Cannabinoid Receptor 2 Suppresses Leukocyte Inflammatory Migration by Modulating the JNK/c-Jun/Alox5 Pathway*

Background: The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. Results: Cnr2 activation down-regulates 5-lipoxygenase (Alox5) expression by suppressing the JNK/c-Jun activation. Conclusion: The Cnr2-JNK-Alox5 axis modulates leukocyte inflammatory migration. Significance: Linking two important regulators in leukocyte inflammatory migration and providing a potential therapeutic strategy for treating human inflammation-associated diseases. Inflammatory migration of immune cells is involved in many human diseases. Identification of molecular pathways and modulators controlling inflammatory migration could lead to therapeutic strategies for treating human inflammation-associated diseases. The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. Through a chemical genetic screen using a zebrafish model for leukocyte migration, we found that both an agonist of the Cnr2 and inhibitor of the 5-lipoxygenase (Alox5, encoded by alox5) inhibit leukocyte migration in response to acute injury. These agents have a similar effect on migration of human myeloid cells. Consistent with these results, we found that inactivation of Cnr2 by zinc finger nuclease-mediated mutagenesis enhances leukocyte migration, while inactivation of Alox5 blocks leukocyte migration. Further investigation indicates that there is a signaling link between Cnr2 and Alox5 and that alox5 is a target of c-Jun. Cnr2 activation down-regulates alox5 expression by suppressing the JNK/c-Jun activation. These studies demonstrate that Cnr2, JNK, and Alox5 constitute a pathway regulating leukocyte migration. The cooperative effect between the Cnr2 agonist and Alox5 inhibitor also provides a potential therapeutic strategy for treating human inflammation-associated diseases.

The activation and directional migration of innate immune cells in response to infection, trauma, toxin, lesion, or autoimmune injury is a classic process associated with inflammation. However, because it is a stressor of the tissue, inflammation must be tightly controlled. Abnormal migration and activation of innate immune cells can also be harmful and has been implicated widely in the progression of many human diseases, such as multiple sclerosis, rheumatoid arthritis, atherosclerosis, and cancer (1)(2)(3)(4)(5). Therefore, systematic identification of molecular pathways that control innate immune cell migration and activation may provide new therapeutic strategies for treating human diseases associated with disordered inflammation.
Chemical genetics is a powerful technology for understanding genetic and biological functions. This approach uncovers unknown molecular pathways and gene functions involving certain biological process through a phenotype-based screen. Compared to forward genetics, chemical genetics is more conditional because of the flexible approach of chemical treatment. In addition this approach identifies lead compounds that serve as a foundation for drug discovery (6 -8).
Zebrafish is a powerful vertebrate model for genetic study of development and diseases. Their small size, optical clarity, and substantial fecundity make zebrafish larva an ideal tool for an in vivo chemical genetic screen. Several chemical genetic screens have already been performed in zebrafish (9 -16). These screens successfully identified new pathways involving in development or disease processes, as well as novel small molecules regulating biological processes.
We have previously established a transgenic zebrafish line, TG(zlyz:EGFP), for tracking the migration of zebrafish leukocytes in response to acute injury (17). In the early stage of TG(zlyz:EGFP) embryos before 36 h-post-fertilization (hpf), 5 the EGFP-positive cells are primitive macrophages, while in the later stage after 48 hpf of embryos, the EGFP-positive cells include both monocytes/macrophages and neutrophils (17). Here we refer these EGFP-positive cells in the TG(zlyz:EGFP) embryos as leukocytes. Leukocyte, including neutrophil and monocyte/macrophage, is the most important part of the innate immune system (18). In tail-transected TG(zlyz:EGFP) zebrafish embryos at 72 hpf, the EGFP-positive leukocytes migrate to the wound. Within 6 h-post-tail-transection (hptt), the number of EGFP-positive leukocytes that aggregate in the wound reaches a maximum, about 45. Thus, the tail-transected TG(zlyz:EGFP) zebrafish provides us not only a visual and quantitative model of immune cell migration and inflammation, but also a useful tool for screening therapeutic compounds to treat human disorders associated with abnormal inflammation.
