Atlas of Fshr Expression from Novel Reporter Mice
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eLife assessment
The development of this mouse model is an important step to establish the role of the FSH Receptor in tissues beyond the reproductive system, and the data provided in this paper are convincing for a role for the FSH receptor in cell systems well beyond the classic reproductive tissues. Such model(s) have long been needed in this field and will provide expanded opportunities to better define FSH biology in vivo in these important target tissues. Ultimately, this model could shed light on FSH biology in women after menopause, when endogenous FSH levels rise dramatically, or in men with hypogonadism when FSH levels are high.
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Abstract
The FSH-FSHR signaling pathway has traditionally been considered an essential regulator in reproductive development and fertility. But there has been emerging evidence of FSHR expression in extragonadal tissues/organs. This poses new questions and long-term debates regarding the physiological role of the FSH-FSHR pathway, and underscores the need for reliable, in vivo analysis of FSHR expression in animal models. However, conventional methods have proven insufficient for examining FSHR expression due to limitations, such as the scarcity of ″reliable″ antibodies, rapid turnover/degradation of transcripts, and a lack of robust in vivo tools. To address this challenge, we developed Fshr-ZsGreen ″knockin″ reporter mice under the control of Fshr endogenous promoter using CRISPR/Cas9 genome-editing technology to append a P2A-ZsGreen targeting vector into a locus between the last exon and the stop codon of Fshr. With this novel genetic tool, we provide a reliable readout of Fshr expression at single-cell resolution level in vivo and in real time. Reporter animals were also subjected to additional analyses, including immunohistochemical staining, ddRT-PCR, and in situ hybridization, to define the accurate expression profile of FSHR in gonadal and extragonadal organs/tissues. Our compelling results not only demonstrated Fshr expression in intragonadal tissues but also, strikingly, unveiled notably increased expression in Leydig cells, osteoblast lineage cells, endothelial cells in vascular structures, and epithelial cells in bronchi of the lung and renal tubes. The genetic decoding of the widespread pattern of Fshr expression highlights its physiological relevance beyond reproduction and fertility, and opens new avenues for therapeutic options for age-related disorders of the bones, lungs, kidneys, and hearts, among other tissues/organs. Exploiting the power of the Fshr knockin reporter animals, this report provides the first comprehensive genetic record of the spatial distribution of FSHR expression, correcting a long-term misconception about Fshr expression and offering prospects for extensive exploration of FSH-FSHR biology.
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eLife assessment
The development of this mouse model is an important step to establish the role of the FSH Receptor in tissues beyond the reproductive system, and the data provided in this paper are convincing for a role for the FSH receptor in cell systems well beyond the classic reproductive tissues. Such model(s) have long been needed in this field and will provide expanded opportunities to better define FSH biology in vivo in these important target tissues. Ultimately, this model could shed light on FSH biology in women after menopause, when endogenous FSH levels rise dramatically, or in men with hypogonadism when FSH levels are high.
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Reviewer #1 (Public Review):
The manuscript describes the development of a mouse model that co-expresses a fluorescent protein ZsGreen) marker in gene fusion with the FSHR gene.
The authors are correct in that there is a lack of reliable antibodies against many of the GPCR family members. The approach is novel and interesting, with the potential to help understand the expression pattern of gonadotropin receptors. There has been a very long debate about the expression of gonadotropin receptors in other tissues other than gonads. While their expression of the FSHR in some of those tissues has been detected by a variety of methods, their physiological, or pathophysiological, function(s) remain elusive.
The authors in this manuscript assume that the expression of ZsGren and the FSHR are equal. While this is correct genetically …
Reviewer #1 (Public Review):
The manuscript describes the development of a mouse model that co-expresses a fluorescent protein ZsGreen) marker in gene fusion with the FSHR gene.
The authors are correct in that there is a lack of reliable antibodies against many of the GPCR family members. The approach is novel and interesting, with the potential to help understand the expression pattern of gonadotropin receptors. There has been a very long debate about the expression of gonadotropin receptors in other tissues other than gonads. While their expression of the FSHR in some of those tissues has been detected by a variety of methods, their physiological, or pathophysiological, function(s) remain elusive.
The authors in this manuscript assume that the expression of ZsGren and the FSHR are equal. While this is correct genetically (transcription->translation) it does not go hand in hand with other posttranslational processes.
(1) One of the shocking observations in this manuscript is the expression of FSHR in Leydig cells. Other observations are in the osteoblasts and endothelial cells as well as epithelial cells in different organs. The expression of ZsGreen in these tissues seems high and one shall start questioning if there are other mechanisms at play here.
First, the turnover of fluorescent proteins is long, longer than 48h, which means that they accumulate at a different speed than the endogenous FSHR This means that ZsGreen will accumulate in time while the FSHR receptor might be degraded almost immediately. This correlated with mRNA expression (by the authors) but does not with the results of other studies in single-cell sequencing (see below).
