SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways
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Abstract
SOX2 expression levels are crucial for the balance between maintenance and differentiation of airway progenitor cells during development and regeneration. Here, we describe patterning of the mouse proximal airway epithelium by SOX21, which coincides with high levels of SOX2 during development. Airway progenitor cells in this SOX2+/SOX21+ zone show differentiation to basal cells, specifying cells for the extrapulmonary airways. Loss of SOX21 showed an increased differentiation of SOX2+ progenitor cells to basal and ciliated cells during mouse lung development. We propose a mechanism where SOX21 inhibits differentiation of airway progenitors by antagonizing SOX2-induced expression of specific genes involved in airway differentiation. Additionally, in the adult tracheal epithelium, SOX21 inhibits basal to ciliated cell differentiation. This suppressing function of SOX21 on differentiation contrasts SOX2, which mainly drives differentiation of epithelial cells during development and regeneration after injury. Furthermore, using human fetal lung organoids and adult bronchial epithelial cells, we show that SOX2+/SOX21+ regionalization is conserved. Lastly, we show that the interplay between SOX2 and SOX21 is context and concentration dependent leading to regulation of differentiation of the airway epithelium.
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###Reviewer #2
In their manuscript titled "SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways" from Eenjes et al. the authors describe a new role for Sox21 in the developing and adult airway epithelium. Building on prior work they observe a unique distribution of Sox21 expression in the developing airways and through careful and elegant immunostaining and over-expression studies they suggest that Sox21 is downstream of a key airway development transcription factor Sox2. They suggest opposing roles for these genes based on analysis of Sox2 and Sox21 heterozygous knock-out mice whereby Sox21 prevents and Sox2 promotes differentiation of immature airway progenitors into basal cells. This raises interesting questions regarding the role of Sox21 in the biology of airway progenitors and the …
###Reviewer #2
In their manuscript titled "SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways" from Eenjes et al. the authors describe a new role for Sox21 in the developing and adult airway epithelium. Building on prior work they observe a unique distribution of Sox21 expression in the developing airways and through careful and elegant immunostaining and over-expression studies they suggest that Sox21 is downstream of a key airway development transcription factor Sox2. They suggest opposing roles for these genes based on analysis of Sox2 and Sox21 heterozygous knock-out mice whereby Sox21 prevents and Sox2 promotes differentiation of immature airway progenitors into basal cells. This raises interesting questions regarding the role of Sox21 in the biology of airway progenitors and the balance of cell types in the airways. In adult mice no obvious differences are detected in Sox21+/- mice in terms of regenerative capacity. In in vitro assays of adult mouse basal cell differentiation (ALI culture) failed to identify obvious differences in basal cell number of differentiation into specific lineages in Sox21+/- cells. The authors report an overall similar pattern of SOX21 expression in a human fetal lung organoid platform. Finally, in human ALI cultures the authors identify SOX21 expression most abundant in paranasal cells suggesting a similar possible role for this genes in differentiating basal cells in humans. Overall, the molecular mechanisms that control airway stem cell maintenance and differentiation are of great interest to the field.
Major concerns:
The development data is clear based on the studies presented. The immunostaining and figures are elegant. It does not appear the Sox21 is necessary for airway development. A key question raised by this work is how important and what precisely is the role of Sox21? For example, overexpression of Sox21 in the developing and adult airways might have been more instructive in answering these key questions rather than under the control of the SPC-promoter.
The adult mouse data and human data seem to overall suggest that SOX2 and SOX21 are upregulated in differentiating cells in vitro and are expressed at lower levels in basal cells but real mechanism or phenotype related to SOX21 function is observed in the SOX21 het mouse cells.
The attempt to assess human fetal airways is admirable. However, sections of fetal lungs would be more relevant. Can the authors address how similar fetal lung cultures are to in vivo airways and at what developmental time point ?
Overall the relevance of SOX21 expression in the adult airways is unclear.
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###Reviewer #1
This is an interesting but also a bit confusing manuscript by Evelien Eenjes and colleagues.
I was wondering whether the seemingly conflicting effect of Sox21 on basal cell differentiation could be due to an increase or decrease in Sox9 positive basal cells. These atypical basal cells have been described in a number of manuscripts.
