3079RESEARCH ARTICLE

INTRODUCTIONNeural tube defects (NTDs) affect ~1 out of 1000 live births (for areview, see Detrait et al., 2005). The causes of NTDs are complex,with both genetic and environmental factors influencing itspenetrance (for a review, see Zohn and Sarkar, 2008).Epidemiological studies have implicated peri-conception maternalnutrition as an important factor influencing the occurrence of NTDs(for a review, see Blom et al., 2006). From these studies, folic acidhas emerged as an important nutrient for normal development, andexperimental data in both human and animal models demonstratethat folic acid fortification can reduce the incidence of NTDs. Asa result of these findings, many countries now fortify the foodsupply with folic acid and observe significant reductions in theincidence of NTDs in their populations (Eichholzer et al., 2006).Other nutrients, such as iron, have also emerged fromepidemiological studies as nutritional factors that are likely toinfluence the penetrance of NTDs (Felkner et al., 2005; Groenen etal., 2004) (for a review, see Czeizel, 2009); however, experimentalevidence demonstrating a clear role for iron in the prevention ofNTDs is lacking.

Iron plays essential roles in development and growth byparticipating in electron transport, DNA synthesis, oxygen transportand reduction-oxidation reactions. It also acts as a co-factor for

many enzymes and regulates translation by binding to iron-responsive elements. Iron levels are tightly regulated, as either irondeficiency or iron overload results in diverse pathologicalconditions (for a review, see Andrews, 1999). Unlike with folicacid, controlled studies in humans to investigate the role of iron insuppressing NTDs have not been carried out. Additionally, mousemodels with disruption of iron homeostasis either suffer from earlyembryonic lethality or exhibit no observable phenotype,presumably owing to functional redundancy, thus precluding theinvestigation of this nutrient in the etiology of NTDs (for a review,see De Domenico et al., 2008).

In the mouse embryo, neural tube closure begins aroundembryonic day 8.5 (E8.5) and is complete by E10.5 when theposterior neuropore finally closes. During this time and until theestablishment of the placenta between E9.5 and E10.5, the visceralendoderm (VE) and yolk sack (YS) provide for the nutritionalsupport (including the uptake of iron) and exchange of wasteproducts between the maternal circulation and the developingembryo (for a review, see Bielinska et al., 1999). Followingimplantation, the conceptus or egg cylinder is shaped like anelongated cup (see Fig. 10). The distal half (from the implantationsite) of the egg cylinder is the epiblast, which will develop into theembryo proper; and the proximal half (the extra-embryonic region)will develop into the placenta. The entire egg cylinder is encasedwithin the VE, a polarized epithelial layer of cells morphologicallyand functionally similar to gut endoderm. The apical surface of theVE has microvilli to allow absorption of nutrients, including iron,from the maternal environment. These nutrients are then transferredto the epiblast on the basal side of the VE. As gastrulationproceeds, the VE is displaced from the distal half of the epiblast asthe definitive endoderm (DE) emerges from the primitive streak.

Development 137, 3079-3088 (2010) doi:10.1242/dev.048744© 2010. Published by The Company of Biologists Ltd

1Center for Neuroscience Research, Children’s Research Institute, Children’s NationalMedical Center, Washington, DC 20010, USA. 2Howard Hughes Medical Institute,Department of Pediatrics, University of Colorado Denver, Aurora, CO 80045, USA.

*Author for correspondence (izohn@cnmcresearch.org)

Accepted 7 July 2010

SUMMARYNeural tube defects (NTDs) are some of the most common birth defects observed in humans. The incidence of NTDs can bereduced by peri-conceptional folic acid supplementation alone and reduced even further by supplementation with folic acid plusa multivitamin. Here, we present evidence that iron maybe an important nutrient necessary for normal development of theneural tube. Following implantation of the mouse embryo, ferroportin 1 (Fpn1) is essential for the transport of iron from themother to the fetus and is expressed in the visceral endoderm, yolk sac and placenta. The flatiron (ffe) mutant mouse line harborsa hypomorphic mutation in Fpn1 and we have created an allelic series of Fpn1 mutations that result in graded developmentaldefects. A null mutation in the Fpn1 gene is embryonic lethal before gastrulation, hypomorphic Fpn1ffe/ffe mutants exhibit NTDsconsisting of exencephaly, spina bifida and forebrain truncations, while Fpn1ffe/KI mutants exhibit even more severe NTDs. Weshow that Fpn1 is not required in the embryo proper but rather in the extra-embryonic visceral endoderm. Our data indicate thatloss of Fpn1 results in abnormal morphogenesis of the anterior visceral endoderm (AVE). Defects in the development of theforebrain 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, wedemonstrate that this loss of forebrain maintenance is due in part to the iron deficiency that results from the absence of fullyfunctional Fpn1.

KEY WORDS: Neural tube defect, Iron deficiency, Forebrain patterning, Mouse, Sl40a1

The iron exporter ferroportin 1 is essential for developmentof the mouse embryo, forebrain patterning and neural tubeclosureJinzhe Mao1, David M. McKean2, Sunita Warrier1, Joshua G. Corbin1, Lee Niswander2 and Irene E. Zohn1,*

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At this time the VE forms one of the two cell layers that make upthe YS, which forms as the extra-embryonic mesoderm migratesbeneath the VE. At the end of gastrulation, the YS assumes the roleof nutrient and waste exchange for the embryo until the placentabegins to take over starting around E9.5.

