Limits...
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.

Show MeSH

Related in: MedlinePlus

Embryonic heart IGF2R protein levels following maternal transmission of Igf2r+m/HUp(A) Two wild-type and a surviving Igf2rHUm/+p littermate at post-natal day 7. (B) Q-RT-PCR of Igf2r mRNA expression using mouse and human allele specific primers. Results are shown for progeny of heterozygote inter-cross to generate homozygote humanised knock-in alleles, compared to heterozygote progeny derived from maternal transmission. (*P<0.05, t-test, **P<0.01, 1 way ANOVA, Dunn’s post-test). Note, origin of the humanised allele in the heterozygote progeny was detected by exon 2–3 human and mouse specific RT-PCR. (C) Upper panel; H and E stained example of whole mouse embryo (E18.5) cross-sections below the level of the tracheal bifurcation showing dilated and enlarged heart in Hum. (Bar 1mm). Lower panels; higher power H and E images (Bar 0.25mm) showing regions visualised by immuno-fluoresence labelling of IGF2R (green), LAMP1 (red, for endosomal compartment localisation) and DAPI (nuclear DNA). Relative IGF2R levels appear reduced including the pericardial regions. (Bar 0.1mm). (D)Western blots of IGF2R in mouse embryos at day E18.5 in the lower limb and heart (n = 2–4). IGF2R levels were normalised to α-tubulin. Levels of IGF2R were reduced in limbs and hearts of embryos with a humanised maternal allele (HUm) derived from a heterozygous inter-cross (one way ANOVA with Tukey’s post-test, **P<0.005, *P<0.05), Levels of protein following maternal allele transmission alone also appear reduced but were not significant (n = 2). (E) Adult body weights (measured at 10 weeks) of surviving offspring resulting from maternal transmission of the humanised allele (Igf2r+m/HUp females crossed to wild-type males). Wild-type males n = 12 Igf2rHUm/+p males n = 1 wild-type females n = 5 Igf2rHUm/+p females n = 2. (C) Adult uteri from wild-type littermate and Igf2rHum/+p surviving female showing dilated, enlarged and fluid filled uterine horns (scale bar = 1cm).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585325&req=5

pone-0057270-g005: Embryonic heart IGF2R protein levels following maternal transmission of Igf2r+m/HUp(A) Two wild-type and a surviving Igf2rHUm/+p littermate at post-natal day 7. (B) Q-RT-PCR of Igf2r mRNA expression using mouse and human allele specific primers. Results are shown for progeny of heterozygote inter-cross to generate homozygote humanised knock-in alleles, compared to heterozygote progeny derived from maternal transmission. (*P<0.05, t-test, **P<0.01, 1 way ANOVA, Dunn’s post-test). Note, origin of the humanised allele in the heterozygote progeny was detected by exon 2–3 human and mouse specific RT-PCR. (C) Upper panel; H and E stained example of whole mouse embryo (E18.5) cross-sections below the level of the tracheal bifurcation showing dilated and enlarged heart in Hum. (Bar 1mm). Lower panels; higher power H and E images (Bar 0.25mm) showing regions visualised by immuno-fluoresence labelling of IGF2R (green), LAMP1 (red, for endosomal compartment localisation) and DAPI (nuclear DNA). Relative IGF2R levels appear reduced including the pericardial regions. (Bar 0.1mm). (D)Western blots of IGF2R in mouse embryos at day E18.5 in the lower limb and heart (n = 2–4). IGF2R levels were normalised to α-tubulin. Levels of IGF2R were reduced in limbs and hearts of embryos with a humanised maternal allele (HUm) derived from a heterozygous inter-cross (one way ANOVA with Tukey’s post-test, **P<0.005, *P<0.05), Levels of protein following maternal allele transmission alone also appear reduced but were not significant (n = 2). (E) Adult body weights (measured at 10 weeks) of surviving offspring resulting from maternal transmission of the humanised allele (Igf2r+m/HUp females crossed to wild-type males). Wild-type males n = 12 Igf2rHUm/+p males n = 1 wild-type females n = 5 Igf2rHUm/+p females n = 2. (C) Adult uteri from wild-type littermate and Igf2rHum/+p surviving female showing dilated, enlarged and fluid filled uterine horns (scale bar = 1cm).

