Direct repression of Nanog and Oct4 by OTX2 modulates the contribution of epiblast-derived cells to germline and somatic lineage.

In mammals, the pre-gastrula proximal epiblast gives rise to primordial germ cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work has shown that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here, we show that, in mice, OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and a deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs also occurs in mutant embryos. We propose that the OTX2-mediated repressive control of Oct4 and Nanog is the basis of the mechanism that determines epiblast contribution to germline and somatic lineage.

Establishment of stable iPS-derived human neural stem cell lines suitable for cell therapies.

Establishing specific cell lineages from human induced pluripotent stem cells (hiPSCs) is vital for cell therapy approaches in regenerative medicine, particularly for neurodegenerative disorders. While neural precursors have been induced from hiPSCs, the establishment of hiPSC-derived human neural stem cells (hiNSCs), with characteristics that match foetal hNSCs and abide by cGMP standards, thus allowing clinical applications, has not been described. We generated hiNSCs by a virus-free technique, whose properties recapitulate those of the clinical-grade hNSCs successfully used in an Amyotrophic Lateral Sclerosis (ALS) phase I clinical trial. Ex vivo, hiNSCs critically depend on exogenous mitogens for stable self-renewal and amplification and spontaneously differentiate into astrocytes, oligodendrocytes and neurons upon their removal. In the brain of immunodeficient mice, hiNSCs engraft and differentiate into neurons and glia, without tumour formation. These findings now warrant the establishment of clinical-grade, autologous and continuous hiNSC lines for clinical trials in neurological diseases such as Huntington's, Parkinson's and Alzheimer's, among others.

Functional Antagonism between OTX2 and NANOG Specifies a Spectrum of Heterogeneous Identities in Embryonic Stem Cells.

Embryonic stem cells (ESCs) cultured in leukemia inhibitory factor (LIF) plus fetal bovine serum (FBS) exhibit heterogeneity in the expression of naive and primed transcription factors. This heterogeneity reflects the dynamic condition of ESCs and their versatility to promptly respond to signaling effectors promoting naive or primed pluripotency. Here, we report that ESCs lacking Nanog or overexpressing Otx2 exhibit an early primed identity in LIF + FBS and fail to convert into 2i-induced naive state. Conversely, Otx2-null ESCs possess naive identity features in LIF + FBS similar to Nanog-overexpressing ESCs and convert poorly into FGF-induced early primed state. When both Nanog and Otx2 are inactivated, ESCs cultured in LIF + FBS exhibit primed identity and weakened ability to convert into naive state. These data suggest that, through mutual antagonism, NANOG and OTX2 specify the heterogeneous identity of ESCs cultured in LIF + FBS and individually predispose them for optimal response to naive or primed inducing factors.

Loss of the Otx2-Binding Site in the Nanog Promoter Affects the Integrity of Embryonic Stem Cell Subtypes and Specification of Inner Cell Mass-Derived Epiblast.

Mouse embryonic stem cells (ESCs) and the inner cell mass (ICM)-derived epiblast exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs) and the postimplantation epiblast exhibit primed pluripotency. Although core pluripotency factors are well-characterized, additional regulators, including Otx2, recently have been shown to function during the transition from naive to primed pluripotency. Here we uncover a role for Otx2 in the control of the naive pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 transcriptional network, we targeted the strongest Otx2-binding site in the Nanog promoter, finding that this site modulates the size of specific ESC-subtype compartments in cultured cells and promotes Nanog expression in vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog regulation thus contributes to the integrity of the ESC state and cell lineage specification in preimplantation development.

Mitochondrial Protection by Exogenous Otx2 in Mouse Retinal Neurons.