To identify chemical inhibitors of leukocyte migration, an in vivo chemical genetic screen was performed with tail-transected TG(zlyz:EGFP) zebrafish embryos. Seven out of 1,262 bioactive compounds with known targets from the Glaxo-SmithKline LOPAC molecular library were identified to suppress the migration of zebrafish leukocytes to the wound after tail transection. Two of these, (R)-(ϩ)-WIN55,212,-2 mesylate (WIN) and AA-861, are a cannabinoid receptor agonist and an Alox5 inhibitor, respectively.
WIN is a non-selective agonist for cannabinoid receptor type 1 (Cnr1) and cannabinoid receptor type 2(Cnr2). Cnr1 and Cnr2 are both G protein-coupled receptors with seven transmembrane ␣-helices. Cnr1 is highly expressed in the central nervous system and less in peripheral tissues (19), while Cnr2 is predominantly expressed in cells of hematopoietic origin in both mice and humans (20,21). Previous studies of the human and mouse cannabinoid system have shown that Cnr2 modulates the inflammatory migration of immune cells through many pathways and participates in the progression of many important autoimmune diseases (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). To confirm our chemical genetic screening, we generated mutant alleles of zebrafish cnr2 (gene encoding Cnr2) and found that leukocyte migration is enhanced by the inactivation of Cnr2, further confirming that Cnr2 acts as a conserved negative inflammatory regulator in the vertebrate.
Alox5 also named 5-lipoxygenase, mainly expressed in leukocytes, is a key enzyme involved in the biosynthesis of leukotrienes, including LTB 4 . Accumulation of leukotrienes aggravates inflammation in many diseases, like atherosclerosis and asthma. LTB 4 , which is known as a potent chemoattractant, has been shown to induce activation and migration of leukocytes (32,33). It has been shown that both alox5 knock-out and Alox5 inhibitor treatment lead to a decrease of in vivo leukocyte inflammatory migration of mice (34).
Recent studies showed that cannabidiol, a non-psychotropic cannabinoid, which has been reported to exert its effect through direct or indirect activation of Cnr2 (35)(36)(37), inhibits tumor cell growth by down-regulating Alox5 (38). Though the studies indicate a possible interaction between Cnr2 and Alox5, the mechanism is unknown. In studying the mechanism by which both WIN and AA-861 suppress zebrafish leukocyte migration, we found that there is a signaling link between Cnr2 and Alox5 and that alox5 is transcriptionally inhibited by Cnr2 activation through JNK/c-Jun inactivation. In addition, our in vivo screen results provide important information about the efficacy as well as the toxicity of the compounds identified, accelerating the development of new therapies for human disorders associated with deregulated leukocyte migration.

MATERIALS AND METHODS
Fish Care-Zebrafish maintenance, breeding, and staging were performed as standard methods. All studies were conducted after review by the Institutional Animal Care and Use Committee at Shanghai Institute of Hematology and in accordance with the GlaxoSmithKline Policy on the Care, Welfare, and Treatment of Laboratory Animals.
Tail Transection-Embryos were subject to tail transection with a sterile scalpel at the same anatomic site posterior to the end of tail circulation, resulting in consistent leukocyte recruitment with the number between 45 Ϯ 5. The dynamic behavior and quantification of chemotactic migration were assessed by fluorescent steromicroscope (Zeiss Lumar V12 steromicroscope equipped with an AxioCam MRC5 digital camera and AxioVision Rel.4.5 software). All experiments were repeated three times with separate batches of embryos.
Chemicals-The LOPAC library from GSK (GlaxoSmithKline) containing 1,262 known bioactive compounds was used for the screen. The compounds for the follow-up experiments, JWH-015, were purchased from Tocris. SP600125 was purchased from Calbiochem. Dexamethasone (DEX) was purchased from Sigma.
Zinc Finger Nuclease (ZFN) Construction-The functional ZFNs for cnr2 were designed with ZiFiT software (zifit.partners.org/ZiFiT/) by the context-dependent assembly (CoDA) approach. And the zinc finger units were synthesized in Shanghai Biosune Biotechnology Co. Ltd.
Flow Cytometry Analysis (FACS)-Embryos (72 hpf) were dissected in PBS with 10% FBS, and digested with 1ϫ trypsin/ EDTA for 30 min at 37°C. Single cell suspension was obtained by centrifugation at 800 ϫ g for 5 min. Cells were then washed twice with PBS/FBS, and passed through a 40-m nylon mesh filter. Fluorescence-activated cell sorting was performed with MoFlo FACS (Dako Cytomation) to obtain a homogenous sample of EGFP-positive cells.