Mentions: Combining the maternal transmitted allele with disruption of the paternal expressed allele of Igf2 (Igf2+m/−p, [28]) resulted in rescue of the peri-natal lethality (Table 1). The surviving pups exhibited post-natal growth identical to Igf2+m/−p, i.e. similar kinetics to wild-type but at 60% of the weight (Fig. 4A). Combining the maternal transmitted allele with disruption of the intron 2 region of the paternal allele of Igf2r (R2Δ+m/−p, [7]) also resulted in rescue of the expected peri-natal lethality (Table 1). The surviving pups also exhibited similar post-natal growth to R2Δ+m/−p (Fig. 4B). These results indicate that the overgrowth phenotype and perinatal lethality were related to the supply of IGF2, and that expression of the murine paternal allele following imprinting disruption was sufficient to rescue the humanised maternal allele. Moreover, in the combined alleles (R2Δ+m/−p, Igf2rHUm/+p) disruption of Airn in intron 2 would have occurred in both alleles, indicating that the humanised allele was probably functioning as a hypomorphic allele independent of Airn. However, when we generated mice homozygous for the humanised allele (Igf2rHUmHUp), we surprisingly observed complete rescue of the post-natal over-growth and mortality phenotype (Table 1). From these latter data, we suggest that the function of a single humanised allele during spermatogenesis may have resulted in an overall reduction in expression of the Airn transcript, potentially resulting in ‘trans’ effects on the paternal transmitted wild-type allele, up-regulating the expression of paternal Igf2r allele (bi-allelic) expression, and therefore compensating for the hypomorphic function of the humanised maternal allele. Overall, there was a partial lethality phenotype associated with over-growth in Igf2rHUm/+p, suggesting that the humanised allele must be less than 50% of the normal wild-type function of a maternal expressed mouse allele of Igf2r (Fig. 5D).


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 IGF2R protein levels following maternal transmission of Igf2r+m/HUp(A) Two wild-type and a surviving Igf2rHUm/+p littermate at post-natal day 7. (B) Q-RT-PCR of Igf2r mRNA expression using mouse and human allele specific primers. Results are shown for progeny of heterozygote inter-cross to generate homozygote humanised knock-in alleles, compared to heterozygote progeny derived from maternal transmission. (*P<0.05, t-test, **P<0.01, 1 way ANOVA, Dunn’s post-test). Note, origin of the humanised allele in the heterozygote progeny was detected by exon 2–3 human and mouse specific RT-PCR. (C) Upper panel; H and E stained example of whole mouse embryo (E18.5) cross-sections below the level of the tracheal bifurcation showing dilated and enlarged heart in Hum. (Bar 1mm). Lower panels; higher power H and E images (Bar 0.25mm) showing regions visualised by immuno-fluoresence labelling of IGF2R (green), LAMP1 (red, for endosomal compartment localisation) and DAPI (nuclear DNA). Relative IGF2R levels appear reduced including the pericardial regions. (Bar 0.1mm). (D)Western blots of IGF2R in mouse embryos at day E18.5 in the lower limb and heart (n = 2–4). IGF2R levels were normalised to α-tubulin. Levels of IGF2R were reduced in limbs and hearts of embryos with a humanised maternal allele (HUm) derived from a heterozygous inter-cross (one way ANOVA with Tukey’s post-test, **P<0.005, *P<0.05), Levels of protein following maternal allele transmission alone also appear reduced but were not significant (n = 2). (E) Adult body weights (measured at 10 weeks) of surviving offspring resulting from maternal transmission of the humanised allele (Igf2r+m/HUp females crossed to wild-type males). Wild-type males n = 12 Igf2rHUm/+p males n = 1 wild-type females n = 5 Igf2rHUm/+p females n = 2. (C) Adult uteri from wild-type littermate and Igf2rHum/+p surviving female showing dilated, enlarged and fluid filled uterine horns (scale bar = 1cm).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585325&req=5

