Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression.

BACKGROUND: Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. METHODS: Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. RESULTS: Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression. CONCLUSIONS: Our results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer's vesicle morphogenesis and spaw expression in the left LPM.

Foxa2 and Hif1ab regulate maturation of intestinal goblet cells by modulating agr2 expression in zebrafish embryos.

Mammalian anterior gradient 2 (AGR2), an endoplasmic reticulum (ER) protein disulfide-isomerase (PDI), is involved in cancer cell growth and metastasis, asthma and inflammatory bowel disease (IBD). Mice lacking Agr2 exhibit decreased Muc2 protein in intestinal goblet cells, abnormal Paneth cell development, ileitis and colitis. Despite its importance in cancer biology and inflammatory diseases, the mechanisms regulating agr2 expression in the gastrointestinal tract remain unclear. In the present study, we investigated the mechanisms that control agr2 expression in the pharynx and intestine of zebrafish by transient/stable transgenesis, coupled with motif mutation, morpholino knockdown, mRNA rescue and ChIP. A 350 bp DNA sequence with a hypoxia-inducible response element (HRE) and forkhead-response element (FHRE) within a region -4.5 to -4.2 kbp upstream of agr2 directed EGFP expression specifically in the pharynx and intestine. No EGFP expression was detected in the intestinal goblet cells of Tg(HREM:EGFP) or Tg(FHREM:EGFP) embryos with mutated HRE or FHRE, whereas EGFP was expressed in the pharynx of Tg(HREM:EGFP), but not Tg(FHREM:EGFP), embryos. Morpholino knockdown of foxa1 (forkhead box A1) reduced agr2 levels in the pharynx, whereas knockdown of foxa2 or hif1ab decreased intestinal agr2 expression and affected the differentiation and maturation of intestinal goblet cells. These results demonstrate that Foxa1 regulates agr2 expression in the pharynx, whereas both Foxa2 and Hif1ab control agr2 expression in intestinal goblet cells to regulate maturation of these cells.

Epicatechin isolated from Tripterygium wilfordii extract reduces tau-GFP-induced neurotoxicity in zebrafish embryo through the activation of Nrf2.

Tau plays important roles in the assembly and stabilization of the microtubule structure to facilitate axonal transport in mammalian brain. The intracellular tau aggregates to form paired helical filaments leading to neurodegenerative disorders, collectively called tauopathies. In our previous report, we established a zebrafish model to express tau-GFP to induce neuronal death, which could be directly traced in vivo. Recently, we used this model to screen 400 herbal extracts and found 45 of them to be effective on reducing tau-GFP-induced neuronal death. One of the effective herbal extracts is the Tripterygium wilfordii stem extract. HPLC analysis and functional assay demonstrated that epicatechin (EC) is the major compound of Tripterygium wilfordii stem extract to decrease the neurotoxicity induced by tau-GFP. Using a luciferase reporter assay in the zebrafish, we confirmed that EC could activate Nrf2-dependent antioxidant responses to significantly increase the ARE-controlled expression of luciferase reporter gene. These data suggest that EC from the Tripterygium wilfordii stem extract could diminish tau-GFP-induced neuronal death through the activation of Nrf2.

Multiple signaling factors and drugs alleviate neuronal death induced by expression of human and zebrafish tau proteins in vivo.

BACKGROUND: The axonal tau protein is a tubulin-binding protein, which plays important roles in the formation and stability of the microtubule. Mutations in the tau gene are associated with familial forms of frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17). Paired helical filaments of tau and extracellular plaques containing beta-amyloid are found in the brain of Alzheimer's disease (AD) patients. RESULTS: Transgenic models, including those of zebrafish, have been employed to elucidate the mechanisms by which tau protein causes neurodegeneration. In this study, a transient expression system was established to express GFP fusion proteins of zebrafish and human tau under the control of a neuron-specific HuC promoter. Approximately ten neuronal cells expressing tau-GFP in zebrafish embryos were directly imaged and traced by time-lapse recording, in order to evaluate the neurotoxicity induced by tau-GFP proteins. Expression of tau-GFP was observed to cause high levels of neuronal death. However, multiple signaling factors, such as Bcl2-L1, Nrf2, and GDNF, were found to effectively protect neuronal cells expressing tau-GFP from death. Treatment with chemical compounds that exert anti-oxidative or neurotrophic effects also resulted in a similar protective effect and maintained human tau-GFP protein in a phosphorylated state, as detected by antibodies pT212 and AT8. CONCLUSIONS: The novel finding of this study is that we established an expression system expressing tau-GFP in zebrafish embryos were directly imaged and traced by time-lapse recording to evaluate the neurotoxicity induced by tau-GFP proteins. This system may serve as an efficient in vivo imaging platform for the discovery of novel drugs against tauopathy.

