Myofibrillogenesis in the developing zebrafish heart: A functional study of tnnt2

Various hypotheses have been proposed to explain the molecule processes of sarcomere assembly, partially due to the lack of systematic genetic studies of sarcomeric genes in an in vivo model. Towards the goal of developing zebrafish as a vertebrate model for this purpose, we characterized myofibrillogenesis in a developing zebrafish heart and went on to examine the functions of cardiac troponin T (tnnt2). We found that sarcomere assembly in zebrafish heart was initiated from a non-striated actin filament network at the perimembrane region, whereas sarcomeric myosin is independently assembled into thick filaments of variable length before integrating into the thin filament network. Compared to Z-discs that are initially aligned to form shorter periodic dots and expanded longitudinally at a later time, M-lines assemble later and have a constant length. Depletion of full-length tnnt2 disrupted the striation of thin filaments and Z-bodies, which sequentially affects the striation of thick filaments and M-lines. Conversely, truncation of a C-terminal troponin complex-binding domain did not affect the striation of these sarcomere sub-structures, but resulted in reduced cardiomyocyte size. In summary, our data indicates that zebrafish are a valuable in vivo model for studying both myofibrillogenesis and sarcomere-based cardiac diseases.     

Distinct signals from the microbiota promote different aspects of zebrafish gut differentiation

All animals exist in intimate associations with microorganisms that play important roles in the hosts' normal development and tissue physiology. In vertebrates, the most populous and complex community of microbes resides in the digestive tract. Here, we describe the establishment of the gut microbiota and its role in digestive tract differentiation in the zebrafish model vertebrate, Danio rerio. We find that in the absence of the microbiota, the gut epithelium is arrested in aspects of its differentiation, as revealed by the lack of brush border intestinal alkaline phosphatase activity, the maintenance of immature patterns of glycan expression and a paucity of goblet and enteroendocrine cells. In addition, germ-free intestines fail to take up protein macromolecules in the distal intestine and exhibit faster motility. Reintroduction of a complex microbiota at later stages of development or mono-association of germ-free larvae with individual constituents of the microbiota reverses all of these germ-free phenotypes. Exposure of germ-free zebrafish to heat-killed preparations of the microbiota or bacterial lipopolysaccharide is sufficient to restore alkaline phosphatase activity but not mature patterns of Gal alpha1,3Gal containing glycans, indicating that the host perceives and responds to its associated microbiota by at least two distinct pathways.     

Apoptosis in the developing zebrafish embryo.

Apoptosis is a major part of the normal development of many organ systems and tissues. The zebrafish (Danio rerio) has become a useful model for studying early development, and recent advances in techniques used to label apoptotic cells have made it possible to visualize apoptotic cells in this model system. We have used the in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) to describe the temporal and spatial distribution of apoptotic cells during normal development of the zebrafish embryo from 12 to 96 h postfertilization. By counting labeled apoptotic cells, we have demonstrated transient high rates of cell death in various structures during development, and we have correlated these peaks with previously described developmental changes in these structures. Our analysis has focused on the nervous system and associated sensory organs including the olfactory organ, retina, lens, cornea, otic vesicle, lateral line organs, and Rohon-Beard neurons. Apoptosis is also described in other non-neural structures such as the notochord, somites, muscle, tailbud, and fins.     

Evidence for a core gut microbiota in the zebrafish

Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status.     

