Peer review of: ABHD4-dependent developmental anoikis safeguards the embryonic brain (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Prof. Katona and colleagues proposed developmental anoikis and the molecular player that 
controls this process. Developmental anoikis was triggered as a result of exposure to different 
types of insults. One is forced delamination of neural progenitor cells by overexpression of Cdh2 
mutant. Another is prenatal alcohol exposure. Concept of “developmental anoikis” that reduces 
errors of development that is elicited by environmental insults is novel. Based on the known 
function in cancer anoikis, and the specific expression in developmental stage, the authors picked 
up Abhd4 as a candidate gene of a controller of this event. The knockout animal did not show 
obvious phenotypes in normal condition. However, under exposure to insults, the aniokis is 
significantly reduced in the knockout. The authors also provide good discussion of potential 
mechanisms how Abhd4 controls developmental aniokis. This article provides very interesting 
model, and I believe the model will be further strengthened by answering the questions below. 
 
Abhd4 expression is remarkably specific to radial glial progenitor cells. Did authors observe 
reduction of cell death only in that cell population (for example, no changes in cortical plate?) in 
the knockout cortex of Abhd4 in cases of delta cdh2 overexpression and FASD model? A related 
comment; is Abhd4 expression altered by prenatal alcohol exposure? For example, do cells other 
than radial glial progenitor cells increase the expression of Abhd4, so that reduction of cell death is 
not limited to radial glial progenitor cells in the knockout? Depending on answers to those 
questions, proposed model may also require revision. 
 
The effects on migration and cell death are obvious in embryonic stages in Adhd4 manipulation. 
Are those effects kept until adulthood and make abnormal adult brain structure, or do cells catch 
up migration? This information is important for discussion about significance of developmental 
aniokis. 
 
Other comments: 
Most of TUNEL labelling did not overlap with GFP. Does cell death happen in cell non autonomous 
manner? 
 
 
 
Reviewer #2: 
Remarks to the Author: 
With this paper the Katona’s lab seeks to introduce a new concept in the field of developmental 
neuroscience: the developmental anoikis as a safeguarding mechanism responsible for the 
elimination of abnormally delaminated apical progenitors (RGPCs) during brain development. The 
authors identified (i) ABDH4 as a crucial molecular player in developmental anoikis and (ii) 
developmental anoikis as one of the possible mechanisms responsible for the elimination of 
abnormally delaminated cells in a pathological condition, fetal alcohol syndrome. 
 
The paper is a pleasant read and the ideas illustrated by the authors bring a fresh perspective in 
the neurogenesis field. I acknowledge that the authors address an issue that is under-represented 
in our field: how robustness is achieved in the context of cortical development and brain 
morphogenesis. 
 
Given the potential of their work, the authors must address the following points to make their 
paper suitable for acceptance and publication in Nature Communication. 
 
MINOR POINTS 
 
- A general remark. Throughout the text, the authors refer to abnormal RGPC delamination and 

AJs disassembly. Strictly speaking, delamination is always paralleled by the cell’s disengagement 
from the AJs belt. What abnormal exactly means? What is different between a normal and an 
abnormal AJ disassembly/delamination? What is then setting the difference? 
The same apply to the use of the word “premature”, referred to delamination. Premature 
compared to what exactly? Do the authors mean a delamination that is not paralleled by a fate 
transition? Or a delamination that is not sync with cell cycle progression? The authors must clarify 
and elaborate conceptually on that. 
 
- Page 5: the authors write “spatially limited destruction…” upon nCadh20-GFP manipulation 
referring to Figure1 a-d. 
However, the panels show a deep and generalized modification of the AJs. The authors must clarify 
this point and make clear if the image is representative of their conclusions. Is the manipulation 
really causing a limited change in the AJ? 
If so, then the authors should choose a picture reflecting this claim. 
Otherwise, the authors should change the sentence to reflect what the picture showed: a 
pronounced and general change in the AJs. 
The same apply to the phalloidin staining shown in Supplementary Figure 1a-f. The phalloidin 
staining at the apical surface is clearly less intense in the nCadh20-GFP -electroporated area 
compared to control electroporation area. This is of course compatible with a junctional 
perturbation, but it suggests that the perturbation is generalized, and not is “spatially limited”. 
 
