Files in this Data Supplement:
SUPPLEMENTAL TABLE S1. Sequences of primers used for qRT-PCR.
SUPPLEMENTAL TABLE S2. siRNAs used for functional analysis of gene activity.
SUPPLEMENTAL TABLE S3. Numbers of genes present and GDNF-regulated in cultures of rat EPCAM+ clump forming germ cells.
SUPPLEMENTAL TABLE S4. Functional clustering of GDNF-regulated genes.
SUPPLEMENTAL TABLE S5. Expression of genes that have been reported to be important for SSC self-renewal, as SSC markers, or for SSC differentiation.
SUPPLEMENTAL TABLE S6. Expression of genes in EPCAM+ clump forming germ cells that have been reported to be important for stem cell function in cells other than SSCs.
SUPPLEMENTAL TABLE S7. Expression of GDNF-regulated genes identified in mouse SSCs [2] in cultured rat EPCAM+ clump forming germ cells."
Supplemental Figure S1. Venn diagrams of GDNF-regulated genes in cultures of rat EPCAM+ clump forming germ cells. A) 113 genes were up-regulated at least 2-fold by GDNF. Expression of 52 of these genes did not recover within 8 h of GDNF replacement; however expression of 61 did, with most by 4 h. B) Many fewer genes (24) were down-regulated by GDNF at least 2-fold.
Supplemental Figure S2. Expression patterns of core ESC pluripotency regulators Sox2, Nanog, and Pou5f1 in cultures of EPCAM+ clump forming germ cells before (hour -18) and after GDNF withdrawal (hour 0), and after GDNF replacement (hour +4) (A, C) and in Day 0 (0), Day 6 (6), Day 15 (15), and adult (Ad) testes, fresh EPCAM+ cells (Ep), and cultured EPCAM+ cells (Cu) (B, D). Sox2 was not expressed in the rat EPCAM+ clump forming germ cells or testis. A) Nanog was expressed, but not GDNF-regulated in cultures of rat EPCAM+ clump forming germ cells; furthermore, (B) Nanog was more highly expressed in Day 0 testes and Day 8-9 EPCAM+ cells than in Day 6, 15, or adult testes. C) Pou5f1 was also expressed, but not GDNF-regulated in cultures of rat EPCAM+ clump forming germ cells. D) Pou5f1 was most highly expressed in day 8-9 EPCAM+ cells; however, this expression decreased in culture. These data indicate that SSC self-renewal does not utilize the same mechanisms as ESC self-renewal. Bars with different letters are significantly different.
Supplemental Figure S3. Validation of gene expression in the microarray by qRT-PCR. Ten GDNF-regulated genes were chosen for further validation by qRT-PCR. The pattern of gene expression was similar for all genes between the microarray (open circles) and the qRT-PCR analysis (closed circles).
Supplemental Figure S4. Model for GDNF regulation of SSC self-renewal in rodents. GDNF signals through GFRA1 and RET initiating SFK signaling mechanisms resulting in the regulation of genes that have been demonstrated to be involved in SSC self-renewal. Molecules that have been implicated in both rat and mouse SSC self-renewal are purple, those only implicated in mice are red, and those only implicated in rats are blue."