Control of nerve cord formation by Engrailed and Gooseberry-Neuro: A multi-step, coordinated process.

One way to better understand the molecular mechanisms involved in the construction of a nervous system is to identify the downstream effectors of major regulatory proteins. We previously showed that Engrailed (EN) and Gooseberry-Neuro (GsbN) transcription factors act in partnership to drive the formation of posterior commissures in the central nervous system of Drosophila. In this report, we identified genes regulated by both EN and GsbN through chromatin immunoprecipitation ("ChIP on chip") and transcriptome experiments, combined to a genetic screen relied to the gene dose titration method. The genomic-scale approaches allowed us to define 175 potential targets of EN-GsbN regulation. We chose a subset of these genes to examine ventral nerve cord (VNC) defects and found that half of the mutated targets show clear VNC phenotypes when doubly heterozygous with en or gsbn mutations, or when homozygous. This strategy revealed new groups of genes never described for their implication in the construction of the nerve cord. Their identification suggests that, to construct the nerve cord, EN-GsbN may act at three levels, in: (i) sequential control of the attractive-repulsive signaling that ensures contralateral projection of the commissural axons, (ii) temporal control of the translation of some mRNAs, (iii) regulation of the capability of glial cells to act as commissural guideposts for developing axons. These results illustrate how an early, coordinated transcriptional control may orchestrate the various mechanisms involved in the formation of stereotyped neuronal networks. They also validate the overall strategy to identify genes that play crucial role in axonal pathfinding.

Neurotoxicity of a Biopesticide Analog on Zebrafish Larvae at Nanomolar Concentrations.

Despite the ever-increasing role of pesticides in modern agriculture, their deleterious effects are still underexplored. Here we examine the effect of A6, a pesticide derived from the naturally-occurring α-terthienyl, and structurally related to the endocrine disrupting pesticides anilinopyrimidines, on living zebrafish larvae. We show that both A6 and an anilinopyrimidine, cyprodinyl, decrease larval survival and affect central neurons at micromolar concentrations. Focusing on a superficial and easily observable sensory system, the lateral line system, we found that defects in axonal and sensory cell regeneration can be observed at much lower doses, in the nanomolar range. We also show that A6 accumulates preferentially in lateral line neurons and hair cells. We examined whether A6 affects the expression of putative target genes, and found that genes involved in apoptosis/cell proliferation are down-regulated, as well as genes reflecting estrogen receptor activation, consistent with previous reports that anilinopyrimidines act as endocrine disruptors. On the other hand, canonical targets of endocrine signaling are not affected, suggesting that the neurotoxic effect of A6 may be due to the binding of this compound to a recently identified, neuron-specific estrogen receptor.

Development vs. behavior: a role for neural adaptation in evolution?

We examine the evolution of sensory organ patterning in the lateral line system of fish. Based on recent studies of how this system develops in zebrafish, and on comparative analyses between zebrafish and tuna, we argue that the evolution of lateral line patterns is mostly determined by variations in the underlying developmental processes, independent of any selective pressure. Yet the development of major developmental innovations is so directly linked to their exploitation that it is hard not to think of them as selected for, i.e., adaptive. We propose that adaptation resides mostly in how the nervous system adjusts to new morphologies to make them functional, i.e., that species are neurally adapted to whatever morphology is provided to them by their own developmental program. We show that recent data on behavioral differences between cave forms (blind) and surface forms (eyed) of the mexican fish Astyanax fasciatus support this view, and we propose that this species might provide a unique opportunity to assess the nature of adaptation and of selection in animal evolution.

Dynamics of axonal regeneration in adult and aging zebrafish reveal the promoting effect of a first lesion.

Axonal regeneration is a major issue in the maintenance of adult nervous systems, both after nerve injuries and in neurodegenerative diseases. However, studying this process in vivo is difficult or even impossible in most vertebrates. Here we show that the posterior lateral line (PLL) of zebrafish is a suitable system to study axonal regeneration in vivo because of both the superficial location and reproducible spatial arrangement of neurons and targets, and the possibility of following reinnervation in live fish on a daily basis. Axonal regeneration after nerve cut has been demonstrated in this system during the first few days of life, leading to complete regeneration within 24 h. However, the potential for PLL nerve regeneration has not been tested yet beyond the early larval stage. We explore the regeneration potential and dynamics of the PLL nerve in adult zebrafish and report that regeneration occurs throughout adulthood. We observed that irregularities in the original branching pattern are faithfully reproduced after regeneration, suggesting that regenerating axons follow the path laid down by the original nerve branches. We quantified the extent of target reinnervation after a nerve cut and found that the latency before the nerve regenerates increases with age. This latency is reduced after a second nerve cut at all ages, suggesting that a regeneration-promoting factor induced by the first cut facilitates regeneration on a second cut. We provide evidence that this factor remains present at the site of the first lesion for several days and is intrinsic to the neurons.

