Faculty Bibliography

Bone morphogenetic proteins (BMPs) are key mediators of dorsoventral patterning in vertebrates and are required for the induction of ventral fates in fish and frogs. A widely accepted model of dorsoventral patterning postulates that a morphogenetic BMP activity gradient patterns cell fates along the dorsoventral axis. Recent work in zebrafish suggests that the role of BMP signaling changes over time, with BMPs required for global dorsoventral patterning during early gastrulation and for tail patterning during late gastrulation and early somitogenesis. Key questions remain about the late phase, including which BMP ligands are required and how the functions of BMPs differ during the early and late gastrula stages. In a screen for dominant enhancers of mutations in the homeobox genes vox and vent, which function in parallel to bmp signaling, we identified an insertion mutation in bmp4. We then performed a reverse genetic screen to isolate a null allele of bmp4. We report the characterization of these two alleles and demonstrate that BMP4 is required during the later phase of BMP signaling for the specification of ventroposterior cell fates. Our results indicate that different bmp genes are essential at different stages. In addition, we present genetic evidence supporting a role for a morphogenetic BMP gradient in establishing mesodermal fates during the later phase of BMP signaling.

STUDY DESIGN: In vivo study of the effect of an injection of recombinant human osteogenic protein-1 into degenerated discs induced by chondroitinase ABC. OBJECTIVE: To investigate the efficacy of an injection of recombinant human osteogenic protein-1 to induce the recovery of disc height, and biochemical and histologic repair, in discs degenerated through enzymatic digestion by chondroitinase ABC. SUMMARY OF THE BACKGROUND DATA: Chondroitinase ABC is currently proposed as a chemonucleolysis agent; however, postchemonucleolysis degeneration is currently unavoidable. Recombinant human OP-1 has been shown to promote extracellular matrix repair in vitro and in vivo. METHODS: Fifty-four adolescent New Zealand white rabbits were used. Four weeks after an initial injection of chondroitinase ABC (10 mU/disc), 5% lactose (10 microL/disc) or recombinant human osteogenic protein-1 (100 microg in 10 microL lactose/disc) was injected. Disc heights were monitored radiographically at 2-week intervals, and rabbits were killed at 6, 8, 12, and 16 weeks after the initial chondroitinase ABC injections. The intervertebral discs were subjected to histologic and biochemical analyses. RESULTS: Significant disc space narrowing was observed in both groups 2 weeks after the injection of chondroitinase ABC. In the chondroitinase ABC/lactose group, this narrowing progressed after the vehicle injection and was sustained for up to 16 weeks. In the chondroitinase ABC/recombinant human osteogenic protein-1 group, the disc height index showed a significant increase at 6 weeks (lactose vs. recombinant human osteogenic protein-1; P < 0.01); this recovery was sustained for up to 16 weeks. The proteoglycan content was higher in the chondroitinase ABC/recombinant human osteogenic protein-1 group than in the chondroitinase ABC/lactose group. However, histologic changes, after the recombinant human osteogenic protein-1 injection, were not observed. CONCLUSIONS: A single injection of recombinant human osteogenic protein-1 into a rabbit disc dramatically reversed the decrease in disc height induced by chondroitinase ABC chemonucleolysis. The recovery was significant and sustained over the next 12 weeks. The therapeutic effects of both chondroitinase ABC chemonucleolysis and recombinant human osteogenic protein-1 injections should be further explored in higher animals before it is applied to humans.

Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are attractive targets for the development of novel cancer therapeutics. Herein, we demonstrate that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains resulting in dramatic induction of auto-ubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis. Additionally, the c-IAP proteins were found to function as regulators of NF-kappaB signaling. Through their ubiquitin E3 ligase activities c-IAP1 and c-IAP2 promote proteasomal degradation of NIK, the central ser/thr kinase in the noncanonical NF-kappaB pathway.

We surveyed native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China, and C. nitschkei, a sympatric species on chestnut trees in Japan, to learn more about the diversity of hypoviruses and other double-stranded (ds) RNA viruses. In a sample of 472 isolates of C. parasitica and 45 isolates of C. nitschkei from six prefectures in Japan, we found 27 containing one or more dsRNAs. Twelve isolates of C. parasitica and two isolates of C. nitschkei were infected with Cryphonectria hypovirus 1 (CHV-1); four of these 12 C. parasitica isolates also contained other dsRNAs that did not hybridize to CHV-1. In China, only one of 85 C. parasitica isolates was CHV-1-infected; no dsRNAs were detected in the other isolates from China. No other known hypoviruses were found in this study. However, we found two previously undescribed dsRNAs in Japan approximately 9kb in size that did not hybridize to each other or to any known dsRNAs from C. parasitica. We also found three additional groups of dsRNAs, one of which represents the genome of a new member of the virus family Chrysoviridae and was found only in C. nitschkei; the other two dsRNAs were found previously in isolates of C. parasitica from Japan or China. The most significant result of this survey is the discovery of novel dsRNAs that can be characterized in future research.

Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) delta plays a central role at various stages of pregnancy, whereas maternal PPARdelta is critical to implantation and decidualization, and embryonic PPARdelta is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARdelta-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARdelta signaling provides evidence that PPARdelta is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans.

The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. Because microRNAs (miRNAs) have major roles in regulating gene expression, we speculated that they participate in directing the highly regulated spatiotemporally expressed genetic network during implantation. Here, we show that two miRNAs, mmu-miR-101a and mmu-miR-199a*, are spatiotemporally expressed in the mouse uterus during implantation coincident with expression of cyclooxygenase-2, a gene critical for implantation. More interestingly, our in vitro gain- and loss-of-function experiments show that cyclooxygenase-2 expression is posttranscriptionally regulated by these two miRNAs. We report on miRNA-mediated regulation of uterine gene expression in the context of implantation. We believe that many other critical genes related to this process are also regulated by miRNAs. Thus, elucidating the physiological roles of uterine miRNAs will help us better understand the genetic control of implantation, the gateway to a successful pregnancy.

