Table of contents

Wnt homologs and neural patterning and differentiation

Cell cycle progression and exit must be precisely patterned during development to generate tissues of the correct size, shape and symmetry. Evidence that dorsal-ventral growth of the developing spinal cord is regulated by a Wnt mitogen gradient. Wnt signaling through the ß-catenin/TCF pathway positively regulates cell cycle progression and negatively regulates cell cycle exit of spinal neural precursors in part through transcriptional regulation of cyclin D1 and cyclin D2. Wnts expressed at the dorsal midline of the spinal cord, Wnt1 and Wnt3a, have mitogenic activity, while more broadly expressed Wnts do not. Several lines of evidence are presented suggesting that dorsal midline Wnts form a dorsal to ventral concentration gradient. A growth gradient that correlates with the predicted gradient of mitogenic Wnts emerges as the neural tube grows, with the proliferation rate highest dorsally, and the differentiation rate highest ventrally. These data are rationalized in a 'mitogen gradient model' that explains how proliferation and differentiation can be patterned across a growing field of cells. Computer modeling demonstrates that this model is a robust and self-regulating mechanism for patterning cell cycle regulation in a growing tissue (Megason, 2002).

The relationship between where different Wnts are expressed in the spinal cord and their mitogenic activities was investigated to determine how their local activities might regulate the morphogenesis of the spinal cord. A number of Wnts are expressed in almost identical patterns in chick and mouse spinal cord. Since only short sections of the chick cDNAs are available, their expression in the mouse was shown and the activities of each mammalian Wnt-member was compared in the chick model. Wnt1 and Wnt3a are both expressed soon after the neural plate forms in the dorsal third of the neural tube. The expression of Wnt1 and Wnt3a is restricted to the dorsal midline of the neural tube by E9.5, and remains only in the dorsal midline throughout morphogenesis of the spinal cord. Wnt3 and Wnt4 are also initially expressed in the dorsal third of the spinal cord, but as development proceeds their expression broadens ventrally to include the entire dorsal half of the ventricular zone. Wnt7a and Wnt7b are initially expressed at low levels throughout much of the ventricular zone and their expression increases at intermediate-ventral levels with time, until they are expressed at high levels throughout the ventral three quarters of the ventricular zone (Megason, 2002).

Interestingly, electroporation in the chick neural tube indicated that only Wnt1 and Wnt3a, the two members whose expression is restricted to the dorsal midline, have mitogenic activity. Wnt3, Wnt4, Wnt7a, and Wnt7b have no effect on proliferation or differentiation of neural precursors relative to empty vector controls. Both Wnt1 and Wnt3a caused an 80% increase in the BrdU labeling index in the ventral three fifths of the ventricular zone and a 50% reduction in differentiation. Surprisingly, Wnt3, which is more similar to Wnt3a (84% amino acid identity) than Wnt3a is to Wnt1 (43% amino acid identity) does not share the mitogenic activity or expression pattern of its close relative (Megason, 2002).

Potential transcriptional targets of mitogenic Wnt signaling in neural precursors were investigated to address the mechanism through which they regulate the cell cycle and to determine the pattern of mitogenic Wnt response. Since the activities of Wnt1 and Wnt3a suggest that they directly impinge on the cell cycle rather than regulate cell fate specification, key components of the cell cycle were screened including cyclins, cyclin dependent kinases (CDKs), and CDK inhibitors (CKIs) as candidate targets. cyclin D1 is expressed throughout the early period of neural tube development in a dorsal to ventral gradient (highest dorsally) in mitotically active medial neural precursors in both mouse and chick. D-type cyclins are key regulators of G1 exit. When the spinal cord is small, the gradient of cyclin D1 expression extends all the way across the ventricular zone but as the spinal cord grows the expression gradient becomes more dorsally restricted relative to the size of the DV axis (Megason, 2002).

To investigate the mechanism by which dorsal midline Wnts regulate the cell cycle of neural precursors, the expression of cell cycle regulators was examined in the neural tube of embryos transfected with Wnt signaling components. Transfection of dominant active ß-catenin upregulates transcription of the G1 cyclins cyclin D1 and cyclin D2 but not the G2/M cyclins cyclin A1 or cyclin B3 in neural precursors. The locations of transfected cells were first determined by visualizing GFP and then the same sections were processed by in situ hybridization. Regions of the neural tube that ectopically express ß-catenin have increased levels of cyclin D1 and cyclin D2. Previous reports have found that ß-catenin signaling upregulates cyclin D1 but not cyclin D2 in cultured cell lines and that the cyclin D1 promoter contains consensus Lef/TCF binding sites required for this activity. The transcriptional regulation of cyclin D1 by Wnt signaling in neural precurors could thus be direct (Megason, 2002).

Ectopic expression of Wnt1 or Wnt3a also upregulated cyclin D1 expression. However, in contrast to dominant active ß-catenin, expression of Wnt1 or Wnt3a only upregulated cyclinD1 at intermediate to ventral levels. These data again suggest that endogenous mitogenic Wnts are saturating at dorsal levels. Ventral precursors have a stronger mitogenic response to ectopic Wnt expression than do dorsal precursors. This dorsal-ventral pattern of mitogenic responsiveness of neural precursors to ectopic expression of Wnt1 correlates with the pattern of cyclin D1 upregulation. If activating Wnt signaling upregulates cyclin D1 transcription, then attenuating Wnt signaling should downregulate cyclin D1 transcription. Accordingly, high levels of expression of dominant negative TCF4 downregulates expression of cyclin D1 (Megason, 2002).

