It had already been established by in situ hybridization that Sall4 transcript is present in mature oocytes and e6.5 implantation sites until mid gestation. Protein expression studies thus spanned all embryonic stages until e10.5 (figure 9). Protein could be visualized from cleavage stage embryos continuously until e8.5 embryos where levels start to decrease. In oocytes as well as in all other cell types examined, Sall4 is localized in the nucleus. High magnification of fluorescent immunostainings shows localization in distinct spots within the nucleus.
Expression can still be detected at two cell stage and is likely due to maternal contribution of protein and mRNA (figure 9A). In cleavage stage embryos, expression of Sall4 is again seen at 16 cell stage and can subsequently be visualized in all blastomeres. Expression of a paternal Sall4-GFP allele also starts at morula stage, e2.5, in all blastomeres, arguing for an onset of zygotic Sall4 expression at the 16 cell stage. Blastocysts at e3.5 start to show higher level of expression in the inner cell mass while the trophoblast still expresses considerable amounts of Sall4. After expanding blastocysts in culture for 24 hours, Sall4 is expressed stronger in the inner cell mass stem cells than in the surrounding trophectoderm but expression is still visible in all cells.
Figure 09: Sall4 expression at periimplantation stages
Analysis of Sall4 expression by immunofluorescence (A-D), immunohistochemistry (E-H) and in situ hybridization (I-L).
Maternal Sall4 protein is still detectable at the two cell stage (A) prior to zygotic transcription. The first zygotic transcrip-tion is visible between 8 and 16 cell stage (B), will be ubiquitously expressed in early blastocysts (C) and localize mainly to the inner cell mass in expanded blastocysts (D). At early postimplantation stages e6.5 (E), e7.5 (F), specific expression in the inner cell mass (ICM) including embryo proper (EP), extraembryonic mesoderm (EM) and exrtaembryonic enco-derm (visceral: VE, parietal:PE) reaches completion (GC: giant cells of the trophoblast; EE: extraembryonic ectoenco-derm). Data obtained by antibody staining were confirmed using in situ hybridization. Sall4 transcript is exclusively found in the inner cell mass of a blastocyst ouwtgrowth in culture (I), an embryo e6.5 (J) and e11.5 (K). H and L show expression in maturing oocytes at P3 (H, top) and eight weeks of age (H, bottom and L);
M) differentiation of wildtype ESCs by withdrawl of LIF and addition of retinoic acid results in a decrease of Sall4 and Oct4 present. Tubulin was used as a loading control.
Sall4
Post implantation at e6.5, expression is seen in embryo proper and primitive endoderm exclusively. All these tissues are ICM derivates. The primitive or extraembryonic endoderm surrounds the epiblast cells and forms an epithelium all around the blastocoel separating apolar epiblast cells from the lumen of the blastula. Sall4 protein abundance is similar in all ICM cell types. At e7.5, all three embryonic germlayers can easily be recognized to express Sall4. Extraembryonically, Sall4 immunoreactivity is visible in allantois and chorion of the extraembryonic mesoderm as well as in visceral and parietal primitive endoderm.
Cells of the trophectoderm lineage comprise two cell types, polar (extraembryonic ectoderm, EE) and mural trophectoderm (giant cells, GC). In the presence of FGF4 (fibroblast growth factor 4) secreted from the ICM, trophoblast cells remain proliferative as polar trophectoderm. Without the stimulus of this growth factor trophoblast cells will undergo endoreplication and form postmitotic, polyploid giant cells (Tanaka et al., 1998). Both cell types do not express Sall4 protein at detectable levels. The expression in the embryo is regulated down upon onset of differentiation at e8.5 but expression is still detected in embryo proper, extraembryonic mesoderm as well as in visceral and parietal primitive endoderm. At this stage, all membranes surrounding the embryo are fully developed and show strong immunoreactivity while the developing placenta still shows only very faint expression. In figure 9 I-L in situ hybridization is visualizing transcripts enzymatically (I, J) or radiographically (K, L). Expression of transcript and protein fully overlaps and confirms the data obtained from immunostaining. In figure 9I, a blastocyst outgrowth (BO) is shown expressing Sall4 in the ICM exclusively. A BO is a blastocyst attached and grown on a surface for five days (see also below).
Sall paralogous proteins show strong correlations of expression of in stem cell pools of ventricular zones of the brain and in early embryos. I next wanted to know if Sall4 expression is linked to an undifferentiated state of pluripotency. If such a connection can be made, expression levels of Sall4 should drop quantitatively upon induction of differentiation in ES cell culture. The transcription factors nanog and Oct4 had previously been shown to be required for stem cell identity. Protein abundance of these stem cell factors is known to drop in differentiation assays of embryonic stem cells (Boyer et al., 2005).
ES cells were plated at very low density on feeder free 10cm culture dishes in ESC medium and kept in culture for seven days. Medium was exchanged every day. Each day, colonies of one more plate were deprived of ESC medium and medium was replaced by differentiation medium containing all-trans retinoic acid at 100 nMol/liter. Removal of LIF from ES cell medium together with the addition of retinoic acid induces differentiation of
31 ESCs (Gribnau et al., 2005). Protein levels of Sall4 and Oct4 were analyzed by western blot.
Equal protein loading was assured by reprobing the membrane against tubulin.
Figure 9M shows strong immunoreactivity against Sall4 and Oct4 protein in an ES cell lysate. Both protein levels decrease upon LIF withdrawal to a similar extend as well as with comparable kinetics. Reduction of Sall4 expression could thus be linked to differentiation in embryonic stem cells. Whether loss of Sall4 is sufficient to inhibit ESC self renewal was then analyzed in vivo as well as ex vivo utilizing the previously generated Sall4-GFP allele.