Placenta and Trophoblast
The placenta and its underlying trophoblast lineage comprise a major developmental checkpoint, critical for mammalian embryonic survival, and sensitive to a surprisingly large number of gene knockouts. This feature and the recent establishment of trophoblast stem (TS) cell cultures make the placenta an ideal genetic platform to study cell differentiation and organogenesis.
PPARγ is one of the many genes proving essential for placental development. Deficiency for PPARγ causes severe defects in the differentiation of the labyrinthine trophoblast, which fails to complete mesenchymal to epithelial transition.This defect results in a failure of the trophoblast to interact with the fetal endothelium, and in turn the inability of fetal blood vessels to form a placental vascular network. By E10.0, PPARγ null embryos can no longer survive, likely due to starvation in utero.
Interestingly, PPARγ null embryos
exhibit severe defects in myocardial development, whereby ventricular subepicardial myocytes differentiate prematurely, leading to thinning of the ventricle. Tetraploid chimeras, which selectively rescue trophoblast defects, eliminated these cardiac anomalies, proving that they are secondary to the placental ones, and revealing a novel developmental dependence of the heart on a functional placenta.
Since PPARγ is a transcription factor, its functions should be fully accounted for in terms of its target genes. We started to pursue PPAR? -inducible genes (gigs) in trophoblasts by subtraction and microarray profiling of cDNAs from wt and PPAR? null placentas. The expression of true gigs should decline in PPARγ null placentas and trophoblast cultures and rise in agonist-treated trophoblasts, with the remaining qualifier being the identification of PPARγ -responsive sequences in the target gene promoter. The most robust gig (gig1) encodes a transmembranal protein prototypic of secretory epithelia, invoking a novel analogy between these epithelial structures and the placenta, and implicating PPARγ as a regulator of this shared aspect.
The gig1 promoter provides a golden opportunity to study the regulation of a natural PPARγ target. Our preliminary transfection studies reveal that the proximal gig1 promoter contains all information sufficient and necessary for activation by PPARγ. Moreover, unlike any target characterized so far, it is specifically activated by PPARγ, but not PPAR α or δ. We further study the molecular basis of this remarkable PPARγ specificity to understand how the three PPARs, which share highly similar DNA-binding properties, evolved to regulate distinct biological responses.
Gig1 regulation is surprisingly complex, requiring cooperativity between two distinct promoter modules, one of which is a low affinity PPARγ -binding site, and the other - a module which does not interact directly with PPARγ, but is nevertheless critical for the response. We continue to analyze this promoter, seeking to identify the individual components involved along with PPARγ in its regulation, and to understand the molecular basis of this transcriptional cooperativity.
In a fresh effort, we started seeking new pathway connections in the trophoblast differentiation program by analyzing the relationships between PPARγ and genes whose deficiency yields comparable placental phenotypes. We test the products of these genes for either potential epistasis or crosstalk with PPARγ and its targets. Our broad working hypothesis is that a finite set of factors regulates a wider set of targets in a combinatorial fashion, and that once identified, these factors and the relationships between them will provide a molecular definition of trophoblast and epithelial differentiation.
