Catherine M. Nolan

Epigenetics and gene expression

Transcription, translation and subsequent protein modification represent the transfer of genetic information from the archival copy of DNA to the short-lived messenger RNA, usually with subsequent production of protein. Although all cells in an organism contain essentially the same DNA, cell types and functions differ because of qualitative and quantitative differences in their gene expression. Thus, control of gene expression is at the heart of differentiation and development. Epigenetic processes, including DNA methylation, histone modification and various RNA-mediated processes, are thought to influence gene expression chiefly at the level of transcription; however, other steps in the process (for example, translation) may also be regulated epigenetically. The following paper will outline the role epigenetics is believed to have in influencing gene expression.

Imprint status of M6P/IGF2R and IGF2 in chickens.

Abstract. Genomic imprinting is a method of gene regulation whereby a gene is expressed in a parent-of-origin-dependent fashion; however, it is hypothesized that imprinting should not occur in oviparous taxa such as birds. Therefore, we examined the allelic expression of two genes in the chicken that are reciprocally imprinted in most mammals, mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and insulin-like growth factor 2 (IGF2). Single nucleotide polymorphisms were identified in these genes, and cDNA was prepared from several tissues of embryos heterozygous for these polymorphisms. Both alleles of M6P/IGF2R and IGF2 were expressed in all tissues examined by RT-PCR. Since the expression of these genes was independent of the parent from which they were inherited, we conclude that neither M6P/IGF2R nor IGF2 are imprinted in the chicken.

Tissue-Specific Inactivation of Murine M6P/IGF2R

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes a multifunctional protein involved in lysosomal enzyme trafficking, fetal organogenesis, tumor suppression, and T cell- mediated immunity. M6P/IGF2R is an imprinted gene in mice with expression only from the maternal allele. Complete knockout of this gene causes neonatal lethality, thus preventing analysis of its multifunctional role postnatally. To help elucidate the biological functions of M6P/IGF2R in adulthood, we generated both complete and tissue-specific M6P/IGF2R knockout mice using the Cre/loxP system. We confirm that complete M6P/IGF2R knockout results in fetal overgrowth and neonatal lethality. In contrast, tissue-specific inactivation of this gene in either the liver or skeletal and cardiac muscle gives rise to viable animals with no obvious phenotype. The successful creation of viable tissue-specific M6P/IGF2R knockout mouse models will now allow for detailed analysis of receptor function in a number of cellular processes including brain development, carcinogenesis, lysosomal trafficking, and T cell-mediated immunity.

Abnormal postnatal maintenance of elevated DLK1 transcript levels in callipyge sheep

The underlying mechanism of the callipyge muscular hypertrophy phenotype in sheep (Ovis aries) is not presently understood. This phenotype, characterized by increased glycolytic type II muscle proportion and cell size accompanied by decreased adiposity, is not visibly detectable until approximately three to eight weeks after birth. The muscular hypertrophy results from a single nucleotide change located at the telomeric end of ovine Chromosome 18, in the region between the imprinted MATERNALLY EXPRESSED GENE 3 (MEG3) and DELTA, DROSOPHILA, HOMOLOG-LIKE 1 (DLK1) genes. The callipyge phenotype is evident only when the mutation is paternally inherited by a heterozygous individual. We have examined the pre- and postnatal expression of MEG3 and DLK1 in sheep of all four possible genotypes in affected and unaffected muscles as well as in liver. Here we show that the callipyge phenotype correlates with abnormally high DLK1 expression during the postnatal period in the affected sheep and that this elevation is specific to the hypertrophy-responsive fast-twitch muscles. These results are the first to show anomalous gene expression that coincides with both the temporal and spatial distribution of the callipyge phenotype. They suggest that the effect of the callipyge mutation is to interfere with the normal postnatal downregulation of DLK1 expression.

IGF-1 receptor mediates differentiation of primary cultures of mouse skeletal myoblasts.

Studies involving immortalized myoblasts suggested that insulin-like growth factors (IGFs) promote differentiation of skeletal muscle, but gene targeting experiments in mice did not provide support for this hypothesis. To address this discrepancy, we examined differentiation of primary cultures of mouse myoblasts. Differentiation was normally unaffected by addition of IGFs to the differentiation medium. However, when we interrupted IGF-mediated signaling, by incubating myoblasts with suramin or with a monoclonal antibody to the IGF-I receptor, differentiation was inhibited. Inhibition was reversed by exogenous IGF-I or IGF-II, but not by insulin. Differentiation was enhanced in myoblasts that were incubated with an inhibitor of the mitogen-activated protein kinase signaling pathway (PD098059) and such cells were responsive to exogenous IGF-I. Our results demonstrate that IGF action contributes to the differentiation of non-immortalized mouse myoblasts and that these cells represent a model system that can be experimentally manipulated to study the molecular events involved in skeletal muscle differentiation.

Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos

The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti-apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf-2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.

Intracellular traffic of the mannose 6-phosphate receptor and its ligands.

The role of the mannose 6-phosphate recognition marker and the phosphomannosyl receptor in the intracellular transport of acid hydrolases is well established. Several details of the process, however, are presently unclear and warrant further investigation (Table 1). The development of in vitro systems for the reconstitution of receptor-mediated endocytosis and intracellular transport, together with the application of the techniques of molecular biology to this field, should lead to major advances in our understanding of the targeting of enzymes to lysosomes.

An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution

Parental genetic conflict may have exploited changes in the coding of a protein loop in a growth factor receptor. Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation. Many mammals have imprinted alleles, where the paternal or maternal version is solely expressed during reproduction. In humans, one such imprinted gene set is the growth promoter insulin-like growth factor 2 (IGF2) and its binding inhibitor, IGF2R mannose 6-phosphate/IGF2 receptor. To avoid parental conflict in fetal growth, imprinting regulates expression of these genes so that expression of IGF2R in the fetus quenches IGF2 and prevents fetus overgrowth through high-affinity binding of IGF2 to IGF2R. Williams et al. (p. 1209) demonstrate that high-affinity binding of IGF2 to IGF2R is present in placental and marsupial mammals; absent in birds and fish; and present, with a 10-fold lower affinity, in monotreme (egg-laying) mammals. The appearance of exonic splicing enhancers in exon 34 of the IGF2R of monotremes appears to have been a key mutational event leading to the establishment of higher affinity, which may have been driven by selection to minimize parental conflict.

Mannose 6-phosphate receptors in an ancient vertebrate, zebrafish

The endosome/lysosome system plays key roles in embryonic development, but difficulties posed by inaccessible mammalian embryos have hampered detailed studies. The accessible, transparent embryos of Danio rerio, together with the genetic and experimental approaches possible with this organism, provide many advantages over rodents. In mammals, mannose 6-phosphate receptors (MPRs) target acid hydrolases to endosomes and lysosomes, but nothing is known of acid hydrolase targeting in zebrafish. Here, we describe the sequence of the zebrafish cation-dependent MPR (CD-MPR) and cation-independent MPR (CI-MPR), and compare them with their mammalian orthologs. We show that all residues critical for mannose 6-phosphate (M6P) recognition are present in the extracellular domains of the zebrafish receptors, and that trafficking signals in the cytoplasmic tails are also conserved. This suggests that the teleost receptors possess M6P binding sites with properties similar to those of mammalian MPRs, and that targeting of lysosomal enzymes by MPRs represents an ancient pathway in vertebrate cell biology. We also determined the expression patterns of the CD-MPR and CI-MPR during embryonic development in zebrafish. Both genes are expressed from the one-cell stage through to the hatching period. In early embryos, expression is ubiquitous, but in later stages, expression of both receptors is restricted to the anterior region of the embryo, covering the forebrain, midbrain and hindbrain. The expression patterns suggest time- and tissue-specific functions for the receptors, with particular evidence for roles in neural development. Our study establishes zebrafish as a novel, genetically tractable model for in vivo studies of MPR function and lysosome biogenesis.

Imprinted expression of the canine IGF2R, in the absence of an anti-sense transcript or promoter methylation

SUMMARY Imprinted genes are epigenetically modified in a parent of origin‐dependent manner, and as a consequence, are differentially expressed. Although the evolution of genomic imprinting is a subject of intense debate, imprinted genes have been studied primarily in mice and humans and in a small number of marsupial mammals. Comparative studies involving rodents and primates are of limited value, as they belong to the same superordinal group of eutherian mammals (Euarchontoglires). On the other hand, comparisons involving marsupials may not be informative, due to phylogenetic distance. Canis familiaris belongs to Laurasiatheria, a sister‐group of Euarchontoglires, and should prove useful in comparative studies of imprinted genes. Using RT‐PCR we demonstrate monoallelic expression of the canine IGF2R in several tissues, including uterus and umbilical cord. In the case of umbilical cord, we identify the expressed allele as maternally derived. The canine IGF2R is thus an imprinted gene. Using bisulfite sequencing, we show that the canine IGF2R resembles the imprinted mouse Igf2r in having a CpG island in intron 2 that is hemi‐methylated. However, it differs from the mouse gene in that maintenance of the monoallelic expression of canine IGF2R does not require expression of an anti‐sense transcript from the paternally derived allele, or methylation of the repressed IGF2R promoter. In these two important features, the imprinted canine gene resembles the imprinted opossum IGF2R. Our data suggest that these features were properties of the ancestral imprinted IGF2R and that the anti‐sense transcript (Air) and promoter methylation observed in mouse are derived features of the mouse Igf2r locus.