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The receptor tyrosine kinase Ret plays a critical role in regulating enteric nervous system (ENS) development. Ret is important for proliferation, migration, and survival of enteric progenitor cells (EPCs). Ret also promotes neuronal fate, but its role during neuronal differentiation and in the adult ENS is less well understood. Inactivating RET mutations are associated with ENS diseases, e.g., Hirschsprung Disease, in which distal bowel lacks ENS cells. Zebrafish is an established model system for studying ENS development and modeling human ENS diseases. One advantage of the zebrafish model system is that their embryos are transparent, allowing visualization of developmental phenotypes in live animals. However, we lack tools to monitor Ret expression in live zebrafish. Here, we developed a new BAC transgenic line that expresses GFP under the ret promoter. We find that EPCs and the majority of ENS neurons express ret:GFP during ENS development. In the adult ENS, GFP+ neurons are equally present in females and males. In homozygous mutants of ret and sox10—another important ENS developmental regulator gene—GFP+ ENS cells are absent. In summary, we characterize a ret:GFP transgenic line as a new tool to visualize and study the Ret signaling pathway from early development through adulthood.

As they form, synapses go through various stages of maturation and refinement. These steps are linked to significant changes in synaptic function, potentially resulting in emergence and maturation of behavioral outputs. Synaptotagmins are calcium-sensing proteins of the synaptic vesicle exocytosis machinery, and changes in Synaptotagmin proteins at synapses have significant effects on vesicle release and synaptic function. Here, we examined the distribution of the synaptic vesicle protein Synaptotagmin 2a (Syt2a) during development of the zebrafish nervous system. Syt2a is widely distributed throughout the midbrain and hindbrain early during larval development but very weakly expressed in the forebrain. Later in development, Syt2a expression levels in the forebrain increase, particularly in regions associated with social behavior, and most intriguingly, around the time social behavior becomes apparent. We provide evidence that Syt2a localizes to synapses onto neurons implicated in social behavior in the ventral forebrain and show that Syt2a is colocalized with tyrosine hydroxylase, a biosynthetic enzyme in the dopamine pathway. Our results suggest a developmentally important role for Syt2a in maturing synapses in the forebrain, coinciding with the emergence of social behavior.

The enteric nervous system (ENS) controls many aspects of intestinal homeostasis, including parameters that shape the habitat of microbial residents. Previously we showed that zebrafish lacking an ENS, due to deficiency of the sox10 gene, develop intestinal inflammation and bacterial dysbiosis, with an expansion of proinflammatory Vibrio strains. To understand the primary defects resulting in dysbiosis in sox10 mutants, we investigated how the ENS shapes the intestinal environment in the absence of microbiota and associated inflammatory responses. We found that intestinal transit, intestinal permeability, and luminal pH regulation are all aberrant in sox10 mutants, independent of microbially induced inflammation. Treatment with the proton pump inhibitor, omeprazole, corrected the more acidic luminal pH of sox10 mutants to wild type levels. Omeprazole treatment also prevented overabundance of Vibrio and ameliorated inflammation in sox10 mutant intestines. Treatment with the carbonic anhydrase inhibitor, acetazolamide, caused wild type luminal pH to become more acidic, and increased both Vibrio abundance and intestinal inflammation. We conclude that a primary function of the ENS is to regulate luminal pH, which plays a critical role in shaping the resident microbial community and regulating intestinal inflammation.Author SummaryThe intestinal microbiota is an important determinant of health and disease and is shaped by the environment of the gut lumen. The nervous system of the intestine, the enteric nervous system (ENS), helps maintain many aspects of intestinal health including a healthy microbiota. We used zebrafish with a genetic mutation that impedes ENS formation to investigate how the ENS prevents pathogenic shifts in the microbiota. We found that mutants lacking an ENS have a lower luminal pH, higher load of pathogenic bacteria, and intestinal inflammation. We showed that correcting the low pH, using the commonly prescribed pharmacological agent omeprazole, restored the microbiota and prevented intestinal inflammation. Conversely, we found that lowering the luminal pH of wild type animals, using the drug acetazolamide, caused expansion of pathogenic bacteria and increased intestinal inflammation. From these experiments, we conclude that a primary function of the ENS is to maintain normal luminal pH, thereby constraining intestinal microbiota community composition and promoting intestinal health.

Why did zebrafish become an important biomedical model? Here I review the background and history leading to the popularity of zebrafish as a genetic model for investigating vertebrate embryonic development and preview how zebrafish research has expanded into other areas.

The zebrafish (Danio rerio) has emerged as a powerful model to study vertebrate development and disease. Its short generation time makes it amenable to genetic manipulation and analysis, and its small size and high fecundity make it especially well suited for large-scale forward genetic and chemical screens. Fast-developing zebrafish embryos are transparent, facilitating live imaging of a variety of developmental processes in wild-type and mutant animals. Published version

Intestinal tract development is a coordinated process involving signaling among the progenitors and developing cells from all three germ layers. Development of endoderm-derived intestinal epithelium has been shown to depend on epigenetic modifications, but whether that is also the case for intestinal tract cell types from other germ layers remains unclear. We found that functional loss of a DNA methylation machinery component, ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1), leads to reduced numbers of ectoderm-derived enteric neurons and severe disruption of mesoderm-derived intestinal smooth muscle. Genetic chimeras revealed that Uhrf1 functions both cell-autonomously in enteric neuron precursors and cell-non-autonomously in surrounding intestinal cells, consistent with what is known about signaling interactions between these cell types that promote one another's development. Uhrf1 recruits the DNA methyltransferase Dnmt1 to unmethylated DNA during replication. Dnmt1 is also expressed in enteric neurons and smooth muscle progenitors. dnmt1 mutants have fewer enteric neurons and disrupted intestinal smooth muscle compared to wildtypes. Because dnmt1;uhrf1 double mutants have a similar phenotype to dnmt1 and uhrf1 single mutants, Dnmt1 and Uhrf1 must function together during enteric neuron and intestinal muscle development. This work shows that genes controlling epigenetic modifications are important to coordinate intestinal tract development, provides the first demonstration that these genes influence development of the ENS, and advances uhrf1 and dnmt1 as potential new Hirschsprung disease candidates.

