Meis2 controls skeletal formation in the hyoid region.

A vertebrate skull is composed of many skeletal elements which display enormous diversity of shapes. Cranial bone formation embodies a multitude of processes, i.e., epithelial-mesenchymal induction, mesenchymal condensation, and endochondral or intramembranous ossification. Molecular pathways determining complex architecture and growth of the cranial skeleton during embryogenesis are poorly understood. Here, we present a model of the hyoid apparatus development in Wnt1-Cre2-induced Meis2 conditional knock-out (cKO) mice. Meis2 cKO embryos develop an aberrant hyoid apparatus-a complete skeletal chain from the base of the neurocranium to lesser horns of the hyoid, resembling extreme human pathologies of the hyoid-larynx region. We examined key stages of hyoid skeletogenesis to obtain a complex image of the hyoid apparatus formation. Lack of Meis2 resulted in ectopic loci of mesenchymal condensations, ectopic cartilage and bone formation, disinhibition of skeletogenesis, and elevated proliferation of cartilage precursors. We presume that all these mechanisms contribute to formation of the aberrant skeletal chain in the hyoid region. Moreover, Meis2 cKO embryos exhibit severely reduced expression of PBX1 and HAND2 in the hyoid region. Altogether, MEIS2 in conjunction with PBX1 and HAND2 affects mesenchymal condensation, specification and proliferation of cartilage precursors to ensure development of the anatomically correct hyoid apparatus.

The Mandibular and Hyoid Arches-From Molecular Patterning to Shaping Bone and Cartilage.

The mandibular and hyoid arches collectively make up the facial skeleton, also known as the viscerocranium. Although all three germ layers come together to assemble the pharyngeal arches, the majority of tissue within viscerocranial skeletal components differentiates from the neural crest. Since nearly one third of all birth defects in humans affect the craniofacial region, it is important to understand how signalling pathways and transcription factors govern the embryogenesis and skeletogenesis of the viscerocranium. This review focuses on mouse and zebrafish models of craniofacial development. We highlight gene regulatory networks directing the patterning and osteochondrogenesis of the mandibular and hyoid arches that are actually conserved among all gnathostomes. The first part of this review describes the anatomy and development of mandibular and hyoid arches in both species. The second part analyses cell signalling and transcription factors that ensure the specificity of individual structures along the anatomical axes. The third part discusses the genes and molecules that control the formation of bone and cartilage within mandibular and hyoid arches and how dysregulation of molecular signalling influences the development of skeletal components of the viscerocranium. In conclusion, we notice that mandibular malformations in humans and mice often co-occur with hyoid malformations and pinpoint the similar molecular machinery controlling the development of mandibular and hyoid arches.

MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus.

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

Neural crest cells require Meis2 for patterning the mandibular arch via the Sonic hedgehog pathway.

Cranial neural crest cells (cNCCs) originate in the anterior neural tube and populate pharyngeal arches in which they contribute to formation of bone and cartilage. This cell population also provides molecular signals for the development of tissues of non-neural crest origin, such as the tongue muscles, teeth enamel or gland epithelium. Here we show that the transcription factor Meis2 is expressed in the oral region of the first pharyngeal arch (PA1) and later in the tongue primordium. Conditional inactivation of Meis2 in cNCCs resulted in loss of Sonic hedgehog signalling in the oropharyngeal epithelium and impaired patterning of PA1 along the lateral-medial and oral-aboral axis. Failure of molecular specification of PA1, illustrated by altered expression of Hand1/2, Dlx5, Barx1, Gsc and other markers, led to hypoplastic tongue and ectopic ossification of the mandible. Meis2-mutant mice thus display craniofacial defects that are reminiscent of several human syndromes and patients with mutations in the Meis2 gene.

The transcriptional regulator MEIS2 sets up the ground state for palatal osteogenesis in mice.