For example - https://www.ncbi.nlm.nih.gov/pubmed/26869074 - it appears from the figures that Sox21 may increase Sox9 expression. Can the authors look at the presence of Sox9 positive basal cells in the trachea of the different mutants?
It would be important to quantify the % of club cells in the corn oil and naphthalene treated tracheas of the different mutants as these are the main cells that should be replaced upon naphthalene injury. I am not sure that people normally look at the number of …
###Reviewer #1
This is an interesting but also a bit confusing manuscript by Evelien Eenjes and colleagues.
I was wondering whether the seemingly conflicting effect of Sox21 on basal cell differentiation could be due to an increase or decrease in Sox9 positive basal cells. These atypical basal cells have been described in a number of manuscripts.
For example - https://www.ncbi.nlm.nih.gov/pubmed/26869074 - it appears from the figures that Sox21 may increase Sox9 expression. Can the authors look at the presence of Sox9 positive basal cells in the trachea of the different mutants?
It would be important to quantify the % of club cells in the corn oil and naphthalene treated tracheas of the different mutants as these are the main cells that should be replaced upon naphthalene injury. I am not sure that people normally look at the number of ciliated cells reappearing after naphthalene injury as ciliated cells are not thought to be killed by naphthalene.
The authors should also look at Sox9 positive basal cells in the tracheas of the corn oil and naphthalene treated ctrl and mutant mice.
Are the intermediate para-basal cells in the human airway differentiation experiment club cells? Why do the authors think these cells are on their way to become ciliated cells? Doesn't Sox21 inhibit ciliated cell differentiation?
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###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on June 1, 2020, follows.
Summary
This is a comprehensive analysis of Sox21 in lung development, homeostasis, and injury, using wide array of methods including mouse human cell-based ALI cultures and organoids. The authors describe a new role for Sox21 downstream of the key airway development transcription factor Sox2. They suggest opposing roles for these genes based on analysis of Sox2 and Sox21 heterozygous knock-out mice whereby Sox21 prevents and Sox2 promotes differentiation of immature airway progenitors into basal cells. This raises interesting questions regarding the role of Sox21 in the biology of airway progenitors and the balance of cell types in the airways. In adult mice no obvious differences are detected in Sox21+/- mice …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on June 1, 2020, follows.
Summary
This is a comprehensive analysis of Sox21 in lung development, homeostasis, and injury, using wide array of methods including mouse human cell-based ALI cultures and organoids. The authors describe a new role for Sox21 downstream of the key airway development transcription factor Sox2. They suggest opposing roles for these genes based on analysis of Sox2 and Sox21 heterozygous knock-out mice whereby Sox21 prevents and Sox2 promotes differentiation of immature airway progenitors into basal cells. This raises interesting questions regarding the role of Sox21 in the biology of airway progenitors and the balance of cell types in the airways. In adult mice no obvious differences are detected in Sox21+/- mice in terms of regenerative capacity. In in vitro assays of adult mouse basal cell differentiation (ALI culture) failed to identify obvious differences in basal cell number of differentiation into specific lineages in Sox21+/- cells. In human ALI cultures the authors identify a similar possible role for Sox21 in differentiating basal cells in humans. Overall, the molecular mechanisms that control airway stem cell maintenance and differentiation are of great interest to the field. However, in its current form the studies remain primarily descriptive and several data inconsistencies remain, which makes it difficult to draw impactful conclusions at the current stage. The potential suitability for eLife would significantly increase with additional mechanistic experiments and data.
Essential Revisions
In particular, the following major points require additional experimental data (details are outlined below):
- The functional Sox21 role in the adult lung is not clear.
Mouse studies would benefit from inclusion of overexpression of Sox21 in the airways
In human airways and the models used (organoids and ALI cultures) gain and loss of function studies are not performed and would also behelpful to further determine the phenotype of the intermediate para-basal cells in the human airway.
Evidence for genetic interaction between Sox21 and Sox2 in the adult lung needs to be expanded.
Presence of Sox9 positive basal cells as additional atypical basal cells needs to be further investigated.
Quantification the % of club cells in the corn oil and naphthalene treated tracheas of the different mutants as these are the main cells that should be replaced upon naphthalene injury.
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