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Maternal transmission of a humanised Igf2r allele results in an Igf2 dependent hypomorphic and non-viable growth phenotype.

Hughes J, Frago S, Bühnemann C, Carter EJ, Hassan AB - PLoS ONE (2013)

Bottom Line: Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality.In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r.This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Tumour Growth Group, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The cation independent mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) functions in the transportation and regulation of insulin-like growth factor 2 (IGF2) and mannose 6-phosphate modified proteins. The relative and specific titration of IGF2 by high affinity binding of IGF2R represents a mechanism that supports the parental conflict theory of genomic imprinting. Imprinting of Igf2 (paternal allele expressed) and Igf2r (maternal allele expressed) arose to regulate the relative supply of both proteins. Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality. In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r. Here we show that the knock-in construct resulted in over-growth when the humanised Igf2r allele was maternally transmitted, a phenotype that was rescued by either paternal transmission of the humanised allele, expression of a wild-type paternal allele or loss of function of Igf2. We also show that expression of IGF2R protein was reduced to less than 50% overall in tissues previously known to be Igf2 growth dependent. This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele. The phenotype following maternal transmission of the humanised allele resulted in overgrowth of the embryo, heart and placenta with partial peri-natal lethality, suggesting that further generation of hypomorphic Igf2r alleles are likely to be at the borderline of maintaining Igf2 dependent viability.

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Embryonic heart Cathepsin L protein levels following maternal transmission of Igf2r+m/HUp(A) Western blots of Cathepsin L in mouse embryos at day E18.5. (B) Densitometry of protein levels normalised to α -tubulin loading control. Levels of cathepsin L were reduced in hearts of embryos with a humanised maternal allele (HUm) (P<0.05).
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pone-0057270-g006: Embryonic heart Cathepsin L protein levels following maternal transmission of Igf2r+m/HUp(A) Western blots of Cathepsin L in mouse embryos at day E18.5. (B) Densitometry of protein levels normalised to α -tubulin loading control. Levels of cathepsin L were reduced in hearts of embryos with a humanised maternal allele (HUm) (P<0.05).

Mentions: In order to investigate the cause of the reduced function of the humanised allele, we next performed quantitative RT-PCR to quantify gene expression (Fig 5B). Maternal transmission of the humanised allele following breeding with a wild-type male resulted in reduced mouse transcript levels compared to wild-type littermate controls. However, when transmitted by both females and males to generate litters containing homozygote humanised alleles, the relative mouse transcript levels appeared higher in all genotypes, supporting the potential for relaxation of paternal allele imprinting. The relative levels of the humanised alleles were higher in homozygotes as expected, but surprisingly, were expressed in addition with mouse transcripts (Fig. 5B). Analysis of heart tissue showed enlargement of the heart with an apparent increase in ventricular muscle (Fig. 5C). Morphometry of serial 5 µM sections of the hearts of E18.5 embryos stained with H&E revealed relatively dilated hearts with increased blood filled spaces within muscle (proportion of muscle to blood filled spaces, Igf2r+m/+p = 0.87, n = 3, Igf2rHUm/+p = 0.73, n = 4, p<0.01) and no evidence for hypertrophy based on the ratio of nuclei to heart muscle (ratio of nuclear to muscle area Igf2r+m/+p = 0.12 n = 3, Igf2rHUm/+p = 0.14 n = 4, NS). Immuno-localisation of IGF2R suggested reduced labelling throughout the heart, including the pericardium, but without gross change in IGF2R cytoplasmic distribution relative to an endosomal marker, LAMP1 (Fig. 5C). We next evaluated the levels of detectable IGF2R using western blots in the limbs and heart of embryos with heterozygote and homozygote humanised alleles. In Igf2rHUm/+p there was significantly less than 50% of the amount of IGF2R compared to WT control littermates, and close to 50% levels in homozygote Igf2rHUm/HUp mice, consistent with the genetic findings (Fig. 5D). These data suggest that the cause of the over-growth phenotype associated with humanisation of the murine Igf2r allele was the reduced IGF2R protein levels (approximately 30% of controls). We failed to detect increased levels of soluble IGF2 in either serum or heart, although increased low molecular weight forms of IGF2 were detected in embryonic limbs in Igf2rHUm/+p (not shown). We also screened a number of lysosomal proteases, and detected reduced levels of cathepsin L in heart muscle of Igf2rHUm/+p embryos, presumably because of the associated reduction in lysosomal mannose 6-phosphate related intra-cellular transport (Fig.6A and B).


Maternal transmission of a humanised Igf2r allele results in an Igf2 dependent hypomorphic and non-viable growth phenotype.

