SUMMARYAdult zebrafish possess a significant ability to regenerate injured heart tissue through proliferation of pre-existing cardiomyocytes, which contrasts with the inability of mammals to do so after the immediate postnatal period. Zebrafish therefore provide a model system in which to study how an injured heart can be repaired. However, it remains unknown what important processes cardiomyocytes are involved in other than partial de-differentiation and proliferation. Here we show that migration of cardiomyocytes to the injury site is essential for heart regeneration. Ventricular amputation induced expression of cxcl12a and cxcr4b, genes encoding a chemokine ligand and its receptor. We found that cxcl12a was expressed in the epicardial tissue and that Cxcr4 was expressed in cardiomyocytes. We show that pharmacological blocking of Cxcr4 function as well as genetic loss of cxcr4b function causes failure to regenerate the heart after ventricular resection. Cardiomyocyte proliferation was not affected but a large portion of proliferating cardiomyocytes remained localized outside the injury site. A photoconvertible fluorescent reporter-based cardiomyocyte-tracing assay demonstrates that cardiomyocytes migrated into the injury site in control hearts but that migration was inhibited in the Cxcr4-blocked hearts. By contrast, the epicardial cells and vascular endothelial cells were not affected by blocking Cxcr4 function. Our data show that the migration of cardiomyocytes into the injury site is regulated independently of proliferation, and that coordination of both processes is necessary for heart regeneration.

We report the development of a modular and optimized thin-sheet laser imaging microscope (TSLIM) for nondestructive optical sectioning of organisms and thick tissues such as the mouse cochlea, zebrafish brain/inner ear, and rat brain at a resolution that is comparable to wide-field fluorescence microscopy. TSLIM optically sections tissue using a thin sheet of light by inducing a plane of fluorescence in transparent or fixed and cleared tissues. Moving the specimen through the thinnest portion of the light sheet and stitching these image columns together results in optimal resolution and focus across the width of a large specimen. Dual light sheets and aberration-corrected objectives provide uniform section illumination and reduce absorption artifacts that are common in light-sheet microscopy. Construction details are provided for duplication of a TSLIM device by other investigators in order to encourage further use and development of this important technology.

BackgroundThe zebrafish is an increasingly popular model for studying many aspects of biology. Recently, ztert, the zebrafish homolog of the mammalian telomerase gene has been cloned and sequenced. In contrast to humans, it has been shown that the zebrafish maintains telomerase activity for much of its adult life and has remarkable regenerative capacity. To date, there has been no longitudinal study to assess whether this retention of telomerase activity equates to the retention of chromosome telomere length through adulthood.Methodology/Principal FindingsWe have systematically analyzed individual organs of zebrafish with regard to both telomere length and telomerase activity at various time points in its adult life. Heart, gills, kidney, spleen, liver, and intestine were evaluated at 3 months, 6 months, 9 months, and 2 years of age by Southern blot analysis. We found that telomeres do not appreciably shorten throughout the lifespan of the zebrafish in any organ. In addition, there was little difference in telomere lengths between organs. Even when cells were under the highest pressure to divide after fin-clipping experiments, telomere length was unaffected. All aged (2 year old) tissues examined also expressed active amounts of telomerase activity as assessed by TRAP assay.Conclusions/SignificanceIn contrast to several other species including humans, the retention of lifelong telomerase and telomeres, as we have reported here, would be necessary in the zebrafish to maintain its tremendous regenerative capacity. The ongoing study of the zebrafish's ability to maintain telomerase activity may be helpful in unraveling the complexity involved in the maintenance (or lack thereof) of telomeres in other species such the mouse or human.

