[Show abstract][Hide abstract] ABSTRACT: Angiopoietin-like proteins (angptls) are capable of ex vivo expansion of mouse and human hematopoietic stem and progenitor cells (HSPCs). Despite this intriguing ability, their mechanism is unknown. In this study, we show that angptl2 overexpression is sufficient to expand definitive HSPCs in zebrafish embryos. Angptl1/2 are required for definitive hematopoiesis and vascular specification of the hemogenic endothelium. The loss-of-function phenotype is reminiscent of the notch mutant mindbomb (mib), and a strong genetic interaction occurs between angptls and notch. Overexpressing angptl2 rescues mib while overexpressing notch rescues angptl1/2 morphants. Gene expression studies in ANGPTL2-stimulated CD34+ cells showed a strong MYC activation signature and myc overexpression in angptl1/2 morphants or mib restored HSPCs formation. ANGPTL2 can increase NOTCH activation in cultured cells and ANGPTL receptor interacted with NOTCH to regulate NOTCH cleavage. Together our data provide insight to the angptl-mediated notch activation through receptor interaction and subsequent activation of myc targets.
[Show abstract][Hide abstract] ABSTRACT: Cranial neural crest (CNC) cells are patterned and coalesce to facial prominences that undergo convergence and extension to generate the craniofacial form. We applied a chemical genetics approach to identify pathways that regulate craniofacial development during embryogenesis. Treatment with the nitric oxide synthase inhibitor 1-(2-[trifluoromethyl] phenyl) imidazole (TRIM) abrogated first pharyngeal arch structures and induced ectopic ceratobranchial formation. TRIM promoted a progenitor CNC fate and inhibited chondrogenic differentiation, which were mediated through impaired nitric oxide (NO) production without appreciable effect on global protein S-nitrosylation. Instead, TRIM perturbed hox gene patterning and caused histone hypoacetylation. Rescue of TRIM phenotype was achieved with overexpression of histone acetyltransferase kat6a, inhibition of histone deacetylase, and complementary NO. These studies demonstrate that NO signaling and histone acetylation are coordinated mechanisms that regulate CNC patterning, differentiation, and convergence during craniofacial morphogenesis.
No preview · Article · Mar 2014 · Chemistry & biology
[Show abstract][Hide abstract] ABSTRACT: The need for scalable strategies to probe the biological consequences of candidate cancer genes has never been more pressing. The zebrafish, with its capacity for high-throughput transgenesis, in vivo imaging and chemical/genetic screening, has ideal features for undertaking this task. Unique biological insights from zebrafish have already led to the identification of novel oncogenic drivers and small molecules being used to treat the human cancer. This review summarizes the recent main findings and describes pertinent areas where the zebrafish can greatly contribute to our understanding of cancer biology and treatment.
Full-text · Article · Dec 2013 · Current opinion in genetics & development
[Show abstract][Hide abstract] ABSTRACT: This article is about resettled Afghan Hazaras in Australia, many of whom are currently undergoing a complex process of transition (from transience into a more stable position) for the first time in their lives. Despite their permanent residency status, we show how resettlement can
be a challenging transitional experience. For these new migrants, we argue that developing a sense of belonging during the transition period is a critical rite of passage in the context of their political and cultural identity. A study of forced migrants such as these, moving out of one transient
experience into another transitional period (albeit one that holds greater promise and permanence) poses a unique intellectual challenge. New understandings about the ongoing, unpredictable consequences of ‘transience’ for refugee communities is crucial as we discover what might
be necessary, as social support structures, to facilitate the process of transition into a distinctly new environment. The article is based on a doctoral ethnographic study of 30 resettled Afghan Hazara living in the region of Dandenong in Melbourne, Australia. Here, we include four of these
participants’ reflections of transition during different phases of their resettlement. These reflections were particularly revealing of the ways in which some migrants deal with change and acquire a sense of belonging to the community. Taking a historical view, and drawing on Bourdieu’s
notion of symbolic social capital to highlight themes in individual experiences of belonging, we show how some new migrants adjust and learn to ‘embody’ their place in the new country. Symbolic social capital illuminates how people access and use resources such as social networks
as tools of empowerment, reflecting how Hazara post-arrival experiences are tied to complex power relations in their everyday social interactions and in their life trajectories as people in transition. We learned that such tools can facilitate the formation of Hazara migrant identities and
are closely tied to their civic community participation, English language development, and orientation in, as well as comprehension of local cultural knowledge and place. This kind of theorization allows refugee, post-refugee and recent migrant narratives to be viewed not merely as static
expressions of loss, trauma or damage, but rather as individual experiences of survival, adaptation and upward mobility.