To address how major a role transcriptional control of D-cyclins plays in the mitogenic response of Wnts, dominant negative and wild-type versions of cyclin D1 were ectopically expressed. Transfection of a dominant negative cyclin D1 construct that forms abortive complexes with the G1 cyclin dependent kinases CDK4 and CDK6 reduce neural precursor expansion but do not block neural cell cycle progression as severely as dominant negative TCF4. Ectopic expression of wild-type cyclin D1 is not sufficient to cause overgrowth of the neural tube as does Wnt1, Wnt3a, and dominant active ß-catenin. Additionally, mice mutant for cyclin D1 have small eyes and reduced body size but are viable. These data show that two key components of the cell cycle, cyclin D1 and cyclin D2, are transcriptional targets of Wnt signaling in neural precursors but suggest other targets are also involved in the mitogenic response of neural precursors to Wnts. Conversely, it is likely that other regulatory elements in addition to the TCF binding sites contribute to the regulation of cyclin D1 in the neural tube, especially its transient expression in apparently exiting cells. Taken together, these results support a model in which Wnt1 and Wnt3a expressed in the dorsal midline form a dorsal to ventral mitogen gradient that controls the graded expression of cell cycle regulators including D-type cyclins through the ß-catenin pathway (Megason, 2002).

Early neural patterning in vertebrates involves signals that inhibit anterior (A) and promote posterior (P) positional values within the nascent neural plate. In this study, the contributions of, and interactions between, retinoic acid (RA), Fgf and Wnt signals have been investigated in the promotion of posterior fates in the ectoderm. Expression and function of cyp26/P450RAI, a gene that encodes retinoic acid 4-hydroxylase, has been examined as a tool for investigating these events. Cyp26 is first expressed in the presumptive anterior neural ectoderm and the blastoderm margin at the late blastula. When the posterior neural gene hoxb1b is expressed during gastrulation, it shows a strikingly complementary pattern to cyp26. Using these two genes, as well as otx2 and meis3 as anterior and posterior markers, it has been shown that Fgf and Wnt signals suppress expression of anterior genes, including cyp26. Overexpression of cyp26 suppresses posterior genes, suggesting that the anterior expression of cyp26 is important for restricting the expression of posterior genes. Consistent with this, knock-down of cyp26 by morpholino oligonucleotides leads to the anterior expansion of posterior genes. Fgf- and Wnt-dependent activation of posterior genes is mediated by RA, whereas suppression of anterior genes does not depend on RA signaling. Fgf and Wnt signals suppress cyp26 expression, while Cyp26, an enzyme that degrades RA, limits the range of RA-mediated posteriorization in the embryo by suppressing the RA signal. Thus, cyp26 has an important role in linking the Fgf, Wnt and RA signals to regulate AP patterning of the neural ectoderm in the late blastula to gastrula embryo in zebrafish (Kudoh, 2002).

In the early Xenopus embryo, the Xiro homeodomain proteins of the Iroquois (Iro) family control the expression of proneural genes and the size of the neural plate. Xiro1 functions as a repressor that is strictly required for neural differentiation, even when the BMP4 pathway is impaired. Xiro1 and Bmp4 repress each other. Consistently, Xiro1 and Bmp4 have complementary patterns of expression during gastrulation. The expression of Xiro1 requires Wnt signaling. Thus, Xiro1 is probably a mediator of the known downregulation of Bmp4 by Wnt signaling (Gomez-Skarmeta, 2001).

The analysis of mutant alleles at the Wnt-1 locus has demonstrated that Wnt-1-mediated cell signaling plays a critical role in development of distinct regions of the embryonic central nervous system (CNS). To determine how these signals participate in the formation of the CNS, this factor was ectopically expressed in the spinal cord under the control of the Hoxb-4 Region A enhancer. Ectopic Wnt-1 expression causes a dramatic increase in the number of cells undergoing mitosis in the ventricular region and a concomitant ventricular expansion. Although this leads to consistent changes in the relative proportions of dorsal and ventral regions, Wnt-1 does not appear to act as a primary patterning signal. Rather, these experiments indicate that Wnt-1 can act as a mitogen in the developing CNS (Dickinson, 1994).

Xwnt-2b is a novel member of the Wnt gene family and is 73-74% similar to human and mouse Wnt-2 proteins. Starting from stage 15, Xwnt-2b transcripts are localized to a non-contiguous stripe in the anterior neural plate of the Xenopus embryo. In the tailbud, Xwnt-2b is expressed along the dorsoanterior side of the prosencephalon-mesencephalon boundary. At the tadpole stages, the brain-specific expression fades, but the total amount of Xwnt-2b mRNA does not decline due to activation of its expression in non-brain areas. Microinjection of Xwnt-2b mRNA into a ventral blastomere of 4-8-cell embryos results in the formation of complete secondary body axes. These results suggest that Xwnt-2b is a member of the axis-inducing Wnts and that it is involved in brain development and in later organogenesis. Other axis inducing Wnts in Xenopus are Xwnt-1, Xwnt-3a, Xwnt-8 and Xwnt-8b (Landesman, 1997).

