Diverse paths for chemoreception in ciliated neurons contacting the cerebrospinal fluid in the spinal cord

Reviewer #1 (Public review):

Summary:

This manuscript examines the expression of putative chemoreceptors in CSF-contacting neurons of the larval zebrafish spinal cord. Using in situ hybridization, the authors show that sstr2a is preferentially expressed in ventral CSF-cNs, whereas grm2a, ptprna, and ldlrad2 are detected in both ventral and dorsolateral CSF-cNs, with additional expression in neighboring cells around the central canal.

Strengths:

The study provides useful anatomical information on the expression of putative chemoreceptors in CSF-contacting neurons. The experiments appear to be carefully performed, and the results are clearly presented with high-quality illustrations and informative schematics.

Weaknesses:

This work remains largely descriptive and based on mRNA expression. Therefore, the proposed roles in chemoreception, ligand sensing, lipid capture, or long-range CSF signaling remain speculative without protein-level or functional validation.

Reviewer #2 (Public review):

Summary:

Verran et al. leverage a previously published RNAseq dataset of zebrafish cerebrospinal fluid contacting neurons (CSF-cNs) to identify potential receptors involved in chemosensory signalling in these neurons. They then validate expression of the identified receptors by hybridization chain reaction (HCR) in zebrafish larvae. This way they uncover potential roles for the somatostatin receptor Sstr2a, metabotropic glutamtate receptor Grm2a, LDL receptor Ldlrad2 and the Phosphatase receptor Ptprna, suggesting the existence of numerous chemo-sensory pathways in CSF-cNs and providing a potential entry point for further investigation.

Strengths:

This is a useful resource; the provided HCR data that demonstrates expression of these receptors in CSF-cNs is convincing, and the finding that CSF-cNs express these receptors is interesting.

Weaknesses:

The overall insight provided by this manuscript is rather limited, essentially just demonstrating the expression of 4 receptors in CSF-cNs, whose expression was predicted to be enriched in these neurons anyway by a previously published dataset.

Reviewer #3 (Public review):

Summary:

The authors aimed to identify new molecular pathways that could enable long-range signaling through the cerebrospinal fluid (CSF), focusing on a specialized class of neurons called CSF-contacting neurons (CSF-cNs) in larval zebrafish.

Strengths:

Anatomical validation of transcriptomic candidates using HCR, providing high-resolution spatial mapping of chemoreceptor expression in CSF-contacting neurons and neighboring spinal cord cells. The work broadens the potential understanding of CSF-cNs and offers a resource for future functional investigations of CSF-mediated signaling.

Weaknesses:

The principal limitation of the study is that the conclusions remain largely transcriptomic and inferential. Although HCR convincingly validates mRNA expression, no protein-level evidence is provided to demonstrate receptor translation or subcellular localization, leaving uncertainty regarding functional receptor availability at the CSF interface. Moreover, the study does not establish whether the proposed ligands are present in the relevant CSF microenvironment or engage the identified receptors in vivo. As such, the functional significance of the proposed chemosensory pathways remains speculative.