Forebrain Marker Research Articles

Neuronal developmental gene and miRNA signatures induced by histone deacetylase inhibitors in human embryonic stem cells.

Human embryonic stem cells (hESCs) may be applied to develop human-relevant sensitive in vitro test systems for monitoring developmental toxicants. The aim of this study was to identify potential developmental toxicity mechanisms of the histone deacetylase inhibitors (HDAC) valproic acid (VPA), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) relevant to the in vivo condition using a hESC model in combination with specific differentiation protocols and genome-wide gene expression and microRNA profiling. Analysis of the gene expression data showed that VPA repressed neural tube and dorsal forebrain (OTX2, ISL1, EMX2 and SOX10)-related transcripts. In addition, VPA upregulates axonogenesis and ventral forebrain-associated genes, such as SLIT1, SEMA3A, DLX2/4 and GAD2. HDACi-induced expression of miR-378 and knockdown of miR-378 increases the expression of OTX2 and EMX2, which supports our hypothesis that HDACi targets forebrain markers through miR-378. In conclusion, multilineage differentiation in vitro test system is very sensitive for monitoring molecular activities relevant to in vivo neuronal developmental toxicity. Moreover, miR-378 seems to repress the expression of the OTX2 and EMX2 and therefore could be a regulator of the development of neural tube and dorsal forebrain neurons.

Activity-dependent expression of neuronal PAS domain-containing protein 4 (npas4a) in the developing zebrafish brain.

In rodents, the Npas4 gene has recently been identified as being an important regulator of synaptic plasticity and memory. Homologs of Npas4 have been found in invertebrate species though their functions appear to be too divergent for them to be studied as a proxy for the mammalian proteins. The aim of this study, therefore, was to ascertain the suitability of the zebrafish as a model organism for investigating the function of Npas4 genes. We show here that the expression and regulation of the zebrafish Npas4 homolog, npas4a, is remarkably similar to that of the rodent Npas4 genes. As in mammals, expression of the zebrafish npas4a gene is restricted to the brain where it is up-regulated in response to neuronal activity. Furthermore, we also show that knockdown of npas4a during embryonic development results in a number of forebrain-specific defects including increased apoptosis and misexpression of the forebrain marker genes dlx1a and shha. Our work demonstrates that the zebrafish is a suitable model organism for investigating the role of the npas4a gene and one that is likely to provide valuable insights into the function of the mammalian homologs. Furthermore, our findings highlight a potential role for npas4a in forebrain development.

Stepwise, non-adherent differentiation of human pluripotent stem cells to generate basal forebrain cholinergic neurons via hedgehog signaling

Basal forebrain cholinergic neurons (bfCNs) which provide innervation to the hippocampus and cortex, are required for memory and learning, and are primarily affected in Alzheimer's Disease (AD), resulting in related cognitive decline. Therefore generation of a source of bfCNs from human pluripotent stem cells (hPSCs) is crucial for in vitro disease modeling and development of novel AD therapies. In addition, for the advancement of regenerative approaches there is a requirement for an accurate developmental model to study the neurogenesis and survival of this population. Here we demonstrate the efficient production of bfCNs, using a novel embryoid body (EB) based non-adherent differentiation (NAdD) protocol. We establish a specific basal forebrain neural stem cell (NSC) phenotype via expression of the basal forebrain transcription factors NKX2.1 and LHX8, as well as the general forebrain marker FOXG1. We present evidence that this lineage is achieved via recapitulation of embryonic events, with induction of intrinsic hedgehog signaling, through the use of a 3D non-adherent differentiation system. This is the first example of hPSC-derived basal forebrain-like NSCs, which are scalable via self-renewal in prolonged culture. Furthermore upon terminal differentiation these basal forebrain-like NSCs generate high numbers of cholinergic neurons expressing the specific markers ChAT, VACht and ISL1. These hPSC-derived bfCNs possess characteristics that are crucial in a model to study AD related cholinergic neuronal loss in the basal forebrain. Examples are expression of the therapeutic target p75NTR, the release of acetylcholine, and demonstration of a mature, and functional electrophysiological profile. In conclusion, this work provides a renewable source of human functional bfCNs applicable for studying AD specifically in the cholinergic system, and also provides a model of the key embryonic events in human bfCN development.

