Motor neurons are dispensable for the assembly of a sensorimotor circuit for gaze stabilization

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RESEARCH ARTICLE Motor neurons are dispensable for the assembly of a sensorimotor circuit for gaze stabilization

eLife Assessment

This important study asks whether motor neurons within the vestibulo-ocular circuit of zebrafish are required to determine the identity, connectivity, and function of upstream premotor neurons. They provide compelling and comprehensive genetic, anatomical and behavioral evidence that the answer is, "No!". This work will be of general interest to developmental neurobiologists and will motivate future studies of whether motor neurons are dispensable for assembly of other sensorimotor neural circuits.

Abstract Sensorimotor reflex circuits engage distinct neuronal subtypes, defined by precise connectivity, to transform sensation into compensatory behavior. Whether and how motor neuron populations specify the subtype fate and/or sensory connectivity of their pre-motor partners remains controversial. Here, we discovered that motor neurons are dispensable for proper connectivity in the vestibular reflex circuit that stabilizes gaze. We first measured activity following vestibular sensation in pre-motor projection neurons after constitutive loss of their extraocular motor neuron partners. We observed normal responses and topography indicative of unchanged functional connectivity between sensory neurons and projection neurons. Next, we show that projection neurons remain anatomically and molecularly poised to connect appropriately with their downstream partners. Lastly, we show that the transcriptional signatures that typify projection neurons develop independently of motor partners. Our findings comprehensively overturn a long-standing model: that connectivity in the circuit for gaze stabilization is retrogradely determined by motor partner-derived signals. By defining the contribution of motor neurons to specification of an archetypal sensorimotor circuit, our work speaks to comparable processes in the spinal cord and advances our understanding of principles of neural development.

Introduction

Developing sensorimotor reflex circuits must precisely connect functional subtypes of neurons to ensure appropriate behavior. For example, withdrawal from noxious stimuli requires maturation of a sensorimotor circuit that uses subtypes of spinal interneurons to transform noxious stimulation into activation of both ipsilateral flexor and contralateral extensor motor neurons. Work over the past forty years has highlighted motor partner populations as possible orchestrators of connectivity in pre-motor reflex circuits.

eLife digest Some external stimuli, such as a painful touch or sudden head movements, can trigger automatic physical responses. These reactions are controlled by sensorimotor circuits which are comprised of three types of neurons. First, sensory neurons detect the external stimulus. They then pass the information to interneurons, which relay the signal to motor neurons that activate the muscles required to produce a prompt physical response.

Sensorimotor circuits form very early in life, but it remains unclear how the three types of neurons involved contribute to one another's development. Previous research suggests that motor neurons send chemical signals 'upstream' to interneurons to help them mature. However, there is conflicting evidence both for and against this hypothesis.

To investigate this theory, Goldblatt et al. studied a sensorimotor circuit found in all vertebrates known as the gaze stabilization reflex. The sensory neurons in this circuit detect head movements and transmit this information, via interneurons, to the motor neurons that control muscles in the eye. This allows gaze to remain stable while the head moves.

Goldblatt et al. used a genetic tool to eliminate the motor neurons involved in the gaze stabilization reflex from zebrafish larvae. This prevented the motor neurons from sending chemical signals upstream to the interneurons and caused the zebrafish larvae to develop eyes that were permanently rotated outward.

However, further experiments revealed that the connections between sensory neurons and interneurons still developed normally despite the absence of motor neurons. The interneurons also expressed the appropriate set of genes for the stage of larval development tested. This suggests that interneurons involved in the gaze stabilization reflex can develop normally without chemical signals from motor neurons.

These findings are a major step towards understanding how sensorimotor circuits develop, and suggest that current models of this process may need to be revised. It remains to be seen whether other sensorimotor circuits can also develop without signals from downstream motor neurons. Gaining more detailed insights into how these circuits develop could also enhance our understanding of certain neurological conditions further down the line.

but controversy remains about the nature of their role. In the spinal cord, molecular perturbations of motor neuron identity have provided evidence both for and against an instructive role in establishing connectivity. Part of this controversy stems from the wide variety of inputs to spinal motor neurons, the molecular and functional heterogeneity of pre-motor interneurons, and their complex roles in gait and posture. Further, transcription factors play multivariate and redundant roles in spinal motor neuron development, such that the effects of molecular perturbations of identity can be masked.

The sensorimotor circuit for vertical gaze stabilization offers a simple framework to evaluate whether and how motor neurons shape pre-motor circuit fate and connectivity. The vertebrate vestibulo-ocular reflex circuit consists of three neuron types - peripheral sensory, central projection, and extraocular motor neurons - that stabilize gaze after head/body tilts. Subtype fate, anatomical connectivity, and function are inextricably linked: directionally-tuned sensory neurons innervate nose-up/nose-down subtypes of projection neurons, which in turn innervate specific motor neurons that selectively control either eyes-down or eyes-up muscles. As both the recipients and origin of directional information, projection neuron fate specification is paramount to proper circuit assembly. Recent work has established the vertical vestibulo-ocular reflex circuit in zebrafish as a model to uncover determinants of fate and connectivity given the ease of optical imaging, abundant tools for genetic perturbations, rapid development, and robust evolutionary conservation.

The current model for vestibulo-ocular reflex circuit development was motivated by pioneering work in chick and formalized by Hans Straka: "[circuit assembly] is accomplished by a specification process that retrogradely transmits post-synaptic target identities to pre-synaptic neurons." In its strongest form, this 'retrograde' model posits a causal role

Developmental Biology

Peripheral-to-central circuit assembly does not require motor partners

Discussion

Transcriptional influences on motor neuron fate specification

Motor neurons: active or passive architects of pre-motor connectivity?

Alternative mechanisms for fate specification and sensory input specificity in projection neurons

Developmental Biology

Conclusion

Materials and methods

Materials availability

Experimental model and subject details

Transgenic lines

Generation of phox two A mutants

Maintenance of phox two A adults

Method details

Identification of phox two A larvae

Birthdating of n three/n four motor neurons

Fluorescent in situ hybridization and imaging

Calcium imaging of tonic and impulse tilt stimuli responses

Retrograde photolabeling of tangential nucleus neurons

Neuron harvesting, dissociation, and flow cytometry

Bulk RNA sequencing

Quantification and statistical analysis

Analysis of calcium imaging experiments

Spatial mapping of tangential nucleus neurons

Alignment, quality control, and differential expression analysis of bulk sequencing data

Developmental Biology

Filtering of bulk sequencing data using a reference single-cell sequencing dataset

Additional statistics

Author contributions

Data availability

Overview

The study examines whether motor neurons influence the connectivity and function of upstream premotor neurons within a zebrafish sensorimotor circuit. Findings reveal that motor neurons are not required for the proper development and function of the circuit, challenging previous models of sensorimotor connectivity.

Key Points

1Motor neurons do not determine the identity or connectivity of premotor neurons in the circuit for gaze stabilization
2Normal activity was observed in premotor projection neurons despite the absence of motor neurons
3The study provides genetic and anatomical evidence that supports the dispensability of motor neurons
4Findings suggest a need to revise existing models of sensorimotor circuit development
5Understanding motor neuron dispensability could inform future studies on other neural circuits.

Details

Authors: Goldblatt et al.
Category: Biology and Natural Sciences

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