Haploinsufficiency of Meis homeobox 2 (MEIS2), encoding a transcriptional regulator, is associated with human cleft palate, and Meis2 inactivation leads to abnormal palate development in mice, implicating MEIS2 functions in palate development. However, its functional mechanisms remain unknown. Here we observed widespread MEIS2 expression in the developing palate in mice. Wnt1Cre -mediated Meis2 inactivation in cranial neural crest cells led to a secondary palate cleft. Importantly, about half of the Wnt1Cre ;Meis2f/f mice exhibited a submucous cleft, providing a model for studying palatal bone formation and patterning. Consistent with complete absence of palatal bones, the results from integrative analyses of MEIS2 by ChIP sequencing, RNA-Seq, and an assay for transposase-accessible chromatin sequencing identified key osteogenic genes regulated directly by MEIS2, indicating that it plays a fundamental role in palatal osteogenesis. De novo motif analysis uncovered that the MEIS2-bound regions are highly enriched in binding motifs for several key osteogenic transcription factors, particularly short stature homeobox 2 (SHOX2). Comparative ChIP sequencing analyses revealed genome-wide co-occupancy of MEIS2 and SHOX2 in addition to their colocalization in the developing palate and physical interaction, suggesting that SHOX2 and MEIS2 functionally interact. However, although SHOX2 was required for proper palatal bone formation and was a direct downstream target of MEIS2, Shox2 overexpression failed to rescue the palatal bone defects in a Meis2-mutant background. These results, together with the fact that Meis2 expression is associated with high osteogenic potential and required for chromatin accessibility of osteogenic genes, support a vital function of MEIS2 in setting up a ground state for palatal osteogenesis.

Wnt/ß-catenin signalling is necessary for gut differentiation in a marine annelid, Platynereis dumerilii.

BACKGROUND: Wnt/ß-catenin (or canonical) signalling pathway activity is necessary and used independently several times for specification of vegetal fate and endoderm, gut differentiation, maintenance of epithelium in adult intestine and the development of gut-derived organs in various vertebrate and non-vertebrate organisms. However, its conservation in later stages of digestive tract development still remains questionable due to the lack of detailed data, mainly from Spiralia. RESULTS: Here we characterize the Pdu-Tcf gene, a Tcf/LEF orthologue and a component of Wnt/ß-catenin pathway from Platynereis dumerilii, a spiralian, marine annelid worm. Pdu-Tcf undergoes extensive alternative splicing in the C-terminal region of the gene generating as many as eight mRNA isoforms some of which differ in the presence or absence of a C-clamp domain which suggests a distinct DNA binding activity of individual protein variants. Pdu-Tcf is broadly expressed throughout development which is indicative of many functions. One of the most prominent domains that exhibits rather strong Pdu-Tcf expression is in the putative precursors of endodermal gut cells which are detected after 72 h post-fertilization (hpf). At day 5 post-fertilization (dpf), Pdu-Tcf is expressed in the hindgut and pharynx (foregut), whereas at 7 dpf stage, it is strongly transcribed in the now-cellularized midgut for the first time. In order to gain insight into the role of Wnt/ß-catenin signalling, we disrupted its activity using pharmacological inhibitors between day 5 and 7 of development. The inhibition of Wnt/ß-catenin signalling led to the loss of midgut marker genes Subtilisin-1, Subtilisin-2, α-Amylase and Otx along with a drop in ß-catenin protein levels, Axin expression in the gut and nearly the complete loss of proliferative activity throughout the body of larva. At the same time, a hindgut marker gene Legumain was expanded to the midgut compartment under the same conditions. CONCLUSIONS: Our findings suggest that high Wnt/ß-catenin signalling in the midgut might be necessary for proper differentiation of the endoderm to an epithelium capable of secreting digestive enzymes. Together, our data provide evidence for the role of Wnt/ß-catenin signalling in gut differentiation in Platynereis.

Tcf7L2 is essential for neurogenesis in the developing mouse neocortex.

