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Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart.

Maddox DM, Manlapat A, Roon P, Prasad P, Ganapathy V, Smith SB - BMC Dev. Biol. (2003)

Bottom Line: Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking.Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy.These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, USA. oxmandm@yahoo.com

ABSTRACT

Background: Folate is essential for cellular proliferation and tissue regeneration. As mammalian cells cannot synthesize folates de novo, tightly regulated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support biosynthesis of purines, pyrimidines, and some amino acids (serine, methionine). Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking. We utilized in situ hybridization and immunolocalization to determine the developmental distribution of RFC message and protein, respectively.

Results: In the mouse, RFC transcripts and protein are expressed in the E10.0 placenta and yolk sac. In the E9.0 to E11.5 mouse embryo RFC is widely detectable, with intense signal localized to cell populations in the neural tube, craniofacial region, limb buds and heart. During early development, RFC is expressed throughout the eye, but by E12.5, RFC protein becomes localized to the retinal pigment epithelium (RPE).

Conclusions: Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy. The mechanism, however, by which folate supplementation ameliorates the occurrence of developmental defects is unclear. The present work demonstrates that RFC is present in placenta and yolk sac and provides the first evidence that it is expressed in the neural tube, craniofacial region, limb buds and heart during organogenesis. These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency.

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In situ hybridization and immunolocalization of RFC during early murine embryonic development. Panels A and C demonstrate expression of RFC mRNA in a section of uterus containing an E10.0 embryo. Panel E demonstrates the absence of background staining when a sense riboprobe for RFC is hybridized with placental sections as a negative control. To determine whether RFC protein may be localized to the same areas that are positive for RFC message during murine embryonic development, immunofluorescence was utilized in E10.0 frozen uterus sections. Panels B and D indeed verify that RFC protein is localized in a manner consistent with the expression pattern of RFC message. Specificity of the RFC antibody is demonstrated by lack of fluorescence in tissues incubated with RFC antibody that had been pre-incubated with blocking peptide (Panel F). L, labyrinth zone; NT, neural tube; JZ, junctional zone; TG, trophoblastic giant cells; YS, yolk sac. Scale bar represents 25 μm in A and E and 4 μm in B, C and D.
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Figure 1: In situ hybridization and immunolocalization of RFC during early murine embryonic development. Panels A and C demonstrate expression of RFC mRNA in a section of uterus containing an E10.0 embryo. Panel E demonstrates the absence of background staining when a sense riboprobe for RFC is hybridized with placental sections as a negative control. To determine whether RFC protein may be localized to the same areas that are positive for RFC message during murine embryonic development, immunofluorescence was utilized in E10.0 frozen uterus sections. Panels B and D indeed verify that RFC protein is localized in a manner consistent with the expression pattern of RFC message. Specificity of the RFC antibody is demonstrated by lack of fluorescence in tissues incubated with RFC antibody that had been pre-incubated with blocking peptide (Panel F). L, labyrinth zone; NT, neural tube; JZ, junctional zone; TG, trophoblastic giant cells; YS, yolk sac. Scale bar represents 25 μm in A and E and 4 μm in B, C and D.

Mentions: In situ hybridization analysis was performed on cryosections of mouse uterus obtained at embryonic day (E)10.0 of gestation. Figure 1A shows that RFC message is expressed in trophoblast cells of all layers of the placenta, with the expression most abundant in the trophoblast cells surrounding blood lacunae. RFC message was expressed also in the trophoblastic giant cells, labyrinth, junctional zone, yolk sac and within the neural tube of the developing embryo (Figure 1A and 1C). Specificity of the staining was verified by hybridization of placental sections with an RFC specific sense probe as a negative control (Figure 1E). The presence of RFC protein in mouse placenta was determined by immunofluorescence using frozen sections of E10.0 day mouse placenta. RFC-specific antibody [3] produced immune reaction in trophoblast cells of all layers of the mouse placenta (Figure 1B). RFC message was detected abundantly in the trophoblast cells surrounding blood lacunae in the labyrinth zone. Immunofluorescence showed that RFC protein was present widely throughout the placenta with particularly intense expression in the trophoblastic giant cells, the yolk sac and within the neural tube of the developing embryo (Figure 1B and 1D). Specificity of the RFC antibody was confirmed by the lack of fluorescence in tissues incubated with RFC antibody previously treated with blocking peptide (Figure 1F).


Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart.

