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Xenopus nuclear factor 7 (xnf7) is a maternally expressed nuclear protein that is retained in the cytoplasm from oocyte maturation until the midblastula transition (MBT). Mutations of the xnf7 phosphorylation sites to glutamic acids (dnxnf7) resulted in the retention of the endogenous protein in the cytoplasm past the MBT, indicating that cytoplasmic retention is a phosphorylation dependent process. In addition, dnxnf7 acted as a dominant negative mutant by keeping the endogenous xnf7 protein in the cytoplasm past the MBT. Overexpression of dnxnf7 in future dorsal blastomeres resulted in a ventralized or posteriorized phenotype in which the embryos lacked anterior structures, while overexpression in ventral blastomeres resulted in dorsalized embryos. dnxnf7 also affected the expression of both dorsal and ventral mesodermal markers. These data suggest that xnf7 functions in dorsal/ventral patterning and that the movement of the protein from the cytoplasm to the nucleus at the MBT is critical for the execution of a genetic program conferring a dorsal or ventral identity to the mesoderm.
FIG. 1. The mutation of phosphorylation sites 1 and 2 to glutamic acid results in retention of xnf7 in the cytoplasm. Synthetic mRNA
from the dnxnf7 mutant was injected into fertilized eggs and analyzed for the presence of exogenous xnf7 using the T7 epitope tag antibody.
(A) Sections of embryos at stage 10 that were injected with the dnxnf7 mRNA. (B) Embryos at stage 10 that were injected with the xnf7-
8 (wild-type) mRNA. The arrows point to the nuclei, and the constructs are diagrammed below. CRD, cytoplasmic retention domain
(stippled box); NLS, nuclear localization signal (dotted circle); Ring-finger and B-box zinc finger domains (open boxes); striped ovals are
phosphorylation sites; open ovals are mutated phosphorylation sites 1 and 2.
FIG. 2. dnxnf7 protein acts as a dominant-negative mutant to retain the endogenous protein in the cytoplasm past the MBT. dnxnf7
mRNA was injected into one of the blastomeres of a two-celled embryo to limit its area of expression in the embryo. The resulting
embryos were allowed to develop to stage 9.5, past the MBT. Embryos were sectioned and alternate serial sections were stained with the
T7 epitope tag antibody (recognizes only the exogenous protein) and the L24 antibody (recognizes both endogenous and exogenous xnf7
protein). (A) A section stained with the T7 antibody showing that the dnxnf7 protein was, as expected, expressed in only a portion of the
embryo. In this section we detected the exogenous dnxnf7 protein in the cytoplasm even though this is after the MBT (inset). (B) A
subsequent serial section from the same embryo that was stained using the L24 antibody. Arrows point to nuclei.
FIG. 3. Dorsalized and ventralized phenotypes developed from embryos injected with the dnxnf7 mRNA. Fertilized eggs were injected
with 1.5 ng of the mutant dnxnf7 mRNA prior to first cleavage. Injected embryos were allowed to develop to various stages to identify
any mutant phenotypes. (A) The appearance of the dorsal lip in both control (xnf7-8 injected) (c) and dnxnf7 mRNA-injected embryos (m).
(B) Appearance of the dorsalized and ventralized phenotypes in dnxnf7-injected embryos. The top diagram is the DAI (dorsal–anterior
index scale taken from Kao and Elinson, 1988). To the left are ventralized embryos of DAI 2–3 while on the right are dorsalized embryos
DAI 7–8. A normal stage 28 embryo is shown for comparison. The arrows point to the cement gland or the anterior portion of the embryo
when the cement gland is absent.
FIG. 4. Injection of dnxnf7 mRNA into the future dorsal and ventral blastomeres produces a range of dorsalized and ventralized
phenotypes. A, C, and E are embryos exhibiting various degrees of dorsalization produced by injection of dnxnf7 mRNA into the
ventral blastomeres of the four-celled embryo. Notice a gradual shortening of the anterior–posterior axis to yield the dorsalized
embryo. These fall between the ranges of DAI 6–7. B, D, and F are embryos resulting from the injection of dnxnf7 mRNA into the
dorsal blastomeres of the four-celled embryo. They range in ventralized phenotypes from DAI 1–3. The arrows point to the cement
gland or the anterior of the embryo when the cement gland is absent as in the ventralized embryos in B, D, and F.
FIG. 5. Overexpression of dnxnf7 does not affect cell viability. Embryos were coinjected with a mixture of dnxnf7 and green fluorescent
protein (GFP) mRNA in the ventral blastomeres. An example of a stage 35 dorsalized embryo is shown in A. B is a diagram of the embryo
showing the distribution of the GFP. Other light areas of the embryo are due to autofluorescence of the yolk (dorsal side up).
FIG. 6. Overexpression of dnxnf7 affects the expression of the pan-mesodermal marker Xbra. In situ hybridization analysis of embryos injected with wild-type xnf7-8 in the dorsal (A) or ventral (C) blastomeres showing Xbra expression at stage 10.5. Xbra expression was also analyzed in embryos injected with dnxnf7 in the future dorsal region (B) and in the future ventral region (D). In addition, we also immunostained embryos using an alkaline phosphatase-conjugated anti-T7 Tag antibody (E and F, red color). (E) Immunostaining of the embryos injected in dorsal blastomeres with wild-type xnf7-8. (F) Embryo injected with dnxnf7 in the dorsal blastomeres. Dorsal side of embryos is up and arrows point to the gap in Xbra expression.
FIG. 7. Dnxnf7 affects the expression of the dorsal mesodermal patterning gene chordin. Embryos injected in the dorsal blastomeres (A) or the ventral blastomeres (C) with wild-type xnf7-8 and analyzed for chordin expression. Embryo injected with dnxnf7 in the future dorsal blastomeres showing a loss of chordin expression (B). Embryo injected with the dnxnf7 into the future ventral blastomeres showing an increase in the chordin domain (D). The arrows in D define the extent of the chordin expression domain. Dorsal side of the embryos is up.
FIG. 8. dnxnf7 overexpression affects the expression of the mesodermal patterning gene Xvent-1. (A) In situ hybridization pattern of Xvent1 expression in uninjected stage 10 embryos. Embryos injected with wild-type xnf7-8 in the dorsal (B) and ventral (C) regions and analyzed for Xvent1 expression. Embryos injected with dnxnf7 in the dorsal (D) region and analyzed for Xvent1 expression. Embryo injected with dnxnf7 in the ventral (E) region and analyzed for Xvent1 expression. (F) Ventral view of the embryo shown in E to show gap in Xvent1 expression.
FIG. 9. Summary of the role of xnf7 in D/V patterning. xnf7 is phosphorylated during oocyte maturation by several different kinases at two sites. This results in its retention in the cytoplasm in a 670-kDa complex (Shou et al., 1996). It remains cytoplasmic until the MBT when it is dephosphorylated and enters the nucleus. We speculate that other components interact with xnf7 in nuclei within different regions of the embryo, resulting in the regulation of genes giving mesoderm a dorsal/ventral character. xnf7 probably functions in parallel with other mesodermal regulatory pathways.