The expression of ZsGreen in Leydig cells seems much higher than in Sertoli cells, this is "disturbing" to put it mildly. This is visible in both the ZsGreen expression and the FISH assay (Figure 2 B-D).
(2) The expression in WAT and BAT is also questionable as the expression of ZsGreen is high everywhere. That makes it difficult to believe that the images are truly informative. For example, the stainings of aorta show the ZsGreen expression where elastin and collagen fibres are - these are not "cells" and therefore are not expressing ZsGreen.
(3) FISH expression (for FSHR) in WT mice is missing.
Also, the tissue sections were stained with the IgG only (neg control) but in practice both the KI and the WT tissues should be stained with the primary and secondary antibodies. The only control that I could think of to truly get a sense of this would be a tagged receptor (N-terminal) that could then be analysed by immunohistochemistry.
(4) The authors also claim:
To functionally prove the presence of FSHR in osteoblasts/osteocytes, we also deleted FSHR in osteocytes using an inducible model. The conditional knockout of FSHR triggered a much more profound increase in bone mass and decrease in fat mass than blockade by FSHR antibodies (unpublished data).This would be a good control for all their images. I think it is necessary to make the large claim of extragonadal expression, as well as intragonadal such as Leydig cells.
(5) Claiming that the under-developed Leydig cells in FSHR KO animals are due to a direct effect of the FSHR, and not via a cross-talk between Sertoli and Leydig cells, is too much of a claim. It might be speculated to some degree but as written at the moment it suggests this is "proven".
(6) We also do not know if this FSHR expressed is a spliced form that would also result in the expression of ZsGreen but in a non-functional FSHR, or whether the FSHR is immediately degraded after expression. The insertion of the ZsGreen might have disturbed the epigenetics, transcription, or biosynthesis of the mRNA regulation.
(7) The authors should go through single-cell data of WT mice to show the existence of the FSHR transcript(s).
For example here:
https://www.nature.com/articles/sdata2018192 -
Reviewer #2 (Public Review):
The authors developed an original knock-in reporter mice line expressing ZSGreen under the control of endogenous FSHR promoter. The existence of FSHR in various extra-gonadal tissues and the physio-pathological consequences indeed remains a debated question and could potentially have an important impact on many high-incidence diseases occurring in menopausal women. Unfortunately, the provided data set lacks crucial controls and therefore does not provide a robust/convincing answer to the above-mentioned question.
Summary:
The authors investigated the expression pattern of the FSHR in the gonads, where its expression has been demonstrated for decades, but also in many extra-gonadal tissues. The question is important since the expression of FSHR outside of the gonads has been increasingly reported and …Reviewer #2 (Public Review):
The authors developed an original knock-in reporter mice line expressing ZSGreen under the control of endogenous FSHR promoter. The existence of FSHR in various extra-gonadal tissues and the physio-pathological consequences indeed remains a debated question and could potentially have an important impact on many high-incidence diseases occurring in menopausal women. Unfortunately, the provided data set lacks crucial controls and therefore does not provide a robust/convincing answer to the above-mentioned question.
Summary:
The authors investigated the expression pattern of the FSHR in the gonads, where its expression has been demonstrated for decades, but also in many extra-gonadal tissues. The question is important since the expression of FSHR outside of the gonads has been increasingly reported and associated with the dramatic increase of circulating FSH after menopause, and has been suggested to play an important role in the advent of multiple diseases occurring with high incidence in post-menopausal women. However, the reality of such extra-gonadal expression of FSHR remains debated, mainly because this receptor is expressed at a low level and because the specificity/affinity of the available anti-FSHR antibodies is questionable.Strengths:
The development of reporter mice expressing ZsGreen fluorescent protein under the control of endogenous FSHR promoter is an original and potentially powerful approach to tackle the problem.Weaknesses:
The data provided are provocative since the FSHR seems to be expressed in all tested tissues. In the testis, for instance, the authors report very high levels of FSHR in interstitial cells and germ cells. In the ovary, there seems to be no difference in FSHR expression between granulosa cells and the other cell types. These findings alone contradict all the knowledge on FSH expression patterns in the gonads that have been accumulated over decades by many independent labs. In view of such results, the validity of the reporter mice line should be questioned thoroughly:(1) Is the FSHR expression pattern affected by the knockin mice (no side-by-side comparison between wt and GSGreen mice, using in situ hybridization and ddRTPCR, at least in the gonads, is provided)?
(2) Is the splicing pattern of the FSHR affected in the knockin compared to wt mice, at least in the gonads?
(3) Are there any additional off-target insertions of GSGreen in these mice?
(4) Are similar results observed in separate founder mice?
(5) How long is GSGreen half-life? Could a very long half-life be a major reason for the extremely large expression pattern observed?
In the absence of answers to these questions, the data produced in extra-gonadal tissues using the same reporter mice, are not convincing and do not support the authors' claims.
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