Ground state depletion microscopy as a tool for studying microglia-synapse interactions.

Ground state depletion followed by individual molecule return microscopy (GSDIM) has been used in the past to study the nanoscale distribution of protein co-localization in living cells. We now demonstrate the successful application of GSDIM to archival human brain tissue sections including from Alzheimer's disease cases as well as experimental tissue samples from mouse and zebrafish larvae. Presynaptic terminals and microglia and their cell processes were visualized at a resolution beyond diffraction-limited light microscopy, allowing clearer insights into their interactions in situ. The procedure described here offers time and cost savings compared to electron microscopy and opens the spectrum of molecular imaging using antibodies and super-resolution microscopy to the analysis of routine formalin-fixed paraffin sections of archival human brain. The investigation of microglia-synapse interactions in dementia will be of special interest in this context.

Diversity in cell motility reveals the dynamic nature of the formation of zebrafish taste sensory organs.

Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud. We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly.

The aPKC/Par3/Par6 Polarity Complex and Membrane Order Are Functionally Interdependent in Epithelia During Vertebrate Organogenesis.

The differential distribution of lipids between apical and basolateral membranes is necessary for many epithelial cell functions, but how this characteristic membrane organization is integrated within the polarity network during ductal organ development is poorly understood. Here we quantified membrane order in the gut, kidney and liver ductal epithelia in zebrafish larvae at 3-11 days post fertilization (dpf) with Laurdan 2-photon microscopy. We then applied a combination of Laurdan imaging, antisense knock-down and analysis of polarity markers to understand the relationship between membrane order and apical-basal polarity. We found a reciprocal relationship between membrane order and the cell polarity network. Reducing membrane condensation by exogenously added oxysterol or depletion of cholesterol reduced apical targeting of the polarity protein, aPKC. Conversely, using morpholino knock down in zebrafish, we found that membrane order was dependent upon the Crb3 and Par3 polarity protein expression in ductal epithelia. Hence our data suggest that the biophysical property of membrane lipid packing is a regulatory element in apical basal polarity.

Calpain Inhibition Is Protective in Machado-Joseph Disease Zebrafish Due to Induction of Autophagy.

The neurodegenerative disease Machado-Joseph disease (MJD), also known as spinocerebellar ataxin-3, affects neurons of the brain and spinal cord, disrupting control of the movement of muscles. We have successfully established the first transgenic zebrafish (Danio rerio) model of MJD by expressing human ataxin-3 protein containing either 23 glutamines (23Q, wild-type) or 84Q (MJD-causing) within neurons. Phenotypic characterization of the zebrafish (male and female) revealed that the ataxin-3-84Q zebrafish have decreased survival compared with ataxin-3-23Q and develop ataxin-3 neuropathology, ataxin-3 cleavage fragments and motor impairment. Ataxin-3-84Q zebrafish swim shorter distances than ataxin-3-23Q zebrafish as early as 6 days old, even if expression of the human ataxin-3 protein is limited to motor neurons. This swimming phenotype provides a valuable readout for drug treatment studies. Treating the EGFP-ataxin-3-84Q zebrafish with the calpain inhibitor compound calpeptin decreased levels of ataxin-3 cleavage fragments, but also removed all human ataxin-3 protein (confirmed by ELISA) and prevented the early MJD zebrafish motor phenotype. We identified that this clearance of ataxin-3 protein by calpeptin treatment resulted from an increase in autophagic flux (indicated by decreased p62 levels and increased LC3II). Cotreatment with the autophagy inhibitor chloroquine blocked the decrease in human ataxin-3 levels and the improved movement produced by calpeptin treatment. This study demonstrates that this first transgenic zebrafish model of MJD is a valuable tool for testing potential treatments for MJD. Calpeptin treatment is protective in this model of MJD and removal of human ataxin-3 through macro-autophagy plays an important role in this beneficial effect.SIGNIFICANCE STATEMENT We have established the first transgenic zebrafish model of the neurodegenerative disease MJD, and identified relevant disease phenotypes, including impaired movement from an early age, which can be used in rapid drug testing studies. We have found that treating the MJD zebrafish with the calpain inhibitor compound calpeptin produces complete removal of human ataxin-3 protein, due to induction of the autophagy quality control pathway. This improves the movement of the MJD zebrafish. Artificially blocking the autophagy pathway prevents the removal of human ataxin-3 and improved movement produced by calpeptin treatment. These findings indicate that induction of autophagy, and removal of ataxin-3 protein, plays an important role in the protective effects of calpain inhibition for the treatment of MJD.

