Pax4-Ghrelin mediates the conversion of pancreatic ε-cells to ß-cells after extreme ß-cell loss in zebrafish.

Pancreatic ε-cells producing ghrelin are one type of endocrine cell found in islets, which have been shown to influence other intra-islet cells, especially in regulating the function of ß cells. However, the role of such cells during ß-cell regeneration is currently unknown. Here, using a zebrafish nitroreductase (NTR)-mediated ß-cell ablation model, we reveal that ghrelin-positive ε-cells in the pancreas act as contributors to neogenic ß-cells after extreme ß-cell loss. Further studies show that the overexpression of ghrelin or the expansion of ε-cells potentiates ß-cell regeneration. Lineage tracing confirms that a proportion of embryonic ε-cells can transdifferentiate to ß-cells, and that the deletion of Pax4 enhances this transdifferentiation of ε-cells to ß-cells. Mechanistically, Pax4 binds to the ghrelin regulatory region and represses its transcription. Thus, deletion of Pax4 derepresses ghrelin expression and causes producing more ghrelin-positive cells, enhancing the transdifferentiation of ε-cells to ß-cells and consequently potentiating ß-cell regeneration. Our findings reveal a previously unreported role for ε-cells during zebrafish ß-cell regeneration, indicating that Pax4 regulates ghrelin transcription and mediates the conversion of embryonic ε-cells to ß-cells after extreme ß-cell loss.

Her4.3+ radial glial cells maintain the brain vascular network through activation of Wnt signaling.

During vascular development, radial glial cells (RGCs) regulate vascular patterning in the trunk and contribute to the early differentiation of the blood-brain barrier. Ablation of RGCs results in excessive sprouting vessels or the absence of bilateral vertebral arteries. However, interactions of RGCs with later brain vascular networks after pattern formation remain unknown. Here, we generated a her4.3 transgenic line to label RGCs and applied the metronidazole/nitroreductase system to ablate her4.3+ RGCs. The ablation of her4.3+ RGCs led to the collapse of the cerebral vascular network, disruption of the blood-brain barrier, and downregulation of Wnt signaling. The inhibition of Wnt signaling resulted in the collapse of cerebral vasculature, similar to that caused by her4.3+ RGC ablation. The defects in the maintenance of brain vasculature resulting from the absence of her4.3+ RGCs were partially rescued by the activation of Wnt signaling or overexpression of Wnt7aa or Wnt7bb. Together, our study suggests that her4.3+ RGCs maintain the cerebral vascular network through Wnt signaling.

Brain vascular damage-induced lymphatic ingrowth is directed by Cxcl12b/Cxcr4a.

After ischemic stroke, promotion of vascular regeneration without causing uncontrolled vessel growth appears to be the major challenge for pro-angiogenic therapies. The molecular mechanisms underlying how nascent blood vessels (BVs) are correctly guided into the post-ischemic infarction area remain unknown. Here, using a zebrafish cerebrovascular injury model, we show that chemokine signaling provides crucial guidance cues to determine the growing direction of ingrown lymphatic vessels (iLVs) and, in turn, that of nascent BVs. The chemokine receptor Cxcr4a is transcriptionally activated in the iLVs after injury, whereas its ligand Cxcl12b is expressed in the residual central BVs, the destinations of iLV ingrowth. Mutant and mosaic studies indicate that Cxcl12b/Cxcr4a-mediated chemotaxis is necessary and sufficient to determine the growing direction of iLVs and nascent BVs. This study provides a molecular basis for how the vessel directionality of cerebrovascular regeneration is properly determined, suggesting potential application of Cxcl12b/Cxcr4a in the development of post-ischemic pro-angiogenic therapies.

