Decoding the activated stem cell phenotype of the neonatally maturing pituitary

  1. Emma Laporte
  2. Florian Hermans
  3. Silke De Vriendt
  4. Annelies Vennekens
  5. Diether Lambrechts
  6. Charlotte Nys
  7. Benoit Cox
  8. Hugo Vankelecom

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    This is a well conducted study on development of neonatal mouse pituitary using multiple ScRNA Seq and organoid culture models. Some weaknesses include accuracy of the language used, lack of data demonstrating the role of Wnt signaling, and lack of follicle-stellate markers and lack of characterization of pituitary defects in IL6 null mice.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

The pituitary represents the endocrine master regulator. In mouse, the gland undergoes active maturation immediately after birth. Here, we in detail portrayed the stem cell compartment of neonatal pituitary. Single-cell RNA-sequencing pictured an active gland, revealing proliferative stem as well as hormonal (progenitor) cell populations. The stem cell pool displayed a hybrid epithelial/mesenchymal phenotype, characteristic of development-involved tissue stem cells. Organoid culturing recapitulated the stem cells’ phenotype, interestingly also reproducing their paracrine activity. The pituitary stem cell-activating interleukin-6 advanced organoid growth, although the neonatal stem cell compartment was not visibly affected in Il6 −/− mice, likely due to cytokine family redundancy. Further transcriptomic analysis exposed a pronounced WNT pathway in the neonatal gland, shown to be involved in stem cell activation and to overlap with the (fetal) human pituitary transcriptome. Following local damage, the neonatal gland efficiently regenerates, despite absence of additional stem cell proliferation, or upregulated IL-6 or WNT expression, all in line with the already high stem cell activation status, thereby exposing striking differences with adult pituitary. Together, our study decodes the stem cell compartment of neonatal pituitary, exposing an activated state in the maturing gland. Understanding stem cell activation is key to potential pituitary regenerative prospects.

Article activity feed

  1. Version published to 10.7554/elife.75742 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    This study uses cutting edge transcriptomics to decode the changes in transcript expression with neonatal development.

    Strengths

    The study is sufficiently detailed so that the reader can evaluate the data and conclusions. Most importantly, the scientist who is able to analyze data from RNA-sequencing such as this will be able to seek answers their own questions about gene ontology and pathways involved in pituitary cell development.

    The study is validated by the use of the organoid cultures, which recapitulate the transcriptome expression of the developing pituitary stem cells. An important strength is the fact that they were able to develop growth media that is optimal for neonatal pituitaries, as the organoid media used by many has been developed for adult cultures. This will be an important addition for many laboratories wishing to study organoid cultures from neonatal pituitary populations.

    The study of the damaged neonatal pituitary (damaged by the ablation of somatotropes) is interesting and shows that the damage focuses on somatotropes and does not ablate stem cells. The study is worth further analysis by those who are interested in the impact of loss of somatotropes on pituitary cell populations.

    The populations subjected to scRNA-seq are available publicly and provide important tools for other researchers who want to decode stem cell activation.

    Weaknesses

    1. The study is best analyzed by individuals who are well versed in bioinformatics approaches or by individuals who have access to this expertise. This is not a major weakness, only a precautionary remark.

    We thank the reviewer for this precautionary remark. For the reviewer’s informaton, during the last couple of years, our group acquired substantial bioinformatic skills to perform such scRNA-seq analyses, as witnessed by our recent publications (Hemeryck et al., 2022; Vennekens et al., 2021). Moreover, our group collaborates with (other) experts in the single-cell bioinformatic field (also listed as co-authors on previous and current papers) and the Leuven Institute for Single Cell Omics (LISCO; https://lisco.kuleuven.be), of which the PI is affiliated member. Moreover, we did our best to explain the bioinformatic findings (using established tools such as SCENIC and CellPhoneDB) as clear as possible to enable researchers, less experienced in single-cell bioinformatics, to understand the study.

    1. This reviewer wonders about the use of the word "vividly" in the title and throughout the manuscript. Clearly these pituitary populations from the 7 day neonatal mice are maturing, however what about the study makes this maturation "vivid". The maturation is fairly ordinary and expected and not any more vivid than any other type of study of neonatal development. Vivid denotes a dynamic state and only one age was chosen for analysis of maturation.

    We understand the reviewer’s concern and agree that ‘vivid’ may not be the most appropriate word (although it sounded so when translated to our native language). Therefore, we removed it (e.g. in title and Abstract), or replaced it throughout the manuscript with ‘active/dynamic’ (or other appropriate words) at the indicated places.

    1. Readers need to recognize that this transcriptome reflects gene activity in the PND 7 mouse and there may be additional changes during the second week of development, especially when prolactin cells begin to differentiate. This is not a major weakness because these types of studies are very expensive (in the US) and one must choose one's model carefully. The rationale for the use of 7 day old neonate could be spelled out (why not 4 or 5 day mice). One might guess however that this has to do with the size of the pituitary which is very tiny in the developing mouse.

    We thank the reviewer for this genuine reflection.