Generation of neurons in the embryonic neocortex is a balanced process of proliferation and differentiation of neuronal progenitor cells. Canonical Wnt signalling is crucial for expansion of radial glial cells in the ventricular zone and for differentiation of intermediate progenitors in the subventricular zone. We detected abundant expression of two transcrtiption factors mediating canonical Wnt signalling, Tcf7L1 and Tcf7L2, in the ventricular zone of the embryonic neocortex. Conditional knock-out analysis showed that Tcf7L2, but not Tcf7L1, is the principal Wnt mediator important for maintenance of progenitor cell identity in the ventricular zone. In the absence of Tcf7L2, the Wnt activity is reduced, ventricular zone markers Pax6 and Sox2 are downregulated and the neuroepithelial structure is severed due to the loss of apical adherens junctions. This results in decreased proliferation of radial glial cells, the reduced number of intermediate progenitors in the subventricular zone and hypoplastic forebrain. Our data show that canonical Wnt signalling, which is essential for determining the neuroepithelial character of the neocortical ventricular zone, is mediated by Tcf7L2.

Polycomb repression complex 2 is required for the maintenance of retinal progenitor cells and balanced retinal differentiation.

Polycomb repressive complexes maintain transcriptional repression of genes encoding crucial developmental regulators through chromatin modification. Here we investigated the role of Polycomb repressive complex 2 (PRC2) in retinal development by inactivating its key components Eed and Ezh2. Conditional deletion of Ezh2 resulted in a partial loss of PRC2 function and accelerated differentiation of Müller glial cells. In contrast, inactivation of Eed led to the ablation of PRC2 function at early postnatal stage. Cell proliferation was reduced and retinal progenitor cells were significantly decreased in this mutant, which subsequently caused depletion of Müller glia, bipolar, and rod photoreceptor cells, primarily generated from postnatal retinal progenitor cells. Interestingly, the proportion of amacrine cells was dramatically increased at postnatal stages in the Eed-deficient retina. In accordance, multiple transcription factors controlling amacrine cell differentiation were upregulated. Furthermore, ChIP-seq analysis showed that these deregulated genes contained bivalent chromatin (H3K27me3+ H3K4me3+). Our results suggest that PRC2 is required for proliferation in order to maintain the retinal progenitor cells at postnatal stages and for retinal differentiation by controlling amacrine cell generation.

The Gene Regulatory Network of Lens Induction Is Wired through Meis-Dependent Shadow Enhancers of Pax6.

Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction.

Tcf7l1 protects the anterior neural fold from adopting the neural crest fate.

The neural crest (NC) is crucial for the evolutionary diversification of vertebrates. NC cells are induced at the neural plate border by the coordinated action of several signaling pathways, including Wnt/ß-catenin. NC cells are normally generated in the posterior neural plate border, whereas the anterior neural fold is devoid of NC cells. Using the mouse model, we show here that active repression of Wnt/ß-catenin signaling is required for maintenance of neuroepithelial identity in the anterior neural fold and for inhibition of NC induction. Conditional inactivation of Tcf7l1, a transcriptional repressor of Wnt target genes, leads to aberrant activation of Wnt/ß-catenin signaling in the anterior neuroectoderm and its conversion into NC. This reduces the developing prosencephalon without affecting the anterior-posterior neural character. Thus, Tcf7l1 defines the border between the NC and the prospective forebrain via restriction of the Wnt/ß-catenin signaling gradient.

Meis2 is essential for cranial and cardiac neural crest development.

BACKGROUND: TALE-class homeodomain transcription factors Meis and Pbx play important roles in formation of the embryonic brain, eye, heart, cartilage or hematopoiesis. Loss-of-function studies of Pbx1, 2 and 3 and Meis1 documented specific functions in embryogenesis, however, functional studies of Meis2 in mouse are still missing. We have generated a conditional allele of Meis2 in mice and shown that systemic inactivation of the Meis2 gene results in lethality by the embryonic day 14 that is accompanied with hemorrhaging. RESULTS: We show that neural crest cells express Meis2 and Meis2-defficient embryos display defects in tissues that are derived from the neural crest, such as an abnormal heart outflow tract with the persistent truncus arteriosus and abnormal cranial nerves. The importance of Meis2 for neural crest cells is further confirmed by means of conditional inactivation of Meis2 using crest-specific AP2α-IRES-Cre mouse. Conditional mutants display perturbed development of the craniofacial skeleton with severe anomalies in cranial bones and cartilages, heart and cranial nerve abnormalities. CONCLUSIONS: Meis2-null mice are embryonic lethal. Our results reveal a critical role of Meis2 during cranial and cardiac neural crest cells development in mouse.

