A microRNA that controls the emergence of embryonic movement

Reviewer #1 (Public Review):

Summary:

This is an experimentally soundly designed work and a very well-written manuscript. There is a very clear logic that drives the reader from one experiment to the next, the experimental design is clearly explained throughout and the relevance of the acquired data is well analyzed and supports the claims made by the authors. The authors made an evident effort to combine imaging, genetic, and molecular data to describe previously unknown early embryonic movement patterns and to identify regulatory mechanisms that control several aspects of it.

Strengths:

The authors develop a new method to analyze, quantitatively, the onset of movement during the latter embryonic stages of Drosophila development. This setup allows for a high throughput analysis of general movement dynamics based on the capture of variations of light intensity reflected by the embryo. This setup is capable of imaging several embryos simultaneously and provides a detailed measure of movement over time, which proves to be very useful for further discoveries in the manuscript. This setup already provides a thorough and quantifiable description of a process that is little known and identifies two different phases during late embryonic movements: a myogenic phase and a neurogenic phase, which they elegantly prove is dependent on neuronal activity by knocking down action potentials across the nervous system.

However, in this system, movement is detected as a whole, and no further description of the type of movement is provided beyond frequency and amplitude; it would be interesting to know from the authors if a more precise description of the movements that take place at this stage can be achieved with this method (e.g. motion patterns across the A-P body axis).

Importantly, this highly quantitative experimental setup is an excellent system for performing screenings of motion regulators during late embryonic development, and its use could be extended to search for different modulators of the process, beyond miRNAs (genetic mutants, drugs, etc.).

Using their newly established motion detection pipeline, the authors identify miR-2b-1 as required for proper larval and embryonic motion, and identify an overall reduction in the quantity of both myogenic and neurogenic movements, as well as an increased frequency in neurogenic movement "pulses".

Focusing on the neurogenic movement phenotype the authors use in situ probes and perform RT-PCR on FACS-sorted CNS cells to unambiguously detect miR-2b-1 expression in the embryonic nervous system. The neurogenic motion defects observed in miR-2b-1 mutant embryos and early larvae can be completely rescued by the expression of ectopic miR-2b-1 specifically in the nervous system, providing solid evidence of the requirement and sufficiency of miR-2b-1 expressed in the nervous system to regulate these phases of movement.

To explore the mechanism through which miR-2b-1 impacts embryonic movement, the authors use a state-of-the-art bioinformatic approach to identify potential targets of miR-2b-1, and find that the expression levels of an uncharacterized gene, CG3638, are indeed regulated by miR-2b-1. Furthermore, they prove that by knocking down the expression of CG3638 in a miR-2b-1 mutant background, the neurogenic embryonic movement defects are rescued, pointing that the repression of CG3638 by miR-2b-1 is necessary for correct motion patterns in wild-type embryos. Therefore, this paper provides the first functional characterization of CG3638, and names this gene Janus.

Finally, the authors aim to discriminate which elements of the embryonic motor system miR-2b-1/Janus are required. Using directed overexpression of miR-2b-1 and Janus knockdown in the motor neurons and the chordotonal (sensory) organs, they prove that the miR-2b-1/Janus regulatory axis is specifically required in the sensory organs to promote normal embryonic and larval movement.

Weaknesses:

The authors do not describe properly how the miRNA screening was performed and just claim that only miR-2b-1 mutants presented a defective motion phenotype in early L1. How many miRNAs were tested, and how candidates were selected is never explicitly mentioned in the text or the Methods section.

The initial screening to identify miRNAs involved in motion behaviors is performed in early larval movement. The logic presented by the authors is clear - it is assumed that early larval movement cannot proceed normally in the absence of previous embryonic motion - and ultimately helped them identify a miRNA required for modulation of embryonic movement. However, it is possible that certain miRNAs play a role in the modulation of embryonic movement while being dispensable for early L1 behaviors. Such regulators might have been missed with the current screening setup.

Although similar changes to those described for the neurogenic phase of embryonic movement are described for the myogenic phase in miR-2b-1 mutants (reduction in motion amplitude), this phenotype goes unexplored. This is not a big issue, as the authors convincingly demonstrate later that miR-2b-1 is specifically required in the nervous system for proper embryonic and larval movement, and the effects of miR-2b-1 on myogenic movement might as well be the focus of future work. However, it will be interesting to discuss here the implications of a reduced myogenic movement phase, especially as miR-2b-1 is specifically involved in regulating the activity of the chordotonal system - which precisely detects early myogenic movements.

FACS-sorting of neuronal cells followed by RT-PCR convincingly detects the presence of miR-2b-1 in the embryonic CNS. However, control of non-neuronal cells would be required to explore whether miR-2b-1 is not only present but enriched in the nervous system compared to other tissues. This is also the case in the miR-2b-1 and Janus expression analysis in the chordotonal organs: a control sample from the motor neurons would help discriminate whether miR-2b-1/Janus regulatory axis is specifically enriched in chordotonal organs or whether both genes are expressed throughout the CNS but operate under a different regulation or requirements for the movement phenotypes.