    The PD7 age was chosen for several reasons. First, the particular age was analyzed in our previous study which showed signs of an activated stem cell state at this age (Gremeaux et al., 2012) (as referenced in the text). Second, and indeed, the neonatal mouse pituitary is very small in size, and to obtain a sufficient number of cells for downstream analyses, we chose the most useful (but still actively maturing) age of PD7. Third, for the damage and regeneration experiments, pups had to be i.p. injected with DT which had to start 3 days before the analysis timepoint at PD7, and PD4 was the limit age in which this could be (most) reliably performed.

    It would certainly be interesting to explore still other ages during the early-postnatal maturation phase (e.g. the second week as suggested, however already focused on in (Russell et al., 2021)), but also in Europe/Belgium, scRNA-seq analyses are very expensive and therefore ages and models must be carefully selected.

    1. It is impossible to remove the posterior pituitary and not also remove the intermediate lobe and the data show clearly that melanotropes were present in the PND 7 mouse as well as the adult.

    Although we aimed at meticulously removing (under the stereomicroscope) the intermediate and posterior lobes, some residual intermediate lobe cells appear to remain attached to the anterior lobe as clear from the scRNA-seq analysis (adult). In the neonatal mouse, the pituitary tissue is still more ‘sticky’, making it technically still more challenging to dissect away the posterior and intermediate lobes. Hence, not only a cluster of melanotropes (as in adults) but also a (very small) cluster of posterior lobe (PL) cells remains present.

  3. eLife

    Evaluation Summary:

    This is a well conducted study on development of neonatal mouse pituitary using multiple ScRNA Seq and organoid culture models. Some weaknesses include accuracy of the language used, lack of data demonstrating the role of Wnt signaling, and lack of follicle-stellate markers and lack of characterization of pituitary defects in IL6 null mice.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Pituitary stem cells are a population of low proliferating and differentiating cells in the mature gland. In contrast, during the early post-natal period, they are much more active. In this manuscript "Decoding the activated stem cell phenotype of the vividly maturing neonatal pituitary", Laporte et al investigate the basis of this activation. To this end, they perform single cell transcriptomic analyses. In addition, the effect of manipulation of IL6 and WNT pathways are investigated in vivo and on pituitary organoids. Differential effects are observed between young and adult cells which may explain the proliferative difference between neonate and adult cells, however further investigations are required. Furthermore, based on the single cell transcriptomic analyses, cross-talks between Wnt-secreting and responding stem cells are suggested; these should be further substantiated. The consequences of acute endocrine cell ablation on stem cell regenerative potential are finally examined. These show that regeneration occurs efficiently in the neonate gland. Despite this, further activation of neonatal stem cells is not observed after acute cell ablation and the mechanisms underlaying regeneration remain unknown. In summary, this is the first report of a single cell transcriptomic analysis of the whole neonatal murine pituitary, allowing analysis of the activated stem cell compartment.

  5. eLife

    Reviewer #2 (Public Review):

    This study uses cutting edge transcriptomics to decode the changes in transcript expression with neonatal development.

    Strengths:

    The study is sufficiently detailed so that the reader can evaluate the data and conclusions. Most importantly, the scientist who is able to analyze data from RNA-sequencing such as this will be able to seek answers their own questions about gene ontology and pathways involved in pituitary cell development.

    The study is validated by the use of the organoid cultures, which recapitulate the transcriptome expression of the developing pituitary stem cells. An important strength is the fact that they were able to develop growth media that is optimal for neonatal pituitaries, as the organoid media used by many has been developed for adult cultures. This will be an important addition for many laboratories wishing to study organoid cultures from neonatal pituitary populations.

    The study of the damaged neonatal pituitary (damaged by the ablation of somatotropes) is interesting and shows that the damage focuses on somatotropes and does not ablate stem cells. The study is worth further analysis by those who are interested in the impact of loss of somatotropes on pituitary cell populations.
    The populations subjected to scRNA-seq are available publicly and provide important tools for other researchers who want to decode stem cell activation.

    Weaknesses:

    1. The study is best analyzed by individuals who are well versed in bioinformatics approaches or by individuals who have access to this expertise. This is not a major weakness, only a precautionary remark.
    2. This reviewer wonders about the use of the word "vividly" in the title and throughout the manuscript. Clearly these pituitary populations from the 7 day neonatal mice are maturing, however what about the study makes this maturation "vivid". The maturation is fairly ordinary and expected and not any more vivid than any other type of study of neonatal development. Vivid denotes a dynamic state and only one age was chosen for analysis of maturation.
    3. Readers need to recognize that this transcriptome reflects gene activity in the PND 7 mouse and there may be additional changes during the second week of development, especially when prolactin cells begin to differentiate. This is not a major weakness because these types of studies are very expensive (in the US) and one must choose one's model carefully. The rationale for the use of 7 day old neonate could be spelled out (why not 4 or 5 day mice). One might guess however that this has to do with the size of the pituitary which is very tiny in the developing mouse.
    4. It is impossible to remove the posterior pituitary and not also remove the intermediate lobe and the data show clearly that melanotropes were present in the PND 7 mouse as well as the adult.

  6. Version published to 10.1101/2022.01.18.476723v1 on bioRxiv