A toolbox to study epidermal cell types in zebrafish.

Epithelia provide a crucial protective barrier for our organs and are also the sites where the majority of carcinomas form. Most studies on epithelia and carcinomas use cell culture or organisms where high-resolution live imaging is inaccessible without invasive techniques. Here, we introduce the developing zebrafish epidermis as an excellent in vivo model system for studying a living epithelium. We developed tools to fluorescently tag specific epithelial cell types and express genes in a mosaic fashion using five Gal4 lines identified from an enhancer trap screen. When crossed to a variety of UAS effector lines, we can now track, ablate or monitor single cells at sub-cellular resolution. Using photo-cleavable morpholino oligonucleotides that target gal4, we can also express genes in a mosaic fashion at specific times during development. Together, this system provides an excellent in vivo alternative to tissue culture cells, without the intrinsic concerns of culture conditions or transformation, and enables the investigation of distinct cell types within living epithelial tissues.

Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia.

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

Loss of laminin alpha 1 results in multiple structural defects and divergent effects on adhesion during vertebrate optic cup morphogenesis.

The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1(UW1)) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1(UW1) mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis.

High-resolution analysis of central nervous system expression patterns in zebrafish Gal4 enhancer-trap lines.

BACKGROUND: The application of the Gal4/UAS system to enhancer and gene trapping screens in zebrafish has greatly increased the ability to label and manipulate cell populations in multiple tissues, including the central nervous system (CNS). However the ability to select existing lines for specific applications has been limited by the lack of detailed expression analysis. RESULTS: We describe a Gal4 enhancer trap screen in which we used advanced image analysis, including three-dimensional confocal reconstructions and documentation of expression patterns at multiple developmental time points. In all, we have created and annotated 98 lines exhibiting a wide range of expression patterns, most of which include CNS expression. Expression was also observed in nonneural tissues such as muscle, skin epithelium, vasculature, and neural crest derivatives. All lines and data are publicly available from the Zebrafish International Research Center (ZIRC) from the Zebrafish Model Organism Database (ZFIN). CONCLUSIONS: Our detailed documentation of expression patterns, combined with the public availability of images and fish lines, provides a valuable resource for researchers wishing to study CNS development and function in zebrafish. Our data also suggest that many existing enhancer trap lines may have previously uncharacterized expression in multiple tissues and cell types.

Mirror movement-like defects in startle behavior of zebrafish dcc mutants are caused by aberrant midline guidance of identified descending hindbrain neurons.

Mirror movements are involuntary movements on one side of the body that occur simultaneously with intentional movements on the contralateral side. Humans with heterozygous mutations in the axon guidance receptor DCC display such mirror movements, where unilateral stimulation results in inappropriate bilateral motor output. Currently, it is unclear whether mirror movements are caused by incomplete midline crossing and reduced commissural connectivity of DCC-dependent descending pathways or by aberrant ectopic ipsilateral axonal projections of normally commissural neurons. Here, we show that in response to unilateral tactile stimuli, zebrafish dcc mutant larvae perform involuntary turns on the inappropriate body side. We show that these mirror movement-like deficits are associated with axonal guidance defects of two identified groups of commissural reticulospinal hindbrain neurons. Moreover, we demonstrate that in dcc mutants, axons of these identified neurons frequently fail to cross the midline and instead project ipsilaterally. Whereas laser ablation of these neurons in wild-type animals does not affect turning movements, their ablation in dcc mutants restores turning movements. Thus, our results demonstrate that in dcc mutants, turns on the inappropriate side of the body are caused by aberrant ipsilateral axonal projections, and suggest that aberrant ipsilateral connectivity of a very small number of descending axons is sufficient to induce incorrect movement patterns.

Sonic hedgehog is indirectly required for intraretinal axon pathfinding by regulating chemokine expression in the optic stalk.