Nrf2 dictates the neuronal survival and differentiation of embryonic zebrafish harboring compromised alanyl-tRNA synthetase.

tRNA synthetase deficiency leads to unfolded protein responses in neuronal disorders; however, its function in embryonic neurogenesis remains unclear. This study identified an aars1cq71/cq71 mutant zebrafish allele that showed increased neuronal apoptosis and compromised neurogenesis. aars1 transcripts were highly expressed in primary neural progenitor cells, and their aberration resulted in protein overloading and activated Perk. nfe2l2b, a paralog of mammalian Nfe2l2, which encodes Nrf2, is a pivotal executor of Perk signaling that regulates neuronal phenotypes in aars1cq71/cq71 mutants. Interference of nfe2l2b in nfe2l2bΔ1/Δ1 mutants did not affect global larval development. However, aars1cq71/cq71;nfe2l2bΔ1/Δ1 mutant embryos exhibited increased neuronal cell survival and neurogenesis compared with their aars1cq71/cq71 siblings. nfe2l2b was harnessed by Perk at two levels. Its transcript was regulated by Chop, an implementer of Perk. It was also phosphorylated by Perk. Both pathways synergistically assured the nuclear functions of nfe2l2b to control cell survival by targeting p53. Our study extends the understanding of tRNA synthetase in neurogenesis and implies that Nrf2 is a cue to mitigate neurodegenerative pathogenesis.

Mdka produced by the activated HSCs drives bipotential progenitor cell redifferentiation during zebrafish biliary-mediated liver regeneration.

BACKGROUND AND AIMS: After extensive hepatocyte loss or impaired hepatocyte proliferation, liver regeneration occurs through trans-differentiation of biliary epithelial cells (BECs), which involves dedifferentiation of biliary epithelial cells into bipotential progenitor cells (BP-PCs) and subsequent redifferentiation of BP-PCs into nascent hepatocytes and biliary epithelial cells. Despite several studies on the redifferentiation process of BP-PCs into nascent hepatocytes, the contributions of nonparenchymal cells in this process remain poorly understood. APPROACH AND RESULTS: Using the zebrafish severe liver injury model, we observed specific expression of midkine a (Mdka) in the activated HSCs through single-cell analyses and fluorescence in situ hybridization. Genetic mutation, pharmacological inhibition, whole-mount in situ hybridizations, and antibody staining demonstrated an essential role of mdka in the redifferentiation of BP-PCs during liver regeneration. Notably, we identified Nucleolin (Ncl), the potential receptor for Mdka, specifically expressed in BP-PCs, and its mutant recapitulated the mdka mutant phenotypes with impaired BP-PC redifferentiation. Mechanistically, the Mdka-Ncl axis drove Erk1 activation in BP-PCs during liver regeneration. Furthermore, overexpression of activated Erk1 partially rescued the defective liver regeneration in the mdka mutant. CONCLUSIONS: The activated HSCs produce Mdka to drive the redifferentiation process of BP-PCs through activating Erk1 during the biliary-mediated liver regeneration, implying previously unappreciated contributions of nonparenchymal cells to this regeneration process.

Macrophages Mediate the Repair of Brain Vascular Rupture through Direct Physical Adhesion and Mechanical Traction.

Hemorrhagic stroke and brain microbleeds are caused by cerebrovascular ruptures. Fast repair of such ruptures is the most promising therapeutic approach. Due to a lack of high-resolution in vivo real-time studies, the dynamic cellular events involved in cerebrovascular repair remain unknown. Here, we have developed a cerebrovascular rupture system in zebrafish by using multi-photon laser, which generates a lesion with two endothelial ends. In vivo time-lapse imaging showed that a macrophage arrived at the lesion and extended filopodia or lamellipodia to physically adhere to both endothelial ends. This macrophage generated mechanical traction forces to pull the endothelial ends and facilitate their ligation, thus mediating the repair of the rupture. Both depolymerization of microfilaments and inhibition of phosphatidylinositide 3-kinase or Rac1 activity disrupted macrophage-endothelial adhesion and impaired cerebrovascular repair. Our study reveals a hitherto unexpected role for macrophages in mediating repair of cerebrovascular ruptures through direct physical adhesion and mechanical traction.

Intestinal precursors avoid being misinduced to liver cells by activating Cdx-Wnt inhibition cascade.

