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The Regulation of Development in the Mouse
Summary: Shirley Tilghman is interested in the function and mechanism of genomic imprinting and genes that regulate embryonic development in the mouse.
For the vast majority of genes, the mother’s and father’s copies are activated and regulated identically. However, for a small class of mammalian genes, only the mother’s or father’s copy is expressed. These genes are imprinted; that is, during the process that generates eggs or sperm, imprinted genes are marked in such a way that the resulting embryo can distinguish the parental origin of the gene and express it accordingly. Our laboratory is trying to understand two aspects of genomic imprinting: its function in mammalian development and its transcriptional mechanism.
Genomic imprinting is difficult to understand from a genetic perspective, because imprinting discards the advantage of having two copies of each gene. We have investigated the evolution of imprinting and have shown that the process exists in placental mammals such as eutherians and marsupials but is absent in other vertebrates, such as chickens. Thus an explanation for the function of imprinting must resolve the question of why it is mammalian specific. One clue comes from observations that many of the ~30 imprinted genes are involved in regulating the growth of the mammalian fetus. Mammals are unique among organisms in that their embryos develop as parasites of the mother, deriving all their nutrients through the placenta; thus it is conceivable that imprinting represents a novel mechanism for ensuring the well-being of both mother and embryo.
We are studying the consequences of disruptions in imprinting in the wild rodent species Peromyscus. We have found dramatic placental and embryonic overgrowth in hybrids between different Peromyscus species that arise from a combination of disruptions in imprinting and a genetic incompatibility between one or more X-linked genes and one or more imprinted genes. The loss of imprinting of paternally expressed genes in these hybrids is widespread and is associated with the most dramatic examples of overgrowth. The genetic incompatibilities bring to mind a model of speciation proposed by Theodosius Dobzhansky in the 1930s whereby genes that are hemizygous—such as those on the X and Y chromosomes—will accumulate allelic variants in reproductively isolated populations that are incompatible with one another when the populations hybridize. We propose that epigenetic phenomena such as imprinting provide an additional mechanism by which recessive incompatibilities are uncovered in mammals and may help to explain the rapid rate of speciation in mammals.
Imprinting disruptions have also been suggested as an explanation for Beckwith-Wiedemann syndrome (BWS), a clinically variable disorder that is characterized by somatic overgrowth, macroglossia, abdominal wall defects, visceromegaly, and an increased susceptibility to childhood tumors. The disease has been linked to a large cluster of imprinted genes at human chromosome 11p15.5. A small fraction of BWS patients have been identified with loss-of-function mutations in the maternally expressed p57KIP2 gene, encoding a G1 cyclin kinase inhibitor. A separate set of patients display loss of imprinting of IGF2, a fetal-specific growth factor that is paternally expressed.
To understand how the same disease can result from misregulation of two linked but unrelated genes, we generated a mouse model for BWS, together with Stephen Elledge (HHMI, Baylor College of Medicine), that harbors a null mutation in p57KIP2 as well as loss of Igf2 imprinting. These mice display many of the characteristics of BWS, including placentomegaly and dysplasia, kidney dysplasia, macroglossia, cleft palate, omphalocele, and polydactyly. Some but not all of the phenotypes have been shown to be Igf2 dependent. In several of the affected tissues, the two imprinted genes appear to act in an antagonistic manner, a finding that may help explain how BWS may result from mutations in either gene.
Our studies into the mechanism of gene silencing of imprinted genes have led us to conclude that there are multiple mechanisms at work. Genes such as the H19 gene, which is expressed exclusively from the maternal chromosome, are silenced by DNA methylation that is inherited from the paternal germline. Igf2, on the other hand, is silenced indirectly through an element that blocks the interaction of the gene with enhancers that lie 100 kilobases away. This enhancer blocker is inactivated by DNA methylation on the paternal chromosome, allowing for expression of the Igf2 gene. Enhancer blockers only function when placed between a gene and an enhancer. Such elements help explain how imprinted genes can be imprinted in some tissues but not in others, as imprinting will depend on the position of transcriptional regulatory elements relative to the epigenetic control region.
During embryogenesis, neural crest cells arise from the dorsal end of the closing neural tube. These cells are pluripotent, giving rise to a wide range of cell types, including neurons and glia of the peripheral nervous system, craniofacial bones and cartilage, smooth muscle cells, and melanocytes. Upon their emergence from the neural tube, neural crest cells undergo extensive proliferation as they migrate along distinct pathways to destinations where they differentiate into specific lineages.
We have been interested in the role of the endothelin receptor B (Ednrb) gene, which encodes a G protein–coupled seven-transmembrane receptor, in this process. Mice that lack Ednrb are white, except for occasional pigmented areas in the head and rump, and die as juveniles from megacolon, a condition that results from the inability of neural crest–derived enteric neurons to colonize the distal colon. To understand the role of Ednrb in development, we generated a mutant mouse in which the bacterial lacZ gene is expressed in place of Ednrb, providing us with an easy way to visualize its expression in vivo. Both melanoblasts and enteric ganglia are formed, but they fail to migrate appropriately.
To determine the specific time in development when the Ednrb gene is required for melanocyte and enteric ganglia development, we engineered two strains of mice in which the gene can be tightly regulated by the administration of doxycycline, a stable tetracycline derivative, in the drinking water. Using these mice, we have shown that the EDNRB-mediated signal is only required at a highly restricted time in development, between embryonic days 10.5 and 12.5, precisely the time that migration is occurring. Moreover, our results suggest that EDNRB is only required for committing melanoblasts into the migratory pathway.
Last updated October 23, 2000
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