In order to identify factors involved in posteriorization of the central nervous system, a functional screen was undertaken in Xenopus animal cap explants that involved coinjecting noggin RNA together with pools of RNA from a chick somite cDNA library. In the course of this screen, a clone was isolated encoding a truncated form of ß-catenin, which induced posterior neural and dorsal mesodermal markers when coinjected with noggin in animal caps. Similar results were obtained with Xwnt-8 and Xwnt-3a, suggesting that these effects are a consequence of activating the canonical Wnt signaling pathway. To investigate whether the activation of posterior neural markers requires mesoderm induction, experiments were performed using a chimeric inducible form of ß-catenin. Activation of this protein during blastula stages results in the induction of both posterior neural and mesodermal markers, while activation during gastrula stages induces only posterior neural markers. This posteriorizing activity occurs by an indirect and noncell-autonomous mechanism requiring FGF signaling (Domingos, 2001).

Interactions between cells help to elaborate pattern within the vertebrate central nervous system (CNS). The genes Wnt-1 and Wnt-3a, which encode members of the Wnt family of cysteine-rich secreted signals, are coexpressed at the dorsal midline of the developing neural tube, coincident with dorsal patterning. Each signal is essential for embryonic development, Wnt-1 for midbrain patterning, and Wnt-3a for formation of the paraxial mesoderm, but the absence of a dorsal neural-tube phenotype in each mutant suggests that Wnt signaling may be redundant. In the absence of both Wnt- and Wnt-3a there is a marked deficiency in neural crest derivatives, which originate from the dorsal neural tube, and a pronounced reduction in dorsolateral neural precursors within the neural tube itself. These phenotypes do not seem to result from a disruption in the mechanisms responsible for establishing normal dorsoventral polarity. Rather, these results are consistent with a model in which local Wnt signaling regulates the expansion of dorsal neural precursors. Given the widespread expression of different Wnt genes in discrete areas of the mammalian neural tube, this may represent a general model for the action of Wnt signaling in the developing CNS (Ikeya, 1997).

Neural patterning occurs during early development, soon after neural induction. In Xenopus, several caudalizing factors transform anterior neural tissue to posterior neural tissue at the open neural plate stages, while other factors are responsible for setting up mediolateral polarity, which becomes the dorsoventral (D-V) axis after neural tube closure. Many Wnt ligands are expressed in the neural tube in distinct anteroposterior (A-P) and D-V domains, implying a function in neural patterning. Xwnt7B induces neural crest markers Xslug and Xtwist in ectodermal explants coinjected with neural inducer noggin and in ectodermal cells neuralized by dissociation. In vivo, Xwnt7B expands the Xtwist expression domain when injected in the animal pole. These results suggest that Wnt members are involved in dorsoventral patterning of the neural tube (Chang, 1998).

Members of the Wnt family of signaling molecules are expressed differentially along the dorsal-ventral axis of the developing neural tube. An examination was performed to determine whether Wnt factors are involved in patterning of the nervous system along this axis. Wnt-1 and Wnt-3a, both of which are expressed in the dorsal portion of the neural tube, can synergize with the neural inducers noggin and chordin in Xenopus animal explants to generate the most dorsal neural structure, the neural crest, as determined by the expression of Krox-20, AP-2, and slug. Overexpression of Wnt-1 or Wnt-3a in the neuroectoderm of whole embryos leads to a dramatic increase of slug and Krox-20-expressing cells, but the hindbrain expression of Krox-20 remains unaffected. Enlargement in the neural crest population can occur even when cell proliferation is inhibited. Wnt-5A and Wnt-8, neither of which is expressed in the dorsal neuroectoderm, fail to induce neural crest markers. Overexpression of glycogen synthase kinase 3, known to antagonize Wnt signaling, blocks the neural-crest-inducing activity of Wnt-3a in animal explants and inhibits neural crest formation in whole embryos. Previous studies indicate that neural crest development can be mediated through a bone morphogenetic protein (BMP)-like signal produced by nonneural ectoderm and by the dorsal neural tube. The level of BMP-4 transcripts are unchanged in animal explants expressing noggin and Wnt-1 or Xwnt-3a, indicating that the Wnt effect described here is probably not mediated by upregulation of BMP-4. However, BMPs are known to up-regulate Wnts in the chicken neural tube, suggesting the possibility that neural crest induction by BMPs may be mediated, in part, by Wnt factors. It is suggested that Wnt-1 and Wnt-3a have a role in patterning the neural tube along its dorsoventral axis and function in the differentiation of the neural crest (Saint-Jeannet, 1997).

Cells in the caudal mesencephalon and rostral metencephalon become organized by signals emanating from the isthmus organizer (IsO). The IsO is associated with the isthmus, a morphological constriction of the neural tube that eventually defines the mesencephalic/ metencephalic boundary (MMB). The transcription factor Lmx1b is expressed and functions in a distinct region of the IsO. Lmx1b expression is maintained by the glycoprotein Fgf8, a signal capable of mediating IsO signaling. Lmx1b, in turn, maintains the expression of the secreted factor Wnt1. These conclusions are substantiated by the following: (1) Lmx1b mRNA becomes localized to the isthmus immediately after Fgf8 initiation, (2) Wnt1 expression is localized to the Lmx1b expression domain, but with slightly later kinetics, (3) Fgf8-soaked beads generate similar domains of expression for Lmx1b and Wnt1 and (4) retroviral-mediated expression of Lmx1b (Lmx1b/RCAS) maintains Wnt1 expression in the mesencephalon. Ectopic Lmx1b is insufficient to alter the expression of a number of other genes expressed at the IsO, suggesting that it does not generate a new signaling center. Instead, if Lmx1b/RCAS-infected brains are allowed to develop longer, changes in mesencephalic morphology are detected. Since both ectopic and endogenous Lmx1b expression occurs in regions of the isthmus undergoing morphological changes, it could normally play a role in this process. Furthermore, a similar phenotype is not observed in Wnt1/RCAS-infected brains, demonstrating that ectopic Wnt1 is insufficient to mediate the effect of ectopic Lmx1b in this assay. Since Wnt1 function has been linked to the proper segregation of mesencephalic and metencephalic cells, it is suggested that Lmx1b and Wnt1 normally function in concert to affect IsO morphogenesis (Adams, 2000).