Full-text · Article · Nov 2013 · Stem Cell Reports
[Show abstract][Hide abstract] ABSTRACT: Melanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process.
To decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAFV600E or NRASQ61K driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C > T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAFV600E and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway.
This is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation.
[Show abstract][Hide abstract] ABSTRACT: Rhabdomyosarcoma is a pediatric malignancy thought to arise from the uncontrolled proliferation of myogenic cells. Here, we have generated models of rhabdomyosarcoma in the zebrafish by inducing oncogenic KRAS(G12D) expression at different stages during muscle development. Several zebrafish promoters were used, including the cdh15 and rag2 promoters, which drive gene expression in early muscle progenitors, and the mylz2 promoter, which is expressed in differentiating myoblasts. The tumors that developed differed in their ability to recapitulate normal myogenesis. cdh15:KRAS(G12D) and rag2:KRAS(G12D) fish developed tumors that displayed an inability to complete muscle differentiation as determined by histological appearance and gene expression analyses. By contrast, mylz2:KRAS(G12D) tumors more closely resembled mature skeletal muscle and were most similar to well-differentiated human rhabdomyosarcoma in terms of gene expression. mylz2:KRAS(G12D) fish showed significantly improved survival compared with cdh15:KRAS(G12D) and rag2:KRAS(G12D) fish. Tumor-propagating activity was enriched in myf5-expressing cell populations within all of the tumor types. Our results demonstrate that oncogenic KRAS(G12D) expression at different stages during muscle development has profound effects on the ability of tumor cells to recapitulate normal myogenesis, altering the tumorigenic capability of these cells.
[Show abstract][Hide abstract] ABSTRACT: Phenotype-driven chemical genetic screens in zebrafish have become a proven approach for both dissection of developmental mechanisms and discovery of potential therapeutics. A library of small molecules can be arrayed into multiwell plates containing zebrafish embryos. The embryo becomes a whole organism in vivo bioassay that can produce a phenotype upon treatment. Screens have been performed that are based simply on the morphology of the embryo. Other screens have scored complex phenotypes using whole mount in situ hybridization, fluorescent transgenic reporters, and even tracking of embryo movement. The availability of many well-characterized zebrafish mutants has also enabled the discovery of chemical suppressors of genetic phenotypes. Importantly, the application of chemical libraries that already contain FDA-approved drugs has allowed the rapid translation of hits from zebrafish chemical screens to clinical trials. WIREs Dev Biol 2012, 1:459-468. doi: 10.1002/wdev.37 For further resources related to this article, please visit the WIREs website.
No preview · Article · May 2012 · Wiley Interdisciplinary Reviews: Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: Adult zebrafish are being increasingly used as a model in cancer and stem cell research. Here we describe an integrated optical system that combines a laser scanning confocal microscope (LSCM) and an in vivo flow cytometer (IVFC) for simultaneous visualization and cell quantification. The system is set up specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish (casper) that have been engineered to be optically transparent. Confocal imaging in this instrument serves the dual purpose of visualizing fish tissue microstructure and an imaging-based guide to locate a suitable vessel for quantitative analysis of circulating cells by IVFC. We demonstrate initial testing of this novel instrument by imaging the transparent adult zebrafish casper vasculature and tracking circulating cells in CD41-GFP/Gata1-DsRed transgenic fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. In vivo measurements allow cells to be tracked under physiological conditions in the same fish over time, without drawing blood samples or sacrificing animals. We also discuss the potential applications of this instrument in biomedical research.
[Show abstract][Hide abstract] ABSTRACT: Zebrafish have become a powerful vertebrate model organism for drug
discovery, cancer and stem cell research. A recently developed
transparent adult zebrafish using double pigmentation mutant, called
casper, provide unparalleled imaging power in in vivo longitudinal
analysis of biological processes at an anatomic resolution not readily
achievable in murine or other systems. In this paper we introduce an
optical method for simultaneous visualization and cell quantification,
which combines the laser scanning confocal microscopy (LSCM) and the in
vivo flow cytometry (IVFC). The system is designed specifically for
non-invasive tracking of both stationary and circulating cells in adult
zebrafish casper, under physiological conditions in the same fish over
time. The confocal imaging part in this system serves the dual purposes
of imaging fish tissue microstructure and a 3D navigation tool to locate
a suitable vessel for circulating cell counting. The multi-color,
multi-channel instrument allows the detection of multiple cell
populations or different tissues or organs simultaneously. We
demonstrate initial testing of this novel instrument by imaging
vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed
transgenic casper fish whose thrombocytes/erythrocytes express the green
and red fluorescent proteins. Circulating fluorescent cell incidents
were recorded and counted repeatedly over time and in different types of
vessels. Great application opportunities in cancer and stem cell
researches are discussed.
