Hierarchical differentiation competence in response to retinoic acid ensures stem cell maintenance during mouse spermatogenesis

Stem cells ensure tissue homeostasis through the production of differentiating and self-renewing progeny. In some tissues, this is achieved by the function of a definitive stem cell niche. However, the mechanisms that operate in mouse spermatogenesis are unknown because undifferentiated spermatogonia (A _undiff) are motile and intermingle with differentiating cells in an ‘open’ niche environment of seminiferous tubules. A _undiff include glial cell line-derived neurotrophic factor receptor α1 (GFRα1) ⁺ and neurogenin 3 (NGN3) ⁺ subpopulations, both of which retain the ability to selfrenew. However, whereas GFRα1 ⁺ cells comprise the homeostatic stem cell pool, NGN3 ⁺ cells show a higher probability to differentiate into KIT ⁺ spermatogonia by as yet unknown mechanisms. In the present study, by combining fate analysis of pulse-labeled cells and a model of vitamin A deficiency, we demonstrate that retinoic acid (RA), which may periodically increase in concentration in the tubules during the seminiferous epithelial cycle, induced only NGN3 ⁺ cells to differentiate. Comparison of gene expression revealed that retinoic acid receptor γ (Rarg) was predominantly expressed in NGN3 ⁺ cells, but not in GFRα1 ⁺ cells, whereas the expression levels of many other RA response-related genes were similar in the two populations. Ectopic expression of RARγ was sufficient to induce GFRα1 ⁺ cells to directly differentiate to KIT ⁺ cells without transiting the NGN3 ⁺ state. Therefore, RARγ plays key roles in the differentiation competence of NGN3 ⁺ cells. We propose a novel mechanism of stem cell fate selection in an open niche environment whereby undifferentiated cells show heterogeneous competence to differentiate in response to ubiquitously distributed differentiation inducing signals.