In the avian hindbrain, the loss of premigratory neural crest cells from rhombomeres 3 and 5 (r3, r5) through programmed cell death contributes to the patterning of emigrant crest cells into three discrete streams. Programmed cell death is induced by the upregulation of Bmp4 and Msx2 in r3 and r5. Secreted frizzled related protein cSFRP2, a WNT antagonist, is expressed in the even-numbered rhombomeres and over-expression of cSfrp2 inhibits Bmp4 expression in r3 and r5, preventing programmed cell death. By contrast, depleting cSFRP2 function in r4 results in elevated levels of Msx2 expression and ectopic programmed cell death, as does overexpression of Wnt1. It is proposed that programmed cell death in the rhombencephalic neural crest is modulated by pre-patterned cSfrp2 expression and a WNT-BMP signaling loop (Ellies, 2000).

The secreted PCD-inducing molecule, while produced by the even-numbered rhombomeres, can apparently induce PCD only in rhombomeres 3 and 5. The even-numbered rhombomeres must therefore either contain an inhibitor against the induction of PCD, or they lack a co-factor required for the PCD inducer function. It is proposed that PCD in the even-numbered rhombomeres is actively inhibited, and that cSFRP2 is the molecule responsible for the inhibition. cSFRP2 has appropriate characteristics as a candidate effector of inhibition: it is expressed specifically in the even-numbered rhombomeres at the critical stages, and gain or reduction of function experiments indicate that cSFRP2 interferes with the PCD program. Gain of cSFRP2 function leads to loss of Bmp4 expression and arrest of PCD in r3 and r5, whereas interference with cSFRP2 function leads to increase of Msx2 expression in the even-numbered rhombomeres and the induction of PCD. It is likely that cSFRP2 functions in the control of PCD via the suppression of WNT signaling (Ellies, 2000).

Several Wnt genes are expressed in the developing hindbrain and are candidates for mediating PCD: Wnt1 and Wnt3a are expressed along the dorsal midline of the hindbrain, whereas Wnt8c (see Drosophila Wnt8) is restricted to the dorsal midline of r4. Together, Wnt1 and Wnt3a have been shown by overexpression or null mutation to function during NC induction; their involvement with NC PCD is therefore difficult to assess. To determine whether or not Wnt3a and/or Wnt1 function to modulate PCD in the chick rhombencephalon, the effect of their overexpression has been analyzed. The embryos show upregulation of PCD in the even-numbered rhombomeres, similar to the antisense reduction of cSFRP2 function. During normal hindbrain development, Wnt1 and Wnt3a expression starts at HH 7, a stage when cSfrp2 expression is ubiquitous. PCD would thus be prevented by cSFRP2 until, at HH 9, cSfrp2 expression is downregulated in r3 and r5. At this stage and rhombomere level, WNT signaling is no longer antagonized and PCD is initiated (Ellies, 2000).

WNT1 is more effective than WNT3a at increasing levels of Msx2 expression and PCD in the neural folds. cSFRP2 antagonizes the dorsalizing activity of WNT1 but not that of WNT3a in Xenopus assays. Both WNT3a and WNT1 could be involved in regulating PCD in r3 and r5. This is consistent with the phenotypes of Wnt mutant mice, which exhibit similar developmental defects to those seen when PCD is arrested, as in Apaf1 mutant mice or caspase inhibition; each of these result in embryos exhibiting incomplete fusion of the neural folds. The effects may often be concealed, since Wnt genes act not only during neurulation and neural crest induction, but also during gastrulation; thus, Wnt mutant mice exhibit severe defects which are associated with this earlier event. Wnt1 mutants show a loss of midbrain and cerebellar structures only (Ellies, 2000 and references therein).

WNT signaling activates dishevelled (DSH), which can then act along two distinct pathways: one of these involves antagonizing ZesteWhite-3 kinase/GSK3beta to stabilize beta-catenin and enable its nuclear translocation; the other pathway, independent of beta-catenin, involves the kinases JNK/SAPK. JNK signaling is involved in inducing cell death in many cell types. Furthermore, Jnk is expressed in the hindbrain of mice, and double Jnk1;Jnk2 mutant mice have reduced PCD in the hindbrain and their neural folds fail to close. Further evidence for beta-catenin-independent WNT signaling in hindbrain NC PCD comes from analyzing other components of the b-catenin pathway, such as TCF4, which is required to bind beta-catenin for nuclear translocation and transcription of target genes. Tcf4 is expressed throughout the hindbrain at equivalent stages in chick, suggesting that during PCD, WNTs may signal through a beta-catenin-independent pathway (Ellies, 2000).

