Imprinted Gene Expression and Phenotype of Bovine Concepti with Bos taurus and Bos indicus Genetics

Epigenetic parent-of-origin effects contribute significantly to phenotypic variation in animals. Imprinted genes, which show differential allelic expression in a parent of-origin dependent manner, are critical regulators of prenatal development. Altered epigenetic modifications of imprinted genes in response to maternal environmental stimuli are believed to impact on prenatal development with long-term consequences for postnatal phenotype, a process which is known as fetal programming. Most studies in the area of fetal programming have focused on the epigenetic link between intrauterine environment and fetal phenotype, and genetic programming of variation in pre- and postnatal growth traits remains largely unexplored. We hypothesised that heterosis, i.e., the superiority of F1 hybrids compared to their parents, in growth traits is programmed prenatally through changes in expression patterns of imprinted genes. The purpose of this thesis was (i) to perform a comparative in silico analysis of promoter-specific transcripts and splice variants of the imprinted IGF2 and IGF2R genes as well as their imprinted regulatory non-coding RNAs, H19 and AIRN, (ii) to analyse expression patterns of all identified transcripts in bovine pre- and postnatal tissues, and (iii) to investigate fetal genetic effects on gene expression and their association with heterotic phenotype. To this end, a bovine model was employed using two genetically and phenotypically distinct subspecies of domesticated cattle, Bos taurus and Bos indicus, and their reciprocal crosses. Real time quantitative PCR was used to quantify expression of IGF2 promoterspecific transcripts, IGF2R, H19 and AIRN, in liver, brain, heart, cotyledon, skeletal muscle, kidney, lung and testis at three developmental stages, Day-48 embryo, Day-153 fetus and 12- month juvenile. Heterotic effects on transcript abundances and expression patterns of imprinted genes and their correlations with fetal body weight and weights of fetal tissues were estimated with general linear models. xiv All studied imprinted genes were subject to developmental control of gene expression with the transcript abundance being downregulated in postnatal tissues. We identified IGF2 promoter-specific transcripts and the bovine orthologue of human P0 promoter, and found a developmental shift in tissue specificity of P0 from fetal skeletal muscle to postnatal liver. Fetal body weight and absolute weights of fetal tissues, except brain, were subject to significant parent-of-origin effects, and were higher in fetal groups with B. taurus maternal genetics compared to B. indicus maternal genetics. Fetal placental weight, lung weight and relative muscle mass showed significant heterosis. Heterosis in fetal placental weight was associated with a polar overdominance imprinting pattern of IGF2R and AIRN in cotyledon with highest expression in B. indicus (sire) × B. taurus (dam) group, whereas the transcript abundance in the other reciprocal was close to purebred groups. In hybrid genetic groups, expression of IGF2R and AIRN in cotyledon was positively correlated with weight of fetal placenta and negatively correlated with placental efficiency, defined as fetal to placental weight ratio. H19 expression in skeletal muscle was significantly affected by the interaction between parental genomes. With respect to significant negative association between H19 expression and muscle mass, the negative heterotic effect on H19 expression may explain the positive heterosis in relative muscle mass. Fetal genetics consistently influenced H19 expression, which in B. indicus was between 1.5 to 2.2-fold higher than in B. taurus in all examined tissues, except brain. A negative relationship was observed between transcript abundance of H19 and weights of fetal tissues, except brain. These results suggest that H19 could be a molecular driver for differential subspecies-specific fetal phenotypes. By in silico search, we found B. indicus-specific nucleotide polymorphisms at CpG sites of the consensus sequence for the first CTCF binding site within the imprinting control region (ICR) located upstream of H19 promoter. This led us to speculate that higher expression of H19 in B. indicus tissues could be attributable to partial or complete relaxation of imprinting resulting from epigenetic changes in the ICR. xv In conclusion, our results provide insight into the interplay between genetics and epigenetics and its consequences for genetic programming of phenotypic variation and heterosis. Significant interaction between parental genomes on expression of H19, a miRNA precursor and master regulator of an imprinted gene network, and negative relationship between H19 expression and fetal muscle mass suggested imprinted genes and miRNA interference as mechanisms for differential effects of maternal and paternal genomes on fetal muscle.Thesis (Ph.D.) -- University of Adelaide, School of Animal & Veterinary Sciences, 201

