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During development of the mouse urogenital complex, the gonads undergo changes in three-dimensional structure, body position and spatial relationship with the mesonephric ducts, kidneys and adrenals. The complexity of genital ridge development obscures potential connections between morphogenesis and gonadal sex determination. To characterize the morphogenic processes implicated in regulating gonad shape and fate, we used whole-embryo tissue clearing and light sheet microscopy to assemble a time course of gonad development in native form and context. Analysis revealed that gonad morphology is determined through anterior-to-posterior patterns as well as increased rates of growth, rotation and separation in the central domain that may contribute to regionalization of the gonad. We report a close alignment of gonad and mesonephric duct movements as well as delayed duct development in a gonad dysgenesis mutant, which together support a mechanical dependency linking gonad and mesonephric duct morphogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Background: The hemangioblast is a bi-potential precursor cell with the capacity to differentiate into hematopoietic and vascular cells. In mouse E7.0-7.5 embryos, the hemangioblast can be identified by a clonal blast colony-forming cell (BL-CFC) assay or single cell OP9 co-culture. However, the ontogeny of the hemangioblast in mid-gestation embryos is poorly defined., Design and Methods: The BL-CFC assay and the OP9 system were combined to illustrate the hemangioblast with lymphomyeloid and vascular potential in the mouse aorta-gonad-mesonephros region. The colony-forming assay, reverse transcriptase polymerase chain reaction analysis, immunostaining and flow cytometry were used to identify the hematopoietic potential, and Matrigel- or OP9-based methods were employed to evaluate endothelial progenitor activity., Results: Functionally, the aorta-gonad-mesonephros-derived BL-CFC produced erythroid/myeloid progenitors, CD19(+) B lymphocytes, and CD3(+)TCRbeta(+) T lymphocytes. Meanwhile, the BL-CFC-derived adherent cells generated CD31(+) tube-like structures on OP9 stromal cells, validating the endothelial progenitor potential. The aorta-gonad-mesonephros-derived hemangioblast was greatly enriched in CD31(+), endomucin(+) and CD105(+) subpopulations, which collectively pinpoints the endothelial layer as the main location. Interestingly, the BL-CFC was not detected in yolk sac, placenta, fetal liver or embryonic circulation. Screening of candidate cytokines revealed that interleukin-3 was remarkable in expanding the BL-CFC in a dose-dependent manner through the JAK2/STAT5 and MAPK/ERK pathways. Neutralizing interleukin-3 in the aorta-gonad-mesonephros region resulted in reduced numbers of BL-CFC, indicating the physiological requirement for this cytokine. Both hematopoietic and endothelial differentiation potential were significantly increased in interleukin-3-treated BL-CFC, suggesting a persistent positive influence. Intriguingly, interleukin-3 markedly amplified primitive erythroid and macrophage precursors in E7.5 embryos. Quantitative polymerase chain reaction analysis demonstrated declined Flk-1 and elevated Scl and von Willebrand factor transcription upon interleukin-3 stimulation, indicating accelerated hemangiopoiesis., Conclusions: The hemangioblast with lymphomyeloid potential is one of the precursors of definitive hematopoiesis in the mouse aorta-gonad-mesonephros region. Interleukin-3 has a regulatory role with regards to both the number and capacity of the dual-potential hemangioblast.

Mesonephric cell contribution to the developing mouse gonad has previously been shown to be male-specific in in vitro culture models. This study shows that somatic cell exchange occurs between fetal mouse mesonephroi and gonads during in vitro culture of assembled tissues. Assembling gonads with cell marker-carrying mesonephroi and vice versa identified cell exchange. Two different cell markers were used. One of the markers was the thymidine analogue bromodeoxyuridine (BrdU), which was added to the culture medium during an initial culture period before in vitro culture of assembled tissues in medium without BrdU. The other was bacterial beta-galactosidase, which is expressed ubiquitously in tissues from the transgenic mouse strain ROSA26. The tissues were taken from either 10.5- or 11.5-day-old mouse fetuses, and the assembled tissues were cultured for 36-60 h. Both cell markers demonstrated the occurrence of a cell exchange in both directions between the mesonephros and the gonad. The cell contribution was found to be minor and independent of the initial age of the tissue and hours in culture. Further, mesonephric cell contribution was not male-specific. Cell exchange between the two tissues may be due to growth between the assembled tissues rather than active signal-regulated migration. It is suggested that in vitro culture models may not be applicable for investigating mesonephric cell contribution to the developing mouse gonad., (Copyright 2001 S. Karger AG, Basel)