Data Analysis and Statistics-Data are expressed as means Ϯ S.E. When differences between two groups were compared, the Student's t-test was used.

Identification of Chemical Inhibitors of Leukocyte Migration
in Response to Acute Injury-An in vivo chemical screen was performed to identify small molecules that inhibit the migration of leukocytes (Fig. 1). A total of 1,262 bioactive compounds were screened with tail-transected TG(zlyz:EGFP) zebrafish embryos. TG(zlyz:EGFP) zebrafish embryos were produced by breeding ten pairs of homozygous TG(zlyz:EGFP) zebrafish, which were enough for two 96-well plates each week. To induce an acute injury, the tails of zebrafish embryos were transected at the anatomic site posterior to the end of tail circulation. Immediately after the transection, three transgenic zebrafish embryos at 72 hpf were placed into each well and incubated with one of the library compounds at a concentration of 10 M. Dexamethasone (DEX, 500 M), which has been proven to inhibit leukocyte migration, was included as a positive control in each of the 96-well plates. Six hours after treatment, the EGFP-positive leukocytes congregated in the tail were quantified in each well, and then compared with those found in DMSO control and DEX control to assess the effect of the compounds in modulating leukocyte migration in response to acute injury.
We initially identified fifteen compounds that inhibit the migration of leukocytes during the original screen and later confirmed seven of them in the following concentration gradient re-testing (supplemental Fig. S1, A and B). The seven compounds were further tested using an in vitro cell migration assay and each of them could efficiently block the chemotactic migration of THP-1, a human acute monocytic leukemia (AML) cell line, in a concentration-dependent manner (supplemental Fig.  S2). The known targets of these compounds are shown in supplemental Table S1. Three of the seven hit compounds, forskolin, thapsigargin, and calcimycin, showed general toxicity in zebrafish embryos. Treatment with 3 M calcimycin killed embryos in the dose-response re-tests, while the lethal concentrations of forskolin and thapsigargin were 10 M (supplemental Fig. S1A). These results show the advantage of performing a chemical screen in a zebrafish model, which provides more information about the toxicity of these molecules and helps identify compounds with more drug development potential than a cell-based chemical screen (supplemental Fig. S1A).
Cnr2 Signaling Negatively Regulates Leukocyte Migration-The treatment of WIN, the Cnr1 and Cnr2 agonist identified in our screen, could block zebrafish leukocyte migration in a timedependent manner (Fig. 2, A and B). Cnr1 has been shown to be widely expressed in the zebrafish nervous system (19). Consistent with findings in mammals (20), we found that cnr2 is highly expressed in EGFP-positive zebrafish leukocytes whereas there was no difference in cnr1 expression between EGFP-negative and EGFP-positive cells (Fig. 2C). We reasoned that the inhibition of leukocyte inflammatory migration to the wound in tailtransected TG(zlyz:EGFP) zebrafish embryos by WIN was mediated by Cnr2, but not Cnr1. We went on to test whether the specific Cnr2 agonist JWH-015 was able to suppress the migration of zebrafish leukocytes (42). We found that, similar to WIN, JWH-015 could also efficiently block the migration of zebrafish leukocytes (Fig. 2, D--F). The activation of Cnr2 signaling by JWH-015 was also shown to effectively block the chemotactic migration of THP-1 cells induced by MCP-1 in vitro (supplemental Fig. S3C). In addition, compared with WIN, JWH-015 showed less toxicity on embryos.
To further confirm the negative role of Cnr2 in regulating zebrafish leukocyte migration, we sought to generate heritable mutation of cnr2 in zebrafish using zinc finger nucleases engineered by the CoDA approach (39). Two potential CoDA ZFN target sites were identified in the exon 2 of cnr2 with the online ZiFiT program (supplemental Fig. S3A). CoDA ZFNs targeting one of the two CoDA sites (gCTCCACAGCactgcGCA-GACGCCc) could efficiently induce a variety of deletions or insertions at the target site (Fig. 3A, supplemental Table S2 and data not shown). Embryos injected with mRNA encoding this pair of ZFNs were raised and screened for founders with the cnr2 mutation. Two different kinds of heritable deletion mutations in cnr2 were identified (Fig. 3B). The mutations caused frameshifts and premature termination codons, generating N-terminal-truncated Cnr2 with only one of the seven transmembrane-spanning domains (Fig. 3C).