Asymmetric growth-limiting development of the female conceptus

IntroductionSex differences in prenatal growth may contribute to sex-dependent programming effects on postnatal phenotype. MethodsWe integrated for the first time phenotypic, histomorphological, clinico-chemical, endocrine and gene expression analyses in a single species, the bovine conceptus at mid-gestation. ResultsWe demonstrate that by mid-gestation, before the onset of accelerated growth, the female conceptus displays asymmetric lower growth compared to males. Female fetuses were smaller with lower ponderal index and organ weights than males. However, their brain:body weight, brain:liver weight and heart:body weight ratios were higher than in males, indicating brain and heart ‘sparing’. The female placenta weighed less and had lower volumes of trophoblast and fetal connective tissue than the male placenta. Female umbilical cord vessel diameters were smaller, and female-specific relationships of body weight and brain:liver weight ratios with cord vessel diameters indicated that the umbilico-placental vascular system creates a growth-limiting environment where blood flow is redistributed to protect brain and heart growth. Clinico-chemical indicators of liver perfusion support this female-specific growth-limiting phenotype, while lower insulin-like growth factor 2 (IGF2) gene expression in brain and heart, and lower circulating IGF2, implicate female-specific modulation of key endocrine mediators by nutrient supply. ConclusionThis mode of female development may increase resilience to environmental perturbations in utero and contribute to sex-bias in programming outcomes including susceptibility to non-communicable diseases

Maternal and paternal genomes differentially affect myofibre characteristics and muscle weights of bovine fetuses at midgestation

Postnatal myofibre characteristics and muscle mass are largely determined during fetal development and may be significantly affected by epigenetic parent-of-origin effects. However, data on such effects in prenatal muscle development that could help understand unexplained variation in postnatal muscle traits are lacking. In a bovine model we studied effects of distinct maternal and paternal genomes, fetal sex, and non-genetic maternal effects on fetal myofibre characteristics and muscle mass. Data from 73 fetuses (Day153, 54% term) of four genetic groups with purebred and reciprocal cross Angus and Brahman genetics were analyzed using general linear models. Parental genomes explained the greatest proportion of variation in myofibre size of Musculus semitendinosus (80–96%) and in absolute and relative weights of M. supraspinatus, M. longissimus dorsi, M. quadriceps femoris and M. semimembranosus (82–89% and 56–93%, respectively). Paternal genome in interaction with maternal genome (P<0.05) explained most genetic variation in cross sectional area (CSA) of fast myotubes (68%), while maternal genome alone explained most genetic variation in CSA of fast myofibres (93%, P<0.01). Furthermore, maternal genome independently (M. semimembranosus, 88%, P<0.0001) or in combination (M. supraspinatus, 82%; M. longissimus dorsi, 93%; M. quadriceps femoris, 86%) with nested maternal weight effect (5–6%, P<0.05), was the predominant source of variation for absolute muscle weights. Effects of paternal genome on muscle mass decreased from thoracic to pelvic limb and accounted for all (M. supraspinatus, 97%, P<0.0001) or most (M. longissimus dorsi, 69%, P<0.0001; M. quadriceps femoris, 54%, P<0.001) genetic variation in relative weights. An interaction between maternal and paternal genomes (P<0.01) and effects of maternal weight (P<0.05) on expression of H19, a master regulator of an imprinted gene network, and negative correlations between H19 expression and fetal muscle mass (P<0.001), suggested imprinted genes and miRNA interference as mechanisms for differential effects of maternal and paternal genomes on fetal muscle.Ruidong Xiang, Mani Ghanipoor-Samami, William H. Johns, Tanja Eindorf, David L. Rutley, Zbigniew A. Kruk, Carolyn J. Fitzsimmons, Dana A. Thomsen, Claire T. Roberts, Brian M. Burns, Gail I. Anderson, Paul L. Greenwood, Stefan Hiendlede

Data Ghanipoor-Samami and Javadmanesh et al. for PLoS ONE 2018.xlsx

Real time qPCR data<br><br><br

Tissue-specific expression profiles of IGF system binding proteins in bovine pre- and postnatal developmental stages.

<p>Abundances of transcripts for <i>IGFBP1</i>, <i>IGFBP2</i>, <i>IGFBP3</i>, <i>IGFBP4</i>, <i>IGFBP5</i>, <i>IGFBP6</i>, <i>IGFBP7</i> and <i>IGFBP8</i> were measured in tissues of Day 48 embryos, Day 153 fetuses and 12–14 month-old juveniles. Placental samples were obtained from Day 48 embryos, Day 153 fetuses and term calves born by Caesarean section (C-section) at Day 277/278 of gestation. Means and standard deviations of means for each transcript and tissue were calculated based on triplicate measures of pooled cDNA comprising up to 60 embryonic cDNA samples, 73 fetal cDNA samples, 5 placental cDNA samples of C-section calves and 17 juvenile cDNA samples. Transcript abundances were calculated by the standard curve method and expressed in relative units, and are presented in logarithmic scale. ‘m’ denotes missing tissue such as kidney that is not yet present in embryos, where transcript abundances could not be determined.</p

Tissue-specific expression profiles of IGF system ligands in bovine pre- and postnatal developmental stages.

