Gap junction mediated bioelectric coordination is required for slow muscle development, organization, and function.

Bioelectrical signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the Connexins. The connexin gene family is large and complex, presenting a challenge in identifying the specific Connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for appropriate slow muscle development and function. Through a combination of mutant analysis and in vivo imaging we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging we find that spinal cord generated neural activity is transmitted to developing slow muscle cells and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization, and that disruption leads to defects in behavior. Our work reveals the molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system_may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role played by GJ communication in coordinating bioelectric signaling during development.

Determination of primary motoneuron identity in developing zebrafish embryos.

The developmental determination of primary motoneurons was investigated by transplanting identified motoneurons in embryonic zebrafish to new spinal cord positions. Some cells moved from the new positions in which they were placed back to their original positions, thus it was difficult to evaluate whether they were determined. Among cells that remained in their new positions, those transplanted about 1 hour before axogenesis developed axonal trajectories that were appropriate for their original soma positions, whereas those transplanted 2 to 3 hours before axogenesis developed morphologies appropriate for their new soma positions. These results suggest that motoneuronal identity is determined before axogenesis.

A genetic linkage map for the zebrafish.

To facilitate molecular genetic analysis of vertebrate development, haploid genetics was used to construct a recombination map for the zebrafish Danio (Brachydanio) rerio. The map consists of 401 random amplified polymorphic DNAs (RAPDs) and 13 simple sequence repeats spaced at an average interval of 5.8 centimorgans. Strategies that exploit the advantages of haploid genetics and RAPD markers were developed that quickly mapped lethal and visible mutations and that placed cloned genes on the map. This map is useful for the position-based cloning of mutant genes, the characterization of chromosome rearrangements, and the investigation of evolution in vertebrate genomes.

The role of interactions in determining cell fate of two identified motoneurons in the embryonic zebrafish.

The role of cellular interactions in determining the fates of two identified motoneurons in the embryonic zebrafish was investigated by transplanting individual motoneurons from labeled donor embryos to unlabeled hosts. The results suggest that although these cells normally adopt different fates, they form an equivalence group in which one fate is primary and the other is secondary. Both cells are able to adopt the primary fate. A cell that has adopted the secondary fate can be induced to switch to the primary fate by ablating the cell that has adopted the primary fate, even many hours after axogenesis. Although interactions between the two cells appear to regulate which cell adopts the secondary fate, these interactions seem to be independent of neuromuscular activity.

Pathway selection by ectopic motoneurons in embryonic zebrafish.

Primary motoneurons in embryonic zebrafish innervate cell-specific muscles. During pathfinding, motoneuronal growth cones encounter three distinct regions: a common pathway, a choice point, and separate cell-specific pathways. To learn whether the order in which these regions are encountered influences pathway choice, we transplanted individual motoneurons to the choice point region. These cells selected their appropriate cell-specific pathways. Thus, the sequence in which pathway regions are encountered may not be important for accurate path-finding, and the cell-specific pathways may be delineated by distinct cues that individual growth cones recognize. Moreover, these cues are unlikely to be general ones, since primary sensory neurons transplanted to the same location do not extend growth cones along the motoneuronal pathways.

The spt-1 mutation alters segmental arrangement and axonal development of identified neurons in the spinal cord of the embryonic zebrafish.

We examined the arrangement and development of identified neurons in zebrafish embryos homozygous for the mutation spt-1, which acts autonomously and specifically to alter the development of precursors of trunk segmented mesoderm, resulting in muscle-deficient myotomes. We found that the mutation alters the morphology, number, and arrangement of identified motoneurons. By transplanting identified motoneurons between wild-type and mutant embryos, we found that the effect of the mutation was nonautonomous. We suggest that the segmental arrangement and proper axonal development of motoneurons may result from interactions with segmented mesoderm.

The growth cones of identified motoneurons in embryonic zebrafish select appropriate pathways in the absence of specific cellular interactions.

Developing motoneurons in zebrafish embryos follow a stereotyped sequence of axonal outgrowth and accurately project their axons to cell-specific target muscles. During axonal pathfinding, an identified motoneuron pioneers the peripheral motor pathway. Growth cones of later motoneurons interact with the pioneer via contact, coupling, and axonal fasciculation. In spite of these interactions, ablation of the pioneer motoneuron does not affect the ability of other identified motoneurons to select the pathways that lead to appropriate target muscles. We conclude that interactions between these cells during pathfinding are not required for accurate pathway selection.

Lateral specification of cell fate during vertebrate development.

Cells within equivalence groups interact via lateral specification to determine cell fates during development in Caenorhabditis elegans and other invertebrates. Populations of cells within the developing zebrafish have features similar to those of invertebrate equivalence groups. In a simple example, two identified zebrafish motoneurons behave as an equivalence pair in which one cell adopts a primary fate and interactions between the cells assign the other cell to a secondary fate. A more complicated situation exists for two initially equivalent populations of zebrafish neural crest cells. We consider whether mechanisms similar to those involved in fate specification within invertebrate equivalence groups also function furing fate specification in vertebrates.

Interactions with identified muscle cells break motoneuron equivalence in embryonic zebrafish.