Background: To understand the basis of nervous system development, we must learn how multipotent progenitors generate diverse neuronal and glial lineages. We addressed this issue in the zebrafish enteric nervous system (ENS), a complex neuronal and glial network that regulates essential intestinal functions. Little is currently known about how ENS progenitor subpopulations generate enteric neuronal and glial diversity. Results: We identified temporally and spatially dependent progenitor subpopulations based on coexpression of three genes essential for normal ENS development: phox2bb, sox10, and ret. Our data suggest that combinatorial expression of these genes delineates three major ENS progenitor subpopulations, (1) phox2bb + /ret- /sox10-, (2) phox2bb + /ret + /sox10-, and (3) phox2bb + /ret + /sox10+, that reflect temporal progression of progenitor maturation during migration. We also found that differentiating zebrafish neurons maintain phox2bb and ret expression, and lose sox10 expression. Conclusions: Our data show that zebrafish enteric progenitors constitute a heterogeneous population at both early and late stages of ENS development and suggest that marker gene expression is indicative of a progenitor's fate. We propose that a progenitor's expression profile reveals its developmental state: “younger” wave front progenitors express all three genes, whereas more mature progenitors behind the wave front selectively lose sox10 and/or ret expression, which may indicate developmental restriction. Developmental Dynamics 245:1081–1096, 2016. © 2016 Wiley Periodicals, Inc.

Fishes of the genus Danio exhibit diverse pigment patterns that serve as useful models for understanding the genes and cell behaviors underlying the evolution of adult form. Among these species, zebrafish D. rerio exhibit several dark stripes of melanophores with sparse iridophores that alternate with light interstripes of dense iridophores and xanthophores. By contrast, the closely related species D. nigrofasciatus has an attenuated pattern with fewer melanophores, stripes and interstripes. Here we demonstrate species differences in iridophore development that presage the fully formed patterns. Using genetic and transgenic approaches we identify the secreted peptide Endothelin-3 (Edn3)—a known melanogenic factor of tetrapods—as contributing to reduced iridophore proliferation and fewer stripes and interstripes in D. nigrofasciatus. We further show the locus encoding this factor is expressed at lower levels in D. nigrofasciatus owing to cis-regulatory differences between species. Finally, we show that functions of two paralogous loci encoding Edn3 have been partitioned between skin and non-skin iridophores. Our findings reveal genetic and cellular mechanisms contributing to pattern differences between these species and suggest a model for evolutionary changes in Edn3 requirements for pigment patterning and its diversification across vertebrates., Author summary Neural crest derived pigment cells generate the spectacular variation in skin pigment patterns among vertebrates. Mammals and birds have just a single skin pigment cell, the melanocyte, whereas ectothermic vertebrates have several pigment cells including melanophores, iridophores and xanthophores, that together organize into a diverse array of patterns. In the teleost zebrafish, Danio rerio, an adult pattern of stripes depends on interactions between pigment cell classes and between pigment cells and their tissue environment. The close relative D. nigrofasciatus has fewer stripes and prior analyses suggested a difference between these species that lies extrinsic to the pigment cells themselves. A candidate for mediating this difference is Endothelin-3 (Edn3), essential for melanocyte development in warm-blooded animals, and required by all three classes of pigment cells in an amphibian. We show that Edn3 specifically promotes iridophore development in Danio, and that differences in Edn3 expression contribute to differences in iridophore complements, and striping, between D. rerio and D. nigrofasciatus. Our study reveals a novel function for Edn3 and provides new insights into how changes in gene expression yield morphogenetic outcomes to effect diversification of adult form.

Resident microbes promote many aspects of host development, although the mechanisms by which microbiota influence host tissues remain unclear. We showed previously that the microbiota is required for allocation of appropriate numbers of secretory cells in the zebrafish intestinal epithelium. Because Notch signaling is crucial for secretory fate determination, we conducted epistasis experiments to establish whether the microbiota modulates host Notch signaling. We also investigated whether innate immune signaling transduces microbiota cues via the Myd88 adaptor protein. We provide the first evidence that microbiota-induced, Myd88-dependent signaling inhibits host Notch signaling in the intestinal epithelium, thereby promoting secretory cell fate determination. These results connect microbiota activity via innate immune signaling to the Notch pathway, which also plays crucial roles in intestinal homeostasis throughout life and when impaired can result in chronic inflammation and cancer.

Deficits in social engagement are diagnostic of multiple neurodevelopmental disorders, including autism and schizophrenia [1]. Genetically tractable animal models like zebrafish (Danio rerio) could provide valuable insight into developmental factors underlying these social impairments, but this approach is predicated on the ability to accurately and reliably quantify subtle behavioral changes. Similarly, characterizing local molecular and morphological phenotypes requires knowledge of the neuroanatomical correlates of social behavior. We leveraged behavioral and genetic tools in zebrafish to both refine our understanding of social behavior and identify brain regions important for driving it. We characterized visual social interactions between pairs of adult zebrafish, and discovered that they perform a stereotyped orienting behavior that reflects social attention [2]. Furthermore, in pairs of fish, the orienting behavior of one individual is the primary factor driving the same behavior in the other individual. We used manual and genetic lesions to investigate the forebrain contribution to this behavior and identified a population of neurons in the ventral telencephalon whose ablation suppresses social interactions, while sparing other locomotor and visual behaviors. These neurons are cholinergic and express the gene encoding the transcription factor Lhx8a, which is required for development of cholinergic neurons in the mouse forebrain [3]. The neuronal population identified in zebrafish lies in a region homologous to mammalian forebrain regions implicated in social behavior such as the lateral septum [4]. Our data suggest that an evolutionarily conserved population of neurons controls social orienting in zebrafish.

Zebrafish first became a model in which to study genetic mechanisms underlying vertebrate development in the early 1980s. Since that time, zebrafish has also become a premier model to study mechanisms underlying a variety of human genetic diseases, organ regeneration, and host-microbe interactions, as well as providing a platform for identifying new therapeutic agents. This article describes features of zebrafish development and seminal discoveries that propelled research on this model forward, providing glimpses into these studies and into future research possibilities.

Summary StatementWe present a new image analysis technique using image velocimetry and spectral analysis that returns quantitative measures of gut contraction strength, frequency, and wave speed that can be used to study gut motility and other cellular movements.AbstractNormal gut function requires rhythmic and coordinated movements that are affected by developmental processes, physical and chemical stimuli, and many debilitating diseases. The imaging and characterization of gut motility, especially regarding periodic, propagative contractions driving material transport, are therefore critical goals. Whereas previous image analysis approaches have successfully extracted properties related to temporal frequency of motility modes, robust measures of contraction magnitude remain elusive. We developed a new image analysis method based on image velocimetry and spectral analysis that reveals temporal characteristics such as frequency and wave propagation speed, while also providing quantitative measures of the amplitude of gut motions. We validate this approach using several challenges to larval zebrafish, imaged with differential interference contrast microscopy. Both acetylcholine exposure and feeding increase frequency and amplitude of motility. Larvae lacking enteric nervous system gut innervation show the same average motility frequency, but reduced and less variable amplitude compared to wild-types. Our image analysis approach enables insights into gut dynamics in a wide variety of developmental and physiological contexts and can also be extended to analyze other types of cell movements.

