SALL4 participates in transcriptional regulatory networks during fetal development of various organs 
and is critical for cell fate decisions and lineage specification 
. In contrast to most organs, expression in the germline is maintained after birth 
and is restricted to pre-meiotic germ cells in rodents, non-human primates and humans 
. Although the exact function of SALL4 in spermatogonia is still unclear, increasing knowledge from studies of embryonic stem cells and hematopoietic stem cells may provide clues about possible mechanisms of SALL4 action in germ cells 
. Here, we analyzed the expression pattern of SALL4 during spermatogenic lineage development and steady-state spermatogenesis in relation to clone size and established markers for undifferentiated and differentiating spermatogonia.
Our results show that SALL4 is expressed by a subpopulation of quiescent gonocytes at PND0, but is expressed by all gonocytes at PND3, which coincides with re-entry into the cell cycle and migration to the seminiferous tubule basement membrane 
. A similar pattern has been reported for neonatal and postnatal germ cells in the marmoset 
. The pool of perinatal gonocytes has two destinies; differentiate to produce the first round of spermatogenesis and establish the initial pool of spermatogonial stem cells 
. Perhaps the heterogeneous SALL4 expression at PND0 distinguishes these two populations. However, by day 7 in our study, when gonocytes reach the basement membrane, SALL4 was expressed in all germ cells. Moreover, the observation that two SALL4 splice variants are expressed differentially during this specific time when the SSC pool is established may suggest that the SALL4A and SALL4B isoforms possess different regulatory functions in germ cells. To our knowledge, this is the first study that identified differential expression of SALL4 isoforms in undifferentiated spermatogonia. It was previously shown that SALL4A and SALL4B can form homo- or heterodimers with distinct DNA target regions regulating either pluripotency genes (SALL4A/B heterodimers, SALL4B/B homodimers) or differentiating/patterning genes (SALL4A/A homodimers) 
. Thus, the presence of SALL4B alone (in gonocytes), or SALL4A and SALL4B (in PND7 spermatogonia) may have implications for the regulation of target genes, perhaps influencing the fate toward stem cell or differentiation to produce the first round of spermatogenesis. However, we were not able to identify subpopulations of spermatogonia at PND7 or 14 with the phenotypes SALL4A only or SALL4B only; rather, all spermatogonia had the SALL4A/B co-stained phenotype. It is possible that the ratio of SALL4A to SALL4B changes on a per cell basis, but this could not be unequivocally confirmed by immunohistochemistry.
We then investigated SALL4 expression during steady-state spermatogenesis. Our data confirmed previous reports of SALL4 co-localization with PLZF, an established marker for undifferentiated spermatogonia, in tissue sections 
. We further show that SALL4 is almost completely co-expressed with a second spermatogonia marker, the RNA-binding protein LIN28. In contrast, GATA4-expressing Sertoli cells did not express SALL4. We observed 16.6 Sertoli cells per tubular cross section, which is comparable to data reported by Oatley and coworkers 
, and using this approach we calculated that approximately 800 SALL4-positive spermatogonia per 1000 Sertoli cells are present in the adult mouse testis. It is important to note that the size of the SALL4-positive spermatogonial population was similar to the population of PLZF-positive and LIN28-positive spermatogonia. Because of the extensive overlap between SALL4, PLZF, and LIN28 expression, and almost identical population size relative to Sertoli cell number, we propose that SALL4 is expressed in undifferentiated spermatogonia.
