Jennifer Rhodes

Knockdown of Zebrafish Fancd2 Causes Developmental Abnormalities via p53-Dependent Apoptosis

Mechanisms underlying the multiple developmental defects observed in Fanconi anemia (FA) patients are not well defined. We have identified the zebrafish homolog of human FANCD2, which encodes a nuclear effector protein that is monoubiquitinated in response to DNA damage, targeting it to nuclear foci where it preserves chromosomal integrity. Fancd2-deficient zebrafish embryos develop defects similar to those found in children with FA, including shortened body length, microcephaly, and microophthalmia, which are due to extensive cellular apoptosis. Developmental defects and increased apoptosis in Fancd2-deficient zebrafish were corrected by injection of human FANCD2 or zebrafish bcl2 mRNA, or by knockdown of p53, indicating that in the absence of Fancd2, developing tissues spontaneously undergo p53-dependent apoptosis. Thus, Fancd2 is essential during embryogenesis to prevent inappropriate apoptosis in neural cells and other tissues undergoing high levels of proliferative expansion, implicating this mechanism in the congenital abnormalities observed in human infants with FA.

Live imaging of chronic inflammation caused by mutation of zebrafish Hai1.

The hallmark of chronic inflammation is the infiltration and persistence of leukocytes within inflamed tissue. Here, we describe the first zebrafish chronic inflammation mutant identified in an insertional mutagenesis screen for mutants that exhibit abnormal tissue distribution of neutrophils. We identified a mutant line with an insertion in the Hepatocyte growth factor activator inhibitor 1 gene (hai1; also known as Spint1) that showed accumulation of neutrophils in the fin. The mutant embryos exhibited inflammation in areas of epidermal hyperproliferation that was rescued by knock-down of the type II transmembrane serine protease Matriptase 1 (also known as St14), suggesting a novel role for Hai1-Matriptase 1 pathway in regulating inflammation. Using time-lapse microscopy of mutant embryos that express GFP from a neutrophil-specific promoter, we found that individual neutrophils in inflamed tissue displayed random motility characterized by periods of pausing alternating with periods of motility. During periods of persistent movement the cells were highly polarized, while the pausing modes were characterized by a loss of cell polarity. In contrast to responses to acute injury, neutrophils did not exhibit clear retrograde chemotaxis or resolution of inflammation in the mutant. These findings illustrate the utility of zebrafish as a new model system to study chronic inflammation and to visualize immune responses with high resolution in vivo.

Control of Hematopoietic Stem Cell Emergence by Antagonistic Functions of Ribosomal Protein Paralogs

It remains controversial whether the highly homologous ribosomal protein (RP) paralogs found in lower eukaryotes have distinct functions and this has not been explored in vertebrates. Here we demonstrate that despite ubiquitous expression, the RP paralogs, Rpl22 and Rpl22-like1 (Rpl22l1) play essential, distinct, and antagonistic roles in hematopoietic development. Knockdown of Rpl22 in zebrafish embryos selectively blocks the development of T lineage progenitors after they have seeded the thymus. In contrast, knockdown of the Rpl22 paralog, Rpl22l1, impairs the emergence of hematopoietic stem cells (HSC) in the aorta-gonad-mesonephros by abrogating Smad1 expression and the consequent induction of essential transcriptional regulator, Runx1. Indeed, despite the ability of both paralogs to bind smad1 RNA, Rpl22 and Rpl22l1 have opposing effects on Smad1 expression. Accordingly, circumstances that tip the balance of these paralogs in favor of Rpl22 (e.g., Rpl22l1 knockdown or Rpl22 overexpression) result in repression of Smad1 and blockade of HSC emergence.

CrkL functions as a nuclear adaptor and transcriptional activator in Bcr-Abl–expressing cells