Heart position in snakes: response to "phylogeny, ecology, and heart position in snakes"

Here we comment on a recent article (Gartner et al. 2010 ) that addresses previous adaptive interpretations of heart position in the context of gravity effects on blood circulation of snakes. The authors used phylogenetically based statistical methods and concluded that both habitat and phylogeny influence heart position, which they contend is relatively more posterior in arboreal compared to terrestrial species. Their result is based on measurements of heart position relative to snout-vent length, rather than total body length as in previous studies. However, gravity acts on the total length of the arterial-venous vasculature, and caudal segments of continuous blood columns cannot be ignored. Arboreal snakes have relatively long tails; therefore anterior hearts appear to be more "posterior" when the position is described relative to a shorter trunk. There is no physiologically valid explanation for the alleged posterior heart position in arboreal snakes, and multiple lines of published evidence to the contrary are ignored. The authors secondarily evaluated their data set with estimates for total body length based on measurements from other taxa. They found no statistical difference between heart position in arboreal versus terrestrial species, yet their article implied otherwise. Gartner et al. ( 2010 ) contrasted "aquatic" and terrestrial species throughout their paper, and they claimed there is no correlation between heart position and habitat among "aquatic and terrestrial species." But they did not include any aquatic species in their data set. Therefore, the article confuses rather than promotes understanding of cardiovascular adaptation to gravity.     

Right heart structural changes are independently associated with exercise capacity in non-severe COPD

Background

Pulmonary hypertension (PH) occurs frequently and results in functional limitation in advanced COPD. Data regarding the functional consequence of PH in less severe COPD are limited. Whether echocardiographic evidence of right sided heart pathology is associated with functional outcomes in patients with non-severe COPD is unknown.

Methods

We evaluated pulmonary function, six minute walk distance, and echocardiography in 74 consecutive patients with non-severe COPD. We performed multivariable linear regression to evaluate the association between right heart echocardiographic parameters and six minute walk distance adjusting for lung function, age, sex, race, and BMI.

Main Results

The mean six minute walk distance was 324±106 meters. All subjects had preserved left ventricular (LV) systolic function (LV ejection fraction 62.3%±6.1%). 54.1% had evidence of some degree of diastolic dysfunction. 17.6% of subjects had evidence of right ventricular enlargement and 36.5% had right atrial enlargement. In univariate analysis RV wall thickness (β = −68.6; p = 0.002), log right atrial area (β = −297.9; p = 0.004), LV mass index (β = −1.3; p = 0.03), E/E'' ratio (β = −5.5; p = 0.02), and degree of diastolic dysfunction (β = −42.8; p = 0.006) were associated with six minute walk distance. After adjustment for co-variables, the associations between right atrial area (log right atrial area β = −349.8; p = 0.003) and right ventricular wall thickness (β = −43.8; p = 0.04) with lower six minute walk distance remained significant independent of forced expiratory volume in one second (FEV1). LV mass index, E/E'' ratio, and degree of diastolic dysfunction were not independent predictors of six minute walk distance.

Conclusion

In patients with non-severe COPD right sided cardiac structural changes are associated with lower six minute walk distance independent of lung function. These findings may indicate that echocardiographic evidence of pulmonary hypertension is present in patients with non-severe COPD and has important functional consequences.     

Unique and conserved aspects of gut development in zebrafish

Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences account for the finding that the zebrafish digestive system is assembled from individual organ anlagen, whereas the digestive anlagen of amniotes arise from a primitive gut tube. Despite differences of organ morphogenesis, conserved molecular programs regulate pharynx, esophagus, liver, and pancreas development in teleosts and mammals. Specifically, we show that zebrafish faust/gata-5 is a functional ortholog of gata-4, a gene that is essential for the formation of the mammalian and avian foregut. Further, extraembryonic gata activity is required for this function in zebrafish as has been shown in other vertebrates. We also show that a loss-of-function mutation that perturbs sonic hedgehog causes defects in the development of the esophagus that parallel those associated with targeted disruption of this gene in mammals. Perturbation of sonic hedgehog also affects zebrafish liver and pancreas development, and these effects occur in a reciprocal fashion, as has been described during mammalian liver and ventral pancreas development. Together, these data define aspects of digestive system development necessary for the characterization of zebrafish mutants. Given the similarities of teleost and mammalian digestive physiology and anatomy, these findings have implications for developmental and evolutionary studies as well as research of human diseases, such as diabetes, liver cirrhosis, and cancer.     