pone-0057270-g005: Embryonic heart IGF2R protein levels following maternal transmission of Igf2r+m/HUp(A) Two wild-type and a surviving Igf2rHUm/+p littermate at post-natal day 7. (B) Q-RT-PCR of Igf2r mRNA expression using mouse and human allele specific primers. Results are shown for progeny of heterozygote inter-cross to generate homozygote humanised knock-in alleles, compared to heterozygote progeny derived from maternal transmission. (*P<0.05, t-test, **P<0.01, 1 way ANOVA, Dunn’s post-test). Note, origin of the humanised allele in the heterozygote progeny was detected by exon 2–3 human and mouse specific RT-PCR. (C) Upper panel; H and E stained example of whole mouse embryo (E18.5) cross-sections below the level of the tracheal bifurcation showing dilated and enlarged heart in Hum. (Bar 1mm). Lower panels; higher power H and E images (Bar 0.25mm) showing regions visualised by immuno-fluoresence labelling of IGF2R (green), LAMP1 (red, for endosomal compartment localisation) and DAPI (nuclear DNA). Relative IGF2R levels appear reduced including the pericardial regions. (Bar 0.1mm). (D)Western blots of IGF2R in mouse embryos at day E18.5 in the lower limb and heart (n = 2–4). IGF2R levels were normalised to α-tubulin. Levels of IGF2R were reduced in limbs and hearts of embryos with a humanised maternal allele (HUm) derived from a heterozygous inter-cross (one way ANOVA with Tukey’s post-test, **P<0.005, *P<0.05), Levels of protein following maternal allele transmission alone also appear reduced but were not significant (n = 2). (E) Adult body weights (measured at 10 weeks) of surviving offspring resulting from maternal transmission of the humanised allele (Igf2r+m/HUp females crossed to wild-type males). Wild-type males n = 12 Igf2rHUm/+p males n = 1 wild-type females n = 5 Igf2rHUm/+p females n = 2. (C) Adult uteri from wild-type littermate and Igf2rHum/+p surviving female showing dilated, enlarged and fluid filled uterine horns (scale bar = 1cm).
Mentions: Combining the maternal transmitted allele with disruption of the paternal expressed allele of Igf2 (Igf2+m/−p, [28]) resulted in rescue of the peri-natal lethality (Table 1). The surviving pups exhibited post-natal growth identical to Igf2+m/−p, i.e. similar kinetics to wild-type but at 60% of the weight (Fig. 4A). Combining the maternal transmitted allele with disruption of the intron 2 region of the paternal allele of Igf2r (R2Δ+m/−p, [7]) also resulted in rescue of the expected peri-natal lethality (Table 1). The surviving pups also exhibited similar post-natal growth to R2Δ+m/−p (Fig. 4B). These results indicate that the overgrowth phenotype and perinatal lethality were related to the supply of IGF2, and that expression of the murine paternal allele following imprinting disruption was sufficient to rescue the humanised maternal allele. Moreover, in the combined alleles (R2Δ+m/−p, Igf2rHUm/+p) disruption of Airn in intron 2 would have occurred in both alleles, indicating that the humanised allele was probably functioning as a hypomorphic allele independent of Airn. However, when we generated mice homozygous for the humanised allele (Igf2rHUmHUp), we surprisingly observed complete rescue of the post-natal over-growth and mortality phenotype (Table 1). From these latter data, we suggest that the function of a single humanised allele during spermatogenesis may have resulted in an overall reduction in expression of the Airn transcript, potentially resulting in ‘trans’ effects on the paternal transmitted wild-type allele, up-regulating the expression of paternal Igf2r allele (bi-allelic) expression, and therefore compensating for the hypomorphic function of the humanised maternal allele. Overall, there was a partial lethality phenotype associated with over-growth in Igf2rHUm/+p, suggesting that the humanised allele must be less than 50% of the normal wild-type function of a maternal expressed mouse allele of Igf2r (Fig. 5D).

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