Tissue-specific models of spinal muscular atrophy confirm a critical role of SMN in motor neurons from embryonic to adult stages.

Spinal muscular atrophy (SMA) is an autosomal recessive disease linked to survival motor neuron (SMN) protein deficiency. While SMN protein is expressed ubiquitously, its deficiency triggers tissue-specific hallmarks, including motor neuron death and muscle atrophy, leading to impaired motor functions and premature death. Here, using stable miR-mediated knockdown technology in zebrafish, we developed the first vertebrate system allowing transgenic spatio-temporal control of the smn1 gene. Using this new model it is now possible to investigate normal and pathogenic SMN function(s) in specific cell types, independently or in synergy with other cell populations. We took advantage of this new system to first test the effect of motor neuron or muscle-specific smn1 silencing. Anti-smn1 miRNA expression in motor neurons, but not in muscles, reproduced SMA hallmarks, including abnormal motor neuron development, poor motor function and premature death. Interestingly, smn1 knockdown in motor neurons also induced severe late-onset phenotypes including scoliosis-like body deformities, weight loss, muscle atrophy and, seen for the first time in zebrafish, reduction in the number of motor neurons, indicating motor neuron degeneration. Taken together, we have developed a new transgenic system allowing spatio-temporal control of smn1 expression in zebrafish, and using this model, we have demonstrated that smn1 silencing in motor neurons alone is sufficient to reproduce SMA hallmarks in zebrafish. It is noteworthy that this research is going beyond SMA as this versatile gene-silencing transgenic system can be used to knockdown any genes of interest, filling the gap in the zebrafish genetic toolbox and opening new avenues to study gene functions in this organism.

Mutations in SIPA1L3 cause eye defects through disruption of cell polarity and cytoskeleton organization.

Correct morphogenesis and differentiation are critical in development and maintenance of the lens, which is a classic model system for epithelial development and disease. Through germline genomic analyses in patients with lens and eye abnormalities, we discovered functional mutations in the Signal Induced Proliferation Associated 1 Like 3 (SIPA1L3) gene, which encodes a previously uncharacterized member of the Signal Induced Proliferation Associated 1 (SIPA1 or SPA1) family, with a role in Rap1 signalling. Patient 1, with a de novo balanced translocation, 46,XY,t(2;19)(q37.3;q13.1), had lens and ocular anterior segment abnormalities. Breakpoint mapping revealed transection of SIPA1L3 at 19q13.1 and reduced SIPA1L3 expression in patient lymphoblasts. SIPA1L3 downregulation in 3D cell culture revealed morphogenetic and cell polarity abnormalities. Decreased expression of Sipa1l3 in zebrafish and mouse caused severe lens and eye abnormalities. Sipa1l3(-/-) mice showed disrupted epithelial cell organization and polarity and, notably, abnormal epithelial to mesenchymal transition in the lens. Patient 2 with cataracts was heterozygous for a missense variant in SIPA1L3, c.442G>T, p.Asp148Tyr. Examination of the p.Asp148Tyr mutation in an epithelial cell line showed abnormal clustering of actin stress fibres and decreased formation of adherens junctions. Our findings show that abnormalities of SIPA1L3 in human, zebrafish and mouse contribute to lens and eye defects, and we identify a critical role for SIPA1L3 in epithelial cell morphogenesis, polarity, adhesion and cytoskeletal organization.