OTX2 (orthodenticle homeobox 2) haplodeficiency causes diverse defects in mammalian visual systems ranging from retinal dysfunction to anophthalmia. We find that the retinal dystrophy of Otx2(+/GFP) heterozygous knockin mice is mainly due to the loss of bipolar cells and consequent deficits in retinal activity. Among bipolar cell types, OFF-cone bipolar subsets, which lack autonomous Otx2 gene expression but receive Otx2 proteins from photoreceptors, degenerate most rapidly in Otx2(+/GFP) mouse retinas, suggesting a neuroprotective effect of the imported Otx2 protein. In support of this hypothesis, retinal dystrophy in Otx2(+/GFP) mice is prevented by intraocular injection of Otx2 protein, which localizes to the mitochondria of bipolar cells and facilitates ATP synthesis as a part of mitochondrial ATP synthase complex. Taken together, our findings demonstrate a mitochondrial function for Otx2 and suggest a potential therapeutic application of OTX2 protein delivery in human retinal dystrophy.

Dickkopf 3 Promotes the Differentiation of a Rostrolateral Midbrain Dopaminergic Neuronal Subset In Vivo and from Pluripotent Stem Cells In Vitro in the Mouse.

Wingless-related MMTV integration site 1 (WNT1)/ß-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons, including the substantia nigra pars compacta (SNc) subpopulation that preferentially degenerates in Parkinson's disease (PD). However, the precise functions of WNT1/ß-catenin signaling in this context remain unknown. Stem cell-based regenerative (transplantation) therapies for PD have not been implemented widely in the clinical context, among other reasons because of the heterogeneity and incomplete differentiation of the transplanted cells. This might result in tumor formation and poor integration of the transplanted cells into the dopaminergic circuitry of the brain. Dickkopf 3 (DKK3) is a secreted glycoprotein implicated in the modulation of WNT/ß-catenin signaling. Using mutant mice, primary ventral midbrain cells, and pluripotent stem cells, we show that DKK3 is necessary and sufficient for the correct differentiation of a rostrolateral mdDA neuron subset. Dkk3 transcription in the murine ventral midbrain coincides with the onset of mdDA neurogenesis and is required for the activation and/or maintenance of LMX1A (LIM homeobox transcription factor 1α) and PITX3 (paired-like homeodomain transcription factor 3) expression in the corresponding mdDA precursor subset, without affecting the proliferation or specification of their progenitors. Notably, the treatment of differentiating pluripotent stem cells with recombinant DKK3 and WNT1 proteins also increases the proportion of mdDA neurons with molecular SNc DA cell characteristics in these cultures. The specific effects of DKK3 on the differentiation of rostrolateral mdDA neurons in the murine ventral midbrain, together with its known prosurvival and anti-tumorigenic properties, make it a good candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD. Significance statement: We show here that Dickkopf 3 (DKK3), a secreted modulator of WNT (Wingless-related MMTV integration site)/ß-catenin signaling, is both necessary and sufficient for the proper differentiation and survival of a rostrolateral (parabrachial pigmented nucleus and dorsomedial substantia nigra pars compacta) mesodiencephalic dopaminergic neuron subset, using Dkk3 mutant mice and murine primary ventral midbrain and pluripotent stem cells. The progressive loss of these dopamine-producing mesodiencephalic neurons is a hallmark of human Parkinson's disease, which can up to now not be halted by clinical treatments of this disease. Thus, the soluble DKK3 protein might be a promising new agent for the improvement of current protocols for the directed differentiation of pluripotent and multipotent stem cells into mesodiencephalic dopaminergic neurons and for the promotion of their survival in situ.

A WNT1-regulated developmental gene cascade prevents dopaminergic neurodegeneration in adult En1(+/-) mice.

