1. Reviewer #2 (Public Review):

    This interesting study from Kurashina et al. examines novel postmitotic roles for transcription factors traditionally considered to specify neuronal cell fate. The paper examines a form of synaptic tiling in C. elegans motor neurons to provide evidence that the unc-4 and unc-37 transcription factors, previously implicated in determining cholinergic motor neuron identity, have additional roles in the regulation of synaptic wiring that are at least partially separable from cell fate specification.

    The authors develop new tools for defining the temporal actions of unc-4 and unc-37 and the clean dissection of the spatiotemporal requirements for unc-4/unc-37 transcriptional regulation is a major advance offered by the study. In particular, the authors demonstrate that unc-4 acts at a later development stage to control synaptic wiring compared with its role in cell fate regulation. Overall, the paper is clearly written and offers new insight into how transcription factors that act to define neuronal identity may have additional roles in specifying aspects of synapse organization. The study falls a little short in clearly defining mechanism of action downstream of unc-4/unc-37 and in describing the relationship of these newly described roles for unc-4 and unc-37 to those previously described.

    The authors use a clever strategy to assess tiling of individual cholinergic motor neurons using DA8 and DA9 as a model, but in some cases observe variable degradation of the RAB-3::GFPnovo, presumably due to weak expression of ZIF1 in some of the mutants. This makes it a little difficult to assess the tiling defects in some of the figures. The residual GFPnovo signal seems to be defined based on colocalization with the more broadly expressed mCherry::RAB-3 marker, but no data is shown for the extent of colocalization in the absence of ZIF1. This analysis would benefit from more explanation.

    The analysis of temporal requirements using ts alleles in combination with the AID system is very convincing and quite informative. The authors clearly show a later requirement for proper tiling, at stages when cell fate determination is expected to be complete. However, it is less clear how these newly defined aspects of unc-4 and unc-37 functions relate to their previously defined roles.

    The authors examine PLX-1::GFP subcellular localization in DA neurons (using cell specific itr-1 promoter) of unc-4 mutants but do not directly examine plx-1 expression levels in DA neurons. This analysis would further solidify links between plx-1 and unc-4 transcriptional regulation.

    Did the authors examine whether degradation of unc-4 and/or unc-37 at much later developmental time points also lead to tiling defects? Is there an ongoing requirement to maintain tiling?

    Did the authors examine whether the unc-4::AID and unc-37::AID animals became uncoordinated subsequent to treatment with auxin analog? Do the tiling defects potentially contribute to locomotor changes?

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  2. Reviewer #1 (Public Review):

    This manuscript by Kurashina et al. describes a novel post-mitotic role in synaptic patterning for a cell fate determining gene, unc-4, and its co-repressor, unc-37. The DA neurons in C. elegans are cholinergic motor-neurons that exhibit unique synaptic tiling of their dorsal axonal segments. Mizumoto et al has previously shown that Semaphorin-Plexin signaling is required to establish the tiling between DA8 and DA9, by functioning in cis in the DA9 neuron. Using temperature sensitive mutant of unc-4, as well as a combination of CRISPR/Cas9 genome editing with the AID system for specific temporal degradation, the authors nicely examine the spatiotemporal requirement of unc-4, and show that unc-4 is required only post-mitotically for synapse tiling, but not during the development of the DA neurons. Interestingly, activity of the corepressor unc-37 is required both during development and postmitotically for correct tiling. unc-4 and unc-37 are suggested to function by inhibiting the canonical wnt signaling. Overall this is an interesting study which sheds light on our understanding of the post-developmental role of cell fate genes in synapse patterning. I only have one major issue that requires some clarification. The authors present in their introduction the results from Kerk et al, regarding the role of unc-4 as a cell fate determining gene for the VAs and DAs. Kerk et al have shown that UNC-4 is specifically required for the expression of DA genes, without affecting ACh pathway genes. Table 1, however, doesn't fully recapitulate the same results and actually shows that unc-4 and unc-37 mutants do not exhibit significant cell fate defects. The authors use these results to argue in the discussion that the synaptic patterning defects can occur independent of the cell fate transformation. The issue of unc-4 as a cell fate determining gene of A type motor neurons needs to be more clearly addressed. The authors should test whether acr-5 expression is elevated in DAs in unc-4 and unc-37 mutants (Winnier 1999, Kerk 2017). In addition, they should also analyze these DB markers in the temp shift experiments (do the VB-DB markers show up in the post embryonic knockout? And if so, will silencing them specifically in DA neurons rescue the tiling defects?). The discussion, accordingly, should also address these issues.

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  3. Evaluation Summary:

    This paper is of potential interest to a broad audience of neuroscientists, as it adds to our growing understanding of transcriptional mechanisms that regulate neural connectivity. Specifically, the paper provides support for the idea that transcriptional pathways previously implicated in neuronal cell fate determination can have independent roles in specifying connectivity between neurons. The study is highly technically innovative and cleverly uses a set of newly developed tools to analyze the developmental time window over which transcriptional activity is required to achieve to proper connectivity. However, the paper falls a little short in defining specific mechanisms involved downstream of the transcription factors themselves.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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