This study indicates an important role for WNT signaling in the induction of PCD in the developing rhombencephalon. Bmp4 and Msx2, both closely associated with PCD, are downstream of WNT signaling. However, WNT signaling alone appears not to be sufficient to cause PCD, since odd-numbered, Wnt-expressing rhombomeres cultured in isolation do not undergo PCD. Culturing r3 in conjunction with r4 in cSFRP2-conditioned medium, culturing r3 in isolation, or removing the PCD inducer originating from the even-numbered rhombomeres, all result in the down-regulation of Bmp4 expression and arrest of r3 PCD. This indicates that the induction of Bmp4 expression (and death) is regulated by the combination of WNT signaling and a PCD-inducing signal diffusing from the even-numbered rhombomeres (Ellies, 2000).

Once Bmp4 expression is initiated, it acts on the odd-numbered rhombomeres to induce Msx2 and PCD. In contrast, when BMP4 protein is added to even-numbered rhombomeres, they do not undergo PCD. Consequently, BMP4 alone is unable to cause PCD in the presence of cSFRP2. Furthermore, Msx2 gain-of-function studies have shown that Msx2 is capable of inducing PCD within the even-numbered rhombomeres. This suggests that another, WNT-dependent factor is required to work in conjunction with BMP4 to initiate the Msx2 and PCD (Ellies, 2000).

It is proposed that the apoptotic program in the rhombencephalon is initiated by WNT signaling. The ubiquitous expression of the WNT antagonist cSfrp2 in the early hindbrain initially prevents PCD. Later in rhombencephalic development, cSfrp2 expression becomes downregulated in the odd-numbered rhombomeres. In precise correlation with cSfrp2 down-regulation, PCD becomes detectable in the odd-numbered rhombomeres. Presumably, the lack of cSFRP2 in the odd-numbered rhombomeres allows WNT to act in combination with the diffusible signal from the even-numbered rhombomeres to induce Bmp4 expression. BMP4, in turn, acts with a WNT-dependent molecule (possibly TCF4 or JNK) to activate Msx2 expression, and thus an autocrine loop induces PCD in the odd-numbered rhombomeres (Ellies, 2000).

Pax3 and Pax7 are transcription factors sharing high sequence identity and overlapping patterns of expression in particular in the dorsal spinal cord. Analysis of Pax3 and Pax7 double mutant mice demonstrates that both genes share redundant functions to restrict ventral neuronal identity in the spinal cord. In their absence, the En1 expression domain is expanded dorsally but that of Evx1 is not affected. At embryonic day 9.5, Wnt4 normally starts to be expressed in the dorsal spinal cord. In the Double Pax mutants, Wnt4 is not expressed in the dorsal spinal cord, while the expression in the ventral spinal cord is only reduced. Thus Pax3 and Pax7 are necessary for the initiation and/or maintenance of Wnt4 expression in the dorsal spinal cord. The expression of En1 is extended dorsally into the Pax7 expression domain in double mutants. Since En1 expression defines V1 interneurons, the expansion of En1 suggests that some dorsally located cells might have acquired the ventral cell fate of the V1 interneuron type. Therefore, one possible role of Pax3 and Pax7 might be to restrict ventral neuronal identity. In addition, Pax3 and Pax7 are expressed in commissural neurons and double mutant embryos exhibit highly reduced ventral commissures. These findings reveal two distinct regulatory pathways for spinal cord neurogenesis, only one of which is dependent on Pax3/7 and 6 (Mansouri, 1998).

In the developing vertebrate CNS, members of the Wnt gene family are characteristically expressed at signaling centers that pattern adjacent parts of the neural tube. To identify candidate signaling centers in the telencephalon, Wnt gene fragments were isolated from cDNA derived from embryonic mouse telencephalon. In situ hybridization experiments demonstrate that one of the isolated Wnt genes, Wnt7a, is broadly expressed in the embryonic telencephalon. By contrast, three others, Wnt3a, 5a and a novel mouse Wnt gene, Wnt2b, are expressed only at the medial edge of the telencephalon, defining the hem of the cerebral cortex. The Wnt-rich cortical hem is a transient, neuron-containing, neuroepithelial structure that forms a boundary between the hippocampus and the telencephalic choroid plexus epithelium (CPe) throughout their embryonic development. Indicating a close developmental relationship between the cortical hem and the CPe, Wnt gene expression is upregulated in the cortical hem both before and just as the CPe begins to form, and persists until birth. In addition, although the cortical hem does not show features of differentiated CPe, such as expression of transthyretin mRNA, the CPe and cortical hem are linked by shared expression of members of the Bmp and Msx gene families. In the extra-toesJ (XtJ) mouse mutant, telencephalic CPe fails to develop. Wnt gene expression is shown to be deficient at the cortical hem in XtJ/XtJ mice, but the expression of other telencephalic developmental control genes, including Wnt7a, is maintained. The XtJ mutant carries a deletion in Gli3, a vertebrate homolog of the Drosophila gene cubitus interruptus (ci), which encodes a transcriptional regulator of the Drosophila Wnt gene, wingless. These observations indicate that Gli3 participates in Wnt gene regulation in the vertebrate telencephalon, and suggest that the loss of telencephalic choroid plexus in XtJ mice is due to defects in the cortical hem that include Wnt gene misregulation (Grove, 1998).