Expression of zebrafish anterior gradient 2 in the semicircular canals and supporting cells of otic vesicle sensory patches is regulated by Sox10.

AGR2 is a member of the protein disulfide isomerase (PDI) family, which is implicated in cancer cell growth and metastasis, asthma, and inflammatory bowel disease. Despite the contributions of this protein to several biological processes, the regulatory mechanisms controlling expression of the AGR2 gene in different organs remain unclear. Zebrafish anterior gradient 2 (agr2) is expressed in several organs, including the otic vesicles that contain mucus-secreting cells. To elucidate the regulatory mechanisms controlling agr2 expression in otic vesicles, we generated a Tg(-6.0 k agr2:EGFP) transgenic fish line that expressed EGFP in a pattern recapitulating that of agr2. Double immunofluorescence studies were used to demonstrate that Agr2 and GFP colocalize in the semicircular canals and supporting cells of all sensory patches in the otic vesicles of Tg(-6.0 k agr2:EGFP) embryos. Transient/stable transgenic analyses coupled with 5'-end deletion revealed that a 100 bp sequence within the -2.6 to -2.5 kbp region upstream of agr2 directs EGFP expression specifically in the otic vesicles. Two HMG-binding motifs were detected in this region. Mutation of these motifs prevented EGFP expression. Furthermore, EGFP expression in the otic vesicles was prevented by knockdown of the sox10 gene. This corresponded with decreased agr2 expression in the otic vesicles of sox10 morphants during different developmental stages. Electrophoretic mobility shift assays were used to show that Sox10 binds to HMG-binding motifs located within the -2.6 to -2.5 kbp region upstream of agr2. These results demonstrate that agr2 expression in the otic vesicles of zebrafish embryos is regulated by Sox10.

Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish.

BACKGROUND: Mutations in mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) that occur during cell proliferation and tumor formation are well described. Information on the roles of MEK2 in these effects is still limited. We established a constitutive MEK2 transgenic zebrafish, Tg(krt14:MEK2S219D-GFP), to elucidate the role of MEK2 in skin tumor formation. RESULTS: We found that both constitutive MEK2 and MEK1 are able to phosphorylate the extracellular signal-regulated kinase 1 (ERK1) protein. Transient expression of constitutive MEK2 and MEK1 in the zebrafish epidermis induced papillary formation at 48 h post-fertilization, but no effects were observed due to the expression of MEK1, MEK2, or the dominant negative form of MEK2. The transgenic zebrafish, Tg(krt14:MEK2S219D-GFP), developed skin papillomas in the epidermis within 6 days post-fertilization (dpf). The phospho-ERK signal was detected in section of skin papillomas in an immunohistochemical experiment. Treatment with 50 µM of the MEK inhibitor, U0126, had significantly decreased the skin papilloma formation in Tg(krt14:MEK2S219D-GFP) zebrafish by 6 dpf. In vitro and in vivo proliferation assay in COS-1 cells and in Tg(krt14:MEK2S219D-GFP) transgenic fish show significantly increased cell number and Ki-67 signaling. CONCLUSION: Our data indicate that MEK2 is sufficient to induce epidermal papilloma formation through MAPK signaling in zebrafish, and this transgenic model can be used as a new platform for drug screening.

Differential regulation of Tetraodon nigroviridis Mx gene promoter activity by constitutively-active forms of STAT1, STAT2, and IRF9.

Induction of interferons (IFNs) produces an innate immune response through activation of the JAK-STAT signaling pathway. Type I IFN signaling activates downstream gene expression through the IFN-stimulated gene factor 3 (ISGF3) complex, while type II IFN (IFN-γ) signaling is mediated through active STAT1 protein. The IFN target gene Mx is involved in the defense against viral infection. However, the mechanism by which Tetraodon (pufferfish) Mx is regulated by IFN signaling has not been identified. In this study, we describe the cloning and expression of Tetraodon STAT1, STAT2, and IFN regulatory factor 9 (IRF9). By combining constitutively-active STAT1 (STAT1-JH1) and STAT2 (STA2-JH1) fusion proteins with IRF9, we demonstrate that a constitutively-active ISGF3 complex increases the transcriptional activity of the Tetraodon Mx promoter via direct binding to two IFN-stimulated response element (ISRE) sites. In addition, a constitutively-active TnIRF9-S2C containing a fusion of the C-terminal region of STAT2 and IRF9 also activated the Mx promoter through binding to the ISRE sites. Furthermore, constitutively-active STAT1-JH1 elevates Mx promoter activity through two IFN gamma-activated sequence (GAS) elements. The Mx promoter is also activated by constitutively-active TnIRF9-S2C and STAT1-JH1 protein, as determined using an in vivo luciferase assay. We conclude that the Tetraodon Mx gene is activated via Type I (IFN-1) and Type II (IFN-γ) signaling. These results provide mechanistic insights into the role of IFN signaling in teleosts, and the in vivo luciferase assay may be suitable as a tool for studying induction and regulation by IFNs in teleost fish.

