Zimp10 is emerging as a transcriptional coregulator in a variety of important cellular pathways, including the AR, transforming growth factor β (TGF-β), and p53 signaling cascades (20
). However, its biological role in vivo still remains unclear. To address this question, we have generated mice with a targeted disruption of the zimp10
gene. In this study, we show that embryos deficient for the Zimp10
allele die around E10.5 due to severe defects in yolk sac vessel organization, indicating that Zimp10 plays an essential role in the extraembryonic vascular development process. Subtle differences also were observed in aortic arch architecture, heart ventricle development, and the number of branching capillaries in the head region in zimp10
knockout embryos. Additionally, cell proliferation was inhibited in zimp10−/−
embryo fibroblasts and in cells with reduced Zimp10 levels. Despite the anemic appearance of the zimp10−/−
embryos, the hematopoietic potential appeared normal in zimp10−/−
yolk sacs. Intriguingly, we provide evidence in this report demonstrating that the most severe defect in zimp10
knockouts is the failure of the yolk sac vascular plexus to form a mature vascular network, which is critical for proper development during embryogenesis.
By around E9.5, embryos no longer can survive by the direct exchange of gasses and nutrients, and thus the establishment of a functional extraembryonic vasculature becomes critical (10
). The identification of a poorly organized yolk sac vasculature in zimp10
knockout embryos suggests that this defect is the primary cause of embryonic death around E10.5. Our results are consistent with those of previous studies showing similar embryonic lethal phenotypes between E9.5 and E10.5 that result from yolk sac defects (7
). Although we have observed that a primitive vascular network of large vessels is present in zimp10−/−
yolk sacs, we demonstrated that the vessels fail to develop from a primitive vascular plexus into a mature vascular network. In addition, fewer branching capillary structures were observed in the head region of zimp10−/−
embryos, and subtle differences in vessel and heart architecture were observed, suggesting that vascular abnormalities may exist in zimp10−/−
embryos as well. The staining of zimp10−/−
yolk sacs with a smooth-muscle actin antibody revealed that smooth-muscle cells are organized in a punctate pattern rather than the expected branching network seen in wild-type yolk sacs. This result provides evidence that a mature vessel network is unable to form in zimp10−/−
yolk sacs, which may be one of the primary factors resulting in the embryonic lethality of zimp10−/−
animals. Although the zimp10
knockout system is the first in vivo system to implicate a potential role for PIAS-like proteins in vascular development, previous studies have shown potential roles for the PIAS proteins in the regulation of angiogenesis in vivo and in smooth-muscle differentiation in cell culture systems through the STAT signaling pathways (2
). Thus, the roles of Zimp10 and other PIAS proteins in angiogenesis and vasculogenesis should be further explored.
Interestingly, disruptions of the Notch and TGF-β pathways in mice result in similar defects in vascular remodeling (6
). In these models, abnormalities are observed around E9.5, and embryonic death occurs by E10.5. In particular, the TGF-β type I receptor knockout mice showed vascular defects very similar to those observed in zimp10−/−
mice. Previous studies have shown a potential link between PIAS proteins and Smad proteins, downstream transducers of TGF-β pathways (23
). Interestingly, our recent works showed that Smad3/4 transcriptional activity is impaired in Zimp10 knockout embryos, implying a regulatory role for Zimp10 in Smad3- and Smad4-mediated transcription (21
). However, we were unable to identify significant differences in the expression of notch components in the zimp10−/−
yolk sacs by RT-PCR in this study (data not shown). Further study of the precise molecular mechanisms by which Zimp10 regulates the development of the vascular system and angiogenesis may help us to understand the interaction between Zimp10 and TGF-β pathways.
