Figure 1.
Differential expression of SALL4 isoforms during postnatal testis development.
(A–D) Co-staining for DAZL (all germ cells, red) and SALL4A/B (green). (A, B) Some SALL4-negative gonocytes (white arrows) were observed at PND 0. Autofluorescence was observed in the interstitium (asterisk). All germ cells strongly express SALL4 at PND 7 (C) and PND 14 (D) (E–H) Co-staining for VASA/DDX4 (all germ cells, red) and SALL4A (green). (E,F) SALL4A was absent from VASA-positive germ cells at PND 0 and 3. SALL4A was strongly expressed in all germ cells at PND 7 (G) and PND 14 (H). Scale bars = 10 µm. (I) Western blot analysis of total testis protein isolated from different postnatal ages showed differential expression of SALL4 isoforms. Protein from R1 mouse embryonic stem cells is shown as positive control. Blots were reprobed with anti-beta actin IgG to ensure equal loading of proteins.
Figure 2.
SALL4A and SALL4B co-expression in juvenile germ cells.
SALL4A and SALL4B are expressed in the same population of germ cells at PND 7 (A–C) and PND 14 (D–F). Scale bars = 10 µm.
Figure 3.
SALL4 is expressed by undifferentiated spermatogonia in postnatal day 7 and postnatal day 14 testes.
(A–F) SALL4 expression is restricted to LIN28 positive undifferentiated spermatogonia at PND 7 and PND 14. (G–L) SALL4 expression is restricted to PLZF positive undifferentiated spermatogonia at PND 7 and PND 14. Scale bars = 10 µm.
Figure 4.
SALL4 is expressed in undifferentiated spermatogonia in adult mouse seminiferous tubules.
Double immunohistochemistry of tissue sections was used to examine the localization of SALL4 in comparison to PLZF (A–D) and LIN28 (E–H), two known markers for undifferentiated spermatogonia. (D, H) Data are expressed as the average number of single or double stained cells per tubular cross section ± SEM. (I–K) SALL4 was not expressed in GATA4-positive Sertoli cells. (L) SALL4, PLZF and LIN28-positive cells were normalized to 1000 Sertoli cells to express the relative size of the respective spermatogonial populations. The basement membrane of each tubule is marked by a dashed line. Scale bars = 10 µm. For quantitative analysis, 100 circular tubule cross sections in testes from at least 3 adult male mice were scored.
Figure 5.
Clonal organization of SALL4 expressing cells in adult mouse seminiferous tubules.
Whole mount seminiferous tubules were isolated from adult animals and subjected to immunofluorescence staining. (A–D) SALL4 was found to be expressed in As, Apr and Aal spermatogonia. Scale bars = 10 µm. (E) Quantification of the frequency of SALL4 positive As, Apr and Aal spermatogonial clones that were observed in adult mouse seminiferous tubules. Data are presented as average ± SEM from three independent experiments. The number of clones counted is indicated in parentheses beneath the graph. Total cell numbers in each clone size were calculated by multiplying the number of clones by the number of cells contained in each clone of a certain size, e.g. 254 As clones = 254 cells, 223 Apr clones = 446 cells, etc. Cell numbers are shown in parentheses.
Figure 6.
SALL4 is co-expressed with PLZF and LIN28 in clones of undifferentiated spermatogonia.
(A–D) SALL4 and PLZF were co-expressed in most undifferentiated spermatogonia. Small fractions of As and Apr spermatogonia showed molecular heterogeneity (*). (E–H) SALL4 also largely overlaps with LIN28 expression, with heterogeneity of expression observed in few As and Apr spermatogonial clones (*). Scale bars = 50 µm. Data obtained from n = 3 animals, presented as average ± SEM. Asym: Number of clones with molecular asymmetry.
Figure 7.
Differential expression of GFRα1 and SALL4 in As and Apr spermatogonia.
Expression of GFRα1 was limited to short chains of Aundiff and more restricted than expression of SALL4. (A–D) Examples of GFRα1 and SALL4 expressing spermatogonial clones in whole mount seminiferous tubules. Scale bars = 10 µm. Panel D was modified from [1] with permission. (E) Quantitative evaluation of GFRα1 and SALL4 expression in clones of undifferentiated spermatogonia. Among As spermatogonia, 50% of cells co-expressed both markers, but substantial populations of SALL4-only and GFRα1-only cells were also observed. The number of SALL4-only cells increased in longer chains, when GFRα1 expression ceased. Asym: Number of clones with molecular asymmetry.
Figure 8.
SALL4 is downregulated in differentiating spermatogonia.
(A) SALL4 and cKIT are co-expressed in chained Aal spermatogonia. (B) SALL4 becomes downregulated as spermatogonia differentiate. In contrast, cKIT expression persists in differentiated germ cells. (C) Quantification of SALL4/cKIT co-staining showed that SALL4 expression decreased in larger clones coincident with increased cKIT expression. Note that cKIT expression in Aal16 remained heterogeneous. Scale bars = 50 µm. Asym: Number of clones with molecular asymmetry.
Figure 9.
Summary of spermatogonial marker expression in mouse spermatogonia.
Graphical summary of quantitative data from Figures 6–8 were used to calculate the ratio of cells expressing each marker within the populations of As, Apr, and Aal spermatogonia. Each bar represents 10% GFRα1-, SALL4-, PLZF-, LIN28- or cKIT- positive cells, respectively, and half bars represent approximately 5% positive cells. For example, co-staining revealed that 71% of As spermatogonia expressed GFRα1, therefore 7 bars (each representing 10%), are shown in the diagram. The heterogeneity of the molecular phenotype of spermatogonia clone sizes is represented by the extent of overlap among bars across marker profiles.