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PREreview of “Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis”

Summary

The manuscript comprises the experimental characterization of cellular processes found on neural progenitor cells in the mouse spinal cord.

To this aim, the authors carry out different approaches involving morphometric measurements of cells and their protrusions. Furthermore, they test the use of drugs disrupting actin dynamics to understand the nature of these processes. Finally, they test the contribution of the processes to the formation of spatial patterns through fast Fourier transform analyses and autocorrelation tests.

Altogether, the results presented in the manuscript contribute to the understanding of the dynamics of these processes, their distribution in the cell and their possible participation in cellular signaling pathways that contribute to the establishment of patterns during neurogenesis.

Major concerns 

• At the beginning of the Results section, the authors refer to the processes as filopodia-like structures, and later in the text they begin to refer to them categorically as filopodia. We consider that this statement should be moderated, since no specific labeling for this type of structure (e.g., anti-MYO10) was performed.

• The manuscript would benefit from clearly stating the bin size in (all) the histograms (Figures 3 and 4).

• The number of embryos and slices is clearly stated only in Figure 3. Please extend it to the rest of the figures. 

• Figure 3D-F presents three plots with the same set of data. However, in figure 3D a value appears above 20 um that is not obviously present in 3F. Please check. Likewise, given that an outlier is mentioned with respect to the protrusion lifetime, it would improve interpretation to have the number of individuals processed.

• Three biological replicates of the same experiment are presented in Figure 6E. One of these replicas has statistically significant differences while the others have none. In Figure 6C these results are shown averaged, while there is no statistical significance. We find it confusing to present the data in this format. We suggest leaving only Figures 6D and E.

• Both in the Introduction and the Discussion section, the authors state the existence of tendencies which are not supported by data. a- “[...] CDC42 inhibitor ML141, leading to a tendency to decrease the distance between high HES5 cell clusters [...]” b- “Finally, altering the protrusion density resulted in a tendency towards smaller HES5 spatial periods (reduced by 20% as measured by Fourier transform), as predicted by our previous mathematical modelling, although the change was not statistically significant.” We strongly suggest either to provide a linear trend estimation (or its non-parametric equivalent) based on the data, or to revise the phrasing.

Minor concerns

• In the Abstract, the authors state “Here, we report that cellular protrusions are present all along the apicobasal axis of individual neural progenitor cells”. However, as they clearly state later in the Introduction this phenomenon was already reported “In mouse cortex neuroepithelia, protrusions containing DLL1 were found in both RGCs and intermediate neural progenitors, however, no functional role for these protrusions was tested (Nelson et al., 2013).” Also, “Protrusions in the mouse spinal cord were recently observed to emanate from floorplate and roofplate cells carrying Shh and Wnt signals and were required for correct dorsal-ventral patterning (Hall et al., 2024)”. We suggest the authors consider changing the phrasing form “we report” to “we confirm”. 

• Radial glial cells (RGCs) and neural precursors are mentioned interchangeably throughout the manuscript. In the discussion and figures both terms are used synonymously. We consider that these terms should be better defined and delimited to enhance the understanding of the manuscript.

• We noticed that scale bars do not appear in the first two panels of Figure 4D. This can easily be solved.

• The authors might remark on the significance of their findings by clearly stating the rationale for choosing E10 embryos.

• Figure 5 and the section “DLL1 is present in neural progenitor protrusions” show that the receptor localizes at the processes. However, it is also true that the immunolocalization of Dll1 is quite ubiquitous, and might be worth discussing. Overall, the relation to Notch signaling is a bit lateral and not dealt with in the manuscript.

• Figure 4B repeats a plot that previously appears in Figure 3D with a density layer added. However, this repetition seems unnecessary as its inclusion in the main text would suffice. 

• A clearer statement of how zones are delimited and its rationale would enhance interpretation of the results. Additionally, including the distance between the soma, processes and basement membrane would provide developmental context. 

• Figures 1B and S1 could improve accessibility by choosing a color-blind friendly palette (e.g., choosing magenta instead of red). 

• Regarding the results shown in figure 4D the authors state in the text that “we found that where protrusions emanate from on RGCs determines how likely non-neighbouring contacts are due to the varying distances they need to traverse.”  We suggest them either to provide a correlation analysis based on the data, or to revise the phrasing.

• The first panel of Figure 4D shows a small cyan arrowhead that might be overlooked. We suggest increasing its value (brightness), or choosing a different hue. 

• Please clarify whether the results shown in Figure 5B are representative of what happens in different individuals. 

Competing interests

The authors declare that they have no competing interests.