The iron exporter ferroportin 1 is essential for development of the mouse embryo, forebrain patterning and neural tube closure

Abstract

Neural tube defects (NTDs) are some of the most common birth defects observed in humans. The incidence of NTDs can be reduced by peri-conceptional folic acid supplementation alone and reduced even further by supplementation with folic acid plus a multivitamin. Here, we present evidence that iron maybe an important nutrient necessary for normal development of the neural tube. Following implantation of the mouse embryo, ferroportin 1 (Fpn1) is essential for the transport of iron from the mother to the fetus and is expressed in the visceral endoderm, yolk sac and placenta. The flatiron (ffe) mutant mouse line harbors a hypomorphic mutation in Fpn1 and we have created an allelic series of Fpn1 mutations that result in graded developmental defects. A null mutation in the Fpn1 gene is embryonic lethal before gastrulation, hypomorphic Fpn1(ffe/ffe) mutants exhibit NTDs consisting of exencephaly, spina bifida and forebrain truncations, while Fpn1(ffe/KI) mutants exhibit even more severe NTDs. We show that Fpn1 is not required in the embryo proper but rather in the extra-embryonic visceral endoderm. Our data indicate that loss of Fpn1 results in abnormal morphogenesis of the anterior visceral endoderm (AVE). Defects in the development of the forebrain in Fpn1 mutants are compounded by defects in multiple signaling centers required for maintenance of the forebrain, including the anterior definitive endoderm (ADE), anterior mesendoderm (AME) and anterior neural ridge (ANR). Finally, we demonstrate that this loss of forebrain maintenance is due in part to the iron deficiency that results from the absence of fully functional Fpn1.

Fig. 1.

Fpn1^ffe/ffe mutant embryos exhibit NTDs. (A-D) Lateral view of wild-type (A,C) and Fpn1^ffe/ffe mutant (B,D) embryos at E12.5 (A,B) and E14.5 (C,D). Mutant embryos are developmentally delayed, and exhibit exencephaly, microphthalmia and generalized edema. Lateral views of embryos with anterior towards the left.

Fig. 2.

Reduction in the expression of molecular markers of the forebrain in Fpn1^ffe/ffe mutants. (A-F) Expression of molecular markers of the forebrain Foxg1 (A,B), Pax6 (C,D) and Six3 (E,F) at E9.5 (A-D) and E8.5 (E,F) in wild-type (A,C,E) and Fpn1^ffe/ffe mutant (B,D,F) embryos. At E9.5, expression of Foxg1 and Pax6 in the telencephalon (T) is reduced and Pax6 expression in the diencephalon (D) is reduced to a lesser extent in Fpn1 mutants compared with sibling controls. (E,F) The reduction in Six3 expression in the telencephalon is evident by E8.5 (n=4). (G,H) Fpn1 mutants exhibit a mild holoprosencephaly evident by a narrower domain of BAT-Gal reporter activation in the dorsal telencephalon of a mutant (H) when compared with wild-type embryo (G) at E9.5. (I-N) Expression of molecular markers of the midbrain-hindbrain boundary (arrows) Wnt1 (I,J), Otx2 (K,L) and Gbx2 (M,N) at E9.5 in wild-type (I,K,M) and Fpn1^ffe/ffe mutant (J,L,N) embryos. (A-D,I-N) Lateral views with anterior towards the left. (E-H) Frontal views.

Fig. 3.

An Fpn^KI allele fails to complement the Fpn1^ffe mutation. (A-H) Lateral view of wild-type (A,C,E), Fpn1^ffe/ffe (B) and Fpn1^ffe/KI mutant (D,F-H) embryos at E10.5 (A-D,G,H) and E9.5 (E,F). Fpn1^ffe/KI mutants are developmentally delayed and exhibit severe NTDs that consists of exencephaly (between arrowheads in B), spina bifida and craniorachischisis. (A-F) Lateral views with anterior towards the left. (G) Dorsal view. (H) Frontal view.

Fig. 4.

Severe reduction in the expression of molecular markers of the telencephalon in Fpn1^ffe/KI mutants. (A-D) Expression of the telencephalic markers (arrows) Foxg1 at E9.5 (A,B) and Six3 at E8.5 (C,D) are reduced in Fpn1^ffe/KI mutants (B,D) when compared with wild-type controls (A,C). (E,F) Expression of the midbrain-hindbrain boundary marker Gbx2 (arrows) is relatively normal in wild-type (E) versus mutant (F) embryos at E9.5. Lateral views of embryos with anterior towards the left.

Fig. 5.

Fpn1 is expressed in the visceral endoderm during gastrulation and neurulation. (A-F) Expression of lacZ-Fpn1 in Fpn1^KI/+ embryos (A-D) or Fpn1 in wild-type embryos (E,F) at E6.5 (A), E7.5 (B,E), E8.5 (C,F) and E9.5 (D) determined by in situ hybridization analysis. Inset shows sectioned embryos, demonstrating expression in the VE lineages. VE, visceral endoderm; M, mesoderm; E, ectoderm. Lateral views of embryos with anterior towards the left.

