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Molecular and cellular determinants of motor asymmetry in zebrafish

Eric J. Horstick, Yared Bayleyen, Harold A. Burgess

Preprint posted on June 11, 2019 https://www.biorxiv.org/content/10.1101/666594v1

Why are we not naturally ambidextrous? - Horstick et al. investigate the causes underlying motor asymmetry

Selected by Amrutha Swaminathan

Background:

Many species show motor asymmetry, which means they have a preference to using one hand/paw over the other. Some brain areas have been associated with this motor bias, but the neuronal mechanisms underlying this bias remain poorly understood. Further, there is almost no molecular understanding of the determination of motor bias. In this study, the authors report the establishment of a developmental model showing robust motor bias. Their results contribute to a better understanding of the neuronal and molecular factors underlying this phenomenon.

Key findings:

In this preprint, Horstick et al. show that zebrafish larvae have a stable left- or right-identity as early as 6 days post fertilization (dpf). The authors have developed an elegant assay, where they observe that when exposed to darkness for 30 seconds, some larvae prefer circular swimming to the right, and others, to the left. Unlike humans, where 90% of the population is right-handed, zebrafish show almost equal distribution of right- and left-preference. The authors have designed careful experiments, and measure this behavior 4 times for each individual to confirm that the motor bias matches across trials.

Further, this attribute is retained for at least 4 days (tested at 10 dpf). In addition to using visual experience (illumination changes), the authors show that acoustic stimuli can also result in the same behavior as visual stimuli. Interestingly, the authors go another step in showing that blind mutants also show this preference by virtue of having deep brain photoreceptors. The authors also show that motor bias is not inherited from the previous generation.

In their quest to identify a mechanism underlying this phenomenon, which is a major caveat in the field, the authors perform a broad screen using the UAS:Gal4 system combined with the bacterial nitroreductase system, which can be used for temporally and spatially controlled cell ablation. By employing numerous Gal4 lines showing varied expression patterns, they specifically ablate Gal4 expressing cells in the brain, and check for persistent handedness in these lines. Of the Gal4 driver lines they screened, the authors found two Gal4 lines: y279 and y375, which show no preferential bias for swimming to either side. Upon analyzing the expression pattern of Gal4 in these lines, they find the posterior tuberculum neurons to overlap between the lines. Validating the role of the posterior tuberculum neurons, ablation of these neurons leads to loss of left/right motor bias. These neurons project to the habenula, and consistently, ablation of the habenula neurons also leads to loss of left/right bias.

In the last part of their study, the authors show using a mutant, that, when Epb41l5, a Notch pathway factor, is mutated, the development of left/right bias is perturbed.

To summarize, the authors demonstrate, using a novel and interesting approach, that young zebrafish larvae show a left/right identity. The neuronal network extending from the posterior tuberculum to the habenula and the Notch signaling pathway are both critical for the determination and maintenance of this motor bias.

Why do I like this work?

As the authors have pointed out in the manuscript, the cellular and molecular basis underlying left-/right-handedness is not well understood, and the study addresses this caveat in the field. Specifically, I liked two features of this study:

  1. I liked the robustness of the observations the authors made in this study. In particular, the results show clearly, using multiple methods, that zebrafish larvae do indeed show left-/right preference. These results also reinforce the developmental nature of the determination of handedness.
  2. The authors take an unbiased and logical approach to identify the neuronal clusters involved in the phenomenon, which gives new insight into the neuronal circuitry.

Future directions/questions for the authors:

  1. The authors show that the posterior tuberculum and habenula neurons are critical for the motor bias. Following this, they shift gears and show that Epb41l5 in the Notch pathway is important to develop the left-/right bias. What is the connection between the circuitry and molecular mechanism?
  2. Do the PT neurons project to other regions? Is it possible that the circuit is larger than just the PT-habenula neurons that the authors show?
  3. Why did the authors choose the y606 mutant?
  4. In multiple instances, it is evident that not all larvae show clear motor bias. Could this be due to a developmental delay? Or greater plasticity in some individuals?
  5. The experiments were performed in TL genetic background of fish. Out of curiosity, do other genetic backgrounds also show similar motor bias?
  6. Do all zebrafish adults show such bias? Is it the same tendency as what they showed when young?

 

Posted on: 17th July 2019

Read preprint (1 votes)




  • 1 comment

    1 month

    Harry and Eric

    Hi Amrutha

    Thanks for your very nice summary of our work! We are especially glad that you appreciated our efforts to build a robust assay as we were our own fiercest critics throughout the project, constantly probing our results from every angle.

    Your first question, regarding how epb41l5 and notch signaling influence the motor bias circuit, is precisely the one we are most interested in for the next part of our studies. We need to examine not only structural properties of the circuit in epb41l5 mutants, but also determine if signaling in the PT or habenula is abnormal and linked to the loss of motor bias. This will be a focus of Eric’s work in his new lab at the University of West Virginia, so if any potential grad students or postdocs are interested – please apply to him!

    PT neurons also project caudally, into the medulla oblongata, a projection that may also influence motor bias. However because (1) the habenula is required for motor bias and (2) severing the PT projection across the habenula commissure lead to a loss of motor bias, we focused on this part of the circuit. We are working on methods that will allow us (and others) to more selectively lesion specific outputs from genetically targeted neurons, so stay tuned.

    We didn’t chose the y606 mutant… it chose us. You asked if motor bias is only present in TL – we’ve tested several other stains and always found robust motor bias in larvae. However a few years ago, the assay abruptly stopped working in our TL wildtype stock. After several months effort, we were able to sort our adult stock into ‘clean’ fish, where the progeny showed normal motor bias, and a contaminated group. We realized that among progeny of the contaminated group, 1/4 had a strong morphological curly-up phenotype, 1/4 had ok motor bias, and 1/2 had no motor bias. So we suspected that the morphological phenotype was recessive, and loss of motor bias the result of haploinsufficiency. So, as we describe in the manuscript, we mapped the curly-up phenotype to epb41l5. This lead to much consternation, as our neighbors, the Chitnis lab have previously worked on this gene and we were concerned that we must have somehow contaminated our stock with their mutation. Yet, mapping the breakpoints of the y606 deletion and of our neighbor’s mutant, revealed that these are actually different alleles. Remarkably then, y606 is a spontaneous small deletion in epb41l5, heterozygous mutations in which are sufficient to disrupt motor bias. Two take-away messages are that (1) relatively subtle behaviors like motor bias may be most susceptible to background gene mutations, and (2) you never know where the next breakthrough may come from – it may just be spontaneous!

    Again, thanks for your terrific summary.

    4

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