We have generated a line of transgenic zebrafish, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of the dopamine transporter (dat) gene. In Tg(dat:EGFP) fish, dopamine (DA) neurons are labeled with GFP, including those in ventral diencephalon (vDC) clusters, amacrine cells in the retina, in the olfactory bulb, in the pretectum, and in the caudal hypothalamus. In the vDC, DA neurons of groups 2-6 are correctly labeled with GFP, based on colocalization analyses. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) treatments induced a modest but significant loss of DA neurons in groups 2-6 of the vDC. This transgenic line will be useful for the study of DA neuron development and in models of DA neuron loss.

To determine the impact of a controlled loss of dopaminergic neurons on locomotor function, we generated transgenic zebrafish, Tg(dat:CFP-NTR), expressing a cyan fluorescent protein-nitroreductase fusion protein (CFP-NTR) under the control of dopamine transporter (dat) cis-regulatory elements. Embryonic and larval zebrafish express the transgene in several groups of dopaminergic neurons, notably in the olfactory bulb, telencephalon, diencephalon and caudal hypothalamus. Administration of the pro-drug metronidazole (Mtz) resulted in activation of caspase 3 in CFP-positive neurons and in a reduction in dat-positive cells by 5 days postfertilization (dpf). Loss of neurons coincided with impairments in global locomotor parameters such as swimming distance, percentage of time spent moving, as well as changes in tail bend parameters such as time to maximal bend and angular velocity. Dopamine levels were transiently decreased following Mtz administration. Recovery of some of the locomotor parameters was observed by 7 dpf. However, the total numbers of dat-expressing neurons were still decreased at 7, 12, or 14 dpf, even though there was evidence for production of new dat-expressing cells. Tg(dat:CFP-NTR) zebrafish provide a model to correlate altered dopaminergic neuron numbers with locomotor function and to investigate factors influencing regeneration of dopaminergic neurons. Behavioral changes and neurochemical deficiencies have been previously described in the zebrafish following administration of neurotoxins such as 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which target the catecholaminergic neuron populations. Although dopaminergic neurons have been shown to regenerate in both the planarian (Nishimura et al. 2011) and salamander (Parish et al. 2007) neurotoxin-based models, this has not been demonstrated in the zebrafish brain. This lack of information may be attributed, in part, to the fact that although 6-hydroxydopamine and MPTP induce both neurochemical and behavioral changes, no dopaminergic neuron loss and cell death are detected in zebrafish (Anichtchik et al. 2004). Indeed, in larval zebrafish, MPTP treatments results in a short-lived reduction in tyrosine hydroxylase (TH) immunoreactivity at 5 and 6 dpf which is recovered by 7 dpf and does not lead to signs of apoptosis or cell death.To address the events that take place following DA neuron loss and to determine whether the ablated neuronal population can be re-established in the brain of larval zebrafish, we have Received March 25, 2015; revised manuscript received May 20, 2015; accepted May 26, 2015. Address correspondence and reprint requests to Dr Marc Ekker, Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa Ottawa, K1N-6N5, Canada. E-mail: mekker@uottawa.ca Abbreviations used: BrdU, 5-bromo-2 0 -deoxyuridine; CFP-NTR, cyan fluorescent protein-nitroreductase fusion protein; EGFP, -enhanced green fluorescent protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydro...

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The fish gill is a multifunctional organ involved in numerous physiological processes, such as gas exchange and sensing of hypoxia by respiratory chemoreceptors, called neuroepithelial cells (NECs). Many studies have focused on zebrafish (Danio rerio) to investigate the structure, function and development of the gills, yet the transcriptomic profile of most gill cells remains obscure. We present the results of a comprehensive transcriptomic analysis of the gills of zebrafish using single-cell RNA sequencing (scRNA‐seq). Gill cells from ETvmat2:EGFP zebrafish were individually labelled before scRNA‐seq library construction using 10× Genomics Chromium technology. 12,819 cells were sequenced with an average depth of over 27,000 reads per cell. We identified a median of 485 genes per cell and 16 cell clusters, including NECs, neurons, pavement cells, endothelial cells and mitochondrion-rich cells. The identity of NECs was confirmed by expression of slc18a2, encoding the vesicular monoamine transporter, Vmat2. Highly differentially-expressed genes in NECs included tph1a, encoding tryptophan hydroxylase, sv2 (synaptic vesicle protein), and proteins implicated in O2 sensing (ndufa4l2a, cox8al and epas1a). In addition, NECs and neurons expressed genes encoding transmembrane receptors for serotonergic, cholinergic or dopaminergic neurotransmission. Differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of acclimation to hypoxia. NECs in the hypoxia group showed high expression of genes involved in cell cycle control and proliferation. The present article provides a complete cell atlas for the zebrafish gill and serves as a platform for future studies investigating the molecular biology and physiology of this organ.