BMP signal attenuates FGF pathway in anteroposterior neural patterning

In vertebrate early development, the neural tissue is specified along the antero-posterior (A-P) axis by the activity of graded patterning signals such as Wnt, Nodal and FGF. Attenuation of these signals has been shown to play critical roles in the determination of anterior neural region, but it remains poorly understood how FGF action is counteracted in the neural plate. Here, we show that BMP signal acts as an antagonist of FGF signaling for AP neural patterning in Xenopus embryo. During the neurula stages, BMP signal was up-regulated in the anterior neural plate, displaying a graded pattern along the AP axis. Inhibition of the late BMP signaling after mid-gastrulation abrogated the expression of anterior neural markers. We found that BMP signaling interfered with FGFs-induced ERK phosphorylation and neural caudalization. This inhibitory action of BMP signal involved repression of the expression of Flrt3, a positive regulator of FGF signaling. Furthermore, the gain- and loss-of-function of Flrt3 inhibited and expanded the expression of forebrain marker genes, respectively. Together, these results demonstrate that BMP signal can down-regulate FGF pathway via inhibition of Flrt3 expression for anterior neural formation, revealing stage-specific roles of BMP signaling and its novel crosstalk with FGF pathway in neural development.

Prevention of β-amyloid induced toxicity in human iPS cell-derived neurons by inhibition of Cyclin-dependent kinases and associated cell cycle events

Alzheimer's disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline due to the selective neuronal loss in the cortex and hippocampus of the brains. Generation of human induced pluoripotent stem (hiPS) cells holds great promise for disease modeling and drug discovery in AD. In this study, we used neurons with forebrain marker expression from two unrelated hiPS cell lines. As both populations of neurons were vulnerable to β-amyloid 1–42 (Aβ1–42) aggregates, a hallmark of AD pathology, we used them to investigate cellular mediators of Aβ1–42 toxicity. We observed in neurons differentiated from both hiPS cell lines that Aβ induced toxicity correlated with cell cycle re-entry and was inhibited by pharmacological inhibitors or shRNAs against Cyclin-dependent kinase 2 (Cdk2). As one of the hiPS cell lines has been developed commercially to supply large quantities of differentiated neurons (iCell® Neurons), we screened a chemical library containing several hundred compounds and discovered several small molecules as effective blockers against Aβ1–42 toxicity, including a Cdk2 inhibitor. To our knowledge, this is the first demonstration of an Aβ toxicity screen using hiPS cell-derived neurons. This study provided an excellent example of how hiPS cells can be used for disease modeling and high-throughput compound screening for neurodegenerative diseases.

Anti-Aβ drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease.

BackgroundAlzheimer's disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline during middle to late adult life. The AD brain is characterized by deposition of amyloid β peptide (Aβ), which is produced from amyloid precursor protein by β- and γ-secretase (presenilin complex)-mediated sequential cleavage. Induced pluripotent stem (iPS) cells potentially provide an opportunity to generate a human cell-based model of AD that would be crucial for drug discovery as well as for investigating mechanisms of the disease.Methodology/Principal FindingsWe differentiated human iPS (hiPS) cells into neuronal cells expressing the forebrain marker, Foxg1, and the neocortical markers, Cux1, Satb2, Ctip2, and Tbr1. The iPS cell-derived neuronal cells also expressed amyloid precursor protein, β-secretase, and γ-secretase components, and were capable of secreting Aβ into the conditioned media. Aβ production was inhibited by β-secretase inhibitor, γ-secretase inhibitor (GSI), and an NSAID; however, there were different susceptibilities to all three drugs between early and late differentiation stages. At the early differentiation stage, GSI treatment caused a fast increase at lower dose (Aβ surge) and drastic decline of Aβ production.Conclusions/SignificanceThese results indicate that the hiPS cell-derived neuronal cells express functional β- and γ-secretases involved in Aβ production; however, anti-Aβ drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation.