OBJECTIVE To identify tyrosine phosphorylated proteins that interact with CrkL in Bcr-Abl-expressing cells and analyze the function of that association. MATERIALS AND METHODS Immunoprecipitation of CrkL was performed on lysates from parental cells (Rat-1, MO7e, or 32D) or Bcr-Abl-expressing cells (Rat-1p185, MO7p210, 32Dp210, K562) followed by immunoblotting for pTyr, Stat5, or CrkL. Interactions were confirmed in vitro using GST-CrkL fusion proteins. Electrophoretic mobility shift assays were performed on K562 nuclear extracts using a beta-casein promoter-derived probe. Supershift analysis was performed with CrkL, Stat5, Stat1, Grb2, and peptide-blocked CrkL and Stat5 antibodies. CrkL localization in Rat-1 and Rat-1p185 cells was detected with indirect immunofluorescence. Transcriptional activation was analyzed in COS7 cells transfected with a Stat-responsive luciferase reporter construct and Bcr-Abl, kinase-defective Bcr-Abl, CrkL, or Grb2. RESULTS We show that, in Bcr-Abl-expressing cells, CrkL+ interacts with tyrosine phosphorylated Stat5. Additionally, in the presence of Bcr-Abl, CrkL is found in the nucleus, can be detected in a Stat5/DNA complex, and increases transcriptional activation from a Stat-responsive reporter construct. CONCLUSION This suggests a novel role for CrkL, functioning as a nuclear adaptor protein that can associate with and activate Stat proteins in Bcr-Abl-expressing cells.

Evolutionary conservation of zebrafish linkage group 14 with frequently deleted regions of human chromosome 5 in myeloid malignancies

Recurring interstitial loss of all or part of the long arm of chromosome 5, del(5q), is a hallmark of myelodysplastic syndrome and acute myeloid leukemia. Although the genes affected by these changes have not been identified, two critically deleted regions (CDRs) are well established. We have identified 76 zebrafish cDNAs orthologous to genes located in these 5q CDRs. Radiation hybrid mapping revealed that 33 of the 76 zebrafish orthologs are clustered in a genomic region on linkage group 14 (LG14). Fifteen others are located on LG21, and two on LG10. Although there are large blocks of conserved syntenies, the gene order between human and zebrafish is extensively inverted and transposed. Thus, intrachromosomal rearrangements and inversions appear to have occurred more frequently than translocations during evolution from a common chordate ancestor. Interestingly, of the 33 orthologs located on LG14, three have duplicates on LG21, suggesting that the duplication event occurred early in the evolution of teleosts. Murine orthologs of human 5q CDR genes are distributed among three chromosomes, 18, 11, and 13. The order of genes within the three syntenic mouse chromosomes appears to be more colinear with the human order, suggesting that translocations occurred more frequently than inversions during mammalian evolution. Our comparative map should enhance understanding of the evolution of the del(5q) chromosomal region. Mutant fish harboring deletions affecting the 5q CDR syntenic region may provide useful animal models for investigating the pathogenesis of myelodysplastic syndrome and acute myeloid leukemia.

Emi1 Maintains Genomic Integrity during Zebrafish Embryogenesis and Cooperates with p53 in Tumor Suppression

ABSTRACT A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase IIα-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.

Muscle degeneration and leukocyte infiltration caused by mutation of zebrafish fad24

Factor for adipocyte differentiation 24 (fad24) is a novel gene that has been implicated in adipocyte differentiation and DNA replication. In a screen for zebrafish mutants that have an abnormal tissue distribution of neutrophils, we identified an insertional allele of fad24, fad24hi1019. Homozygous fad24hi1019 larvae exhibit muscle degeneration accompanied by leukocyte infiltration. Muscle degeneration was extensive and included tissue apoptosis and disorganized, poorly striated muscle fibers. Blocking apoptosis using pan‐caspase inhibitors resulted in decreased neutrophil recruitment into the body of the larva, suggesting a causative link between apoptosis and leukocyte infiltration. These findings suggest that zebrafish is a powerful genetic model system to address the interplay between muscle degeneration and leukocyte infiltration, and indicate that tissue apoptosis may contribute to neutrophil recruitment in some inflammatory states. Developmental Dynamics 238:86–99, 2009.

Ddx18 is essential for cell-cycle progression in zebrafish hematopoietic cells and is mutated in human AML.

In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18(hi1727/+) embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies.

A Pak1/Erk Signaling Module Acts through Gata6 to Regulate Cardiovascular Development in Zebrafish

Proper neural crest development and migration is critical during embryonic development, but the molecular mechanisms regulating this process remain incompletely understood. Here, we show that the protein kinase Erk, which plays a central role in a number of key developmental processes in vertebrates, is regulated in the developing neural crest by p21-activated protein kinase 1 (Pak1). Furthermore, we show that activated Erk signals by phosphorylating the transcription factor Gata6 on a conserved serine residue to promote neural crest migration and proper formation of craniofacial structures, pigment cells, and the outflow tract of the heart. Our data suggest an essential role for Pak1 as an Erk activator, and Gata6 as an Erk target, during neural crest development.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.