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1.  Use of PCR Template-Derived Probes Prevents Off-Target Whole Mount In Situ Hybridization in Transgenic Zebrafish 
Zebrafish  2012;9(2):85-89.
Abstract
Transgenic zebrafish have been utilized for in vivo analysis of cell behaviors using advanced imaging techniques, for analyzing spatiotemporal gene regulation, and for targeted mis-expression of transgenes. The Tg(fli1a:EGFP)y1 vascular reporter has been particularly useful for examining the development of blood and lymphatic vessels, but it has been suggested that whole-mount in situ hybridization may result high background staining in this line, potentially limiting its usefulness. Here, we show that off-target hybridization of plasmid vector-derived probes to tissues expressing transgenes occurs in a number of different commonly used transgenic lines as a result of multiple cloning site sequences present in the cloning vectors, suggesting this may be a more general problem. However, we also show that this problem is easily avoided by performing in situ hybridization using probes synthesized from PCR templates lacking vector sequences.
doi:10.1089/zeb.2011.0731
PMCID: PMC3388497  PMID: 22715949
2.  LYMPHATIC DEVELOPMENT 
The lymphatic system is essential for fluid homeostasis, immune responses, and fat absorption, and is involved in many pathological processes, including tumor metastasis and lymphedema. Despite its importance, progress in understanding the origins and early development of this system has been hampered by lack of defining molecular markers and difficulties in observing lymphatic cells in vivo and performing genetic and experimental manipulation of the lymphatic system. Recent identification of new molecular markers, new genes with important functional roles in lymphatic development, and new experimental models for studying lymphangiogenesis has begun to yield important insights into the emergence and assembly of this important tissue. This review focuses on the mechanisms regulating development of the lymphatic vasculature during embryogenesis.
doi:10.1002/bdrc.20155
PMCID: PMC2755610  PMID: 19750516
3.  reg6 is required for branching morphogenesis during blood vessel regeneration in zebrafish caudal fins 
Developmental biology  2003;264(1):263-274.
Postnatal neovascularization is essential for wound healing, cancer progression, and many other physiological functions. However, its genetic mechanism is largely unknown. In this report, we study neovascularization in regenerating adult zebrafish fins using transgenic fish that express EGFP in blood vessel endothelial cells. We first describe the morphogenesis of regenerating vessels in wild-type animals and then the phenotypic analysis of a genetic mutation that disrupts blood vessel regeneration. In wild-type zebrafish caudal fins, amputated blood vessels heal their ends by 24 h postamputation (hpa) and then reconnect arteries and veins via anastomosis, to resume blood flow at wound sites by 48 hpa. The truncated vessels regenerate by first growing excess vessels to form unstructured plexuses, resembling the primary capillary plexuses formed during embryonic vasculogenesis. Interestingly, this mode of vessel growth switches by 8 days postamputation (dpa) to growth without a plexus intermediate. During blood vessel regeneration, vessel remodeling begins during early plexus formation and continues until the original vasculature pattern is reestablished at ~35 dpa. Temperature-sensitive mutants for reg6 have profound defects in blood vessel regeneration. At the restrictive temperature, reg6 regenerating blood vessels first fail to make reconnections between severed arteries and veins, and then form enlarged vascular sinuses rather than branched vascular plexuses. Reciprocal temperature-shift experiments show that reg6 function is required throughout plexus formation, but not during later growth. Our results suggest that the reg6 mutation causes defects in branch formation and/or angiogenic sprouting.
PMCID: PMC3665419  PMID: 14623247
Zebrafish; Regeneration; Angiogenesis; Plexus; Branching; Anastomosis
4.  The Effect of Stocking Densities on Reproductive Performance in Laboratory Zebrafish (Danio rerio) 
Zebrafish  2011;8(3):141-146.
Abstract
Despite the growing popularity of the zebrafish model system, the optimal husbandry conditions for this animal are not well defined. The aim of this study was to examine the effect of stocking density on reproductive performance in zebrafish. In this study, undertaken by eight different zebrafish facilities, clutches of at least 200 wild-type zebrafish embryos from a single pairwise mating were produced at each participating institution and subsequently reared according to “in-house protocols” until they were 14 weeks old. Fish were then randomly assigned into treatment groups with balanced sex ratios and densities of 3, 6, or 12 fish/L. After a 1-month acclimation period, fish were spawned in pair crosses every 2 weeks for 3 months, for a total of six spawning dates. The number of viable and nonviable embryos produced in each clutch were counted at 1 day postfertilization. Although there was a great deal of variability in clutch size and percent spawning success among laboratories, there were no significant differences in average clutch size, spawning success, or percent viable among the treatment densities. These data suggest that using stocking densities as high as 12 fish/L does not have a negative impact on performance, when measured by reproductive performance.
doi:10.1089/zeb.2011.0688
PMCID: PMC3180766  PMID: 21854208
5.  Vascular Development in the Zebrafish 
The zebrafish has emerged as an excellent vertebrate model system for studying blood and lymphatic vascular development. The small size, external and rapid development, and optical transparency of zebrafish embryos are some of the advantages the zebrafish model system offers. Multiple well-established techniques have been developed for imaging and functionally manipulating vascular tissues in zebrafish embryos, expanding on and amplifying these basic advantages and accelerating use of this model system for studying vascular development. In the past decade, studies performed using zebrafish as a model system have provided many novel insights into vascular development. In this article we discuss the amenability of this model system for studying blood vessel development and review contributions made by this system to our understanding of vascular development.
