Regenerative failure of intrahepatic biliary cells in Alagille syndrome rescued by elevated Jagged/Notch/Sox9 signaling.

Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.

Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy.

Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.

Attenuating the Epidermal Growth Factor Receptor-Extracellular Signal-Regulated Kinase-Sex-Determining Region Y-Box 9 Axis Promotes Liver Progenitor Cell-Mediated Liver Regeneration in Zebrafish.

BACKGROUND AND AIMS: The liver is a highly regenerative organ, but its regenerative capacity is compromised in severe liver injury settings. In chronic liver diseases, the number of liver progenitor cells (LPCs) correlates proportionally to disease severity, implying that their inefficient differentiation into hepatocytes exacerbates the disease. Moreover, LPCs secrete proinflammatory cytokines; thus, their prolonged presence worsens inflammation and induces fibrosis. Promoting LPC-to-hepatocyte differentiation in patients with advanced liver disease, for whom liver transplantation is currently the only therapeutic option, may be a feasible clinical approach because such promotion generates more functional hepatocytes and concomitantly reduces inflammation and fibrosis. APPROACH AND RESULTS: Here, using zebrafish models of LPC-mediated liver regeneration, we present a proof of principle of such therapeutics by demonstrating a role for the epidermal growth factor receptor (EGFR) signaling pathway in differentiation of LPCs into hepatocytes. We found that suppression of EGFR signaling promoted LPC-to-hepatocyte differentiation through the mitogen-activated ERK kinase (MEK)-extracellular signal-regulated kinase (ERK)-sex-determining region Y-box 9 (SOX9) cascade. Pharmacological inhibition of EGFR or MEK/ERK promoted LPC-to-hepatocyte differentiation as well as genetic suppression of the EGFR-ERK-SOX9 axis. Moreover, Sox9b overexpression in LPCs blocked their differentiation into hepatocytes. In the zebrafish liver injury model, both hepatocytes and biliary epithelial cells contributed to LPCs. EGFR inhibition promoted the differentiation of LPCs regardless of their origin. Notably, short-term treatment with EGFR inhibitors resulted in better liver recovery over the long term. CONCLUSIONS: The EGFR-ERK-SOX9 axis suppresses LPC-to-hepatocyte differentiation during LPC-mediated liver regeneration. We suggest EGFR inhibitors as a proregenerative therapeutic drug for patients with advanced liver disease.

Sox9b is a mediator of retinoic acid signaling restricting endocrine progenitor differentiation.

Centroacinar cells (CACs) are ductal Notch-responsive progenitors that in the larval zebrafish pancreas differentiate to form new islets and ultimately contribute to the majority of the adult endocrine mass. Uncovering the mechanisms regulating CAC differentiation will facilitate understanding how insulin-producing ß cells are formed. Previously we reported retinoic acid (RA) signaling and Notch signaling both regulate larval CAC differentiation, suggesting a shared downstream intermediate. Sox9b is a transcription factor important for islet formation whose expression is upregulated by Notch signaling in larval CACs. Here we report that sox9b expression in larval CACs is also regulated by RA signaling. Therefore, we hypothesized that Sox9b is an intermediate between both RA- and Notch-signaling pathways. In order to study the role of Sox9b in larval CACs, we generated two cre/lox based transgenic tools, which allowed us to express full-length or truncated Sox9b in larval CACs. In this way we were able to perform spatiotemporal-controlled Sox9b gain- and loss-of-function studies and observe the subsequent effect on progenitor differentiation. Our results are consistent with Sox9b regulating CAC differentiation by being a downstream intermediate of both RA- and Notch-signaling pathways. We also demonstrate that adult zebrafish with only one functional allele of sox9b undergo accelerated ß-cell regeneration, an observation consistent with sox9b regulating CAC differentiation in adults.

The Caudal Skeleton of the Zebrafish, Danio rerio, from a Phylogenetic Perspective: A Polyural Interpretation of Homologous Structures.