Successful axon pathfinding requires both correct patterning of tissues, which will later harbor axonal tracts, and precise localization of axon guidance cues along these tracts at the time of axon outgrowth. Retinal ganglion cell (RGC) axons grow towards the optic disc in the central retina, where they turn to exit the eye through the optic nerve. Normal patterning of the optic disc and stalk and the expression of guidance cues at this choice point are necessary for the exit of RGC axons out of the eye. Sonic hedgehog (Shh) has been implicated in both patterning of ocular tissue and direct guidance of RGC axons. Here, we examine the precise spatial and temporal requirement for Hedgehog (Hh) signaling for intraretinal axon pathfinding and show that Shh acts to pattern the optic stalk in zebrafish but does not guide RGC axons inside the eye directly. We further reveal an interaction between the Hh and chemokine pathways for axon guidance and show that cxcl12a functions downstream of Shh and depends on Shh for its expression at the optic disc. Together, our results support a model in which Shh acts in RGC axon pathfinding indirectly by regulating axon guidance cues at the optic disc through patterning of the optic stalk.

A complex choreography of cell movements shapes the vertebrate eye.

Optic cup morphogenesis (OCM) generates the basic structure of the vertebrate eye. Although it is commonly depicted as a series of epithelial sheet folding events, this does not represent an empirically supported model. Here, we combine four-dimensional imaging with custom cell tracking software and photoactivatable fluorophore labeling to determine the cellular dynamics underlying OCM in zebrafish. Although cell division contributes to growth, we find it dispensable for eye formation. OCM depends instead on a complex set of cell movements coordinated between the prospective neural retina, retinal pigmented epithelium (RPE) and lens. Optic vesicle evagination persists for longer than expected; cells move in a pinwheel pattern during optic vesicle elongation and retinal precursors involute around the rim of the invaginating optic cup. We identify unanticipated movements, particularly of central and peripheral retina, RPE and lens. From cell tracking data, we generate retina, RPE and lens subdomain fate maps, which reveal novel adjacencies that might determine corresponding developmental signaling events. Finally, we find that similar movements also occur during chick eye morphogenesis, suggesting that the underlying choreography is conserved among vertebrates.

Motoneurons are essential for vascular pathfinding.

The neural and vascular systems share common guidance cues that have direct and independent signaling effects on nerves and endothelial cells. Here, we show that zebrafish Netrin 1a directs Dcc-mediated axon guidance of motoneurons and that this neural guidance function is essential for lymphangiogenesis. Specifically, Netrin 1a secreted by the muscle pioneers at the horizontal myoseptum (HMS) is required for the sprouting of dcc-expressing rostral primary motoneuron (RoP) axons and neighboring axons along the HMS, adjacent to the future trajectory of the parachordal chain (PAC). These axons are required for the formation of the PAC and, subsequently, the thoracic duct. The failure to form the PAC in netrin 1a or dcc morphants is phenocopied by laser ablation of motoneurons and is rescued both by cellular transplants and overexpression of dcc mRNA. These results provide a definitive example of the requirement of axons in endothelial guidance leading to the parallel patterning of nerves and vessels in vivo.

Extraocular ectoderm triggers dorsal retinal fate during optic vesicle evagination in zebrafish.

Dorsal retinal fate is established early in eye development, via expression of spatially restricted dorsal-specific transcription factors in the optic vesicle; yet the events leading to initiation of dorsal fate are not clear. We hypothesized that induction of dorsal fate would require an extraocular signal arising from a neighboring tissue to pattern the prospective dorsal retina, however no such signal has been identified. We used the zebrafish embryo to determine the source, timing, and identity of the dorsal retina-inducing signal. Extensive cell movements occur during zebrafish optic vesicle morphogenesis, however the location of prospective dorsal cells within the early optic vesicle and their spatial relationship to early dorsal markers is currently unknown. Our mRNA expression and fate mapping analyses demonstrate that the dorsolateral optic vesicle is the earliest region to express dorsal specific markers, and cells from this domain contribute to the dorsal retinal pole at 24 hpf. We show that three bmp genes marking dorsal retina at 25 hpf are also expressed extraocularly before retinal patterning begins. We identified gdf6a as a dorsal initiation signal acting from the extraocular non-neural ectoderm during optic vesicle evagination. We find that bmp2b is involved in dorsal retina initiation, acting upstream of gdf6a. Together, this work has identified the nature and source of extraocular signals required to pattern the dorsal retina.

Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification.

