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  1. Evaluation Summary:

    This study presents novel experimental data from a mutant mouse model lacking microglia (Pu.1-/- mouse line), indicating that these cells have an important role in the embryonic establishment of critical neural circuits in the brainstem generating breathing motor behavior in mice. This paper is of interest to scientists within the field of microglia as well as respiratory neurobiology as it provides original key information about a new role of microglia in the embryonic development of respiratory circuits. Overall, the data are clearly presented and rigorous. Some of the conclusions should be toned down as the data in another microglia depletion model do not support some claims of the paper.

    (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 and Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    This study presents novel experimental data from a mutant mouse model lacking microglia (Pu.1-/- mouse line) which indicates that these cells have an important role in the embryonic establishment of critical neural circuits in the brainstem generating breathing motor behavior in mice. Microglia are known to have important roles in shaping neural circuit assembly during development by controlling cell death, synapse refining, neurogenesis, and axon tract formation, but such roles have not been examined in the development of functional respiratory circuits. The authors examined the anatomical and functional characteristics of two main respiratory neuronal groups-in the embryonic parafacial (epF) and the preBötzinger complex (preBötC) regions that operate together in the developing brainstem to generate the rhythmic neural signals that are necessary to establish normal breathing behavior and ensure survival at birth. They present evidence that these respiratory networks become functional at typical developmental stages in the absence of microglia, but exhibit anomalies in rhythm generation (slower respiratory rhythm) and the mutants are unable to sustain breathing behavior at birth, consistent the observed neonatal death. Their data suggest that these deficits are associated with reduced cell numbers and abnormal rhythmogenesis in epF, and reduced commissural axonal projections of the preBötC circuits responsible for generating inspiratory rhythm.

    Strengths of this study include the authors' use of the Pu.1-/- mutant in combination with technically well-executed, novel anatomical reconstruction of distributions of microglia in the developing hindbrain, neuronal activity imaging in the epF of the embryonic brainstem in vitro, and electrophysiological recording approaches in slices to assess aspects of the anatomical and functional status of the epF and preBötC relative to the control wild type mice. They also examine inspiratory drive transmission to phrenic motoneurons in vitro to assess the functional status of spinal respiratory motor output critical for breathing behavior at birth. Furthermore, their behavioral measurements by plethysmography document show that late-term (E18.5) Pu.1-/- embryos are unable to sustain breathing activity ex utero, which is consistent with the observed neonatal death of the mutants.

    A limitation of the study is that the microglia-related mechanisms involved in regulating cell numbers in epF and the proper bilateral connectivity of preBötC circuits have not been investigated. Therefore it remains unknown if the reduced cell numbers in epF in the Pu.1-/- mutant is a defect, for example, of neurogenesis/neuronal migration or abnormal control of cell death, and if the defect of preBötC connectivity is actually related to the aggregation of microglia along the midline (possibly affecting commissural axonal tract formation), as the authors suggest.

  3. Reviewer #2 (Public Review):

    In this manuscript by Cabirol et al., the authors examine the role of murine microglia, the brain resident macrophages, in the development of embryonic respiratory networks within the hindbrain. They show that embryonic microglia are enriched in facial and hypoglossal nuclei where motoneurons are located while they are poorly present or devoid in the facial nucleus of the parafacial respiratory group (epF) and in the nucleus ambiguous of the preBotzinger (preBotC) complex. They also reported a specific enrichment of microglia around the hindbrain midline. To decipher the potential involvement of microglia in embryonic respiratory networks, the authors use the Pu.1-/- mouse model that lacks microglia and does not survive at birth. They first report that epF cells are reduced in mutant conditions and using calcium imaging, they reveal that mutant mice exhibit fewer epF cells rhythmically active associated with a decreased rhythm frequency in the epF at E14.5 and in the preBotC at E16.5 while their chemosensitivity appears preserved. They further show that the commissural projections that connect these two nuclei are affected in mutants presumably leading to their inter-network synchronicity decreased. Last, the authors record phrenic motor axons at E18.5 and show a decreased frequency of bursts associated with increased duration although their synchronicity is not impacted. Finally, they performed a C-section showing that most of the Pu.1-/- embryos were not able to induce breathing behavior. Taken together, the authors conclude that microglia are required for the embryonic development of respiratory circuit wiring. Subsequently, they propose that through this specific role, microglia could contribute to the neonatal death observed in Pu.1-/- mice.
    Deciphering the role of microglia in the development of respiratory circuits is original and timely, and provides key findings for our understanding of microglial embryonic function which remains poorly described. The manuscript is well written and the results presented are of interest. The conclusions of this paper are mostly well supported by data, but some aspects need to be clarified.

    1. Pu.1 is expressed in hematopoietic stem cells and common lymphoid progenitors impacting not only microglia but also meningeal, choroid plexus, and perivascular macrophages, as well as peripheral immune cells. To date, the FIRE mice (Rojo et al., 2019) have a more specific microglia depletion without drastically affecting meningeal, choroid plexus, and perivascular cells, and importantly, there are no reported neonatal deaths. Although microglia certainly partially participate in this phenotype, how can the authors explain that in other specific depletion models of microglia, there are no effects on neonatal death?
      Additionally, while Pu.1-/- model is quite a useful tool to investigate embryonic microglia functions, it prevents the analysis of subsequent long-term consequences on the circuitry due to neonatal death.

    2. The authors are referring to microglia using Iba1 or GFP staining in the cx3cr1gfp mouse line but one limitation is that they both stain microglia, meningeal, choroid plexus, and perivascular macrophages contrary to P2ry12 that specifically labels microglia in the brain parenchyma.

    3. The kinetics of microglia colonization of the different nuclei in the brainstem is not precisely described.

    4. The characterization of the VII and epF nuclei is incomplete (size, density of cells...).

    5. This works raises new intriguing data on a new role of microglia during development but it remains unclear which actors at a cellular level are implicated.

    6. While the discussion part is nicely addressing different hypotheses on the underlying mechanisms of the observed effects, the description of the respiratory system in particular regarding what is known about synchronicity and the cellular actors involved is not explained sufficiently.

  4. Reviewer #3 (Public Review):

    This excellent paper is of interest to developmental brain scientists in general and especially those interested in the development of the vital brainstem circuitry that is necessary for postnatal life. The manuscript provides substantial new insight into the crucial role of microglial in the formation of functional neural circuits. Overall, the data are properly controlled, analysed, and presented although other potential functional deficits in the microglia deficient mice (Pu.1-/-) could be discussed.

    Microglia, brain-resident macrophages, play key roles during prenatal development in defining neural circuitry function, ensuring proper synaptic wiring, and maintaining homeostasis.

    The thorough and well-designed experiments, analysis, and presentation of the results from wild-type and microglia-deficient embryonic and early postnatal mice are convincing. The authors clearly show how microglia deficient mice exhibit lower respiratory activity fewer embryonic active respiration-related neurons as well as less connectivity. Thus their claim that microglia are crucial for vital respiration-related neural networks to function properly is convincing.

    Further understanding of the role of microglia in brain and brainstem development is important, since environmental pathogens that affect microglia function, may contribute to susceptibility to developmental disorders associated with altered synapse numbers and dysfunctional neural networks.

    The paper does not describe any other malformations, that might contribute to the immediate or close to immediate postnatal death of newborn pups.
    Please add some more references/discussion or data to underline that the deficits that you show are a major contributor to immediate postnatal death.
    Are there any signs of Peripheral deficits; eg upper airway, heart, or lung anatomical /functional abnormalities that might contribute to the immediate postnatal death?