During coordinated development of two neighboring organs from the same germ layer, how precursors of one organ resist the inductive signals of the other to avoid being misinduced to wrong cell fate remains a general question in developmental biology. The liver and anterior intestinal precursors located in close proximity along the gut axis represent a typical example. Here we identify a zebrafish leberwurst (lbw) mutant with a unique hepatized intestine phenotype, exhibiting replacement of anterior intestinal cells by liver cells. lbw encodes the Cdx1b homeoprotein, which is specifically expressed in the intestine, and its precursor cells. Mechanistically, in the intestinal precursors, Cdx1b binds to genomic DNA at the regulatory region of secreted frizzled related protein 5 (sfrp5) to activate sfrp5 transcription. Sfrp5 blocks the mesoderm-derived, liver-inductive Wnt2bb signal, thus conferring intestinal precursor cells resistance to Wnt2bb. These results demonstrate that the intestinal precursors avoid being misinduced toward hepatic lineages through the activation of the Cdx1b-Sfrp5 cascade, implicating Cdx/Sfrp5 as a potential pharmacological target for the manipulation of intestinal-hepatic bifurcations, and shedding light on the general question of how precursor cells resist incorrect inductive signals during embryonic development.

Rngtt governs biliary-derived liver regeneration initiation by transcriptional regulation of mTORC1 and Dnmt1 in zebrafish.

In cases of end-stage liver diseases, the proliferation of existing hepatocytes is compromised, a feature of human chronic liver disease, in which most hepatocytes are dysfunctional. So far, liver transplantation represents the only curative therapeutic solution for advanced liver diseases, and the shortage of donor organs leads to high morbidity and mortality worldwide. The promising treatment is to prompt the biliary epithelial cells (BECs) transdifferentiation. However, the critical factors governing the initiation of BEC-derived liver regeneration are largely unknown. The zebrafish has advantages in large-scale genetic screens to identify the critical factors involved in liver regeneration. Here, we combined N-ethyl-N-nitrosourea screen, positional cloning, transgenic lines, antibody staining, and in situ hybridization methods and identified a liver regeneration defect mutant ( lrd ) using the zebrafish extensive liver injury model. Through positional cloning and genomic sequencing, we mapped the mutation site to rngtt . Loss of rngtt leads to the defects of BEC dedifferentiation, bipotential progenitor cell activation, and cell proliferation in the initiation stage of liver regeneration. The transdifferentiation from BECs to hepatocytes did not occur even at the late stage of liver regeneration. Mechanically, Rngtt transcriptionally regulates the attachment of mRNA cap to mTOR complex 1 (mTORC1) components and dnmt1 to maintain the activation of mTORC1 and DNA methylation in BECs after severe liver injury and prompt BEC to hepatocyte conversion. Furthermore, rptor and dnmt1 mutants displayed the same liver regeneration defects as rngtt mutation. In conclusion, our results suggest Rngtt is a new factor that initiates BEC-derived liver regeneration.

Formimidoyltransferase cyclodeaminase prevents the starvation-induced liver hepatomegaly and dysfunction through downregulating mTORC1.

The liver is a crucial center in the regulation of energy homeostasis under starvation. Although downregulation of mammalian target of rapamycin complex 1 (mTORC1) has been reported to play pivotal roles in the starvation responses, the underpinning mechanisms in particular upstream factors that downregulate mTORC1 remain largely unknown. To identify genetic variants that cause liver energy disorders during starvation, we conduct a zebrafish forward genetic screen. We identify a liver hulk (lvh) mutant with normal liver under feeding, but exhibiting liver hypertrophy under fasting. The hepatomegaly in lvh is caused by enlarged hepatocyte size and leads to liver dysfunction as well as limited tolerance to starvation. Positional cloning reveals that lvh phenotypes are caused by mutation in the ftcd gene, which encodes the formimidoyltransferase cyclodeaminase (FTCD). Further studies show that in response to starvation, the phosphorylated ribosomal S6 protein (p-RS6), a downstream effector of mTORC1, becomes downregulated in the wild-type liver, but remains at high level in lvh. Inhibition of mTORC1 by rapamycin rescues the hepatomegaly and liver dysfunction of lvh. Thus, we characterize the roles of FTCD in starvation response, which acts as an important upstream factor to downregulate mTORC1, thus preventing liver hypertrophy and dysfunction.

A single-cell-resolution fate map of endoderm reveals demarcation of pancreatic progenitors by cell cycle.