Making mosaic fish primordia by focal electroporation.

The lateral line is a mechanosensory system that comprises a set of discrete sense organs called neuromasts, which are arranged in reproducible patterns on the surface of fish and amphibians. The posterior component of the system, the posterior lateral line (PLL), comprises the neuromasts on the body and tail. It develops from the migrating primordium and so can be used to examine various aspects of neural development, including the control of long-range, collective cell migration and the mechanisms underlying the establishment of appropriate connectivity. Mosaic animals are those in which one or a few cells differ genetically from all others. Several methods have been developed to generate mosaic zebrafish, which can be used in long-term fate mapping or lineage tracing experiments if the progenitor cell stably expresses a reporter gene such as green fluorescent protein. Mosaic analysis can also be used to evaluate the cell autonomy of a given mutation, confronting mutant and wild-type cells, or two different types of mutant cells, in morphogenetic mosaics. Here we present an application of electroporation, which is designed to generate mosaics in defined parts of the developing zebrafish PLL. The method is based on the idea of focal electroporation, a technique developed to introduce dyes and constructs into cells within intact tissues. Current is forced into the embryo by sealing the tip of the electrode against the enveloping cells (periderm or ectoderm depending on the developmental stage, or mantle cells in the case of neuromasts).

Labeling hair cells and afferent neurons in the posterior lateral-line system of zebrafish.

The lateral line is a mechanosensory system that comprises a set of discrete sense organs called neuromasts, which are arranged in reproducible patterns on the surface of fish and amphibians. The posterior component of the system, the posterior lateral line, comprises the neuromasts on the body and tail. Each neuromast has a core of mechanosensory hair cells, each of which is depolarized by water motion in one direction and hyperpolarized by motion in the other direction, thereby enabling fish to extract information from the movements of water around their body. Neuromasts are innervated by a few afferent neurons (usually two, but sometimes more), which have their cell bodies clustered in cranial ganglia and project their central axons to the hindbrain, where they extend longitudinally along all rhombomeres. Hair cells are readily labeled by small cationic styryl pyridinium dyes such as DiASP. Afferent fibers are also progressively labeled with this dye, presumably by trans-synaptic uptake. Adjusting the dye concentration and incubation time can lead to the labeling of the entire afferent system, thereby providing a fast and easy method for visualizing the central projection in the hindbrain of live fish. The simplicity of the method makes it potentially useful for screens based on forward or reverse genetic approaches. Here we present protocols for labeling hair cells in live zebrafish and for labeling afferent neurons in zebrafish embryos.

Labeling second-order sensory neurons in the posterior lateral-line system of zebrafish.

The lateral line is a mechanosensory system that comprises a set of discrete sense organs called neuromasts, which are arranged in reproducible patterns on the surface of fish and amphibians. The posterior component of the system, the posterior lateral line (PLL), comprises the neuromasts on the body and tail and has its ganglion just posterior to the otic vesicle. The peripheral location of the PLL system makes it accessible and easily visualized by imaging methods. Neuromasts are innervated by a few afferent neurons (usually two, but sometimes more), which have their cell bodies clustered in cranial ganglia and project their central axons to the hindbrain, where they extend longitudinally along all rhombomeres. Positively charged lipophilic carbocyanine dyes, such as DiI, have traditionally been used to label neurons, as described here. This method is especially useful for the analysis of PLL innervation because injection of the dye into a neuromast leads to specific labeling of the afferent neurons. The method can also be used to follow the flow of PLL information to higher central nervous system levels by first labeling the central projection of chosen afferent neurons and then making a second injection of DiI within the synaptic field to label the second-order neurons that extend dendrites to this field.

Time-lapse analysis of primordium migration during the development of the fish lateral line.