Inflammatory migration of leukocytes was observed within 6 hptt in cnr2 Ϫ/Ϫ zebrafish embryos at 72 hpf. As expected when a negative regulator was inactivated, more leukocytes were recruited to the wound in cnr2 Ϫ/Ϫ embryos, in contrast to embryos treated with JWH-015 (Fig. 2, E and F; Fig. 3, D and E). To identify whether the inhibitory effect of JWH-015 is through the activation of Cnr2, we went on to activate Cnr2 in WT and cnr2 Ϫ/Ϫ embryos and found that the treatment of JWH-015 could not reverse the congregation of GFP-positive cells of cnr2 Ϫ/Ϫ embryos (Fig. 3, F and G), which indicated that the increase of inflammatory migration is caused by the inactiva-

Cnr2-JNK-Alox5 Axis in Leukocyte Migration
tion of Cnr2 in cnr2 Ϫ/Ϫ embryos. Consistent with the effect of Cnr2 inactivation in zebrafish embryos, siRNA-mediated knockdown of Cnr2 in the THP-1 cell line also led to a significant increase of chemotactic migration in vitro (supplemental Fig. S3, B and C). Our results demonstrated that activation of Cnr2 signaling inhibits leukocyte migration while inhibition of Cnr2 signaling enhances recruitment of leukocytes in response to acute inflammation, implicating that Cnr2 serves as an important negative regulator in the leukocyte inflammatory migration response.
Regulation of Leukocyte Inflammatory Migration by Alox5-AA-861 (AA), a competitive inhibitor of Alox5 (43), was identified in our screen to efficiently inhibit leukocyte migration in zebrafish in a dose-dependent manner (supplemental Fig. S1A). Time-lapse fluorescence photographs taken within 6 hptt show a severe impairment of the recruitment of leukocytes to the wound in TG(zlyz:EGFP) zebrafish embryos treated with 20 M AA-861 (Fig. 4, A and B). To confirm the effect of AA-861 in inhibiting leukocyte migration through Alox5 specifically, we used morpholino technology to knockdown alox5 in vivo and found that knocking down alox5 blocks migration of zebrafish leukocytes to the wound within 6 hptt (Fig. 4, C and D). To confirm the specificity of alox5 morpholino, we co-injected alox5 mRNA and alox5 morpholino and found that alox5 mRNA could revise the phenotype of alox5 morphants by a dose-dependent manner (supplemental Fig. S4). In addition, we found that alox5 mRNA is markedly up-regulated in EGFPpositive leukocytes from tail-transected TG(zlyz:EGFP) zebrafish embryos at 6 hptt (Fig. 4G). These data indicate that Alox5 is a responder to acute wound and that it positively regulates leukocyte migration.
Interaction between Cnr2 and Alox5 Pathways in Regulation of Leukocyte Inflammatory Migration-Because both Cnr2 agonist and Alox5 inhibitor can modulate leukocyte migration, we analyzed the potential interaction between the two pathways in our in vivo model of leukocyte inflammatory migration. We found that the increased recruitment of leukocytes to the tail wound of cnr2 Ϫ/Ϫ embryos were almost completely rescued by blocking of the Alox5 pathway mediated with alox5 morpholino (Fig. 4, E and F). These data indicate that the Cnr2 signaling and the Alox5 pathway are connected and that Alox5 functions downstream of Cnr2. We also found that mRNA levels of alox5 were decreased in EGFP-positive leukocytes of TG(zlyz:EGFP) zebrafish at 6 hptt after Cnr2 signaling was activated by JWH-015 (Fig. 4G). Similarly, the Alox5 protein level was also downregulated in THP-1 cells by JWH-015 induced activation of Cnr2 (Fig. 5A). These results indicate that the Alox5 pathway is a downstream target of the Cnr2 signaling in regulating the migration of leukocytes.
We also found that combined treatment with WIN and AA-861 elicited significantly more inhibition of the recruitment of leukocytes to the wound in tail-transected zebrafish embryos than either of the two compounds alone (Fig. 4, H and  I), indicating targeting both parts of the pathway is a more effective anti-inflammatory therapy. Similar cooperative effect was observed between the selective Cnr2 agonist, JWH-015, and AA-861 (supplemental Fig. S3, D and E).