Motor neuron-expressed microRNAs 218 and their enhancers are nested within introns of Slit2/3 genes.

miR218-1 and miR218-2 are embedded in introns of SLIT2 and SLIT3, respectively, an arrangement conserved throughout vertebrate genomes. Both miR218 genes are predicted to be transcribed in the same orientation as their host genes and were assumed to be spliced from Slit2/3 primary transcripts. In zebrafish miR218 is active in cranial nerve motor nuclei and spinal cord motor neurons, while slit2 and slit3 are expressed predominantly in the midline. This differential expression pattern suggested independent regulation of miR218 genes by distinct enhancers. We tested conserved noncoding elements for regulatory activity by reporter gene transgenesis in zebrafish. Two human enhancers, 76 kb and 130 kb distant from miR218-2, were identified that drove GFP expression in zebrafish in an almost complete miR218 expression pattern. In the zebrafish slit3 locus, two enhancers with identical activity were discovered. In human SLIT2 one enhancer 52 kb upstream of miR218-1 drove an expression pattern very similar to the enhancers of miR218-2. This establishes that miR218-1/-2 regulatory units are nested within SLIT2/3 and that they are duplicates of an ancestral single locus. Due to the strong activity of the enhancers, unique transgenic lines were created that facilitate morphological and gene functional genetic experiments in motor neurons.

BAC transgenic zebrafish reveal hypothalamic enhancer activity around obesity associated SNP rs9939609 within the human FTO gene.

Single Nucleotide Polymorphisms in FTO intron 1 have been associated with obesity risk, leading to the hypothesis that FTO is the obesity-related gene. However, other studies have shown that the FTO gene is part of the regulatory domain of the neighboring IRX3 gene and that enhancers in FTO intron 1 regulate IRX3. While Irx3 activity was shown to be necessary in the hypothalamus for the metabolic function of Irx3 in mouse, no enhancers with hypothalamic activity have been demonstrated in the risk-associated region within FTO. In order to identify potential enhancers at the human FTO locus in vivo, we tested regulatory activity in FTO intron 1 using BAC transgenesis in zebrafish. A minimal gata2 promoter-GFP cassette was inserted 1.3 kb upstream of the obesity associated SNP rs9939609 in a human FTO BAC plasmid. In addition to the previously identified expression domains in notochord and kidney, human FTO BAC:GFP transgenic zebrafish larvae expressed GFP in the ventral posterior tuberculum, the posterior hypothalamus and the anterior brainstem, which are also expression domains of zebrafish irx3a. In contrast, an in-frame insertion of a GFP cassette at the FTO start codon resulted in weak ubiquitous GFP expression indicating that the promoter of FTO does likely not react to enhancers located in the obesity risk-associated region.

Chemical genetics unveils a key role of mitochondrial dynamics, cytochrome c release and IP3R activity in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. The subcellular mechanisms of DMD remain poorly understood and there is currently no curative treatment available. Using a Caenorhabditis elegans model for DMD as a pharmacologic and genetic tool, we found that cyclosporine A (CsA) reduces muscle degeneration at low dose and acts, at least in part, through a mitochondrial cyclophilin D, CYN-1. We thus hypothesized that CsA acts on mitochondrial permeability modulation through cyclophilin D inhibition. Mitochondrial patterns and dynamics were analyzed, which revealed dramatic mitochondrial fragmentation not only in dystrophic nematodes, but also in a zebrafish model for DMD. This abnormal mitochondrial fragmentation occurs before any obvious sign of degeneration can be detected. Moreover, we demonstrate that blocking/delaying mitochondrial fragmentation by knocking down the fission-promoting gene drp-1 reduces muscle degeneration and improves locomotion abilities of dystrophic nematodes. Further experiments revealed that cytochrome c is involved in muscle degeneration in C. elegans and seems to act, at least in part, through an interaction with the inositol trisphosphate receptor calcium channel, ITR-1. Altogether, our findings reveal that mitochondria play a key role in the early process of muscle degeneration and may be a target of choice for the design of novel therapeutics for DMD. In addition, our results provide the first indication in the nematode that (i) mitochondrial permeability transition can occur and (ii) cytochrome c can act in cell death.

Copy number variants in patients with intellectual disability affect the regulation of ARX transcription factor gene.

