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Cell-tracking pipeline reveals how motor circuits are built

A sophisticated imaging pipeline has been developed to track neurons in early-stage zebrafish embryos over time and space. It reveals how newborn neurons come together to build a spinal cord capable of locomotion.
Kristen P. D’Elia is in the Departments of Otolaryngology and of Neuroscience and Physiology, Neuroscience Institute, New York University Langone Medical Center, New York, New York 10016, USA.

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David Schoppik is in the Departments of Otolaryngology and of Neuroscience and Physiology, Neuroscience Institute, New York University Langone Medical Center, New York, New York 10016, USA.
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Where a person comes from and what they do are often considered key parts of their identity. Similarly, neurons can be categorized by both their developmental history and their role in the nervous system. But, just as knowing someone’s job title does not necessarily tell you what part they play in a team at work, knowing what role a neuron has does not mean that we understand how it comes together with other diverse neuron types to form circuits — for instance, to permit movement. Writing in Cell, Wan et al.1 describe an imaging protocol that will help researchers determine how neural circuits form. They use their method to comprehensively chart motor-circuit assembly and emerging function in the spinal cord of zebrafish.

In vertebrate embryos, the first neuronal circuits to respond to sensory information and orchestrate movement are found in the spine2. These motor circuits are assembled from dozens of molecularly specialized types of neuron. Nonetheless, this is a relatively simple set-up, making it a useful system for studying how neuronal circuits come together to produce behaviour — in this case, muscles contracting in distinct patterns.

Wan et al. set out to study the formation of these early motor circuits in zebrafish embryos (Fig. 1). This research group has long been at the forefront of in vivo microscopy, pioneering light-sheet microscopy techniques that can illuminate all of the individual cells that make up developing organisms such as zebrafish without harming them. Zebrafish are well suited to such studies because they are small, transparent and develop rapidly.

Figure 1 | Tracking the building blocks of a circuit. Wan et al.1 have developed an imaging and computational pipeline to track neurons of the zebrafish spinal cord, from their ‘birth’ 6 hours after embryo fertilization until they begin to show the coordinated activity of a motor circuit at 22 hours. The authors traced newly born sister cells (derived from the same immediate ancestor, indicated by different shades of the same colour). By 17 hours, the cells have migrated to their mature positions and adopted molecular characteristics of either motor neurons (star-shaped cell) or interneurons (circular cell). By 22 hours, the cells become wired into coordinated circuits (inset). Motor neurons are the first to become active, and the authors showed that they then imprint their activity onto other neurons (dotted arrows), leading these neurons to adopt the same activity pattern.

The researchers imaged zebrafish from 6 hours after embryo fertilization, when spinal neurons first arise from their progenitors, to 22 hours after fertilization, when the patterns of neuronal activity that trigger tail movements begin. The imaging process generated vast libraries of images that Wan and colleagues processed to extract information about the location of individual cells over time. In addition, the authors optimized their microscope design to allow them to measure emergent patterns of functional activity from individual cells. The result was a data set that enabled the group to track the organization and function of every cell in the zebrafish spinal cord throughout early development.

Motor neurons and interneurons are key neuron types in spinal motor circuits. The former are responsible for triggering muscle-fibre contraction and the latter coordinate signalling within and between circuits3 (for example, to ensure alternating left–right movements during swimming). Motor neurons have often been thought of as passive cells controlled by upstream interneuron inputs, whereas interneurons had been thought to be the driving force behind the assembly and function of spinal motor circuits4. But over the past few years, evidence has emerged that both developing5 and mature6 motor neurons can control their connections to interneurons, and even control interneuron activity. In zebrafish, motor neurons are the first spinal neurons to display spontaneous activity patterns7. As a circuit develops, neurons often first become active on their own, and then coordinate their activity with that of other neurons. Wan et al. therefore asked whether this activity originates in the motor neurons themselves, or reflects interneuron control.

The authors found that select motor neurons seem to impose their own activity on neighbouring motor neurons and interneurons, producing pairs of cells that have the same activity patterns. Thus, the earliest patterns of collective activity are initiated by motor neurons. This finding adds to the emerging picture of motor neurons as a fundamental driver of spinal-cord development. Consistent with previous findings, the authors also confirmed that interneurons coordinate the global patterns of activity necessary at later developmental stages for tail movement.

One theory of neural development states that cells that have a shared ancestry are destined to have common connectivity, and to perform similar roles in a circuit8. Evidence for such determinism remains contentious, reflecting the challenge of tracing related neurons as they migrate9. But Wan and colleagues were able to investigate this issue, thanks to their ability to comprehensively track cells over time.

The authors examined the activity of sister neurons — those that shared an immediate ancestor. In line with ideas of determinism, sister neurons that ended up in close proximity to one another were more likely than unrelated neurons to be co-active. But, intriguingly, most sibling pairs did not remain close to one another. Indeed, sister neurons were just as likely to migrate to opposite sides of the spinal cord, where they would participate in different phases of movement. Thus, ancestry can explain only a small part of functional organization. That said, Wan and colleagues’ study is limited to the earliest part of development, well before zebrafish hatch and swim freely. It will be interesting to re-evaluate questions of ancestral determinism over longer periods of time.

Another limitation of the authors’ technique is that their cutting-edge microscope is best suited to small model organisms. It would be interesting to analyse whether their findings also apply to more-complex organisms. However, current microscopes cannot be used for such purposes.

Notably, the group that performed the study (and the Janelia Research Campus in Ashburn, Virginia, at which it works) is committed to providing access to the microscope used in the current work. In addition, the authors’ data and analysis pipelines are available to download. Thus, other researchers can further assess the relationship between the developmental history and function outlined in the current study.

Advances in the transcriptional profiling of single cells have revealed remarkable variability among neurons10, making circuit development ever-more fascinating but incredibly challenging to fully understand. Until we have a greater understanding of the molecular logic that enables neurons to form motor circuits, our ability to prevent, diagnose and treat disorders of movement will remain limited. The apparatus and analysis pipeline developed by Wan et al. present a technically demanding but demonstrably fruitful path towards better grasping how a neuron’s birth shapes its future role in a circuit.

Nature 576, 46-47 (2019)

doi: 10.1038/d41586-019-03492-6

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