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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, placental and heart growth following maternal transmission of Igf2r+m/HUp.(A) Total body weights at E14.5 (WT n = 4, Igf2rHUm/+p n = 3), E17.5 (WT n = 12, Igf2rHUm/+p n = 6) and E18.5 (WT n = 10, Igf2rHUm/+p n = 9) showing gain in weight of Igf2rHUm/+p. (B) Placental weights at E14.5 (WT n = 4, Igf2rHUm/+p n = 3) E17.5 (WT n = 12, Igf2rHUm/+p n = 6) and E18.5 (WT n = 10, Igf2rHUm/+p n = 9). At E14.5 P = 0.0148, at E17.5 P = NS, at E18.5 P = 0.0037. (C) Heart weights at E18.5 (WT n = 11, Igf2rHUm/+p n = 6) showing gain in weight of hearts of Igf2rHUm/+p. *P<0.05, **P<0.01, ***P<0.001. Whiskers ± standard deviation. Paired t-test.
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pone-0057270-g003: Embryonic, placental and heart growth following maternal transmission of Igf2r+m/HUp.(A) Total body weights at E14.5 (WT n = 4, Igf2rHUm/+p n = 3), E17.5 (WT n = 12, Igf2rHUm/+p n = 6) and E18.5 (WT n = 10, Igf2rHUm/+p n = 9) showing gain in weight of Igf2rHUm/+p. (B) Placental weights at E14.5 (WT n = 4, Igf2rHUm/+p n = 3) E17.5 (WT n = 12, Igf2rHUm/+p n = 6) and E18.5 (WT n = 10, Igf2rHUm/+p n = 9). At E14.5 P = 0.0148, at E17.5 P = NS, at E18.5 P = 0.0037. (C) Heart weights at E18.5 (WT n = 11, Igf2rHUm/+p n = 6) showing gain in weight of hearts of Igf2rHUm/+p. *P<0.05, **P<0.01, ***P<0.001. Whiskers ± standard deviation. Paired t-test.

Mentions: Previously reported germline Igf2r loss of function identified a placental and embryonic overgrowth (120–135%) phenotype starting around E12.5 [12], [13], [14]. We analysed embryonic growth following timed matings using maternal transmission of the humanised allele. By E14.5, Igf2rHUm/+p embryos, placentae and hearts were all significantly heavier than littermate controls with the relative overgrowth in the range of 105–120% at each time point (Fig. 3A, B and C). By E18.5, the ratio of heart to overall body weight was approximately 0.0073 in Igf2rHUm/+p embryos compared to 0.006 in Igf2r+m/+p littermate controls (not shown, NS), suggesting that the heart growth phenotype was not grossly disproportionate.


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, placental and heart growth following maternal transmission of Igf2r+m/HUp.(A) Total body weights at E14.5 (WT n = 4, Igf2rHUm/+p n = 3), E17.5 (WT n = 12, Igf2rHUm/+p n = 6) and E18.5 (WT n = 10, Igf2rHUm/+p n = 9) showing gain in weight of Igf2rHUm/+p. (B) Placental weights at E14.5 (WT n = 4, Igf2rHUm/+p n = 3) E17.5 (WT n = 12, Igf2rHUm/+p n = 6) and E18.5 (WT n = 10, Igf2rHUm/+p n = 9). At E14.5 P = 0.0148, at E17.5 P = NS, at E18.5 P = 0.0037. (C) Heart weights at E18.5 (WT n = 11, Igf2rHUm/+p n = 6) showing gain in weight of hearts of Igf2rHUm/+p. *P<0.05, **P<0.01, ***P<0.001. Whiskers ± standard deviation. Paired t-test.
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Related In: Results  -  Collection

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pone-0057270-g003: Embryonic, placental and heart growth following maternal transmission of Igf2r+m/HUp.(A) Total body weights at E14.5 (WT n = 4, Igf2rHUm/+p n = 3), E17.5 (WT n = 12, Igf2rHUm/+p n = 6) and E18.5 (WT n = 10, Igf2rHUm/+p n = 9) showing gain in weight of Igf2rHUm/+p. (B) Placental weights at E14.5 (WT n = 4, Igf2rHUm/+p n = 3) E17.5 (WT n = 12, Igf2rHUm/+p n = 6) and E18.5 (WT n = 10, Igf2rHUm/+p n = 9). At E14.5 P = 0.0148, at E17.5 P = NS, at E18.5 P = 0.0037. (C) Heart weights at E18.5 (WT n = 11, Igf2rHUm/+p n = 6) showing gain in weight of hearts of Igf2rHUm/+p. *P<0.05, **P<0.01, ***P<0.001. Whiskers ± standard deviation. Paired t-test.
Mentions: Previously reported germline Igf2r loss of function identified a placental and embryonic overgrowth (120–135%) phenotype starting around E12.5 [12], [13], [14]. We analysed embryonic growth following timed matings using maternal transmission of the humanised allele. By E14.5, Igf2rHUm/+p embryos, placentae and hearts were all significantly heavier than littermate controls with the relative overgrowth in the range of 105–120% at each time point (Fig. 3A, B and C). By E18.5, the ratio of heart to overall body weight was approximately 0.0073 in Igf2rHUm/+p embryos compared to 0.006 in Igf2r+m/+p littermate controls (not shown, NS), suggesting that the heart growth phenotype was not grossly disproportionate.

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