disorganized CtAs was increased in the hindbrain of sgIIb mutant embryos. Time-lapse imaging indicated that depletion of sgIIb alone in zebrafish could lead to severe defects of CtA sprouting and pathfinding. However, some CtAs recovered to their normal appearance at 52 hpf in the sgIIb mutant embryos. This may be due to functional compensation by other angiogenic proteins, which may also act via MAPK or PI3K/AKT pathways (Kubota et al., 2005; Yu et al., 2010; Rossi et al., 2015). Brain morphology and expression of neuronal markers such as HuC, neurod4, and robo3 in rhombomeres or rhombomere boundaries (Park et al., 2000; Challa et al., 2001; Wang et al., 2003) remained normal. Neurons co-expressing sgIIb and camk2d2 also appeared normal in sgIIb mutant embryos. Injection of SNb mRNA could partly rescue the defects of CtA caused by the sgIIb mutation. Our cell transplantation experiments showed that sgIIb was non-cell autonomous and required for CtA development. Thus, sgIIb specifically participates in neurovascular modeling in the hindbrain of developing zebrafish. Despite the different physiological functions of the vascular and nervous systems, blood vessels are often aligned with nerves, and the two systems share some common molecular mechanisms (Park et al., 2004; Carmeliet and Tessier-Lavigne, 2005). Axon guidance molecules including netrins, ephrins, slits, semaphorins, and their receptors are cues in vascular morphogenesis (Carmeliet and Tessier-Lavigne, 2005). They participate in blood vessel navigation by either repelling or attracting the filopodia of tip ECs. We found that sgIIb is mainly expressed in the CNS of zebrafish embryos and co-expressed with camk2d2, one member of CaMKII family. As reported previously, the CaMKII family functions in growth cone guidance (Wen et al., 2004). The sgIIb-expressing cells were aligned with CtAs in the hindbrain. Previous studies demonstrated that SN is released from neurons (Kirchmair et al., 1994) and also acts as a direct angiogenic cytokine in vitro (Kirchmair et al., 2004b), suggesting the presence of a putative SN receptor on ECs. Defects of CtA sprouting and pathfinding in sgIIb mutant embryos imply that sgIIb is involved in vascular guidance in the hindbrain. Notch and VEGF signalings are fundamental for angiogenesis in physiological and pathological conditions. It has been demonstrated that VEGF signaling is stimulatory for angiogenic sprouting, while Notch signaling is inhibitory (Zhong et al., 2000, 2001; Covassin et al., 2006b; Suchting et al., 2007). In the hindbrain, Notch and VEGF signalings also play important roles in arterial–venous identity, which is regarded as an essential process of vascular development (Lawson and Weinstein, 2002; Bussmann et al., 2011). We found that the expression of Notch and VEGF pathway-related genes was not affected in sgIIb mutant brain ECs and arterial–venous identity of hindbrain was normal. Immunohistochemistry with an anti-PH3 antibody indicated that the percentage of proliferative ECs was decreased in CtAs of sgIIb mutant embryos. The MAPK and PI3K/AKT pathways play important roles in the proliferation and migration of ECs. Using loss-of-function and gain-of-function methods, we found that MAPK and PI3K/AKT pathways were inhibited in sgIIb mutant embryos in vivo. Injection of SNb, Myr-Akt1 or MEK1 mRNA, delivery of the activator ARA, and EC-specific expression of Myr-Akt1 or MEK1 could restore the function of MAPK and PI3K/AKT pathways and partially rescue CtA developmental defects in sgIIb mutant embryos. This is consistent with previous in vitro reports showing that culturing HUVECs and endothelial progenitor cells (EPCs) with SN could activate the MAPK system and the PI3K/AKT pathway (Kirchmair et al., 2004a, b). Our results indicate that the MAPK system and PI3K/AKT pathway mediate the effects sgIIb on neurovascular development. However, there are conflicting reports about whether the effects of SN on MAPK and PI3K/AKT pathway are dependent on VEGF. In HUVECs, the activation of MAPK and PI3K/AKT pathway by SN is not dependent on VEGF (Kirchmair et al., 2004b). Results obtained from HCAECs indicate that SN could promote binding of VEGF to its co-receptors heparin and neuropilin-1, demonstrating the role of SN in VEGF signaling (Albrecht-Schgoer et al., 2012). Our data provide evidence that VEGF pathway-related genes and VEGF-dependent arterial−venous identity are not altered in sgIIb−/− mutants. In summary, we have provided the first in vivo evidence that sgIIb plays a critical role in neurovascular modeling in the hindbrain that is mediated by activation of the MAPK and PI3K/AKT pathways. Cells expressing sgIIb are found in close association with vascular markers in the hindbrain; therefore, these sgIIb-positive cells are the likely source of SgIIb and/or SNb that controls the development of these CtAs. Ischemic stroke and neurodegenerative diseases such as Alzheimer’s disease are associated with abnormal angiogenesis in the brain. It is significant that SN promotes neuroprotection in mouse models of stroke (Shyu et al., 2008) and that proteomic markers for SgII are significantly decreased in CSF in Alzheimer’s disease patients (Spellman et al., 2015). Here, we show that injection of SNb mRNA could rescue the CtA defects in sgIIb mutant embryos. Targeting the SgII system may therefore represent a new avenue for the treatment of vascular defects in the CNS. Materials and methods Zebrafish care and maintenance AB strain zebrafish (Danio rerio) and their embryos were raised and maintained under standard conditions at 28.5°C. Embryos were staged as previously described (Kimmel et al., 1995). Tg(kdrl:EGFP) zebrafish were obtained from the China Zebrafish Resource Center (CZRC, Wuhan, China). The experiments involving zebrafish were performed under the approval of the Institutional Animal Care and Use Committee of the Institute of Hydrobiology, Chinese Academy of Sciences. Establishment of mutant zebrafish lines The paired TALENs for sgIIa or sgIIb were constructed using the golden gate method as described previously (Cermak et al., 2011; Liu et al., 2014). The final TALEN plasmids were linearized using Not1. TALEN mRNAs were synthesized using the mMessage mMACHINE SP6 Kit (Ambion, Inc.) and purified by LiCl precipitation. To generate mutant zebrafish lines, 250−500 pg TALEN mRNAs were microinjected into one-cell stage WT zebrafish embryos. Injected embryos were raised to adulthood and then outcrossed with WT fish to identify founders that transmitted mutations through the germ line. Mutations were genotyped by competitive PCR (Liu et al., 2014) and confirmed by sequencing. Plasmids and mRNA preparations Plasmids containing sequences of myristoylated Akt1 and Mek1(S218E, S222D) were purchased from Wuhan Miaolingbio Bioscience & Technology and Wuhan Genecreate Bioengineering, respectively. The full-length sequence of myristoylated Akt1 and Mek1(S218E, S222D) was cloned into a pCS2+ vector or into a Tol2-Kdrl promoter vector (a gift from Dr Jiulin Du, Institute of Neuroscience, Chinese Academy of Sciences, China) linked with mCherry via a P2A peptide sequence. Capped mRNAs were synthesized using the mMessage mMACHINE SP6 Kit (Ambion) and purified by LiCl precipitation. Embryo mounting, confocal microscopy, and image processing For in vivo confocal imaging, embryos were mounted in 1% low-melt agarose after anaesthetized in 168 mg/L tricaine. Fluorescence photomicrographs were collected with laser scanning confocal microscope (Zeiss LSM710). Time-lapse movies were prepared using the software program ZEN (Zeiss). Egg water containing tricaine and propylthiouracil was warmed to ~28°C and circulated during the observation period, z-stacks were collected at 10-min intervals. Images were processed using Adobe Photoshop CS3 Extended. The numbers of total CtAs, normal CtAs, and disorganized CtAs were counted using a fluoroscope (Leica M250) combined with laser scanning confocal microscope (Zeiss LSM710). The length of CtAs and midbrain vasculature was calculated with ImageJ analysis software (NIH). The unpaired two-tailed Student’s t-test as implemented in GraphPad Prism 5 was used to analyze the data. WISH and double-fluorescence in situ hybridization sgIIa, sgIIb, camk2d2, kdrl, cdh5, dab2, flt1, flt4, hey2, dll4, tal2, gad67 sequences were cloned by PCR amplification, PCR products were subcloned into the pMD18-T vector (TAKARA). Digoxigenin-labeled antisense probes for sgIIa, sgIIb, cdh5, dab2, flt1, flt4, hey2, dll4 were synthesized using DIG RNA Labeling Mix (Roche Diagnostics, Mannheim, Germany). WISH was carried out as described previously (Thisse and Thisse, 2008). Fluorescein-labeled antisense probes for camk2d2, kdrl, tal2, gad67 were synthesized using