BMP signalling facilitates transit amplification in the developing chick and human cerebellum

Reviewer #1 (Public Review):

Summary:

Rook et al examined the role of BMP signaling in cerebellum development, using chick as a model alongside human tissue samples. They first examined p-SMADs and found differences between the species, with human samples retaining high p-SMAD after foliation, while in chick, BMP signaling appears to decrease following foliation. To understand the role of BMP during early development, they then used early chick embryos to modulate BMP, using either a constitutively active BMP regulator to increase BMP signaling or overexpressing the negative intracellular BMP regulator to decrease BMP signaling. After validating the constructs in ovo, the authors then examined GNP morphology and migration. They then determined whether the effects were cell autonomous.

Strengths:

The experiments were well-designed and well-controlled. The figures were extremely clear and convincing, and the accompanying drawings help orient the reader to easily understand the experimental set up. These studies also help clarify the role of BMP at different stages of cerebellum development, suggesting early BMP signaling is required for dorsalization, not rhombic lip induction, and that later BMP signaling is needed to regulate the timing of migration and maturation of granule neurons.

Weaknesses:

While these studies certainly hint that BMP modulation may affect tumor growth, this was not explicitly tested here. Future studies are required to generalize the functional role of BMP signaling in normal cerebellum development to malignant growth.

Reviewer #2 (Public Review):

Summary:

This is a fundamental and elegant study showing the role of BMP signaling in cerebellar development. This is an important question because there are multiple diseases, including aggressive childhood cancers, which involve granule cell precursors. Thus understanding of the factors that govern the formation of the granule cell layer is important both from a basic science and a disease perspective.

Overall, the manuscript is clear and well-written. The figures are extremely clear, wonderfully informative, and overall quite beautiful.

Figures 1-3 show the experimental design and report how BMP activity is altered over development in both the chick and the human developing cerebellum. Both data is very impressive and convincing.

They then go on to modulate BMP activity in the developing chick, using a complex electroporation paradigm that allows them to label cells with GFP as well as with cell-specific reporters of BMP activity levels. They bidirectionally modulate BMP levels and then can look at both cell-specific and non-specific alterations in the formation of the external and internal granule cell layer, across different developmental timepoints. These are really elegant and rigorous experiments, as they look at both sagittal and transverse sections to collect this data. This makes the data extremely compelling. With these rigorous techniques, they show that BMP signaling serves more than one function across development: it is involved in the initial tangential migration from the rhombic lip, but at a later time, both up- and down-regulation of BMP activity reduces density of amplifying cells in the external granule cell layer.

Strengths:

Overall, I think the paper is interesting and important and the data is strong. The use of both chick and human tissue strengthens the findings. They are extremely rigorous, analyzing data from multiple planes at multiple ages, which also really strengthens their findings. The dual electroporation approach is extremely elegant, providing beautiful visual representations of their findings.

Weaknesses:

I find no significant weaknesses.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

(1.1) I thought the manuscript was very clear. While I realize the authors included the reference to medulloblastoma in the introduction based on previous reviewer comments, I think this speculation is better left in the discussion.

Whilst we appreciate the reviewers feedback here, we felt it was important to include a reference to medulloblastoma and developmental disorders associated with the cerebellum to put this work into a broader context.

We removed the sentence “Medulloblastoma can be a consequence of uncontrolled proliferation of granule cell progenitors, with BMP overexpression being a potential therapeutic avenue to inhibit this proliferation” to limit the speculation in this statement.

(1.2) line 81: It would be better to cite the 2 original papers (Hendrikes et al 2022, Smith et al 2022) rather than the Phoenix commentary article. I'm not sure the Phoenix article needs to be cited at all within this paper.

We have cited the two suggested papers and removed the citation to Phoenix et al.

(1.3) line 102: confusing sentence with the unexpected separation of do and not: "the same conditional deletions of BMP pathway elements that fail to block early granule cell specification at the rhombic lip do result not in a larger cerebellum as might be expected, but either have no affect".

We thank the reviewer for pointing out this error and have corrected the text to “do not result in a larger cerebellum”.

(1.4) line 133: inconsistent acronyms (for example, W9 vs pcw9).

This has been corrected to PCW in all occurrences.

(1.5) line 139: coronal vs transverse? it seems like you show transverse sectioning but refer to it as coronal in the text.

We thank the reviewer for highlighting this and have corrected the text to “transverse”.

(1.6) fig 2C: would it be possible to provide a similar inset as 2D?

We thank the reviewer for this suggestion and have added the insets in 2C. We agree that this is now clearer and more consistent with the rest of the figure.

(1.7) line 368/369/435/436 missing arrows.

The arrows have been re-added- it appears that they did not show up on the uploaded PDF.

(1.8) line 517 missing word: rhombic-lip-derived.

This typo has been corrected.

Reviewer #2 (Public Review):

(2.1) Fig. 3 M Why are there asterisks both above and below the brackets?

This was a formatting error that has now been corrected.

(2.2) Fig. 8. The arrows (BMP up and BMP down) are touching the right ")" in the figure, which makes it hard to read.

This was also a formatting issue which has been corrected.

(2.3) Fig. 4 and 8 legends. There are spaces in the text which I believe are for arrows to be inserted "(BMP )", but the arrows have been omitted in the PDF that I read.

This is the same as reviewer 1’s comment- these have been re-added to the text and appears to have been an issue with the PDF upload.

(2.4) Fig. 3 legend gets very hard to read at the end, where it seems some punctuation is missing.

We have re-worded the legend for Fig. 3 to make it easier to read.

(2.5) Significant figures in some of the text are probably too much given the accuracy at which they can be measured with.

We appreciate the reviewer’s concerns here, however these were added in response to the original reviewer’s request to “provide some additional support to otherwise qualitative observations”.