The dopaminergic neurons of the basal ganglia play critical roles in CNS function and human disease, but specification of dopamine neuron phenotype is poorly understood in vertebrates. We performed an in vivo screen in zebrafish to identify dopaminergic neuron enhancers, in order to facilitate studies on the specification of neuronal identity, connectivity, and function in the basal ganglia. Based primarily on identification of conserved non-coding elements, we tested 54 DNA elements from four species (zebrafish, pufferfish, mouse, and rat), that included 21 genes with known or putative roles in dopaminergic neuron specification or function. Most elements failed to drive CNS expression or did not express specifically in dopaminergic neurons. However, we did isolate a discrete enhancer from the otpb gene that drove specific expression in diencephalic dopaminergic neurons, although it did not share sequence conservation with regulatory regions of otpa or other dopamine-specific genes. For the otpb enhancer, regulation of expression in dopamine neurons requires multiple elements spread across a large genomic area. In addition, we compared our in vivo testing with in silico analysis of genomic regions for genes involved in dopamine neuron function, but failed to find conserved regions that functioned as enhancers. We conclude that regulation of dopaminergic neuron phenotype in vertebrates is regulated by dispersed regulatory elements.

Gal80 intersectional regulation of cell-type specific expression in vertebrates.

Characterization and functional manipulation of specific groups of neurons in the vertebrate central nervous system (CNS) remains a major hurdle for understanding complex circuitry and functions. In zebrafish, the Gal4/UAS system has permitted expression of transgenes and enhancer trap screens, but is often limited by broad expression domains. We have developed a method for cell-type specific expression using Gal80 inhibition of Gal4-dependent expression. We show that native Gal4 is able to drive strong expression, that Gal80 can inhibit this expression, and that overlapping Gal4 and Gal80 expression can achieve "intersectional" expression in spatially and genetically defined subsets of neurons. We also optimize Gal80 for expression in vertebrates, track Gal80 expression with a co-expressed fluorescent marker, and use a temperature-sensitive allele of Gal80 to temporally regulate its function. These data demonstrate that Gal80 is a powerful addition to the genetic techniques available to map and manipulate neural circuits in zebrafish.

Shutdown corner, a large deletion mutant isolated from a haploid mutagenesis screen in zebrafish.

Morphogenesis, the formation of three-dimensional organ structures, requires precise coupling of genetic regulation and complex cell behaviors. The genetic networks governing many morphogenetic systems, including that of the embryonic eye, are poorly understood. In zebrafish, several forward genetic screens have sought to identify factors regulating eye development. These screens often look for eye defects at stages after the optic cup is formed and when retinal neurogenesis is under way. This approach can make it difficult to identify mutants specific for morphogenesis, as opposed to neurogenesis. To this end, we carried out a forward genetic, small-scale haploid mutagenesis screen in zebrafish (Danio rerio) to identify factors that govern optic cup morphogenesis. We screened ∼100 genomes and isolated shutdown corner (sco), a mutant that exhibits multiple tissue defects and harbors a ∼10-Mb deletion that encompasses 89 annotated genes. Using a combination of live imaging and antibody staining, we found cell proliferation, cell death, and tissue patterning defects in the sco optic cup. We also observed other phenotypes, including paralysis, neuromuscular defects, and ocular vasculature defects. To date, the largest deletion mutants reported in zebrafish are engineered using CRISPR-Cas9 and are less than 300 kb. Because of the number of genes within the deletion interval, shutdown corner [Df(Chr05:sco)z207] could be a useful resource to the zebrafish community, as it may be helpful for gene mapping, understanding genetic interactions, or studying many genes lost in the mutant.

Slit1a inhibits retinal ganglion cell arborization and synaptogenesis via Robo2-dependent and -independent pathways.

Upon arriving at their targets, developing axons cease pathfinding and begin instead to arborize and form synapses. To test whether CNS arborization and synaptogenesis are controlled by Slit-Robo signaling, we followed single retinal ganglion cell (RGC) arbors over time. ast (robo2) mutant and slit1a morphant arbors had more branch tips and greater arbor area and complexity compared to wild-type and concomitantly more presumptive presynaptic sites labeled with YFP-Rab3. Increased arborization in ast was phenocopied by dominant-negative Robo2 expressed in single RGCs and rescued by full-length Robo2, indicating that Robo2 acts cell-autonomously. Time-lapse imaging revealed that ast and slit1a morphant arbors stabilized earlier than wild-type, suggesting a role for Slit-Robo signaling in preventing arbor maturation. Genetic analysis showed that Slit1a acts both through Robo2 and Robo2-independent mechanisms. Unlike previous PNS studies showing that Slits promote branching, our results show that Slits inhibit arborization and synaptogenesis in the CNS.

