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The maintenance of pluripotency in mammalian embryonic stem cells depends upon the expression of regulatory genes like Oct3/4 and Sox2. While homologues of these genes are also characterized in non-mammalian vertebrates, like birds, amphibians and fish, existence and function of developmental pluripotency associated genes (Dppa) in lower vertebrates have not yet been reported. Here we describe a Dppa2/4-like gene, XDppa2/4, in Xenopus. The protein contains a SAP domain and a conserved C-terminal region. Overexpression of XDppa2/4, murine Dppa2 or Dppa4 produces similar phenotypes (defects in blastopore closure), while injection of XDppa2/4 morpholino generates a loss of blastopore closure and neural fold formation. Embryos die up to tailbud stage. mDppa2 (but not mDppa4) rescues blastopore closure and neurulation defects caused by XDppaMO, but does not prevent subsequent death of embryos. Although XDppa2/4 exhibits a Dppa-like expression pattern and is indispensable for embryogenesis, analyses of various marker genes make its role as a pluripotency factor rather unlikely. Both the gain and loss of function effects until the end of neurulation are caused by the conserved C-terminal region but not by the SAP domain. The SAP domain is required for association of XDppa2/4 to chromatin and for embryonic survival at later stages of development suggesting epigenetic programming events.
Fig. 2. Temporal and spatial expression of XDppa2/4 during early development and in adult tissues of Xenopus laevis. (A) Expression was quantified by real time RT-PCR in total embryos at different stages of development (Nieuwkoop and Faber, 1967) or (B) in different adult tissues. Values in histograms are presented as relative units normalized to XDppa2/4 expression at stage 2 or in gut, respectively. (C) XDppa2/4 whole mount in situ hybridization of embryos at various stages of development (stages 2â 34). The staining pattern demonstrates an enrichment of transcripts within the animal half. Note the loss of XDppa2/4 during neurulation.
Fig. 3. Results of representative loss and gain of XDppa2/4 function experiments. (A) Depletion of XDppa2/4 leads to defects during gastrulation and lack of neurulation. Injection of XDppaMO gives rise to a wide-open blastopore at stage 11.5. The blastopore remains open until stage 18 and neural fold formation is missing. Most of the embryos die before the end of neurulation and disaggregate. Co-injection of 50 ng XDppaMO and 600 pg mut-XDppa2/4 RNA rescues development and restores the wild type. As controls, unmanipulated embryos and embryos injected with control MO (ctrlMO) are shown. (B) Gain of function studies with XDppa2/4, mDppa2 or mDppa4 RNA result in comparable phenotypes. Overexpression of 400 pg XDppa2/4 reveals defects in blastopore closure. At stage 17, the embryos form a neural fold but still show an open blastopore. At tailbud stage, XDppa2/4 gain of function results in reduction of body axis, tail structures and open neural folds. Overexpressions of mouse mDppa2 (800 pg) and mDppa4 (100 pg) lead to similar phenotypes including failure of blastopore closure, open neural fold and axis reduction. Uninjected embryos are shown as controls.
Fig. 4. Whole mount in situ hybridization of gain and loss of function embryos. Embryos were injected with XDppa2/4, mDppa2, mDppa4 RNA or XDppaMO into both blastomeres at 2-cell stage. Whole mount in situ hybridization was performed at stage 11 for Xbra, BMP4, MyoD, Gsc, XSox2, Xema and XSox17a. Uninjected embryos or embryos injected with ctrlMO show the expected wild type expression pattern of marker genes at stage 11. Orientation of embryos is given. For the vegetal view the dorsal side is always located at top and for the lateral view the dorsal side is located at the left side.
Fig. 5. Blastopore closure and neural fold formation in XDppaMO injected embryos are rescued by mDppa2. The open blastopore and the lack of neural fold formation caused by XDppaMO are rescued by co-injection of mDppa2 mRNA giving rise to normal embryos. The wild type expression pattern of markers Xbra (ventral view), Chordin and XSox2 (dorsal view), which are delocalised or downregulated after loss of function, is restored at stage 17. Unmanipulated embryos and embryos injected with ctrlMO are shown as neurulating wild type controls (left columns).
Fig. 6. The SAP domain is dispensable for the gain as well as for the loss of function phenotypes. (A) Schematic representation of XDppa2/4 deletion constructs used in overexpression studies. Numbers represent positions of amino acids. The SAP domain is shown in yellow and the DCR domain in turquoise. L represents the linker region. (B) Results of overexpression of mutant transcripts. Vegetal view on uninjected control embryo at stage 11.5 and embryos injected with a total of 800 pg XDppa2/4, δSAP, δDCR, δSAPδDCR, DCR, SAP + L + DCR, aa1–65, aa66–240 and δN-term RNA into two blastomeres at the 2-cell stage. (C) Rescue of the loss of function (MO) phenotype by co-injection of mut-δSAP RNA. Dorsal view of embryos at stage 18. Embryos injected with 50 ng MO or with 650 pg mut-δSAP suffer from gastrulation defects and lack of neurulation. Neural fold formation is rescued by co-injection of 550 pg mut-XDppa2/4 or 600 pg mut-δSAP RNA.
Fig. 7. Subcellular (A) and subnuclear (B) localization of XDppa2/4 deletion mutants. (A) Transfections of HeLa cell lines with indicated EGFP-fused XDppa2/4 deletion constructs show differences in subcellular localization. Constructs encoding EGFP alone or wild type XDppa2/4 were transfected as controls. (B) Only XDppa2/4 proteins with an intact SAP domain reveal chromatin association as shown by co-staining with histone H2B-mRuby (red) and DAPI (blue). (C) Results of subcellular localization experiments in comparison with phenotypes obtained after overexpression of the respective deletion constructs. A diffuse localization within the nucleus was interpreted as indication that the respective mutant is not associated with chromatin.