Murine inner cell mass-derived lineages depend on Sall4 function

Abstract

Sall4 is a mammalian Spalt transcription factor expressed by cells of the early embryo and germ cells, an expression pattern similar to that of both Oct4 and Sox2, which play essential roles during early murine development. We show that the activity of Sall4 is cell-autonomously required for the development of the epiblast and primitive endoderm from the inner cell mass. Furthermore, no embryonic or extraembryonic endoderm stem cell lines could be established from Sall4-deficient blastocysts. In contrast, neither the development of the trophoblast lineage nor the ability to generate trophoblast cell lines from murine blastocysts was impaired in the absence of Sall4. These data establish Sall4 as an essential transcription factor required for the early development of inner cell mass-derived cell lineages.

Fig. 1.

Early Sall4 protein expression and marker analysis on Sall4 mutant blastocysts Sall4 antibody staining. Sall4 protein is visible in cells at the 8- to 16-cell stage (A), to become enriched within cells of the ICM (B). At 6.5 dpc Sall4 protein is detected in ICM derivatives including epiblast, visceral, and parietal endoderm (C). Blastocysts from Sall4^H2bEGFP intercrosses (F) display different GFP fluorescence intensity and can be sorted into strong (G Left), medium (G Center), and no GFP signal (G Right). Bright GFP fluorescent blastocysts do not express Sall4 RNA as shown by RT-PCR using actin as a control and do not show Sall4 immunostaining (D and E). Indirect immunofluorescence and RT-PCR marker analysis on WT and Sall4 mutant expanded blastocysts does not reveal any obvious differences (I–O). Nanog (epiblast marker) (J and M), Troma1 (trophectoderm/epithelial marker) (J and M), Oct4 (ICM marker) (K and N), Cdx2 (trophectoderm marker) (L and O), and Sox2 (L and O) are shown. DAPI counterstaining is blue.

Fig. 2.

Sall4^H2bEGFP mutant embryos lack ICM derivatives. Shown are paraffin sections of PAS-stained, presorted, and reimplanted control (A) and mutant (B) embryos at 5.5 dpc. Marker RNA in situ analysis was performed on reimplanted, pregenotyped embryos at 5.5 dpc (WT, C–F; mutant, G–J). Sall4 mutant embryos do not form epiblast or primitive endoderm derivatives. Shown are Oct4 (epiblast maker) (C and G) and Gata6 (primitive endoderm marker) (F and J). Disorganized extraembryonic tissue is present in Sall4 mutant implantation sites as shown by positive staining for Cdx2 (E and I). Giant cells are clearly visible in H19 staining (D and H).

Fig. 3.

Sall4 mutant blastocysts show defective ICM development in culture. Phase contrast of WT (A–C) and Sall4 mutant blastocyst (D–F) outgrowths cultured in standard ES cell medium plus LIF. Sall4 mutant ICMs initially showed a small outgrowth, which did not expand further under prolonged culture conditions. Only few endoderm cells were seen compared with WT controls (arrowhead marks endoderm cells, and arrow marks giant cells). Proliferation in mutant Sall4 outgrowths was nearly not detectable as shown by phospho-HistoneH3 immunostaining after 5 days of in vitro culture (G and M). Marker RNA in situ hybridization (H–J and N–P) and immunostaining (K, L, Q, and R) analysis on day-5 blastocyst outgrowths are shown. Note increased H19 staining in Sall4 mutant blastocyst outgrowths (N) compared with WT controls (H). Also, increased numbers of Cdx2-immunopositive cells are seen in mutant blastocyst outgrowths compared with controls (L and R). Few cells still express Gata6 (I and O) and Hnf4 (J and P). Rarely, Oct4-immunopositive cells can be detected (K and Q).

Fig. 4.

Sall4 mutant ICMs give rise to trophectoderm cultures. (A) Phase contrast of ICM outgrowths in ES cell medium after immunosurgery. Sall4 mutant ICM outgrowths do not expand significantly but undergo a change in cell morphology toward a giant cell-like appearance in contrast to WT ICMs. (B) Immunostaining marker analysis of ICM outgrowth in ES cell medium after immunosurgery. Sall4 mutant ICM cells are positive for the trophectoderm markers Cdx2 and Cdh3 in immunostainings. (C) Phase contrast of ICM outgrowth in TS medium plus FGF4 after immunosurgery. Both WT and Sall4 mutant ICMs show robust outgrowths under these conditions. Whereas the control culture displays lattice-like structures characteristic for primitive endoderm after FGF4 withdrawal, the Sall4 mutant outgrowths differentiate into giant cells. (D) PCR genotyping using primers 1F, 2F, and 1R of primitive endoderm and giant cell cultures shown in C demonstrating that TS cell cultures and giant cells are Sall4-negative. (E) RT-PCR marker analysis of cell cultures shown in C demonstrating that cells generated from Sall4 mutant ICM outgrowths express, consistent with their morphology, trophectoderm lineage markers in contrast to the control cultures. PE, primitive endoderm; TC, trophectoderm cells; GC, giant cells.

Fig. 5.

Cell-autonomous requirement of Sall4 in the epiblast and primitive endoderm. (A) Sall4 heterozygous and mutant morula stage embryos were sorted according to their fluorescence intensity and aggregated with morulae heterozygous for ROSA26. Successful contribution of Sall4 mutant cells to the ICM of chimeric blastocysts was checked by using GFP/Oct4 double staining. (B and C) Whole-mount β-galactosidase staining. (B) Representative embryo recovered from control population showing various degrees of chimerism in different organs. (C) Representative embryo recovered from Sall4 homozygous mutant/ROSA26 morula aggregations being completely blue. (D) Schematic drawing of the Sox2-Cre deletion region in green. (E) Epiblast-specific deletion of Sall4 leads to an arrest of embryonic development at the late primitive streak stage before somite formation. (F) ES cell–embryo chimera analysis injecting WT ES cells into Sall4 mutant blastocysts. In this situation WT lacZ⁺ ES cells (blue) contribute only to the epiblast. Injection of ROSA26 ES cells with lacZ inserted into the ROSA locus or R1 ES cells carrying a CAG-LacZ expression module in the ROSA locus into Sall4 homozygous mutant blastocysts. (G and H) Nuclear fast red counterstained paraffin sections of whole-mount β-galactosidase-stained 6.5-dpc embryos. (G) Representative embryo of the control group where blue cells can be seen only in the epiblast. (H) The most advanced embryo found from Sall4 homozygous mutant blastocysts injected with WT ES cells. Note that the epiblast consists of only blue cells.