In addition to its role in supporting the nutritional requirementsof the embryo, the VE provides patterning information to establishthe anterior-posterior axis of the fetus. The VE in the posteriorregion of the embryo is required for induction of the hematopoieticlineage (Belaoussoff et al., 1998). The amnionless gene is requiredin the VE for development of the non-midline trunk derivatives ofthe primitive streak, including the lateral plate, intermediate andparaxial mesoderm (Tomihara-Newberger et al., 1998). Perhaps themost well-characterized signaling center in the VE is the anteriorvisceral endoderm (AVE), which is required for induction of thehead and positioning of the primitive streak at the posterior end ofthe embryo (for a review, see Martinez-Barbera and Beddington,2001; Srinivas, 2006). By E5.5, reciprocal interactions between theepiblast and the VE induce the formation of a specialized group ofVE cells at the distal tip of the egg cylinder called the distalvisceral endoderm (DVE). Following its induction, the DVEmigrates to one side of the egg cylinder, forming the AVE anddefining the anterior pole of the embryo. The DVE and AVEexpress a panel of transcription and secreted factors, includingHex1, Lim1 and Cerl, which are required for the properspecification, migration and signaling activities of this organizingcenter.

Following gastrulation, several signaling centers in the embryoproper maintain and refine the anterior-posterior pattern of theneuroectoderm (for a review, see Martinez-Barbera andBeddington, 2001). Signals from the AVE induce an unstable pre-forebrain, which is stabilized by interaction with the anteriordefinitive endoderm (ADE) and anterior mesendoderm (AME).Both the ADE and AME promote neuralization and protect theanterior neural tissue from caudalizing signals secreted from thenode. For example, Hex1 is expressed in the ADE and deletion ofHex1 in the epiblast results in forebrain truncations (MartinezBarbera et al., 2000). Similarly, Foxa2 is expressed in the AMEunderlying the prospective forebrain and, in its absence, theforebrain is reduced (Hallonet et al., 2002). Another importantsignaling center for stabilizing and refining the anterior forebrainis the anterior neural ridge (ANR) at the rostral tip of the neuralplate (for a review, see Martinez-Barbera and Beddington, 2001;Rubenstein et al., 1998). Fgf8 is expressed in the ANR where it isessential for the induction of Foxg1, a telencephalic markerexpressed beginning around E8.5 (Shimamura and Rubenstein,1997).

In an ENU mutagenesis screen to identify genes required forneural tube closure, we isolated the flatiron (ffe) mouse mutant linethat carries a point mutation in the Scl40a1 gene encoding the irontransporter ferroportin 1 (Fpn1) (Zohn et al., 2007). Fpn1 is theonly iron exporter in eukaryotes and is expressed in the VE,placenta and intestine, where it plays an essential role in theabsorption of iron (Donovan et al., 2000; Donovan et al., 2005).The ffe mutation results in altered cell surface localization of thetransporter and reduces its ability to transport iron out of the cell(Zohn et al., 2007). Our previous study demonstrated that the ffemouse provides a model for the iron overload disorderHemochromatosis Type IV in heterozygous animals (Zohn et al.,2007). Here, we characterize the neural tube closure and forebrainpatterning defects observed in homozygous mutant embryos. Wedemonstrate that Fpn1 is required in the extra-embryonic lineage

for normal development of the neural tube. NTDs in Fpn1 mutantsare due to both a cell-autonomous requirement for Fpn1 in the VE,presumably to prevent the accumulation of iron in these cells, anda non-autonomous requirement for Fpn1 in the establishment ofsignaling centers in the epiblast that refine and maintain the initialanterior-posterior pattern of the neural tube. Significantly, our dataindicate that this cell non-autonomous role for Fpn1 is probablyrelated to its iron transport activity and to the severe iron deficiencythat is predicted to result in the embryo in its absence.

MATERIALS AND METHODSMouse strains and genotypingThe Fpn1ffe mouse line was generated by ENU mutagenesis and genotypedas described (Zohn et al., 2007). MORE- (Tallquist and Soriano, 2000),Sox2-Cre (Hayashi et al., 2002) and BAT-Gal (Maretto et al., 2003)transgenic lines were purchased from Jackson Laboratories and genotypedas indicated. The Ttr-Cre transgenic line was generously provided by DrAnna-Katerina Hadjantonakis and genotyped as described (Kwon andHadjantonakis, 2009; Kwon et al., 2008). The Fpn1KI mouse line wasgenerated by Deltagen, purchased from Jackson Laboratories andgenotyped as indicated (http://jaxmice.jax.org/strain/005840.html). Forconditional deletion experiments, the Fpn1flox allele was generouslyprovided by Dr Nancy Andrews and genotyped as described (Donovan etal., 2005). All mouse lines were crossed onto the C3H/HeJ geneticbackground for 2-5 generations before crossing with the Fpn1ffe allele.

Embryo culture with iron chelatorsEmbryos were dissected from deciduas at E7.5 and cultured by standardtechniques (Nagy et al., 2003). Briefly, embryo dissections were carried outin culture media (DMEM (#11054-020, GIBCO) supplemented with 1�Pen/strep (#15070-063, GIBCO), Glutamax (#350505-061, GIBCO) andHEPES (#15620-080, GIBCO). Embryos were dissected with the YS intactand the ectoplacental cone still attached to uterine tissue. Followingdissection, embryos were transferred to roller bottles containing completeculture media [culture media supplemented with 50% whole embryoculture rat serum (BT-4520, Harlan)] that was spiked with either water (10ml, vehicle control) or 1,2 Dimethyl-3-hydroxy-4-pyridone (200 mM;ACROS #30652-11-0). Prior to the addition of embryos, the completeculture media was equilibrated in a roller culture apparatus (BTCengineering) at 37°C with a constant flow of gas (90% N2, 5% O2 and 5%CO2) for at least 2 hours. Embryos were cultured in the roller cultureapparatus at 37°C with a constant flow of gas for ~36 hours until theydeveloped to E8.5.