<p>Abundances of global <i>IGF1</i> transcript and splice variants <i>IGF1</i> class 1 and 2, global <i>IGF2</i> transcript and promoter and splice variant-specific <i>IGF2</i>-P0, <i>IGF2</i>-P1e2, <i>IGF2</i>-P1e3, <i>IGF2</i>-P2e4, <i>IGF2</i>-P2e5, <i>IGF2</i>-P3 and <i>IGF2</i>-P4 transcript were measured in tissues of Day 48 embryos, Day 153 fetuses and 12–14 month-old juveniles. Placental samples were obtained from Day 48 embryos, Day 153 fetuses and term calves born by Caesarean section (C-section) at Day 277/278 of gestation. Means and standard deviations of means for each transcript and tissue were calculated based on triplicate measures of pooled cDNA comprising up to 60 embryonic cDNA samples, 73 fetal cDNA samples, 5 placental cDNA samples of C-section calves and 17 juvenile cDNA samples. Transcript abundances were calculated by the standard curve method and expressed in relative units, and are presented in logarithmic scale. ‘m’ denotes missing tissue such as kidney that is not yet present in embryos, where transcript abundances could not be determined.</p

Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants.

<p>The exon-intron structure of bovine insulin/insulin-like growth factor 2 (<i>INS/IGF2</i>, GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters (<i>IGF2</i>-P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human <i>IGF2</i>-P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine <i>IGF2</i>-P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 (<i>IGF2</i>-P1e2) and exon 3 (<i>IGF2</i>-P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for <i>IGF2</i>-P1e2) and exons 3 and 8 (for <i>IGF2</i>-P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since <i>IGF2</i>-transcript P1 exon 2 is part of the first exonic region of transcript <i>IGF2</i>-P0, and exon 3 is present in both <i>IGF2-</i>P0 and <i>IGF2-</i>P1 transcripts, the <i>IGF2</i>-P1e2 and -P1e3 amplicons could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from <i>IGF2-</i>P2 promoter which comprise leading exon 4 (<i>IGF2</i>-P2e4) or leading exons 4 and 5 (<i>IGF2</i>-P2e5) as well as the protein coding exons. The forward primer for <i>IGF2</i>-P2e4 was designed to span the junction of exons 4 and 8, and for <i>IGF2</i>-P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify <i>IGF2</i>-P3 and <i>IGF2</i>-P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200466#pone.0200466.s003" target="_blank">S3 Table</a></b>.</p

Tissue-specific expression profiles of IGF system receptors in bovine pre- and postnatal developmental stages.

<p>Abundances of global <i>IR</i> transcript and splice variants <i>IR</i>-A and <i>IR</i>-B, <i>IGF1R</i> and <i>IGF2R</i> were measured in tissues of Day 48 embryos, Day 153 fetuses and 12–14 month-old juveniles. Placental samples were obtained from Day 48 embryos, Day 153 fetuses and term calves born by Caesarean section (C-section) at Day 277/278 of gestation. Means and standard deviations of means for each transcript and tissue were calculated based on triplicate measures of pooled cDNA comprising up to 60 embryonic cDNA samples, 73 fetal cDNA samples, 5 placental cDNA samples of C-section calves and 17 juvenile cDNA samples. Transcript abundances were calculated by the standard curve method and expressed in relative units, and are presented in logarithmic scale. ‘m’ denotes missing tissue such as kidney that is not yet present in embryos, where transcript abundances could not be determined.</p

Tissue-specific expression profiles of long non-coding RNAs associated with the IGF system in bovine pre- and postnatal developmental stages.

<p>Abundances of <i>H19</i> and <i>AIRN</i> transcript were measured in tissues of Day 48 embryos, Day 153 fetuses and 12–14 month-old juveniles. Placental samples were obtained from Day 48 embryos, Day 153 fetuses and term calves born by Caesarean section (C-section) at Day 277/278 of gestation. Means and standard deviations of means for each transcript and tissue were calculated based on triplicate measures of pooled cDNA comprising up to 60 embryonic cDNA samples, 73 fetal cDNA samples, 5 placental cDNA samples of C-section calves and 17 juvenile cDNA samples. Transcript abundances were calculated by the standard curve method and expressed in relative units, and are presented in logarithmic scale. ‘m’ denotes missing tissue such as kidney that is not yet present in embryos, where transcript abundances could not be determined.</p

Relative contribution of promoter and splice variant-specific <i>IGF2</i> transcripts to global <i>IGF2</i> transcript abundance in fetal tissues and placenta.

<p><i>IGF2</i>-P0, <i>IGF2</i>-P3 and <i>IGF2</i>-P4 are percent transcript abundance derived from P0, P3 and P4 promoters, respectively. Splice variants of promoter P2 transcript are <i>IGF2</i>-P2e4 with untranslated leader exon 4 and <i>IGF2</i>-P2e5 with untranslated leader exons 4 and 5. Estimated means are from 73 fetal cDNA samples per tissue and 95% confidence intervals are detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200466#pone.0200466.s009" target="_blank">S9 Table</a></b>. Transcript abundances were calculated by the standard curve method, normalized with reference genes and expressed in relative units. The relative contribution of each promoter-specific transcript to global <i>IGF2</i> transcript abundance was calculated by Johnson’s Relative Weight procedure.</p