Fig. 6.

Fpn1 is required in the extra-embryonic lineage. (A,F) Schematics illustrating the lineage-restricted expression of (A) epiblast-specific Cre-recombinase (MORE-Cre or Sox2-Cre) when compared with (F) VE-specific Cre-recombinase (Ttr-Cre). (B-E) Conditional deletion of Fpn1 using either MORE-Cre (B,C) or Sox2-Cre (D,E) drivers in the epiblast lineage results in morphologically normal embryos in Fpn1^ffe/flox;MORE-Cre (not shown), Fpn^flox/KI;MORE-Cre (C), Fpn1^ffe/flox;Sox2-Cre (not shown) and Fpn^flox/KI;Sox2-Cre (E) embryos at E10.5 compared with sibling controls (B,D). Fpn1^ffe/flox;Sox2-Cre embryos are smaller than wild-type controls. (G-J) By contrast, conditional deletion of Fpn1 with the VE-specific Ttr-Cre (H,J) resulted in exencephaly (between arrowheads in H) in Fpn1^flox/KI;Ttr-Cre embryo (H; n=6) at E10.5 and reduction in Six3 expression at E8.5 compared with sibling control embryo (G,I). Lateral views of embryos with anterior towards the left.

Fig. 7.

Diffuse and ectopic AVE and neural induction in Fpn1^ffe/KI mutants. (A-D) Expression of Cerl in E6.5 wild-type (A,C) and Fpn1^ffe/KI mutant (B,D) embryos shows ectopic (asterisk) and diffuse localization of the AVE in the anterior region in Fpn^flox/KI mutant embryos (n=3). (E-H) Ectopic expression of Six3 immediately following its induction at E7.5 in Fpn1^ffe/KI (F,H, indicated asterisks) when compared with wild-type (E,G) embryos (n=6). (A,B,E,F) Lateral views of embryo with anterior towards the left. (C,D,G,H) Frontal views: G and H show the same embryos as in E and F, respectively.

Fig. 8.

Fpn1^ffe/KI mutants exhibit defects in multiple signaling centers within the epiblast that maintain and refine the anterior forebrain. (A,B) Expression of Cerl is reduced in the anterior definitive endoderm (ADE) but not the AVE (arrow) of Fpn1^ffe/KI mutants at E7.5 (n=3). (C,D) Expression of Shh does not extend to the anterior limit of the neural plate in Fpn1^ffe/KI mutant (bracket; D) at E8.5 when compared with wild-type embryo (C). (E,F) Fgf8 expression in the anterior neural ridge (ANR) is also reduced at E8.5 in Fpn1^ffe/KI mutants (arrows). (A,B,E,F) Lateral views of embryos with anterior towards the left. (C,D) Frontal view.

Fig. 9.

Wild-type embryos cultured in iron chelators phenocopies the forebrain defects of Fpn1 mutants. (A,B) Six3 expression in wild-type (A) and Fpn1^ffe/ffe mutant (B) embryos at E8.5. (C,D) Six3 and Krox20 expression in wild-type embryos cultured in the presence of water (vehicle control; C) or 200 μM of the iron chelator 1,2-dimethyl-3-hydroxy-4-pyridone (D). Four out of five iron chelator-treated embryos show reduced Six3 expression compared with those treated with vehicle control.

Fig. 10.

Model for the role of Fpn1 in anterior neural development. In wild-type embryos (top), multiple transporters (pink) are involved in the uptake of iron (yellow) from the maternal environment into the VE (blue). In the VE, ferritin (green) binds to free iron to buffer its oxidative potential. Fpn1 (red) is the only exporter capable of transporting iron out of the VE and to the developing epiblast. Mutation of Fpn1 (bottom) would result in accumulation of iron in the VE and iron deficiency in the epiblast owing to reduced transport of iron to the developing embryo. In wild-type embryos (top), by E5.5 the DVE (pink) is induced in the distal region of the egg cylinder. By E6.5, the AVE migrates to the anterior region of the embryo where it participates in induction of the anterior neuroectoderm (green). By E7.5, the VE has been replaced by definitive endoderm (white) and the prospective forebrain (green) begins to express markers of anterior neural identity such as Six3. By E8.5 the neural domain is further stabilized and refined. In Fpn1 mutants (bottom), the AVE is diffusely localized to the anterior region of the embryo resulting in the diffuse and ectopic induction of tissue of anterior neural character at E7.5. However, this anterior neural tissue is not maintained, because by E8.5 the expression of markers of the forebrain are disproportionately reduced. Between E7.5 and E8.5, iron that is transported from the VE to the embryo is required to maintain forebrain development.