fluorescein RNA Labeling Mix (Roche Diagnostics, Mannheim, Germany). Double-fluorescence in situ hybridization was performed as described (Lauter et al., 2011). Primers used can be found in Supplementary Table S1. RT-PCR and RT-qPCR Heads of 36 hpf zebrafish embryos were cut off using a pair of forceps. ECs dissociated from 300−400 heads of Tg(kdrl:EGFP) and TG2 (sgIIb−/−;Tg(kdrl:EGFP)) were sorted using the FACSAriaTM III (BD Biosciences) as described previously (Covassin et al., 2006a). Total RNA was extracted from the sorted ECs or 40 zebrafish embryos (WT, sgIIa−/−, sgIIb−/−, sgIIa−/−/sgIIb−/−) at desired stages using TRIzol reagent (Invitrogen) and resuspended in diethyl-pyrocarbonate (DEPC)-treated water. The quality of extracted RNA was confirmed by UV spectrophotometer and agarose gel electrophoresis. Total RNA was reverse-transcribed into cDNA using ReverTra Ace M-MLV (TOYOBO) with random primers. For RT-PCR, the cDNA samples were PCR-amplified using gene-specific primers as listed in Supplementary Table S2. RT-qPCR was carried out on a Roche LightCycler 480 real-time PCR system using 2× SYBR green real-time PCR mix (TOYOBO) and primers as listed in Supplementary Table S3. All values were normalized to the level of β-actin mRNA. Western blotting Embryos were de-yolked as described (Link et al., 2006). The Total Protein Extraction Kit (Sangon Biotech) was used to lyse zebrafish embryos and adult zebrafish pituitaries according to the manufacturer’s instructions. The proteins of each lysate were separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and trans-blotted onto nitrocellulose membrane (Millipore), probed with indicated primary antibodies against phospho p44/42 MAPK (phospho-Erk1/2, 1:2000), p44/42 MAPK (total Erk1/2, 1:1000), p-AKT(1:2000), total AKT (1:1000) (Cell Signaling Technology), SNa (1:1000) (Zhao et al., 2006a), β-actin (Bioss Company, 1:1500), and VEGFA (Beyotime Biotechnology, 1:500). Then the membrane was washed in phosphate-buffered saline (PBS) containing Tween-20 (PBST) for 4 × 5 min, incubated with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies, and detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The signals were visualized using ImageQuant LAS 4000 mini system (GE Healthcare). Immunoblots were analyzed with ImageJ analysis software (NIH). Cell transplantation experiments Cell transplantation methods were similar to those described previously (Siekmann and Lawson, 2007) with some modifications. Tg(kdrl:EGFP) (WTEGFP) or sgIIb−/−;Tg(kdrl:EGFP) (TG2) embryos at one-cell stage were injected with tetramethylrhodamine isothiocyanate–dextran (Sigma) as a lineage tracer, and some TG2 embryos were co-injected with SNb mRNA (TG2SNb mRNA) or RFP mRNA (TG2RFP mRNA). These injected embryos were subsequently used as donors. Non-transgenic WT or sgIIb mutant (sgIIb−/− or TG2) embryos were used as hosts. Both donor and host embryos were dechorionated by forceps and subsequently cultured in plate covered with agarose. Cell transplantation was performed when donor and host embryos developed at the sphere stage (~4 hpf). Approximately 20−40 donor cells were transplanted into the margin of each host embryo. Five groups of cell transplantation experiments were carried out: WTEGFP > WT; TG2 > WT; WTEGFP > sgIIb−/−; TG2SNb mRNA > TG2, and TG2RFP mRNA > TG2. Transplanted cells were assessed by visualization of EGFP expression or tetramethylrhodamine isothiocyanate–dextran in host embryos. Numbers of chimeras with EGFP-positive CtAs and chimeras with EGFP-positive ECs in the hindbrain were counted using a fluoroscope (Leica M250) combined with laser scanning confocal microscope (Zeiss LSM710). Whole-mount immunohistochemistry Whole-mount immunohistochemistry was performed using phospho p44/42 MAPK (phospho-Erk1/2, 1:1000, CST) as primary antibody and DyLight 488 goat anti-mouse IgG (Abbkine, 1:200) as secondary antibody. Briefly, dechorionated zebrafish embryos were fixed in 4% PFA in PBS overnight at 4°C, dehydrated in 100% methanol for 15 min at room temperature, and stored at −20°C in 100% methanol for at least 2 h before use. Embryos were permeabilized in acetone (−20°C) for 10 min, washed with PBS containing Triton X-100 (PBST), incubated with the primary antibody overnight at 4°C, and then washed extensively and incubated with the secondary antibodies overnight at 4°C. Embryos were washed extensively again and mounted in glycerol. ImageJ (NIH) was used to measure the p-ERK density (signal intensity per pixel) in CtAs. Statistical analysis Statistical analysis was performed using unpaired two-tailed Student’s t-test as implemented in GraphPad Prism 5 (GraphPad Software Inc.). Data are presented as mean ± SEM. Acknowledgements We are grateful to Ms Fang Zhou (Analytical & Testing Center, IHB, CAS) for providing confocal services and Professors Marie-Andrée Akimenko (University of Ottawa) and Jingwei Xiong (Peking University) for their valuable discussion and critical reading of the manuscript. Funding This work was supported by the National Natural Science Foundation of China (31325026 and 31721005 to W.H.), Natural Sciences and Engineering Research Council of Canada (to V.L.T.), and the University of Ottawa International Research Acceleration Program (to V.L.T. and W.H.). Conflict of interest none declared. References Albrecht-Schgoer , K. , Schgoer , W. , Holfeld , J. , et al. . ( 2012 ). 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Google Scholar Crossref Search ADS PubMed © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

Secretogranin-II plays a critical role in zebrafish neurovascular modeling

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Oxford University Press
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© The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
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1674-2788
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10.1093/jmcb/mjy027
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Abstract

Abstract Secretoneurin (SN) is a neuropeptide derived from specific proteolytic processing of the precursor secretogranin II (SgII). In zebrafish and other teleosts, there are two paralogs named sgIIa and sgIIb. Our results showed that neurons expressing sgIIb were aligned with central arteries in the hindbrain, demonstrating a close neurovascular association. Both sgIIb−/− and sgIIa−/−/sgIIb−/− mutant embryos were defective in hindbrain central artery development due to impairment of migration and proliferation of central artery cells. Further study revealed that sgIIb is non-cell autonomous and required for central artery development. Hindbrain arterial and venous network identities were not affected in sgIIb−/− mutant embryos, and the mRNA levels of Notch and VEGF pathway-related genes were not altered. However, the activation of MAPK and PI3K/AKT pathways was inhibited in sgIIb−/− mutant embryos. Reactivation of MAPK or PI3K/AKT in endothelial cells could partially rescue the central artery developmental defects in the sgIIb mutants. This study provides the first in vivo evidence that sgIIb plays a critical role in neurovascular modeling of the hindbrain. Targeting the SgII system may, therefore, represent a new avenue for the treatment of vascular defects in the central nervous system. Secretogranin-II, secretoneurin, TALENs, neurovascular, zebrafish Introduction The development of the vascular system occurs by two processes: vasculogenesis and angiogenesis. Vasculogenesis is the de novo assembly of the first blood vessels, whereas angiogenesis is the coordinated growth of endothelial cells (ECs) from the pre-existing vasculature (Risau, 1997). Both processes are essential for the maintenance of tissue growth and organ function in development (Carmeliet, 2003a). Numerous congenital or acquired diseases are associated with pathological vasculogenesis or angiogenesis (Folkman, 1995; Carmeliet and Jain, 2000; Psaltis and Simari, 2015). Among them, brain tumors, ischemic stroke, and neurodegenerative diseases including Alzheimer’s disease are associated with abnormal brain angiogenesis (Krupinski et al., 1994; Zlokovic, 2005; Kim and Lee, 2009). Therapeutic is possible for the treatment of such diseases (Jain, 2001; Chu and Wang, 2012; Wong et al., 2016). However, some of the larger clinical trials using single angiogenic factors have not corroborated the exciting early results (Krichavsky and Losordo, 2011), but combinations of multiple angiogenic factors may be a more promising approach. It is therefore necessary to uncover new pro-angiogenic and anti-angiogenic factors and to have a comprehensive understanding of the mechanism of angiogenesis. Many ligands and their receptors are involved in the regulation of angiogenesis. These