The structure of the caudal skeleton of extant teleost fishes has been interpreted in two different ways. In a diural interpretation, a caudal skeleton is composed of two centra articulated with one to six hypurals. Most subsequent authors have followed this interpretation. In contrast, a polyural interpretation considers the teleost fin to be derived from a fully metameristic ancestral bauplan originally composed of a one-to-one relationship between neural arches, centra (when present), and hypurals. Three different interpretations of the identity and homology of skeletal components of the caudal skeleton of the teleost fish Danio rerio have been proposed, two from a diural perspective and one from a polyural perspective. We examine each caudal skeletal component of Danio rerio from both a developmental and phylogenetic perspective. We propose that a polyural interpretation of structures is consistent with the current interpretation of the basal neopterygian caudal fin for this model organism rather than the older diural interpretation that does not take into account the metamerism observed in caudal structures during development. The polyural interpretation suggests several shared evolutionary innovations of major clades that would remain undiscovered under the older diural naming paradigm and makes the terminology of the parts of the caudal fin of Danio rerio strictly comparable to more basal fishes.

brca2 in zebrafish ovarian development, spermatogenesis, and tumorigenesis.

Humans with inherited mutations in BRCA2 are at increased risk for developing breast and ovarian cancer; however, the relationship between BRCA2 mutation and these cancers is not understood. Studies of Brca2 mutation by gene targeting in mice are limited, given that homozygous Brca2 mutation typically leads to early embryonic lethality. We established a zebrafish line with a nonsense mutation in brca2 exon 11 (brca2(Q658X)), a mutation similar in location and type to BRCA2 mutations found in humans with hereditary breast and ovarian cancer. brca2(Q658X) homozygous zebrafish are viable and survive to adulthood; however, juvenile homozygotes fail to develop ovaries during sexual differentiation. Instead, brca2(Q658X) homozygotes develop as infertile males with meiotic arrest in spermatocytes. Germ cell migration to the embryonic gonadal ridge is unimpaired in brca2(Q658X) homozygotes; thus, failure of ovarian development is not due to defects in early establishment of the embryonic gonad. Homozygous tp53 mutation rescues ovarian development in brca2(Q658X) homozygous zebrafish, reflecting the importance of germ cell apoptosis in gonad morphogenesis. Adult brca2(Q658X) homozygous zebrafish are predisposed to testicular neoplasias. In addition, tumorigenesis in multiple tissues is significantly accelerated in combination with homozygous tp53 mutation in both brca2(Q658X) homozygous and brca2(Q658X) heterozygous zebrafish. These studies reveal critical roles for brca2 in ovarian development and tumorigenesis in reproductive tissues.

Incomplete splicing, cell division defects, and hematopoietic blockage in dhx8 mutant zebrafish.

BACKGROUND: Vertebrate hematopoiesis is a complex developmental process that is controlled by genes in diverse pathways. To identify novel genes involved in early hematopoiesis, we conducted an ENU (N-ethyl-N-nitrosourea) mutagenesis screen in zebrafish. The mummy (mmy) line was investigated because of its multiple hematopoietic defects. RESULTS: Homozygous mmy embryos lacked circulating blood cell types and were dead by 30 hr post-fertilization (hpf). The mmy mutants did not express myeloid markers and had significantly decreased expression of progenitor and erythroid markers in primitive hematopoiesis. Through positional cloning, we identified a truncation mutation in dhx8 in the mmy fish. dhx8 is the zebrafish ortholog of the yeast splicing factor prp22, which is a DEAH-box RNA helicase. mmy mutants had splicing defects in many genes, including several hematopoietic genes. mmy embryos also showed cell division defects as characterized by disorganized mitotic spindles and formation of multiple spindle poles in mitotic cells. These cell division defects were confirmed by DHX8 knockdown in HeLa cells. CONCLUSIONS: Together, our results confirm that dhx8 is involved in mRNA splicing and suggest that it is also important for cell division during mitosis. This is the first vertebrate model for dhx8, whose function is essential for primitive hematopoiesis in developing embryos.

EWSR1 prevents the induction of aneuploidy through direct regulation of Aurora B.

