The ATG initiation codon is located within exon 3. The locations of some restriction enzymes are indicated. C , PCR analysis of embryos from timed matings of Nor-1 heterozygous inter-crosses. D , whole mount in situ hybridization of wild type embryos at embryonic day 7. This data indicated that homozygosity for the Nor-1 mutation caused embryonic lethality. The Chi square test showed a value of This was larger than the 0. Thus, the observed numbers of wild type to Nor-1 heterozygous offspring were statistically significantly different from the expected 1 to 2 ratio.
This data suggests that partial loss of Nor-1 may also cause embryos lethality at some frequency Table I. To determine the age of embryonic death, we first analyzed embryonic day 3. Therefore loss of Nor-1 does not appear to affect pre-implantation events. We then examined embryos at later days of gestation. At E6. At E7. The most severely affected class were greatly reduced in size as compared with their wild type littermates.
By E8. The smaller, morphologically abnormal embryos at E7. Interestingly, at E8. These embryos looked phenotypically like E7. Embryos in this category appeared to have formed neural folds but had underdeveloped trunk regions.
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This is consistent with the earlier findings Table I that heterozygosity at the Nor-1 locus may cause lethality at some low frequency. Analysis of Nordeficient embryos. Morphologically mutant embryos showed an absence of Nor-1 staining as compared with the wild type embryos Fig. Hybridization probes corresponding to a number of different developmental markers were used note: experiments with each marker were done at least three times with similar results.
Brachyury is a transcription factor that belongs to the Tbox family of molecules 29 — Brachyury expression continues along the proximal-distal axis marking the migration of the primitive streak. In situ hybridization of E7. Its signal is reduced in the distal tip of Nor-1 homozygous mutant embryos i. Fgf8 is another gene known to be important in early gastrulation events.
Fgf8 is a secreted growth factor whose expression in the mouse embryo correlates with the production and movement of mesoderm 24 , Fgf8 expression is seen at the onset of gastrulation at E6.
Similar to Brachyury, Fgf8 expression is found in early primitive streak and is maintained as the streak moves anteriorly toward the prospective node. Our in situ analysis of Nor-1 mutant embryos shows that Fgf8 expression can be detected but appears to be expanded somewhat, indicating possible disregulation of fgf8 expression when Nor-1 is absent Fig.
We also examined the expression profile of Tbx6, another member of the T-box family of transcription factors. Tbx6 is known to be expressed in the primitive streak, paraxial, and axial mesoderm but not in the node 27 , Wild type embryos show Tbx6 staining extending along the entire length of the primitive streak toward the distal tip of the embryo.
These expression profiles suggest that anterior and lateral movement of mesoderm do occur in the absence of Nor-1, but abnormal expression of Tbx6 suggests a delayed formation of anterior mesoderm.
Formation of the primitive streak is one of the earliest events in gastrulation, followed by the movement of cells to the distal portion of the embryo culminating in the formation of the structure known as the node, or the organizer. Transplantation experiments in mouse have shown that the node is capable of secondary axis formation 35 , In our studies of Nor-1 mutant mice we looked at expression patterns of a number of markers for the node to determine whether this structure is made.
As the organizer is a derivative of the migrating mesoderm, and because the Nor-1 mutants appear to be deficient in anterior mesoderm it was important to ascertain whether the Nor-1 mutants were capable of generating this structure. We tested a number of known markers of the node, including Lim1, Hnf3b, and noggin. Lim1 is a homeodomain containing protein, loss of which leads to embryonic lethality and dramatic defects in head formation 26 , 37 as well as gastrulation. In wild type embryos, lim1 antisense probe stains most of the anterior primitive streak-derived tissues but is down-regulated after E7.
In the retarded Nordeficient embryo, there is staining of the distal tip of the embryo, but the faint staining of the lateral mesoderm seen in wild type embryo is absent Fig. Additional future studies are needed to clarify this issue. Its expression can be found in the node at E7. This is also true in more severely affected embryos Fig. Staining with Sonic Hedgehog, another marker for axial mesoderm, revealed similar findings data not shown.
The anterior visceral endoderm is also important for patterning of the mouse embryo and is involved in restriction of posterior signals Otx2 mutant mice are embryonic lethal at the time of gastrulation and also show defects in head formation 28 , 35 , These data suggest that Nor-1 function is necessary for the formation of normal anterior mesoderm and its derivatives.
Nordeficient Embryos Exhibit Defects of Cell Migration during Gastrulation —Cell movement during gastrulation is very important for proper development of the embryo, such that abnormal or defective migration often leads to early lethality. The molecular aspects of this process have been investigated by a number of different groups, and several key molecules have been identified.
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One molecule previously mentioned is Fgf8. Loss of Fgf8 leads to lethality at E8. These mutants exhibit an accumulation of mesoderm at the proximal posterior region of the streak 33 and are unable to form anterior mesoderm. Another gene shown to be involved in this process is the embryonic ectoderm development gene eed.
Loss of function mutation of eed in mice shows an increase in the production of extra embryonic mesoderm at the expense of embryonic anterior mesoderm Data from this study suggests that the Nor-1 gene may also play a role in distribution of early mesodermal cells within the streak.
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Hematoxylin and eosin staining of paraffin-embedded serial transverse sections of 7. Accumulation of cells at the proximal posterior of the epiblast can be seen in Nordeficient embryos, leading to a narrowing of the cavity within the embryo compare Fig. Sagittal sections of Nor-1 mutant embryos also show the accumulation of mesoderm midway along the proximal-distal axis in the primitive streak Fig.
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In some cases, allocation of mesoderm seems irregular, such that one side of the embryo appears to preferentially have more mesoderm than the other Fig. This accumulation of mesoderm is strikingly similar to that seen in fgf8-null embryos, whereby embryos appear to make nascent mesoderm; however, these newly formed cells are not distributed normally or not able to migrate efficiently away from the streak.
follow Alternatively, there might be abnormal growth of the mesodermal cells. Transverse and sagittal sections of E7. Among the three Nur77 family members, Nur77 has been studied extensively for its role in T cell apoptosis 9. Constitutive or over-expression of Nur77 in thymocytes or several non-lymphoid cell lines leads to apoptosis. In addition, expression of a Nur77 dominant negative protein in T cells can inhibit apoptosis accompanying T cell selection 14 , Genetic analysis of in vitro plant tissue culture responses and regeneration capacities.
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