Truncated radial glia as a common precursor in the late corticogenesis of gyrencephalic mammals

Reviewer #1 (Public Review):

In this manuscript, Bilgic et al aim to identify the progenitor types (and their specific progeny) that underlie the expanded nature of gyrencephalic brains. To do this, they take a comparative scRNAseq (single cell transcriptomics) approach between neurodevelopment of the gyrencephalic ferret, and previously published primary human brain and organoid data.

They first improve gene annotations of the ferret genome and then collect a time series of scRNAseq data of 6 stages of the developing ferret brain spanning both embryonic and post-natal development. Among the various cell types they identify are a small proportion of truncated radial glial cells (tRGs), a population known to be enriched in humans and macaques that emerges late in neurogenesis as the RGC scaffold splits into an oRGC that contact the pial surface and a tRG that contacts the ventricular surface. They find that the tRGs consist of three distinct subpopulations two of which are committed to ependymal and astroglial fates.

By integrating these data with publicly available data of developing human brains and human brain organoids they make some important observations. Human and ferret tRGs have very similar transcriptional states, suggesting that the human tRGs too give rise to ependymal and astroglial fates. They also find that the current culture conditions of human brain organoids seem to lack tRGs, something that will need to be addressed if they are to be used to study tRGs. While the primary human data set did contain tRGs, the stage or the region sampled were likely not appropriate, and therefore, the number of cells they could retrieve was low.

The authors have spent considerable efforts in improving gene modeling of the ferret genome, which will be important for the field. They've generated valuable time series data for the developing ferret brain, and have proposed the lineal progeny for the tRGs in the human brain. Whether tRGs actually do give rise to the ependymal and astrogial fates needs to be validated in future studies.

Reviewer #2 (Public Review):

Bilgic et al first explored cellular diversity in the developing cerebral cortex of ferret, honing in on progenitor cell diversity by employing FACS sorting of HES5-positive cells. They have generated a novel single cell transcriptomic dataset capturing the diversity of cells in the developing ferret cerebral cortex, including diverse radial glial and excitatory neuron populations. Unexpectedly, this analysis revealed the presence of CRYAB-positive truncated radial glia previously described only in humans. Using bioinformatic analyses, the investigators proposed that truncated radial glia produce ependymal cells, astrocytes, and to a lesser degree, neurons. Of particular interest to the field, they identify enriched expression of FOXJ1 in late truncated radial glia strongly indicating that towards the end of neurogenesis, these cells likely give rise to ependymal cells. This study represents a major advancement in the field of cortical development and a valuable dataset for future studies of ferret cortical development.

Author Response

Summary of reviewers recommendations.

Reviewer 1

Point# 1. Make a new section in the text with a figure about the improvement of the genomic information (gene modeling) of ferrets ".

Point# 2. the references to figure S8A, B alright (line 306)?

Point# 3. Revise the main text and figure legends of the section dealing with the human-ferret comparison for clarity.

Reviewer 2

Point# 4. Weaken (change the text from “conclusive” to suggestive” ) the expression that we identified that tRG become ependymal cells, because we have not demonstrated the morphological transformation of a tRG cell into an ependymal cell, which is practically difficult although we have shown morphological change in terms of the single-cilia to multi-cilia form transition (Fig. S6A).

Point# 5. Correct several typos throughout the manuscript that I would recommend fixing for example, page 5 line 123 says "OLIGO2" instead of “OLIG2.

Provisional revision plan and our responses.

Point #1 The new section for the improvement of gene models will be made by transferring the part of methods to the main text and Fig S2B,C to new Figure 1 with one schematic panel.

Point #2; We cited (Bhaduri et al., 2020) as a reference in the figure S8A , while "Bhaduri et, al, 2021” was cited in the text. Which is correct? We will correct this, by choosing the correct one. Descriptions are indeed poor regarding Fig. S8A and S8B in the text as well as in the legends.

Point #3 : We will describe the methods of comparison between ferrets and humans more thoroughly, by adding definition of words such as gene scores, subtype scores in the main text. (as well, the explanation of (Figure S3C) will be improved. ). Legends for Fig. 6 are too simple. So we would explain more in these legends. Explanations of analysis and figures, which we made, responding to the reviewer comments of “review commons” are generally not easy to understand with too short explanations, comparing with complexity of figures and contents, let’s say, Figure S8A-D. We will give more explanations for each of panel in Figure S8A-D, and E and F.

Point #4; The authors' response to this point goes like this; we totally agree that we need to genetically labeling (knocking in the Cryab gene) to prove “tRG cells differentiate ependymal cells”. We tried many times but eventually failed. We have partially show single-cilia to multi-cilia transition which is characteristic to epithelial-ependymal transition. This process appears to take a long time and therefore, morphological tracing by time-lapse imaging in tissue culture is not a realistic way, Therefore, we weakened the conclusion; it is "highly likely" that tRG cells differentiate to be ependymal cells.

Point#5: We will survey typos-> correct them, by all authors read the manuscript carefully again.