During inflammation, the proper inflammatory infiltration of neutrophils is crucial for the host to fight against infections and remove damaged cells and detrimental substances. IL-1β and NADPH oxidase–mediated reactive oxygen species (ROS) have been implicated to play important roles in this process. However, the cellular and molecular basis underlying the actions of IL-1β and ROS and their relationship during inflammatory response remains undefined. In this study, we use the zebrafish model to investigate these issues. We find that, similar to that of NADPH oxidase–mediated ROS signaling, the Il-1β–Myd88 pathway is required for the recruitment of neutrophils, but not macrophages, to the injury-induced inflammatory site, whereas it is dispensable for bacterial-induced inflammation. Interestingly, the Il-1β–Myd88 pathway is independent of NADPH oxidase–mediated ROS signaling and critical for the directional migration, but not the basal random movement, of neutrophils. In contrast, the NADPH oxidase–mediated ROS signaling is required for both basal random movement and directional migration of neutrophils. We further document that ectopic expression of Il-1β in zebrafish induces an inflammatory disorder, which can be suppressed by anti-inflammatory treatment. Our findings reveal that the Il-1β–Myd88 axis and NADPH oxidase–mediated ROS signaling are two independent pathways that differentially regulate neutrophil migration during sterile inflammation. In addition, Il-1β overexpressing Tg(hsp70:^mil-1β_eGFP;lyz:DsRed2)hkz10t;nz50 transgenic zebrafish provides a useful animal model for the study of chronic inflammatory disorder and for anti-inflammatory drug discovery.

Ikzf1 is a Krüppel-like zinc-finger transcription factor that plays indispensable roles in T and B cell development. Although the function of Ikzf1 has been studied extensively, the molecular mechanism underlying T lymphopoiesis remains incompletely defined during the embryonic stage. Here we report that the genetic ablation of ikzf1 in mutant zebrafish resulted in abrogated embryonic T lymphopoiesis. This was ascribed to impaired thymic migration, proliferation, and differentiation of hematopoietic stem/progenitor cells (HSPCs). Ccr9a and Irf4a, two indispensable factors in T lymphopoiesis, were the direct targets of Ikzf1 and were absent in the ikzf1 mutants. Genetic deletion of either ccr9a or irf4a in the corresponding mutant embryos led to obvious T cell development deficiency, which was mainly caused by disrupted thymic migration of HSPCs. Restoration of ccr9a in ikzf1 mutants obviously promoted HSPC thymus homing. However, the HSPCs then failed to differentiate into T cells. Additional replenishment of irf4a efficiently induced HSPC proliferation and T cell differentiation. Our findings further demonstrate that Ikzf1 regulates embryonic T lymphopoiesis via Ccr9 and Irf4 and provide new insight into the genetic network of T lymphocyte development.

The mechanisms underlying gut development, especially peristalsis, are widely studied topics. However, the causes of gut peristalsis-related diseases, especially Opioid-Induced Bowel Dysfunction (OIBD) disorder, have not been well defined. Therefore, our study used zebrafish, a popular model for studying both gut development and peristalsis, and DCFH-DA, a dye that clearly labels the live fish gut lumen, to characterize the formation process of gut lumen as well as the gut movement style in vivo. By applying Loperamide Hydrochloride (LH), the μ-opioid receptor-specific agonist, we established an OIBD-like zebrafish model. Our study found that acetylcholine (ACh) was a key transmitter that derepressed the phenotype induced by LH. Overall, the study showed that the antagonistic role of ACh in the LH-mediated opioid pathway was evolutionarily conserved; moreover, the OIBD-like zebrafish model will be helpful in the future dissection of the molecular pathways involved in gut lumen development and pathology.

The mechanisms that ensure fertilization of egg by a sperm are not fully understood. In all teleosts, a channel called the ‘micropyle’ is the only route of entry for sperm to enter and fertilize the egg. The micropyle forms by penetration of the vitelline envelope by a single specialized follicle cell, the micropylar cell. The mechanisms underlying micropylar cell specification and micropyle formation are poorly understood. Here, we show that an effector of the Hippo signaling pathway, the Transcriptional co-activator with a PDZ-binding domain (Taz), plays crucial roles in micropyle formation and fertilization in zebrafish (Danio rerio). Genome editing mutants affecting taz can grow to adults. However, eggs from homozygous taz females are not fertilized even though oocytes in mutant females are histologically normal with intact animal-vegetal polarity, complete meiosis and proper ovulation. We find that taz mutant eggs have no micropyle. Taz protein is specifically enriched in mid-oogenesis in the micropylar cell located at the animal pole of wild type oocyte, where it might regulate the cytoskeleton. Taz protein and micropylar cells are not detected in taz mutant ovaries. Our work identifies a novel role for the Hippo/Taz pathway in micropylar cell specification in zebrafish, and uncovers the molecular basis of micropyle formation in teleosts. In many fish, sperm enters eggs through a specialized channel called the ‘micropyle’. The micropyle is formed by a special follicle cell, the ‘micropylar cell’, which sits on the top of the developing egg during oogenesis, and forms the sperm entry canal. The underlying mechanisms of this process are unknown. We find that Taz, an effector of an important signaling pathway, the Hippo pathway, is specifically enriched in micropylar cells in zebrafish, and regulates formation of the micropyle. Loss of Taz function in females results in no micropylar cells, failure to form a micropyle on eggs, which are consequently, not fertilized. Our study identifies a new role for the Hippo/Taz pathway in cell fate specification in the ovary, and reveals a potential mechanism for forming the sperm entry port. Similar mechanisms might operate in other fish as well.

Emergency granulopoiesis occurs in response to severe microbial infection. However, whether and how other blood components, particularly monocytes/macrophages and their progenitors, including hematopoietic stem/progenitor cells (HSPCs), participate in the process and the underlying molecular mechanisms remain unknown. In this study, we challenged zebrafish larvae via direct injection of Escherichia coli into the bloodstream, which resulted in systemic inoculation with this microbe. The reaction of hematopoietic cells, including HSPCs, in the caudal hematopoietic tissue was carefully analysed. Both macrophages and neutrophils clearly expanded following the challenge. Thus, emergency myelopoiesis, including monopoiesis and granulopoiesis, occurred following systemic bacterial infection. The HSPC reaction was dependent on the bacterial burden, manifesting as a slight increase under low burden, but an obvious reduction following the administration of an excessive volume of bacteria. Pu.1 was important for the effective elimination of the microbes to prevent excessive HSPC apoptosis in response to stress. Moreover, Pu.1 played different roles in steady and emergency monopoiesis. Although Pu.1 was essential for normal macrophage development, it played suppressive roles in emergency monopoiesis. Overall, our study established a systemic bacterial infection model that led to emergency myelopoiesis, thereby improving our understanding of the function of Pu.1 in this scenario.