Zebrafish embryos are small, optically transparent, and develop rapidly. They are an ideal vertebrate for studying blood and lymphatic vascular development.
doi:10.1101/cshperspect.a006684
PMCID: PMC3331685  PMID: 22553495
6.  Endothelial cells promote migration and proliferation of enteric neural crest cells via β1 integrin signaling 
Developmental biology  2009;330(2):263-272.
Enteric neural crest-derived cells (ENCCs) migrate along the intestine to form a highly organized network of ganglia that comprises the enteric nervous system (ENS). The signals driving the migration and patterning of these cells are largely unknown. Examining the spatiotemporal development of the intestinal neurovasculature in avian embryos, we find endothelial cells (ECs) present in the gut prior to the arrival of migrating ENCCs. These ECs are patterned in concentric rings that are predictive of the positioning of later arriving crest-derived cells, leading us to hypothesize that blood vessels may serve as a substrate to guide ENCC migration. Immunohistochemistry at multiple stages during ENS development reveals that ENCCs are positioned adjacent to vessels as they colonize the gut. A similar close anatomic relationship between vessels and enteric neurons was observed in zebrafish larvae. When EC development is inhibited in cultured avian intestine, ENCC migration is arrested and distal aganglionosis results, suggesting that ENCCs require the presence of vessels to colonize the gut. Neural tube and avian midgut were explanted onto a variety of substrates, including components of the extracellular matrix and various cell types, such as fibroblasts, smooth muscle cells, and endothelial cells. We find that crest-derived cells from both the neural tube and the midgut migrate avidly onto cultured endothelial cells. This EC-induced migration is inhibited by the presence of CSAT antibody, which blocks binding to β1 integrins expressed on the surface of crest-derived cells. These results demonstrate that ECs provide a substrate for the migration of ENCCs via an interaction between β1 integrins on the ENCC surface and extracellular matrix proteins expressed by the intestinal vasculature. These interactions may play an important role in guiding migration and patterning in the developing ENS.
doi:10.1016/j.ydbio.2009.03.025
PMCID: PMC2690696  PMID: 19345201
enteric nervous system; endothelial cells; blood vessels; Hirschsprung’s disease; integrins; avian; zebrafish
7.  Big fish in the genome era 
doi:10.1093/bfgp/eln057
PMCID: PMC2722260  PMID: 19109307
8.  Loss of unc45a precipitates arteriovenous shunting in the aortic arches 
Developmental biology  2008;318(2):258-267.
Aortic arch malformations are common congenital disorders that are frequently of unknown etiology. To gain insight into the factors that guide branchial aortic arch development, we examined the process by which these vessels assemble in wild type zebrafish embryos and in kurzschlusstr12 (kus tr12) mutants. In wild type embryos, each branchial aortic arch first appears as an island of angioblasts in the lateral pharyngeal mesoderm, then elaborates by angiogenesis to connect to the lateral dorsal aorta and ventral aorta. In kustr12 mutants, angioblast formation and initial sprouting are normal, but aortic arches 5 and 6 fail to form a lumenized connection to the lateral dorsal aorta. Blood enters these blind-ending vessels from the ventral aorta, distending the arteries and precipitating fusion with an adjacent vein. This arteriovenous malformation (AVM), which shunts nearly all blood directly back to the heart, is not genetically programmed, as its formation correlates with blood flow and aortic arch enlargement. By positional cloning, we have identified a nonsense mutation in unc45a in kustr12 mutants. Our results are the first to ascribe a role for Unc45a, a putative myosin chaperone, in vertebrate development, and identify a novel mechanism by which an AVM can form.
doi:10.1016/j.ydbio.2008.03.022
PMCID: PMC2483962  PMID: 18462713
aortic arches; pharyngeal arches; unc45a; zebrafish; arteriovenous malformation; kurzschluss
9.  Combinatorial interaction between CCM pathway genes precipitates hemorrhagic stroke 
Disease models & mechanisms  2008;1(4-5):275-281.
