Junctional Adhesion Molecule (JAM)-C recruitment of Pard3 and drebrin to cell contacts initiates neuron-glia recognition and layer-specific cell sorting in developing cerebella

Reviewer #1 (Public Review):

This study by Hallada et al. reported the detailed characterization of cis and trans-binding of JAM-C in mediating the developmental migration of CGNs, combining ex vivo cultures, time-lapse imaging, and mathematical analyses. Overall, the study was comprehensively carried out, and the conclusion is important in our understanding of the signaling mechanism of cerebellar development.

Weaknesses:

Several technical concerns need to be clarified.

(1) The efficiency of shRNA knockdown of endogenous JAM-C. The entire study was based on the assumption that the endogenous wild-type JAM-C was depleted to the extent that it would not influence the observed phenotypes. However, this point requires verification, particularly in the ex vivo cultures.

(2) The expression levels of mutant JAM-C proteins. It is unclear whether the exogenous expression of mutant JAM-C proteins would be comparable to that of the endogenous JAM-C. In addition, the levels of exogenously expressed JAM-C may likely alter over the time course of experiments, e.g., in some experiments over 48 hours.

(3) The resolution of imaging methods. Different imaging methods were utilized in the study, and it is essential to clearly state the resolution of each imaging dataset (e.g., 0.2 x 0.2 um per pixel). This information is crucial to assess the reliability of observed phenotypes, which in some cases were relatively unimpressive.

Reviewer #2 (Public Review):

Summary:

Lamination is a layered neuronal arrangement that provides a basic frame to establish functional connectivity in the brain. The formation of a layered structure requires a highly coordinated interaction between migrating neurons and the developing microenvironment. Earlier studies revealed that to reach specific locations, migrating neurons typically follow various morphogen gradients. Here, Hallada et al. showed that cerebellar granule neurons (CGNs) could navigate via adhesive interaction with Junctional Adhesion Molecule C (JAM-C) followed by recruitment and distribution of intercellular partners (Pard3 and debris) at the contact sites. These results show that neuronal migration could be structured by specific interactions with adhesion molecules and spatial re-arrangements of downstream effectors.

Strengths:

The authors concluded that cis/trans binding sites of JAM-C on CGNs are crucial for contact formation with cerebellar glial cells (Bergman glial cells, BGs) and recruitment of Pard3 and drebrin to contact sites. This conclusion was based on the data obtained utilizing several advanced tools and technical approaches, such as cutting-edge microscopy, detailed visualization of cell-cell recognition, and a new correlation analysis.

Weaknesses:

(1) Despite multiple advanced methodologies, the study has weaknesses related primarily to the lack of specific evidence in support of findings and data interpretation issues. For example, it is unclear how JAM-C-mediated adhesion facilitates the entry of CGNs into the cerebellar molecular layer (ML). The authors described that CGN-CGN JAM recognition recruits more Pard3 and drebrin compared to CGN-BG recognition, which could increase the dwelling time of CGNs before moving to ML. However, such a mechanism does not explain what would initiate the entry of CGNs into ML. Perhaps the authors could provide a detailed explanation of this phenomenon in the Discussion (but certainly not in the Abstract). Also, the authors could consider revising the content of the Abstract, emphasizing their findings, and leaving out the speculations.

(2) To allow for comparison, it would be very helpful to indicate specific numerical values for each data point throughout the manuscript. For example, the authors stated that a change in instantaneous migration angle due to JAM-C silencing negatively affects CGNs movement to the ML (Figure 2) and that spatial distribution of negative JAM-Drebrin correlation is altered at CGN-CGN contacts (Figure 7). However, without specific values, it remains unclear what the magnitude of the discussed changes is or whether they were actually significant. It was not certainly straightforward to make specific conclusions based on graphical presentation alone.

Reviewer #3 (Public Review):

Summary:

This study elucidated the mechanism controlling the switch from parallel migration to radial migration during the development of cerebellar granule cells by analyzing the behavior of cell-type-specific JAM-mediated adhesion and the downstream factors that promote migration. The research represents a detailed analysis, employing probes to capture cell recognition events between different cell types, a co-culture system (monolayer culture and slice imaging), and imaging techniques, building upon the authors' prior research on JAM-Pard3 interactions. As a result, the authors found that:

(1) JAM-C-mediated interactions between granule cells (GCNs) are formed earlier and are more robust than JAM-C/JAM-B interactions between GCNs and glia;

(2) Recruitment of migration-promoting factors Pard3/Drebrin by JAM interactions is more efficient in GCN-GCN (JAM-C/JAM-C) interactions; and

(3) The distribution pattern of Pard3/Drebrin differs between GCN-GCN and GCN-Glia interactions, as revealed by detailed imaging analysis.

Consequently, the authors discovered that these differences contribute to a time lag between parallel and radial migration, which serves as a temporal checkpoint sorting mature cerebellar granule cells.

Strengths:

Cell migration is a commonly observed phenomenon in neural development. It is crucial for sorting specific cell populations and positioning them appropriately to develop proper neural circuits. While the regulation of these migrations is known to be mediated by secreted guidance factors, this study demonstrates that combinations of cell adhesion molecules (JAM) mediate cell type-specific interactions that contribute to the timing control of cell migration. This finding significantly advances our understanding of the mechanisms governing cell migration in neural development.

Weaknesses:

The author's hypothesis has been validated using in vitro systems. While in vitro systems allow for a more detailed design of experimental parameters, validation in vivo would still be necessary to demonstrate whether the temporal checkpoint of migration mediated by cell-cell interactions works. For example, knockout of JAM-C in cerebellar granule cells could be considered for such validation. Furthermore, the behavioral analysis of these mutant mice would be interesting.

Additionally, the author's observation that recruitment patterns of Pard3 and Drebrin at adhesive sites vary between interacting cell pairs is intriguing and suggests exciting implications. It would be highly informative if the relationship between these differences and ML entry timing could be demonstrated.