The mechanisms that regulate patterning and growth of the developing cerebral cortex remain unclear. Suggesting a role for Wnt signaling in these processes, multiple Wnt genes are expressed in selective patterns in the embryonic cortex. The role of Wnt-3a signaling at the caudomedial margin of the developing cerebral cortex, the site of hippocampal development, has been examined. Wnt-3a acts locally to regulate the expansion of the caudomedial cortex, from which the hippocampus develops. In mice lacking Wnt-3a, caudomedial cortical progenitor cells appear to be specified normally, but then underproliferate. By mid-gestation, the hippocampus is missing or represented by tiny populations of residual hippocampal cells. Thus, Wnt-3a signaling is crucial for the normal growth of the hippocampus. It is suggested that the coordination of growth with patterning may be a general role for Wnts during vertebrate development (Lee, 2000).

The developing vertebrate mesencephalon shows a rostrocaudal gradient in the expression of a number of molecular markers and in the cytoarchitectonic differentiation of the tectum, where cells cease proliferating and differentiate in a rostral to caudal progression. Tissue grafting experiments have implicated cell signaling by the mesencephalic-metencephalic (mid-hindbrain) junction (or isthmus) in orchestrating these events. The role of Wnt-1 and FGF8 (Drosophila homolog: Branchless) signaling has been explored in the regulation of mesencephalic polarity. FGF8 is expressed in cardiac mesoderm underlying the presumptive mesencephalic/metencephalic region and may play a role in mesencephalic induction. Fgf8 is also expressed in the neural plate itself, in the most rostral metencephalon. Wnt-1 is expressed in the caudal mesencephalon. Wnt-1 regulates Fgf8 expression in the adjacent metencephalon, most likely via a secondary mesencephalic signal. Ectopic expression of Fgf8 in the mesencephalon is sufficient to activate expression of Engrailed-2 and ELF-1, two genes normally expressed in a decreasing caudal to rostral gradient in the posterior mesencephalon. ELF-1 is a ligand for a EPH-like receptor tyrosine kinase expressed in rostrocaudally increasing gradients across the caudal tectum and may function to inhibit temporal axon ingrowth and/or to attract nasal axons. Ectopic expression of Engrailed-1, a functionally equivalent homolog of En-2 is sufficient to activate ELF-1 expression by itself. These results indicate the existence of a molecular hierarchy in which FGF8 signaling establishes the graded expression of En-2 within the tectum (Lee, 1997).

In mammals, Pax-6 is expressed in several discrete domains of the developing CNS and has been implicated in neural development, although its precise role remains elusive. A novel Small eye rat strain (rSey2) was found with phenotypes similar to mouse and rat Small eye. Analyses of the Pax-6 gene reveals one base (C) insertion in an exon encoding the region downstream of the paired box of the Pax-6 gene (resulting in the rSey2 mutation), resulting in the generation of a truncated protein due to the frame shift. rSey2/rSey2 mutant rats exhibit abnormal development of motor neurons in the hindbrain. The Islet-1-positive motor neurons are generated just ventral to the Pax-6-expressing domain, both in the wild-type and mutant embryos. However, two somatic motor (SM) nerves, the abducens and hypoglossal nerves, are missing in homozygous embryos. No SM-type axonogenesis (ventrally growing) is found in the mutant postotic hindbrain, though branchiomotor and visceral motor (BM/VM)-type axons (dorsally growing) are observed within the neural tube. To discover whether the identity of these motor neuron subtypes is changed in the mutant, expression of LIM homeobox genes Islet-1, Islet-2 and Lim-3 were examined. At the postotic levels of the hindbrain, SM neurons express all the three LIM genes, whereas BM/VM-type neurons are marked by Islet-1 only. In the Pax-6 mutant hindbrain, Islet-2 expression is specifically missing, which results in the loss of the cells harboring the post-otic hindbrain SM-type LIM code (Islet-1 + Islet-2 + Lim-3). Expression of Wnt-7b, which overlaps with Pax-6 in the ventrolateral domain of the neural tube, is also specifically missing in the mutant hindbrain, while it remained intact in the dorsal non-overlapping domain. These results strongly suggest that Pax-6 is involved in the specification of subtypes of hindbrain motor neurons, presumably through the regulation of Islet-2 and Wnt-7b expression. Since Islet-2 and Pax-6 expression domains do not overlap, is suggested that Islet-2 expression is regulated by indirecly by Pax-6 via Wnt-7b. This makes evolutionary sense as it is known that Drosophila paired regulates wingless and Wnt-1 expression in the midbrain/hindbrain boundary is controlled by Pax-2,5, 8. Wnt-7b may be involved in either specification of neuronal subdypes or axon pathfinding (Osumi, 1997).

In cerebellum development the formation of synapses between granule cells and their synaptic partners occurs after cerebellar granule cells (GCs) have migrated from the proliferative outer layer of the postnatal cerebellum, the external granular layer. GCs migrate downward while their axons are left behind in the forming of the molecular layer. Upon arrival in the internal granular layer, GCs begin to make contacts with their synaptic partners, the Purkinje cells (PCs) and mossy fiber (MF) axons. After contact, GCs induce cytoskeletal changes in both PCs and MFs. The formation of the PC dendritic tree depends on GCs and the relatively smooth surface of MF growth cones becomes multilobulated on contact with GCs. This structure, called a glomerular rosette, represents a multisynaptic region (Lucas, 1997 and references).