EWSR1 (Ewing sarcoma breakpoint region 1) was originally identified as a part of an aberrant EWSR1/FLI1 fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the EWSR1/FLI1 fusion gene in the tumor genome, the cell loses one wild type EWSR1 allele. Our previous study demonstrated that the loss of ewsr1a (homologue of human EWSR1) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the tp53 mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using an Auxin Inducible Degron (AID) system. When both EWSR1 genes of DLD-1 cell were tagged with mini-AID at its 5'-end using a CRISPR/Cas9 system, treatment of the (AID-EWSR1/AID-EWSR1) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the EWSR1 knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at Kinetochore proximal centromere compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of EWSR1-mCherry and EWSR1:R565A-mCherry (a mutant that has low affinity for Aurora B) in the (AID-EWSR1/AID-EWSR1) DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 prevents the induction of lagging chromosomes, and of aneuploidy through the interaction with Aurora B.

Loss of Ewing sarcoma EWS allele promotes tumorigenesis by inducing chromosomal instability in zebrafish.

The Ewing sarcoma family of tumors expresses aberrant EWSR1- (EWS) fusion genes that are derived from chromosomal translocation. Although these fusion genes are well characterized as transcription factors, their formation leaves a single EWS allele in the sarcoma cells, and the contribution that the loss of EWS makes towards disease pathogenesis is unknown. To address this question, we utilized zebrafish mutants for ewsa and tp53. The zebrafish tp53(M214K)(w/m) line and the ewsa(w/m), zygotic ewsa(m/m), and Maternal-Zygotic (MZ) ewsa(m/m) lines all displayed zero to low incidence of tumorigenesis. However, when the ewsa and tp53 mutant lines were crossed with each other, the incidence of tumorigenesis drastically increased. Furthermore, 27 hour post fertilization (hpf) MZ ewsa(m/m) mutant embryos displayed a higher incidence of aberrant chromosome numbers and mitotic dysfunction compared to wildtype zebrafish embryos. Consistent with this finding, tumor samples obtained from ewsa(m/m);tp53(w/m) zebrafish displayed loss of heterozygosity (LOH) for the wildtype tp53 locus. These results suggest that wildtype Ewsa inhibits LOH induction, possibly by maintaining chromosomal stability. We propose that the loss of ewsa promotes tumorigenesis, and EWS deficiency may contribute to the pathogenesis of EWS-fusion-expressing sarcomas.

Reduced O-GlcNAcase expression promotes mitotic errors and spindle defects.

Alterations in O-GlcNAc cycling, the addition and removal of O-GlcNAc, lead to mitotic defects and increased aneuploidy. Herein, we generated stable O-GlcNAcase (OGA, the enzyme that removes O-GlcNAc) knockdown HeLa cell lines and characterized the effect of the reduction in OGA activity on cell cycle progression. After release from G1/S, the OGA knockdown cells progressed normally through S phase but demonstrated mitotic exit defects. Cyclin A was increased in the knockdown cells while Cyclin B and D expression was reduced. Retinoblastoma protein (RB) phosphorylation was also increased in the knockdown compared to control. At M phase, the knockdown cells showed more compact spindle chromatids than control cells and had a greater percentage of cells with multipolar spindles. Furthermore, the timing of the inhibitory tyrosine phosphorylation of Cyclin Dependent Kinase 1 (CDK1) was altered in the OGA knockdown cells. Although expression and localization of the chromosomal passenger protein complex (CPC) was unchanged, histone H3 threonine 3 phosphorylation was decreased in one of the OGA knockdown cell lines. The Ewing Sarcoma Breakpoint Region 1 Protein (EWS) participates in organizing the CPC at the spindle and is a known substrate for O-GlcNAc transferase (OGT, the enzyme that adds O-GlcNAc). EWS O-GlcNAcylation was significantly increased in the OGA knockdown cells promoting uneven localization of the mitotic midzone. Our data suggests that O-GlcNAc cycling is an essential mechanism for proper mitotic signaling and spindle formation, and alterations in the rate of O-GlcNAc cycling produces aberrant spindles and promotes aneuploidy.

Ewing sarcoma ewsa protein regulates chondrogenesis of Meckel's cartilage through modulation of Sox9 in zebrafish.