Genetic interaction between Pax6 and ß-catenin in the developing retinal pigment epithelium.

Wnt/ß-catenin signaling plays an essential role in the retinal pigment epithelium (RPE) determination. Since activity of Pax6 (together with Pax2) is also required for the RPE determination, we investigated a possible genetic interaction between Pax6 and Wnt/ß-catenin signaling pathway by analyzing Pax6, ß-catenin, and Pax6/ß-catenin conditional knockout mice. Although Pax6 inactivation alone had no impact on initial specification determined by the expression of Mitf and Otx2, melanin pigmentation was reduced in the RPE. This suggests that along with Mitf and Otx2, Pax6 is required for the full differentiation of RPE. Reporter gene assays in vitro suggest that hypopigmentation is at least in part due to the direct regulation of genes encoding enzymes involved in melanin synthesis by Pax6, Mitf, and ß-catenin. The RPE of a ß-catenin/Pax6 double mutant was differentiated into the neural retina; however, the tissue was thinner than that of the conditional ß-catenin mutant due to reduced proliferation. Together, our data demonstrate that Pax6 is required for the RPE differentiation by regulating pigmentation and accountable for hyperproliferation in the transdifferentiated RPE. In this context, Pax6 appears to function as a pleiotropic regulator, directing development of ocular tissues in concert with the signaling pathway and, at the same time, regulating expression of structural component of the eye, such as shielding pigment.

Ectopic activation of Wnt/ß-catenin signaling in lens fiber cells results in cataract formation and aberrant fiber cell differentiation.

The Wnt/ß-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/ß-catenin signaling during lens fiber cell differentiation. To activate Wnt/ß-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of ß-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/ß-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/ß-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency.

Generation of mRx-Cre transgenic mouse line for efficient conditional gene deletion in early retinal progenitors.

During mouse eye development, all retinal cell types are generated from the population of retina-committed progenitors originating from the neuroepithelium of the optic vesicle. Conditional gene inactivation provides an efficient tool for studying the genetic basis of the developing retina; however, the number of retina-specific Cre lines is limited. Here we report generation of the mRx-Cre BAC transgenic mouse line in which the expression of Cre recombinase is controlled by regulatory sequences of the mouse Rx gene, one of the earliest determinants of retinal development. When mRx-Cre transgenic mice were crossbred with the ROSA26R or ROSA26R-EYFP reporter lines, the Cre activity was observed in the optic sulcus from embryonic day 8.5 onwards and later in all progenitors residing in the neuroepithelium of the optic cup. Our results suggest that mRx-Cre provides a unique tool for functional genetic studies in very early stages of retinal development. Moreover, since eye organogenesis is dependent on the inductive signals between the optic vesicle and head surface ectoderm, the inductive ability of the optic vesicle can be analyzed using mRx-Cre transgenic mice.

A novel tankyrase small-molecule inhibitor suppresses APC mutation-driven colorectal tumor growth.