Wnt-7a is expressed in cerebellar granule cell neurons as they begin to extend processes and form synapses. Wnt-7a increases axonal spreading and branching in cultured granule cells. Moreover, Wnt-7a increases the levels of synapsin I, a presynaptic protein involved in synapse formation and function. Lithium mimics Wnt-7a in granule cells by inhibiting GSK-3beta, a component of the Wnt signaling pathway. These results suggest a direct effect of Wnt-7a in the regulation of neuronal cytoskeleton and synapsin I in granule cell neurons. It is suggested that Wnt-7a made by GCs could induce axonal remodeling on neighboring GCs and other neurons by modifying the activity of GSK-3beta during the formation of cerebellar synapses (Lucas, 1998a).

In the developing spinal cord, signals from the roof plate are required for the development of three classes of dorsal interneuron: D1, D2, and D3, listed dorsal to ventral. Absence of Wnt1 and Wnt3a, normally expressed in the roof plate, leads to diminished development of D1 and D2 neurons and a compensatory increase in D3 neuron populations. This occurs without significantly altered expression of BMP and related genes in the roof plate. Moreover, Wnt3a protein induces expression of D1 and D2 markers in the isolated medial region of the chick neural plate, and Noggin does not interfere with this induction. Thus, Wnt signaling plays a critical role in the specification of cell types for dorsal interneurons (Muroyama, 2002).

Sensory axons from dorsal root ganglia neurons are guided to spinal targets by molecules differentially expressed along the dorso-ventral axis of the neural tube. NT-3-responsive muscle afferents project ventrally, cease extending, and branch upon contact with motoneurons (MNs), their synaptic partners. WNT-3 has been identified as a candidate molecule that regulates this process. Wnt-3 is expressed by MNs of the lateral motor column at the time when MNs form synapses with sensory neurons. WNT-3 increases branching and growth cone size while inhibiting axonal extension in NT-3- but not NGF-responsive axons. Ventral spinal cord secretes factors with axonal remodeling activity for NT-3-responsive neurons. This activity is present at limb levels and is blocked by a WNT antagonist. It is proposed that WNT-3, expressed by MNs, acts as a retrograde signal that controls terminal arborization of muscle afferents (Krylova, 2002).

Pattern formation and growth must be tightly coupled during embryonic development. In vertebrates, however, little is known of the molecules that serve to link these two processes. Bone morphogenetic proteins (BMP) coordinate the acquisition of pattern information and the stimulation of proliferation in the embryonic spinal neural tube. BMP and transforming growth factor-β superfamily (TGFβ) function was blocked in the chick embryo using Noggin, a BMP antagonist, and siRNA against Smad4. BMPs/TGFβs are shown to be necessary to regulate pattern formation and the specification of neural progenitor populations in the dorsal neural tube. BMPs also serve to establish discrete expression domains of Wnt ligands, receptors, and antagonists along the dorsal-ventral axis of the neural tube. Using the extracellular domain of Frizzled 8 to block Wnt signaling and Wnt3a ligand misexpression to activate WNT signaling, it has been demonstrated that the Wnt pathway acts mitogenically to expand the populations of neuronal progenitor cells specified by BMP. Thus, BMPs, acting through WNTs, couple patterning and growth to generate dorsal neuronal fates in the appropriate proportions within the neural tube (Chesnutt, 2004).

These studies led to an integrated model for neural patterning and growth. BMP/TGFβs regulate the expression of D/V patterning genes, as well as WNT signaling components, and WNTs function as mitogenic signals regulating neural tube growth. As such, BMP/TGFβs are the key signals in the coordination of patterning and growth in the dorsal neural tube. Loss of BMP/TGFβ function alters D/V pattern, eliminates Wnt1 and Wnt3a, and reduces proliferation because of reduced WNT activity. The results utilizing activated BMPR show expanded domains of midline Wnt1 and Wnt3a expression. This coupled with the mitogenic activity of WNTs suggest that the increased proliferation in the activated BMPR-Ia transgenic mouse is likely a direct consequence of the robust expansion of Wnt1. On the ventral side of the neural tube, SHH may serve an analogous role in the coordination of patterning and growth. Indeed, Pax6, known to be under SHH control in the spinal cord, is required for expression of sFRP2 and Wnt7b. BMPs may similarly control dorsal Wnt expression boundaries through Pax7 activation and Pax6 repression. The intersection of the BMP/TGFb and WNT signaling pathways appears to coordinate not only the specification of cell fate, but also the maintenance of proper growth, as well as appropriate cell cycle exit to ensure the correct organization of the spinal cord (Chesnutt, 2004).

Wnt-5a strongly regulates dopaminergic neuron differentiation by inducing phosphorylation of Dishevelled (Dvl). This study identifies additional components of the Wnt-5a-Dvl pathway in dopaminergic cells. Using in vitro gain-of-function and loss-of-function approaches, it is shown that casein kinase 1 (CK1) δ and CK1epsilon are crucial for Dvl phosphorylation by non-canonical Wnts. In response to Wnt-5a, CK1epsilon binds Dvl and is subsequently phosphorylated. Moreover, in response to Wnt-5a or CK1epsilon, the distribution of Dvl changed from punctate to an even appearance within the cytoplasm. The opposite effect was induced by a CK1epsilon kinase-dead mutant or by CK1 inhibitors. As expected, Wnt-5a blocked the Wnt-3a-induced activation of β-catenin. However, both Wnt-3a and Wnt-5a activated Dvl2 by a CK1-dependent mechanism in a cooperative manner. Finally, it was show that CK1 kinase activity is necessary for Wnt-5a-induced differentiation of primary dopaminergic precursors. Thus, these data identify CK1 as a component of Wnt-5a-induced signalling machinery that regulates dopaminergic differentiation, and suggest that CK1δ/epsilon-mediated phosphorylation of Dvl is a common step in both canonical and non-canonical Wnt signalling (Bryja, 2007).