Sustaining a balanced intestinal microbial community is critical for maintaining intestinal health and preventing chronic inflammation. The gut is a highly dynamic environment, subject to periodic waves of peristaltic activity. We hypothesized that this dynamic environment is a prerequisite for a balanced microbial community and that the enteric nervous system (ENS), a chief regulator of physiological processes within the gut, profoundly influences gut microbiota composition. We found that zebrafish lacking an ENS due to a mutation in the Hirschsprung disease gene, sox10, develop microbiota-dependent inflammation that is transmissible between hosts. Profiling microbial communities across a spectrum of inflammatory phenotypes revealed that increased levels of inflammation were linked to an overabundance of pro-inflammatory bacterial lineages and a lack of anti-inflammatory bacterial lineages. Moreover, either administering a representative anti-inflammatory strain or restoring ENS function corrected the pathology. Thus, we demonstrate that the ENS modulates gut microbiota community membership to maintain intestinal health., Author summary Intestinal health depends on maintaining a balanced microbial community within the highly dynamic environment of the intestine. Every few minutes, this environment is rocked by peristaltic waves of muscular contraction and relaxation through a process regulated by the enteric nervous system (ENS). We hypothesized that normal, healthy intestinal microbial communities are adapted to this dynamic environment, and that their composition would become perturbed without a functional ENS. To test this idea, we used a model organism, the zebrafish, with a genetic mutation that prevents formation of the ENS. We found that some mutant individuals without an ENS develop high levels of inflammation, whereas other mutants have normal intestines. We profiled the intestinal bacteria of inflamed and healthy mutants and found that the intestines of inflamed individuals have an overabundance of pro-inflammatory bacterial lineages, lack anti-inflammatory bacterial lineages, and are able to transmit inflammation to individuals with a normally functioning ENS. Conversely, we were able to prevent inflammation in the ENS mutants by either administering a representative anti-inflammatory bacterial strain or restoring ENS function. From these experiments, we conclude that the ENS modulates intestinal microbiota community membership to maintain intestinal health.

All animals are ecosystems with resident microbial communities, referred to as microbiota, which play profound roles in host development, physiology, and evolution. Enabled by new DNA sequencing technologies, there is a burgeoning interest in animal–microbiota interactions, but dissecting the specific impacts of microbes on their hosts is experimentally challenging. Gnotobiology, the study of biological systems in which all members are known, enables precise experimental analysis of the necessity and sufficiency of microbes in animal biology by deriving animals germ-free (GF) and inoculating them with defined microbial lineages. Mammalian host models have long dominated gnotobiology, but we have recently adapted gnotobiotic approaches to the zebrafish (Danio rerio), an important aquatic model. Zebrafish offer several experimental attributes that enable rapid, large-scale gnotobiotic experimentation with high replication rates and exquisite optical resolution. Here we describe detailed protocols for three procedures that form the foundation of zebrafish gnotobiology: derivation of GF embryos, microbial association of GF animals, and long-term, GF husbandry. Our aim is to provide sufficient guidance in zebrafish gnotobiotic methodology to expand and enrich this exciting field of research.

A central problem in development is how fates of closely related cells are segregated. Lineally related motoneurons (MNs) and interneurons (INs) express many genes in common yet acquire distinct fates. For example, in mouse and chick Lhx3 plays a pivotal role in the development of both cell classes. Here, we utilize the ability to recognize individual zebrafish neurons to examine the roles of Lhx3 and its paralog Lhx4 in the development of MNs and ventral INs. We show that Lhx3 and Lhx4 are expressed by post-mitotic axial MNs derived from the MN progenitor (pMN) domain, p2 domain progenitors and by several types of INs derived from pMN and p2 domains. In the absence of Lhx3 and Lhx4, early-developing primary MNs (PMNs) adopt a hybrid fate, with morphological and molecular features of both PMNs and pMN-derived Kolmer–Agduhr′ (KA′) INs. In addition, we show that Lhx3 and Lhx4 distinguish the fates of two pMN-derived INs. Finally, we demonstrate that Lhx3 and Lhx4 are necessary for the formation of late-developing V2a and V2b INs. In conjunction with our previous work, these data reveal that distinct transcription factor families are deployed in post-mitotic MNs to unequivocally assign MN fate and suppress the development of alternative pMN-derived IN fates.

Origin of fish pigment cell for pattern Zebrafish stripes arise from the interactions of pigment cells: black melanophores, iridescent iridophores, and yellow-orange xanthophores. Melanophores and iridophores develop from nerve-associated stem cells, but the origin of xanthophores is unclear. Two studies now reveal that adult xanthophores originate from xanthophores in embryonic and larval fish, when they proliferate to cover the skin before the arrival of black and silver cells in a striped arrangement. Mahalwar et al. show that xanthophores change their final shape and color depending on their location. In black cells, xanthophores appear faint and stellate, but in silver cells, they are bright and compact. Precise superposition creates the blue and golden colors. McMenamin et al. observe the loss of pigment in embryonic xanthophores and the later reappearance in the adult. They show that redifferentiation depends on the thyroid hormone that also limits melanophore population expansion. Science , this issue p. 1362 and p. 1358

To characterize fluorescent enteric neurons labeled for expression of cytoplasmic markers in zebrafish mutants, we developed a new MATLAB-based program that can be trained by user input. We used the program to count enteric neurons and to analyze co-expression of the neuronal marker, Elavl, and the neuronal subtype marker, serotonin, in 3D confocal image stacks of dissected whole-mount zebrafish intestines. We quantified the entire population of enteric neurons and the serotonergic subpopulation in specific regions of the intestines of gutwrencher mutant and wild-type sibling larvae. We show a marked decrease in enteric neurons in gutwrencher mutants that is more severe at the caudal end of the intestine. We also show that gutwrencher mutants have the same number of serotonin-positive enteroendocrine cells in the intestine as wild types.