The protracted and age-dependent degeneration of dopamine (DA)-producing neurons of the Substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) in the mammalian midbrain is a hallmark of human Parkinson's Disease (PD) and of certain genetic mouse models of PD, such as mice heterozygous for the homeodomain transcription factor Engrailed 1 (En1(+/-) mice). Neurotoxin-based animal models of PD, in contrast, are characterized by the fast and partly reversible degeneration of the SNc and VTA DA neurons. The secreted protein WNT1 was previously shown to be strongly induced in the neurotoxin-injured adult ventral midbrain (VM), and to protect the SNc and VTA DA neurons from cell death in this context. We demonstrate here that the sustained and ectopic expression of Wnt1 in the SNc and VTA DA neurons of En1(+/Wnt1) mice also protected these genetically affected En1 heterozygote (En1(+/-)) neurons from their premature degeneration in the adult mouse VM. We identified a developmental gene cascade that is up-regulated in the adult En1(+/Wnt1) VM, including the direct WNT1/ß-catenin signaling targets Lef1, Lmx1a, Fgf20 and Dkk3, as well as the indirect targets Pitx3 (activated by LMX1A) and Bdnf (activated by PITX3). We also show that the secreted neurotrophin BDNF and the secreted WNT modulator DKK3, but not the secreted growth factor FGF20, increased the survival of En1 mutant dopaminergic neurons in vitro. The WNT1-mediated signaling pathway and its downstream targets BDNF and DKK3 might thus provide a useful means to treat certain genetic and environmental (neurotoxic) forms of human PD.

Embryonic defects and growth alteration in mice with homozygous disruption of the Patz1 gene.

PATZ1 is an emerging cancer-related gene coding for a POZ/AT-hook/kruppel Zinc finger transcription factor, which is lost or misexpressed in human neoplasias. Here, we investigated its role in development exploring wild-type and Patz1-knockout mice during embryogenesis. We report that the Patz1 gene is ubiquitously expressed at early stages of development and becomes more restricted at later stages, with high levels of expression in actively proliferating neuroblasts belonging to the ventricular zones of the central nervous system (CNS). The analysis of embryos in which Patz1 was disrupted revealed the presence of severe defects in the CNS and in the cardiac outflow tract, which eventually lead to a pre-mature in utero death during late gestation or soon after birth. Moreover, the Patz1-null mice showed a general growth retardation, which was consistent with the slower growth rate and the increased susceptibility to senescence of Patz1(-/-) mouse embryonic fibroblasts (MEFs) compared to wild-type controls. Therefore, these results indicate a critical role of PATZ1 in the control of cell growth and embryonic development.

Cellular heterogeneity during embryonic stem cell differentiation to epiblast stem cells is revealed by the ShcD/RaLP adaptor protein.

The Shc family of adaptor proteins are crucial mediators of a plethora of receptors such as the tyrosine kinase receptors, cytokine receptors, and integrins that drive signaling pathways governing proliferation, differentiation, and migration. Here, we report the role of the newly identified family member, ShcD/RaLP, whose expression in vitro and in vivo suggests a function in embryonic stem cell (ESC) to epiblast stem cells (EpiSCs) transition. The transition from the naïve (ESC) to the primed (EpiSC) pluripotent state is the initial important step for ESCs to commit to differentiation and the mechanisms underlying this process are still largely unknown. Using a novel approach to simultaneously assess pluripotency, apoptosis, and proliferation by multiparameter flow cytometry, we show that ESC to EpiSC transition is a process involving a tight coordination between the modulation of the Oct4 expression, cell cycle progression, and cell death. We also describe, by high-content immunofluorescence analysis and time-lapse microscopy, the emergence of cells expressing caudal-related homeobox 2 (Cdx2) transcription factor during ESC to EpiSC transition. The use of the ShcD knockout ESCs allowed the unmasking of this process as they presented deregulated Oct4 modulation and an enrichment in Oct4-negative Cdx2-positive cells with increased MAPK/extracellular-regulated kinases 1/2 activation, within the differentiating population. Collectively, our data reveal ShcD as an important modulator in the switch of key pathway(s) involved in determining EpiSC identity.

Fgf15-mediated control of neurogenic and proneural gene expression regulates dorsal midbrain neurogenesis.