Protein-coding mutations in the transcription factor-encoding gene ARX cause various forms of intellectual disability (ID) and epilepsy. In contrast, variations in surrounding non-coding sequences are correlated with milder forms of non-syndromic ID and autism and had suggested the importance of ARX gene regulation in the etiology of these disorders. We compile data on several novel and some already identified patients with or without ID that carry duplications of ARX genomic region and consider likely genetic mechanisms underlying the neurodevelopmental defects. We establish the long-range regulatory domain of ARX and identify its brain region-specific autoregulation. We conclude that neurodevelopmental disturbances in the patients may not simply arise from increased dosage due to ARX duplication. This is further exemplified by a small duplication involving a non-functional ARX copy, but with duplicated enhancers. ARX enhancers are located within a 504-kb region and regulate expression specifically in the forebrain in developing and adult zebrafish. Transgenic enhancer-reporter lines were used as in vivo tools to delineate a brain region-specific negative and positive autoregulation of ARX. We find autorepression of ARX in the telencephalon and autoactivation in the ventral thalamus. Fluorescently labeled brain regions in the transgenic lines facilitated the identification of neuronal outgrowth and pathfinding disturbances in the ventral thalamus and telencephalon that occur when arxa dosage is diminished. In summary, we have established a model for how breakpoints in long-range gene regulation alter the expression levels of a target gene brain region-specifically, and how this can cause subtle neuronal phenotypes relating to the etiology of associated neuropsychiatric disease.

Using zebrafish transgenesis to test human genomic sequences for specific enhancer activity.

We detail an approach for the identification of human tissue-specific transcriptional enhancers involving three steps: delineation of search space around a locus or target gene, in silico identification and size definition of putative candidate sequences, and testing through several independent genomic insertions in a transgenic zebrafish reporter assay. Candidate sequences are defined through evolutionary conservation, transcription factor binding and chromatin marks (e.g. ENCODE data) and are amplified from genomic DNA, cloned into basal promoter:fluorescent protein reporter vectors based on the Tol2 transposon system and are microinjected into fertilized zebrafish eggs. After raising injected founders to sexual maturity, fluorescent screening identifies positive founder fish whose offspring undergo a detailed expression analysis to determine tissue specificity and reproducibility of specific enhancers.

Long-range gene regulation links genomic type 2 diabetes and obesity risk regions to HHEX, SOX4, and IRX3.

Genome-wide association studies identified noncoding SNPs associated with type 2 diabetes and obesity in linkage disequilibrium (LD) blocks encompassing HHEX-IDE and introns of CDKAL1 and FTO [Sladek R, et al. (2007) Nature 445:881-885; Steinthorsdottir V, et al. (2007) Nat. Genet 39:770-775; Frayling TM, et al. (2007) Science 316:889-894]. We show that these LD blocks contain highly conserved noncoding elements and overlap with the genomic regulatory blocks of the transcription factor genes HHEX, SOX4, and IRX3. We report that human highly conserved noncoding elements in LD with the risk SNPs drive expression in endoderm or pancreas in transgenic mice and zebrafish. Both HHEX and SOX4 have recently been implicated in pancreas development and the regulation of insulin secretion, but IRX3 had no prior association with pancreatic function or development. Knockdown of its orthologue in zebrafish, irx3a, increased the number of pancreatic ghrelin-producing epsilon cells and decreased the number of insulin-producing beta-cells and glucagon-producing alpha-cells, thereby suggesting a direct link of pancreatic IRX3 function to both obesity and type 2 diabetes.

Exonic remnants of whole-genome duplication reveal cis-regulatory function of coding exons.

Using a comparative genomics approach to reconstruct the fate of genomic regulatory blocks (GRBs) and identify exonic remnants that have survived the disappearance of their host genes after whole-genome duplication (WGD) in teleosts, we discover a set of 38 candidate cis-regulatory coding exons (RCEs) with predicted target genes. These elements demonstrate evolutionary separation of overlapping protein-coding and regulatory information after WGD in teleosts. We present evidence that the corresponding mammalian exons are still under both coding and non-coding selection pressure, are more conserved than other protein coding exons in the host gene and several control sets, and share key characteristics with highly conserved non-coding elements in the same regions. Their dual function is corroborated by existing experimental data. Additionally, we show examples of human exon remnants stemming from the vertebrate 2R WGD. Our findings suggest that long-range cis-regulatory inputs for developmental genes are not limited to non-coding regions, but can also overlap the coding sequence of unrelated genes. Thus, exonic regulatory elements in GRBs might be functionally equivalent to those in non-coding regions, calling for a re-evaluation of the sequence space in which to look for long-range regulatory elements and experimentally test their activity.

Cis-regulatory characterization of sequence conservation surrounding the Hox4 genes.