Most colorectal cancers (CRC) are initiated by mutations of APC, leading to increased ß-catenin-mediated signaling. However, continued requirement of Wnt/ß-catenin signaling for tumor progression in the context of acquired KRAS and other mutations is less well-established. To attenuate Wnt/ß-catenin signaling in tumors, we have developed potent and specific small-molecule tankyrase inhibitors, G007-LK and G244-LM, that reduce Wnt/ß-catenin signaling by preventing poly(ADP-ribosyl)ation-dependent AXIN degradation, thereby promoting ß-catenin destabilization. We show that novel tankyrase inhibitors completely block ligand-driven Wnt/ß-catenin signaling in cell culture and display approximately 50% inhibition of APC mutation-driven signaling in most CRC cell lines. It was previously unknown whether the level of AXIN protein stabilization by tankyrase inhibition is sufficient to impact tumor growth in the absence of normal APC activity. Compound G007-LK displays favorable pharmacokinetic properties and inhibits in vivo tumor growth in a subset of APC-mutant CRC xenograft models. In the xenograft model most sensitive to tankyrase inhibitor, COLO-320DM, G007-LK inhibits cell-cycle progression, reduces colony formation, and induces differentiation, suggesting that ß-catenin-dependent maintenance of an undifferentiated state may be blocked by tankyrase inhibition. The full potential of the antitumor activity of G007-LK may be limited by intestinal toxicity associated with inhibition of Wnt/ß-catenin signaling and cell proliferation in intestinal crypts. These results establish proof-of-concept antitumor efficacy for tankyrase inhibitors in APC-mutant CRC models and uncover potential diagnostic and safety concerns to be overcome as tankyrase inhibitors are advanced into the clinic.

A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice.

Increased nuclear accumulation of ß-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/ß-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the ß-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the ß-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the ß-catenin destruction complex, followed by increased degradation of ß-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of ß-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/ß-catenin signaling through inhibiting the PARP domain of TNKS1/2.

Mouse Tcf3 represses canonical Wnt signaling by either competing for ß-catenin binding or through occupation of DNA-binding sites.

Tcf3 acts as a transcription factor controlling gene expression in canonical Wnt signaling. In this study we show that mouse Tcf3 represses canonical Wnt signaling in mouse neural stem cells and in human HEK 293 cells. We demonstrate that mouse Tcf3 mediates repression of both moderate and high levels of canonical Wnt signaling, by either competing with other members of the Tcf/Lef family for binding to ß-catenin, or for binding to DNA. We observed that the repressor activity of mouse Tcf3 was only relieved effectively upon simultaneous disruption of both mechanisms. Immunofluorescence of transfected HEK 293 cells showed co-localization of ß-catenin and Tcf3 in the nucleus of cells transfected with full-length Tcf3, but not in cells transfected with N-terminal deleted versions. A direct physical interaction between ß-catenin and Tcf3 in the nucleus was confirmed by co-immunoprecipitation studies. The inhibitory ß-catenin/Tcf3 interface was independent of the ability of Tcf3 to directly interact with DNA.

Characterization and functional analysis of the 5'-flanking promoter region of the mouse Tcf3 gene.

Tcf3 is a nuclear mediator of canonical Wnt signaling which functions in many systems as a repressor of target gene transcription. In this study, we have cloned and characterized a 6.7 kb fragment of the 5'-flanking promoter region of the mouse Tcf3 gene. In silico analysis of the promoter sequence identified the existence of GC boxes and CpG islands, but failed to identify any TATA box. In addition, the promoter sequence contained putative binding sites for multiple transcription factors, including a few known to regulate the function of mTcf3. Of those, we confirmed functional binding sites for NFκB and Oct1 using a luciferase assay and ChIP. In vitro analysis revealed five potential transcription start sites which resulted in a 298 base pair 5'-untranslated region upstream of the mTcf3 translation start site ATG. Using a luciferase assay, we analyzed the activity of the cloned promoter fragment in embryonically derived neural stem cells. The luciferase activity of a 3.5 kb core promoter fragment (-3243/+211) showed up to 40-fold increased activity compared to the empty vector. Addition of sequences 5' to the 3.5 kb core promoter fragment resulted in a 20-fold drop in luciferase activity, indicating the presence of further upstream repressive elements. In vivo analysis of a 4.5 kb promoter fragment (-4303/+211) driving, the expression of EGFP in mouse embryos highly resembled endogenous expression of mTcf3.

In vitro differentiation of mouse embryonic stem cells into neurons of the dorsal forebrain.

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10-12 day incubation. Activation of Wnt signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical Wnt signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex.