Origin, specification and differentiation of a rare supporting-like lineage in the developing mouse gonad
Posted on: 29 September 2021
Preprint posted on 17 September 2021
Article now published in Science Advances at http://dx.doi.org/10.1126/sciadv.abm0972
Go big or Go-nad: building a single-cell transcriptomic atlas of the developing mouse gonads.
Selected by Martin Estermann
Categories: cell biology, developmental biology, genomics, molecular biology
Background:
Gonads can differentiate into two completely different organs: testes in males and ovaries in females. During early embryonic development, male and female gonads are indistinguishable. They contain the same cell types and have a similar transcriptome. During the process of sex determination, each of the gonadal cell types commits into an ovarian or testicular fate. In mouse, this process starts around embryonic day 11.5 (E11.5) with the upregulation of the Y-linked gene Sry in male supporting cells, initiating the differentiation towards Sertoli cells by upregulating Sox9, Fgf9 and Amh. In females, the lack of Sry expression results in the stabilization of the Wnt4/b-catenin signalling pathway, which results in pre-granulosa cell differentiation and upregulation of the transcription factor Foxl2. These supporting cells will then coordinate the differentiation of the remaining gonadal cell types, resulting in a fully functional ovary or testis. In testis, a network of interconnecting tubules, the rete testis, connects the seminiferous tubules with the efferent ductules, transporting the sperm to the epididymis. In females, the rete ovarii is homologous to the rete testis, although its physiological function is unclear. The embryonic origin, molecular regulation and development of the rete testis and ovarii is poorly understood.
Single-cell RNA sequencing (scRNA-seq) technologies have improved the analysis of gene expression across heterogenous cell types, allowing an unbiased analysis of the cell identity. We are closer to being able to precisely define gene expression at the single cell level. In this research, whole gonadal single-cell RNA-seq was performed on male and female mouse gonads, from E10.5 to E16.5. This research generated a transcriptomic atlas of the developing male and female gonads, identifying a novel cell population involved in rete testis/ovarii formation.
Key findings
1) Identification of a novel Pax8 positive supporting like cell population
Single-cell RNA sequencing was performed on male and female E10.5 and E11.5 Nr5a1-GFP mice urogenital ridges containing both gonadal and mesonephric tissue, and on E12.5, E13.5 and E16.5 XX and XY gonads. In total, more than 90,000 cells were sequenced, making it the largest embryonic gonadal dataset till date. Clustering analysis identified 43 transcriptionally distinct clusters and 19 gonadal cell types, including epithelial, supporting, steroidogenic, interstitial, mesonephric, immune and germ cells.
Among them, a novel supporting like cell population was identified. These cells express somatic gonadal cell markers including Nr5a1, Wt1, and Gata4, as well as Pax8, a well-known mesonephric tubule and rete testis/ovarii marker. Additionally, this cell type expresses Sox9, a supporting male (Sertoli) marker, as well as Wnt4, a granulosa (female) cell marker. PAX8 immunofluorescence in E11.5 male gonads confirmed the presence of these cell types in the mesonephros-gonad border, colocalizing with GATA4 and partially with SOX9 (Figure 1).
Fig. 1. PAX8 positive cells localize in the gonadal-mesonephros border. PAX8, GATA4 and SOX9 whole mount immunofluorescence in XY E11.5 genital ridges. White box indicates magnification area, dashed line delineates the gonadal-mesonephros border. Scale bar = 50um (Preprint Fig. 4).
2) Pax8 positive cells contribute to the rete testi/ovarii and supporting cells
To address the contribution of this novel supporting-like cell population, lineage tracing was performed using a transgenic mouse. This mouse expresses RFP under the control of the Pax8 promoter. XX and XY gonads were collected at E13.5, E16.5 and at birth (P0), cleared and 3D imaged using lightsheet fluorescence microscopy (Fig. 2A). In males, Pax8 positive derived cells clustered in the rete testis, at the gonad cranio-dorsal region. In females, RFP positive cells were also detected in the anterior-dorsal region of the gonad. In both gonads, RFP positive cells were also identified scattered within the gonad, but in higher proportions in males than in females. These Pax8 derived gonadal cells colocalized with the Sertoli cell marker AMH in males (Fig. 2B) and with the granulosa cell marker FOXL2 in females (Fig. 2B). This suggests that Pax8 positive cells give rise to the rete testi and ovarii, and some of gonadal supporting cells.
Fig. 2. Pax8 positive cells lineage tracing. (A) 3D reconstruction of Pax8-derived cells (magenta) in E13.5, E16.5 and P0 male and female gonads. (B) Colocalization of Pax8-derived cells (magenta) with the Sertoli cell marker AMH (green) in males or granulosa marker FOXL2 (green) in females. White arrows indicate colocalization of both markers. White box indicates magnification area. (Preprint Fig. 5).
3) WNT4 but not retinoic acid signalling regulates rete testis differentiation
In E13.5 gonads, in both sexes, Wnt4 expression is localized in the antero-dorsal part, where both rete testis and rete ovarii forms. This suggests that Wnt4 signalling might be important for rete testis and rete ovarii development. In Wnt4 knockout testis, there were fewer Pax8 positive cells, lacking rete testis formation. In contrast, testis lacking retinoic acid signalling (lacking all 3 isotypes of retinoic acid receptors) showed normal rete testis and rete ovarii development. Taken together these results suggest that WNT4 but not retinoic acid signalling is important for rete testis development.
Why I choose this paper:
As a person working in gonadal development and sex differentiation, this is an interesting paper to me. Not only is it the largest single cell dataset in mammalian embryonic gonads, but it also involves the discovery of a previously uncharacterized cell population involved in one of the least studied processes, the development of rete testis and rete ovarii. This publication has the potential to become the mouse embryonic gonadal atlas. This research might have only scratched the surface of this database by focusing only in this uncharacterized Pax8 positive population, and I don’t doubt that deeper analysis of this large dataset will impact the way we understand gonadal development and differentiation.
Future directions / questions for the authors:
- It is interesting that these cells express Pax8 (a mesonephric tubule marker) and are located in the mesonephric-gonad border. From the Pax8 lineage tracing it looks like they are migrating into the gonad from the mesonephros. Are those cells migrating from the mesonephros or derived from the coelomic epithelium? Do you see GFP (Nr5a1) expression on them (3D imaging and scRNA-seq data)?
- Why do you think this population remained uncharacterized until now? Why do you think this population was not identified in previous gonadal scRNA-seq?
doi: https://doi.org/10.1242/prelights.30761
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