Midbrain and forebrain patterning delivers immunocytochemically and functionally similar populations of neuropeptide Y containing GABAergic neurons

Neurons differentiated in vitro from embryonic stem cells (ESCs) have the potential to serve both as models of disease states and in drug discovery programs. In this study, we use sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF-8) to enrich for forebrain and midbrain phenotypes from mouse ESCs. We then investigate, using Ca 2+ imaging and [ 3H]-GABA release studies, whether the GABAergic neurons produced exhibit distinct functional phenotypes. At day 24 of differentiation, reverse transcriptase-PCR showed the presence of both forebrain (Bf-1, Hesx1, Pgc-1α, Six3) and midbrain (GATA2, GATA3) selective mRNA markers in developing forebrain-enriched cultures. All markers were present in midbrain cultures except for Bf-1 and Pgc-1α. Irrespective of culture conditions all GABA immunoreactive neurons were also immunoreactive to neuropeptide Y (NPY) antibodies. Forebrain and midbrain GABAergic neurons responded to ATP (1 mM), l-glutamate (30 μM), noradrenaline (30 μM), acetylcholine (30 μM) and dopamine (30 μM), with similar elevations of intracellular Ca 2+([Ca 2+] i ). The presence of GABA A and GABA B antagonists, bicuculline (30 μM) and CGP55845 (1 μM), increased the elevation of [Ca 2+] i in response to dopamine (30 μM) in midbrain, but not forebrain GABAergic neurons. All agonists, except dopamine, elicited similar [ 3H]-GABA release from forebrain and midbrain cultures. Dopamine (30 μM) did not stimulate significant [ 3H]-GABA release in midbrain cultures, although it was effective in forebrain cultures. This study shows that differentiating neurons toward a midbrain fate restricts the expression of forebrain markers. Forebrain differentiation results in the expression of forebrain and midbrain markers. All GABA + neurons contain NPY, and show similar agonist-induced elevations of [Ca 2+] i and [ 3H]-GABA release. This study indicates that the pharmacological phenotype of these particular neurons may be independent of the addition of the patterning factors that direct neurons toward forebrain and midbrain fates.

Anterior neural development requires Del1, a matrix-associated protein that attenuates canonical Wnt signaling via the Ror2 pathway

During early embryogenesis, the neural plate is specified along the anterior-posterior (AP) axis by the action of graded patterning signals. In particular, the attenuation of canonical Wnt signals plays a central role in the determination of the anterior brain region. Here, we show that the extracellular matrix (ECM) protein Del1, expressed in the anterior neural plate, is essential for forebrain development in the Xenopus embryo. Overexpression of Del1 expands the forebrain domain and promotes the formation of head structures, such as the eye, in a Chordin-induced secondary axis. Conversely, the inhibition of Del1 function by a morpholino oligonucleotide (MO) represses forebrain development. Del1 also augments the expression of forebrain markers in neuralized animal cap cells, whereas Del1-MO suppresses them. We previously reported that Del1 interferes with BMP signaling in the dorsal-ventral patterning of the gastrula marginal zone. By contrast, we demonstrate here that Del1 function in AP neural patterning is mediated mainly by the inhibition of canonical Wnt signaling. Wnt-induced posteriorization of the neural plate is counteracted by Del1, and the Del1-MO phenotype (posteriorization) is reversed by Dkk1. Topflash reporter assays show that Del1 suppresses luciferase activities induced by Wnt1 and beta-catenin. This inhibitory effect of Del1 on canonical Wnt signaling, but not on BMP signaling, requires the Ror2 pathway, which is implicated in non-canonical Wnt signaling. These findings indicate that the ECM protein Del1 promotes forebrain development by creating a local environment that attenuates the cellular response to posteriorizing Wnt signals via a unique pathway.

Sonic hedgehog expression during Xenopus laevis forebrain development

We have analyzed the developing expression pattern of x-Shh in the Xenopus forebrain, interpreting the results within the framework of the neuromeric model to assess evolutionary trends and clues. To achieve this goal, we have characterized phenotypically the developing x-Shh expressing forebrain subdivisions and neurons by means of the combination of in situ hybridization for x-Shh and immunohistochemistry for the detection of forebrain essential regulators and markers, such as the homeodomain transcription factors Islet 1, Orthopedia, NKX2.1 and NKX2.2 and tyrosine hydroxylase. Substantial evidence was found for x-Shh expression in the telencephalic commissural preoptic area and this is strongly correlated with the presence of a pallidum and/or a basal telencephalic cholinergic system. In the diencephalon, x-Shh was demonstrated in the zona limitans intrathalamica and the x-Shh expressing cells were extended into the prethalamus. Throughout development and in the adult hypothalamic x-Shh expression was strong in basal regions but, in addition, in the alar suprachiasmatic region. The findings obtained in the forebrain of Xenopus revealed a largely conserved pattern of Shh expression among tetrapods. However, interesting differences were also noted that could be related to evolutive changes in forebrain organization.

