Sox9 marks limbal stem cells and is required for asymmetric cell fate switch in the corneal epithelium

Reviewer #1 (Public Review):

Summary:

The authors wanted to identify genes that are critical for regulating the asymmetric fates of limbal stem cells and their transit amplified progeny in the central cornea. To this end, they utilized an in vivo cell cycle reporter to isolate proliferating basal cells from the anterior ocular surface epithelium and performed single-cell RNA-seq. This strategy revealed distinct basal cell identities with unique expression profiles of structural genes and transcription factors. The authors then focused on the Sox9 transcription factor implicated in stem cell regulation. It was differentially expressed between limbal stem cells and their progeny in the central cornea. Lineage tracing analysis confirmed that Sox9 marks long-lived limbal stem cells. Conditional deletion of Sox9 led to abnormal differentiation and squamous metaplasia in the central cornea. The authors suggest that Sox9 is required for the switch to asymmetric fate and commitment toward differentiation, as transit cells exit the limbal niche. By inhibiting the terminal differentiation of corneal progenitors and forcing them into continuous symmetric divisions, the Sox9 loss-of-function phenotype was replicated.

Strengths:

Thus, the paper shows the important role of Sox9 in the spatial regulation of asymmetric fate in the corneal epithelium and its proliferation and cell differentiation. The work is elegantly done using several models that converge on the main conclusions. It is very novel and delineates a new player in determining corneal epithelial cell fate. The experiments are well done, and the data are credible.

Weaknesses:

This reviewer has some minor concerns mostly related to data interpretation and the use of the LSC term.

Reviewer #2 (Public Review):

Summary, strengths, and weaknesses:

This article by Rice et al focuses on the study of limbal epithelial stem cells (LESCs). To obtain high resolution of stem/progenitor cell populations by single-cell RNA sequencing (scRNA-seq), the authors enriched the stem/progenitors by capturing GFP+ epithelial cells undergoing mitosis using the Cyclin-B-GFP transgene. The key novelty in the paper is that they identified Sox9 as a new LSC gene that is important for the health of the cornea. They show that Sox9 is expressed by LSCs at the mRNA and protein level, and that the Sox9-GFP transgene is useful to identify LSCs in live animals. They performed lineage tracing of Sox9-Cre mice that clearly demonstrated that Sox9+ cells are true LSCs. Further, Sox9-knockouts display severe opacification of the cornea, accompanied by the transformation of the central cornea into hyperplastic epidermis - a phenotype similar to previously described Notch1 conditional knockout (cKO). By studying the lifetimes of Notch dominant-negative transgenic individual basal corneal epithelial cells, they suggest that the thickening of the central zone in the Notch model is due to the loss of asymmetric division, and from that, they infer the phenotype of the Sox2-KO. In other words, it is suggested that the increased rate and type of division in the central Sox9-null cornea produce this dramatic epithelial thickening phenotype. This claim makes sense but suggests a role for the Notch, not Sox9, and this role is relevant for the central corneal epithelial cells, and not in LESCs. So I suggest considering revising the conclusions (title of the paper) on this aspect. This would not affect the impact of the paper.

In general, I believe that this is a very interesting and novel study that will be of interest to a broad readership. The methodology and study design are robust and elegant.

However, in some cases, typos, text modifications, additional controls, and new experiments are suggested.