Analysis of mutant phenotypesWhole-mount RNA in situs were performed as described (Zohn et al.,2006) using the following probes: Foxg1 (Tao and Lai, 1992), Pax6(Grindley et al., 1995), Six3 (Oliver et al., 1995), Wnt1 (Parr et al., 1993),Otx2 (Ang et al., 1994), Gbx2 (Bouillet et al., 1995), Cerl (Belo et al.,1997), Lim1 (Barnes et al., 1994), Fgf8 (Crossley and Martin, 1995), Shh(Echelard et al., 1993) and Krox20 (Wilkinson et al., 1989). Fpn1 transcriptexpression was determined using an antisense RNA probe synthesized fromIMAGE clone: 2647365 or by expression of the lacZ transcript in Fpn1KI/+

embryos. All in situs were performed on at least two mutant and twocontrol embryos, with similar results unless otherwise indicated in thefigure legend.

RESULTSHypomorphic Fpnffe/ffe mutant embryos exhibitNTDsWe have previously isolated the flatiron mutation in ferroportin 1(Fpnffe) from an ENU mutagenesis screen to identify genesrequired for neural tube closure (Zohn et al., 2005; Zohn et al.,2007). Fpnffe/ffe mutants exhibit NTDs on a C3H/HeJ geneticbackground, including exencephaly and spina bifida, as well asdevelopmental delays, microphthalmia and severe iron deficiency

RESEARCH ARTICLE Development 137 (18)

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(Fig. 1; data not shown). In addition, the size of the forebrain inFpnffe/ffe mutants appeared smaller than that of wild-type controls,an observation confirmed by examination of the expression ofmolecular markers of the forebrain (Fig. 2). At E9.5, Foxg1 isexpressed in the telencephalon of wild-type embryos and itsexpression is reduced in Fpnffe/ffe mutants. Pax6 is also reduced inthe telencephalon and less so in the diencephalon of Fpnffe/ffe

mutants. Reductions in telencephalic marker expression are evidentby E8.5, as shown by decreased Six3 expression in Fpnffe/ffe

mutants when compared with sibling controls. Fpnffe/ffe mutants

also exhibit a mild holoprosencephaly evident at E9.5 by the BAT-Gal reporter that marks a narrower Wnt-activation domain in thedorsal midline of a narrower telencephalon in Fpnffe/ffe mutants. Incontrast to the anterior forebrain defects, the expression domainsof Wnt1, Otx2 and Gbx2 indicate that the midbrain/hindbrainboundary is properly positioned along the anterior-posterior axis ofthe embryo (Fig. 2).

An Fpn1 null allele fails to complement theFpn1ffe mutationUsing a positional cloning strategy, the ffe phenotype was mappedto a 2.6 Mb interval on mouse chromosome 1 that contains 16transcripts, including the Slc40a1 gene encoding the Fpn1 protein(Zohn et al., 2007). Our previous studies identified a point mutationin the Fpn1 gene in ffe mutants that changes a conserved histidineto an arginine (H32R), and demonstrated that this amino acidsubstitution results in a reduction in the iron export activity of Fpn1(Zohn et al., 2007). However, the involvement of the Fpn1 gene inneural tube closure and forebrain patterning was unexpected. Asthe possibility remained that NTD phenotypes were due tomutation of another gene in this 2.6 Mb interval, we obtained atargeted b-galactosidase knock-in allele of Fpn1 that expresses lacZfrom the Fpn1 locus (Fpn1KI) and used it in a complementation testcross. Although a null allele of Fpn1 is embryonic lethal beforeneural tube closure (Donovan et al., 2005), transheterozygousFpn1ffe/KI mutant embryos exhibit NTDs, which were more severethan in Fpn1ffe/ffe mutants (Fig. 3). Whereas Fpn1ffe/ffe mutantsprimarily exhibit exencephaly and, rarely, spina bifida, Fpn1ffe/KI

mutant embryos consistently exhibit both. In addition, in manyembryos the neural tube failed to close along the entireanterior/posterior neural axis, resulting in a condition known ascraniorachischisis (Fig. 3G). Furthermore, Fpn1ffe/KI mutants aremore significantly developmentally delayed when compared withwild-type or Fpn1ffe/ffe mutants. For example, by E9.5, Fpn1ffe/KI

mutants fail to turn and are markedly smaller than littermatecontrols (Fig. 3E,F). Forebrain truncations in Fpn1ffe/KI mutants are

3081RESEARCH ARTICLEFerroportin in neural development

Fig. 1. Fpn1ffe/ffe mutant embryos exhibit NTDs. (A-D) Lateral viewof wild-type (A,C) and Fpn1ffe/ffe mutant (B,D) embryos at E12.5 (A,B)and E14.5 (C,D). Mutant embryos are developmentally delayed, andexhibit exencephaly, microphthalmia and generalized edema. Lateralviews of embryos with anterior towards the left.

