Yolk-sac–derived macrophages regulate fetal testis vascularization and morphogenesis

DeFalco, Tony; Bhattacharya, Indrashis; Williams, Alyna V.; Sams, Dustin M.; Capel, Blanche

doi:10.1073/pnas.1400057111

Cited by 172 publications

(211 citation statements)

References 60 publications

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“…The testis interstitium harbors the steroidogenic Leydig cells, peritubular myoid cells, macrophages, vasculature, and other uncharacterized cell types such as fibroblasts and vascularassociated cells (Brennan and Capel, 2004;DeFalco et al, 2014). In the mouse, steroidogenic Leydig cells consist of two populations based on the time of their appearance: fetal and adult Leydig cells (Benton et al, 1995;Huhtaniemi and Pelliniemi, 1992).…”

Section: Introductionmentioning

confidence: 99%

Mapping lineage progression of somatic progenitor cells in the mouse fetal testis

2016

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Testis morphogenesis is a highly orchestrated process involving lineage determination of male germ cells and somatic cell types. Although the origin and differentiation of germ cells are known, the developmental course specific for each somatic cell lineage has not been clearly defined. Here, we construct a comprehensive map of somatic cell lineage progression in the mouse testis. Both supporting and interstitial cell lineages arise from WT1 + somatic progenitor pools in the gonadal primordium. A subpopulation of WT1 + progenitor cells acquire SOX9 expression and become Sertoli cells that form testis cords, whereas the remaining WT1 + cells contribute to progenitor cells in the testis interstitium. Interstitial progenitor cells diversify through the acquisition of HES1, an indication of Notch activation, at the onset of sex determination. HES1 + interstitial progenitors, through the action of Sertoli cell-derived Hedgehog signals, become positive for GLI1. The GLI1 + interstitial cells eventually develop into two cell lineages: steroid-producing fetal Leydig cells and non-steroidogenic cells. The fetal Leydig cell population is restricted by Notch2 signaling from the neighboring somatic cells. The non-steroidogenic progenitor cells retain their undifferentiated state during fetal stage and become adult Leydig cells in post-pubertal testis. These results provide the first lineage progression map that illustrates the sequential establishment of somatic cell populations during testis morphogenesis.

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Section: Introductionmentioning

confidence: 99%

Mapping lineage progression of somatic progenitor cells in the mouse fetal testis

2016

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“…The ex vivo whole organ droplet method allows the researcher to administer pharmacological reagents easily to fetal organs in a very small volume of culture media. To examine the effects of vascularization and vascular remodeling on testis differentiation and morphogenesis, we used the small-molecule inhibitor TKI II, a reagent that disrupts testis vascular development 3 by blocking the activity of VEGF receptors; the formation of fetal testis architecture occurs in a vascular-dependent manner, acting through vascular endothelial growth factor A (VEGFA) 11,12 . While vascularization of the testis is critical for the export of testosterone that drives virilization of the embryo, it is also a major driver of testis cord morphogenesis: previous work has shown that when VEGFA signaling is blocked at or prior to E11.5, Sertoli cells fail to partition out from surrounding interstitial cells and no cord structures form 3,12 .…”

Section: Representative Resultsmentioning

confidence: 99%

“…One significant advantage of culturing fetal gonads is that small-molecule inhibitors can readily access the whole organ by simple diffusion. DeFalco et al have shown that utilizing this ex vivo droplet culture method in conjunction with smallmolecule inhibitors can be used to study signaling processes and interactions occurring during gonad development 3 ; these processes would be difficult to examine in vivo due to technical challenges (e.g., passage of drugs through the placenta or lethality of affecting multiple organs using genetic or pharmacological approaches). .…”

Section: Introductionmentioning

confidence: 99%

Using <em>Ex Vivo</em> Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Potter

DeFalco

2015

JoVE

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Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development. Video LinkThe video component of this article can be found at

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“…The obvious differences between these two models was that mixed culture conditions allowed direct cellcell contact, whereas co-culture conditions allowed shared medium contact but considerable distance between fetal Leydig and remaining cells. Recently, other interstitial cells have emerged as critical players that together with endothelial cells organize testis morphogenesis and regulate fetal Leydig cell differentiation [22,57,58]. Thus, although our mixed culture system failed to recapitulate native cell-cell interactions, direct interactions were still present to facilitate more efficient conversion to testosterone.…”

Section: Maintenance Of Fetal Leydig Cell Activity Over Time In Culturementioning

confidence: 97%

Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

Carney

Muszynski

Strotman

et al. 2014

Biology of Reproduction

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Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3-5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cellcell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture.

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Yolk-sac–derived macrophages regulate fetal testis vascularization and morphogenesis

Cited by 172 publications

References 60 publications

Mapping lineage progression of somatic progenitor cells in the mouse fetal testis

Mapping lineage progression of somatic progenitor cells in the mouse fetal testis

Using <em>Ex Vivo</em> Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

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