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  1. Evaluation Summary:

    This paper will be of interest to developmental biologists who study the gene regulatory mechanisms necessary for induction and maintenance of postmitotic neuronal identity. The study generated a useful resource of genomic data and provided new insights into the dynamic regulation of accessible chromatin regions in post-mitotic serotonin (5-HT) neurons of the mouse hindbrain. This work proposes two transcription factors (Pet1, Lmx1b) as necessary for establishment and maintenance of accessible chromatin regions in serotonin (5-HT) neurons. The study is a major technical achievement but some of the central claims are not yet fully demonstrated.

    (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. Reviewer #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    While it is known for a long time that transcription factors control the identity of individual neuron types by acting either as activators or repressors of gene expression, very little is known about the mechanisms controlling chromatin accessibility in post-mitotic neurons. Most studies on this topic are done in vitro due to the low abundance of many post-mitotic neuron types in vivo. However, the current study bypasses this limitation by employing cutting-edge methods (e.g., single cell ATAC-Seq, RNA-Seq, ChIPmentation) to establish maps of accessible chromatin and cis-regulatory elements in the context of developing, postmitotic serotonergic (5-HT) neurons of the mouse hindbrain. Major conclusions include:

    1. Diverse, single-cell chromatin landscapes are stablished early in postmitotic Pet1 neurons and likely account for the transcriptomic heterogeneity of adult 5-HT neuron subtypes.

    2. By comparing chromatin accessibility across different developmental stages, they found that chromatin remodeling is highly dynamic following cell cycle exit but later stabilizes.

    3. The terminal selector-type transcription factor Pet1 reorganizes chromatin accessibility of genes necessary for serotonin (5-HT) biosynthesis.

    4. The early euchromatin landscape of 5-HT neurons is dynamic and requires continuous Pet1 activity for maintenance of chromatin accessibility.

    5. The terminal selector-type transcription factor Lmx1b has a broader impact than Pet1 on serotonin (5-HT) neuron chromatin.

    The conclusions are supported by the experimental evidence.

    Strengths:

    Technically, this is a tour-de-force effort. The study employs cutting-edge molecular, genetic and biochemical methods - in vivo - to study the molecular mechanisms underlying serotonergic neuron maturation in wild-type and conditional mouse mutants for Pet1 and Lmx1b. Essential controls are included and the authors provide all necessary information to help the reader understand how the analysis of all datasets was performed.

    Conceptually, this study advances the field by shedding light into the poorly understood mechanisms governing chromatin accessibility in maturing neuron types. First, the authors found that heterogenous chromatin landscapes are established early during development in Pet-1-neurons, possibly setting the stage for the generation of distinct subtypes of 5-HT neurons. Second, they propose a new function for terminal selector-type transcription factors. That is, Pet1 and Lmx1b control 5-HT neuron maturation not only through sequence-specific activation of terminal effector genes, but also by reorganizing accessible chromatin at cis-regulatory regions in 5-HT neurons.

    Weaknesses:

    The mechanistic details of how Pet1 and Lmx1b open chromatin remain elusive.

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  3. Reviewer #2 (Public Review):

    Zhang et al examined regulatory regions controlling expression of genes in embryonic serotonergic neurons. They mapped accessible chromatin regions and gene expression using bulk and single cell analysis, performed chromatin immunoprecipitation studies of Pet-1 and Lmx1b transcription factors and investigated effects of Pet1 and Lmx1b loss of function on chromatin accessibility. The study generated a useful resource of genomic data and provided new insights into the dynamic regulation of accessible chromatin regions in postmitotic neurons.

    Despite the wealth of new genomic data, the focus of the manuscript is somewhat elusive. The goal seems to be to study the mechanisms that control chromatin accessibility in postmitotic serotonergic neurons during their subtype diversification. The authors provide in depth analysis of Lmx1b and Pet1 binding and effects of knockouts of these factors on chromatin accessibility. The authors also rely on conditional deletion of these TFs to address whether the factors are required to "maintain" TACs. Unfortunately, analysis of these conditional mice is limited to one timepoint, which complicates interpretation of the data. It is also unclear why the authors do not show more global analysis of gene expression changes in the pet1/Lmx1b DKO.

    In summary, while the manuscript provides important new data, the model how serotonergic neuron specification is controlled by Pet1 and Lmx1b remains unclear. Most importantly, the interesting single cell data are not well integrated with transcription factor function and with temporal changes in chromatin accessibility. Overall, the manuscript will benefit from clearer writing, explicit stating what hypotheses are being tested and better explanation of the rationale for the performed experiments.

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  4. Reviewer #3 (Public Review):

    In this manuscript Zhang et al. perform an extensive analysis on the chromatin accessibility state of serotonergic neurons at different stages in development, in wild type and Pet1 and Lmx1b mutant backgrounds, two transcription factors directly involved in the terminal differentiation of serotonergic neurons.

    Authors also present data on epigenetic marks on these neurons and single cell ATACseq and scRNAseq data for the wildtype e14.5 embryonic stage.

    There is an impressive amount of data that nicely shows the dynamics of chromatin accessibility along serotonergic neuron maturation and the diversity of chromatin states in different 5HT neuron subtypes.

    Most of the analysis reinforces known roles for Pet1 and Lmx1b however, it does not significantly increase our understanding of important questions that remain unknown for serotonergic specification and could be of broad intestest: 1) how is sustained expression of Pet1 and Lmx1b combined with additional mechanism to regulate dynamic chromatin landscapes and gene expression during serotonergic maturation?; 2) Are Pet1 and Lmx1b TFs involved in subtype diversity of serotonergic neurons? 3) If so, how is broad Pet1 and Lmx1b expression translated into gene specific defects in 5HT neuron subtypes? 4) Is Pet1 and Lmx1b binding dynamic along postmitotic serotonergic development? 5) How do Pet1 and Lmx1b mediate repression of gene expression?

    Addressing some of these questions might require additional experimental approaches and experiments.

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