Stage-specific effects of retinoic acid on gene expression during forebrain development

Treatment of early gastrula- and neurula-staged Xenopus embryos with all- trans retinoic acid (RA) results in truncation of the anterior structures of the forebrain and head. The extent of truncation is dependent upon both the stage of immersion and the RA concentration used. As a method to investigate genes important during early forebrain regionalization, late gastrula and neurula embryos were immersed for 2 h within low (1 × 10 −9 M to 5 × 10 −8 M) concentrations of RA. Embryos were allowed to develop to tadpole stages and forebrain marker genes were assessed for any alteration in patterns of expression. Comparisons of controls to experimental groups indicated that the greatest sensitivity to low levels of RA occurred just before the initial expression of the forebrain-specific markers investigated. We concluded that forebrain regionalization and gene expression occurred in the following order: Xotx2 → Xsix3 → Xrx (& Xfez1) → Xbf1 → Xemx1. Xsix3 seems to be very important for the initial parcellation of telencephalon, retinal and diencephalon areas.

Abnormal laminar position and dendrite development of interneurons in the reeler forebrain

The majority of cortical and hippocampal interneurons originate in the subcortical telencephalon and migrate tangentially into pallial regions before settling in various cortical layers. The molecular cues that regulate final positioning of specific interneurons in cortical structures have not yet been identified. The positioning of radially migrating principal neurons of the cortex and hippocampus depends upon Reelin, an extracellular protein expressed near the pial surface during embryonic development that is absent in reeler mutant mice. To determine whether the layer specification of interneurons, like that of principal neurons, requires Reelin, we crossed reeler with transgenic mice that contain Green Fluorescent Protein (GFP)-expressing Inhibitory Neurons (GINs). These neurons express basal forebrain markers Dlx1/2 in normal and reeler mice. In normal mice, GINs express Reelin and are localized to specific layers of the cortex and hippocampus. In reeler mutant mice, we show that GINs migrate normally into the pallium, but fail to acquire proper layer position. Double labeling experiments indicate that the neurochemical profile of these interneurons is not generally altered in reeler mice. However, the extension of their cellular processes is abnormal. Quantitative analysis of GINs in the cortex revealed that they are hypertrophic, bearing longer neuritic branches than normal. Thus, the lack of Reelin signaling results in abnormal positioning and altered morphology of forebrain interneurons.

Abnormal laminar position and dendrite development of interneurons in the reeler forebrain

The majority of cortical and hippocampal interneurons originate in the subcortical telencephalon and migrate tangentially into pallial regions before settling in various cortical layers. The molecular cues that regulate final positioning of specific interneurons in cortical structures have not yet been identified. The positioning of radially migrating principal neurons of the cortex and hippocampus depends upon Reelin, an extracellular protein expressed near the pial surface during embryonic development that is absent in reeler mutant mice. To determine whether the layer specification of interneurons, like that of principal neurons, requires Reelin, we crossed reeler with transgenic mice that contain Green fluorescent protein (GFP)-expressing Inhibitory Neurons (GINs). These neurons express basal forebrain markers Dlx1/2 in normal and reeler mice. In normal mice, GINs express Reelin and are localized to specific layers of the cortex and hippocampus. In reeler mutant mice, we show that GINs migrate normally into the pallium, but fail to acquire proper layer position. Double labeling experiments indicate that the neurochemical profile of these interneurons is not generally altered in reeler mice. However, the extension of their cellular processes is abnormal. Quantitative analysis of GINs in the cortex revealed that they are hypertrophic, bearing longer neuritic branches than normal. Thus, the lack of Reelin signaling results in abnormal positioning and altered morphology of forebrain interneurons.