Zinc finger protein 219-like (ZNF219L) and Sox9a regulate synuclein-γ2 (sncgb) expression in the developing notochord of zebrafish.

Zebrafish synuclein-γ2 (sncgb) has been reported to be expressed specifically in the notochord. However, the mechanism by which the sncgb gene promoter is regulated has not been described. In this paper, we demonstrate that Zinc finger protein 219-like (ZNF219L) and sox9a are involved in the regulation of sncgb gene expression. Furthermore, we observed that over-expression of both ZNF219L and Sox9a resulted in increased sncgb expression. In addition, ZNF219L is physically associated with Sox9a, and simultaneous morpholino knockdown of znf219L and sox9a caused a synergistic decrease of sncgb expression in the notochord. Taken together, our results reveal that coordination of ZNF219L with Sox9a is involved in the regulation of notochord-specific expression of sncgb.

IRF9-Stat2 Fusion Protein as an Innate Immune Inducer to Activate Mx and Interferon-Stimulated Gene Expression in Zebrafish Larvae.

Virus infection often causes large amounts of mortality during teleost larvae stage. Strong induction of innate immunity to increase survival rates of teleost larvae has been less reported. In this study, we present a zebrafish IRF9-Stat2 fusion protein (zIRF9-S2C) as a strong innate immunity inducer and characterized induction of interferon-stimulated genes (ISGs) in zebrafish larvae. zIRF9-S2C could mimic IFN-stimulated gene factor 3 (ISGF3) complex to constitutively activate transcription of Mx promoter through IFN-stimulatory element (ISRE) sites. Mutation of two ISRE sites on Mx promoter reduced transactivation activities of Mx promoter induced by zIRF9-S2C. An electrophoretic mobility shift assay experiment shows that zIRF9-S2C could directly bind to two ISRE sites of Mx promoter. Induction of transactivation of Mx promoter by zIRF9-S2C shows significantly higher activity than by zebrafish IFN1 (zIFN1), IFNγ (zIFNγ), and Tetraodon IRF9-S2C (TnIRF9-S2C). zIRF9-S2C raises transcription of Mxa, Mxb, Mxc, Ifnφ1, Ifnφ2, and Ifnφ3 in zebrafish liver ((ZFL) cell line) cells and zebrafish larvae. Collectively, we suggest that IRF9-S2C could activate transcription of ISGs with species-specific recognition and could be an innate immunity inducer in teleost larvae.

Murine tribbles homolog 2 deficiency affects erythroid progenitor development and confers macrocytic anemia on mice.

Tribbles homolog 2 (Trib2) is a member of Tribbles protein pseudokinases and involves in apoptosis, autoimmunity, cancer, leukemia and erythropoiesis, however, the physiological function of Trib2 in hematopoietic system remains to be elucidated. Here, we report that Trib2 knockout (KO) mice manifest macrocytic anemia and increase of T lymphocytes. Although Trib2 deficient RBCs have similar half-life as the control RBCs, Trib2 KO mice are highly vulnerable to oxidant-induced hemolysis. Endogenous Trib2 mRNA is expressed in early hematopoietic progenitors, erythroid precursors, and lymphoid lineages, but not in mature RBCs, myeloid progenitors and granulocytes. Consistently, flow cytometric analysis and in vitro colony forming assay revealed that deletion of Trib2 mainly affected erythroid lineage development, and had no effect on either granulocyte or megakaryocyte lineages in bone marrow. Furthermore, a genetic approach using double knockout of Trib2 and C/ebpα genes in mice suggested that Trib2 promotes erythropoiesis independent of C/ebpα proteins in vivo. Finally, ectopic expression of human Trib2 in zebrafish embryos resulted in increased expression of erythropoiesis-related genes and of hemoglobin. Taking all data together, our results suggest that Trib2 positively promotes early erythrocyte differentiation and is essential for tolerance to hemolysis.

Zebrafish cyclin Dx is required for development of motor neuron progenitors, and its expression is regulated by hypoxia-inducible factor 2α.