Inhibition of Leukocyte Migration by Cnr2
Activation via JNK-MAPK signaling cascades play a critical role in both immune cell development and the immune response to pathogens. Cnr2 signaling has been shown to regulate three major MAPKs, including extracellular signal-regulated protein kinases (ERK), p38 MAPK, and c-Jun NH 2 -terminal kinases (JNK) (29, 44 -48). Our previous studies have shown that JNK is involved in leukocyte migration induced by acute injury (17). We went on to investigate whether Cnr2 inhibits leukocyte migration by regulating JNK activity. We found that JNK phosphorylation of whole embryo is increased in response to acute injury, whereas such increase is abolished by the activation of Cnr2 signaling by JWH-015 in tail-transected zebrafish at 6 hptt (Fig. 6A). Similarly, the phosphorylation of JNK is inhibited by JWH-015 in THP-1 cells in vitro (Fig. 5, C and D). These data suggest that inhibition of the inflammatory migration of leukocytes by Cnr2 activation acts, at least in part, by preventing JNK activation. To further test this idea, we checked the effect of a constitutively active JNK on regulating leukocyte migration. Previous studies have shown that fusion of MKK7 to JNK1 causes a constitutive activation of JNK (49). Based on the high conservation of the JNK pathway between zebrafish and mammals (50, 51), we generated a human MKK7-JNK1 fusion gene (Fig. 6B). We injected the zlyz-MKK7-JNK1 plasmid into TG(zlyz:EGFP) zebrafish embryos to induce constitutive JNK phosphorylation in EGFP-positive cells. Strikingly, constitutively activated JNK efficiently rescued the block of leukocyte migration in tail-transected zebrafish embryos by JWH-015 (Fig. 6, C  and D). In addition, the JNK specific inhibitor SP600125 reversed the increased congregation of GFP-positive leukocytes at the wound in cnr2 Ϫ/Ϫ embryos (Fig. 6, E and F). These results demonstrate that Cnr2 activation inhibits JNK activation, which underlies the inhibitory effect on leukocyte inflammatory migration by the Cnr2 agonist.
Transcriptional Suppression of alox5 via c-Jun Inactivation by Cnr2 Signaling-Because both JNK and Alox5 function downstream of Cnr2, it is possible that Alox5 expression is regulated by JNK activation. We tested this possibility and found that the JNK-specific inhibitor SP600125 down-regulated the expression of Alox5 in THP-1 cells (Fig. 5, E and F). c-Jun regulates the transcription of genes widely involved in cell migration, wound healing, and inflammation, and its activity is regulated by JNK (52)(53)(54). Decreased c-Jun-targeted gene transcription might be responsible for blocking leukocyte migration by Cnr2 activation. We tested whether alox5 is transcriptionally regulated by c-Jun, which in turn is activated by JNK in zebrafish. We first analyzed the promoter 4.0 kb upstream of the coding sequence region of alox5 with the online transcription factor predict software Alibaba 2.1 (www. gene-regulation.com/pub/programs.html#alibaba2) to look for potential transcription factor binding sites. We identified 3 putative AP-1 binding sites (Fig. 7A). E-ChIP was performed to test whether phosphorylated c-Jun (p-c-Jun) could bind directly to these sites in response to acute wound in vivo (Fig.  7B). As expected, there was little or no level of p-c-Jun binding to the AP-1 site I, II, and III at 0 hptt. In contrast, the binding  activity of p-c-Jun to AP-1 site II and III, but not I, was markedly increased at 6 hptt. Interestingly, the increased binding activity of p-c-Jun to AP-1 site II or III could be completely abolished by either JWH-015-induced Cnr2 activation or SP600125-mediated inhibition of JNK. In addition, constitutive JNK activity up-regulated the mRNA level of alox5 in zebrafish EGFP-positive leukocytes at 6 hptt and could efficiently rescue the transcriptional inhibition by JWH-015-induced Cnr2 activation (Fig. 7C). These results demonstrate that alox5 is transcriptionally regulated by p-JNK activated c-Jun, and that Cnr2 signaling acts as an important negative regulator in leukocyte inflammatory migration by inhibiting the JNK-c-Jun-Alox5 axis.