No preview · Article · Feb 2012 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: Tissue or cell transplantation has been an extremely valuable technique for studying developmental potential of certain cell population, dissecting cell-environment interaction relationship, identifying stem cells, and many other applications. One key technical requirement for performing transplantation assay is the capability of distinguishing the transplanted donor cells from the endogenous host cells, and tracing the donor cells over time. Zebrafish has emerged as an excellent model organism for performing transplantation assay, thanks to the transparency of embryos during development and even certain adults. Using transgenic techniques and fast-evolving imaging technology, fluorescence-labeled donor cells can be easily identified and studied in vivo. In this chapter, we will first discuss the rationale of different types of zebrafish transplantation in both embryos and adults, and then focus on detailed methods of three types of transplantation: blastula/gastrula transplantation for mosaic analysis, stem cell transplantation, and tumor transplantation.
No preview · Article · Dec 2011 · Methods in cell biology
[Show abstract][Hide abstract] ABSTRACT: Disruption of ribosomal proteins is associated with hematopoietic phenotypes in cell culture and animal models. Mutations in ribosomal proteins are seen in patients with Diamond Blackfan anemia, a rare congenital disease characterized by red cell aplasia and distinctive craniofacial anomalies. A zebrafish screen uncovered decreased hematopoietic stem cells in embryos with mutations in ribosomal protein rps29. Here, we determined that rps29(-/-) embryos also have red blood cell defects and increased apoptosis in the head. As the p53 pathway has been shown to play a role in other ribosomal protein mutants, we studied the genetic relationship of rps29 and p53. Transcriptional profiling revealed that genes upregulated in the rps29 mutant are enriched for genes upregulated by p53 after irradiation. p53 mutation near completely rescues the rps29 morphological and hematopoietic phenotypes, demonstrating that p53 mediates the effects of rps29 knockdown. We also identified neuronal gene orthopedia protein a (otpa) as one whose expression correlates with rps29 expression, suggesting that levels of expression of some genes are dependent on rps29 levels. Together, our studies demonstrate a role of p53 in mediating the cellular defects associated with rps29 and establish a role for rps29 and p53 in hematopoietic stem cells and red blood cell development.
Preview · Article · Nov 2011 · Experimental hematology
[Show abstract][Hide abstract] ABSTRACT: The molecular events underlying the progression of T-lymphoblastic lymphoma (T-LBL) to acute T-lymphoblastic leukemia (T-ALL) remain elusive. In our zebrafish model, concomitant overexpression of bcl-2 with Myc accelerated T-LBL onset while inhibiting progression to T-ALL. The T-LBL cells failed to invade the vasculature and showed evidence of increased homotypic cell-cell adhesion and autophagy. Further analysis using clinical biopsy specimens revealed autophagy and increased levels of BCL2, S1P1, and ICAM1 in human T-LBL compared with T-ALL. Inhibition of S1P1 signaling in T-LBL cells led to decreased homotypic adhesion in vitro and increased tumor cell intravasation in vivo. Thus, blockade of intravasation and hematologic dissemination in T-LBL is due to elevated S1P1 signaling, increased expression of ICAM1, and augmented homotypic cell-cell adhesion.
[Show abstract][Hide abstract] ABSTRACT: Chemical genetic screening can be described as a discovery approach in which chemicals are assayed for their effects on a defined biological system. The zebrafish, Danio rerio, is a well-characterized and genetically tractable vertebrate model organism that produces large numbers of rapidly developing embryos that develop externally. These characteristics allow for flexible, rapid and scalable chemical screen design using the zebrafish. We describe a protocol for screening compounds from a chemical library for effects on early zebrafish development using an automated in situ based read-out. As screenings are carried out in the context of a complete, developing organism, this approach allows for a more comprehensive analysis of the range of a chemical's effects than that provided by, for example, a cell culture-based or in vitro biochemical assay. Using a 24-h chemical treatment, one can complete a round of screening in 6 d.
[Show abstract][Hide abstract] ABSTRACT: Advantageous organismal and technical attributes of the zebrafish are being increasingly applied to study cancer biology. Along with other tumor models, zebrafish that develop melanomas have been generated. In both genetics and phenotype, zebrafish melanomas are strikingly similar to their human counterparts. For this reason, studies in the zebrafish are poised to make significant contributions to melanoma biology. In this review, we summarize important features of human melanoma and discuss how the zebrafish can be used to address many questions that remain unanswered about this devastating disease.