IGF2R (A & C) and IGF1R (E & G) protein in the bilaminar (BYS; A & E) and trilaminar (TYS; C & G) yolk sac at day 25

Appropriate IgG antibody negative controls for IGF2R and IGF1R antibodies are shown (B, D, F, & H). IGF2R staining was strongest in the trophoblast (Tr), with lighter staining in the yolk sac endoderm (En) and little or no staining in the mesenchyme (Me) surrounding vitelline vessels (Vv). IGF2R staining was localised in the cytoplasm and cell membrane. IGF1R stained all yolk sac cell types. Both antibodies also stained the uterine epithelium and some stromal cells in the endometrium (Endo). Scale bar is shown at the bottom left of each image.<p><b>Copyright information:</b></p><p>Taken from "Expression and protein localisation of in the marsupial placenta"</p><p>http://www.biomedcentral.com/1471-213X/8/17</p><p>BMC Developmental Biology 2008;8():17-17.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2276195.</p><p></p

Intensity of staining to IGF2 and IGF2R antibodies in the yolk sac trophoblast during the final third of gestation (A)

The intensity of staining was measured subjectively as described in the experimental procedures. The bilaminar (BYS) (shaded bars) and trilaminar yolk sac (TYS) (open bars) of matched samples were assessed independently. Samples were grouped into days 19–21 (n = 4), 22–24 (n = 5), and 25–26 (n = 4). Staining intensity to the IGF2 antibody was consistently stronger in the TYS, especially at days 25–26. Staining by the IGF2 antibody was notably lighter at days 19–21 than later stages (days 22 to 26). Staining by the IGF2R antibody did not differ notably between the bilaminar and trilaminar yolk sac, nor were there marked differences corresponding to developmental stage. Intensity of staining by IGF2 and IGF2R antibodies in yolk sac cells (B). Staining intensity was noticeably higher in the trophoblast (Tr) (stippled bars) than in the yolk sac endoderm (En) (striped bars) of the bilaminar (BYS) and trilaminar (TYS) for IGF2, but not IGF2R. The staining intensity represents the average for fetal stages between days 19 and 26 (n = 13). Light background staining with the IgG antibody negative control in the yolk sac endoderm was taken into account when judging the staining intensity of the yolk sac endoderm for IGF2 and IGF2R antibodies.<p><b>Copyright information:</b></p><p>Taken from "Expression and protein localisation of in the marsupial placenta"</p><p>http://www.biomedcentral.com/1471-213X/8/17</p><p>BMC Developmental Biology 2008;8():17-17.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2276195.</p><p></p

IGF2 protein in the bilaminar (A and B) and trilaminar (D and E) yolk sac at day 25–26 of gestation

IgG negative controls for the bilaminar (C) and trilaminar (F) yolk sac. Staining was strongest in the trophoblast (Tr), but some endodermal (En) cells of the yolk sac placenta also stained. Staining was generally stronger in the trilaminar yolk sac and in both portions of the yolk sac staining increased later in gestation (see Fig. 2). Strong staining can be seen in the uterine epithelium (Ep) immediately adjacent to the bilaminar (avascular) yolk sac placenta. There was little staining in the mesenchyme (Me) and endothelium of large vitelline vessels (Vv) of the trilaminar (vascular) yolk sac placenta. Some stromal (St) and endothelial cells (Ed) in the maternal endometrium also stained (G and IgG negative H), as did the uterine epithelium (Ep) and some endometrial glands (Gl). Scale bar is shown at the bottom left of each image.<p><b>Copyright information:</b></p><p>Taken from "Expression and protein localisation of in the marsupial placenta"</p><p>http://www.biomedcentral.com/1471-213X/8/17</p><p>BMC Developmental Biology 2008;8():17-17.</p><p>Published online 20 Feb 2008</p><p>PMCID:PMC2276195.</p><p></p

<i>IGF2</i> mRNA expression from each promoter (P1, P2 and P3) relative to a reference gene in various wallaby tissues.

<p>(<b>A</b>) <i>IGF2</i> mRNA expression relative to <i>GAPDH</i> in the Liver, (B) <i>IGF2</i> mRNA expression relative to <i>GAPDH</i> in the brain, (C) <i>IGF2</i> mRNA expression relative to <i>B-ACTIN</i> in the mammary gland, (D) <i>IGF2</i> mRNA expression relative to <i>B-ACTIN</i> in the bilaminar yolk sac and (E) <i>IGF2</i> mRNA expression relative to 18S in the trilaminar yolk Sac. (A, B) Expression from P2 in the pouch young (PY) is significantly higher than expression from P1 and P3 (* P<0.05) in the liver (A) and brain (B). Except for P1 in the liver, <i>IGF2</i> is expressed at a higher level in the pouch young then in the adult (<i>a</i>, P<0.01) in both the liver and the brain (if adult hypothalamus expression is representative of total adult brain expression). (C, D, E) P2-<i>IGF2</i> is also the predominantly expressed transcript in the mammary gland (* P<0.05) (C) and in the bilaminar (D) and trilaminar (E) placenta (a,b,c have significantly different means, P<0.01). There was no difference in the expression levels of any of the transcripts between non-lactating and lactating mammary glands (C).</p

<i>IGF2</i> mRNA expression in the adult tammar.