The lateral line is a mechanosensory system that comprises a set of discrete sense organs called neuromasts, which are arranged in reproducible patterns on the surface of fish and amphibians. The system is used by these animals to extract information from the movements of water around their body, providing them with a sense of "touch-at-a-distance" that is involved in most aspects of fish behavior. Each neuromast has a core of mechanosensory hair cells, each of which is depolarized by water motion in one direction and hyperpolarized by motion in the other direction. Based on the position of their ganglion, two components of the lateral-line system can be distinguished: the anterior lateral-line (ALL) system comprises the neuromasts on the head and has its ganglion just anterior to the otic vesicle, the posterior lateral-line (PLL) system comprises the neuromasts on the body and tail and has its ganglion just posterior to the otic vesicle. The peripheral location of the PLL system makes it accessible and easily visualized by imaging methods. The PLL develops from the migrating primordium and so can be used to examine various aspects of neural development, including the control of long-range, collective cell migration and the mechanisms underlying the establishment of appropriate connectivity. Here we discuss imaging methods for exploring these processes.

Labeling defined cells or subsets of cells in zebrafish by Kaede photoconversion.

The lateral line is a mechanosensory system that comprises a set of discrete sense organs called neuromasts, which are arranged in reproducible patterns on the surface of fish and amphibians. The posterior component of the system, the posterior lateral line (PLL), comprises the neuromasts on the body and tail and its peripheral location makes the PLL accessible and easily visualized by imaging methods. The PLL develops from the migrating primordium and so can be used to examine various aspects of neural development, including the control of long-range, collective cell migration and the mechanisms underlying the establishment of appropriate connectivity. As the PLL develops, the transition from the simple, eight-neuromast-long embryonic system to the juvenile pattern of four lines extending anteroposteriorly and counting about 60 neuromasts involves three processes. First, two new primordia form that also migrate anteroposteriorly, one (primII) along the lateral myoseptum and the other (primD) along the dorsal myoseptum, depositing proneuromasts and interneuromast cells in a manner similar to the embryonic primordium. Second, displacement of differentiated neuromasts occurs along the dorsoventral axis, and third, local proliferation of interneuromast cells occurs to form additional neuromasts. Here we describe the use of Kaede photoconversion to label a chosen cell, or subset of cells, in the migrating primordium. This method can be used to study migration at the level of a single cell, to track cell lineages, or even to determine patterns of innervation.

Wnt/Dkk negative feedback regulates sensory organ size in zebrafish.

Correct organ size must involve a balance between promotion and inhibition of cell proliferation. A mathematical model has been proposed in which an organ is assumed to produce its own growth activator as well as a growth inhibitor [1], but there is as yet no molecular evidence to support this model [2]. The mechanosensory organs of the fish lateral line system (neuromasts) are composed of a core of sensory hair cells surrounded by nonsensory support cells. Sensory cells are constantly replaced and are regenerated from surrounding nonsensory cells [3], while each organ retains the same size throughout life. Moreover, neuromasts also bud off new neuromasts, which stop growing when they reach the same size [4, 5]. Here, we show that the size of neuromasts is controlled by a balance between growth-promoting Wnt signaling activity in proliferation-competent cells and Wnt-inhibiting Dkk activity produced by differentiated sensory cells. This negative feedback loop from Dkk (secreted by differentiated cells) on Wnt-dependent cell proliferation (in surrounding cells) also acts during regeneration to achieve size constancy. This study establishes Wnt/Dkk as a novel mechanism to determine the final size of an organ.

Innervation is required for sense organ development in the lateral line system of adult zebrafish.

Superficial mechanosensory organs (neuromasts) distributed over the head and body of fishes and amphibians form the "lateral line" system. During zebrafish adulthood, each neuromast of the body (posterior lateral line system, or PLL) produces "accessory" neuromasts that remain tightly clustered, thereby increasing the total number of PLL neuromasts by a factor of more than 10. This expansion is achieved by a budding process and is accompanied by branches of the afferent nerve that innervates the founder neuromast. Here we show that innervation is essential for the budding process, in complete contrast with the development of the embryonic PLL, where innervation is entirely dispensable. To obtain insight into the molecular mechanisms that underlie the budding process, we focused on the terminal system that develops at the posterior tip of the body and on the caudal fin. In this subset of PLL neuromasts, bud neuromasts form in a reproducible sequence over a few days, much faster than for other PLL neuromasts. We show that wingless/int (Wnt) signaling takes place during, and is required for, the budding process. We also show that the Wnt activator R-spondin is expressed by the axons that innervate budding neuromasts. We propose that the axon triggers Wnt signaling, which itself is involved in the proliferative phase that leads to bud formation. Finally, we show that innervation is required not only for budding, but also for long-term maintenance of all PLL neuromasts.