Small heat shock proteins are necessary for heart migration and laterality determination in zebrafish

Small heat shock proteins (sHsps) regulate cellular functions not only under stress, but also during normal development, when they are expressed in organ-specific patterns. Here we demonstrate that two small heat shock proteins expressed in embryonic zebrafish heart, hspb7 and hspb12, have roles in the development of left–right asymmetry. In zebrafish, laterality is determined by the motility of cilia in Kupffer's vesicle (KV), where hspb7 is expressed; knockdown of hspb7 causes laterality defects by disrupting the motility of these cilia. In embryos with reduced hspb7, the axonemes of KV cilia have a 9+0 structure, while control embyros have a predominately 9+2 structure. Reduction of either hspb7 or hspb12 alters the expression pattern of genes that propagate the signals that establish left–right asymmetry: the nodal-related gene southpaw (spaw) in the lateral plate mesoderm, and its downstream targets pitx2, lefty1 and lefty2. Partial depletion of hspb7 causes concordant heart, brain and visceral laterality defects, indicating that loss of KV cilia motility leads to coordinated but randomized laterality. Reducing hspb12 leads to similar alterations in the expression of downstream laterality genes, but at a lower penetrance. Simultaneous reduction of hspb7 and hspb12 randomizes heart, brain and visceral laterality, suggesting that these two genes have partially redundant functions in the establishment of left–right asymmetry. In addition, both hspb7 and hspb12 are expressed in the precardiac mesoderm and in the yolk syncytial layer, which supports the migration and fusion of mesodermal cardiac precursors. In embryos in which the reduction of hspb7 or hspb12 was limited to the yolk, migration defects predominated, suggesting that the yolk expression of these genes rather than heart expression is responsible for the migration defects.     

The nucleolar GTP-binding proteins Gnl2 and nucleostemin are required for retinal neurogenesis in developing zebrafish

Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells such as stem and cancer cells and is involved in the control of cell cycle progression. Both depletion and overexpression of NS result in stabilization of the tumor suppressor p53 protein in vitro. Although it has been previously suggested that NS has p53-independent functions, these to date remain unknown. Here, we report two zebrafish mutants recovered from forward and reverse genetic screens that carry loss of function mutations in two members of this nucleolar protein family, Guanine nucleotide binding-protein-like 2 (Gnl2) and Gnl3/NS. We demonstrate that these proteins are required for correct timing of cell cycle exit and subsequent neural differentiation in the brain and retina. Concomitantly, we observe aberrant expression of the cell cycle regulators cyclinD1 and p57kip2. Our models demonstrate that the loss of Gnl2 or NS induces p53 stabilization and p53-mediated apoptosis. However, the retinal differentiation defects are independent of p53 activation. Furthermore, this work demonstrates that Gnl2 and NS have both non-cell autonomously and cell-autonomous function in correct timing of cell cycle exit and neural differentiation. Finally, the data suggest that Gnl2 and NS affect cell cycle exit of neural progenitors by regulating the expression of cell cycle regulators independently of p53.     

Genetic control of single lumen formation in the zebrafish gut

Most organs consist of networks of interconnected tubes that serve as conduits to transport fluid and cells and act as physiological barriers between compartments. Biological tubes are assembled through very diverse developmental processes that generate structures of different shapes and sizes. Nevertheless, all biological tubes invariably possess one single lumen. The mechanisms responsible for single lumen specification are not known. Here we show that zebrafish mutants for the MODY5 and familial GCKD gene tcf2 (also known as vhnf1) fail to specify a single lumen in their gut tube and instead develop multiple lumens. We show that Tcf2 controls single lumen formation by regulating claudin15 and Na+/K+-ATPase expression. Our in vivo and in vitro results indicate that Claudin15 functions in paracellular ion transport to specify single lumen formation. This work shows that single lumen formation is genetically controlled and appears to be driven by the accumulation of fluid.