The latest stages of mammalian gestation entail an accelerated growth phase, which is critical for optimal neonatal birth weight and preparedness for life outside the womb. While it is apparent that the fuel source for this massive growth spurt is circulating maternal metabolites, it is less clear whether placental storage of metabolic fuels is essential for this process. Insights into this process emerge from our studies of the nuclear receptor PPARy, an essential regulator of placental development and metabolism. We found that the placenta- enriched transcription cofactor LCoR (Ligand-dependent CoRepressor) is a coactivator (Not a corepressor) of PPARy, on the target gene Mud both in vitro and in vivo. Molecular genetic analyses revealed that LCoR- deficient neonates die within hours of birth. These neonates do not exhibit obvious anatomic anomalies, but display limp forelimbs, lethargy, and failure to suckle. Importantly, while exhibiting normal growth until mid- gestation, Lcor-null embryos are significantly growth-restricted at term, alongside significant enlargement of the placenta. Histological analyses reveal that Lcor-null placentas retain a substantially higher number of placental glycogen trophoblasts (GlyT) compared to their WT counterparts, providing a plausible explanation to their larger mass. Importantiy, both PPARy, and LCoR are critical for placental expression of the glucagon receptor gene, Gcgr, suggesting that Lcor-null placentas accumulate glycogen due to failure to respond to the glycogenolytic signal of glucagon. Our findings suggest that LCoR regulates export of glycogen-derived fuel from placental GlyT to the embryo, and that its loss restricts the growth of the late fetus by limiting its access to this fuel. We further surmise that this severe deprivation of placental fuel sources at the last stages of gestation underlies the fatal, lethargic state of Lcor-null neonates. Accordingly, our overarching hypothesis is that transcriptional and developmental regulation of placental glycogen stores plays an essential role in late embryonic growth and neonatal survival.
Three specific aims driven by this hypothesis will dissect the role and regulation of placental glycogen stores in late embryogenesis:
Aim 1. Determine the outcomes of glycogen trophoblast ablation.
Aim 2. Define the lineage-specific funcfions of LCoR in the placenta and the embryo.
Aim 3. Dissect the molecular mechanisms of placental gene regulation by LCoR and PPARy. Relevance Growth restriction, stillbirth, neonatal mortality and infant morbidity due to gestational problems are a serious public health issue. This project will fill major knowledge gaps in this area by defining novel transcriptional and developmental mechanisms that regulate key fuel storage and metabolism processes in the placenta that affect the maturation and survival of the embryo and neonate. PHS 398 (Rev. 6/09) Page 109 Program Director/Principal Investigator (Last, First, Middle): SADOVSKY, YOEL Project II: Introducfion We thank the reviewers for their enthusiastic reception of this project. Below we address their main concerns. 1. Feasibility and time span of the effort to generate the targeted mouse strains proposed in Aims 1 and 2. Gene targeting is a major staple of our laboratory. We possess the full infrastructure to perform it and are extremely well versed in it. To date we have successfully generated over 15 targeted mouse strains without a single failure. We have just successfully completed recombineering the Lcor-/7 targeting construct for Aim 2, including all quality control steps. We plan to introduce it shortly into ES cells, and expect germ-line chimeras to be available even before this application is re-reviewed. Work on the conditional GlyT death allele proposed in Aim 1 should be as logical and efficient, and will commence as soon as we secure sufficient funds for its execution. Having generously received the already live-tested targeting cassette from Dr. Martinez-Barbera further facilitates this task. In addition, we have by now re-derived and started to backcross the Tpbpa-Cre mice received a few months ago from Dr. Cross, which will play a key role in both Aims. Over the years, we introduced many improvements that significantly reduce the time and labor tolls of gene targeting. Upon moving to MWRI, we further enhanced our capabilities by incorporating recombineering technology, which is now fully operational as an MWRI core. Recombineering allows us to design and construct a typical targeting vector within 4-8 weeks;our high-throughput 96-well targeted ES cell screening methods allow the routine completion of homologous recombination in ES cells within another 4-6 weeks, with moderate hands-on work. Importantly, I personally instruct all staff and trainees performing gene targeting in my laboratory and closely supervise every single step of vector design, construction and targeting into ES. To complement these efforts we have established in collaboration with Dr. Chaillet a weekly blastocyst microinjection operation, which has been functioning seamlessly over the last year to produce multiple chimeric mice and embryos for both laboratories. As this effort transitions into the Program's Animal Core, we are ensured that any targeted ES clone generated in this project will be injected without delays. Together, our lab takes approximately 31 weeks to establish a new mouse strain, from conception of the plan to confirmation of germ-line transmission. Dr. Chaillet's expert collaboration on these methodologies is an additional safeguard against potential hurdles. I hope the reviewers favorably recognize our progress since the last submission and our ability to generate the proposed mice diligently and accurately, based on our impeccable track record with this methodology and our meticulous approach to its execution. 2. Choice of cell lines for Gcgr promoter analysis. To address this concern we supplemented these cell choices with primary human trophoblasts (PHTs). Dr. Sadovsky's laboratory has long-standing expertise in this area. They routinely produce these cells, and have the requisite expertise in their transfection, ensuring the technical success of this aspect. Moreover, this change will further strengthen the ties between the laboratories participating in this Program Project. 3. Potential effects of PPARy phosphorylation. Previously, we found no impact of PPARyl phosphorylation site mutations on the activity of Mud promoter. Nevertheless, in response to this critique, we revised Aim 3 to include a comprehensive plan for analyzing the potential impact of PPARy phosphorylation on the activity of Gcg/'promoter, using mutant expression vectors and additional reagents, including mutant mice. 4. Secondary glycogen stores are not well addressed. There is currently no published evidence of secondary glycogen stores in the mouse placenta beyond GlyT. We apologize for not clarifying that this notion was raised as an entirely theoretical scenario when considering potential pitfalls of the GlyT ablation experiment. We need to perform this experiment in order to find out whether such a putative scenario is viable. 5. Why we did not: (a) expand our examinations to human placenta, (b) Examine the relationship to diabetic pregnancy or the effects of imprinted genes in relation to Project I, and (c) Use pharmacological interventions to modulate glycogen deposition? (a) We chose to focus on mouse molecular genetics for Aims 1 and 2, because of its power to dissect molecular perturbations in vivo and our long-standing expertise in this methodology;such studies cannot be performed with the same definitive rigor in human placental explants. Nevertheless, the Gcgr promoter studies in Aim 3 will be performed in primate and human cell lines, including PHTs, extending the relevance of the work to these species, (b) Relationships to diabetic pregnancy or imprinting are probably relevant. However, they prematurely broaden the questions at hand and might therefore substantially detract from the analytical focus of our work, (c) The strength of our work is the targeted, feto-placental specific genetic perturbations. There are no proven ways to target individual embryos with pharmacological interventions. These will likely affect both uterine and general maternal physiology, prohibiting precise inference of their effects on placental glycogen storage and release. 6. Partition of tasks between Drs Shalom-Barak &Pallerla. Neither scientist is key personnel. Since the original submission of the application Dr Pallerla left our laboratory and we are currently recruiting a replacement. We have extended Dr Shalom-Barak's role to recombineering, to align with her expertise in recombinant DNA technology (see Budget Justification). PHS 398 (Rev. 6/09) Page 110
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