In mammals, stromal cell-derived factor-1 (SDF-1) promotes hematopoietic cell mobilization and migration. Although the zebrafish, Danio rerio, is an emerging model for studying hematopoietic cell transplantation (HCT), the role of SDF-1 in the adult zebrafish has yet to be determined. We sought to characterize sdf-1 expression and function in the adult zebrafish in the context of HCT. In situ hybridization of adult zebrafish organs shows sdf-1 expression in kidney tubules, gills, and skin. Radiation up-regulates sdf-1 expression in kidney to nearly 4-fold after 40 Gy. Assays indicate that zebrafish hematopoietic cells migrate toward sdf-1, with a migration ratio approaching 1.5 in vitro. A sdf-1a:DsRed2 transgenic zebrafish allows in vivo detection of sdf-1a expression in the adult zebrafish. Matings with transgenic reporters localized sdf-1a expression to the putative hematopoietic cell niche in proximal and distal renal tubules and collecting ducts. Importantly, transplant of hematopoietic cells into myelosuppressed recipients indicated migration of hematopoietic cells to sdf-1a-expressing sites in the kidney and skin. We conclude that sdf-1 expression and function in the adult zebrafish have important similarities to mammals, and this sdf-1 transgenic vertebrate will be useful in characterizing the hematopoietic cell niche and its interactions with hematopoietic cells. (Blood. 2011;118(3):766-774) IntroductionThe zebrafish, Danio rerio, is emerging as a useful model organism for the study of hematopoietic cell transplantation (HCT). 1,2 Its fecundity makes high experimental sample numbers logistically feasible, it is amenable to high-throughput chemical screens for hematopoietic effects of drugs and small molecules, 3 and its optical transparency offers excellent opportunities for direct visualization of cell migration events and in vivo fluorescence detection of donor-derived hematopoietic reconstitution. The true extent to which the zebrafish will prove useful for experimental HCT studies, however, will ultimately depend on the degree to which the mechanisms that result in HCT success in mammals are conserved in the zebrafish.In mammals, the chemokine stromal cell-derived factor-1 (SDF-1; CXCL12) and its cognate receptor CXCR4 have been strongly implicated in the homing and reconstitution that that occurs during HCT. 4-6 SDF-1 is expressed in many organs throughout the body, including the spleen, thymus, skin, heart, lung, kidney, and bone marrow. 7,8 The widespread anatomic distribution of SDF-1 expression has been shown to play roles in diverse migration and retention events of a wide array of hematopoietic cells, including migration of mast cell precursors in the skin, 9 retention of myeloid cells in extramedullary locations, 10 migration of megakaryocytes in the bone marrow after radiation, 11 migration of hematopoietic stem cells (HSCs) to regions of injury, 12 and HSC migration to bone marrow both during homeostasis as well as during HCT. 13,14 In the bone marrow cavity, cells expressing high levels o...

Down syndrome is frequently associated with complex difficulties in oromotor development, feeding, and swallowing. However, the muscle phenotypes underlying these deficits are unclear. We tested the hypotheses that the Ts65Dn mouse model of DS has significantly altered myosin heavy chain (MyHC) isoform profiles of the muscles involved in feeding and swallowing, as well as reductions in the speed of these movements during behavioral assays. SDS-PAGE, immunofluorescence, and qRT-PCR were used to assess MyHC isoform expression in pertinent muscles, and functional feeding and swallowing performance were quantified through videofluoroscopy and mastication assays. We found that both the anterior digastric (ADG) and posterior digastric (PDG) muscles in 11-day old and 5–6 week old Ts65Dn groups showed significantly lower MyHC 2b protein levels than in age-matched euploid control groups. In videofluoroscopic and videotape assays used to quantify swallowing and mastication performance, 5–6 week old Ts65Dn and euploid controls showed similar swallow rates, inter-swallow intervals, and mastication rates. In analysis of adults, 10–11 week old Ts65Dn mice revealed significantly less MyHC 2b mRNA expression in the posterior digastric, but not the anterior digastric muscle as compared with euploid controls. Analysis of MyHC 2b protein levels across an adult age range (10–53 weeks of age) revealed lower levels of MyHC 2b protein in the PDG of Ts65Dn than in euploids, but similar levels of MyHC 2b in the ADG. Cumulatively, these results indicate biochemical differences in some, but not all, muscles involved in swallowing and jaw movement in Ts65Dn mice that manifest early in post-natal development, and persist into adulthood. These findings suggest potential utility of this model for future investigations of the mechanisms of oromotor difficulties associated with Down syndrome.