include Notch/Delta-like pathway (Siekmann and Lawson, 2007; Wang et al., 2014), VEGF pathway (Olsson et al., 2006), Wnt pathway (Dejana, 2010), shh pathway (Pola et al., 2001), angiopoietin (Suri et al., 1996), and netrins (Wilson et al., 2006) have been shown to be important. It is noteworthy that blood vessels are often aligned with nerves and display similar branching patterns. Some of these pathways or ligand–receptor complexes have been shown to act in parallel on both vascular and neural cells, demonstrating the interdependence and functional connection of the two systems sharing similar regulatory mechanisms (Larrivée et al., 2009). It may accelerate the discovery of mechanistic insights to realize that vascular system and nervous system use some common genetic pathways (Carmeliet, 2003b). Secretogranin-II (SgII) is mainly distributed in dense-core vesicles of many neurons and endocrine cells, and is overexpressed in some neuroendocrine tumors (Mahata et al., 1991). The neuropeptide secretoneurin (SN) is a short conserved peptide (31−43 amino acids) derived from the larger SgII precursor protein (~600 amino acids) by prohormone convertase-mediated processing (Fischer-Colbrie et al., 1995; Trudeau et al., 2012). While several potential peptides may arise from SgII processing, SN is the only highly abundant neuropeptide with known biological activities (Fischer-Colbrie et al., 1995). Various physiological roles have been assigned to SN (Trudeau et al., 2012), including those related to reproduction (Zhao et al., 2009), neuroinflammation (You et al., 1996), and neurotransmitter release (Reinisch et al., 1993). In the rodent brain, SN is found predominantly in the phylogenetically older parts, overlapping partly but not completely with established neurotransmitter or peptidergic systems (Fischer-Colbrie et al., 1995; Trudeau et al., 2012). Supporting this, in the goldfish, SN exhibits a more restricted central nervous system (CNS) distribution mainly to the preoptic magnocellular neurons co-expressing nonapetides in the oxytocin/vasopressin family, certain hypothalamic nuclei, and posterior projections to hindbrain structures (Canosa et al., 2011). Best described are the pro-angiogenic effects of SN. Synthetic SN can promote capillary tube formation in human umbilical vein ECs (HUVECs) in vitro (Kirchmair et al., 2004b) and induce neovascularization in the mouse cornea in vivo (Kirchmair et al., 2004b). The mechanism for the promotion of angiogenesis by SN may be not the same in different blood vessels. In vitro results show that the activation of mitogen-activated protein kinases (MAPK) by SN is dependent on vascular endothelial growth factor (VEGF) in human coronary artery ECs (HCAECs) (Albrecht-Schgoer et al., 2012), while in HUVECs, the angiogenic effects by SN are VEGF-independent (Kirchmair et al., 2004b). To date, most of the studies on angiogenesis have been carried out using exogenously applied SN. However, little is known about the effect of the endogenous SgII precursor protein or the SN neuropeptide on blood vessel formation as there are currently no sgII knockout animal models, and human mutations have not yet been identified. The aim of our study, therefore, was to investigate the role of sgII during early developmental stages. In zebrafish and other teleost fishes, there exist two paralogous genes, sgIIa and sgIIb that generate SNa and SNb peptides, respectively (Zhao et al., 2010). In this study, we have generated sgIIa−/−, sgIIb−/−, and sgIIa−/−/sgIIb−/− mutant zebrafish lines using transcription activator-like effector nucleases (TALENs), and found that sgIIb−/− and sgIIa−/−/sgIIb−/− mutants have specific defects in the development of hindbrain central arteries (CtAs). SgIIb plays a critical role in zebrafish neurovascular modeling that is mediated by MARK and PI3K/AKT signaling in vivo. Results sgIIb is expressed in the central nervous system of zebrafish embryos and sgIIb-expressing neurons and central arteries are aligned in the hindbrain We have established the expression pattern of sgIIb in wild-type (WT) zebrafish embryos by semi-quantitative reverse-transcriptase PCR (RT-PCR) and whole-mount in situ hybridization (WISH) technique. sgIIb mRNA levels were low at 10 h post-fertilization (hpf), then increased over the 14−24 hpf period, and stabilized after 36 hpf (Figure 1A). The WISH results revealed that sgIIb was mainly expressed in the CNS at 24 and 36 hpf, and concentrated in the brain by 45 hpf (Figure 1B). Then we examined the relationship between sgIIb-positive cells and Calcium/calmodulin-dependent protein kinase II delta 2 (Camk2d2)-expressing cells in the hindbrain. Camk2d2 belongs to the CaMKII family that functions in neuronal growth cone guidance and synaptic plasticity, among other functions (Mayford et al., 1995; Wen et al., 2004). Double-fluorescence in situ hybridization revealed colocalization of sgIIb and camk2d2 in the hindbrain (Figure 1C). To investigate the positional relationship between sgIIb-expressing cells and the vascular system in the hindbrain, double-fluorescence in situ hybridization with probes for sgIIb and the vascular marker kdrl (kinase insert domain receptor-like) was performed in 36−45 hpf embryos. Our results showed that sgIIb-expressing cells were aligned with the growing CtAs at 36−39 hpf (Figure 1D−I, P−R). At 42−45 hpf, sgIIb was widely distributed around CtAs in the hindbrain (Figure 1J−O, S−U). The expression of sgIIa was different from that of sgIIb. Firstly, sgIIa was detectable by 10 hpf (Supplementary Figure S1A), ~4 h earlier than sgIIb (Figure 1A). Thereafter, sgIIa expression increased gradually until it stabilized at ~24 hpf. sgIIa was highly expressed in the forebrain, midbrain, and ventral part of the neural tube but barely expressed in the hindbrain (Supplementary Figure S1B), which contrasts significantly with the abundance of sgIIb transcripts in this region (Figure 1B). Double-fluorescence WISH revealed that sgIIa-positive cells co-expressed the GABAergic neuron marker tal2 (basic helix-loop-helix transcription factor) and gad67, the mRNA encoding the GABA-synthesizing enzyme glutamic acid decarboxylase 67 (Supplementary Figure S1C). Figure 1 View largeDownload slide The developmental expression pattern of sgIIb in the CNS of zebrafish. (A) RT-PCR analysis for temporal expression of sgIIb mRNA during embryogenesis and early larval developmental stages. M, molecular size marker; C, no template control. (B) WISH of sgIIb in 24, 36, and 45 hpf zebrafish embryos. (C−U) Mapping expression of sgIIb in relation to camk2d2 and kdrl in the zebrafish hindbrain with double-fluorescence in situ hybridization. (C) Confocal imaging for sgIIb and camk2d2 mRNA at 36 hpf. camk2d2-positive cells co-express sgIIb (white arrowheads). (D−O) Maximal intensity projection of a confocal z-stack for sgIIb and kdrl mRNA. kdrl-positive cells are aligned with sgIIb-expressing cells (white arrowheads). (P−U) Single confocal planes showing sgIIb and kdrl mRNA. Embryos were examined at 36 hpf (D−F, P−R), 39 hpf (G−I), 42 hpf (J−L, S−U), and 45 hpf (M−O). Scale bar, 100 μm. Figure 1 View largeDownload slide The developmental expression pattern of sgIIb in the CNS of zebrafish. (A) RT-PCR analysis for temporal expression of sgIIb mRNA during embryogenesis and early larval developmental stages. M, molecular size marker; C, no template control. (B) WISH of sgIIb in 24, 36, and 45 hpf zebrafish embryos. (C−U) Mapping expression of sgIIb in relation to camk2d2 and kdrl in the zebrafish hindbrain with double-fluorescence in situ hybridization. (C) Confocal imaging for sgIIb and camk2d2 mRNA at 36 hpf. camk2d2-positive cells co-express sgIIb (white arrowheads). (D−O) Maximal intensity projection of a confocal z-stack for sgIIb and kdrl mRNA. kdrl-positive cells are aligned with sgIIb-expressing cells (white arrowheads). (P−U) Single confocal planes showing sgIIb and kdrl mRNA. Embryos were examined at 36 hpf (D−F, P−R), 39 hpf (G−I), 42 hpf (J−L, S−U), and 45 hpf (M−O). Scale bar, 100 μm. Establishment of sgII mutant zebrafish lines with TALENs To investigate the role of sgII during neurovascular development in vivo, two pairs of TALENs were designed for the zebrafish sgIIa and sgIIb genes. The TALEN target sites of sgIIa and sgIIb were both chosen following the ATG start site and in front of SNa and SNb domains (Figure 2A and C). The sgIIa heterozygote with 7-bp deletion and 2-bp insertion (−7,+2 bp) or just 7-bp deletion (−7 bp) and the sgIIb heterozygote with a 7-bp insertion and 5-bp (−5,+7 bp) deletion or just 10-bp deletion (−10 bp) were screened out and further used to establish the sgIIa−/− and sgIIb−/− homozygous mutant line (Figure 2A and C). All these mutant lines resulted in open reading