The balanced proliferation and cell cycle exit of neural progenitors, by generating the appropriate amount of postmitotic progeny at the correct time and in the proper location, is required for the establishment of the highly ordered structure of the adult brain. Little is known about the extrinsic signals regulating these processes, particularly in the midbrain. Fibroblast growth factor (Fgf) 15, the mouse ortholog of FGF19 and member of an atypical Fgf subfamily, is prominently expressed in the dorsolateral midbrain of the midgestational mouse embryo. In the absence of Fgf15, dorsal midbrain neural progenitors fail to exit the cell cycle and to generate the proper amount of postmitotic neurons. We show here that this is due to the altered expression of inhibitory/neurogenic and proneural/neuronal differentiation helix-loop-helix transcription factor (TF) genes. The expression of Id1, Id3, and Hes5 was strongly increased and ectopically expanded, whereas the expression of Ascl1 (Mash1), Neurog1 (Ngn1) and Neurog2 (Ngn2) was strongly decreased and transcription of Neurod1 (NeuroD) was completely abolished in the dorsolateral midbrain of Fgf15(-/-) mice. These abnormalities were not caused by the mis-expression of cell cycle regulatory proteins such as cyclin-dependent kinase inhibitors or retinoblastoma proteins. Furthermore, human FGF19 promotes cell cycle exit of murine dorsal neural progenitors in vitro. Therefore, our data suggest that Fgf15 is a crucial signaling molecule regulating the postmitotic transition of dorsal neural progenitors and thus the initiation and proper progression of dorsal midbrain neurogenesis in the mouse, by controlling the expression of neurogenic and proneural TFs.

Expression of the brain transcription factor OTX1 occurs in a subset of normal germinal-center B cells and in aggressive Non-Hodgkin Lymphoma.

The roles in brain development. Previous studies have shown the association between OTX2 and OTX1 with anaplastic and desmoplastic medulloblastomas, respectively. Here, we investigated OTX1 and OTX2 expression in Non-Hodgkin Lymphoma (NHL) and multiple myeloma. A combination of semiquantitative RT-PCR, Western blot, and immunohistochemical analyses was used to measure OTX1 and OTX2 levels in normal lymphoid tissues and in 184 tumor specimens representative of various forms of NHL and multiple myeloma. OTX1 expression was activated in 94% of diffuse large B-cell lymphomas, in all Burkitt lymphomas, and in 90% of high-grade follicular lymphomas. OTX1 was undetectable in precursor-B lymphoblastic lymphoma, chronic lymphocytic leukemia, and in most marginal zone and mantle cell lymphomas and multiple myeloma. OTX2 was undetectable in all analyzed malignancies. Analysis of OTX1 expression in normal lymphoid tissues identified a subset of resting germinal center (GC) B cells lacking PAX5 and BCL6 and expressing cytoplasmic IgG and syndecan. About 50% of OTX1(+) GC B cells co-expressed CD10 and CD20. This study identifies OTX1 as a molecular marker for high-grade GC-derived NHL and suggests an involvement of this transcription factor in B-cell lymphomagenesis. Furthermore, OTX1 expression in a subset of normal GC B cells carrying plasma cell markers suggests its possible contribution to terminal B-cell differentiation.

Anterior-posterior graded response to Otx2 controls proliferation and differentiation of dopaminergic progenitors in the ventral mesencephalon.