The gut microbiota is a complex consortium of microorganisms with the ability to influence important aspects of host health and development. Harnessing this “microbial organ” for biomedical applications requires clarifying the degree to which host and bacterial factors act alone or in combination to govern the stability of specific lineages. To address this issue, we combined bacteriological manipulation and light sheet fluorescence microscopy to monitor the dynamics of a defined two-species microbiota within a vertebrate gut. We observed that the interplay between each population and the gut environment produces distinct spatiotemporal patterns. As a consequence, one species dominates while the other experiences sudden drops in abundance that are well fit by a stochastic mathematical model. Modeling revealed that direct bacterial competition could only partially explain the observed phenomena, suggesting that a host factor is also important in shaping the community. We hypothesized the host determinant to be gut motility, and tested this mechanism by measuring colonization in hosts with enteric nervous system dysfunction due to a mutation in the ret locus, which in humans is associated with the intestinal motility disorder known as Hirschsprung disease. In mutant hosts we found reduced gut motility and, confirming our hypothesis, robust coexistence of both bacterial species. This study provides evidence that host-mediated spatial structuring and stochastic perturbation of communities can drive bacterial population dynamics within the gut, and it reveals a new facet of the intestinal host–microbe interface by demonstrating the capacity of the enteric nervous system to influence the microbiota. Ultimately, these findings suggest that therapeutic strategies targeting the intestinal ecosystem should consider the dynamic physical nature of the gut environment., Live imaging of a model intestinal microbiota reveals that enteric neural function and peristalsis, combined with the spatial structure of microbial communities, can drive competition between bacterial species., Author Summary Hundreds of microbial species thrive within the gut of humans and other animals, where they can influence the health of their host in profound ways. The factors that shape the composition of the resident gut microbiota are not well understood, but identifying them represents an important step toward developing treatments for diseases associated with microbial imbalances. Current experimental approaches poorly capture spatial and temporal aspects of microbial interactions within the gut, and yet these features may hold clues to what determines the composition of the microbiota. To address this issue, we used state-of-the-art live imaging to track two bacterial species within the intestine of a model vertebrate host, the zebrafish. We observed strikingly different interplay between the spatial organization of each population and the intestine’s peristaltic activity. As a result, one species dominates while the other experiences sudden drops in abundance, the dynamics of which are predicted by a stochastic mathematical model. From this work, we conclude that the composition of indigenous microbial communities may, in part, be shaped by a combination of the physical intestinal environment and the spatial structure of bacterial populations.

The enteric nervous system (ENS) forms intimate connections with many other intestinal cell types, including immune cells and bacterial consortia resident in the intestinal lumen. In this review, we highlight contributions of the zebrafish model to understanding interactions among these cells. Zebrafish is a powerful model for forward genetic screens, several of which have uncovered genes previously unknown to be important for ENS development. More recently, zebrafish has emerged as a model for testing functions of genes identified in human patients or large-scale human susceptibility screens. In several cases, zebrafish studies have revealed mechanisms connecting intestinal symptoms with other, seemingly unrelated disease phenotypes. Importantly, chemical library screens in zebrafish have provided startling new insights into potential effects of common drugs on ENS development. A key feature of the zebrafish model is the ability to rear large numbers of animals germ free or in association with only specific bacterial species. Studies utilizing these approaches have demonstrated the importance of bacterial signals for normal intestinal development. These types of studies also show how luminal bacteria and the immune system can contribute to inflammatory processes that can feedback to influence ENS development. The excellent optical properties of zebrafish embryos and larvae, coupled with the ease of generating genetically marked cells of both the host and its resident bacteria, allow visualization of multiple intestinal cell types in living larvae and should promote a more in-depth understanding of intestinal cell interactions, especially interactions between other intestinal cell types and the ENS.

To understand the molecular mechanisms of development it is essential to be able to turn genes on and off at will and in a spatially restricted fashion. Morpholino oligonucleotides (MOs) are very common tools used in several model organisms with which it is possible to block gene expression. Recently developed photo-activated MOs allow control over the onset of MO activity. However, deactivation of photo-cleavable MO activity has remained elusive. Here, we describe photo-cleavable MOs with which it is possible to activate or de-activate MO function by UV exposure in a temporal and spatial manner. We show, using several different genes as examples, that it is possible to turn gene expression on or off both in the entire zebrafish embryo and in single cells. We use these tools to demonstrate the sufficiency of no tail expression as late as tailbud stage to drive medial precursor cells towards the notochord cell fate. As a broader approach for the use of photo-cleavable MOs, we show temporal control over gal4 function, which has many potential applications in multiple transgenic lines. We demonstrate temporal manipulation of Gal4 transgene expression in only primary motoneurons and not secondary motoneurons, heretofore impossible with conventional transgenic approaches. In another example, we follow and analyze neural crest cells that regained sox10 function after deactivation of a photo-cleavable sox10-MO at different time points. Our results suggest that sox10 function might not be critical during neural crest formation.

Dorsal root ganglion (DRG) sensory neurons transmit all somatosensory information from the trunk region of the body. erbb3 mutant zebrafish do not form DRG neurons because the neural crest cells that generate them migrate aberrantly. Here we report that homozygous erbb3 mutants appear to swim and feed normally, and that they survive through adulthood, despite never forming DRG neurons. The source of sensory compensation in adult erbb3 mutants remains unknown, although it may be from lateral line ganglion neuromasts which are reduced, but present, in erbb3 mutants. We also provide new information about the development of DRG neurons in wild-type juvenile zebrafish.

Physical interaction between the transmembrane proteins Delta and Notch allows only a subset of neural precursors to become neurons, as well as regulating other aspects of neural development. To examine the localization of Delta protein during neural development, we generated an antibody specific to zebrafish DeltaA (Dla). Here, we describe for the first time the subcellular localization of Dla protein in distinct puncta at cell cortex and/or membrane, supporting the function of Dla in direct cell–cell communication. In situ RNA hybridization and immunohistochemistry revealed dynamic, coordinated expression patterns of dla mRNA and Dla protein in the developing and adult zebrafish nervous system. Dla expression is mostly excluded from differentiated neurons and is maintained in putative precursor cells at least until larval stages. In the adult brain, dla mRNA and Dla protein are expressed in proliferative zones normally associated with stem cells. Developmental Dynamics 238:3226–3336, 2009. © 2009 Wiley-Liss, Inc.

One of the most significant problems facing developmental biologists who do not work on an organism with well-developed genetics - and even for some who do - is how to inhibit the action of a gene of interest during development so as to learn about its normal biological function. A widely adopted approach is to use antisense technologies, and especially morpholino antisense oligonucleotides. In this article, we review the use of such reagents and present examples of how they have provided insights into developmental mechanisms. We also discuss how the use of morpholinos can lead to misleading results, including off-target effects, and we suggest controls that will allow researchers to interpret morpholino experiments correctly.