DISCUSSION
In this report, we conducted the first large scale chemical screen on tail-transected TG(zlyz:EGFP) zebrafish embryos. We identified seven chemical compounds with immunomodulatory activity and elucidated the importance of the Cnr2-JNK-Alox5 pathway in regulating leukocyte inflammatory migration.
The block of Cnr2 signaling by cnr2 mutation induced an abnormal increase in leukocyte inflammatory migration, suggesting that Cnr2 signaling is activated by endocannabinoid in the normal inflammation process and acts as an important modulator of inflammation. The increased number of congregated leukocytes in the wound of cnr2 Ϫ/Ϫ embryos phenocopies the increased congregation of Cnr2-deficient T cells in the CNS of the autoimmune encephalomyelitis (EAE) mouse model (55), suggesting that our in vivo inflammation model is highly conserved with mammalian inflammation-associated disease processes. The zebrafish inflammation model would be useful to dissect the exact phenotype of the Cnr2-deficient immune cell.
It has been shown that Cnr2 signaling and the Alox5 pathway are both involved in the regulation of leukocyte migration (22)(23)(24). Previous studies showed that cannabidiol inhibits tumor cell growth by down-regulating the activity and expression of Alox5 (38), and it was also shown that there is a cooperative antineurotoxic effect between a selective Cnr2 agonist (JWH-015) and a specific Alox5 inhibitor (REV 5901) in THP-1 cells in vitro (56). Though these studies indicate a possible interaction between Cnr2 and Alox5, our findings demonstrate that Cnr2 signaling directly regulates Alox5 expression during the process of leukocyte inflammatory migration.
As the most highly activated target of JNK, activated c-Jun has been reported to directly trans-activate many key genes involved in the inflammation process (17,52,53). Here we demonstrate that alox5 is a direct target of c-Jun and that it could be transcriptionally up-regulated by phosphorylated c-Jun. As described in supplemental Fig. S5, in the process of acute inflammation in response to tail-transection, JNK is activated by phosphorylation, which in turn activates the transcription factor c-Jun. P-c-Jun subsequently binds to the AP-1 binding site in the promoter region upstream of the transcription start site of alox5 and trans-activates alox5 expression. Considering the modulation of Alox5 in immune cell migration, the JNK-c-Jun-Alox5 axis, at least in part, contributes to the in vivo leukocyte inflammatory migration response. It had been demonstrated that another Cnr2-specific agonist GP1a could decrease the level of intracellular cAMP (30), and other studies also indicated that cAMP leads to phosphorylation of JNK (57). So it is possible that Cnr2 activation inhibits the phosphorylation of JNK and subsequently inhibits the transcription of alox5 through reducing the level of cAMP. Our finding that the JNKc-Jun-Alox5 axis could be efficiently blocked by Cnr2 signaling activation is consistent with the previous observation that Cnr2 activation inhibits human tumor cell proliferation by downregulating the phosphorylation of JNK (58).
The cooperative effect between the Cnr2 agonist and the Alox5 inhibitor of leukocyte inflammatory migration implicates a new strategy for developing anti-inflammation therapies. The Cnr2 agonist WIN has shown great potential in delaying the progress of multiple sclerosis (59). Suppression of Alox5 pathway is also efficient in therapy of asthma (32,33). Investigating the effect of a combined therapy with Cnr2 agonist and Alox5 inhibitor would be valuable in treating human diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis, asthma, or even cancer.
Several successful chemical genetic screens have been performed in zebrafish to identify suppressors and pathways of oncogene function, angiogenesis, or lipid metabolism (14,60,61). Combined with the fact that many well-established antiinflammatory drugs work well in zebrafish (17,62,63), our screen results provide evidence for highly conserved signaling pathways involved in immune cell migration and inflammation. The targets of the seven hit compounds in our screen fall into different cell signaling pathways, some of which have been implicated in inflammation regulation in mouse or human, including Ca 2ϩ signaling, the PI3K-AKT pathway, cannabinoid signaling, and the Alox5 pathway.
Considering the obvious toxic effects of the three compounds, forskolin, thapsigargin, and calcimycin on zebrafish embryos, these compounds may not be suitable for further antiinflammatory drug development or may at least need to be modified for further investigation. Together, results of our in vivo chemical genetic screen with tail-transected TG(zlyz: EGFP) embryos show an efficient way to dissect cell migration pathways and to identify lead compounds with immunomodulatory activity.