Ewing sarcoma is the second most common skeletal (bone and cartilage) cancer in adolescents, and it is characterized by the expression of the aberrant chimeric fusion gene EWS/FLI1. Wild-type EWS has been proposed to play a role in mitosis, splicing and transcription. We have previously shown that EWS/FLI1 interacts with EWS, and it inhibits EWS activity in a dominant manner. Ewing sarcoma is a cancer that specifically develops in skeletal tissues, and although the above data suggests the significance of EWS, its role in chondrogenesis/skeletogenesis is not understood. To elucidate the function of EWS in skeletal development, we generated and analyzed a maternal zygotic (MZ) ewsa/ewsa line because the ewsa/wt and ewsa/ewsa zebrafish appeared to be normal and fertile. Compared with wt/wt, the Meckel's cartilage of MZ ewsa/ewsa mutants had a higher number of craniofacial prehypertrophic chondrocytes that failed to mature into hypertrophic chondrocytes at 4 days post-fertilization (dpf). Ewsa interacted with Sox9, which is the master transcription factor for chondrogenesis. Sox9 target genes were either upregulated (ctgfa, ctgfb, col2a1a, and col2a1b) or downregulated (sox5, nog1, nog2, and bmp4) in MZ ewsa/ewsa embryos compared with the wt/wt zebrafish embryos. Among these Sox9 target genes, the chromatin immunoprecipitation (ChIP) experiment demonstrated that Ewsa directly binds to ctgfa and ctgfb loci. Consistently, immunohistochemistry showed that the Ctgf protein is upregulated in the Meckel's cartilage of MZ ewsa/ewsa mutants. Together, we propose that Ewsa promotes the differentiation from prehypertrophic chondrocytes to hypertrophic chondrocytes of Meckel's cartilage through inhibiting Sox9 binding site of the ctgf gene promoter. Because Ewing sarcoma specifically develops in skeletal tissue that is originating from chondrocytes, this new role of EWS may provide a potential molecular basis of its pathogenesis.

Ewing sarcoma EWS protein regulates midzone formation by recruiting Aurora B kinase to the midzone.

Ewing sarcoma is a malignant bone cancer that primarily occurs in children and adolescents. Eighty-five percent of Ewing sarcoma is characterized by the presence of the aberrant chimeric EWS/FLI1 fusion gene. Previously, we demonstrated that an interaction between EWS/FLI1 and wild-type EWS led to the inhibition of EWS activity and mitotic dysfunction. Although defective mitosis is considered to be a critical step in cancer initiation, it is unknown how interference with EWS contributes to Ewing sarcoma formation. Here, we demonstrate that EWS/FLI1- and EWS-knockdown cells display a high incidence of defects in the midzone, a midline structure located between segregating chromatids during anaphase. Defects in the midzone can lead to the failure of cytokinesis and can result in the induction of aneuploidy. The similarity among the phenotypes of EWS/FLI1- and EWS siRNA-transfected HeLa cells points to the inhibition of EWS as the key mechanism for the induction of midzone defects. Supporting this observation, the ectopic expression of EWS rescues the high incidence of midzone defects observed in Ewing sarcoma A673 cells. We discovered that EWS interacts with Aurora B kinase, and that EWS is also required for recruiting Aurora B to the midzone. A domain analysis revealed that the R565 in the RGG3 domain of EWS is essential for both Aurora B interaction and the recruitment of Aurora B to the midzone. Here, we propose that the impairment of EWS-dependent midzone formation via the recruitment of Aurora B is a potential mechanism of Ewing sarcoma development.

Ewing sarcoma fusion protein EWSR1/FLI1 interacts with EWSR1 leading to mitotic defects in zebrafish embryos and human cell lines.

The mechanism whereby the fusion of EWSR1 with the ETS transcription factor FLI1 contributes to malignant transformation in Ewing sarcoma remains unclear. We show that injection of human or zebrafish EWSR1/FLI1 mRNA into developing zebrafish embryos leads to mitotic defects with multipolar and disorganized mitotic spindles. Expression of human EWSR1/FLI1 in HeLa cells also results in mitotic defects, along with mislocalization of Aurora kinase B, a key regulator of mitotic progression. Because these mitotic abnormalities mimic those observed with the knockdown of EWSR1 in zebrafish embryos and HeLa cells, we investigated whether EWSR1/FLI1 interacts with EWSR1 and interferes with its function. EWSR1 coimmunoprecipitates with EWSR1/FLI1, and overexpression of EWSR1 rescues the mitotic defects in EWSR1/FLI1-transfected HeLa cells. This interaction between EWSR1/FLI1 and EWSR1 in Ewing sarcoma may induce mitotic defects leading to genomic instability and subsequent malignant transformation.