frame-shift mutants of sgIIa or sgIIb gene, and thus generating truncated proteins with no SN peptides (Figure 2B and D). The sgIIa−/−/sgIIb−/− homozygous mutant line (sgIIa−/−−7,+2 bp;sgIIb−/− −5,+7 bp) was obtained by crossing the sgIIa−/− homozygote (−7,+2 bp) with the sgIIb−/− homozygote (−5,+7 bp). All crossings were performed using in vitro fertilization. Both the real-time quantitative PCR (RT-qPCR) and WISH results revealed that mRNA levels of sgIIa and sgIIb were significantly decreased in sgIIa (Figure 2E and F) and sgIIb (Figure 2E and G) mutant embryos, respectively, compared to WT, indicating a mechanism of nonsense-mediated mRNA decay (Chang et al., 2007). Since the pituitary is a major production site of SgIIa protein and SN peptide, we monitored SN immunoreactivity in adults to determine the effects of mutations on protein production using a well-characterized polyclonal antibody (Zhao et al., 2006b) that recognizes zebrafish SNa but not SNb (Supplementary Figure S2). Two bands corresponding to the full-length precursor (~62.2 kDa) and an intermediate fragment (~59 kDa) were observed in WT zebrafish pituitaries. Our results revealed that SgIIa precursor protein or proteolitically processed SNa-immunoreactive fragments were not detectable in the adult pituitary gland of sgIIa−/− mutant and sgIIa−/−/sgIIb−/− double mutant fish. The ~30 kDa SNa-immunoreactive fragment of SgIIa was dramatically increased in adult sgIIb−/− mutant fish (Figure 2H), suggesting a compensatory expression of sgIIa in adult sgIIb−/− mutant fish. Figure 2 View largeDownload slide Mutation of sgIIb causes defects of CtA development in vivo. (A and C) The location of the TALEN-binding sites (underlined) on zebrafish sgIIa or sgIIb gene and two mutant lines of TALEN-targeted sgIIa alleles (A) or sgIIb alleles (C). Deletions and insertions are indicated by dashes and red letters, respectively. (B and D) Schematic representation of the putative WT SgIIa or SgIIb protein and two mutated SgIIa proteins (B) or SgIIb proteins (D). (E) WISH of WT, sgIIa−/− mutant, and sgIIb−/− mutant embryos. Antisense probes against sgIIa and sgIIb were visualized at 36 hpf. Scale bar, 100 μm. (F and G) Relative mRNA level of sgIIa (F) or sgIIb (G) in 36 hpf sgIIa−/− mutant or sgIIb−/− mutant embryos, respectively, as measured by RT-qPCR. Data shown are mean ± SEM of three independent experiments. Statistical significance was assessed using the two-tailed Student’s t-test. (H) Western blotting analysis of the pituitary samples from WT, sgIIa−/− mutant, sgIIb−/− mutant, and sgIIa−/−/sgIIb−/− mutant adults at 120 dpf. (I) Schematic vascular modeling in the hindbrain of 36 hpf and 45 hpf WT zebrafish embryos. PHBCs are in blue, BA is in red, and CtAs are in green. (J) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP), sgIIb−/−;Tg(kdrl:EGFP) (TG2), and sgIIa−/−/sgIIb−/−;Tg(kdrl:EGFP) (TG3) zebrafish hindbrain. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. Embryos were examined at 36, 39, 42, 45, and 48 hpf. Scale bar, 50 μm. (K−N) Quantitative analysis of CtAs in Tg(kdrl:EGFP), TG2, and TG3 zebrafish hindbrains. Numbers of total CtAs (K), normal CtAs (L), and disorganized CtAs (M) and total length of CtAs (N) were determined. Embryos were 45 and 48 hpf. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. Figure 2 View largeDownload slide Mutation of sgIIb causes defects of CtA development in vivo. (A and C) The location of the TALEN-binding sites (underlined) on zebrafish sgIIa or sgIIb gene and two mutant lines of TALEN-targeted sgIIa alleles (A) or sgIIb alleles (C). Deletions and insertions are indicated by dashes and red letters, respectively. (B and D) Schematic representation of the putative WT SgIIa or SgIIb protein and two mutated SgIIa proteins (B) or SgIIb proteins (D). (E) WISH of WT, sgIIa−/− mutant, and sgIIb−/− mutant embryos. Antisense probes against sgIIa and sgIIb were visualized at 36 hpf. Scale bar, 100 μm. (F and G) Relative mRNA level of sgIIa (F) or sgIIb (G) in 36 hpf sgIIa−/− mutant or sgIIb−/− mutant embryos, respectively, as measured by RT-qPCR. Data shown are mean ± SEM of three independent experiments. Statistical significance was assessed using the two-tailed Student’s t-test. (H) Western blotting analysis of the pituitary samples from WT, sgIIa−/− mutant, sgIIb−/− mutant, and sgIIa−/−/sgIIb−/− mutant adults at 120 dpf. (I) Schematic vascular modeling in the hindbrain of 36 hpf and 45 hpf WT zebrafish embryos. PHBCs are in blue, BA is in red, and CtAs are in green. (J) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP), sgIIb−/−;Tg(kdrl:EGFP) (TG2), and sgIIa−/−/sgIIb−/−;Tg(kdrl:EGFP) (TG3) zebrafish hindbrain. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. Embryos were examined at 36, 39, 42, 45, and 48 hpf. Scale bar, 50 μm. (K−N) Quantitative analysis of CtAs in Tg(kdrl:EGFP), TG2, and TG3 zebrafish hindbrains. Numbers of total CtAs (K), normal CtAs (L), and disorganized CtAs (M) and total length of CtAs (N) were determined. Embryos were 45 and 48 hpf. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. Mutation of sgIIb causes defects of CtA development in vivo The sgIIa−/−, sgIIb−/−, and sgIIa−/−/sgIIb−/− mutant zebrafish lines were crossed with the Tg(kdrl:EGFP) line that expresses green fluorescent protein (GFP) in the vascular system. Their progeny was raised to adulthood and intercrossed to establish three homozygous mutant lines expressing GFP in vascular system, i.e. TG1: sgIIa−/−;Tg(kdrl:EGFP), TG2: sgIIb−/−;Tg(kdrl:EGFP), and TG3: sgIIa−/−/sgIIb−/−;Tg(kdrl:EGFP). CtAs are a set of vessels penetrating the hindbrain; they grow from the primordial hindbrain channels (PHBCs) to the basilar artery (BA) and interconnect the PHBCs and BA (Figure 2I). The percentage of embryos with CtA defects was significantly increased in TG2 embryos (Supplementary Figure S3A). In vivo confocal imaging results showed specific defects of hindbrain CtA development in TG2 and TG3 embryos at 36, 39, 42, 45, and 48 hpf (Figure 2J). Normal CtAs are those extend to BA along an inverted V pattern, as shown previously (Fujita et al., 2011), while disorganized CtAs are those do not migrate along an inverted V pattern. Compared to the Tg(kdrl:EGFP) line, we found that the number of normal CtAs, the number of total CtAs, and the total length of CtAs were all significantly decreased (Figure 2K, L, and N), whereas the number of disorganized CtAs increased (Figure 2M) in TG2 and TG3 embryos at 45 and 48 hpf, respectively. The appearance of the CtA defects in TG2 and TG3 embryos was highly similar (Figure 2J−N). In addition, the overall morphology and survival rate were not affected in sgIIb−/− mutant embryos (Supplementary Figure S4A and B). Other blood vessels outside the hindbrain remained normal in TG2 embryos (Figure 3A−J). Midbrain vasculature was statistically analyzed following methods in a previous report (Chen et al., 2012). Total length and segment number of midbrain vasculature were not affected in TG2 embryos (Figure 3I and J). These data demonstrate that sgIIb is critical for neurovascular modeling specifically in the hindbrain. sgIIb and camk2d2 co-expressing neuronal cells were not affected in the hindbrain of sgIIb−/− mutant embryos (Figure 3L). Brain morphology and the expression of the neuronal markers HuC, neurod4, and robo3 in rhombomeres or rhombomere boundaries (Park et al., 2000; Challa et al., 2001; Wang et al., 2003) also remained normal (Figure 3K, M−O), demonstrating that CtA defects in sgIIb mutants are not an indirect consequence of changes in brain growth or patterning. To examine the key roles of sgIIb in CtA development more precisely, we carried out time-lapse imaging from 36 to 45 hpf using Tg(kdrl:EGFP) and TG2. Our results showed that some CtAs failed to sprout or connect to the BA in TG2 embryos that lack SgIIb (Supplementary Video S1) compared with Tg(kdrl:EGFP) (Supplementary Video S2). By 52 hpf in TG2 and TG3 embryos, the number of disorganized CtAs still remained somewhat high (Supplementary Figure S5C), but some CtAs that did not extend to BA at 48 hpf recovered to their normal appearance (Supplementary Videos S3 and S4, Figure S5A and B). Defects were also visualized using WISH for cadherin 5 (cdh5) in the hindbrain vasculature of 45 hpf sgIIb−/− mutant embryos (Supplementary Figure S3B), an observation consistent with the results from 45 hpf TG2 or TG3 embryos. Importantly, sgIIb knockdown by morpholino injections caused similar defects of CtA development (Supplementary Figures S3C, S6A and B) as noted with the sgIIb knockout experiments. In contrast, CtA development was not affected in TG1 embryos (Supplementary Figure S7A). Since sgIIa is weakly expressed in the hindbrain of zebrafish embryos (Supplementary Figure