Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing development.

Spermatogenesis is maintained by a pool of spermatogonial stem cells (SSCs). Analyses of the molecular profile of SSCs have revealed the existence of subsets, indicating that the stem cell population is more heterogeneous than previously believed. However, SSC subsets are poorly characterized. In rodents, the first steps in spermatogenesis have been extensively investigated, both under physiological conditions and during the regenerative phase that follows germ cell damage. In the widely accepted model, the SSCs are type Asingle (As) spermatogonia. Here, we tested the hypothesis that As spermatogonia are phenotypically heterogeneous by analyzing glial cell line-derived neurotrophic factor (GDNF) family receptor a1 (GFRA1) expression in whole-mounted seminiferous tubules, via cytofluorimetric analysis and in vivo colonogenic assays. GFRA1 is a coreceptor for GDNF, a Sertoli cell-derived factor essential for SSC self-renewal and proliferation.Morphometric analysis demonstrated that 10% of As spermatogonia did not express GFRA1 but were colonogenic, as shown by germ cell transplantation assay. In contrast, cells selected for GFRA1 expression were not colonogenic in vivo. In human testes, GFRA1 was also heterogeneously expressed in Adark and in Apale spermatogonia, the earliest spermatogonia. In vivo 5-bromo-2 0 -deoxyuridine administration showed that both GFRA1 1 and GFRA1 2As spermatogonia were engaged in the cell cycle, a finding supported by the lack of long-term label-retaining As spermatogonia. GFRA1 expression was asymmetric in 5% of paired cells, suggesting that As subsets may be generated by asymmetric cell division. Our data support the hypothesis of the existence of SSC subsets and reveal a previously unrecognized heterogeneity in the expression profile of As spermatogonia in vivo.

In mammals, spermatogenesis is maintained by spermatogonial stem cells (SSC). In their niche, SSC divide to self-maintain and to produce a transit-amplifying population that eventually enters the meiotic cycle to give rise to spermatozoa. The low number of SSC and the lack of specific markers hinder their isolation and enrichment. Stem cells in several adult tissues can be identified by using their verapamil-sensitive Hoechst dye-effluxing properties, which define the characteristic "side population" (SP). Here we show, by multicolor flow cytometric analysis, that immature mouse testis contains a "side-population" (T-SP), which is Sca-1pos, Ep-CAMpos, EE2 pos, alpha6-integrin pos, and alpha(v)-integrin neg. A 13-fold enrichment in SSC activity was observed when sorted T-SP cells from ROSA 26 mice were transplanted in busulfan-treated mouse testis. Whereas an incomplete range of spermatogenic stages was encountered two months after transplantation of unsorted testicular cells, the transplantation of T-SP cells generated all associations of mouse germ cells representing the full range of spermatogenic stages. These data suggest that Hoechst staining and cell sorting might provide a novel approach to SSC enrichment in mammals.

The action of activin-A on Sertoli and spermatogonial cell proliferation during early postnatal life was studied by using in vitro organ culture of testis fragments from 9-day-old rats. Activin significantly stimulated 3H-thymidine incorporation into testis fragments cultured for 3 days in the presence of FSH, whereas it had no effect in the absence of the hormone. This effect was dose dependent in the range 10-200 ng/ml and was specifically inhibited by the activin-binding protein, follistatin. The effect of activin upon proliferation of different testicular cells was studied in detail by 5-bromo-2'-deoxyuridine-labeling fragments at the end of in vitro culture and then determining percentages of different labeled cells on immunostained histological sections. Concomitant treatment with FSH and activin, but not with FSH or activin alone, significantly stimulated Sertoli cell proliferation but markedly depressed that of differentiating type A spermatogonia. In contrast, proliferative activity of undifferentiated type A spermatogonia was independent of activin, irrespective of the presence of FSH. The effect of donor animal age was then investigated by culturing fragments derived from 3- and 18-day-old rats for 3 days. An age-related response was evident. Sertoli cell proliferation was stimulated by FSH alone in fragments from 3-day-old rats, activin having no apparent effect at this age. In contrast, none of the hormones tested either alone or in combination was effective in 18-day-old animals. These results demonstrate that activin acts with FSH in maintaining mitotic potentiality of Sertoli cells in a defined phase of their maturation path, when their proliferative activity is approaching the final arrest. These findings suggest that activin may be an important local factor in regulating Sertoli cell number and that the mitosis of differentiating spermatogonia subsides during Sertoli cell proliferation.