Ewing sarcoma protein ewsr1 maintains mitotic integrity and proneural cell survival in the zebrafish embryo.

BACKGROUND: The Ewing sarcoma breakpoint region 1 gene (EWSR1), also known as EWS, is fused to a number of different partner genes as a result of chromosomal translocation in diverse sarcomas. Despite the involvement of EWSR1 in these diverse sarcomas, the in vivo function of wild type EWSR1 remains unclear. PRINCIPAL FINDINGS: We identified two zebrafish EWSR1 orthologues, ewsr1a and ewsr1b, and demonstrate that both genes are expressed maternally, and are expressed ubiquitously throughout zebrafish embryonic development. Morpholino induced knockdown of both zebrafish ewsr1 genes led to mitotic defects with multipolar or otherwise abnormal mitotic spindles starting from the bud stage (10 hour post-fertilization (hpf)). The abnormalities in mitotic spindles were followed by p53-mediated apoptosis in the developing central nervous system (CNS) leading to a reduction in the number of proneural cells, disorganization of neuronal networks, and embryonic lethality by 5 days post-fertilization. siRNA silencing of EWSR1 in Hela cells resulted in mitotic defects accompanied by apoptotic cell death, indicating that the role of EWSR1 is conserved between zebrafish and human. CONCLUSIONS: Ewsr1 maintains mitotic integrity and proneural cell survival in early zebrafish development.

Perturbation of rRNA synthesis in the bap28 mutation leads to apoptosis mediated by p53 in the zebrafish central nervous system.

Zebrafish is a powerful vertebrate model system for using forward genetics to elucidate mechanisms of early development. We have used chemical mutagenesis to screen for mutants that show defects in the CNS. Here we describe the isolation of the bap28 mutation that leads to abnormalities in the brain starting at midsomitogenesis stages. Mutant embryos display excess apoptosis primarily in the central nervous system (CNS) and die by days 6-7 after fertilization. The mutation was positionally cloned and shown to affect a gene that encodes a large protein with high similarity to the uncharacterized human protein BAP28 and lower similarity to yeast Utp10. Utp10 is a component of a nucleolar U3 small nucleolar RNA-containing RNP complex that is required for transcription of ribosomal DNA and for processing of 18 S rRNA. We show that zebrafish Bap28 likewise is required for rRNA transcription and processing, with a major effect on 18 S rRNA maturation. We suggest that bap28 is required for cell survival in the CNS through its role in rRNA synthesis and processing. Inhibition of p53 protein expression in bap28 mutants led to embryos with morphologically normal appearance, suggesting that p53 is involved in triggering apoptosis in the bap28 mutant CNS. The bap28 mutation provides a genetic approach to study the role of ribosome biogenesis in the development of a vertebrate embryo.

TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a clonal disease that evolves through the accrual of genetic rearrangements and/or mutations within the dominant clone. The TEL-AML1 (ETV6-RUNX1) fusion in precursor-B (pre-B) ALL is the most common genetic rearrangement in childhood cancer; however, the cellular origin and the molecular pathogenesis of TEL-AML1-induced leukemia have not been identified. To study the origin of TEL-AML1-induced ALL, we generated transgenic zebrafish expressing TEL-AML1 either ubiquitously or in lymphoid progenitors. TEL-AML1 expression in all lineages, but not lymphoid-restricted expression, led to progenitor cell expansion that evolved into oligoclonal B-lineage ALL in 3% of the transgenic zebrafish. This leukemia was transplantable to conditioned wild-type recipients. We demonstrate that TEL-AML1 induces a B cell differentiation arrest, and that leukemia development is associated with loss of TEL expression and elevated Bcl2/Bax ratio. The TEL-AML1 transgenic zebrafish models human pre-B ALL, identifies the molecular pathways associated with leukemia development, and serves as the foundation for subsequent genetic screens to identify modifiers and leukemia therapeutic targets.