- Regarding the effect of the manipulation on the basal endfeet. I believe this data is crucial in the 
clarification of the specificity of the cell biological effects of the experimental manipulation. The 
authors must show the data now parked in Supplementary Figure 1j-k in the main figure, possibly 
in Figure 1. 
Furthermore, given previous literature on the dynamics of the basal endfeet (Yokota et al, 2010), 
it would be important to define if any morphological change happens at the basal endfoot. I am 
not asking the author to generate new data, but rather to show the data they already have in a 
quantitative manner. For example, the authors should show a classification/quantification of the 
structure of the basal end feet as in Yokota et al, 2010, Figure 2 (club like vs branched end feet). 
In addition, the authors should add a quantification of the data shown in Supplementary Figure 
1m-n. 
 
- Page 5. The delaminated progenitors generated upon nCadh20-GFP retain the basal process, 
Pax6 expression and show an increased apoptosis 
(i) which is the % of delaminated cells that are also apoptotic? 
(ii) For those cells that are not apoptotic: do these delaminated cells correspond to bRG/oRG cells? 
The authors must address this point. 
I would suggest doing 3D reconstruction of their STORM data (or other suitable data sets) to 
highlight the complete morphology of the manipulated cells in the SVZ. Additionally, I recommend 
a staining with p-Vim to understand if there are more cells in SVZ retaining the basal process in 
mitosis, as hallmark of bRGs. 
 
- As for scRNAseq public libraries: it would be informative for the readers to show the actual 
data/plots for the ABHD4 expression in different cell types in the mentioned libraries. These data 
can be added as or in a supplementary figure. 
 
- Based on the data shown by the authors, the main role of ABDH4 seems to be associated with its 
downregulation. Based in the author’s claim, ABDH4 downregulation takes place immediately after 
delamination. This point is interesting and it is crucial in defining the mechanism of action of 
ABDH4. However, I think the claim is not fully supported by the data. It would be very informative 
to show an immnunostaining to define the localization of ABDH4. I am actually surprised that, 
given their experience with high resolution microscopy, the authors limit their attention the 
mRNAs, without investigating the protein localization. Please clarify if this is technical issue related 
to the Ab availability. Is ABDH4 -both at the mRNa and protein level- associated with the AJs or to 

the asymmetric partitioning of ABDH4 during RGPC mitosis? That could provide a cellular 
mechanism for the fast downregulation of ABDH4 in delaminated cells. Investigating this point 
would bring the entire paper to a higher level. 
 
- Page 7-8: the observation of the inverse correlation of ABHD4 with Tbr2 mRNA is interesting. 
However, I urge the authors to consider their data in light of previously published work. Of note, it 
has been demonstrated that aRGs cells already express Tbr2 mRNA (see Florio et al, 2015). So, 
Tbr2 mRNA is not a specific marker for delaminated IPs, as it is present also in not a delaminated 
IPs, and more importantly also in aRG cells (see Wilsch-Brauninger et al, 2012 and Florio et al, 
2015). In addition, the authors mentioned that they consider cells in the VZ. Again, the VZ 
localization is not indicative of a certain fate, as VZ contain APs, and IPs, both non-delaminated 
and delaminated. It is not clear if the cells analyzed by the authors are retaining or not the apical 
contact. I think the authors should run an additional quantification in which they score separately 
cells with and without the apical contact. That will help with the interpretation of the graphs shown 
in Figure panel X, that in its present state is hard to interpret and not particularly convincing. 
 
 
MINOR POINTS 
 
- Page 65-66: the model should be added as last panel to the main figure. 
 
- Page 3: the word INTRODUCTION is missing. 
 
- the paper address the issue of how robustness is achieved in the context of cortical development 
and brain morphogenesis. I would find extremely interesting if the authors could elaborate more 
on that in the discussion part.