Temporal transcription factors determine circuit membership by permanently altering motor neuron-to-muscle synaptic partnerships

Reviewer #1:

Drosophila[…] 1) As the title of the manuscript highlights it, the main message of this manuscript is that temporal transcription factors determine circuit membership by permanently altering motor neuron-to-muscle synaptic partnerships. However, the manuscript devotes much space to understanding the role of cardinal transcription factors, e.g. Figure 1 is entirely devoted to this question. While the results shown are solid, and related to our understanding of how different transcription factor classes determine circuit membership, the authors do not identify what the proposed compensatory mechanisms are. Therefore, these results do not contribute to the conceptual message of the paper. The authors should remove this section and develop these results for a future manuscript specifically addressing the role of cardinal factors and the nature of the compensatory mechanism.

We appreciated reviewer #1’s recommendation to omit Figure 1 and therefore omitted Figure 1. We are looking forward to using our data from Figure 1 as a launch point into investigating how the system is able to correct itself and describing the compensatory/corrective mechanisms we proposed in our initial submission.

Throughout the manuscript, the description of biological events should be more precise. For instance: "Our data suggest that in the Drosophila motor system, motor neurons are born knowing which circuit they will populate and that this information is provided to them by temporal transcription factors acting in the neuroblast". It ignores the multiple cellular and molecular interactions that establish neuronal morphologies and circuitries.

We removed the sentence with “born knowing”. Throughout the text we have improved our description of biological events. We are happy to make more adjustments, if specifics are provided. Thank you for this comment.

Reviewer #2:

[…] My only comments have to do with models and terminology used by the authors.

1) It is unclear to me whether the use of the words "circuit" and "circuit membership" (which wasn't used in the previous paper) are justified here. For me, a circuit is a set of interconnected neurons that perform a particular function or functions. Based on the readouts they use, I think the authors are studying muscle targeting and NMJ formation, not the assembly of a neural circuit and I find the use of these terms as distracting and unnecessarily showy.

Thank you for this comment. We were not intending to be either distracting or showy!

First, we agree with this definition of “circuit”, especially the reference to a set of neurons that perform a particular function. It is exactly the link to FUNCTION that is the reason we use the term circuit here. Second, our data, together with the body of work from the field shows that temporal transcription factors (TTFs) control multiple cell biological events, which include, but are not limited to muscle targeting and NMJ formation. (e.g., TTFs also regulate early molecular identities neurons from the lineage, and cause the neuroblast to generate neurons that leave the CNS at times when the lineage is normally producing interneurons). So, in our mind using the terms muscle targeting and NMJ formation is too limited for our meaning. Additionally, we wanted to avoid any implication that TTFs are functioning in neurons and at the NMJ.

To address this comment we have limited our use of the term “circuit membership” to subsections of the Introduction and Discussion. We have removed the word “circuit” in the Results section entirely. Instead, we use terms “motor neuron-to-muscle synapses” and “synaptic partner selection”.

2) The same is true for the phrase "cardinal transcription factor", a term that for me would refer to a set of key TFs that set up the body plan, not subordinate TFs downstream of temporal TFs. There is also a term already in the literature used (not just by us!) for this class of TFs: morphology TFs (Enriquez et al., 2015, others). To use a new term (again, not used previously as far as I can tell), especially one that doesn't obviously fit the muscle targeting function studied here, the authors should better justify this decision.

At the suggestion of reviewer 1, we removed Figure 1 data and the accompanying background in the Introduction, so the term “cardinal transcription factors” is no longer used. Nonetheless, we would like to take this opportunity to make a case that morphology and cardinal TFs are distinct.

We agree that there are commonalities between morphology TFs and cardinal TFs. Both are likely to be subordinate to TTFs. Both are expressed transiently in post-mitotic neurons. But these commonalities do not necessarily mean that morphology and cardinal TFs should be grouped together. Morphology TFs, as beautifully laid out by Enriquez et al., are involved, combinatorially, in specifying unique morphological features among a set of neurons that are somewhat similar (e.g., motor neurons). As reviewer 1 points out they do not have cross repressive interactions. In contrast, Cardinal TFs are those that are expressed in groups of neurons that have similar features (Jessell, 2000; Briscoe et al., 2000; Arber, 2012; Lu et al., 2015, Zhang, et al., 2017, many more). This terminology is most often found when referring to groups of interneurons in spinal cord and neo-cortex. But cardinal TFs are generally highly conserved homeodomain TFs, which are also expressed in subsets of neurons in Drosophila nerve cord (e.g., nkx6, eve, hb9, etc.). Carindal TFs, unlike morphology TFs are involved in specifying a set of common neuronal features. Further, they are engaged in cross repressive interactions because they are thought to subdivide groups of neurons into distinct neuronal subtypes (i.e., cardinal classes).

We now cite Enriquez et al., 2015 in the “Conclusion” subsection of the Discussion, and mention that looking at Morphology TFs will be an important future direction.

It would be extremely interesting and useful to have a discussion about terminology surrounding different types of TFs offline.

3) I'm not sure the data presented here suggest the "compensation" model evoked by the authors. What is shown is that changing the tTFs, but not more subordinate TFs, alters muscle targeting late, but that both sets of TFs affect neural identity and targeting in the embryo. Can the authors rule out the more parsimonious explanation that there are other TFs that execute the targeting/morphology functions at the late larval stage? This is different than 'compensation', which suggests that early errors can be corrected by some ill-defined mechanism. At the very least, the authors should consider raising alternative explanations to the Discussion.

At the suggestion of reviewer 1, we removed the data that lead to our “compensation model”. We plan to use this data for a more detailed investigation in the future. Thank you for raising this point.