Deciphering Intercellular Communication in the Cerebellar External Granule Layer: Insights into Non-Classical Connections in Neural Development

PONE-D-26-16284

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“Institut Pasteur (Pasteur Institute): Chiara Zurzolo, Big Brain Theory Program; Institut du Cerveau et de la Moelle Epinière (ICM): Laurence Cathala, Big Brain Theory Program; Agence Nationale de la Recherche (ANR): Chiara Zurzolo, ANR-16-CONV-0005; Fondation pour la Recherche Médicale (FRM): Chiara Zurzolo, EQU202103012630; Agence Nationale de la Recherche (ANR): Chiara Zurzolo, AAPG2023 UnProSec 2024-2027; Fondation pour la Recherche Médicale (FRM): Chiara Zurzolo, MND202310017892; Conseil régional d'Ile-de-France: Gabriel Kaddour; DIM ELICIT: Gabriel Kaddour; Fondation pour la Recherche Médicale (FRM): Gabriel Kaddour, Financement de Fin de thèse”

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Additional Editor Comments:

This manuscript investigates the presence and nature of intercellular communications in the EGL of the developing cerebellum, an ideal system for this purpose, as it is populated by proliferating progenitors, post-mitotic neurons, and migrating neurons. The study is conceived as an extension of an earlier one to properly characterize “non-canonical” connections, including tunneling nanotubes (TNTs).

By using a combination of sparse genetic labeling, live imaging, and automated CNN-based segmentation to study delicate and transient structures that are usually challenging to preserve and identify in vivo, the study goes beyond in vitro models to identify and distinguish various types of intercellular connections (ICs) in a highly dynamic mammalian tissue environment. When measuring cell populations and connections, this multimodal technique ensures high technical precision.

The key finding is that not all reported connections can be explained by cytokinetic bridges (CBs), and that cell-to-cell connections during brain development may include a different form of communication called TNT-like connections. In particular, clonally linked (87%) and non-clonally related (13%) cells appear to generate TNT-like connections de novo, offering a nuanced perspective on how these structures might promote communication across various cell lineages.

Despite a thorough and precise description, some questions remain about the nature and function of these novel communication modalities. The term "TNT-like," rather than definitive identification, results from the authors' reliance on "negative controls"—the absence of CB/IB markers—and morphological criteria, given the lack of molecular markers for TNTs. Accordingly, the absence of conclusive evidence of cargo transfer (such as organelle or protein exchange) via these structures does not rule out the possibility that some structures are only closed-ended filopodia or cytonemes. These limitations persist even after the authors' thorough and careful revision, primarily due to the technical constraints of live imaging. For instance, attempts to detect cytoplasmic transfer using photoconvertible proteins (Dendra2) failed due to tissue density and light refraction, casting doubt on the "open-ended" nature of these connections, as suggested by earlier EM evidence. Advances in presently available methodologies will make this type of research easier.

The study is deemed worthy of publication because it: i) is methodologically sound and correct and offers a rigorous interpretation of the results without overstatements; ii) adds a novel insight about cell interactions that likely control cell fate during neural development, which can help a better understanding of neurodevelopmental disorders, among other things.

I suggest the following small change:

- Introduction, line 49: the term “pathway” is not appropriate here, as the authors refer to cell-to-cell communication modalities.

Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: This is an automated report for PONE-D-26-16284. This report was solicited by the PLOS One editorial team and provided by ScreenIT.

ScreenIT is an independent group of scientists developing automated tools that analyze academic papers. A set of automated tools screened your submitted manuscript and provided the report below. Each tool was created by your academic colleagues with the goal of helping authors. The tools look for factors that are important for transparency, rigor and reproducibility, and we hope that the report might help you to improve reporting in your manuscript. Within the report you will find links to more information about the items that the tools check. These links include helpful papers, websites, or videos that explain why the item is important. While our screening tools aim to improve and maintain quality standards they may, on occasion, miss nuances specific to your study type or flag something incorrectly. Each tool has limitations that are described on the ScreenIT website. The tools screen the main file for the paper; they are not able to screen supplements stored in separate files. Please note that the Academic Editor had access to these comments while making a decision on your manuscript. The Academic Editor may ask that issues flagged in this report be addressed. If you would like to provide feedback on the ScreenIT tool, please email the team at ScreenIt@bih-charite.de. If you have questions or concerns about the review process, please contact the PLOS One office at plosone@plos.org.

Reviewer #2: It is a pleasure to review this excellently revised paper which has responded to thorough reviews by 3 recent reviewers and was extensively revised to meet their criticisms/suggestions. For this reviewer, it is an exciting paper because it presents methods, and demonstrates their use, to study developing tunneling nanotubes (TNTs) in living samples of mouse pups. The use of electroporation to introduce drug-controlled constructs only expressed in neural precursors, into the developing cerebellum allows tissue to be collected and studied in organ culture for short periods. The use of a variety of differently-labelled antibodies to detect mitotic stages and distinguish intercellular bridges remaining from cytokinesis from TNTs, has allowed a method to watch them grow. While some of these may be cytonemes capable of cargo transfer, v,s, TNTs with signaling capabilities, further studies will clarify this point. This is a great advance from the use of scanning em which only allows static views (fixatives for the usual light microscopy mostly destroy them).

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Reviewer #1: No

Reviewer #2: No

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Attachments

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Submitted filename: report_10.1101+2025.08.11.669634.pdf