An intimate discourse between the blastocyst and uterus is essential for successful implantation. However, the molecular basis of this interaction is not clearly understood. Exploiting genomic Hbegf mutant mice, we show here that maternal deficiency of heparin-binding EGF-like growth factor (HB-EGF) defers on-time implantation, leading to compromised pregnancy outcome. We also demonstrate that amphiregulin, but not epiregulin, partially compensates for the loss of HB-EGF during implantation. In search of the mechanism of this compensation, we found that reduced preimplantation estrogen secretion from ovarian HB-EGF deficiency is a cause of sustained expression of uterine amphiregulin before the initiation of implantation. To explore the significance specifically of uterine HB-EGF in implantation, we examined this event in mice with conditional deletion of uterine HB-EGF and found that this specific loss of HB-EGF in the uterus still defers on-time implantation without altering preimplantation ovarian estrogen secretion. The observation of normal induction of uterine amphiregulin surrounding the blastocyst at the time of attachment in these conditional mutant mice suggests a compensatory role of amphiregulin for uterine loss of HB-EGF, preventing complete failure of pregnancy. Our study provides genetic evidence that HB-EGF is critical for normal implantation. This finding has high clinical relevance, because HB-EGF signaling is known to be important for human implantation.

Immunophilin FKBP52 serves as a cochaperone to govern normal progesterone (P(4)) receptor (PR) function. Using Fkbp52(-/-) mice, we show intriguing aspects of uterine P(4)/PR signaling during pregnancy. Implantation failure is the major phenotype found in these null females, which is conserved on both C57BL6/129 and CD1 backgrounds. However, P(4) supplementation rescued implantation and subsequent decidualization in CD1, but not C57BL6/129, null females. Surprisingly, experimentally induced decidualization in the absence of blastocysts failed in Fkbp52(-/-) mice on either background even with P(4) supplementation, suggesting that embryonic signals complement uterine signaling for this event. Another interesting finding was that while P(4) at higher than normal pregnancy levels conferred PR signaling sufficient for implantation in CD1 null females, these levels were inefficient in maintaining pregnancy to full term. However, elevating P(4) levels further restored PR signaling to a level optimal for successful term pregnancy with normal litter size. Collectively, the results show that the indispensability of FKBP52 in uterine P(4)/PR signaling is a function of genetic disparity and is pregnancy stage specific. Since there is evidence for a correlation between P(4) supplementation and reduced risks of P(4)-resistant recurrent miscarriages and remission of endometriosis, these findings have clinical implications for genetically diverse populations of women.

Reduced litter sizes in mice missing pentraxin 3 (Ptx3) have been attributed to fertilization failure. However, our global gene expression studies showed high uterine Ptx3 expression at the implantation site in mice, suggesting its role in blastocyst implantation. We initiated molecular and genetic studies in mice to explore the importance of uterine Ptx3 in this process. We found that Ptx3 is expressed in a unique and transient fashion at implantation sites. With the initiation of implantation on midnight of Day 4 of pregnancy, Ptx3 is expressed exclusively in stromal cells at the site of blastocysts. On Day 5, its expression is more intense in decidualizing stromal cells, but it disappears on Day 6. The expression again becomes evident in the deciduum on Day 7, followed by a more robust expression on Day 8, particularly at the antimesometrial pole. From Day 9, with the initiation of placentation, Ptx3 expression becomes undetectable. These results suggest a role for PTX3 in implantation and decidualization. Indeed, deletion of Ptx3 results in both compromised implantation and decidualization. Interleukin 1B (IL1B), a known inducer of Ptx3, is also transiently expressed in stromal cells at the implantation site, suggesting that IL1B is an inducer of uterine Ptx3 expression. In fact, uterine Ptx3 expression follows that of Il1b induced by lipopolysaccharide treatment on Day 7 of pregnancy. Collectively, these findings provide evidence for an important role for PTX3 in implantation and decidualization. This study has clinical implications, since PTX3 is expressed in the receptive endometrium, and trophoblast cells influence decidual Ptx3 expression in humans.

Embryo implantation is absolutely dependent on the preparation of the uterus to the receptive stage and attainment by the blastocyst of implantation competency. Co-ordinated effects of progesterone and oestrogen are essential for these processes and determine the window of implantation. In rodents, a generalized stromal edema occurs before blastocyst attachment followed by uterine luminal closure. This leads to apposition of the blastocyst trophectoderm against the luminal epithelium and ultimately attachment. Progesterone is essential for luminal closure, which must occur for successful implantation. More importantly, progesterone is critical for almost every stage of pregnancy, including ovulation, fertilization, implantation, decidualization and pregnancy maintenance. Progesterone exerts its effects on target tissues primarily via nuclear progesterone receptor (PR) whose optimal activity is potentiated by an immunophilin co-chaperone, FK-506 binding protein 4 (FKBP52). While mice lacking PR are infertile due to complete failure of ovulation, fertilization, and implantation, female mice with targeted deletion of the Fkbp52 gene are infertile specifically because of implantation failure resulting from compromised uterine receptivity. This review highlights the evolution of knowledge about progesterone signalling during early pregnancy. Future studies are likely to provide a better understanding of FKBP52-PR signalling in promoting uterine receptivity for implantation and may reveal new targets for improving infertility.