Developmental origin of a major difference in sensory patterning between zebrafish and bluefin tuna.

The posterior lateral line system (PLL) of teleost fish comprises a number of mechanosensory organs arranged in defined patterns on the body surface. Embryonic patterns are largely conserved among teleosts, yet adult patterns are highly diverse. Although changes in pattern modify the perceptual abilities of the system, their developmental origin remains unknown. Here we compare the processes that underlie the formation of the juvenile PLL pattern in Thunnus thynnus, the bluefin tuna, to the processes that were elucidated in Danio rerio, the zebrafish. In both cases, the embryonic PLL comprises five neuromasts regularly spaced along the horizontal myoseptum, but the juvenile PLL comprises four roughly parallel anteroposterior lines in zebrafish, whereas it is a simple dorsally arched line in tuna fish. We examined whether this difference involves evolutionary novelties, and show that the same mechanisms mediate the transition from embryonic to juvenile patterns in both species. We conclude that the marked difference in juveniles depends on a single change (dorsal vs. ventral migration of neuromasts) in the first days of larval life.

Patterning the nervous system through development and evolution.

We report presentations and discussions at a meeting held in May 2010 in the small village of Minerve, in the south of France. The meeting was devoted mostly but not exclusively to patterning in the nervous system, with an emphasis on two model organisms, Drosophila Melanogaster and Danio rerio. Among the major issues presented were fear and its neuroanatomy, life in darkness, patterning of sensory systems, as well as fundamental issues of neural connectivity, including the role of lineage in neural development. Talks on large-scale patterning and re-patterning, and on the mouse as a third model system, concluded the meeting.

Glial cell line-derived neurotrophic factor defines the path of developing and regenerating axons in the lateral line system of zebrafish.

How the peripheral axons of sensory neurons are guided to distant target organs is not well understood. Here we examine this question in the case of the posterior lateral line (PLL) system of zebrafish, where sensory organs are deposited by a migrating primordium. Sensory neurites accompany this primordium during its migration and are thereby guided to their prospective target organs. We show that the inactivation of glial cell line-derived neurotrophic factor (GDNF) signaling leads to defects of innervation and that these defects are due to the inability of sensory axons to track the migrating primordium. GDNF signaling is also used as a guidance cue during axonal regeneration following nerve cut. We conclude that GDNF is a major determinant of directed neuritic growth and of target finding in this system, and we propose that GDNF acts by promoting local neurite outgrowth.

Lef1 controls patterning and proliferation in the posterior lateral line system of zebrafish.

The embryonic development of the posterior lateral line of zebrafish involves the migration from head to tail of a primordium comprising approximately 100 cells, and the deposition at regular intervals of presumptive mechanosensory organs (neuromasts). Migration depends on the presence of chemokine SDF1 along the pathway, and on the asymmetrical distribution of chemokine receptors CXCR4 and CXCR7 in the primordium. Primordium polarization depends on Wnt signaling in the leading region. Here, we examine the role of a major effector of Wnt signaling, lef1, in this system. We show that, although its inactivation has no overt effect on the expression of cxcr4b and cxcr7b, lef1 contributes to their control. We also show that cell proliferation, which ensures constant primordium size despite successive rounds of cell deposition, is reduced upon lef1 inactivation. Because of this defect, the primordium runs short of cells and vanishes before the line has been completed. We conclude that lef1-mediated Wnt signaling is involved in various aspects of primordium migration, although part of this implication is masked by a high level of developmental redundancy.

Development of the posterior lateral line system in Thunnus thynnus, the Atlantic blue-fin tuna, and in its close relative Sarda sarda.

The lateral line system of amphibians and fish comprises a large number of individual mechanosensory organs, the neuromasts, and their sensory neurons. The pattern of neuromasts varies markedly between species, yet the embryonic pattern is highly conserved from the relatively basal zebrafish, Danio rerio, to more derived species. Here we examine in more detail the development of the posterior lateral line (PLL) in embryos and early larvae of one of the most derived fish species, the blue-fin tuna Thunnus thynnus, and of its close relative, the Atlantic bonito Sarda sarda. We show that the basic features of embryonic PLL development, including the migratory properties of the PLL primordium, the patterning of neuromasts and their innervation, are largely conserved between zebrafish and tuna. However, Thunnus and Sarda embryos differ from Danio in three respects: the larger size of the neuromast cupula, the capability of mature neuromasts to migrate dorsally, and the presence of a single, precisely located terminal neuromast.