Thyroid hormones (THs) play an important role in vertebrate development; however, the underlying mechanisms of their actions are still poorly understood. Zebrafish (Danio rerio) is an emerging vertebrate model system to study the roles of THs during development. In general, the response to THs relies on closely related proteins and mechanisms across vertebrate species, however some species-specific differences exist. In contrast to mammals, zebrafish has two TRalpha genes (thraa, thrab). Moreover, the zebrafish thraa gene expresses a TRalpha isoform (TRalphaA1) that differs from other TRs by containing additional C-terminal amino acids. C-terminal extensions, called "F domains", are common in other members of the nuclear receptor superfamily and modulate the response of these receptors to hormones. Here we demonstrate that the F-domain constrains the transcriptional activity of zebrafish TRalpha by altering the selectivity of this receptor for certain coactivator binding motifs. We found that the F-domain of zebrafish TRalphaA1 is encoded on a separate exon whose inclusion is regulated by alternative splicing, indicating a regulatory role of the F-domain in vivo. Quantitative expression analyses revealed that TRalphaA1 is primarily expressed in reproductive organs whereas TRalphaB and the TRalphaA isoform that lacks the F-domain (TRalphaA1-2) appear to be ubiquitous. The relative expression levels of these TRalpha transcripts differ in a tissue-specific manner suggesting that zebrafish uses both alternative splicing and differential expression of TRalpha genes to diversify the cellular response to THs.

An intact enteric nervous system is required for normal gastrointestinal tract function. Several human conditions result from decreased innervation by enteric neurons; however, the genetic basis of enteric nervous system development and function is incompletely understood. In an effort to increase our understanding of the mechanisms underlying enteric nervous system development, we screened mutagenized zebrafish for changes in the number or distribution of enteric neurons. We also established a motility assay and rescreened mutants to learn whether enteric neuron number is correlated with gastrointestinal motility in zebrafish. We describe mutations isolated in our screen that affect enteric neurons specifically, as well as mutations that affect other neural crest derivatives or have pleiotropic effects. We show a correlation between the severity of enteric neuron loss and gastrointestinal motility defects. This screen provides biological tools that serve as the basis for future mechanistic studies.

In addition to rapid signaling, electrical activity provides important cues to developing neurons. Electrical activity relies on the function of several different types of voltage-gated ion channels. Whereas voltage-gated Ca2+ channel activity regulates several aspects of neuronal differentiation, much less is known about developmental roles of voltage-gated Na+ channels, essential mediators of electrical signaling. Here, we focus on the zebrafish Na+ channel isotype, Nav1.6a,which is encoded by the scn8a gene. A restricted set of spinal neurons, including dorsal sensory Rohon-Beard cells, two motoneuron subtypes with different axonal trajectories, express scn8a during embryonic development. CaP, an early born primary motoneuron subtype with ventrally projecting axons expresses scn8a, as does a class of secondary motoneurons with axons that project dorsally. To test for developmental roles of scn8a, we knocked down Nav1.6a protein using antisense morpholinos. Na+ channel protein and current amplitudes were reduced in neurons that express scn8a. Furthermore,Nav1.6a knockdown altered axonal morphologies of some but not all motoneurons. Dorsally projecting secondary motoneurons express scn8aand displayed delayed axonal outgrowth. By contrast, CaP axons developed normally, despite expression of the gene. Surprisingly, ventrally projecting secondary motoneurons, a population in which scn8a was not detected,displayed aberrant axonal morphologies. Mosaic analysis indicated that effects on ventrally projecting secondary motoneurons were non cell-autonomous. Thus,voltage-gated Na+ channels play cell-autonomous and non cell-autonomous roles during neuronal development.

The expression of LIM homeobox genes islet1 and islet2 is tightly regulated during development of zebrafish primary motoneurons. All primary motoneurons express islet1 around the time they exit the cell cycle. By the time primary motoneurons undergo axogenesis, specific subtypes express islet1, whereas other subtypes express islet2,suggesting that these two genes have different functions. Here, we show that Islet1 is required for formation of zebrafish primary motoneurons; in the absence of Islet1, primary motoneurons are missing and there is an apparent increase in some types of ventral interneurons. We also provide evidence that Islet2 can substitute for Islet1 during primary motoneuron formation. Surprisingly, our results demonstrate that despite the motoneuron subtype-specific expression patterns of Islet1 and Islet2, the differences between the Islet1 and Islet2 proteins are not important for specification of the different primary motoneuron subtypes. Thus, primary motoneuron subtypes are likely to be specified by factors that act in parallel to or upstream of islet1 and islet2.

Retinoic acid signaling is important for patterning the central nervous system, paired appendages, digestive tract, and other organs. To begin to investigate retinoic acid signaling in zebrafish, we determined orthologies between zebrafish and tetrapod retinoic acid receptors (Rars) and examined the expression patterns of rar genes during embryonic development. Analysis of phylogenies and conserved syntenies showed that the three cloned zebrafish rar genes include raraa and rarab, which are co-orthologs of tetrapod Rara, and rarg, which is the zebrafish ortholog of tetrapod Rarg. We did not, however, find an ortholog of Rarb. RNA in situ hybridization experiments showed that rarab and rarg, are maternally expressed. Zygotic expression of raraa occurs predominantly in the hindbrain, lateral mesoderm, and tailbud. Zygotic expression of rarab largely overlaps that of raraa, except that in later stages rarab is expressed more broadly in the brain and in the pectoral fin bud and pharyngeal arches. Zygotic expression of zebrafish rarg also overlaps the other two genes, but it is expressed more strongly in the posterior hindbrain beginning in late somitogenesis as well as in neural crest cells in the pharyngeal arches. Thus, these three genes have largely overlapping expression patterns and a few gene-specific expression domains. Knowledge of these expression patterns will guide the interpretation of the roles these genes play in development.

In avians and mice, trunk neural crest migration is restricted to the anterior half of each somite. Sclerotome has been shown to play an essential role in this restriction; the potential role of other somite components in specifying neural crest migration is currently unclear. By contrast, in zebrafish trunk neural crest, migration on the medial pathway is restricted to the middle of the medial surface of each somite. Sclerotome comprises only a minor part of zebrafish somites, and the pattern of neural crest migration is established before crest cells contact sclerotome cells, suggesting other somite components regulate the pattern of zebrafish neural crest migration. Here, we use mutants to investigate which components regulate the pattern of zebrafish trunk neural crest migration on the medial pathway. The pattern of trunk neural crest migration is aberrant in spadetail mutants that have very reduced somitic mesoderm, in no tail mutants injected with spadetail morpholino antisense oligonucleotides that entirely lack somitic mesoderm and in somite segmentation mutants that have normal somite components but disrupted segment borders. Fast muscle cells appear dispensable for patterning trunk neural crest migration. However, migration is abnormal in Hedgehog signaling mutants that lack slow muscle cells, providing evidence that slow muscle cells regulate the pattern of trunk neural crest migration. Consistent with this idea, surgical removal of adaxial cells, which are slow muscle precursors, results in abnormal patterning of neural crest migration;normal patterning can be restored by replacing the ablated adaxial cells with ones transplanted from wild-type embryos.