Fig. 2. Reduction in the expression of molecularmarkers of the forebrain in Fpn1ffe/ffe mutants.(A-F) Expression of molecular markers of theforebrain Foxg1 (A,B), Pax6 (C,D) and Six3 (E,F) atE9.5 (A-D) and E8.5 (E,F) in wild-type (A,C,E) andFpn1ffe/ffe mutant (B,D,F) embryos. At E9.5,expression of Foxg1 and Pax6 in the telencephalon(T) is reduced and Pax6 expression in thediencephalon (D) is reduced to a lesser extent in Fpn1mutants compared with sibling controls. (E,F) Thereduction in Six3 expression in the telencephalon isevident by E8.5 (n=4). (G,H) Fpn1 mutants exhibit amild holoprosencephaly evident by a narrowerdomain of BAT-Gal reporter activation in the dorsaltelencephalon of a mutant (H) when compared withwild-type embryo (G) at E9.5. (I-N) Expression ofmolecular markers of the midbrain-hindbrainboundary (arrows) Wnt1 (I,J), Otx2 (K,L) and Gbx2(M,N) at E9.5 in wild-type (I,K,M) and Fpn1ffe/ffe

mutant (J,L,N) embryos. (A-D,I-N) Lateral views withanterior towards the left. (E-H) Frontal views.

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also more pronounced than in wild-type or Fpn1ffe/ffe mutants (Figs3 and 4). Expression of telencephalic specific markers Foxg1 andSix3 at E9.5 and E8.5, respectively, are significantly reduced whencompared with wild type or even Fpn1ffe/ffe mutants (compare Fig.2 with Fig. 4). Similar to Fpn1ffe/ffe mutants, positioning of themidbrain-hindbrain boundary, which is marked by Gbx2expression, is relatively normal in Fpn1ffe/KI mutants. These resultsindicate that Fpn1 is required for neural tube closure along theentire anterior-posterior axis and that development of the forebrainis most sensitive to Fpn1 loss.

Fpn1 is required in the visceral endoderm fordevelopment of the neural tubePrevious studies indicated that Fpn1 is expressed in thesyncytiotrophoblast cells of the placenta and visceral endoderm atE7.5 (Donovan et al., 2000; Donovan et al., 2005), but acomprehensive analysis of Fpn1 expression has not been carriedout during neurulation. Therefore, we analyzed Fpn1 expression atE6.5, 7.5, 8.5 and 9.5 of mouse development by detectingexpression of lacZ in the FpnKI/+ embryos (Fig. 5). Expression ofFpn1 at E6.5 is restricted to the VE surrounding the developingepiblast. At E7.5, as the VE is displaced by the DE, Fpn1-drivenlacZ expression remains exclusively in the VE-derived cell layerof the YS surrounding the middle region of the egg cylinder. ByE8.5 and E9.5, strong staining is detected in the YS and weakstaining in ventral regions of the embryo proper (Fig. 5C,D).Similar results were obtained when an in situ probe against Fpn1was used in wild-type embryos (Fig. 5E,F). Sectioning of YSstained for Fpn1-lacZ expression reveals expression exclusively inthe cell layer derived from the VE not the extra-embryonicmesoderm. As the major sites of Fpn1 expression were notdetected in the embryo proper, these results suggest that NTDs inFpn1 mutants are probably due to a cell non-autonomous role forFpn1 in the VE.

To investigate the hypothesis that Fpn1 is required in the VElineage for neural tube closure, we performed conditional geneablation experiments using Cre-deleter lines to remove Fpn1 in eitherthe epiblast (MORE- and Sox2-Cre) or visceral endoderm (Ttr-Cre)lineages. Previous studies have demonstrated that deletion of Fpn1using the MORE-Cre transgenic deleter strain on a mixed B6;129genetic background resulted in the birth of morphologically normal

embryos (Donovan et al., 2005). These Fpn1flox/flox;MORE-Creanimals die within a couple of weeks of birth owing to severe irondeficiency that results from an inability to absorb iron throughintestinal enterocytes. However, our previous studies suggest that thepenetrance of NTDs in Fpn1ffe mutants depends on the geneticbackground (Zohn et al., 2007), so the possibility remained that thelack of a NTDs is due to genetic background effects. In addition, asCre-mediated recombination by the MORE-Cre driver occurs in amosaic pattern (Hayashi et al., 2002; Tallquist and Soriano, 2000),NTDs were potentially not observed because of incomplete Cre-mediated recombination of the Fpn1flox allele. To investigate thesepossibilities, we performed the conditional deletion experiments onthe C3H background using the Sox2-Cre transgenic deleter strain that

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Fig. 3. An FpnKI allele fails to complement theFpn1ffe mutation. (A-H) Lateral view of wild-type(A,C,E), Fpn1ffe/ffe (B) and Fpn1ffe/KI mutant (D,F-H)embryos at E10.5 (A-D,G,H) and E9.5 (E,F). Fpn1ffe/KI

mutants are developmentally delayed and exhibitsevere NTDs that consists of exencephaly (betweenarrowheads 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 markersof the telencephalon in Fpn1ffe/KI mutants. (A-D) Expression of thetelencephalic markers (arrows) Foxg1 at E9.5 (A,B) and Six3 at E8.5(C,D) are reduced in Fpn1ffe/KI mutants (B,D) when compared with wild-type controls (A,C). (E,F) Expression of the midbrain-hindbrain boundarymarker Gbx2 (arrows) is relatively normal in wild-type (E) versus mutant(F) embryos at E9.5. Lateral views of embryos with anterior towards theleft. D

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allows complete recombination in the same cell types as MORE-Cre(Hayashi et al., 2002; Tallquist and Soriano, 2000). Deletion of Fpn1with either the MORE- or Sox2-Cre drivers resulted in embryoswithout NTDs (Fig. 6). Furthermore, identical results were obtainedwhen Fpn1ffe/flox or Fpn1flox/KI alleles were used in this experiment(data not shown). Fpn1ffe/flox;Sox2-Cre and Fpn1flox/KI;Sox2-Creembryos were smaller than littermate controls (Fig. 6D,E) probablyowing to a role for Fpn1 in the epiblast lineages. Nevertheless, asthese smaller embryos did not exhibit NTDs, these experimentssuggest that Fpn1 is required in the VE and not the epiblast duringneural tube closure. To further test the idea that Fpn1 is required inthe VE for neural tube closure, the Ttr-Cre transgenic deleter strainwas used to inactivate Fpn1 in the VE lineage. Ttr-Cre is expressedin a mosaic fashion in the VE by pre-primitive streak stages (Kwonand Hadjantonakis, 2009). Significantly, Fpn1flox/KI;Ttr-Cre embryosexhibited exencephaly and forebrain truncations similar to thatobserved in hypomorphic Fpn1ffe/ffe mutants (Fig. 6G-J). Theseresults confirm that Fpn1 is required cell non-autonomously in theVE lineage for neural tube closure and forebrain patterning.