Synaptic activity and activity-dependent competition regulates axon arbor maturation, growth arrest, and territory in the retinotectal projection.

In the retinotectal projection, synapses guide retinal ganglion cell (RGC) axon arbor growth by promoting branch formation and by selectively stabilizing branches. To ask whether presynaptic function is required for this dual role of synapses, we have suppressed presynaptic function in single RGCs using targeted expression of tetanus toxin light-chain fused to enhanced green fluorescent protein (TeNT-Lc:EGFP). Time-lapse imaging of singly silenced axons as they arborize in the tectum of zebrafish larvae shows that presynaptic function is not required for stabilizing branches or for generating an arbor of appropriate complexity. However, synaptic activity does regulate two distinct aspects of arbor development. First, single silenced axons fail to arrest formation of highly dynamic but short-lived filopodia that are a feature of immature axons. Second, single silenced axons fail to arrest growth of established branches and so occupy significantly larger territories in the tectum than active axons. However, if activity-suppressed axons had neighbors that were also silent, axonal arbors appeared normal in size. A similar reversal in phenotype was observed when single TeNT-Lc:EGFP axons are grown in the presence of the NMDA receptor antagonist MK801 [(+)-5-methyl-10,11- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate]. Although expansion of arbor territory is prevented when neighbors are silent, formation of transient filopodia is not. These results suggest that synaptic activity by itself regulates filopodia formation regardless of activity in neighboring cells but that the ability to arrest growth and focusing of axonal arbors in the target is an activity-dependent, competitive process.

Analysis of the astray/robo2 zebrafish mutant reveals that degenerating tracts do not provide strong guidance cues for regenerating optic axons.

During formation of the optic projection in astray/robo2 mutant zebrafish, optic axons exhibit rostrocaudal pathfinding errors, ectopic midline crossing and increased terminal arbor size. Here we show that these errors persist into adulthood, even when robo2 function is conditionally reduced only during initial formation of the optic projection. Adult errors include massive ectopic optic tracts in the telencephalon. During optic nerve regeneration in astray/robo2 animals, these tracts are not repopulated and ectopic midline crossing is reduced compared with unlesioned mutants. This is despite a comparable macrophage/microglial response and upregulation of contactin1a in oligodendrocytes of entopic and ectopic tracts. However, other errors, such as expanded termination areas and ectopic growth into the tectum, were frequently recommitted by regenerating optic axons. Retinal ganglion cells with regenerating axons reexpress robo2 and expression of slit ligands is maintained in some areas of the adult optic pathway. However, slit expression is reduced rostral and caudal to the chiasm, compared with development and ubiquitous overexpression of Slit2 did not elicit major pathfinding phenotypes. This shows that (1) there is not an efficient correction mechanism for large-scale pathfinding errors of optic axons during development; (2) degenerating tracts do not provide a strong guidance cue for regenerating optic axons in the adult CNS, unlike the PNS; and (3) robo2 is less important for pathfinding of optic axons during regeneration than during development.

nev (cyfip2) is required for retinal lamination and axon guidance in the zebrafish retinotectal system.

In the zebrafish retinotectal system, retinal ganglion cells (RGCs) project topographically along anterior-posterior (A-P) and dorsal-ventral (D-V) axes to innervate their primary target, the optic tectum. In the nevermind (nev) mutant, D-V positional information is not maintained by dorsonasal retinal axons as they project through the optic tract to the tectum. Here we present a detailed phenotypic analysis of the retinotectal projection in nev and show that dorsonasal axons do eventually find their correct location on the tectum, albeit after taking a circuitous path. Interestingly, nev seems to be specifically required for retinal axons but not for several non-retinal axon tracts. In addition, we find that nev is required both cell autonomously and cell nonautonomously for proper lamination of the retina. We show that nev encodes Cyfip2 (Cytoplasmic FMRP interacting protein 2) and is thus the first known mutation in a vertebrate Cyfip family member. Finally, we show that CYFIP2 acts cell autonomously in the D-V sorting of dorsonasal RGC axons in the optic tract. CYFIP2 is a highly conserved protein that lacks known domains or structural motifs but has been shown to interact with Rac and the fragile-X mental retardation protein, suggesting intriguing links to cytoskeletal dynamics and RNA regulation.

