In a manner akin to electrical cables, nerves transmit signals throughout the body to control physical responses. This function depends on specialized cells called neurons, which extend long protrusions (axons) from their cell body that form synapses with cells in distant locations (Figure 1). Neuronal networks are often organized into topographic maps, meaning that the spatial organization of the neuron cell bodies mirrors the spatial organization of their axon targets, which can be located in a completely different region of the body. How these topographic maps form during development is only partially understood.
The Moens Laboratory in the Basic Sciences Division studies the development of neuronal networks that connect the brain to the muscles of the head and neck. Using zebrafish as a model organism, they are investigating how vagus motor neurons originating in the hindbrain innervate pharyngeal arches, the developmental precursors of structures in the throat. As explained by Dr. Cecilia Moens, “before we started this work, it was known that anterior vagus neurons target anterior muscles and posterior neurons target posterior muscles, but how?” Previous work in the field focused on the role of spatial patterning, in which gene expression gradients across a group of neurons control axon localization in a “point-to-point matching system”. However, spatial patterning is unlikely to be the only mechanism in play during topographic map formation because “the genome encodes only a limited number of axon guidance molecules—not nearly enough to uniquely guide each motor nerve to its appropriate target,” stated Dr. Moens.
Dr. Moens and a graduate student in her lab, Gabrielle Barsh, hypothesized that topographic map formation might be governed not only by spatial factors, but by temporal ones as well. Prior studies in other model organisms identified correlations between temporal patterning and topographic mapping, but efforts to establish causality were hampered by experimental constraints. By taking advantage of their abilities to image zebrafish development in real time and to transplant single neurons into different temporal environments, Ms. Barsh and Dr. Moens, along with post-doctoral fellow Dr. Adam Isabella, were able to directly test their hypothesis. Their results, recently published in Current Biology, establish that topographic connectivity in the zebrafish hindbrain is regulated in parallel by both spatial and temporal factors.
To observe innervation of the pharyngeal arches (PA) by the axons of vagus motor neurons (abbreviated mX neurons, for motor neurons of cranial nerve X), the researchers used cell type-specific expression constructs to label PA cells and mX neurons with different fluorescent proteins (Figure 2). This approach revealed that mX axons emerge sequentially from the hindbrain in an anterior-to-posterior order, innervating anterior PAs first and posterior PAs later.
By reducing the amount of the mX-specific construct injected into the embryo, the authors could label single mX neurons to show that those located in anterior positions only innervate anterior PAs, while neurons in the posterior region only innervate posterior PAs. The investigators also transferred single neurons to different positions and observed which PAs they targeted. Transplanted neurons had a strong tendency to innervate PAs corresponding to the transplanted location, not the original location, confirming that neuron position drives axon targeting.
How does the location of an mX neuron determine which PA its axon targets? A likely player in this process was the hox gene family, a class of transcription factors known to be involved in spatial patterning throughout the vertebrate body. Hox genes are expressed in gradients across tissues, and the Moens lab determined that the hox5 gradient specifically overlaps the region of the zebrafish hindbrain in which the mX neurons are located: hox5 genes are expressed in the posterior region but not the anterior region.
In order to test whether hox5 expression drives posterior PA targeting, the authors injected a construct expressing both hox5 and a fluorescent protein into single-cell stage embryos. This method generates embryos with hox5-expressing neurons scattered randomly throughout the vagus region, allowing the researchers to ask whether hox5+ neurons target posterior PAs regardless of their own position on the anterior-posterior axis. Indeed, they observed that axons originating from hox5+ neurons were strongly biased toward innervating posterior PAs.
The authors next asked whether temporal factors might also be at play in regulating axon targeting. They observed that the timing of axon initiation varies as a function of location; axons emerge at 30 hours post-fertilization (hpf) from anterior neurons but not until 38 hpf from posterior neurons, even though mX neurons along the anterior-posterior axis are all the same age. To confirm a causal link between neuron position and the timing of axon initiation, the authors applied their transplantation method to show that neurons transplanted from anterior to posterior positions delayed axon formation, while posterior-to-anterior transplantation caused axons to form earlier.
So, anterior axons form earlier, but is this the reason that they innervate anterior PAs? If this is the case, then anterior neurons transferred from a young donor to a similar position in an older recipient embryo should innervate more posterior targets than they normally would. Indeed, the investigators observed exactly this bias. Importantly, transplantation did not cause the cells to turn on hox5, suggesting that regulation of axon initiation time is independent of hox5-driven spatial patterning. To confirm that late axon initiation in posterior neurons is not driven by hox5, the authors again employed their single-cell injection method to scatter hox5-expressing neurons along the anterior-posterior axis. They observed that hox5-expressing neurons in anterior positions still initiated axons early, indicating that hox5 expression is insufficient to delay axon formation.
Together, the Moens lab’s results reveal that, in addition to hox5-dependent spatial patterning, temporal regulation of axon initiation is a key determinant of topographic map formation in the zebrafish hindbrain. “This finding represents a major departure from how people generally think about topographic mapping,” said Dr. Moens. Much work remains to understand how the temporal and spatial mechanisms interact with each other. In addition, future research in the Moens lab will focus on identifying molecular cue(s) that regulate the timing of axon outgrowth and guidance of late-arriving axons to posterior targets.
Barsh GR, Isabella AJ, Moens CB. (2017). Vagus Motor Neuron Topographic Map Determined by Parallel Mechanisms of hox5 Expression and Time of Axon Initiation. Current Biology. 27(24):3812-3825.
This research was supported by the National Institutes of Health