Fpn1 is required for proper morphogenesis of theAVEFpn1 is expressed on the basal surface of the VE, where it isrequired for transport of iron out of these cells to the epiblast(Donovan et al., 2005). Mutation of Fpn1 would result in twolikely outcomes that could explain NTDs in Fpn1 mutants (see Fig.10): (1) iron would accumulate in the visceral endoderm, possibly

disrupting signaling centers in the VE that pattern the embryo;and/or (2) iron is not transported to the embryo resulting in severeiron deficiency. As Fpn1 mutants exhibit anterior truncations andthe AVE is required for induction of this anterior tissue, weinvestigated whether forebrain truncations are due to alterations inAVE formation or function by examining expression of molecularmarkers of the AVE in Fpn1ffe/KI mutants. As shown in Fig. 7, theAVE appears to be properly specified in Fpn1 mutants, as evidentby the expression of Cerl in the AVE at E6.5; however, theexpression domain of Cerl appears diffusely positioned in theanterior region of mutant embryos. These results indicate thatmorphogenesis of the AVE in Fpn1 mutants is abnormal, possiblyowing to the accumulation of iron that is predicted to occur in theVE in the absence of Fpn1 exporter activity.

Secretion of factors from the AVE is required for induction ofthe most anterior neural tissue (for a review, see Martinez-Barbera and Beddington, 2001; Srinivas, 2006). To determinewhether the mispositioned AVE can induce the anterior neuralplate, expression of Six3 was examined at early neural plate

3083RESEARCH ARTICLEFerroportin in neural development

Fig. 5. Fpn1 is expressed in the visceral endoderm duringgastrulation and neurulation. (A-F) Expression of lacZ-Fpn1 inFpn1KI/+ 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 hybridizationanalysis. Inset shows sectioned embryos, demonstrating expression inthe 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) whencompared 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 normalembryos in Fpn1ffe/flox;MORE-Cre (not shown), Fpnflox/KI;MORE-Cre (C),Fpn1ffe/flox;Sox2-Cre (not shown) and Fpnflox/KI;Sox2-Cre (E) embryos atE10.5 compared with sibling controls (B,D). Fpn1ffe/flox;Sox2-Creembryos are smaller than wild-type controls. (G-J) By contrast,conditional deletion of Fpn1 with the VE-specific Ttr-Cre (H,J) resulted inexencephaly (between arrowheads in H) in Fpn1flox/KI;Ttr-Cre embryo (H;n=6) at E10.5 and reduction in Six3 expression at E8.5 compared withsibling control embryo (G,I). Lateral views of embryos with anteriortowards the left.

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stages when its expression can first be detected. As shown inFig. 7, expression of Six3 is restricted to the most anterior aspectof the neural plate in wild-type embryos, whereas, in Fpn1mutants, in addition to a reduced intensity of Six3 expression inthe prospective forebrain, Six3 expression is induced morecaudally in mutant embryos. These results indicate that both theproperly and improperly localized AVE cells in Fpn1 mutantsare capable of inducing anterior neural identity. Although theexpression of Six3 in its normal domain is slightly reduced inFpn1 mutants when compared with wild-type controls at thisstage, by E8.5 expression of Six3 in the telencephalon isdisproportionately reduced (Fig. 4C,D), suggesting thatmaintenance of this pre-forebrain is compromised in Fpn1mutants. Furthermore, the ectopically induced anterior neuraltissue failed to be maintained as ectopic Six3 expression was notobserved caudally at E8.5 in Fpn1 mutants.

Following the initial induction of the anterior neural tissue bythe AVE, additional epiblast-derived signaling centers maintainand refine this pattern (for a review, see Martinez-Barbera andBeddington, 2001). To determine whether deletion of Fpn1results in cell non-autonomous effects on the specification andor activity of these signaling centers, the expression of molecularmarkers of the anterior definitive endoderm (ADE; Cerl),anterior neural ridge (ANR; Fgf8) and the anteriormesoendoderm (AME; Shh) were examined at the appropriatestages. As shown in Fig. 8, while expression of Cerl appearsequivalent in the AVE of wild-type and mutant embryos at E7.5,expression in the ADE is reduced. Expression of Shh and itsdownstream target Ptch1 in the AME at E8.25 reveals that thisorganizing center does not extend to the anterior limit of theneural plate in mutant embryos (Fig. 8C,D and data not shown).This loss of Shh signaling in the forebrain region is consistentwith the holoprosencephaly observed in Fpn1 mutants (Fig.2G,H). Similarly, expression of Fgf8 at E8.5 marks a muchsmaller ANR in Fpn1 mutants (Fig. 8E,F). Together, theseresults indicate that in addition to abnormal induction of theanterior neural domain by the AVE, forebrain defects in Fpn1mutants are compounded by defects in the maintenance andrefinement of this already smaller pre-forebrain owing to defectsin the ADE, AME and ANR signaling.