S1B), and the sgIIa mRNA level remained unchanged in sgIIb mutant embryos (Supplementary Figure S7B and C), sgIIa is unlikely to participate in the development of hindbrain CtAs. Figure 3 View largeDownload slide sgIIb is critical for neurovascular modeling specifically in the hindbrain. (A−H) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP) and TG2 embryos at 45 hpf. (A−D) Lateral views of Tg(kdrl:EGFP) and TG2 vasculature in the head (A and B) and trunk (C and D). (E and G) Dorsal views of Tg(kdrl:EGFP) and TG2 vasculature in the anterior head region. (F and H) Lateral views of magnified Tg(kdrl:EGFP) and TG2 vasculature in the trunk region. (I and J) Quantitative analysis of midbrain vasculature in Tg(kdrl:EGFP) and TG2 zebrafish lines. (K) Brain morphology of WT and sgIIb−/− mutant embryos at 45 hpf (dorsal view). (L−O) Maximal intensity projection of a confocal z-stack for sgIIb mRNA together with camk2d2 (L), HuC (M), neurod4 (N), and robo3 (O) mRNA at 36 hpf WT and sgIIb−/− mutant embryos using double-fluorescence in situ hybridization (lateral view). DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; h, hindbrain; ISV, intersegmental vessel; m, midbrain; MCeV, mid-cerebral vein; mhb, midbrain−hindbrain boundary; MsV, mesencephalic vein; MtA, metencephalic artery; PCV, posterior cardinal vein. Scale bar, 50 μm. Figure 3 View largeDownload slide sgIIb is critical for neurovascular modeling specifically in the hindbrain. (A−H) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP) and TG2 embryos at 45 hpf. (A−D) Lateral views of Tg(kdrl:EGFP) and TG2 vasculature in the head (A and B) and trunk (C and D). (E and G) Dorsal views of Tg(kdrl:EGFP) and TG2 vasculature in the anterior head region. (F and H) Lateral views of magnified Tg(kdrl:EGFP) and TG2 vasculature in the trunk region. (I and J) Quantitative analysis of midbrain vasculature in Tg(kdrl:EGFP) and TG2 zebrafish lines. (K) Brain morphology of WT and sgIIb−/− mutant embryos at 45 hpf (dorsal view). (L−O) Maximal intensity projection of a confocal z-stack for sgIIb mRNA together with camk2d2 (L), HuC (M), neurod4 (N), and robo3 (O) mRNA at 36 hpf WT and sgIIb−/− mutant embryos using double-fluorescence in situ hybridization (lateral view). DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; h, hindbrain; ISV, intersegmental vessel; m, midbrain; MCeV, mid-cerebral vein; mhb, midbrain−hindbrain boundary; MsV, mesencephalic vein; MtA, metencephalic artery; PCV, posterior cardinal vein. Scale bar, 50 μm. sgIIb is non-cell autonomous and is required for migration and proliferation of central artery ECs in vivo To date, the only known bioactive peptide generated from the SgII precursor is SN. When we injected SNb mRNA into one-cell stage embryos of the TG2 line, the defects of CtA development observed in TG2 embryos could be partly rescued (Figure 4A−C). No ectopic sprouting of blood vessel was observed in Tg(kdrl:EGFP) following SNb mRNA injection (Supplementary Figure S8A−D). Cell transplantation experiments were carried out to investigate whether sgIIb is non-cell autonomous in CtA development. Donor Tg(kdrl:EGFP) or TG2 embryos (donor-derived ECs were marked with GFP) were injected with Tritc-dextran (to mark all donor-derived cells) at the one-cell stage. WT and sgIIb−/− embryos were used as hosts. Cell transplantation results showed that the percentage of embryos with EGFP-positive CtAs was higher in WT hosts, irrespective of which donor embryos were used (Figure 4D and F). More specifically, percentages of embryos with EGFP-positive CtAs were 70% (14/20) in WT–WT group and 61 % (11/18) in the sgIIb−/−–WT group (Figure 4F), indicating that transplanted sgIIb−/− cells could develop to CtAs at a similar degree with WT cells in WT embryo hosts. Importantly, sgIIb expression was detected in camk2d2-positive neurons but not the CtAs (Figure 1C and P−U), indicating that the source of SgIIb/SNb is not the ECs, and thus any effect should be non-cell autonomous. Only 13% (3/23) of the chimeras had EGFP-positive CtAs in WT-sgIIb−/− group (Figure 4F), suggesting that CtA development from WT cells was rather poor, when surrounded by sgIIb−/− cells in the recipient embryos. Moreover, when SNb mRNA-injected TG2 cells were transplanted into areas adjacent to CtAs in TG2 embryos, CtA defects in the sgIIb mutants could be partially rescued (Figure 4E and G). Transplanted cells were not EGFP-positive, demonstrating that transplanted cells were not CtA ECs, while CtA defects could be rescued by these non-ECs. Thus, it can be confirmed that the role of SgIIb/SNb in CtA development was non-cell autonomous. Furthermore, the duration of CtA migration from PHBC to BA was significantly longer in sgIIb mutant fish (Figure 4H), indicating that CtA migration speed was affected by deletion of sgIIb. Anti-phosphorylated histone H3 (PH3) antibody was used to detect the cells in M-phase of the cell cycle. Whole-mount immunohistochemistry results indicated that the percentage of PH3-positive ECs within CtAs was significantly decreased in sgIIb−/− mutant embryos (Figure 4I and N), while the percentage of PH3-positive non-ECs in the hindbrain was not affected (Figure 4J and N). The percentage of PH3-positive cells within PHBC, trunk, forebrain, and midbrain was also not affected in sgIIb−/− mutant embryos (Figure 4K−M and O−Q). Therefore, sgIIb was required for EC proliferation in zebrafish CtAs specifically. These findings demonstrate that sgIIb is non-cell autonomous and required for migration and proliferation of CtA ECs in vivo. Figure 4 View largeDownload slide sgIIb is non-cell autonomously required for migration and proliferation of CtA ECs in vivo. (A) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP) and TG2 embryos injected with SNb or RFP mRNA. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. (B and C) Quantitative analysis of CtAs in the hindbrain of Tg(kdrl:EGFP)+RFP mRNA, TG2+RFP mRNA, and TG2+SNb mRNA-injected zebrafish. Numbers of normal CtAs (B) and disorganized CtAs (C) were counted in 45 hpf zebrafish hindbrain. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. (D) Maximal intensity projection of a confocal z-stack of chimeras at 45 hpf. White arrowheads indicate CtAs. (E) Maximal intensity projection of a confocal z-stack of chimeras at 45 hpf. (F) Percentage of chimeras with EGFP-positive CtAs. Numbers in the bracket of each group represent the number of chimeras with EGFP-positive CtAs/the number of chimeras with EGFP-positive ECs in the hindbrain. (G) Quantitative analysis of normal CtAs in 45 hpf TG2SNb mRNA > TG2 and TG2RFP mRNA > TG2 embryos. (H) Quantitative analysis of CtA migration duration in Tg(kdrl:EGFP) and TG2 embryos. (I−Q) Percentage of PH3-positive ECs and non-ECs in 42 hpf Tg(kdrl:EGFP) and TG2 embryos. The percentage of PH3-positive ECs in CtA (I and N), PHBC (K and O), forebrain and midbrain (L and P), and trunk (M and Q). The percentage of PH3-positive non-ECs in the hindbrain (J). Data shown are mean ± SEM (n = the number of embryos analyzed). DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel; MCeV, mid-cerebral vein; MsV, mesencephalic vein. Scale bar, 50 μm. Figure 4 View largeDownload slide sgIIb is non-cell autonomously required for migration and proliferation of CtA ECs in vivo. (A) Maximal intensity projection of a confocal z-stack of Tg(kdrl:EGFP) and TG2 embryos injected with SNb or RFP mRNA. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. (B and C) Quantitative analysis of CtAs in the hindbrain of Tg(kdrl:EGFP)+RFP mRNA, TG2+RFP mRNA, and TG2+SNb mRNA-injected zebrafish. Numbers of normal CtAs (B) and disorganized CtAs (C) were counted in 45 hpf zebrafish hindbrain. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. (D) Maximal intensity projection of a confocal z-stack of chimeras at 45 hpf. White arrowheads indicate CtAs. (E) Maximal intensity projection of a confocal z-stack of chimeras at 45 hpf. (F) Percentage of chimeras with EGFP-positive CtAs. Numbers in the bracket of each group represent the number of chimeras with EGFP-positive CtAs/the number of chimeras with EGFP-positive ECs in the hindbrain. (G) Quantitative analysis of normal CtAs in 45 hpf TG2SNb mRNA > TG2 and TG2RFP mRNA > TG2 embryos. (H) Quantitative analysis of CtA migration duration in Tg(kdrl:EGFP) and TG2 embryos. (I−Q) Percentage of PH3-positive ECs and non-ECs in 42 hpf Tg(kdrl:EGFP) and TG2 embryos. The percentage of PH3-positive ECs in CtA (I and N), PHBC (K and O), forebrain and midbrain (L and P), and trunk (M and Q). The percentage of PH3-positive non-ECs in the hindbrain (J). Data shown are mean ± SEM (n = the number of embryos analyzed). DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel; MCeV, mid-cerebral vein; MsV, mesencephalic vein. Scale bar, 50 μm. Mutation of sgIIb does not affect arterial−venous identity or Notch and VEGF pathways in the hindbrain The establishment of arterial–venous identity is essential in the development of blood vessels (Lawson and Weinstein, 2002). We therefore examined artery- and vein-specific markers in the hindbrain of 36 hpf embryos by WISH. The expression of vein-specific markers (dab2 and flt4) and artery-specific markers (hey2, flt1, and dll4) was not affected in sgIIb mutant compared to WT embryos (Figure 5A and B). Double-fluorescence in situ hybridization revealed that the artery-specific marker flt1 was still expressed in CtAs of sgIIb mutant embryos (Figure 5C). Thus, CtA defects in sgIIb mutants are not associated with changes to arterial–venous identity in the hindbrain. It has been reported that Notch and VEGF pathways are critical for angiogenesis (Jakobsson et al., 2009; Blanco and Gerhardt, 2013). Fluorescence-activated cell sorting was used to obtain ECs from Tg(kdrl:EGFP) and TG2 embryonic heads. The mRNA level of Notch and VEGF pathway-related genes was determined using RT-qPCR. As shown in Supplementary Figures S9 and S10A, the mRNA levels of Notch pathway-related genes (including notch1a, notch1b, notch2, dll4, hey2, hey1, hey6, dlc, dld) and VEGF pathway-related genes (including vegfaa, vegfab, vegfb, vegfc, vegfd, flt1, kdrl, flt4, nrp1a, nrp1b) in ECs were not affected by mutation of sgIIb. VEGFA protein level in sgIIb mutant embryos was also not changed (Supplementary Figure S10C and D). vegfaa dominantly functions in VEGFa signaling and is also expressed in neural cells in addition to ECs (Wild et al., 2017). The expression of vegfaa was not affected in the sgIIb mutant hindbrain, as determined using WISH (Supplementary Figure S10B). These results indicate that Notch and VEGF pathways are not affected in the hindbrain of sgIIb mutant fish. Figure 5 View largeDownload slide Mutation of sgIIb inhibits the activation of MAPK and PI3K/AKT pathways. (A and B) WISH was performed in 36 hpf WT and sgIIb mutant zebrafish embryos with venous markers dab2 and flt4 (A) and arterial markers hey2, flt1, and dll4 (B). Black arrowheads indicate the expression of venous markers and red arrowheads indicate the expression of venous markers in the hindbrain. (C) Confocal imaging for kdrl and flt1 mRNA in WT and sgIIb mutant zebrafish embryos at 36 hpf. White arrowheads point to sprouting CtAs (kdrl-positive) that co-express flt1. (D) Whole-mount immunohistochemistry for p-ERK1/2 expression in 36 hpf WT and sgIIb−/− mutant embryos. The frequency of embryos with the indicated phenotypes is shown in the bracket of each group. Scale bar, 100 μm. (E) Western blots for phospho-AKT (p-AKT), total AKT, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2 expression in 36 hpf WT and sgIIb−/− mutant embryos. Actin was used as a loading control. (F) Single confocal planes of EGFP and p-ERK1/2 in 42 hpf Tg(kdrl:EGFP) and TG2 embryonic hindbrain (lateral view). Scale bar, 50 μm. (G) Magnified views of single confocal planes of EGFP-positive CtAs and p-ERK1/2 from dashed box in F. Dashed areas delineate CtAs that co-express EGFP and p-ERK1/2. Scale bar, 25 μm. (H and I) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (J) Relative density of p-ERK signals at CtAs in 42 hpf Tg(kdrl:EGFP) and TG2 embryos. Data shown are mean ± SEM (n = the number of CtAs analyzed). Figure 5 View largeDownload slide Mutation of sgIIb inhibits the activation of MAPK and PI3K/AKT pathways. (A and B) WISH was performed in 36 hpf WT and sgIIb mutant zebrafish embryos with venous markers dab2 and flt4 (A) and arterial markers hey2, flt1, and dll4 (B). Black arrowheads indicate the expression of venous markers and red arrowheads indicate the expression of venous markers in the hindbrain. (C) Confocal imaging for kdrl and flt1 mRNA in WT and sgIIb mutant zebrafish embryos at 36 hpf. White arrowheads point to sprouting CtAs (kdrl-positive) that co-express flt1. (D) Whole-mount immunohistochemistry for p-ERK1/2 expression in 36 hpf WT and sgIIb−/− mutant embryos. The frequency of embryos with the indicated phenotypes is shown in the bracket of each group. Scale bar, 100 μm. (E) Western blots for phospho-AKT (p-AKT), total AKT, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2 expression in 36 hpf WT and sgIIb−/− mutant embryos. Actin was used as a loading control. (F) Single confocal planes of EGFP and p-ERK1/2 in 42 hpf Tg(kdrl:EGFP) and TG2 embryonic hindbrain (lateral view). Scale bar, 50 μm. (G) Magnified views of single confocal planes of EGFP-positive CtAs and p-ERK1/2 from dashed box in F. Dashed areas delineate CtAs that co-express EGFP and p-ERK1/2. Scale bar, 25 μm. (H and I) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (J) Relative density of p-ERK signals at CtAs in 42 hpf Tg(kdrl:EGFP) and TG2 embryos. Data shown are mean ± SEM (n = the number of CtAs analyzed). The role of sgIIb in neurovascular modeling is mediated by MAPK and PI3K/AKT signaling in vivo Previous studies have demonstrated that MAPK and PI3K/AKT pathways participate in the proliferation and migration of ECs (Mavria et al., 2006; Karar and Maity, 2011). We examined the MAPK and PI3K/AKT pathways in sgIIb mutant embryos with defects of CtA development. The expression levels of p-ERK1/2 and p-AKT in sgIIb mutant embryos were much lower than that in WT, while the levels of total ERK1/2 and total AKT were unchanged (Figure 5D, E, H, and I). Whole-mount immunohistochemistry and confocal imaging results showed that p-ERK1/2 was expressed in CtAs (Figure 5F and G; Supplementary Figure S11). Furthermore, the relative density of p-ERK signal was significantly decreased in CtAs of TG2 embryos (Figure 5F, G, and J). These results suggest that the activation of MAPK and PI3K/AKT was inhibited in the CtAs of sgIIb mutants. Accordingly, when we injected SNb mRNA into one-cell stage sgIIb mutant embryos, the expression levels of p-ERK1/2 and p-AKT were significantly increased at 36 hpf (Figure 6A−C), while the levels of total ERK1/2 and total AKT remained unchanged. N-arachidonoyl-L-serine (ARA) is a known activator of MAPK and PI3K/AKT pathways (Milman et al., 2006). Delivery of ARA (50 μM) increased p-ERK1/2 and p-AKT levels in sgIIb mutant embryos (Figure 6E−G). Furthermore, CtA developmental defects in TG2 fish could be partially rescued by ARA treatment (Figure 6D, H, and I). The constitutively active forms of the protein Mek1(S218E, S222D) (Mansour et al., 1994) or myristoylated Akt1 (Kubota et al., 2005) were specifically expressed in ECs through injection of 50 pg Tol2-kdrl-Mek1-P2A-mCherry-Tol2 or Tol2-kdrl-Myr-Akt1-P2A-mCherry-Tol2. We found that upregulated MAPK or PI3K/AKT activity in ECs partially rescued CtA defects in sgIIb mutant embryos (Figure 6K and L). To detect whether upregulation of MAPK or PI3K/AKT signal can rescue proliferation defects in sgIIb mutant embryos, whole-mount immunohistochemistry with PH3 antibody was conducted after the injection of 75 pg Mek1(S218E, S222D) or myristoylated Akt1 mRNA. As shown in Figure 6M and N, the percentage of PH3-positive ECs within sgIIb mutant CtAs was significantly increased with Mek1(S218E, S222D) or myristoylated Akt1 mRNA injection. These findings demonstrate that the role of sgIIb in neurovascular modeling is mediated by MAPK and PI3K/AKT signaling in CtA ECs. Figure 6 View largeDownload slide The role of sgIIb in neurovascular modeling is mediated by MAPK and PI3K/AKT signaling in CtA ECs. (A) Western blots for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 in 36 hpf sgIIb−/− mutant and sgIIb−/− mutant embryos injected with SNb mRNA. Actin was used as a loading control. (B and C) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (D) Maximal intensity projection of a confocal z-stack of 45 hpf Tg(kdrl:EGFP) or TG2 embryos incubated with DMSO or ARA. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. Scale bar, 50 μm. (E) Western blots for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 in 36 hpf sgIIb−/− mutant embryos incubated with DMSO or ARA. Actin was used as a loading control. (F and G) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (H and I) Quantitative analysis of CtAs in Tg(kdrl:EGFP) + ARA, TG2 + DMSO, and TG2 + ARA zebrafish hindbrains. Numbers of normal CtAs (H) and disorganized CtAs (I) were counted in 45 hpf zebrafish hindbrains. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. (J) The Tol2 transposon cassettes of the control (Tol2-kdrl-mCherry-Tol2), myristoylated Akt1 (Tol2-kdrl-Myr-Akt1-P2A-mCherry-Tol2), and Mek1 (Tol2-kdrl-Mek1-P2A-mCherry-Tol2) expression plasmids. (K) Maximal intensity projection of a confocal z-stack of 45 hpf Tg(kdrl:EGFP) or TG2 embryos injected with control, myristoylated Akt1, or Mek1 expression vector. (L) Percentage of mCherry-positive normal CtAs. Numbers in the bracket of each group represent the number of mCherry-positive normal CtAs/the number of mCherry-positive total CtAs. (M and N) Percentage of PH3-positive ECs in 45 hpf Tg(kdrl:EGFP) or TG2 embryo CtAs injected with RFP mRNA, Myr-Akt1 mRNA, or Mek1 mRNA. Figure 6 View largeDownload slide The role of sgIIb in neurovascular modeling is mediated by MAPK and PI3K/AKT signaling in CtA ECs. (A) Western blots for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 in 36 hpf sgIIb−/− mutant and sgIIb−/− mutant embryos injected with SNb mRNA. Actin was used as a loading control. (B and C) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (D) Maximal intensity projection of a confocal z-stack of 45 hpf Tg(kdrl:EGFP) or TG2 embryos incubated with DMSO or ARA. White arrowheads indicate normal CtAs connected to BA. Red arrowheads indicate disorganized CtAs. Scale bar, 50 μm. (E) Western blots for p-AKT, total AKT, p-ERK1/2, and total ERK1/2 in 36 hpf sgIIb−/− mutant embryos incubated with DMSO or ARA. Actin was used as a loading control. (F and G) Statistical analysis of the relative levels of p-AKT/AKT and p-ERK/ERK from three independent western blotting experiments. (H and I) Quantitative analysis of CtAs in Tg(kdrl:EGFP) + ARA, TG2 + DMSO, and TG2 + ARA zebrafish hindbrains. Numbers of normal CtAs (H) and disorganized CtAs (I) were counted in 45 hpf zebrafish hindbrains. Data shown are mean ± SEM (n = the number of embryos analyzed). Statistical significance was assessed using the two-tailed Student’s t-test. (J) The Tol2 transposon cassettes of the control (Tol2-kdrl-mCherry-Tol2), myristoylated Akt1 (Tol2-kdrl-Myr-Akt1-P2A-mCherry-Tol2), and Mek1 (Tol2-kdrl-Mek1-P2A-mCherry-Tol2) expression plasmids. (K) Maximal intensity projection of a confocal z-stack of 45 hpf Tg(kdrl:EGFP) or TG2 embryos injected with control, myristoylated Akt1, or Mek1 expression vector. (L) Percentage of mCherry-positive normal CtAs. Numbers in the bracket of each group represent the number of mCherry-positive normal CtAs/the number of mCherry-positive total CtAs. (M and N) Percentage of PH3-positive ECs in 45 hpf Tg(kdrl:EGFP) or TG2 embryo CtAs injected with RFP mRNA, Myr-Akt1 mRNA, or Mek1 mRNA. Discussion The neuropeptide SN, processed from SgII, has multiple physiological functions (Zhao et al., 2009; Albrecht-Schgoer et al., 2012; Trudeau et al., 2012). It has been reported that synthetic SN can promote angiogenesis in vitro and in vivo (Kirchmair et al., 2004b). Therefore, the function of SN in angiogenesis has attracted considerable interest in recent years, yet the role of SN in CNS-specific angiogenesis has remained unknown until now. To our knowledge, no other early developmental roles for sgII have been reported previously. To investigate the role of sgII in CNS-specific angiogenesis, we generated sgII mutant zebrafish lines with GFP expressed in the vascular system. Our in vivo confocal imaging results showed that the number of normal CtAs was decreased while the number of disorganized CtAs was increased in the hindbrain of sgIIb mutant embryos. Time-lapse imaging indicated that depletion of sgIIb alone in zebrafish could lead to severe defects of CtA sprouting and pathfinding. However, some CtAs recovered to their normal appearance at 52 hpf in the sgIIb mutant embryos. This may be due to functional compensation by other angiogenic proteins, which may also act via MAPK or PI3K/AKT pathways (Kubota et al., 2005; Yu et al., 2010; Rossi et al., 2015). Brain morphology and expression of neuronal markers such as HuC, neurod4, and robo3 in rhombomeres or rhombomere boundaries (Park et al., 2000; Challa et al., 2001; Wang et al., 2003) remained normal. Neurons co-expressing sgIIb and camk2d2 also appeared normal in sgIIb mutant embryos. Injection of SNb mRNA could partly rescue the defects of CtA caused by the sgIIb mutation. Our cell transplantation experiments showed that sgIIb was non-cell autonomous and required for CtA development. Thus, sgIIb specifically participates in neurovascular modeling in the hindbrain of developing zebrafish. Despite the different physiological functions of the vascular and nervous systems, blood vessels are often aligned with nerves, and the two systems share some common molecular mechanisms (Park et al., 2004; Carmeliet and Tessier-Lavigne, 2005). Axon guidance molecules including netrins, ephrins, slits, semaphorins, and their receptors are cues in vascular morphogenesis (Carmeliet and Tessier-Lavigne, 2005). They participate in blood vessel navigation by either repelling or attracting the filopodia of tip ECs. We found that sgIIb is mainly expressed in the CNS of zebrafish embryos and co-expressed with camk2d2, one member of CaMKII family. As reported previously, the CaMKII family functions in growth cone guidance (Wen et al., 2004). The sgIIb-expressing cells were aligned with CtAs in the hindbrain. Previous studies demonstrated that SN is released from neurons (Kirchmair et al., 1994) and also acts as a direct angiogenic cytokine in vitro (Kirchmair et al., 2004b), suggesting the presence of a putative SN receptor on ECs. Defects of CtA sprouting and pathfinding in sgIIb mutant embryos imply that sgIIb is involved in vascular guidance in the hindbrain. Notch and VEGF signalings are fundamental for angiogenesis in physiological and pathological conditions. It has been demonstrated that VEGF signaling is stimulatory for angiogenic sprouting, while Notch signaling is inhibitory (Zhong et al., 2000, 2001; Covassin et al., 2006b; Suchting et al., 2007). In the hindbrain, Notch and VEGF signalings also play important roles in arterial–venous identity, which is regarded as an essential process of vascular development (Lawson and Weinstein, 2002; Bussmann et al., 2011). We found that the expression of Notch and VEGF pathway-related genes was not affected in sgIIb mutant brain ECs and arterial–venous identity of hindbrain was normal. Immunohistochemistry with an anti-PH3 antibody indicated that the percentage of proliferative ECs was decreased in CtAs of sgIIb mutant embryos. The MAPK and PI3K/AKT pathways play important roles in the proliferation and migration of ECs. Using loss-of-function and gain-of-function methods, we found that MAPK and PI3K/AKT pathways were inhibited in sgIIb mutant embryos in vivo. Injection of SNb, Myr-Akt1 or MEK1 mRNA, delivery of the activator ARA, and EC-specific expression of Myr-Akt1 or MEK1 could restore the function of MAPK and PI3K/AKT pathways and partially rescue CtA developmental defects in sgIIb mutant embryos. This is consistent with previous in vitro reports showing that culturing HUVECs and endothelial progenitor cells (EPCs) with SN could activate the MAPK system and the PI3K/AKT pathway (Kirchmair et al., 2004a, b). Our results indicate that the MAPK system and PI3K/AKT pathway mediate the effects sgIIb on neurovascular development. However, there are conflicting reports about whether the effects of SN on MAPK and PI3K/AKT pathway are dependent on VEGF. In HUVECs, the activation of MAPK and PI3K/AKT pathway by SN is not dependent on VEGF (Kirchmair et al., 2004b). Results obtained from HCAECs indicate that SN could promote binding of VEGF to its co-receptors heparin and neuropilin-1, demonstrating the role of SN in VEGF signaling (Albrecht-Schgoer et al., 2012). Our data provide evidence that VEGF pathway-related genes and VEGF-dependent arterial−venous identity are not altered in sgIIb−/− mutants. In summary, we have provided the first in vivo evidence that sgIIb plays a critical role in neurovascular modeling in the hindbrain that is mediated by activation of the MAPK and PI3K/AKT pathways. Cells expressing sgIIb are found in close association with vascular markers in the hindbrain; therefore, these sgIIb-positive cells are the likely source of SgIIb and/or SNb that controls the development of these CtAs. Ischemic stroke and neurodegenerative diseases such as Alzheimer’s disease are associated with abnormal angiogenesis in the brain. It is significant that SN promotes neuroprotection in mouse models of stroke (Shyu et al., 2008) and that proteomic markers for SgII are significantly decreased in CSF in Alzheimer’s disease patients (Spellman et al., 2015). Here, we show that injection of SNb mRNA could rescue the CtA defects in sgIIb mutant embryos. Targeting the SgII system may therefore represent a new avenue for the treatment of vascular defects in the CNS. Materials and methods Zebrafish care and maintenance AB strain zebrafish (Danio rerio) and their embryos were raised and maintained under standard conditions at 28.5°C. Embryos were staged as previously described (Kimmel et al., 1995). Tg(kdrl:EGFP) zebrafish