Distinct effects of caudalizing factors on regional specification of embryonic stem cell-derived neural precursors

Recent embryological studies have implicated several “caudalizing factors” in the caudal specification of the central nervous system (CNS). In this study, we have examined the effects of three candidate caudalizing factors on neural precursors induced from embryonic stem (ES) cells by the stromal cell-derived inducing activity (SDIA) method. Among retinoic acid (RA), Wnt and FGF signals, RA causes the strongest level of caudalization: inducing suppression of forebrain differentiation and promotion of caudal CNS specification. Obvious suppression of the telencephalic marker Bf1 and that of the forebrain marker Otx2 occur at 2×10 −8 and 2×10 −7 M, respectively. Activation of the caudal marker genes such as Hoxb9 is observed in a dose-dependent manner over the range of 2×10 −9–2×10 −6 M. Suppression of the forebrain genes has a narrow critical period of RA response during the early culture phase. In contrast, significant induction of the caudal genes is evoked by a 1-day exposure to RA at any time between days 3 and 8. RA treatment not only induces caudal specification but also inhibits differentiation of ventral CNS tissues, particularly of floor plate cells. FGF4 induces partial caudalization while Wnt-3A exhibits weak caudalizing activities only in the presence of RA. These findings provide useful information on the proper selection of combination of signaling molecules, doses and timing for steering ES cell differentiation by caudalizing factors into caudal neural fates.

Isthmus-to-midbrain transformation in the absence of midbrain-hindbrain organizer activity.

In zebrafish acerebellar (ace) embryos, because of a point mutation in fgf8, the isthmic constriction containing the midbrain-hindbrain boundary (MHB) organizer fails to form. The mutants lack cerebellar development by morphological criteria, and they appear to have an enlarged tectum, showing no obvious reduction in the tissue mass at the dorsal mesencephalic/metencephalic alar plate. To reveal the molecular identity of the tissues located at equivalent rostrocaudal positions along the neuraxis as the isthmic and cerebellar primordia in wild-types, we undertook a detailed analysis of ace embryos. In ace mutants, the appearance of forebrain and midbrain specific marker genes (otx2, dmbx1, wnt4) in the caudal tectal enlargement reveals a marked rostralized gene expression profile during early somitogenesis, followed by the lack of early and late cerebellar-specific gene expression (zath1/atoh1, gap43, tag1/cntn2, neurod, zebrin II). The Locus coeruleus (LC) derived from rostral rhombomere 1 is also absent in the mutants. A new interface between otx2 and epha4a suggests that the rostralization stops at the caudal part of rhombomere 1. The mesencephalic basal plate is also affected in the mutant embryos, as indicated by the caudal expansion of the diencephalic expression domains of epha4a, zash1b/ashb, gap43 and tag1/cntn2, and by the dramatic reduction of twhh expression. No marked differences are seen in cell proliferation and apoptotic patterns around the time the rostralization of gene expression becomes evident in the mutants. Therefore, locally distinct cell proliferation and cell death is unlikely to be the cause of the fate alteration of the isthmic and cerebellar primordia in the mutants. Dil cell-lineage labeling of isthmic primordial cells reveals that cells, at the location equivalent of the wild-type MHB, give rise to caudal tectum in ace embryos. This suggests that a caudalto-rostral transformation leads to the tectal expansion in the mutants. Fgf8-coated beads are able to rescue morphological MHB formation, and elicit the normal molecular identity of the isthmic and cerebellar primordium in ace embryos. Taken together, our analysis reveals that cells of the isthmic and cerebellar primordia acquire a more rostral, tectal identity in the absence of the functional MHB organizer signal Fgf8.

Pygopus is required for embryonic brain patterning in Xenopus

We have identified two Xenopus mRNAs that encode proteins homologous to a component of the Wnt/β-catenin transcriptional machinery known as Pygopus. The predicted proteins encoded by both mRNAs share the same structural properties with human Pygo-2, but with Xpygo-2α having an additional 21 N-terminal residues. Xpygo-2α messages accumulate in the prospective anterior neural plate after gastrulation and then are localized to the nervous system, rostral to and including the hindbrain. Xpygo-2β mRNA is expressed in oocytes and early embryos but declines in level before and during gastrulation. In late neurula, Xpygo-2β mRNA is restricted to the retinal field, including eye primordia and prospective forebrain. A C-terminal truncated mutant of Xpygo-2 containing the N-terminal Homology Domain (NHD) caused both axis duplication when injected at the 2-cell stage and inhibition of anterior neural development when injected in the prospective head, mimicking the previously described effects of Wnt-signaling activators. Inhibition of Xpygo-2α and Xpygo-2β by injection of gene-specific antisense morpholino oligonucleotides into prospective anterior neurectoderm caused brain defects that were prevented by coinjection of Xpygo-2 mRNA. Both Xpygo-2α and Xpygo-2β morpholinos reduced the eye and forebrain markers Xrx-1, Xpax-6, and XBF-1, while the Xpygo-2α morpholino also eliminated expression of the mid–hindbrain marker En-2. The differential expression and regulatory activities of Xpygo-2α/β in rostral neural tissue indicate that they represent essential components of a novel mechanism for Wnt signaling in regionalization of the brain.