Genes belonging to the Nkx, Gsh and Msx families are expressed in similar dorsovental spatial domains of the insect and vertebrate central nervous system (CNS), suggesting the bilaterian ancestor used this genetic program during CNS development. We have investigated the significance of these similar expression patterns by testing whether Nkx6 proteins expressed in ventral CNS of zebrafish and flies have similar functions. In zebrafish, Nkx6.1 is expressed in early-born primary and later-born secondary motoneurons. In the absence of Nkx6.1, there are fewer secondary motoneurons and supernumerary ventral interneurons, suggesting Nkx6.1 promotes motoneuron and suppresses interneuron formation. Overexpression of fish or fly Nkx6 is sufficient to generate supernumerary motoneurons in both zebrafish and flies. These results suggest that one ancestral function of Nkx6 proteins was to promote motoneuron development.

We provide the first analysis of how a segmentally reiterated pattern of neurons is specified along the anteroposterior axis of the vertebrate spinal cord by investigating how zebrafish primary motoneurons are patterned. Two identified primary motoneuron subtypes, MiP and CaP, occupy distinct locations within the ventral neural tube relative to overlying somites, express different genes and innervate different muscle territories. In all vertebrates examined so far, paraxial mesoderm-derived signals specify distinct motoneuron subpopulations in specific anteroposterior regions of the spinal cord. We show that signals from paraxial mesoderm also control the much finer-grained segmental patterning of zebrafish primary motoneurons. We examined primary motoneuron specification in several zebrafish mutants that have distinct effects on paraxial mesoderm development. Our findings suggest that in the absence of signals from paraxial mesoderm, primary motoneurons have a hybrid identity with respect to gene expression, and that under these conditions the CaP axon trajectory may be dominant.

Specification of spinal cord neurons is regulated by several different transcription factors. In this study, we analyze expression and regulation of the transcription factor iro3 in zebrafish spinal cord. In addition to its broad expression in the progenitor domain of intermediate spinal cord, iro3 is also expressed in postmitotic ventral neurons, starting at early somitogenesis stages. Initially, this expression is only in two primary motoneurons, CaP and VaP, but by 24 hr postfertilization, iro3 is expressed by all classes of zebrafish spinal motoneurons as well as by a ventral interneuron called VeLD. iro3 expression in the progenitor domain of intermediate spinal cord is regulated independently from its expression in ventral neurons. Hedgehog (Hh) signaling is unnecessary for iro3 expression in intermediate spinal cord, but it is required to repress iro3 expression in the progenitor domain of ventral spinal cord. We also show that the basic helix-loop-helix transcription factor Olig2 is required for repression of iro3 expression in the progenitor domain of ventral spinal cord. We discuss our findings in the context of previous studies, suggesting that iro3 represses formation of motoneurons and promotes formation of interneurons.

The ability of an animal to carry out its normal behavioral repertoire requires generation of an enormous diversity of neurons and glia. The relative simplicity of the spinal cord makes this an especially attractive part of the nervous system for addressing questions about the development of vertebrate neural specification and function. The last decade has witnessed an explosion in our understanding of spinal cord development and the functional interactions among spinal cord neurons and glia. Cellular, genetic, molecular, physiological and behavioral studies in zebrafish have all been important in providing insights into questions that remained unanswered by studies from other vertebrate model organisms. This is the case because many zebrafish spinal neurons can be individually identified and followed over time in living embryos and larvae. In this review, we discuss what is currently known about the cellular, genetic and molecular mechanisms involved in specifying distinct cell types in the zebrafish spinal cord and how these cells establish the functional circuitry that mediates particular behaviors. We start by describing the early signals and morphogenetic movements that form the nervous system, and in particular, the spinal cord. We then provide an overview of the cell types within the spinal cord and describe how they are specified and patterned. We begin ventrally with floor plate and proceed dorsally, through motoneurons and oligodendrocytes, interneurons, astrocytes and radial glia, spinal sensory neurons and neural crest. We next describe axon pathfinding of spinal neurons. Finally, we discuss the roles of particular spinal cord neurons in specific behaviors.

Rohon-Beard cells are large, mechanosensory neurons located in the dorsal spinal cord of anamniote vertebrates. In most species studied to date, these cells die during development. We followed labeled Rohon-Beard cells in living zebrafish embryos and found that they degenerate slowly, over many days. During degeneration, the soma shrinks and finally disappears, and the processes become beady in appearance and finally break apart, but they do not retract. Zebrafish Rohon-Beard cells apparently fragment their DNA, as revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) labeling, before undergoing degenerative morphologic changes. We also followed the development of labeled dorsal root ganglion neurons, as they are developing at the same stages that Rohon-Beard cells are degenerating. We found that, although axons of both cell types extend into similar regions, Rohon-Beard cells degenerate normally in mutants lacking dorsal root ganglia, providing evidence that interactions between the two cell types are not responsible for Rohon-Beard cell degeneration. Developmental Dynamics 229:30–41,2004. © 2003 Wiley-Liss, Inc.

The understanding of vertebrate development has advanced considerably in recent years, primarily due to the study of a few model organisms. The zebrafish, the newest of these models, has risen to prominence because both genetic and experimental embryological methods can be easily applied to this animal. The combination of approaches has proven powerful, yielding insights into the formation and function of individual tissues, organ systems and neural networks, and into human disease mechanisms. Here, we provide a personal perspective on the history of zebrafish research, from the assembly of the first genetic and embryological tools through to sequencing of the genome.

The segmental heritage of all vertebrates is evident in the character of the vertebral column. And yet, the extent to which direct translation of pattern from the somitic mesoderm and de novo cell and tissue interactions pattern the vertebral column remains a fundamental, unresolved issue. The elements of vertebral column pattern under debate include both segmental pattern and anteroposterior regional specificity. Understanding how vertebral segmentation and anteroposterior positional identity are patterned requires understanding vertebral column cellular and developmental biology. In this study, we characterized alignment of somites and vertebrae, distribution of individual sclerotome progeny along the anteroposterior axis and development of the axial skeleton in zebrafish. Our clonal analysis of zebrafish sclerotome shows that anterior and posterior somite domains are not lineage-restricted compartments with respect to distribution along the anteroposterior axis but support a ‘leaky’ resegmentation in development from somite to vertebral column. Alignment of somites with vertebrae suggests that the first two somites do not contribute to the vertebral column. Characterization of vertebral column development allowed examination of the relationship between vertebral formula and expression patterns of zebrafish Hox genes. Our results support co-localization of the anterior expression boundaries of zebrafish hoxc6 homologs with a cervical/thoracic transition and also suggest Hox-independent patterning of regionally specific posterior vertebrae.