Intracerebral hemorrhage (ICH) is a particularly severe form of stroke whose etiology remains poorly understood, with a highly variable appearance and onset of the disease (Felbor et al., 2006; Frizzell, 2005; Lucas et al., 2003). In humans, mutations in any one of three “CCM” genes causes an autosomal dominant genetic ICH disorder characterized by Cerebral Cavernous Malformations. Recent evidence highlighting multiple interactions between the three CCM gene products and other proteins regulating endothelial junctional integrity suggests that minor deficits in these other proteins could potentially predispose to or help to initiate CCM, and that combinations of otherwise silent genetic deficits in CCM and interacting proteins might explain some of the variability in penetrance and expressivity of human ICH disorders. Here, we test this idea by combined knockdown of CCM pathway genes in zebrafish. Reducing the function of rap1b, a Ras GTPase effector protein for CCM1/KRIT1, disrupts endothelial junctions in vivo and in vitro, showing it is a critical player in the CCM pathway. Importantly, very minor reduction of Rap1b in combination with similar small reduction in other CCM pathway genes results in high incidence of ICH. These findings support the idea that minor polygenic deficits in the CCM pathway can strongly synergize to initiate ICH.
doi:10.1242/dmm.000513
PMCID: PMC2590810  PMID: 19093037
10.  Combinatorial function of ETS transcription factors in the developing vasculature 
Developmental biology  2006;303(2):772-783.
Members of the ETS family of transcription factors are among the first genes expressed in the developing vasculature, but loss-of-function experiments for individual ETS factors in mice have not uncovered important early functional roles for these genes. However, multiple ETS factors are expressed in spatially and temporally overlapping patterns in the developing vasculature, suggesting possible functional overlap. We have taken a comprehensive approach to exploring the function of these factors during vascular development by employing the genetic and experimental tools available in the zebrafish to analyze four ETS family members expressed together in the zebrafish vasculature; fli1, fli1b, ets1, and etsrp. We isolated and characterized an ENU-induced mutant with defects in trunk angiogenesis and positionally cloned the defective gene from this mutant, etsrp. Using the etsrp morpholinos targeting each of the four genes, we show that the four ETS factors function combinatorially during vascular and hematopoietic development. Reduction of etsrp or any of the other genes alone results in either partial or no defects in endothelial differentiation, while combined reduction in the function of all four genes causes dramatic loss of endothelial cells. Our results demonstrate that combinatorial ETS factor function is essential for early endothelial specification and differentiation.
doi:10.1016/j.ydbio.2006.10.030
PMCID: PMC1859867  PMID: 17125762
zebrafish; ETS transcription factors; intersegmental vessels; vascular development; angiogenesis
11.  Genetic determinants of hyaloid and retinal vasculature in zebrafish 
Background
The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish.
Results
We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO), subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development.
Conclusion
Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease.
doi:10.1186/1471-213X-7-114
PMCID: PMC2169232  PMID: 17937808
12.  Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos 
Molecular and Cellular Biology  2003;23(22):8295-8305.
Gata1 is a prototype transcription factor that regulates hematopoiesis, yet the molecular mechanisms by which Gata1 transactivates its target genes in vivo remain unclear. We previously showed, in transgenic zebra fish, that Gata1 autoregulates its own expression. In this study, we characterized the molecular mechanisms for this autoregulation by using mutations in the Gata1 protein which impair autoregulation. Of the tested mutations, replacement of six lysine residues with alanine (Gata1KA6), which inhibited self-association activity of Gata1, reduced the Gata1-dependent induction of reporter gene expression driven by the zebra fish gata1 hematopoietic regulatory domain (gata1 HRD). Furthermore, overexpression of wild-type Gata1 but not Gata1KA6 rescued the expression of Gata1 downstream genes in vlad tepes, a germ line gata1 mutant fish. Interestingly, both GATA sites in the double GATA motif in gata1 HRD were critical for the promoter activity and for binding of the self-associated Gata1 complex, whereas only the 3′-GATA site was required for Gata1 monomer binding. These results thus provide the first in vivo evidence that the ability of Gata1 to self-associate critically contributes to the autoregulation of the gata1 gene.
doi:10.1128/MCB.23.22.8295-8305.2003
PMCID: PMC262353  PMID: 14585986
13.  ApoB-containing lipoproteins regulate angiogenesis by modulating expression of VEGF receptor 1 
Nature medicine  2012;18(6):967-973.
Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.
doi:10.1038/nm.2759
PMCID: PMC3959651  PMID: 22581286
14.  Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line 
Background
Because of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals), supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans.
Results
In an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an egfp transgenic stable fish line that trapped tnks1bp1, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(tnks1bp1:EGFP) line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells.
Conclusions
We present a Tg(tnks1bp1:EGFP) stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general.
doi:10.1186/1471-213X-12-6
PMCID: PMC3305402  PMID: 22273551
Regeneration; hair cells; progenitor cells; lateral line; zebrafish; supporting cells; accessory cells; microarrays; Tnk1bp1

Results 1-14 (14)