Forebrain defects in Fpn1 mutants are partiallydue to iron deficiencyAlthough the cell-autonomous effect of Fpn1 loss on AVE functionmay be due to the accumulation of iron in the VE that leads to theiraltered migration, we reasoned that the cell non-autonomous effectof Fpn1 loss on ADE, AME and ANR function and the resultingfailure to maintain the anterior neural tissue was probably due to areduction in iron transported to these cells. To test the hypothesisthat iron deficiency can result in loss of anterior neural character,wild-type embryos were cultured from E7.5 to E8.5 in the presenceor absence of an iron chelator (200 mM; 1,2-Dimethyl-3-hydroxy-4-pyridone) and subjected to in situ hybridization analysis toexamine expression of the prospective telencephalic marker Six3.As shown in Fig. 9, culture of wild-type embryos in water (vehiclecontrol) did not affect expression of Six3 in the telencephalon.Strikingly, culture of wild-type embryos in the presence of the ironchelator during this crucial period phenocopies the forebraintruncation observed in Fpn1ffe/ffe mutants. These results suggest thatforebrain truncations in Fpn1 mutants are due, at least in part, tothe reduced transport of iron across the VE to the developingembryo.

DISCUSSIONHere we analyzed the developmental pathology leading to NTDsin embryos that are mutant for the iron exporter Fpn1. We havecreated an allelic series of Fpn1 mutations that reveal gradeddevelopmental defects depending on the residual activity of theiron transport activity. A null mutation in Fpn1 results inembryonic lethality before E7.5 (Donovan et al., 2005). Ahypomorphic mutation in Fpn1 (Fpn1ffe) results in NTDsconsisting primarily of exencephaly and forebrain truncations.Transheterozygous Fpn1 mutants (Fpn1ffe/KI) exhibit more severeNTDs that consisted of exencephaly, craniorachischisis and moredrastic forebrain truncations. The expression pattern of Fpn1during neurulation suggests that it is required in the VE, anobservation that was confirmed by conditional deletion of Fpn1 inthe VE lineage, resulting in neural tube and forebrain defects. Fpn1function in the VE is essential for proper morphogenesis of theAVE, as the AVE is diffusely localized in the anterior region ofmutant embryos. Consequently, anterior neural character, as

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Fig. 7. Diffuse and ectopic AVE and neuralinduction in Fpn1ffe/KI mutants. (A-D) Expression ofCerl in E6.5 wild-type (A,C) and Fpn1ffe/KI mutant(B,D) embryos shows ectopic (asterisk) and diffuselocalization of the AVE in the anterior region inFpnflox/KI mutant embryos (n=3). (E-H) Ectopicexpression of Six3 immediately following its inductionat E7.5 in Fpn1ffe/KI (F,H, indicated asterisks) whencompared with wild-type (E,G) embryos (n=6).(A,B,E,F) Lateral views of embryo with anteriortowards the left. (C,D,G,H) Frontal views: G and Hshow the same embryos as in E and F, respectively.

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determined by Six3 expression at E7.5, is induced to a slightlylesser degree in its normal domain but also ectopically in morecaudal regions of the embryo. However, this anterior neural tissueis not maintained, as Six3 expression by E8.5 is disproportionatelyreduced. Our data suggest that failure to maintain anterior neuralidentity is due to reduction of a number of signaling centers,including the ADE, AME and ANR. Finally, the failure to maintainanterior neural tissue identity is due to loss of iron transport activityof Fpn1 from the VE to the embryo, as anterior truncations arephenocopied by culture of wild-type embryos in iron chelatorsduring a crucial period (E7.5 to E8.5) of forebrain maintenance.

Diffuse induction and improper maintenance ofthe forebrain in Fpn1 mutantsThe analysis of multiple mouse mutants with forebrain truncationsindicate that this defect can result from a number of differentmechanisms that fall into two general groups. The first group ofmutants fail to induce the anterior neural domain because of defectsin the AVE, whereas in the second group of mutants the forebrainis induced but not maintained (for a review, see Martinez-Barberaand Beddington, 2001). Within the second group, a failure tomaintain the forebrain can be due to defects in the ADE, AME orANR. Our data suggest that forebrain truncations in Fpn1 mutants

are due to disruptions in all of these mechanisms. As schematizedin Fig. 10, the AVE in Fpn1 mutants is formed and induces the pre-forebrain domain; however, the AVE in mutant embryos isdiffusely localized within the anterior region of the embryo. Thepresence of scattered AVE cells results in diffuse and ectopicinduction of anterior neural character. Despite induction of only aslightly weaker anterior neural domain, by E8.5, Six3 expression isvery much reduced, indicating that Fpn1 function is also requiredfor maintenance of the anterior neural domain. The failure tomaintain anterior neural character appears to be due to reductionsin multiple signaling centers, including the ADE, AME and ANR.Specifically, the failure to maintain Six3 expression is due in partto defects in the ADE, as Cerl expression in the ADE is reducedcompared with control embryos. Furthermore, the AME does notextend to the normal anterior limit in Fpn1 mutants, as shown bythe lack of expression of Shh in the most anterior portion of theneural plate. This reduction of AME signaling is likely to beresponsible for the mild holoprosencephaly observed in Fpn1mutant embryos, as Shh signaling from the AME is required forsplitting the forebrain into the bilateral cerebral hemispheres (for areview, see McMahon et al., 2003). In addition, Fgf8 expression inthe ANR is also reduced in Fpn1 mutants at E8.5, which probablyalso contributes to the reduction of forebrain markers, particularlyFoxg1 in Fpn1 mutants. As Fpn1 is not expressed in the embryoproper throughout neurulation and deletion of Fpn1 in theembryonic lineage does not result in NTDs, the requirement forFpn1 in the establishment of these signaling centers is probablysecondary to the requirement for Fpn1 in transporting iron acrossthe VE to the developing embryo. Importantly, the failure tomaintain anterior neural identity is probably due to the irondeficiency that is predicted to result from the loss of Fpn1 activity.This is supported by the observation that in Fpn1 mutants forebraintruncations can be phenocopied by culturing wild-type embryos inthe presence of iron chelators from E7.5 to E8.5, precisely whenthe anterior neural tissue fails to be maintained in Fpn1 mutants.