Origin and early development of the posterior lateral line system of zebrafish.

The lateral line system of teleosts has recently become a model system to study patterning and morphogenesis. However, its embryonic origins are still not well understood. In zebrafish, the posterior lateral line (PLL) system is formed in two waves, one that generates the embryonic line of seven to eight neuromasts and 20 afferent neurons and a second one that generates three additional lines during larval development. The embryonic line originates from a postotic placode that produces both a migrating sensory primordium and afferent neurons. Nothing is known about the origin and innervation of the larval lines. Here we show that a "secondary" placode can be detected at 24 h postfertilization (hpf), shortly after the primary placode has given rise to the embryonic primordium and ganglion. The secondary placode generates two additional sensory primordia, primD and primII, as well as afferent neurons. The primary and secondary placodes require retinoic acid signaling at the same stage of late gastrulation, suggesting that they share a common origin. Neither primary nor secondary neurons show intrinsic specificity for neuromasts derived from their own placode, but the sequence of neuromast deposition ensures that neuromasts are primarily innervated by neurons derived from the cognate placode. The delayed formation of secondary afferent neurons accounts for the capability of the fish to form a new PLL ganglion after ablation of the embryonic ganglion at 24 hpf.

Estrogen receptor ESR1 controls cell migration by repressing chemokine receptor CXCR4 in the zebrafish posterior lateral line system.

The primordium that generates the embryonic posterior lateral line of zebrafish migrates from the head to the tip of the tail along a trail of SDF1-producing cells. This migration critically depends on the presence of the SDF1 receptor CXCR4 in the leading region of the primordium and on the presence of a second SDF1 receptor, CXCR7, in the trailing region of the primordium. Here we show that inactivation of the estrogen receptor ESR1 results in ectopic expression of cxcr4b throughout the primordium, whereas ESR1 overexpression results in a reciprocal reduction in the domain of cxcr4b expression, suggesting that ESR1 acts as a repressor of cxcr4b. This finding could explain why estrogens significantly decrease the metastatic ability of ESR-positive breast cancer cells. ESR1 inactivation also leads to extinction of cxcr7b expression in the trailing cells of the migrating primordium; this effect is indirect, however, and due to the down-regulation of cxcr7b by ectopic SDF1/CXCR4 signaling in the trailing region. Both ESR1 inactivation and overexpression result in aborted migration, confirming the importance of this receptor in the control of SDF1-dependent migration.

Dermal morphogenesis controls lateral line patterning during postembryonic development of teleost fish.

The lateral line system displays highly divergent patterns in adult teleost fish. The mechanisms underlying this variability are poorly understood. Here, we demonstrate that the lateral line mechanoreceptor, the neuromast, gives rise to a series of accessory neuromasts by a serial budding process during postembryonic development in zebrafish. We also show that accessory neuromast formation is highly correlated to the development of underlying dermal structures such as bones and scales. Abnormalities in opercular bone morphogenesis, in endothelin 1-knockdown embryos, are accompanied by stereotypic errors in neuromast budding and positioning, further demonstrating the tight correlation between the patterning of neuromasts and of the underlying dermal bones. In medaka, where scales form between peridermis and opercular bones, the lateral line displays a scale-specific pattern which is never observed in zebrafish. These results strongly suggest a control of postembryonic neuromast patterns by underlying dermal structures. This dermal control may explain some aspects of the evolution of lateral line patterns.

Postembryonic development of the posterior lateral line in the zebrafish.

The posterior lateral line (PLL) of zebrafish comprises seven to eight sense organs at the end of embryogenesis, arranged in a single antero-posterior line that extends along the horizontal myoseptum from the ear to the tip of the tail. At the end of larval life, four antero-posterior lines extend on the trunk and tail, comprising together around 60 sense organs. The embryonic pattern is largely conserved among teleosts, although adult patterns are very diverse. Here we describe the transition from embryonic to juvenile pattern in the zebrafish, to provide a framework for understanding how the diversity of adult patterns comes about. We show that the four lines that extend over the adult body originate from latent precursors laid down by migrating primordia that arise during embryogenesis. We conclude that, in zebrafish, the entire development of the PLL system up to adulthood can be traced back to events that took place during the first 2 days of life. We also show that the transition from embryonic to adult pattern involves few distinct operations, suggesting that the diversity of patterns among adult teleosts may be due to differential control of these few operations acting upon common embryonic precursors.