Cyclins play a central role in cell-cycle regulation; in mammals, the D family of cyclins consists of cyclin D1, D2, and D3. In Xenopus, only homologs of cyclins D1 and D2 have been reported, while a novel cyclin, cyclin Dx (ccndx), was found to be required for the maintenance of motor neuron progenitors during embryogenesis. It remains unknown whether zebrafish possess cyclin D3 or cyclin Dx. In this study, we identified a zebrafish ccndx gene encoding a protein which can form a complex with Cdk4. Through whole-mount in situ hybridization, we observed that zccndx mRNA is expressed in the motor neurons of hindbrain and spinal cord during development. Analysis of a 4-kb promoter sequence of the zccndx gene revealed the presence of HRE sites, which can be regulated by HIF2α. Morpholino knockdown of zebrafish Hif2α and cyclin Dx resulted in the abolishment of isl1 and oligo2 expression in the precursors of motor neurons, and also disrupted axon growth. Overexpression of cyclin Dx mRNA in Hif2α morphants partially rescued zccndx expression. Taken together, our data indicate that zebrafish cyclin Dx plays a role in maintaining the precursors of motor neurons.

Modulation of p53 and met expression by Krüppel-like factor 8 regulates zebrafish cerebellar development.

Krüppel-like factor 8 (Klf8) is a zinc-finger transcription factor implicated in cell proliferation, and cancer cell survival and invasion; however, little is known about its role in normal embryonic development. Here, we show that Klf8 is required for normal cerebellar development in zebrafish embryos. Morpholino knockdown of klf8 resulted in abnormal cerebellar primordium morphology and the induction of p53 in the brain region at 24 hours post-fertilization (hpf). Both p53-dependent reduction of cell proliferation and augmentation of apoptosis were observed in the cerebellar anlage of 24 hpf-klf8 morphants. In klf8 morphants, expression of ptf1a in the ventricular zone was decreased from 48 to 72 hpf; on the other hand, expression of atohla in the upper rhombic lip was unaffected. Consistent with this finding, Purkinje cell development was perturbed and granule cell number was reduced in 72 hpf-klf8 morphants; co-injection of p53 MO(sp) or klf8 mRNA substantially rescued development of cerebellar Purkinje cells in klf8 morphants. Hepatocyte growth factor/Met signaling is known to regulate cerebellar development in zebrafish and mouse. We observed decreased met expression in the tectum and rhombomere 1 of 24 hpf-klf8 morphants, which was largely rescued by co-injection with klf8 mRNA. Moreover, co-injection of met mRNA substantially rescued formation of Purkinje cells in klf8 morphants at 72 hpf. Together, these results demonstrate that Klf8 modulates expression of p53 and met to maintain ptf1a-expressing neuronal progenitors, which are required for the appropriate development of cerebellar Purkinje and granule cells in zebrafish embryos.

A novel zinc finger protein 219-like (ZNF219L) is involved in the regulation of collagen type 2 alpha 1a (col2a1a) gene expression in zebrafish notochord.

The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly expressed in the zebrafish midbrain-hindbrain boundary, hindbrain, and notochord during development. The znf219L morpholino knockdown caused partial abnormal notochord phenotype and reduced expression of endogenous col2a1a in the notochord specifically. In addition, ZNF219L could recognize binding sites with GGGGG motifs and trigger augmented activity of the col2a1a promoter in a luciferase assay. Furthermore, in vitro binding experiments revealed that ZNF219L recognizes the GGGGG motifs in the promoter region of the zebrafish col2a1a gene through its sixth and ninth zinc finger domains. Taken together, our results reveal that ZNF219L is involved in regulating the expression of col2a1a in zebrafish notochord specifically.

Disruption of murine mp29/Syf2/Ntc31 gene results in embryonic lethality with aberrant checkpoint response.

Human p29 is a putative component of spliceosomes, but its role in pre-mRNA is elusive. By siRNA knockdown and stable overexpression, we demonstrated that human p29 is involved in DNA damage response and Fanconi anemia pathway in cultured cells. In this study, we generated p29 knockout mice (mp29(GT/GT)) using the mp29 gene trap embryonic stem cells to study the role of mp29 in DNA damage response in vivo. Interruption of mp29 at both alleles resulted in embryonic lethality. Embryonic abnormality occurred as early as E6.5 in mp29(GT/GT) mice accompanied with decreased mRNA levels of α-tubulin and Chk1. The reduction of α-tubulin and Chk1 mRNAs is likely due to an impaired post-transcriptional event. An aberrant G2/M checkpoint was found in mp29 gene trap embryos when exposed to aphidicolin and UV light. This embryonic lethality was rescued by crossing with mp29 transgenic mice. Additionally, the knockdown of zfp29 in zebrafish resulted in embryonic death at 72 hours of development postfertilization (hpf). A lower level of acetylated α-tubulin was also observed in zfp29 morphants. Together, these results illustrate an indispensable role of mp29 in DNA checkpoint response during embryonic development.