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Fig. 8. Fpn1ffe/KI mutants exhibit defects in multiple signalingcenters within the epiblast that maintain and refine the anteriorforebrain. (A,B) Expression of Cerl is reduced in the anterior definitiveendoderm (ADE) but not the AVE (arrow) of Fpn1ffe/KI mutants at E7.5(n=3). (C,D) Expression of Shh does not extend to the anterior limit ofthe neural plate in Fpn1ffe/KI mutant (bracket; D) at E8.5 whencompared with wild-type embryo (C). (E,F) Fgf8 expression in theanterior neural ridge (ANR) is also reduced at E8.5 in Fpn1ffe/KI mutants(arrows). (A,B,E,F) Lateral views of embryos with anterior towards theleft. (C,D) Frontal view.

Fig. 9. Wild-type embryos cultured in iron chelators phenocopiesthe forebrain defects of Fpn1 mutants. (A,B) Six3 expression inwild-type (A) and Fpn1ffe/ffe mutant (B) embryos at E8.5. (C,D) Six3 andKrox20 expression in wild-type embryos cultured in the presence ofwater (vehicle control; C) or 200mM of the iron chelator 1,2-dimethyl-3-hydroxy-4-pyridone (D). Four out of five iron chelator-treated embryosshow reduced Six3 expression compared with those treated withvehicle control.

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Effect of Fpn1 mutation on iron homeostasis inthe early embryoPrevious studies of the expression pattern of Fpn1 andcharacterization of knockout mouse models have demonstrated acrucial role for Fpn1 in absorption of iron across the intestinalepithelium, the placenta and VE (Donovan et al., 2000; Donovanet al., 2005). Fpn1 protein is expressed on the basal surface ofintestinal epithelium, where it is required for the transport ofdietary iron absorbed by these cells into the bloodstream. Forexample, conditional deletion of Fpn1 in the intestine resulted iniron overload in the duodenal enterocytes, owing to the failure torelease iron into the blood stream in the absence of Fpn1 (Donovanet al., 2005). As in the intestinal enterocytes, iron is taken up by theVE from the maternal environment by a number of irontransporters and is exported from these cells to the embryo by theFpn1 protein (Fig. 10). Consequently, similar to duodenalenterocytes, mutation of Fpn1 in the VE should also result in theaccumulation of iron in these cells. However, iron levels in the VEare below the detection limit of traditional histological stainingprotocols such as Prussian Blue, as we have not been able to detectstaining using this technique in the VE (data not shown). Elevatediron levels in the VE could result in the creation of reactive oxygenspecies and oxidative stress, potentially disrupting the activity oforganizing centers within the VE. In support of this model, weobserved defects in morphogenesis of the AVE such that although

the specification and signaling from the AVE in Fpn1 mutantsappears normal, the AVE is diffusely localized to the anteriorregion of the embryo. This results in diffuse and ectopic inductionof anterior neural markers a day later in development.

Another predicted consequence of reduction of Fpn1 exporteractivity in the VE would be a lack of iron transported to thedeveloping embryo resulting in iron deficiency (Fig. 10). Our dataindicate that iron deficiency also plays a role in forebrain defectsin Fpn1 mutants as culture of wild-type embryos between E7.5 andE8.5 can phenocopy these forebrain defects. Meanwhile, signalingcenters within the epiblast that maintain anterior forebrain characterare affected in a non-cell autonomous fashion. These include theAME, ADE and ANR. Together, these data fit a model (Fig. 10)where both iron overload in the AVE and iron deficiency in theembryo proper contribute to the forebrain defects observed in Fpn1mutants.

The Fpn1ffe mouse model provides a uniqueopportunity to study the effects of iron deficiencyon early neural developmentPrevious studies of mouse models of iron homeostasis have failedto provide insight into the requirement for iron during earlydevelopment of the neural tube as these mutations either providetoo severe or too mild defects in iron uptake. For example, Fpn1-null mice exhibit embryonic lethality before neural tube closure

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Fig. 10. Model for the role of Fpn1 in anterior neural development. In wild-type embryos (top), multiple transporters (pink) are involved in theuptake 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 inaccumulation 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 itparticipates in induction of the anterior neuroectoderm (green). By E7.5, the VE has been replaced by definitive endoderm (white) and the prospectiveforebrain (green) begins to express markers of anterior neural identity such as Six3. By E8.5 the neural domain is further stabilized and refined. In Fpn1mutants (bottom), the AVE is diffusely localized to the anterior region of the embryo resulting in the diffuse and ectopic induction of tissue of anteriorneural character at E7.5. However, this anterior neural tissue is not maintained, because by E8.5 the expression of markers of the forebrain aredisproportionately reduced. Between E7.5 and E8.5, iron that is transported from the VE to the embryo is required to maintain forebrain development.