<p>RT-PCR expression profile after 35 cycles of each <i>IGF2</i> mRNA transcript in a range of adult tammar tissues compared to <i>GAPDH</i>. The expression was strongest from P2, with expression of all three transcripts in the brain, eye, tongue, liver and mammary gland. No expression was detected in the No Template Control (NTC).</p

Genomic structure of tammar IGF2 including 3 TSS and comparison with mouse, human and opossum genomes.

<p>Opossum (clone XX-223O16 ch5∶102112-82113), human (ch11∶2156597-2176597) and mouse (ch7∶149841559-149861559), <i>IGF2</i> base genomes were aligned to tammar <i>IGF2</i> (clone MEKBa-346C2∶62540-82539). VISTA pairwise alignments using a 100 bp sliding window was performed between each species and tammar. Pink peaks represent areas of conservation of 70% over a minimum of 100 bp between tammar and opossum and 55% over a minimum of 50 bp between tammar and human or mouse. A dotted line indicates a region with 70% conservation between P1 and P2 in both marsupials and eutherians. A schematic of each <i>IGF2</i> gene is located above the VISTA plot to show relative location of the non-coding exons (open box), including the non-coding exons transcribed with the P0 TSS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041690#pone.0041690-Monk1" target="_blank">[12]</a>, and coding exons (black box). Location of CpG sites are represented by vertical black bars below the VISTA plot.</p

Allelic expression from the three promoters in various adult and pouch young tissues.

<p>Direct sequencing of two single nucleotide polymorphisms (snp1 and snp2) was used to determine the allelic expression of each of the <i>IGF2</i> transcripts in adult and pouch young (PY) tissues. In a 12 day old female pouch young monoallelic expression was detected from all 3 promoters in the brain and liver, except P1 in the liver was expressed from both alleles. In a 72 day old male pouch young, biased to biallelic expression was detected in the brain while the liver expression was similar to the day 12 pouch young. In the adult, both alleles were detected from all three TSS in two liver samples and monoallelic expression was detected in three mammary gland (MG) samples. Placental samples had biallelic expression in the bilaminar region (BYS) in all samples tested for P1 and P2 and 2 of the 3 samples tested for P3. Biased to monoalleleic expression was observed in the trilaminar region (TYS) in all samples tested for P1, 3 of the 4 samples for P2, and 2 of the 3 samples for P3.</p

A box plot showing the percent of total sequence masked by SINEs (dark blue), LINEs (light blue), LTR elements (purple), DNA elements (pink), simple repeats (teal), and low complexity regions (pale green) in intron 1, 1b, 9, 10, and 14 of (A)

The percent sequence coverage for each element over the entire region from is shown by open circles. There was significantly less sequence masked by LTR and DNA elements in tammar compared to both mouse and human, while there was significantly more sequence occupied by low complexity regions (*). There was significantly less sequence occupied by LINEs in mouse compared to human (**). There was a large range in the sequence covered by LINEs in all species, but particularly in human and mouse. The percent sequence occupied by different types of repetitive elements in the region from , in all of , and in introns 9, 10, 1, 1b, and 14 assessed separately (B). The relative percentage of sequence occupied by LINEs is noticeably more than the percentage occupied by LINEs in any other intron from KCNQ1 and this increase was significant for mouse (*). No other significant differences in the relative amounts of sequence covered by different types of elements was seen between regions within species. However, there was also a noticeable increase in the relative amount of simple repeats in intron 9 of tammar compared to other introns in the tammar.<p><b>Copyright information:</b></p><p>Taken from "Evolution of the CDKN1C-KCNQ1 imprinted domain"</p><p>http://www.biomedcentral.com/1471-2148/8/163</p><p>BMC Evolutionary Biology 2008;8():163-163.</p><p>Published online 29 May 2008</p><p>PMCID:PMC2427030.</p><p></p

Methylation at the tammar <i>IGF2</i> transcription start sites.

<p>Location of CpG sites are represented by black bars below the schematic of the tammar <i>IGF2</i> gene. Bisulphite sequencing of the <i>IGF2</i> CpG islands (CGI) at each of the transcription start sites (CGI1, CGI2 and CGI3) and at exon 3 (CGI4) in the pouch young and adult liver, in the mammary gland, whole yolk sac placenta and in the bilaminar and trilaminar regions of the yolk sac placenta. Black circles represent a methylated CpG site and open circles represent an unmethylated CpG site. Each row represents the methylation pattern of a separate DNA fragment from the same sample. Only CGI3 and CGI4 were significantly methylated in all tissues. CGI4 appears to be differentially methylated in the placenta. However, sequence polymorphisms that might distinguish parental copies of CGI4 were not found among the available stocks of tammar placental samples.</p

Identifying tammar <i>IGF2</i> transcription start sites.

<p>5′-RACE amplification was performed on (A) liver and (B) placenta RNA samples. (A) Three bands first identified in the liver (black arrows) were visible in both tissue types. (B) Additional exon 1B transcription start sites (TSS) were identified in the placenta. (C) Aligning the cloned products to the gDNA sequence identified the location of each of the non-coding exons 1A, 1B and 1C (white boxes) in relation to the coding exons 2, 3 and 4 (black boxes) followed by an untranslated region of unknown length (dashed box). TSSs are indicated by turned arrows. Two single nucleotide polymorphisms (black triangles) identified in the untranslated region were used for imprint analysis (snp1 and snp2). Diagram is not to scale.</p