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 (Aundiff) are motile and intermingle with differentiating cells in an ‘open’ niche environment of seminiferous tubules. Aundiff include glial cell line-derived neurotrophic factor receptor α1 (GFRα1)+ and neurogenin 3 (NGN3)+ subpopulations, both of which retain the ability to self-renew. 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.


Generation of CAG-CAT-3xFLAG-Rarg transgenic mice
A BamHI-XbaI fragment containing the Rarg coding sequence from a FANTOM cDNA clone G370007N15 (DNAFORM) and a fragment encoding a 3xFLAG-tag from pCMV-3Tag-6 (Agilent Technologies) were inserted into the plasmid pCAG-CAT (Kawamoto et al., 2000) to generate pCAG-CAT-3xFLAG-Rarg. A fragment containing the CAG-CAT-3xFLAG-Rarg region was microinjected into the pronuclei of single-cell embryos of C57BL/6J×C57BL/6J mice to produce transgenic mice (Laboratory Animal Resource Center, University of Tsukuba) in accordance with the University of Tsukuba's Guide for the Care and Use of Laboratory Animals with the approval of its Institutional Review Board.

Development | Supplementary Material
Accession #BC054378 (Invitrogen, Life Technologies) was used to prepare a Gfra1 probe. Stages of the seminiferous epithelium were judged from the adjacent section stained with periodic acid-Schiff (PAS) hematoxylin.

IF analysis
Whole-mount IF of seminiferous tubules was performed according to a published method (Nakagawa et al., 2010). After the testes were dissected in PBS, untangled seminiferous tubules were fixed for 3 hours in 4% PFA in PBS and attached to an MAS-coated glass slide (Matsunami Glass). The samples were then dehydrated using a methanol series and rehydrated in TBST (TBS containing 0.1% Tween 20). After the samples were washed twice for 10 minutes each in TBST, they were saturated for 1 hour in TNB blocking buffer (PerkinElmer) containing 4% donkey serum (Jackson ImmunoResearch) and 0.01% Hoechst 33342 (Invitrogen, Life Technologies). Samples were next incubated at 4°C for 12 hours with primary antibody in TNB blocking buffer containing donkey serum at the dilutions indicated below and washed three times in TBST for 15 minutes each.
Tissues were then incubated with secondary antibody for 1 hour at room temperature. The observations and measurements were performed using a BX51 upright fluorescence microscope equipped with a DP72 CCD camera (Olympus) or using a Leica TCS SP8 Confocal System.

Development | Supplementary Material
Immunohistochemistry For immunostaining paraffin sections, testes were fixed in 4% PFA-PBS for 12 hours at 4°C, dehydrated, and then embedded in paraffin. Deparaffinized sections were blocked with blocking reagent (PerkinElmer) and incubated with an anti-RARγ1 rabbit monoclonal antibody (1:800). The immunoreaction was visualized using a biotin-conjugated anti-rabbit secondary antibody (Vector labs) in combination with an ABC Kit (Vector labs) and a DAB substrate Kit (Vector labs). Finally, the sections were stained with hematoxylin (Wako) to visualize nuclei.

Microarray analysis of gene expression
Total RNA was extracted using the RNeasy Micro Kit (Qiagen). RNA was converted to cDNA, and cRNA was then amplified using the Low Input Quick Amp Labeling Kit (Agilent Technologies).
Cyanine 3-CTP-labeled cRNAs were combined and hybridized to the SurePrint G3 Mouse GE 8x60K Microarray (Agilent Technologies) using a Gene Expression Hybridization Kit (Agilent Technologies). After hybridization, arrays were washed and dried according to the manufacturer's instructions. Arrays were scanned using an Agilent Technologies Scanner (G2505C and G2565CA) with the default settings for 8 × 60k-format one-color arrays. Images were analyzed using Feature Extraction ver.10.7.3.1 (Agilent Technologies). Three samples from different animals were used to analyze each spermatogonial fraction (for NGN3+ cells, samples from two mice were pooled). Data preparation and statistical analysis were performed using Gene Spring v12.0.0.0 (Silicon Genetics).
Data correction was performed with the threshold raw signals set to 1.0, percent shift to the 75th percentile as normalization algorithm, and no baseline transformation. Genes selected for display in  Table S2 are associated with the following gene ontogeny (GO) terms: retinoic acid receptor activity, retinoic acid receptor binding, retinoic acid receptor signaling pathway, regulation of retinoic acid receptor signaling pathway, retinoic acid 4-hydroxylase histone acetyltransferase complex, mediator complex, histone deacetylase complex, ligand-dependent chromatin remodeling, and ATP-dependent chromatin remodeling.

Quantitative RT-PCR
Total RNA was isolated using an mirVana miRNA Isolation Kit (Ambion, Life Technologies) and reverse-transcribed using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR primed