A progenitor cell could generate a certain type or multiple types of descendant cells during embryonic development. To make all the descendant cell types and developmental trajectories of every single progenitor cell clear remains an ultimate goal in developmental biology. Characterizations of descendant cells produced by each uncommitted progenitor for a full germ layer represent a big step toward the goal. Here, we focus on early foregut endoderm, which generates foregut digestive organs, including the pancreas, liver, foregut, and ductal system, through distinct lineages. Using unbiased single-cell labeling techniques, we label every individual zebrafish foregut endodermal progenitor cell out of 216 cells to visibly trace the distribution and number of their descendant cells. Hence, single-cell-resolution fate and proliferation maps of early foregut endoderm are established, in which progenitor regions of each foregut digestive organ are precisely demarcated. The maps indicate that the pancreatic endocrine progenitors are featured by a cell cycle state with a long G1 phase. Manipulating durations of the G1 phase modulates pancreatic progenitor populations. This study illustrates foregut endodermal progenitor cell fate at single-cell resolution, precisely demarcates different progenitor populations, and sheds light on mechanistic insights into pancreatic fate determination.

Calcineurin controls proximodistal blastema polarity in zebrafish fin regeneration.

Planarian flatworms regenerate their heads and tails from anterior or posterior wounds and this regenerative blastema polarity is controlled by Wnt/ß-catenin signaling. It is well known that a regeneration blastema of appendages of vertebrates such as fish and amphibians grows distally. However, it remains unclear whether a regeneration blastema in vertebrate appendages can grow proximally. Here, we show that a regeneration blastema in zebrafish fins can grow proximally along the proximodistal axis by calcineurin inhibition. We used fin excavation in adult zebrafish to observe unidirectional regeneration from the anterior cut edge (ACE) to the posterior cut edge (PCE) of the cavity and this unidirectional regeneration polarity occurs as the PCE fails to build blastemas. Furthermore, we found that calcineurin activities in the ACE were greater than in the PCE. Calcineurin inhibition induced PCE blastemas, and calcineurin hyperactivation suppressed fin regeneration. Collectively, these findings identify calcineurin as a molecular switch to specify the PCE blastema of the proximodistal axis and regeneration polarity in zebrafish fin.

Epidemiological characteristics of paediatric Omicron infection during the outbreak of SARS-CoV-2 infection during March-May in 2022 in Shanghai, China.

This study aims to understand the epidemiological characteristics of SARS-CoV-2 infection in the paediatric population during the outbreak of the Omicron variant in Shanghai. We retrospectively analysed the population-based epidemiological characteristics and clinical outcome of SARS-CoV-2 Omicron variant infection in children in Minhang District, Shanghai, based on the citywide surveillance system during the outbreak period in 2022 (March to May). During this time, a total of 63,969 cases of SARS-CoV-2 infection were notified in Minhang District, out of which 4,652 (7.3%) were children and adolescents <18 years. The incidence rate of SARS-CoV-2 infections in children was 153 per 10,000. Of all paediatric cases, 50% reported to be clinically symptomatic within 1-3 days after PCR confirmation by parents or themselves, with 36.3% and 18.9% of paediatric cases reporting fever and cough. Also, 58.4% of paediatric cases had received at least one dose of the COVID-19 vaccine and 52.1% had received two doses of the COVID-19 vaccination. Our findings are informative for the implementation of appropriate measures to protect children from the threat of SARS-CoV-2 infection.

Functional screening of congenital heart disease risk loci identifies 5 genes essential for heart development in zebrafish.

Congenital heart disease (CHD) is the most common birth defect worldwide and a main cause of perinatal and infant mortality. Our previous genome-wide association study identified 53 SNPs that associated with CHD in the Han Chinese population. Here, we performed functional screening of 27 orthologous genes in zebrafish using injection of antisense morpholino oligos. From this screen, 5 genes were identified as essential for heart development, including iqgap2, ptprt, ptpn22, tbck and maml3. Presumptive roles of the novel CHD-related genes include heart chamber formation (iqgap2 and ptprt) and atrioventricular canal formation (ptpn22 and tbck). While deficiency of maml3 led to defective cardiac trabeculation and consequent heart failure in zebrafish embryos. Furthermore, we found that maml3 mutants showed decreased cardiomyocyte proliferation which caused a reduction in cardiac trabeculae due to inhibition of Notch signaling. Together, our study identifies 5 novel CHD-related genes that are essential for heart development in zebrafish and first demonstrates that maml3 is required for Notch signaling in vivo.

Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9.

Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing.

Farnesoid X Receptor Is Required for the Redifferentiation of Bipotential Progenitor Cells During Biliary-Mediated Zebrafish Liver Regeneration.

BACKGROUND AND AIMS: Liver regeneration after extreme hepatocyte loss occurs through transdifferentiation of biliary epithelial cells (BECs), which includes dedifferentiation of BECs into bipotential progenitor cells (BPPCs) and subsequent redifferentiation into nascent hepatocytes and BECs. Although multiple molecules and signaling pathways have been implicated to play roles in the BEC-mediated liver regeneration, mechanisms underlying the dedifferentiation-redifferentiation transition and the early phase of BPPC redifferentiation that is pivotal for both hepatocyte and BEC directions remain largely unknown. APPROACH AND RESULTS: The zebrafish extreme liver damage model, genetic mutation, pharmacological inhibition, transgenic lines, whole-mount and fluorescent in situ hybridizations and antibody staining, single-cell RNA sequencing, quantitative real-time PCR, and heat shock-inducible overexpression were used to investigate roles and mechanisms of farnesoid X receptor (FXR; encoded by nuclear receptor subfamily 1, group H, member 4 [nr1h4]) in regulating BPPC redifferentiation. The nr1h4 expression was significantly up-regulated in response to extreme liver injury. Genetic mutation or pharmacological inhibition of FXR was ineffective to BEC-to-BPPC dedifferentiation but blocked the redifferentiation of BPPCs to both hepatocytes and BECs, leading to accumulation of undifferentiated or less-differentiated BPPCs. Mechanistically, induced overexpression of extracellular signal-related kinase (ERK) 1 (encoded by mitogen-activated protein kinase 3) rescued the defective BPPC-to-hepatocyte redifferentiation in the nr1h4 mutant, and ERK1 itself was necessary for the BPPC-to-hepatocyte redifferentiation. The Notch activities in the regenerating liver of nr1h4 mutant attenuated, and induced Notch activation rescued the defective BPPC-to-BEC redifferentiation in the nr1h4 mutant. CONCLUSIONS: FXR regulates BPPC-to-hepatocyte and BPPC-to-BEC redifferentiations through ERK1 and Notch, respectively. Given recent applications of FXR agonists in the clinical trials for liver diseases, this study proposes potential underpinning mechanisms by characterizing roles of FXR in the stimulation of dedifferentiation-redifferentiation transition and BPPC redifferentiation.

Macrophage-Derived IL-1ß Regulates Emergency Myelopoiesis via the NF-κB and C/ebpß in Zebrafish.

Myeloid phagocytes, neutrophils in particular, are easily consumed when they fight against a large number of invading microbes. Hence, they require efficient and constant replenishment from their progenitors via the well-orchestrated emergency myelopoiesis in the hematopoietic organs. The cellular and molecular details of the danger-sensing and warning processes to activate the emergency myelopoiesis are still under debate. In this study, we set up a systemic infection model in zebrafish (Danio rerio) larvae via circulative administration of LPS. We focused on the cross-talk of macrophages with myeloid progenitors in the caudal hematopoietic tissue. We revealed that macrophages first detected LPS and sent out the emergency message via il1ß The myeloid progenitors, rather than hematopoietic stem and progenitor cells, responded and fulfilled the demand to adapt myeloid expansion through the synergistic cooperation of NF-κB and C/ebpß. Our study unveiled a critical role of macrophages as the early "whistle blowers" to initiate emergency myelopoiesis.

The effector of Hippo signaling, Taz, is required for formation of the micropyle and fertilization in zebrafish.

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.

Sulfamethoxazole induces brain capillaries toxicity in zebrafish by up-regulation of VEGF and chemokine signalling.