Induction and patterning of the telencephalon in Xenopus laevis.

We report an analysis of the tissue and molecular interplay involved in the early specification of the forebrain, and in particular telencephalic, regions of the Xenopus embryo. In dissection/recombination experiments, different parts of the organizer region were explanted at gastrula stage and tested for their inducing/patterning activities on either naive ectoderm or on midgastrula stage dorsal ectoderm. We show that the anterior dorsal mesendoderm of the organizer region has a weak neural inducing activity compared with the presumptive anterior notochord, but is able to pattern either neuralized stage 10.5 dorsal ectoderm or animal caps injected with BMP inhibitors to a dorsal telencephalic fate. Furthermore, we found that a subset of this tissue, the anterior dorsal endoderm, still retains this patterning activity. At least part of the dorsal telencephalic inducing activities may be reproduced by the anterior endoderm secreted molecule cerberus, but not by simple BMP inhibition, and requires the N-terminal region of cerberus that includes its Wnt-binding domain. Furthermore, we show that FGF action is both necessary and sufficient for ventral forebrain marker expression in neuralized animal caps, and possibly also required for dorsal telencephalic specification. Therefore, integration of organizer secreted molecules and of FGF, may account for patterning of the more rostral part of Xenopus CNS.

Disruption of Early Events in Thalamocortical Tract Formation in Mice Lacking the Transcription Factors Pax6 or Foxg1

Early events in the formation of the thalamocortical tract remain poorly understood. Recent work has suggested that thalamocortical axons follow a path pioneered by transient thalamic afferents originating from the medial part of the ventral telencephalon. We studied the development of these transient afferents and the thalamocortical tract in mutant mice lacking transcription factors normally expressed in the dorsal thalamus or ventral telencephalon. Pax6 is expressed in the dorsal thalamus, but not in the medial part of the ventral telencephalon, and the thalamocortical tract fails to form in Pax6(-/-) embryos. We found that transient thalamic afferents from the ventral telencephalon do not form in Pax6(-/-) embryos; this may contribute to the failure of their thalamocortical development. The distribution of Pax6(-/-) cells in Pax6(-/-)<--> Pax6(+/+) chimeras supports conclusions drawn from forebrain marker gene expression that Pax6 is not required for the normal development of the medial part of the ventral telencephalon but is required in the dorsal thalamus. Failure of the transient afferent pathway to develop is therefore likely a cell nonautonomous defect reflecting primary defects in the thalamus. We then examined the formation of thalamic afferents and efferents in Foxg1(-/-) embryos, which lack recognizable ventral telencephalic structures. In these embryos thalamic efferents navigate correctly through the thalamus but fail to turn laterally into the telencephalon, whereas other axons are able to cross the diencephalic/telencephalic boundary. Our results support a role for the ventral telencephalon in guiding the early development of the thalamocortical tract and identify a new role for the transcription factor Pax6 in regulating the ability of the thalamus to attract ventral telencephalic afferents.

Loss of basal forebrain P75(NTR) immunoreactivity in subjects with mild cognitive impairment and Alzheimer's disease.

The long-held belief that degeneration of the cholinergic basal forebrain was central to Alzheimer's disease (AD) pathogenesis and occurred early in the disease process has been questioned recently. In this regard, changes in some cholinergic basal forebrain (CBF) markers (e.g. the high affinity trkA receptor) but not others (e.g., cortical choline acetyltransferase [ChAT] activity, the number of ChAT and vesicular acetylcholine transporter-immunoreactive neurons) suggest specific phenotypic changes, but not frank neuronal degeneration, early in the disease process. The present study examined the expression of the low affinity p75 neurotrophin receptor (p75(NTR)), an excellent marker of CBF neurons, in postmortem tissue derived from clinically well-characterized individuals who have been classified as having no cognitive impairment (NCI), mild cognitive impairment (MCI), and mild AD. Relative to NCI individuals, a significant and similar reduction in the number of nucleus basalis p75(NTR)-immunoreactive neurons was seen in individuals with MCI (38%) and mild AD (43%). The number of p75(NTR)-immunoreactive nucleus basalis neurons was significantly correlated with performance on the Mini-Mental State Exam, a Global Cognitive Test score, as well as some individual tests of working memory and attention. These data, together with previous reports, support the concept that phenotypic changes, but not frank neuronal degeneration, occur early in cognitive decline. Although there was no difference in p75(NTR) CBF cell reduction between MCI and AD, it remains to be determined whether these findings lend support to the hypothesis that MCI is a prodromal stage of AD.