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Genetic mutations and environmental toxins are known to affect mitochondrial health and have been implicated in the progressive degeneration of dopaminergic neurons in Parkinson's disease. To visualize mitochondria in dopaminergic neurons of live zebrafish, we used the regulatory elements of the dopamine transporter (dat) gene to target a reporter, mCherry, after fusion with the mitochondrial localizing signal (MLS) of Tom20. Immunoblot analysis of mitochondrial and cytosolic fractions from Tg(dat:tom20 MLS-mCherry) larvae shows that mCherry is efficiently targeted to the mitochondria. Confocal imaging of live fish was carried out from 1 day postfertilization (dpf) to 9 dpf. We also colocalized dat mRNA expression with the mCherry protein in the olfactory bulb (OB), subpallium (SP), pretectum (Pr), diencephalic clusters 2 and 3 (DC2/3), caudal hypothalamus (Hc), locus coeruleus (LC), anterior preoptic area (POa), retinal amacrine cells (RAC), caudal hypothalamus (Hc), and preoptic area (PO). Treating Tg(dat:tom20 MLS-mCherry) larvae with the dopaminergic neurotoxin MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) at 2 or 3 dpf resulted in a decrease in mCherry fluorescence in the pretectum, olfactory bulb, subpallium, diencephalic clusters 2 and 3, and the caudal hypothalamus. Labeling of mitochondria in nigrostriatal dopaminergic neurons of zebrafish could allow their visualization in vivo following genetic or pharmacological manipulations.

Many genes associated with familial Parkinson's disease contribute to mitochondrial morphology and function. Some of these genes, for example, Pink1 and Parkin, are part of a common pathway. The presenilin-associated rhomboid-like (PARL) gene was recently linked to familial Parkinson's disease. The PARL gene product is found in the inner mitochondrial membrane and cleaves the optic atrophy 1 protein, involved in mitochondrial morphology and apoptosis. In Drosophila, the PARL-related rhomboid-7 gene acts upstream of pink1 and parkin. However, such a genetic relationship is still unknown in vertebrates. Here, we show that the zebrafish genome comprises two parl paralogs: parla and parlb. Morpholino-mediated loss of parla and/or parlb function resulted in mild neurodegeneration, as evidenced by a lower density of dopaminergic neurons. Patterning of dopaminergic neurons was also perturbed in the ventral diencephalon. Morphants exhibited extensive cell death throughout the entire body as well as increased larval mortality. The morphant phenotype could be rescued by injection of human PARL mRNA, but not catalytically inactive PARL, suggesting functional conservation between the human and zebrafish proteins. More importantly, the zebrafish pink1 mRNA as well as the human PINK1 mRNA, but not kinase-dead nor Parkinson's disease-linked mutant PINK1 mRNA, also rescued the morphant phenotype, providing evidence that Parl genes may function upstream of Pink1, as part of a conserved pathway in vertebrates.

RationalEndothelial damage plays a central role in acute lung injury, and regeneration of lung vascular endothelium is required for its resolution in preclinical models.ObjectivesWe sought to define the cellular and molecular mechanisms underlying lung microvascular regeneration in acute lung injury induced by lung endothelial cell ablation.MethodsTransgenic mice were created expressing endothelial-targeted human diphtheria toxin receptor. Changes in lung cell populations and gene expression profiles were determined using single-cell RNA sequencing of dissociated lung cells (10x Genomics) at baseline (day 0) and days 3, 5 and 7 days after lung endothelial cell ablation.Measurements and Main ResultsIntratracheal instillation of diphtheria toxin resulted in ablation of ∼70% of lung endothelial cells, producing severe acute lung injury, with complete resolution by 7 days. Single cell analysis revealed 8 distinct endothelial cell clusters, including type-A capillary endothelial cells which were characterized by the unique expression of apelin at baseline. Diphtheria toxin-induced ablation resulted in the emergence of novel stem-like endothelial cells in the transitional ‘general’ capillary type-B endothelial population at day 3, characterized by the de novo expression of apelin. This was followed by the appearance of proliferative endothelial cells at day 5 expressing apelin receptor and Forkhead box M1 which were responsible for replenishment of all depleted endothelial cell populations. Treatment with an apelin receptor antagonist prevented recovery post DT resulting in excessive mortality.ConclusionsTargeted endothelial cell ablation revealed a remarkable regenerative capacity of the lung microvasculature orchestrated by newly emergent apelin-expressing endothelial stem-like cells primed for endothelial repair.