Transcriptional profiling of extraocular motor neurons reveals sim1a as a candidate strabismus-related gene

Reviewer #1 (Public review):

This study adds important data identifying how ocular motor neurons are transcriptionally specified and identifies additional genes important in ocular motor neuron function. The evidence supporting the claims is convincing, with bulk and single-cell RNA sequencing as well as functional testing of the vestibulo-ocular reflex. This work will be of interest to developmental biologists and eye movement specialists.

Gershowitz, Hamling, et al investigate genes that specify specific cell populations within cranial motor nuclei III and IV, which control eye movements, by bulk and single-cell RNA sequencing, confirmatory in situ hybridization, and functional studies of vestibulo-ocular reflex in knock-out animals. They take advantage of the timing difference in the generation of dorsal versus ventral cells to selectively mark early-born (dorsal) vs late-born (ventral) cells using the Kaede photolabile protein. They used bulk RNASeq to identify differentially expressed genes between the two populations (which innervate different extraocular muscles). They next used single-cell RNASeq to further identify specific subpopulations of motor neurons and identify 3 main clusters, which broadly map to dorsal CNIII, CNIV, and ventral CNIII. They show that the differentially expressed genes identify subpopulations of neurons, rather than reflecting temporal changes related to cell age via a series of in situ hybridizations across ages. Finally, they show that knock-out of Sim1a, which is unregulated in dorsal nIII neurons, leads to decreased vestibulo-ocular reflex, despite a normal number of neurons in nIII. They tested the knock-out of two other differentially expressed genes, nav2a and onecut1, but found both normal cell number and normal vestibulo-ocular reflex.

The conclusions of this paper are well supported by the data. As the authors acknowledge, additional experiments would add to the interpretation. Since the Sim1a mutants have normal cell numbers, the authors hypothesize that axon guidance may be disrupted, leading to the phenotype. This could be relatively easily assessed using the Isl1-GFP transgenic line and examining innervation patterns in the extraocular muscles. Additionally, testing horizontal eye movements and eye movements in response to visual, rather than vestibular, inputs would further refine the phenotypes and perhaps identify eye movement abnormalities in the mutant fish with normal VOR.

More information on why these specific genes were prioritized for functional testing would be helpful, as it is unclear why these three genes were the top candidates.

The authors should also include a discussion of other subtypes of oculomotor neurons, beyond which muscle they innervate. For example, there are oculomotor neurons that form single neuromuscular junctions on fast, singly-innervated fibers, and there is a separate pool of motor neurons that innervate the slow, multiply-innervated fibers. It would be interesting to note if there were any gene expression differences within the clusters that might represent this subdivision of neurons.

This data is likely to be of great use to the field in further studies of cranial motor neuron biology.

Reviewer #2 (Public review):

Summary:

The goal of the work is to identify genes that are uniquely expressed in subsets of eye muscle-innervating motor neurons, as a way to identify candidate genes for strabismus, a congenital vision disorder in humans. The author's previous work identified birth-order differences that correlate with the positions of neurons in the oculomotor (cranial nerve III) motor nucleus. Here, they use Kaede photoconversion to distinguish early- from late-born neurons and identified transcriptional differences between them by bulk RNA sequencing of FACS-sorted cells. Separately, they used single-cell RNA-Seq to sequence the transcriptomes of 89 extraocular motor neurons. They find signatures of early-born mIII, late-born mIII, and mIV neurons. While there is some overlap in gene expression, some of the differentially expressed genes are confirmed by HCR as being unique to one of these three populations of extraocular motor neurons.

The authors test the functions of three differentially expressed genes in the vestibulo-ocular reflex by measuring the speed of rotation of the eye in response to the larval fish being tilted 15° from horizontal. One mutant, in the sim1a transcription factor, has markedly slowed responses. Although this is a global knock-out, the authors argue that this defect in the vestibulo-ocular reflex is due to a loss of sim1a function specifically in dorsal mIII neurons because sim1a is not expressed in the two upstream neurons in the vestibulo-ocular reflex circuit.

Strengths:

(1) This is the first time that transcriptional differences between and within extraocular muscle-innervating neurons have been described during development. In identifying differentially expressed genes that correspond with anatomical, functional, and temporal subdivisions of these neurons, they support the idea that gene expression programs established early in development underlie the functional differences amongst these neurons.

(2) The combination of bulk RNA-Seq and single-cell RNA-Seq strengthens the identification of sim1a-expressing early-born mIII neuron subtype.

(3) The work identifies candidate genes for strabismus.

Weaknesses:

(1) The authors show that sim1a is only expressed in mIII neurons and no other cells in the vestibulo-ocular reflex, as evidence that the phenotype in sim1a mutants is due to loss of its expression specifically in mIII neurons. However, as the authors note in the discussion, sim1a has other functions in zebrafish, including global calcium homeostasis via specification of the corpuscles of Stannius. The loss of this, or of some other sim1a function, could be indirectly responsible for the slow vestibulo-ocular response in sim1a mutants.

(2) The authors perform the vestibulo-ocular response test in sim1a mutants at 7 dpf, which is within a day of when the mutants die, raising the concern that the slowed response is due to a dire systemic condition. The argument that nav2 mutants also die at 7 dpf but have a normal response is weak, since death does not always take a single course.

(3) The evaluation of the sim1a mutant phenotype is limited to the vestibulo-ocular reflex. The authors do not explore whether the oculomotor neuron innervation of target extraocular muscles is affected in sim1a mutants.