Studies in tissue sections do not provide information about the topographical arrangement of spermatogonial clones (As
), which might be related to stage of differentiation. It is therefore important to correlate the molecular phenotype of spermatogonia with their clonal organization by whole-mount seminiferous tubule immunofluorescence staining. Using this approach, we observed SALL4 in As
spermatogonia. Occasionally, clones with an odd number of cells (Aal3
) were observed. These could be the result of clone fragmentation, a phenomenon that has been described by Nakagawa et al. 
and that was proposed as a mechanism to replenish the spermatogonial pool. Further, extensive co-localization studies in whole-mount samples revealed significant co-expression of SALL4 with PLZF and LIN28 in the majority of Aundiff
Germ cell differentiation is regulated by endocrine and paracrine factors that signal either to the somatic component of the seminiferous tubules, the Sertoli cells, or directly to spermatogonial stem cells to elicit self-renewing or differentiation divisions. A key factor for SSC survival and renewal both in vivo and in vitro is glial cell line-derived neurotrophic factor (GDNF) 
that is secreted by Sertoli cells 
and binds to the RET/GFRα1 receptor complex 
on undifferentiated spermatogonia including SSCs 
. Accordingly, the presence of GFRα1 on the cell surface of putative SSCs is well characterized and was found to be restricted predominantly to As
. In contrast to the PLZF and LIN28 results, we observed a high degree of heterogeneity among As
spermatogonia in SALL4 and GFRα1 co-staining experiments. Grisanti et al. reported that 10% of the PLZF-expressing As
spermatogonia are GFRα1-negative and that the stem cell activity in this population is greater than in GFRα1 positive cells, as determined by transplantation assay 
. In the study mentioned above, Grisanti et al. also observed that ~5% of PLZF-positive Apr
spermatogonia had asymmetrical GFRα1 expression. In our study, we rarely observed asymmetrical clones (either SALL4 asymmetric or GFRα1 or LIN28 asymmetric, <1% of clones). The reason that we observed slightly fewer asymmetric clones than previously reported could be that Grisanti et al. made counts from images, while we counted clones directly on the microscope, which allowed us to follow clones through different focal planes.
Here, we found that only half of the As population co-expressed GFRα1 and SALL4, while the other half consisted of SALL4 only or GFRα1 only cells. The biological function and the actual stem cell potential in these different spermatogonial populations is not known and will have to be investigated in future studies, perhaps using transgenic tools.
We next performed co-staining experiments for SALL4 and the cKIT receptor tyrosine kinase, which is a marker for differentiating germ cells. cKit becomes upregulated as Aal
transition to A1 spermatogonia, possibly through de-repression of cKit gene expression in the absence of PLZF 
. In this study, we observed cKIT expression in a minor fraction of As
, which increased in larger clones and was associated with a corresponding decrease in SALL4 expression in larger clones. Interestingly, a very small proportion of Apr
clones expressed cKIT only (without SALL4), suggesting that the Aal
to A1 transition might sometimes occur from small clones, as previously described in the Chinese hamster 
. While the majority of Aal16
clones expressed cKIT (either with or without SALL4), we did observe Aal16
SALL4+ clones that were cKIT negative.
Hobbs et al. recently demonstrated a direct interaction of SALL4 and PLZF in transfected HEK293 cells and reported that the two factors can displace each other from their cognate chromatin locations depending on their relative protein levels 
. PLZF is a repressor of the differentiation factor, cKIT, and the authors proposed that SALL4 might act as an important spermatogonial differentiation factor that functions by removing PLZF from its cognate targets (e.g. cKIT). Does this suggest that the SALL4+/PLZF- As and Apr spermatogonia express cKIT and are destined to differentiate? In this regard, triple staining experiments would be very informative, but are difficult to execute due to availability of compatible antibodies and differences in optimal staining conditions for each marker.
Taken together, our data from cross-section and whole mount preparations of seminiferous tubules corroborate earlier reports of molecular heterogeneity among Aundiff
spermatogonia, which may indicate functional heterogeneity among clone sizes 
. The heterogeneity can only be observed when co-staining with more than one marker is performed, therefore caution is required for the interpretation of experiments that use only a single marker as a denominator for Aundiff
spermatogonia. In this study, we report that SALL4 is a molecular marker for undifferentiated As
spermatogonia, a population that includes spermatogonial stem cells, and that it can be used in combination with other molecular markers to dissect spermatogonial lineage development and steady state spermatogenesis in the postnatal testis.