In zebrafish, cells at the lateral edge of the neural plate become Rohon-Beard primary sensory neurons or neural crest. Delta/Notch signaling is required for neural crest formation. ngn1 is expressed in primary neurons; inhibiting Ngn1 activity prevents Rohon-Beard cell formation but not formation of other primary neurons. Reducing Ngn1 activity in embryos lacking Delta/Notch signaling restores neural crest formation, indicating Delta/Notch signaling inhibits neurogenesis without actively promoting neural crest. Ngn1 activity is also required for later development of dorsal root ganglion sensory neurons; however, Rohon-Beard neurons and dorsal root ganglion neurons are not necessarily derived from the same precursor cell. We propose that temporally distinct episodes of Ngn1 activity in the same precursor population specify these two different types of sensory neurons.

The ability of an animal to move and to interact with its environment requires that motoneurons correctly innervate specific muscles. Although many genes that regulate motoneuron development have been identified, our understanding of motor axon branching remains incomplete. We used transcriptional expression profiling to identify potential candidate genes involved in development of zebrafish identified motoneurons. Here we focus on inab, an intermediate filament encoding gene dynamically expressed in a subset of motoneurons as well as in an identified interneuron. We show that inab is necessary for proper axon morphology of a specific motoneuron subtype.

Sonic hedgehog (Shh) is crucial for motoneuron development in chick and mouse. However, zebrafish embryos homozygous for a deletion of the shh locus have normal numbers of motoneurons, raising the possibility that zebrafish motoneurons may be specified differently. Unlike other vertebrates, zebrafish express three hh genes in the embryonic midline: shh, echidna hedgehog (ehh) and tiggywinkle hedgehog (twhh). Therefore, it is possible that Twhh and Ehh are sufficient for motoneuron formation in the absence of Shh. To test this hypothesis we have eliminated, or severely reduced, all three Hh signals using mutations that directly or indirectly reduce Hh signaling and antisense morpholinos. Our analysis shows that Hh signals are required for zebrafish motoneuron induction. However, each of the three zebrafish Hhs is individually dispensable for motoneuron development because the other two can compensate for its loss. Our results also suggest that Twhh and Shh are more important for motoneuron development than Ehh.

Vertebrate pigment cells are derived from neural crest, a tissue that also forms most of the peripheral nervous system and a variety of ectomesenchymal cell types. Formation of pigment cells from multipotential neural crest cells involves a number of common developmental processes. Pigment cells must be specified; their migration, proliferation, and survival must be controlled and they must differentiate to the final pigment cell type. We previously reported a large set of embryonic mutations that affect pigment cell development from neural crest (R. N. Kelsh et al., 1996, Development 123, 369–389). Based on distinctions in pigment cell appearance between mutants, we proposed hypotheses as to the process of pigment cell development affected by each mutation. Here we describe the cloning and expression of an early zebrafish melanoblast marker, dopachrome tautomerase. We used this marker to test predictions about melanoblast number and pattern in mutant embryos, including embryos homozygous for mutations in the colourless, sparse, touchdown, sunbleached, punkt, blurred, fade out, weiss, sandy, and albino genes. We showed that in homozygous mutants for all loci except colourless and sparse, melanoblast number and pattern are normal. colourless mutants have a pronounced decrease in melanoblast cell number from the earliest stages and also show poor melanoblast differentiation and migration. Although sparse mutants show normal numbers of melanoblasts initially, their number is reduced later. Furthermore, their distribution indicates a defect in melanoblast dispersal. These observations permit us to refine our model of the genetic control of melanophore development in zebrafish embryos.

We examined the role of Delta signaling in specification of two derivatives in zebrafish neural plate: Rohon-Beard spinal sensory neurons and neural crest. deltaA-expressing Rohon-Beard neurons are intermingled with premigratory neural crest cells in the trunk lateral neural plate. Embryos homozygous for a point mutation in deltaA, or with experimentally reduced Delta signaling, have supernumerary Rohon-Beard neurons, reduced trunk-level expression of neural crest markers and lack trunk neural crest derivatives. Fin mesenchyme, a putative trunk neural crest derivative, is present in deltaA mutants, suggesting it segregates from other neural crest derivatives as early as the neural plate stage. Cranial neural crest derivatives are also present in deltaA mutants, revealing a genetic difference in regulation of trunk and cranial neural crest development.

Primary motoneurons, the earliest developing spinal motoneurons in zebrafish, have highly stereotyped axon projections. Although much is known about the development of these neurons, the molecular cues guiding their axons have not been identified. In a screen designed to reveal mutations affecting motor axons, we isolated two mutations in the stumpy gene that dramatically affect pathfinding by the primary motoneuron, CaP. In stumpy mutants, CaP axons extend along the common pathway, a region shared by other primary motor axons, but stall at an intermediate target, the horizontal myoseptum, and fail to extend along their axon-specific pathway during the first day of development. Later, most CaP axons progress a short distance beyond the horizontal myoseptum, but tend to stall at another intermediate target. Mosaic analysis revealed that stumpy function is needed both autonomously in CaP and non-autonomously in other cells. stumpy function is also required for axons of other primary and secondary motoneurons to progress properly past intermediate targets and to branch. These results reveal a series of intermediate targets involved in motor axon guidance and suggest that stumpy function is required for motor axons to progress from proximally located intermediate targets to distally located ones.

The zebrafish fkd6 gene is a marker for premigratory neural crest. In this study, we analyze later expression in putative glia of the peripheral nervous system. Prior to neural crest migration, fkd6 expression is downregulated in crest cells. Subsequently, expression appears initially in loose clusters of cells in positions corresponding to cranial ganglia. Double labelling with a neuronal marker shows that fkd6-expressing cells are not differentiated neurones and generally lie peripheral to neurones in ganglia. Later, expression appears associated with the posterior lateral line and other cranial nerves. For the posterior lateral line nerve, we show that fkd6-labeling extends caudally along this nerve in tight correlation with lateral line primordium migration and axon elongation. Expression in colourless mutant embryos is consistent with these cells being satellite glia and Schwann cells.