Maddox DM, Manlapat A, Roon P, Prasad P, Ganapathy V, Smith SB - BMC Dev. Biol. (2003)

In situ hybridization and immunolocalization of RFC during early murine embryonic development. Panels A and C demonstrate expression of RFC mRNA in a section of uterus containing an E10.0 embryo. Panel E demonstrates the absence of background staining when a sense riboprobe for RFC is hybridized with placental sections as a negative control. To determine whether RFC protein may be localized to the same areas that are positive for RFC message during murine embryonic development, immunofluorescence was utilized in E10.0 frozen uterus sections. Panels B and D indeed verify that RFC protein is localized in a manner consistent with the expression pattern of RFC message. Specificity of the RFC antibody is demonstrated by lack of fluorescence in tissues incubated with RFC antibody that had been pre-incubated with blocking peptide (Panel F). L, labyrinth zone; NT, neural tube; JZ, junctional zone; TG, trophoblastic giant cells; YS, yolk sac. Scale bar represents 25 μm in A and E and 4 μm in B, C and D.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC184426&req=5

Figure 1: In situ hybridization and immunolocalization of RFC during early murine embryonic development. Panels A and C demonstrate expression of RFC mRNA in a section of uterus containing an E10.0 embryo. Panel E demonstrates the absence of background staining when a sense riboprobe for RFC is hybridized with placental sections as a negative control. To determine whether RFC protein may be localized to the same areas that are positive for RFC message during murine embryonic development, immunofluorescence was utilized in E10.0 frozen uterus sections. Panels B and D indeed verify that RFC protein is localized in a manner consistent with the expression pattern of RFC message. Specificity of the RFC antibody is demonstrated by lack of fluorescence in tissues incubated with RFC antibody that had been pre-incubated with blocking peptide (Panel F). L, labyrinth zone; NT, neural tube; JZ, junctional zone; TG, trophoblastic giant cells; YS, yolk sac. Scale bar represents 25 μm in A and E and 4 μm in B, C and D.
Mentions: In situ hybridization analysis was performed on cryosections of mouse uterus obtained at embryonic day (E)10.0 of gestation. Figure 1A shows that RFC message is expressed in trophoblast cells of all layers of the placenta, with the expression most abundant in the trophoblast cells surrounding blood lacunae. RFC message was expressed also in the trophoblastic giant cells, labyrinth, junctional zone, yolk sac and within the neural tube of the developing embryo (Figure 1A and 1C). Specificity of the staining was verified by hybridization of placental sections with an RFC specific sense probe as a negative control (Figure 1E). The presence of RFC protein in mouse placenta was determined by immunofluorescence using frozen sections of E10.0 day mouse placenta. RFC-specific antibody [3] produced immune reaction in trophoblast cells of all layers of the mouse placenta (Figure 1B). RFC message was detected abundantly in the trophoblast cells surrounding blood lacunae in the labyrinth zone. Immunofluorescence showed that RFC protein was present widely throughout the placenta with particularly intense expression in the trophoblastic giant cells, the yolk sac and within the neural tube of the developing embryo (Figure 1B and 1D). Specificity of the RFC antibody was confirmed by the lack of fluorescence in tissues incubated with RFC antibody previously treated with blocking peptide (Figure 1F).

Bottom Line: Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking.Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy.These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, USA. oxmandm@yahoo.com

ABSTRACT

Background: Folate is essential for cellular proliferation and tissue regeneration. As mammalian cells cannot synthesize folates de novo, tightly regulated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support biosynthesis of purines, pyrimidines, and some amino acids (serine, methionine). Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking. We utilized in situ hybridization and immunolocalization to determine the developmental distribution of RFC message and protein, respectively.

Results: In the mouse, RFC transcripts and protein are expressed in the E10.0 placenta and yolk sac. In the E9.0 to E11.5 mouse embryo RFC is widely detectable, with intense signal localized to cell populations in the neural tube, craniofacial region, limb buds and heart. During early development, RFC is expressed throughout the eye, but by E12.5, RFC protein becomes localized to the retinal pigment epithelium (RPE).

Conclusions: Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy. The mechanism, however, by which folate supplementation ameliorates the occurrence of developmental defects is unclear. The present work demonstrates that RFC is present in placenta and yolk sac and provides the first evidence that it is expressed in the neural tube, craniofacial region, limb buds and heart during organogenesis. These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency.

Show MeSH
Related in: MedlinePlus