Hughes J, Frago S, Bühnemann C, Carter EJ, Hassan AB - PLoS ONE (2013)

Embryonic heart Cathepsin L protein levels following maternal transmission of Igf2r+m/HUp(A) Western blots of Cathepsin L in mouse embryos at day E18.5. (B) Densitometry of protein levels normalised to α -tubulin loading control. Levels of cathepsin L were reduced in hearts of embryos with a humanised maternal allele (HUm) (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585325&req=5

pone-0057270-g006: Embryonic heart Cathepsin L protein levels following maternal transmission of Igf2r+m/HUp(A) Western blots of Cathepsin L in mouse embryos at day E18.5. (B) Densitometry of protein levels normalised to α -tubulin loading control. Levels of cathepsin L were reduced in hearts of embryos with a humanised maternal allele (HUm) (P<0.05).
Mentions: In order to investigate the cause of the reduced function of the humanised allele, we next performed quantitative RT-PCR to quantify gene expression (Fig 5B). Maternal transmission of the humanised allele following breeding with a wild-type male resulted in reduced mouse transcript levels compared to wild-type littermate controls. However, when transmitted by both females and males to generate litters containing homozygote humanised alleles, the relative mouse transcript levels appeared higher in all genotypes, supporting the potential for relaxation of paternal allele imprinting. The relative levels of the humanised alleles were higher in homozygotes as expected, but surprisingly, were expressed in addition with mouse transcripts (Fig. 5B). Analysis of heart tissue showed enlargement of the heart with an apparent increase in ventricular muscle (Fig. 5C). Morphometry of serial 5 µM sections of the hearts of E18.5 embryos stained with H&E revealed relatively dilated hearts with increased blood filled spaces within muscle (proportion of muscle to blood filled spaces, Igf2r+m/+p = 0.87, n = 3, Igf2rHUm/+p = 0.73, n = 4, p<0.01) and no evidence for hypertrophy based on the ratio of nuclei to heart muscle (ratio of nuclear to muscle area Igf2r+m/+p = 0.12 n = 3, Igf2rHUm/+p = 0.14 n = 4, NS). Immuno-localisation of IGF2R suggested reduced labelling throughout the heart, including the pericardium, but without gross change in IGF2R cytoplasmic distribution relative to an endosomal marker, LAMP1 (Fig. 5C). We next evaluated the levels of detectable IGF2R using western blots in the limbs and heart of embryos with heterozygote and homozygote humanised alleles. In Igf2rHUm/+p there was significantly less than 50% of the amount of IGF2R compared to WT control littermates, and close to 50% levels in homozygote Igf2rHUm/HUp mice, consistent with the genetic findings (Fig. 5D). These data suggest that the cause of the over-growth phenotype associated with humanisation of the murine Igf2r allele was the reduced IGF2R protein levels (approximately 30% of controls). We failed to detect increased levels of soluble IGF2 in either serum or heart, although increased low molecular weight forms of IGF2 were detected in embryonic limbs in Igf2rHUm/+p (not shown). We also screened a number of lysosomal proteases, and detected reduced levels of cathepsin L in heart muscle of Igf2rHUm/+p embryos, presumably because of the associated reduction in lysosomal mannose 6-phosphate related intra-cellular transport (Fig.6A and B).

Bottom Line: Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality.In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r.This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Tumour Growth Group, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The cation independent mannose 6-phosphate/insulin-like growth factor 2 receptor (IGF2R) functions in the transportation and regulation of insulin-like growth factor 2 (IGF2) and mannose 6-phosphate modified proteins. The relative and specific titration of IGF2 by high affinity binding of IGF2R represents a mechanism that supports the parental conflict theory of genomic imprinting. Imprinting of Igf2 (paternal allele expressed) and Igf2r (maternal allele expressed) arose to regulate the relative supply of both proteins. Experiments in the mouse have established that loss of the maternal allele of Igf2r results in disproportionate growth and peri-natal lethality. In order to systematically investigate the consequences of loss of function and of hypomorphic alleles of Igf2r on growth functions, we introduced a conditional human IGF2R exon 3-48 cDNA into the intron 2 region of murine Igf2r. Here we show that the knock-in construct resulted in over-growth when the humanised Igf2r allele was maternally transmitted, a phenotype that was rescued by either paternal transmission of the humanised allele, expression of a wild-type paternal allele or loss of function of Igf2. We also show that expression of IGF2R protein was reduced to less than 50% overall in tissues previously known to be Igf2 growth dependent. This occurred despite the detection of mouse derived peptides, suggesting that trans-splicing of the knock-in human cDNA with the endogenous maternal mouse Igf2r allele. The phenotype following maternal transmission of the humanised allele resulted in overgrowth of the embryo, heart and placenta with partial peri-natal lethality, suggesting that further generation of hypomorphic Igf2r alleles are likely to be at the borderline of maintaining Igf2 dependent viability.

Show MeSH
Related in: MedlinePlus