Hox genes are key regulators of anterior-posterior axis patterning and have a major role in hindbrain development. The zebrafish Hox4 paralogs have strong overlapping activities in hindbrain rhombomeres 7 and 8, in the spinal cord and in the pharyngeal arches. With the aim to predict enhancers that act on the hoxa4a, hoxb4a, hoxc4a and hoxd4a genes, we used sequence conservation around the Hox4 genes to analyze all fish:human conserved non-coding sequences by reporter assays in stable zebrafish transgenesis. Thirty-four elements were functionally tested in GFP reporter gene constructs and more than 100 F1 lines were analyzed to establish a correlation between sequence conservation and cis-regulatory function, constituting a catalog of Hox4 CNEs. Sixteen tissue-specific enhancers could be identified. Multiple alignments of the CNEs revealed paralogous cis-regulatory sequences, however, the CNE sequence similarities were found not to correlate with tissue specificity. To identify ancestral enhancers that direct Hox4 gene activity, genome sequence alignments of mammals, teleosts, horn shark and the cephalochordate amphioxus, which is the most basal extant chordate possessing a single prototypical Hox cluster, were performed. Three elements were identified and two of them exhibited regulatory activity in transgenic zebrafish, however revealing no specificity. Our data show that the approach to identify cis-regulatory sequences by genome sequence alignments and subsequent testing in zebrafish transgenesis can be used to define enhancers within the Hox clusters and that these have significantly diverged in their function during evolution.

Live analysis of endodermal layer formation identifies random walk as a novel gastrulation movement.

During gastrulation, dramatic movements rearrange cells into three germ layers expanded over the entire embryo [1-3]. In fish, both endoderm and mesoderm are specified as a belt at the embryo margin. Mesodermal layer expansion is achieved through the combination of two directed migrations. The outer ring of precursors moves toward the vegetal pole and continuously seeds mesodermal cells inside the embryo, which then reverse their movement in the direction of the animal pole [3-6]. Unlike mesoderm, endodermal cells internalize at once and must therefore adopt a different strategy to expand over the embryo [7, 8]. With live imaging of YFP-expressing zebrafish endodermal cells, we demonstrate that in contrast to mesoderm, internalized endodermal cells display a nonoriented/noncoordinated movement fit by a random walk that rapidly disperses them over the yolk surface. Transplantation experiments reveal that this behaviour is largely cell autonomous, induced by TGF-beta/Nodal, and dependent on the downstream effector Casanova. At midgastrulation, endodermal cells switch to a convergence movement. We demonstrate that this switch is triggered by environmental cues. These results uncover random walk as a novel Nodal-induced gastrulation movement and as an efficient strategy to transform a localized cell group into a layer expanded over the embryo.

Cis-regulation and chromosomal rearrangement of the fgf8 locus after the teleost/tetrapod split.

The complex expression pattern of fibroblast growth factor 8 (Fgf8) and the cellular responses dependent on concentration of its mRNA in vertebrates suggest that Fgf8 should be tightly controlled at the transcriptional level. We found zebrafish conserved noncoding elements (CNEs) with pan-vertebrate as well as fish-specific orthologous sequences from across 200 kb of the zebrafish fgf8a genomic regulatory block to direct reporter expression in patterns consistent with the expression pattern of fgf8a. These included elements from inside the introns of the skin-specific slc2a15a and the ubiquitously expressed fbxw4 bystander genes. The fgf8a/fbxw4 gene pair, which has remained joined throughout three whole genome duplications in chordate evolution, is inverted in teleost genomes, but CNEs across both evolutionary breakpoints showed specific activity. While some CNEs directed highly reproducible expression patterns, others were subject to variation but showed, in a subset of transgenes, expression in the apical ectodermal ridge, the anterior boundaries of somites and the midbrain-hindbrain boundary, specific Fgf8 signaling domains, suggesting that their activity may be context specific. A human element with tetrapod-specific orthologous sequences directed reporter expression to the vasculature, possibly corresponding to a tetrapod innovation. We conclude that fgf8a transcriptional regulation employs pan-vertebrate and teleost-specific enhancers dispersed over three genes in the zebrafish genome.

Systematic human/zebrafish comparative identification of cis-regulatory activity around vertebrate developmental transcription factor genes.