Neural crest forms four major categories of derivatives: pigment cells, peripheral neurons, peripheral glia, and ectomesenchymal cells. Some early neural crest cells generate progeny of several fates. How specific cell fates become specified is still poorly understood. Here we show that zebrafish embryos with mutations in the colourless gene have severe defects in most crest-derived cell types, including pigment cells, neurons and specific glia. In contrast, craniofacial skeleton and medial fin mesenchyme are normal. These observations suggest that colourless has a key role in development of non-ectomesenchymal neural crest fates, but not in development of ectomesenchymal fates. Thus, the cls mutant phenotype reveals a segregation of ectomesenchymal and non-ectomesenchymal fates during zebrafish neural crest development. The combination of pigmentation and enteric nervous system defects makes colourless mutations a model for two human neurocristopathies, Waardenburg-Shah syndrome and Hirschsprung’s disease.

Background: Fate mapping studies have shown that progenitor cells of three vertebrate embryonic midline structures – the floorplate in the ventral neural tube, the notochord and the dorsal endoderm – occupy a common region prior to gastrulation. This common region of origin raises the possibility that interactions between midline progenitor cells are important for their specification prior to germ layer formation. Results: One of four known zebrafish homologues of the Drosophila melanogaster cell–cell signaling gene Delta, deltaA ( dlA ), is expressed in the developing midline, where progenitor cells of the ectodermal floorplate, mesodermal notochord and dorsal endoderm lie close together before they occupy different germ layers. We used a reverse genetic strategy to isolate a missense mutation of dlA, dlA dx2 , which coordinately disrupts the development of floorplate, notochord and dorsal endoderm. The dlA dx2 mutant embryos had reduced numbers of floorplate and hypochord cells; these cells lie above and beneath the notochord, respectively. In addition, mutant embryos had excess notochord cells. Expression of a dominant-negative form of Delta protein driven by mRNA microinjection produced a similar effect. In contrast, overexpression of dlA had the opposite effect: fewer trunk notochord cells and excess floorplate and hypochord cells. Conclusion: Our results indicate that Delta signaling is important for the specification of midline cells. The results are most consistent with the hypothesis that developmentally equivalent midline progenitor cells require Delta-mediated signaling prior to germ layer formation in order to be specified as floorplate, notochord or hypochord.

The vertebrate spinal cord consists of a large number of different cell types in close proximity to one another. The identities of these cells appear to be specified largely by information acquired from their local environments. We report here that local cell-cell interactions, mediated by zebrafish homologues of the Drosophila melanogaster neurogenic gene, Delta, regulate specification of diverse neuronal types in the ventral spinal cord. We describe identification of a novel zebrafish Delta gene expressed specifically in the nervous system and show, by expressing a dominant negative form of Delta protein in embryos, that Delta proteins mediate lateral inhibition in the zebrafish spinal cord. Furthermore, we find that Delta function is important for specification of a variety of spinal cord neurons, suggesting that lateral inhibition serves to diversify neuronal fate during development of the vertebrate spinal cord.

In zebrafish there are two populations of motoneurons, primary and secondary, that are temporally separate in their development. To determine if midline cells play a role in the specification of these neurons, we analyzed both secondary and primary motoneurons in mutants lacking floor plate, notochord, or both floor plate and notochord. Our data show that the specification of secondary motoneurons, those most similar to motoneurons in birds and mammals, depends on the presence of either a differentiated floor plate or notochord. In the absence of both of these structures, secondary motoneurons fail to form. In contrast, primary motoneurons, early developing motoneurons found in fish and amphibians, can develop in the absence of both floor plate and notochord. A spatial correspondence is found between secondary motoneurons andsonic hedgehog-expressing floor plate and notochord. In contrast, primary motoneuronal specification depends on the presence ofsonic hedgehogin gastrula axial mesoderm, the tissue that will give rise to the notochord. These results suggest that both primary and secondary motoneurons are specified by signals from midline tissues, but at very different stages of embryonic development.

During zebrafish development, identified motoneurons innervate cell-specific regions of each trunk myotome. One motoneuron, CaP, extends an axon along the medial surface of the ventral myotome. To learn how this pathway is established during development, the CaP axon was used as an assay to ask whether other regions of the myotome were permissive for normal CaP pathfinding. Native myotomes were replaced with donor myotomes in normal or reversed dorsoventral orientations and CaP pathfinding was assayed. Ventral myotomes were permissive for CaP axons, even when they were taken from older embryos, suggesting that the CaP pathway remained present on ventral myotome throughout development. Dorsal myotomes from young embryos were also permissive for CaP axons, however, older dorsal myotomes were non-permissive, showing that permissiveness of dorsal myotome for normal CaP pathfinding diminished over time. This process appears to depend on signals from the embryo, since dorsal myotomes matured in vitro remained permissive for CaP axons. Genetic mosaics between wild-type and floating head mutant embryos revealed notochord involvement in dorsal myotome change of permissiveness. Dorsal and ventral myotomes from both younger and older floating head mutant embryos were permissive for CaP axons. These data suggest that initially both dorsal and ventral myotomes are permissive for CaP axons but as development proceeds, there is a notochord-dependent decrease in permissiveness of dorsal myotome for CaP axonal outgrowth. This change participates in restricting the CaP pathway to the ventral myotome and thus to neuromuscular specificity.

Vertebrate embryos display segmental patterns in many trunk structures, including somites and peripheral nervous system elements. Previous work in avian embryos suggests a role for somite-derived sclerotome in segmental patterning of the peripheral nervous system. We investigated sclerotome development and tested its role in patterning motor axons and dorsal root ganglia in embryonic zebrafish. Individual somite cells labeled with vital fluorescent dye revealed that some cells of a ventromedial cell cluster within each somite produced mesenchymal cells that migrated to positions expected for sclerotome. Individual somites showed anterior/posterior distinctions in several aspects of development: (1) anterior ventromedial cluster cells produced only sclerotome, (2) individual posterior ventromedial cluster cells produced both sclerotome and muscle, and (3) anterior sclerotome migrated earlier and along a more restricted path than posterior sclerotome. Vital labeling showed that anterior sclerotome colocalized with extending identified motor axons and migrating neural crest cells. To investigate sclerotome involvement in peripheral nervous system patterning, we ablated the ventromedial cell cluster and observed subsequent development of peripheral nervous system elements. Primary motor axons were essentially unaffected by sclerotome ablation, although in some cases outgrowth was delayed. Removal of sclerotome did not disrupt segmental pattern or development of dorsal root ganglia or peripheral nerves to axial muscle. We propose that peripheral nervous system segmentation is established through interactions with adjacent paraxial mesoderm which develops as sclerotome in some vertebrate species and myotome in others.