Two zebrafish motoneurons, CaP and VaP, are initially developmentally equivalent; later, CaP innervates ventral muscle, whereas VaP dies. Current models suggest that vertebrate motoneuron death results from failure to compete for limited, target-derived trophic support. In contrast, we provide evidence that zebrafish ventral muscle can support both CaP and VaP survival. However, VaP's growth cone is prevented from extending into ventral muscle by CaP-dependent interactions with identified muscle fibers, the muscle pioneers; this interaction breaks the initial equivalence of CaP and VaP. Thus, the processes mediating VaP death are more complex than failure to compete for trophic support, and may be important for correct spatial patterning.

Image velocimetry and spectral analysis enable quantitative characterization of larval zebrafish gut motility.

BACKGROUND: Normal 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. Previous image analysis approaches have successfully extracted properties related to the temporal frequency of motility modes, but robust measures of contraction magnitude, especially from in vivo image data, remain challenging to obtain. METHODS: 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 motion. KEY RESULTS: 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. CONCLUSIONS & INFERENCES: 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.

Delta-mediated specification of midline cell fates in zebrafish embryos.

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, dlAdx2, which coordinately disrupts the development of floorplate, notochord and dorsal endoderm. The dlAdx2 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.

Best practices for germ-free derivation and gnotobiotic zebrafish husbandry.

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.

Patterning motoneurons in the vertebrate nervous system.

Vertebrate motoneurons show considerable diversity in their soma locations, axonal trajectories and innervation targets. Results from studies of a variety of vertebrate species as well as fruit-flies are elucidating the mechanisms by which this diversity is generated. Motoneuron subpopulations appear to be defined by combinations of transcription factor genes expressed in distinct spatiotemporal patterns in both motoneuron progenitors and postmitotic motoneurons. Notochord-derived signals can induce motoneuron formation, paraxial-mesoderm-derived signals can pattern motoneuron subpopulations along the rostrocaudal body axis, and local signals within the neural tube can regulate the number and time at which motoneurons form. Additional, later signals can promote formation of proper central circuitry and motoneuron survival. The identification of the genes and signals responsible for regulating these processes should help to provide a more-detailed understanding of motoneuron patterning.

Genetic and molecular analyses of motoneuron development.

Motoneurons have distinct identities and muscle targets. Recent classical and molecular genetic studies in flies and vertebrates have begun to elucidate how motoneuron identities and target specificities are established. Many of the same molecules participate in the guidance of both vertebrate and fly motor axons. It is less clear, however, whether the same molecular mechanisms establish vertebrate and fly motoneuron identities.

Zebrafish as a model for understanding enteric nervous system interactions in the developing intestinal tract.

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.

Expression of zebrafish fkd6 in neural crest-derived glia.

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.

Delta-Notch signaling and lateral inhibition in zebrafish spinal cord development.

BACKGROUND: Vertebrate neural development requires precise coordination of cell proliferation and cell specification to guide orderly transition of mitotically active precursor cells into different types of post-mitotic neurons and glia. Lateral inhibition, mediated by the Delta-Notch signaling pathway, may provide a mechanism to regulate proliferation and specification in the vertebrate nervous system. We examined delta and notch gene expression in zebrafish embryos and tested the role of lateral inhibition in spinal cord patterning by ablating cells and genetically disrupting Delta-Notch signaling. RESULTS: Zebrafish embryos express multiple delta and notch genes throughout the developing nervous system. All or most proliferative precursors appeared to express notch genes whereas subsets of precursors and post-mitotic neurons expressed delta genes. When we ablated identified primary motor neurons soon after they were born, they were replaced, indicating that specified neurons laterally inhibit neighboring precursors. Mutation of a delta gene caused precursor cells of the trunk neural tube to cease dividing prematurely and develop as neurons. Additionally, mutant embryos had excess early specified neurons, with fates appropriate for their normal positions within the neural tube, and a concomitant deficit of late specified cells. CONCLUSIONS: Our results are consistent with the idea that zebrafish Delta proteins, expressed by newly specified neurons, promote Notch activity in neighboring precursors. This signaling is required to maintain a proliferative precursor population and generate late-born neurons and glia. Thus, Delta-Notch signaling may diversify vertebrate neural cell fates by coordinating cell cycle control and cell specification.

Advanced life support vs basic life support field care: an outcome study.

OBJECTIVE: To determine whether the provision of advanced life support (ALS) field care has any impact on patient outcome in the urban Canadian environment. METHODS: A convenience cohort study was conducted of all emergent ambulance transfers of adults to an urban Canadian hospital from May 22 to July 31, 1996. Data were collected from ambulance call reports regarding presenting complaint and field interventions applied, and from hospital records regarding time in the ED, hospital length of stay (LOS), and discharge disposition. Patient outcomes were compared within 7 presenting complaint groups (chest pain, altered level of consciousness, shortness of breath, abdominal pain, motor vehicle crash, falls, and other) by field care level: level 1--BLS (basic life support) vs levels 2 and 3--ALS. RESULTS: The study population consisted of 1,397 patients. No significant differences were seen between BLS and ALS patients on baseline demographics. ED triage score did not depend on field care level for any group, implying that those in the ALS group were not inherently sicker. Outcome measures (ED LOS, admission rates, and hospital LOS) showed no significant differences between BLS and ALS for each presenting complaint group. Discharge dispositions were analyzed by chi2 but were not varied enough to allow reliable analysis. Observation of trends suggested no difference between BLS and ALS. CONCLUSIONS: There was no beneficial impact on the measured patient outcomes found in association with the provision of ALS vs BLS field care in Metropolitan Toronto for patients who were brought to a nontrauma center.