Meso-diencephalic dopaminergic (mdDA) neurons control voluntary movement, cognition and the reward response, and their degeneration is associated with Parkinson's disease (PD). Prospective cell transplantation therapies for PD require full knowledge of the developmental pathways that control mdDA neurogenesis. We have previously shown that Otx2 is required for the establishment of the mesencephalic field and molecular code of the entire ventral mesencephalon (VM). Here, we investigate whether Otx2 is a specific determinant of mesencephalic dopaminergic (mesDA) neurogenesis by studying mouse mutants that conditionally overexpress or lack Otx2. Our data show that Otx2 overexpression in the VM causes a dose-dependent and selective increase in both mesDA progenitors and neurons, which correlates with a remarkable and specific enhancement in the proliferating activity of mesDA progenitors. Consistently, lack of Otx2 in the VM specifically affects the proliferation of Sox2+ mesDA progenitors and causes their premature post-mitotic transition. Analysis of the developmental pathway that controls the differentiation of mesDA neurons shows that, in the absence of Otx2, the expression of Lmx1a and Msx1, and the proneural genes Ngn2 and Mash1 is not activated in Sox2+ mesDA progenitors, which largely fail to differentiate into Nurr1+ mesDA precursors. Furthermore, proliferation and differentiation abnormalities exhibit increasing severity along the anterior-posterior (AP) axis of the VM. These findings demonstrate that Otx2, through an AP graded effect, is intrinsically required to control proliferation and differentiation of mesDA progenitors. Thus, our data provide new insights into the mechanism of mesDA neuron specification and suggest Otx2 as a potential target for cell replacement-based therapeutic approaches in PD.

Serotonin hyperinnervation abolishes seizure susceptibility in Otx2 conditional mutant mice.

The homeobox-containing transcription factor Otx2 is crucially involved in fate determination of midbrain neurons. Mutant mice, in which Otx2 was conditionally inactivated by a Cre recombinase expressed under the transcriptional control of the Engrailed1 (En1) gene (En1(cre/+); Otx2(flox/flox)), show a reduced number of dopaminergic neurons and an increased number of serotonergic neurons in the ventral midbrain. Despite these developmental anatomical alterations, En1(cre/+); Otx2(flox/flox) adult mice display normal motor function. Here, we further investigated the neurological consequences of Otx2 inactivation in adult En1(cre/+); Otx2(flox/flox) mice. Adult En1(cre/+); Otx2(flox/flox) mice showed increased serotonin (5-HT) levels in the pons, ventral midbrain, hippocampus (CA3 subfield), and cerebral cortex, as indicated by HPLC and immunohistochemistry. Conversely, SERT (5-HT transporter) levels were decreased in conditional mutant brains. As a consequence of this increased 5-HT hyperinnervation, En1(cre/+); Otx2(flox/flox) mice were resistant to generalized seizures induced by the glutamate agonist kainic acid (KA). Indeed, prolonged pretreatment of En1(cre/+); Otx2(flox/flox) mice with the 5-HT synthesis inhibitor para-chlorophenylalanine (pCPA) restored brain 5-HT content to control levels, fully reestablishing KA seizure susceptibility. Accordingly, c-fos mRNA induction after KA was restricted to the hippocampus in En1(cre/+); Otx2(flox/flox) mice, whereas a widespread c-fos mRNA labeling was observed throughout the brain of En1(cre/+); Otx2(flox/flox) mice pretreated with pCPA. These results clearly show that increased brain 5-HT levels are responsible for seizure resistance in En1(cre/+); Otx2(flox/flox) mice and confirm the important role of 5-HT in the control of seizure spread.

Altered dopaminergic innervation and amphetamine response in adult Otx2 conditional mutant mice.

Here, we have investigated the neurological consequences of restricted inactivation of Otx2 in adult En1(cre/+); Otx2(flox/flox) mice. In agreement with the crucial role of Otx2 in midbrain patterning, the mutants had a substantial reduction in tyrosine hydroxylase containing neurons. Although the reduction in the number of DAergic neurons was comparable between the SNc and the VTA, we found an unexpected selectivity in the deinnervation of the terminal fields affecting preferentially the ventral striatum and the olfactory tubercle. Interestingly, the mutants showed no abnormalities in exploratory activity or motor coordination. However, the absence of normal DA tone generated significant alterations in DA D1-receptor signalling as indicated by increased mutant striatal levels of phosphorylated DARPP-32 and by an altered motor response to amphetamine. Therefore, we suggest that the En1(cre/+); Otx2(flox/flox) mutant mouse model represents a genetic tool for investigating molecular and behavioural consequences of developmental neuronal dysfunction in the DAergic system.

A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo.

Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinson's disease.

