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.

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.

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.

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.

Mutations in the stumpy gene reveal intermediate targets for zebrafish motor axons.

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.

Pathfinding by identified zebrafish motoneurons in the absence of muscle pioneers.

To identify the cellular cues that guide zebrafish neuronal growth cones to their targets, we examined interactions between identified motor growth cones and identified muscle fibers and tested whether these fibers were required for growth cone navigation. Caudal primary motoneurons (CaPs) and middle primary motoneurons (MiPs) are identified motoneurons that innervate cell-specific regions of the myotome. Growth cones of both cells initially extend along a common pathway and then pause at a set of identified muscle fibers, called muscle pioneers, before diverging along cell-specific pathways. Muscle pioneers are intermediate targets of both CaP and MiP (Westerfield et al., 1986; Liu and Westerfield, 1990); both motoneurons extend their growth cones directly to the muscle pioneers on which the first functional neuromuscular contacts form, suggesting that muscle pioneers may provide guidance information to these growth cones. We tested this idea by ablating muscle pioneers and observing the resulting motor axonal trajectories. Both CaP and MiP ultimately formed normal axonal arbors after muscle pioneer ablation, showing that muscle pioneers are unnecessary for formation of correct axonal trajectories; however, although final cellular morphology was correct in the absence of muscle pioneers, MiP growth cones branched abnormally or extended ventrally beyond the common pathway. Ablation of CaP and the muscle pioneers together increased the aberrant behavior of the MiP growth cone. Our results provide evidence that an intermediate target, the muscle pioneers, affects motor axonal extension without altering target choice, suggesting that other cues also contribute to proper pathway navigation.

Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation.

We have examined the development of specific muscle fiber types in zebrafish axial muscle by labeling myogenic precursor cells with vital fluorescent dyes and following their subsequent differentiation and fate. Two populations of muscle precursors, medial and lateral, can be distinguished in the segmental plate by position, morphology and gene expression. The medial cells, known as adaxial cells, are large, cuboidal cells adjacent to the notochord that express myoD. Surprisingly, after somite formation, they migrate radially away from the notochord, becoming a superficial layer of muscle cells. A subset of adaxial cells develop into engrailed-expressing muscle pioneers. Adaxial cells differentiate into slow muscle fibers of the adult fish. We have named the lateral population of cells in the segmental plate, lateral presomitic cells. They are smaller, more irregularly shaped and separated from the notochord by adaxial cells; they do not express myoD until after somite formation. Lateral presomitic cells remain deep in the myotome and they differentiate into fast muscle fibers. Thus, slow and fast muscle fiber types in zebrafish axial muscle arise from distinct populations of cells in the segmental plate that develop in different cellular environments and display distinct behaviors.

Pathfinding by zebrafish motoneurons in the absence of normal pioneer axons.

Individually identified primary motoneurons of the zebrafish embryo pioneer cell-specific peripheral motor nerves. Later, the growth cones of secondary motoneurons extend along pathways pioneered by primary motor axons. To learn whether primary motor axons are required for pathway navigation by secondary motoneurons, we ablated primary motoneurons and examined subsequent pathfinding by the growth cones of secondary motoneurons. We found that ablation of the primary motoneuron that pioneers the ventral nerve delayed ventral nerve formation, but a normal-appearing nerve eventually formed. Therefore, the secondary motoneurons that extend axons in the ventral nerve were able to pioneer that pathway in the absence of the pathway-specific primary motoneuron. In contrast, in the absence of the primary motoneuron that normally pioneers the dorsal nerve, secondary motoneurons did not pioneer a nerve in the normal location, instead they formed dorsal nerves in an atypical position. This difference in the ability of these two groups of motoneurons to pioneer their normal pathways suggests that the guidance rules followed by their growth cones may be very different. Furthermore, the observation that the atypical dorsal nerves formed in a consistent incorrect location suggests that the growth cones of the secondary motoneurons that extend dorsally make hierarchical pathway choices.