Regionalisation of anterior neuroectoderm and its competence in responding to forebrain and midbrain inducing activities depend on mutual antagonism between OTX2 and GBX2.

The anterior neural ridge (ANR), and the isthmic organiser (IsO) represent two signalling centres possessing organising properties necessary for forebrain (ANR) as well as midbrain and rostral hindbrain (IsO) development. An important mediator of ANR and IsO organising property is the signalling molecule FGF8. Previous work has indicated that correct positioning of the IsO and Fgf8 expression in this domain is controlled by the transcription factors Otx2 and Gbx2. In order to provide novel insights into the roles of Otx2 and Gbx2, we have studied mutant embryos carrying different dosages of Otx2, Otx1 and Gbx2. Embryos deficient for both OTX2 and GBX2 proteins (hOtx1(2)/hOtx1(2); Gbx2(-/-)) show abnormal patterning of the anterior neural tissue, which is evident at the presomite-early somite stage prior to the onset of Fgf8 neuroectodermal expression. Indeed, hOtx1(2)/hOtx1(2); Gbx2(-/-) embryos exhibit broad co-expression of early forebrain, midbrain and rostral hindbrain markers such as hOtx1, Gbx2, Pax2, En1 and Wnt1 and subsequently fail to activate forebrain and midbrain-specific gene expression. In this genetic context, Fgf8 is expressed throughout the entire anterior neural plate, thus indicating that its activation is independent of both OTX2 and GBX2 function. Analysis of hOtx1(2)/hOtx1(2); Gbx2(-/-) and Otx1(+/-); Otx2(+/-) mutant embryos also suggests that FGF8 cannot repress Otx2 without the participation of GBX2. Finally, we report that embryos carrying a single strong hypomorphic Otx2 allele (Otx2(lambda)) in an Otx2 and Gbx2 null background (Otx2(lambda)/-; Gbx2(-/-)) recover both the headless phenotype exhibited by Otx2(lambda)/- embryos and forebrain- and midbrain-specific gene expression that is not observed in hOtx1(2)/hOtx1(2); Gbx2(-/-) mutants. Together, these data provide novel genetic evidence indicating that OTX2 and GBX2 are required for proper segregation of early regional identities anterior and posterior to the mid-hindbrain boundary (MHB) and for conferring competence to the anterior neuroectoderm in responding to forebrain-, midbrain- and rostral hindbrain-inducing activities.

Xenopus Enhancer of Zeste (XEZ); an anteriorly restricted polycomb gene with a role in neural patterning.

We have identified the Xenopus homologue of Drosophila Enhancer of Zeste using a differential display strategy designed to identify genes involved in early anterior neural differentiation. XEZ codes for a protein of 748 amino acids that is very highly conserved in evolution and is 96% identical to both human and mouse EZ(H)2. In common with most other Xenopus Pc-G genes and unlike mammalian Pc-G genes, XEZ is anteriorly restricted. Zygotic expression of XEZ commences during gastrulation, much earlier than other anteriorly localized Pc-G genes; expression is restricted to the anterior neural plate and is confined later to the forebrain, eyes and branchial arches. XEZ is induced in animal caps overexpressing noggin; up-regulation of XEZ therefore represents a response to inhibition of BMP signalling in ectodermal cells. We show that the midbrain/hindbrain junction marker En-2,and hindbrain marker Krox-20, are target genes of XEZ and that XEZ functions to repress these anteroposterior marker genes. Conversely, XEZ does not repress the forebrain marker Otx-2. XEZ overexpression results in a greatly thickened floor of the forebrain. These results implicate an important role for XEZ in the patterning of the nervous system.