Otx2 regulates subtype specification and neurogenesis in the midbrain.

The transcription factor Otx2 is required to determine mesencephalic versus metencephalic (cerebellum/pons) territory during embryogenesis. This function of Otx2 primarily involves positioning and maintaining the mid-hindbrain organizer at the border between midbrain and anterior hindbrain. Otx2 expression is maintained long after this organizer is established. We therefore generated conditional mutants of Otx2 using the Cre/loxP system to study later roles during rostral brain development. For inactivation of Otx2 in neuronal progenitor cells, we crossed Otx2(flox/flox) animals with Nestin-Cre transgenic animals. In Nestin-Cre/+; Otx2(flox/flox) embryos, Otx2 activity was lost from the ventral midbrain starting at embryonic day 10.5 (E10.5). In these mutant embryos, the mid-hindbrain organizer was properly positioned at E12.5, although Otx2 is absent from the midbrain. Hence, the Nestin-Cre/+; Otx2(flox/flox) animals represent a novel mouse model for studying the role of Otx2 in the midbrain, independently of abnormal development of the mid-hindbrain organizer. Our data demonstrate that Otx2 controls the development of several neuronal populations in the midbrain by regulating progenitor identity and neurogenesis. Dorsal midbrain progenitors ectopically expressed Math1 and generate an ectopic cerebellar-like structure. Similarly, Nkx2.2 ectopic expression ventrally into tegmentum progenitors is responsible for the formation of serotonergic neurons and hypoplasia of the red nucleus in the midbrain. In addition, we discovered a novel role for Otx2 in regulating neurogenesis of dopaminergic neurons. Altogether, these results demonstrate that Otx2 is required from E10.5 onward to regulate neuronal subtype identity and neurogenesis in the midbrain.

Unsuspected role of the brain morphogenetic gene Otx1 in hematopoiesis.

Otx1 belongs to the paired class of homeobox genes and plays a pivotal role in brain development. Here, we show that Otx1 is expressed in hematopoietic pluripotent and erythroid progenitor cells. Moreover, bone marrow cells from mice lacking Otx1 exhibit a cell-autonomous impairment of the erythroid compartment. In agreement with these results, molecular analysis revealed decreased levels of erythroid genes that include the SCL and GATA-1 transcription factors. Accordingly, a gain of function of SCL rescues the erythroid deficiency in Otx1-/- mice. Taken together, our findings indicate a function for Otx1 in the regulation of blood cell production.

Folic acid prevents exencephaly in Cited2 deficient mice.

Cited2 (also Mrg1/p35srj) is a member of a new conserved gene family that is expressed during mouse development and in adult tissues. In order to investigate the function of Cited2 during mouse embryogenesis, we introduced a null mutation into the Cited2 locus. Cited2(-/-) mutants died at late gestation and exhibited heart defects and exencephaly, arising from defective closure of the midbrain (MB) and hindbrain. Initiation of neural tube closure at the forebrain-midbrain (FB-MB) boundary, an essential step for closure of the cranial neural tube, was impaired in the Cited2(-/-) mutants. Gene marker analysis using in situ hybridization revealed that the patterning of the anterior neural plate and head mesenchyme was little affected or normal in the Cited2(-/-) embryos. However, Cited2 was required for the survival of neuroepithelial cells and its absence led to massive apoptosis in dorsal neuroectoderm around the FB-MB boundary and in a restricted transverse domain in the hindbrain. Treatment with folic acid significantly reduced the exencephalic phenotype in the Cited2(-/-) embryos both in vivo and in vitro. However, assessment of folate metabolism revealed no defect in the Cited2(-/-) mutants, and the elevated apoptosis observed in the neuroepithelium of the Cited2(-/-) mutants was apparently not decreased by folic acid supplementation. To our knowledge, the Cited2 mouse represents the first genetic model in which folic acid can prevent a defect in neural tube closure by a mechanism other than the neutralization of a defect in folate homeostasis.