were obtained from the China Zebrafish Resource Center (CZRC, Wuhan, China). The experiments involving zebrafish were performed under the approval of the Institutional Animal Care and Use Committee of the Institute of Hydrobiology, Chinese Academy of Sciences. Establishment of mutant zebrafish lines The paired TALENs for sgIIa or sgIIb were constructed using the golden gate method as described previously (Cermak et al., 2011; Liu et al., 2014). The final TALEN plasmids were linearized using Not1. TALEN mRNAs were synthesized using the mMessage mMACHINE SP6 Kit (Ambion, Inc.) and purified by LiCl precipitation. To generate mutant zebrafish lines, 250−500 pg TALEN mRNAs were microinjected into one-cell stage WT zebrafish embryos. Injected embryos were raised to adulthood and then outcrossed with WT fish to identify founders that transmitted mutations through the germ line. Mutations were genotyped by competitive PCR (Liu et al., 2014) and confirmed by sequencing. Plasmids and mRNA preparations Plasmids containing sequences of myristoylated Akt1 and Mek1(S218E, S222D) were purchased from Wuhan Miaolingbio Bioscience & Technology and Wuhan Genecreate Bioengineering, respectively. The full-length sequence of myristoylated Akt1 and Mek1(S218E, S222D) was cloned into a pCS2+ vector or into a Tol2-Kdrl promoter vector (a gift from Dr Jiulin Du, Institute of Neuroscience, Chinese Academy of Sciences, China) linked with mCherry via a P2A peptide sequence. Capped mRNAs were synthesized using the mMessage mMACHINE SP6 Kit (Ambion) and purified by LiCl precipitation. Embryo mounting, confocal microscopy, and image processing For in vivo confocal imaging, embryos were mounted in 1% low-melt agarose after anaesthetized in 168 mg/L tricaine. Fluorescence photomicrographs were collected with laser scanning confocal microscope (Zeiss LSM710). Time-lapse movies were prepared using the software program ZEN (Zeiss). Egg water containing tricaine and propylthiouracil was warmed to ~28°C and circulated during the observation period, z-stacks were collected at 10-min intervals. Images were processed using Adobe Photoshop CS3 Extended. The numbers of total CtAs, normal CtAs, and disorganized CtAs were counted using a fluoroscope (Leica M250) combined with laser scanning confocal microscope (Zeiss LSM710). The length of CtAs and midbrain vasculature was calculated with ImageJ analysis software (NIH). The unpaired two-tailed Student’s t-test as implemented in GraphPad Prism 5 was used to analyze the data. WISH and double-fluorescence in situ hybridization sgIIa, sgIIb, camk2d2, kdrl, cdh5, dab2, flt1, flt4, hey2, dll4, tal2, gad67 sequences were cloned by PCR amplification, PCR products were subcloned into the pMD18-T vector (TAKARA). Digoxigenin-labeled antisense probes for sgIIa, sgIIb, cdh5, dab2, flt1, flt4, hey2, dll4 were synthesized using DIG RNA Labeling Mix (Roche Diagnostics, Mannheim, Germany). WISH was carried out as described previously (Thisse and Thisse, 2008). Fluorescein-labeled antisense probes for camk2d2, kdrl, tal2, gad67 were synthesized using fluorescein RNA Labeling Mix (Roche Diagnostics, Mannheim, Germany). Double-fluorescence in situ hybridization was performed as described (Lauter et al., 2011). Primers used can be found in Supplementary Table S1. RT-PCR and RT-qPCR Heads of 36 hpf zebrafish embryos were cut off using a pair of forceps. ECs dissociated from 300−400 heads of Tg(kdrl:EGFP) and TG2 (sgIIb−/−;Tg(kdrl:EGFP)) were sorted using the FACSAriaTM III (BD Biosciences) as described previously (Covassin et al., 2006a). Total RNA was extracted from the sorted ECs or 40 zebrafish embryos (WT, sgIIa−/−, sgIIb−/−, sgIIa−/−/sgIIb−/−) at desired stages using TRIzol reagent (Invitrogen) and resuspended in diethyl-pyrocarbonate (DEPC)-treated water. The quality of extracted RNA was confirmed by UV spectrophotometer and agarose gel electrophoresis. Total RNA was reverse-transcribed into cDNA using ReverTra Ace M-MLV (TOYOBO) with random primers. For RT-PCR, the cDNA samples were PCR-amplified using gene-specific primers as listed in Supplementary Table S2. RT-qPCR was carried out on a Roche LightCycler 480 real-time PCR system using 2× SYBR green real-time PCR mix (TOYOBO) and primers as listed in Supplementary Table S3. All values were normalized to the level of β-actin mRNA. Western blotting Embryos were de-yolked as described (Link et al., 2006). The Total Protein Extraction Kit (Sangon Biotech) was used to lyse zebrafish embryos and adult zebrafish pituitaries according to the manufacturer’s instructions. The proteins of each lysate were separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and trans-blotted onto nitrocellulose membrane (Millipore), probed with indicated primary antibodies against phospho p44/42 MAPK (phospho-Erk1/2, 1:2000), p44/42 MAPK (total Erk1/2, 1:1000), p-AKT(1:2000), total AKT (1:1000) (Cell Signaling Technology), SNa (1:1000) (Zhao et al., 2006a), β-actin (Bioss Company, 1:1500), and VEGFA (Beyotime Biotechnology, 1:500). Then the membrane was washed in phosphate-buffered saline (PBS) containing Tween-20 (PBST) for 4 × 5 min, incubated with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies, and detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The signals were visualized using ImageQuant LAS 4000 mini system (GE Healthcare). Immunoblots were analyzed with ImageJ analysis software (NIH). Cell transplantation experiments Cell transplantation methods were similar to those described previously (Siekmann and Lawson, 2007) with some modifications. Tg(kdrl:EGFP) (WTEGFP) or sgIIb−/−;Tg(kdrl:EGFP) (TG2) embryos at one-cell stage were injected with tetramethylrhodamine isothiocyanate–dextran (Sigma) as a lineage tracer, and some TG2 embryos were co-injected with SNb mRNA (TG2SNb mRNA) or RFP mRNA (TG2RFP mRNA). These injected embryos were subsequently used as donors. Non-transgenic WT or sgIIb mutant (sgIIb−/− or TG2) embryos were used as hosts. Both donor and host embryos were dechorionated by forceps and subsequently cultured in plate covered with agarose. Cell transplantation was performed when donor and host embryos developed at the sphere stage (~4 hpf). Approximately 20−40 donor cells were transplanted into the margin of each host embryo. Five groups of cell transplantation experiments were carried out: WTEGFP > WT; TG2 > WT; WTEGFP > sgIIb−/−; TG2SNb mRNA > TG2, and TG2RFP mRNA > TG2. Transplanted cells were assessed by visualization of EGFP expression or tetramethylrhodamine isothiocyanate–dextran in host embryos. Numbers of chimeras with EGFP-positive CtAs and chimeras with EGFP-positive ECs in the hindbrain were counted using a fluoroscope (Leica M250) combined with laser scanning confocal microscope (Zeiss LSM710). Whole-mount immunohistochemistry Whole-mount immunohistochemistry was performed using phospho p44/42 MAPK (phospho-Erk1/2, 1:1000, CST) as primary antibody and DyLight 488 goat anti-mouse IgG (Abbkine, 1:200) as secondary antibody. Briefly, dechorionated zebrafish embryos were fixed in 4% PFA in PBS overnight at 4°C, dehydrated in 100% methanol for 15 min at room temperature, and stored at −20°C in 100% methanol for at least 2 h before use. Embryos were permeabilized in acetone (−20°C) for 10 min, washed with PBS containing Triton X-100 (PBST), incubated with the primary antibody overnight at 4°C, and then washed extensively and incubated with the secondary antibodies overnight at 4°C. Embryos were washed extensively again and mounted in glycerol. ImageJ (NIH) was used to measure the p-ERK density (signal intensity per pixel) in CtAs. Statistical analysis Statistical analysis was performed using unpaired two-tailed Student’s t-test as implemented in GraphPad Prism 5 (GraphPad Software Inc.). Data are presented as mean ± SEM. Acknowledgements We are grateful to Ms Fang Zhou (Analytical & Testing Center, IHB, CAS) for providing confocal services and Professors Marie-Andrée Akimenko (University of Ottawa) and Jingwei Xiong (Peking University) for their valuable discussion and critical reading of the manuscript. Funding This work was supported by the National Natural Science Foundation of China (31325026 and 31721005 to W.H.), Natural Sciences and Engineering Research Council of Canada (to V.L.T.), and the University of Ottawa International Research Acceleration Program (to V.L.T. and W.H.). Conflict of interest none declared. References Albrecht-Schgoer , K. , Schgoer , W. , Holfeld , J. , et al. . ( 2012 ). 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Google Scholar Crossref Search ADS PubMed © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Journal of Molecular Cell BiologyOxford University Press

Published: Oct 1, 2018

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