Reviewer #2 (Public Review):

Summary:
The manuscript, "A microRNA that controls the emergence of embryonic movement" by Menzies, Chagas, and Alonso provides evidence that Drosophila miR-2b-1 is expressed in neurons and controls the expression of the predicted chloride channel CG3638, here named "Janus". Loss of the miRNA leads to movement phenotypes that can be rescued by downregulation of Janus; using specific drivers, the authors show that a larval movement phenotype (slower movement) can be rescued by knockdown of Janus in the chordotonal organs, suggesting that the increase in Janus found in the chordotonal organs is likely the root of the movement defects. Overall, I found the data presented in the manuscript of reasonable quality and are well enough supported by the presented data. That being said, I do have a few problems with the manuscript, mostly stemming from what I feel is an inflated presentation of the importance of the findings.

Strengths:
The genetic and phenotypic analysis seems to be correct. The nicest part of the manuscript is the connection between the loss of a miRNA and finding its likely target in generating a phenotype. The authors also develop some protocols for the analysis of the movement phenotypes which may be useful for others.

Weaknesses:
As I mentioned above, I felt the presentation was a bit overstated. The authors present their data in a way that focuses on movement, the emergence of movement, and how their miRNA of interest is at the center of this topic. I only point to the title and name that they wish to give the target of their miRNA to emphasize this point. "Janus" the god of movement and change. The results and discussion section starts with a paragraph saying, "Movement is the main output of the nervous system... how developing embryos manage to organise the necessary molecular, cellular, and physiological processes to initiate patterned movement is still unknown. Although it is clear that the genetic system plays a role, how genes control the formation, maturation and function of the cellular networks underlying the emergence of motor control remains poorly understood." While there is nothing inherently untrue about these statements, it is a question of levels of understanding. One can always argue that something in biology is still unknown at a certain level. However, one could also argue that much is known about the molecular nature of movement. Next, I am not sure how much this work impacts the area of study regarding the emergence of movement. The authors show that a reduction of a miRNA can affect something about certain neurons, that affects movement. The early movements, although slightly diminished, still emerge. Thus, their work only suggests that the function of some neurons, or perhaps the development of these neurons may impact the early movements. This is not new as it was known already from early work from the Bate lab.

Later larval movements were also shown to be modified in the miRNA mutants and were traced to "janus" overexpression in the chordotonal organs. As neurons are quite sensitive to the levels of Cl- and Janus is thought to be a Cl- channel, this could lead to a slight dysfunction of the chordotonal neurons. So, based on this, the work suggests that dysfunction of the chordotonal organs could impact larval movement. This was, of course, already known. The novelty of this work is in the genes being studied (important or not). We now know that miR 2b-1 and Janus are expressed in the early neurons and larval chordotonal neurons and their removal is consistent with a role for these genes in the functioning of these neurons. This is not to trivialize these findings, simply to state that these results are not significantly changing our overall understanding of movement and the emergence of movement. I would call it a stretch to say that this miRNA 'controls' the emergence of movement, as in the title.

Finally, the name Janus should be changed as it is already being used. A quick scan of flybase shows that there is a Janus A and B in flies (phosphatases) and I am surprised the authors did not check this. I was initially worried about the Janus kinase (JAK) when I performed the search. While I understand that none are only called Janus, studies of the jan A and B genes refer to the locus as the janus region, which could lead to confusion. The completely different molecular functions of the genes relative to CG3638 add to the confusion. Thus, I ask that the authors change the name of CG3638 to something else.

Author Response

We are very pleased to hear the overall positive views and constructive criticisms of eLife Editors and Reviewers on our work. In particular, we appreciate their comments highlighting the value of our new pipeline for high-throughput quantification of fly embryonic movement and the positive views of reviewers and editors that our data on the roles of miR-2b-1 in embryonic movement are well supported.

Regarding Reviewer 1, we thank them for their positive comments that our work is experimentally sound and well-written, their kind words on the value of our new embryonic movement pipeline, and their overall appreciation of the quality, scope, and significance of our work. In a revised version of the manuscript we will consider discussing and addressing some of the interesting points raised by Rev1.

Turning to the comments by Rev2, we are grateful to them for their recognition of the novelty of our miRNA findings and appreciation of the utility of our novel quantitative pipeline for assessing embryonic movement. Nonetheless, we politely – but strongly – disagree with their suggestion that the findings are inflated by our language. For example, they criticise our use of the verb ‘control’, yet this is a standard textbook term in molecular biology to describe biological processes regulated by genetic factors: given that miR-2b-1 regulates movement patterns during embryogenesis, to say that miR-2b-1 ‘controls’ embryonic movement in the Drosophila embryo is reasonable and in line with the language used in the field. It is not inflation. In connection to other comments, in a revised manuscript we will propose a different name for the gene here described as Janus to avoid annotation issues at FlyBase due to other, unrelated genes that include this word as part of their names.