Three-dimensional and molecular brain atlas of the hagfish reveals the evolutionary origin and early diversification of the vertebrate brain

Reviewer #1 (Public review):

Summary:

The manuscript presents a three-dimensional and molecular atlas of the adult hagfish brain to investigate the evolutionary origin and early diversification of vertebrate brain organization. Using whole-brain tissue clearing, light-sheet microscopy, and computational reconstruction, the authors generate a high-resolution 3D anatomical model of the hagfish brain. They complement this structural analysis with gene-expression profiling of neurotransmitter systems and receptors, including glutamatergic, GABAergic, cholinergic, serotonergic, and dopaminergic markers.

Strengths:

Together, the work aims to establish a modern neuroanatomical reference for the hagfish. Given the phylogenetic importance of hagfish as one of two extant species of cyclostomes (the other being lamprey), and the fact that the hagfish brain has barely been studied in contrast to the lamprey, the atlas provides a foundational resource and should be of interest to evolutionary and comparative neurobiology.

Weaknesses:

However, there are several places where both data presentation and the narrative can be improved and clarified, and particularly some of the homology and evolutionary claims seem to be superlative and need to be toned down. I present more detailed comments below:

(1) The authors spend too much effort trying to convince readers of the monophyly of hagfish and lamprey to stress its importance for evolutionary comparisons. This is now well accepted; instead, there could be more details on some of the specific, unique features of the hagfish brain relevant to a comparative atlas. For instance, the unusual fusion of the telencephalon anteriorly with the olfactory bulb and posteriorly with the diencephalon (Wicht and Northcutt, 1992), the degenerate visual system, the absence of the pineal gland, and the oculomotor system can be discussed in reference to the generated atlas and examined marker expression in related structures and their possible identity.

(2) The assertion that the MGE is absent in the lamprey is incorrect based on Sugahara et al. (2016; 2017), who identified lamprey paralogues of Nkx2.1/2.4 that are expressed in the ventral subpallium. This should be corrected.

(3) The major contribution of this study, in my mind, is the "three-dimensional atlas" of the hagfish brain. However, the atlas itself is not presented; A video of the 3D reconstructed Nissl-stained hagfish brain would be an important data resource and should be added. Annotations of forebrain, midbrain and hindbrain regions and constituent major structures can also be illustrated, which will be a useful resource.

(4) In the pallium, there seems to be an inner GABAergic cell layer and inner and outer glutamatergic cell layers, as noticed in lampreys (Suryanarayana et al., 2017). What are the overall proportions of glutamatergic and GABA neurons? In the images, it does seem that vGlut neurons are present in both P2 and P4, while there appear to be more GAD neurons in P4.

(5) As a general comment, homology claims should be toned down throughout the manuscript. This would at least require some connectivity data or transcriptomic analysis for any possible suggestions; the current data, with few markers, are insufficient for any reasonable comparisons.

(6) Expression of Pax6 and AChE is not sufficient to suggest a cerebellum-like structure. While it is true that embryonic Pax6 expression in the rhombic lip of the hagfish embryo is more comparable to other vertebrates than lamprey, and the presence of a rudimentary cerebellum-like structure would be of great interest, the evidence is too limited for such claims and should be toned down.

(7) Again, expression of Tbr1 and GAD1 in NCvl neurons does not suggest that these could be hippocampal neurons. One would at least need to rule out expression of prethalamic markers and demonstrate the presence of pallial markers through transcriptomic data (as in Lamanna et al., 2023).

(8) Presence of GABAergic neurons in the striatum - is there any data on expression of dopamine receptors, particularly given the seeming loss of the D2 receptor subtype in the hagfish?

Reviewer #2 (Public review):

Summary:

The work of Harada and collaborators fills an important gap in our knowledge of neuronal identities in the adult hagfish brain. There is essentially no modern, cell-type-level characterisation of neuronal identity in the hagfish brain yet. Existing data are limited to classical neuroanatomy (e.g. Nieuwenhuys) and sparse transmitter/gene-expression studies, mostly in embryos (e.g. work from the Kuratani lab). This study reveals a very broad peculiar pattern of dopaminergic identities and a strikingly unusual pattern of serotonergic transmission, with serotonergic cell bodies present in the telencephalon, which is uncommon for vertebrates and contrasts with previous reports (e.g., Kadota, 1991).

Strengths:

The three-dimensional reconstruction of the brain, including the ventricular system, is novel and very useful. Most of the neurotransmitter identity patterns presented here have not been previously described, and those that were published earlier, such as the serotonergic system (e.g. Kadota, Nieuwenhuys, Wicht), are old and would clearly benefit from re-evaluation using more modern approaches.

Weaknesses:

Neurotransmitter identities are highly relevant for interpreting the possible presence of LGE/MGE territories in hagfish (e.g. GABAergic patterns), for characterising the raphe nuclei (e.g. serotonergic system), and for refining our understanding of the central prosencephalic complex in relation to other vertebrate brain architectures. However, the authors do not address these points and overlook recent evidence from the amphioxus brain that could help interpret their results in an evolutionary context. Overall, the results are insufficiently discussed in relation to the current state of the art.

The study would clearly benefit from complementary gene expression profiling to place these neurotransmitter patterns within a broader framework of brain partitions, to enable more direct comparisons with other vertebrates, and, importantly, to interpret them in relation to the prosomeric model. Furthermore, the work lacks appropriate controls for the in situ hybridization experiments; Datx2 does not show any expression, so there is currently no evidence that this probe is functional. Including such controls would also strengthen the overall description of the dopaminergic system, especially given that the expression patterns of the different genes analysed appear very diffuse and somewhat random.