Pan-vertebrate developmental cis-regulatory elements are discernible as highly conserved noncoding elements (HCNEs) and are often dispersed over large areas around the pleiotropic genes whose expression they control. On the loci of two developmental transcription factor genes, SOX3 and PAX6, we demonstrate that HCNEs conserved between human and zebrafish can be systematically and reliably tested for their regulatory function in multiple stable transgenes in zebrafish, and their genomic reach estimated with confidence using synteny conservation and HCNE density along these loci. HCNEs of both human and zebrafish function as specific developmental enhancers in zebrafish. We show that human HCNEs result in expression patterns in zebrafish equivalent to those in mouse, establishing zebrafish as a suitable model for large-scale testing of human developmental enhancers. Orthologous human and zebrafish enhancers underwent functional evolution within their sequence and often directed related but non-identical expression patterns. Despite an evolutionary distance of 450 million years, one pax6 HCNE drove expression in identical areas when comparing zebrafish vs. human HCNEs. HCNEs from the same area often drive overlapping patterns, suggesting that multiple regulatory inputs are required to achieve robust and precise complex expression patterns exhibited by developmental genes.

Regulatory divergence of the duplicated chromosomal loci sox11a/b by subpartitioning and sequence evolution of enhancers in zebrafish.

We used the classic example of the duplicated zebrafish sox11a and -b loci to test the duplication, degeneration, complementation (DDC) model of genome evolution through whole genome duplication. While recent reports have demonstrated sub-partitioning of regulatory sequences in duplicated regions, a comparison of the regulatory capabilities of extant regulatory sequences derived from ancient ancestral elements has been scarce. Consistent with the DDC model, we find that ancestral regulatory elements distributed over several hundred kb were lost in either one or the other zebrafish duplicate, leading to subpartitioning. However, regulatory sequences kept as duplicates near both sox11 co-orthologs diverged in sequence from each other and from human elements and in the regulatory patterns they drive in transgenic zebrafish. Evolutionary analysis of the loci suggested that both zebrafish protein coding sox11 orthologs have been maintained by purifying selection, and have evolved at comparable rates, indicative of non-diverged protein functions. The duplicated regulatory elements, conversely, evolved with different divergence rates and degrees of subfunctionalization. These data show that regulatory evolution of gene expression patterns occurred both through differential loss as well as through regulatory sequence evolution in zebrafish versus human genomes.

Genomic regulatory blocks in vertebrates and implications in human disease.

Despite a recent explosion in the production of vertebrate genome sequence data and large-scale efforts to completely annotate the human genome, we still have scant knowledge of the principles that built vertebrate genomes in evolution, and of genome architecture and its functional significance. We review approaches using bioinformatics, zebrafish transgenesis, and recent findings in the molecular basis of gene regulation and tie these in with mechanisms for the maintenance of long-range conserved synteny across all vertebrate genomes. Specifically, we discuss the recently discovered genomic regulatory blocks which we argue are principal units of vertebrate genome evolution and serve as the foundations onto which evolutionary innovations are built through sequence evolution and insertion of new cis-regulatory elements. We subsequently discuss how these arrangements relate to common human heritable diseases and their significance in disease causality.

Identification and real-time imaging of a myc-expressing neutrophil population involved in inflammation and mycobacterial granuloma formation in zebrafish.

By enhancer trap screening we identified a transgenic zebrafish line showing leukocyte-specific YFP expression during late embryo and early larval development. Its enhancer detection insertion was mapped near a novel member of the myc proto-oncogene family, encoding transcription factors known to be important for regulating human myelopoiesis. Characterization of the zebrafish myc family showed that only this particular myc gene is strongly expressed in leukocytes. To identify the myc/YFP-expressing cell type, we re-examined specificity of described myeloid markers by multiplex fluorescent in situ hybridization, showing that lcp1 can be considered as a general leukocyte marker, csf1r as a macrophage-specific marker, and mpx and lyz as neutrophil-specific markers. Subsequent colocalization analysis defined the YFP-positive cells as a subset of the neutrophil population. Using real-time confocal imaging we demonstrate that these cells migrate to sites of inflammation and are involved in innate immune responses towards infections, including Mycobacterium marinum-induced granuloma formation.