As we have reported previously, Zimp10 is a PIAS-like protein and shares structural and functional similarities with members of the PIAS family. The biological roles of PIAS proteins have been investigated in knockout mouse models. Disruption of the PIASx
genes did not show significant defects in mice (30
). In contrast, PIAS1 knockout mice display partial perinatal lethality before E17.5, and mice that survive are 20 to 40% smaller than wild-type littermates (22
). In addition, these mice displayed enhanced interferon-mediated antiviral activity and increased protection against pathogenic infection. Given the fact that the PIAS proteins are very similar structurally, it is possible that other members of the PIAS family compensate for the function of PIAS1x and PIAS1y in their knockout mice. The creation of knockouts with multiple PIAS protein deletions may provide useful information in this regard. The phenotype of zimp10
null mice appears more severe than those observed for PIAS knockouts. These data suggest a unique and important role for Zimp10 in early development, which may be distinct from and cannot be compensated for by other PIAS proteins. While zimp10
knockout results in embryonic lethality by E10.5 with 100% penetrance, intriguingly, we observed that Zimp10 heterozygotes are born at less than the expected Mendelian ratio. It is currently unclear at what stage heterozygote lethality might occur, although several litters have been examined from E9.5 to E15.5 with no observable difference between wild-type and heterozygous embryos (data not shown). It will be very interesting to further identify the lethality of the heterozygotes, which may help us to further understand the biological role of Zimp10 in development.
Vascular development requires the coordinated activation of several transcription factors through a variety of different pathways, including the TGF-β, Notch, VEGF, Wnt, and BMP pathways (5
). The widespread implementation of knockout mouse models has led to the identification of a subset of transcription factors that are essential for vasculogenesis (28
). To examine the precise role of Zimp10 in vascular development, we used a microarray approach to examine the transcription profiles of zimp10+/+
yolk sacs. A number of cDNA sequences encoding proteins that may contribute to vascular development were identified to be downregulated in zimp10
knockout yolk sacs, including Fra-1, PPARγ, AP2γ, GATA-2, and GATA-3 (Table ). Using RT-PCR, we further confirmed the reduction of Fra-1 expression in Zimp10 knockouts. Previous studies have shown that fra-1
knockout mice display defects in the placental and yolk sac vasculature and die at around E10 (32
). Using fra-1
promoter/reporter constructs, we further confirmed a regulatory role for Zimp10 in the fra-1
promoter in human HEK293 cells (Fig. ). In addition, quantitative RT-PCR experiments revealed that the fra-1
transcript is downregulated in HEK293 cells infected with a Zimp10-specific shRNA construct (Fig. ). Interestingly, Zimp7, a homolog of Zimp10, also affected the activity of the fra-1
promoter constructs in the in vitro reporter assays. However, as shown in this paper, Zimp7 appears to be unable to functionally compensate for Zimp10 in vivo. These observations suggest that Zimp7 is able to functionally compensate for Zimp10 in certain contexts, but that it may depend on the temporal and/or tissue-specific expression of the zimp7
gene. Indeed, our previous work has shown that Zimp7 and Zimp10 have different expression patterns in human tissues (9
). Alternatively, differential requirements for transcriptional cofactors that are essential for vascular development may explain this discrepancy. Studies are ongoing to clarify the functional interplay between Zimp10 and Zimp7 in the vasculature. Since Zimp10 has been identified to act as a transcriptional coregulator, our data implicate a possible mechanism by which Zimp10 regulates vascular development through the modulation of Fra-1 transcription.
In conclusion, this study demonstrates an important role for Zimp10 in development. Although we originally identified Zimp10 as an AR coactivator, our current data have elucidated a primary role for Zimp10 in vascular development in vivo. Specifically, homozygous disruption of the zimp10 allele results in defects in the remodeling of the yolk sac vascular plexus into a functional network of mature branching vessels and capillaries. This defect may be due, in part, to reduced expression of fra-1, which appears to be a novel Zimp10 target gene. Further study of the molecular mechanisms by which Zimp10 regulates vasculogenesis and angiogenesis should provide more insight into the role of Zimp10 in development and disease.