Interactions In Zebrafish Research Articles

Chapter 4 - Study of Host–Microbe Interactions in Zebrafish

All animals are ecosystems, home to diverse microbial populations. Animal-associated microbes play important roles in the normal development and physiology of their hosts, but can also be agents of infectious disease. Traditionally, mice have been used to study pathogenic and beneficial associations between microbes and vertebrate animals. The zebrafish is emerging as a valuable new model system for host-microbe interaction studies, affording researchers with the opportunity to survey large populations of hosts and to visualize microbe-host associations at a cellular level in living animals. This chapter provides detailed protocols for the analysis of zebrafish-associated microbial communities, the derivation and husbandry of germ-free zebrafish, and the modeling of infectious disease in different stages of zebrafish development via different routes of inoculation. These protocols offer a starting point for researchers to address a multitude of questions about animals' coexistence with microorganisms.

Sleep–wake regulation and hypocretin–melatonin interaction in zebrafish

In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep-wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr-/-). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr-/- pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr-/- fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr-/- fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation.

Who Benefits from Granulomas, Mycobacteria or Host?

By investigating host-pathogen interactions in zebrafish using intravital imaging, Davis and Ramakrishnan (2009) provide evidence that aggregates of immune cells known as granulomas, long thought to constrain mycobacterial infection, may instead facilitate its spread.

Miles-apart-Mediated regulation of cell–fibronectin interaction and myocardial migration in zebrafish

The migration of myocardial precursor cells towards the embryonic midline underlies the formation of the heart tube and is a key process of heart organogenesis. The zebrafish mutation miles-apart (mil), which affects the gene encoding a sphingosine-1-phosphate receptor, is characterized by defective migration of myocardial precursor cells and results in the formation of two laterally positioned hearts, a condition known as cardia bifida. The mechanism that disrupts myocardial migration in mil mutants remains largely unclear. To investigate how mil regulates this process, here we analyze the interactions between mil and other mediators of myocardial migration. We show that mil function is associated with the other known cardia bifida locus, natter/fibronectin (nat/fn), which encodes fibronectin, a major component of the extracellular matrix, in the control of myocardial migration. By using a primary culture system of embryonic zebrafish cells, we also show that signaling from the sphingosine-1-phosphate receptor regulates cell-fibronectin interactions in zebrafish. In addition, localized inhibition and activation of cell-fibronectin interactions during the stages of myocardial migration reveal that the temporal regulation of cell-fibronectin interaction by mil is required for proper myocardial migration. Our study reveals novel functional links between sphingosine-1-phosphate receptor signaling and cell-fibronectin interaction in the control of myocardial migration during zebrafish heart organogenesis.

Cone Survival Despite Rod Degeneration in XOPS-mCFP Transgenic Zebrafish

In animal models of retinitis pigmentosa, rod photoreceptor degeneration eventually leads to loss of cone photoreceptors. The purpose of this study was to characterize a transgenic model of rod degeneration in zebrafish. Zebrafish transgenic for XOPS-mCFP, a membrane-targeted form of cyan fluorescent protein driven by the Xenopus rhodopsin promoter, were generated by plasmid injection. Immunohistochemistry was used to detect cell type, proliferation, and TUNEL markers in larval and adult retinas. Rod- and cone-specific transcripts were detected by RT-PCR. Visual responses in transgenic adults were measured by electroretinogram. The XOPS promoter directed specific expression of mCFP in rods by 55 hours post fertilization (hpf). Rods in XOPS-mCFP heterozygotes began dying at 3.5 days post fertilization (dpf) and were almost completely absent by 5 dpf. A few rods were observed at the retinal margin, and numerous immature rods were observed in the outer nuclear layer (ONL) of transgenic adults. Apoptosis was increased in the ONL of larval and adult transgenic animals, and an elevation of rod precursor proliferation in adults was observed. ERG analysis confirmed that rod responses were absent in this line. Cone morphology and electrophysiology appeared normal in transgenic animals up to 7 months of age. The XOPS-mCFP transgene causes selective degeneration of rods without secondary loss of cones in animals up to 7 months of age. This raises important questions about the significance of rod-cone interactions in zebrafish and their potential as a model of human inherited retinal degenerations.