Regulation of zebrafish skeletogenesis by ext2/dackel and papst1/pinscher.

Mutations in human Exostosin genes (EXTs) confer a disease called Hereditary Multiple Exostoses (HME) that affects 1 in 50,000 among the general population. Patients with HME have a short stature and develop osteochondromas during childhood. Here we show that two zebrafish mutants, dackel (dak) and pinscher (pic), have cartilage defects that strongly resemble those seen in HME patients. We have previously determined that dak encodes zebrafish Ext2. Positional cloning of pic reveals that it encodes a sulphate transporter required for sulphation of glycans (Papst1). We show that although both dak and pic are required during cartilage morphogenesis, they are dispensable for chondrocyte and perichondral cell differentiation. They are also required for hypertrophic chondrocyte differentiation and osteoblast differentiation. Transplantation analysis indicates that dak(-/-) cells are usually rescued by neighbouring wild-type chondrocytes. In contrast, pic(-/-) chondrocytes always act autonomously and can disrupt the morphology of neighbouring wild-type cells. These findings lead to the development of a new model to explain the aetiology of HME.

In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells.

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.

Netrin-DCC, Robo-Slit, and heparan sulfate proteoglycans coordinate lateral positioning of longitudinal dopaminergic diencephalospinal axons.

Longitudinal axons provide connectivity between remote areas of the nervous system. Although the molecular determinants driving commissural pathway formation have been well characterized, mechanisms specifying the formation of longitudinal axon tracts in the vertebrate nervous system are largely unknown. Here, we study axon guidance mechanisms of the longitudinal dopaminergic (DA) diencephalospinal tract. This tract is established by DA neurons located in the ventral diencephalon and is thought to be involved in modulating locomotor activity. Using mutant analysis as well as gain of function and loss of function experiments, we demonstrate that longitudinal DA axons navigate by integrating long-range signaling of midline-derived cues. Repulsive Robo2/Slit signaling keeps longitudinal DA axons away from the midline. In the absence of repulsive Robo2/Slit function, DA axons are attracted toward the midline by DCC (deleted in colorectal cancer)/Netrin1 signaling. Thus, Slit-based repulsion counteracts Netrin-mediated attraction to specify lateral positions of the DA diencephalospinal tract. We further identified heparan sulfate proteglycans as essential modulators of DA diencephalospinal guidance mechanisms. Our findings provide insight into the complexity of positioning far-projecting longitudinal axons and allow us to provide a model for DA diencephalospinal pathfinding. Simultaneous integrations of repulsive and attractive long-range cues from the midline act in a concerted manner to define lateral positions of DA longitudinal axon tracts.

Temporal regulation of Ath5 gene expression during eye development.

During central nervous system development the timing of progenitor differentiation must be precisely controlled to generate the proper number and complement of neuronal cell types. Proneural basic helix-loop-helix (bHLH) transcription factors play a central role in regulating neurogenesis, and thus the timing of their expression must be regulated to ensure that they act at the appropriate developmental time. In the developing retina, the expression of the bHLH factor Ath5 is controlled by multiple signals in early retinal progenitors, although less is known about how these signals are coordinated to ensure correct spatial and temporal pattern of gene expression. Here we identify a key distal Xath5 enhancer and show that this enhancer regulates the early phase of Xath5 expression, while the proximal enhancer we previously identified acts later. The distal enhancer responds to Pax6, a key patterning factor in the optic vesicle, while FGF signaling regulates Xath5 expression through sequences outside of this region. In addition, we have identified an inhibitory element adjacent to the conserved distal enhancer region that is required to prevent premature initiation of expression in the retina. This temporal regulation of Xath5 gene expression is comparable to proneural gene regulation in Drosophila, whereby separate enhancers regulate different temporal phases of expression.