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(Donovan et al., 2005), illustrating the essential role for Fpn1 inthe delivery of iron to the embryo through the VE. In contrast tothe crucial requirement for Fpn1 in transport of iron from the VEto the embryo, multiple different proteins can uptake iron into theVE. Mutant mouse models of iron uptake proteins do not exhibitdefects in neurulation nor does iron deficiency manifest until laterin embryogenesis when significantly more iron is needed forgrowth of the fetus. For example, in most cells, transferrin-boundiron from the bloodstream enters the cell by binding to transferrinreceptor. Transferrin receptor mutants either exhibit no embryonicphenotypes (Trfr2–/–) or do not exhibit defects in neuraldevelopment until after 10.5 (Trfr–/–), when metabolic needsincrease (Levy, 1999; Fleming, 2002). The multi-ligand endocyticreceptor complex that includes cubilin (Cubn) and megalin (Lrp2)is expressed in the VE and mediates endocytosis of a number ofligands, including folic acid, Vitamin B12, cholesterol and lipids,in addition to transferrin bound to iron (for a review, see Kozyrakiand Gofflot, 2007). Interestingly, megalin-null mutant embryosexhibit exencephaly and forebrain defects reminiscent of thoseexhibited by Fpn1ffe/KI mutants (Spoelgen et al., 2005; Willnow etal., 1996). However, the fact that megalin mediates transfer ofnumerous nutrients important for neural tube closure and thatconditional ablation of the gene encoding megalin only in theepiblast lineage also results in NTDs (Spoelgen et al., 2005)suggests a possible different role from that of Fpn1. As the Fpn1ffe

mutation incompletely impairs the ability of Fpn1 to transport ironacross the VE, this hypomorphic allele provides a uniqueopportunity to examine the effects of iron deficiency in Fpn1ffe/ffe

and Fpn1ffe/KI mutants within a crucial range where embryogenesiscan proceed but neural tube closure and forebrain patterning areaffected. Furthermore, we have found that the degree ofmalformation is directly proportional to the amount of functionalFpn1 expressed and theoretically the amount of iron transportedacross the VE. For example, neural tube closure and anteriorneural patterning are more profoundly affected in Fpn1ffe/KI

mutants compared with Fpn1ffe/ffe mutants. Furthermore, ouranalysis of the mechanism of neural pattering defects in thismouse model has provided valuable insight into the developmentalprocesses most sensitive to iron deficiency.

The analysis of Fpn1 mutants in zebrafish has also failed todemonstrate a role for Fpn1 in early embryogenesis. Two loss-of-function fpn1 (slc40a1 – Zebrafish Information Network)mutations have been identified in the zebrafish anemia mutantweissherbst (weh) (Donovan et al., 2000). In zebrafish, fpn1 isexpressed in the YS and is required for the transport of iron fromthe maternally derived yolk stores to the embryonic circulation.Consequently, weh mutants develop hypochromic anemia but failto develop defects earlier in embryogenesis. These interspeciesdifferences in the severity of the phenotype may be due todifferences in maternal contribution between zebrafish andmouse. The timing of when the embryo needs to completely relyon the mutant zygotic product would depend on the stability ofmaternally deposited proteins and transcripts. In the mouse,maternal contribution of transcripts and proteins is minimal andfollowing implantation essentially non-existent. Therefore, it ispossible that the differences in the phenotypes of Fpn1 mutantsin zebrafish versus the mouse are due to differences in maternalcontribution. These species-specific differences in maternalcontribution could explain the early embryonic lethality inFpn1 null mutants and profound patterning defects in Fpn1ffe/KI

versus the lack of early developmental defects in zebrafish wehmutants.

Iron deficiency and NTDs?Our data suggest that iron deficiency is at least partially responsiblefor the forebrain truncations observed in Fpn1 mutants; however,whether defects in neural tube closure are also due to irondeficiency remains to be determined. Data in humans suggest thatthis hypothesis is likely. Of significance, women of childbearingage are disproportionately affected with iron deficiency. Someretrospective epidemiological studies have suggested a linkbetween iron deficiency and the increased risks for a NTD-affectedpregnancy (Felkner et al., 2005; Groenen et al., 2004).Furthermore, clinical trials suggest that peri-conception vitaminsupplementation that includes iron in addition to folic acid canlower the risk of NTDs further than folic acid supplementationalone (for a review, see Czeizel, 2009). These data suggest thatalthough folic acid is an essential nutrient for preventing NTDs,other nutrients such as iron may also play an important role. Inhumans, neural tube closure occurs during the third and fourthweeks of gestation, before many women are aware of thepregnancy. Our data suggest that the VE may play an essential rolein ‘loading’ the embryo with iron to allow for successful neuraltube closure, implying that supplementation with both folic acidand iron prior to conception may reduce the incidence of NTD-affected pregnancies even further.

AcknowledgementsWe are grateful to Drs Anna-Katerina Hadjantonakis and Nancy Andrews forproviding the Ttr-Cre and Fpn1flox alleles, respectively. The Fpn1ffe allele wasgenerated in the Sloan-Kettering Institute Mouse Mutagenesis Project incollaboration with Kathryn Anderson (U01-HD43478) and Gordon Fischell. Wethank Kathryn Anderson, Anamaris Colberg-Poley, Jerry Kaplan and Ivana DeDomenico for helpful discussions. We also thank Gerald Rosenthal, LoriBulwith and Andrew Pollock for technical assistance. This work was partiallysupported by a Basal O’Conner Award from the March of Dimes, by a YoungInvestigator Award from the Spina Bifida Association and by R01-HD058629 toI.E.Z. L.A.N. is a HHMI investigator. Deposited in PMC for release after 6months.

Competing interests statementThe authors declare no competing financial interests.

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