Sulfamethoxazole (SMX) is a widespread broad-spectrum bacteriostatic antibiotic. Its residual is frequently detected in the water and may therefore bioaccumulate in the brain of aquatic organisms via blood circulation. Brain capillaries toxicity is very important for brain development. However, little information is available in the literature to show the toxicity of SMX to brain development. To study the SMX's brain toxic effects and the related mechanisms, we exposed zebrafish embryos to SMX at different concentrations (0 ppm, 1 ppm, 25 ppm, 100 ppm and 250 ppm) and found that high concentration (250 ppm) of SMX would not only caused an abnormal in malformation rate, hatching rate, body length and survival rate of zebrafish embryos, but also lead to brain oedema. In addition, SMX also induced cerebral ischaemia, aggravates oxidative stress, and changes genes related to oxidative stress (sod1, cat, gpx4, and nrf2). Furthermore, ischaemia caused by SMX could promote ectopic angiogenesis in brain via activating the angiogenesis-related genes (vegfab, cxcr4a, cxcl12b) from 24 h to 53 h. Inhibition of VEGF signalling by SU5416, or inhibition of chemokine downstream PI3K signalling by LY294002, could rescue the brain capillaries toxicity and brain oedema induced by SMX. Our results provide new evidence for the brain toxicity of SMX and its residual danger in the environment and aquatic organisms.

Mypt1 regulates Bmp signaling to promote embryonic exocrine pancreas growth in zebrafish.

Myosin phosphatase targeting subunit 1 (Mypt1) is the regulatory subunit of myosin phosphatase which dephosphorylates the light chain of myosin II to inhibit its contraction. Although biochemical properties of Mypt1 have been characterized in detail, its biological functions in organisms are not well understood. The zebrafish mypt1 sq181 allele was found defective in the ventral pancreatic bud and extrapancreatic duct development, resulting in dysplasia of exocrine pancreas. In mypt1 sq181 mutant, the early growth of the ventral pancreatic bud was initiated but failed to expand due to impaired cell proliferation and increased cell apoptosis. As Mypt1 is essential for cell migration, the loss-of-function of Mypt1 in the mutant disrupted the lateral plate mesoderm migration during gut looping, therefore, altering the Bmp2a expression pattern within it, and eventually leading to impaired Bmp signaling in the adjacent exocrine pancreas. Overexpression of bmp2a could rescue the development of exocrine pancreas, suggesting that the impaired Bmp2a signaling is responsible for the pancreatic development defects. Bmp2a has been reported to promote the early specification of the ventral pancreatic bud, and our study reveals that it continues to serve as a cell proliferation/survival signal to ensure pancreatic bud growth properly in zebrafish.

Improved Tol2-mediated enhancer trap identifies weakly expressed genes during liver and ß cell development and regeneration in zebrafish.

The liver and pancreas are two major digestive organs, and among the different cell types in them, hepatocytes and the insulin-producing ß cells have roles in both health and diseases. Accordingly, clinicians and researchers are very interested in the mechanisms underlying the development and regeneration of liver and pancreatic ß cells. Gene and enhancer traps such as the Tol2 transposon-based system are useful for identifying genes potentially involved in developmental processes in the zebrafish model. Here, we developed a strategy that combines a Tol2-mediated enhancer trap and the Cre/loxP system by using loxP-flanked reporters driven by ß cell- or hepatocyte-specific promoters and the upstream activating sequence (UAS)-driving Cre. Two double-transgenic reporter lines, Tg(ins:loxP-CFPNTR-loxP-DsRed; 10×UAS:Cre, cryaa:Venus) and Tg(fabp10:loxP-CFPNTR-loxP-DsRed; 10×UAS:Cre, cryaa:Venus), were established to label pancreatic ß cells and hepatocytes, respectively. These two double-transgenic lines were each crossed with the Tol2-enhancer trap founder lines to screen for and identify genes expressed in the ß cell and hepatocytes during development. This trap system coupled with application of nitroreductase (NTR)/metronidazole (Mtz)-mediated cell ablation could identify genes expressed during regeneration. Of note, pilot enhancer traps captured transiently and weakly expressed genes such as rab3da and ensab with higher efficiencies than traditional enhancer trap systems. In conclusion, through permanent genetic labeling by Cre/loxP, this improved Tol2-mediated enhancer trap system provides a promising method to identify transiently or weakly expressed, but potentially important, genes during development and regeneration.