CK1δ/epsilon have previously been reported to be a Dvl-phosphorylating kinase acting in the β-catenin pathway. A recent report describes that overexpression of CK1epsilon potentiates canonical Wnt signalling and diminishes JNK activation induced by Dvl1 overexpression. This finding led to the suggestion that CK1epsilon modulates the signalling specificity of Dvl towards β-catenin. CK1δ/epsilon also mediate non-canonical signalling, suggesting that canonical or non-canonical specificities are not determined by CK1epsilon but rather by the ligand. Moreover, the previously findings could be alternatively explained by the fact that CK1epsilon-mediated phosphorylation diminishes the activity of axin in the MEKK1-JNK pathway at the expense of its function in canonical Wnt signalling. Although the involvement of CK1δ/epsilon in the signal transduction of a non-canonical Wnt has not been demonstrated to date, a role of CK1δ/epsilon in other biological processes driven by non-canonical Wnts has been described. These include convergent extension movements in Xenopus and functions regulated by planar cell polarity pathway in Drosophila (Klein, 2006; Strutt, 2006). Thus, these data, together with published reports, support the notion that CK1epsilon mediates non-canonical Wnt signalling. Interestingly, a recent report by Takada (2005) suggests that, in Drosophila cells another CK1 isoform, CK1alpha is playing a similar role to the one described here for CK1δ/epsilon in Wnt-5a-driven phosphorylation of Dvl. Thus, it remains to be investigated whether the involvement of individual CK1 isoforms in Dvl phosphorylation differs among species (Bryja, 2007).

The non-neural ectoderm is divided into neural plate border and epidermal cells. At early blastula stages, Wnt and BMP signals interact to induce epidermal fate, but when and how cells initially acquire neural plate border fate remains poorly defined. Evidence is provided in chick that the specification of neural plate border cells is initiated at the late blastula stage and requires both Wnt and BMP signals. The results indicate, however, that at this stage BMP signals can induce neural plate border cells only when Wnt activity is blocked, and that the two signals in combination generate epidermal cells. Evidence that Wnt signals do not play an instructive role in the generation of neural plate border cells, but promote their generation by inducing BMP gene expression, which avoids early simultaneous exposure to the two signals and generates neural plate border instead of epidermal cells. Thus, specification of neural plate border cells is mediated by a novel Wnt-regulated BMP-mediated temporal patterning mechanism (Patthey, 2009).

The floor plate is a key organizer that controls the specification of neurons in the central nervous system. This study shows a new role of the floor plate: segmental pattern formation of the vertebral column. Analysis of a spontaneous medaka mutant, fused centrum (fsc), which exhibits fused centra and the absence of the intervertebral ligaments, revealed that fsc encodes wnt4b, which was expressed exclusively in the floor plate. In fsc mutants, it was found that wnt4b expression was completely lost in the floor plate and that abnormal conversion of the intervertebral ligament cells into osteoblasts appeared to cause a defect of the intervertebral ligaments. The establishment of the transgenic rescue lines and mosaic analyses allowed the conclusion to be drawn that production of wnt4b by floor plate cells is essential for the segmental patterning of the vertebral column. These findings provide a novel perspective on the mechanism of vertebrate development (Inohaya, 2010).

FGFs and Wnts are important morphogens during midbrain development, but their importance and potential interactions during neurogenesis are poorly understood. This study employed a combination of genetic and pharmacological manipulations in zebrafish to show that during neurogenesis FGF activity occurs as a gradient along the anterior-posterior axis of the dorsal midbrain and directs spatially dynamic expression of the Hairy gene her5. As FGF activity diminishes during development, Her5 is lost and differentiation of neuronal progenitors occurs in an anterior-posterior manner. Mathematical models were generated to explain how Wnt and FGFs direct the spatial differentiation of neurons in the midbrain through Wnt regulation of FGF signalling. These models suggested that a negative-feedback loop controlled by Wnt is crucial for regulating FGF activity. Sprouty genes were tested as mediators of this regulatory loop using conditional mouse knockouts and pharmacological manipulations in zebrafish. These reveal that Sprouty genes direct the positioning of early midbrain neurons and are Wnt responsive in the midbrain. A model is proposed in which Wnt regulates FGF activity at the isthmus by driving both FGF and Sprouty gene expression. This controls a dynamic, posteriorly retracting expression of her5 that directs neuronal differentiation in a precise spatiotemporal manner in the midbrain (Dyer, 2014).

Mesodermal Wnt signaling organizes the neural plate via Meis3

In vertebrates, canonical Wnt signaling controls posterior neural cell lineage specification. Although Wnt signaling to the neural plate is sufficient for posterior identity, the source and timing of this activity remain uncertain. Furthermore, crucial molecular targets of this activity have not been defined. This study identified the endogenous Wnt activity and its role in controlling an essential downstream transcription factor, Meis3. Wnt3a is expressed in a specialized mesodermal domain, the paraxial dorsolateral mesoderm, which signals to overlying neuroectoderm. Loss of zygotic Wnt3a in this region does not alter mesoderm cell fates, but blocks Meis3 expression in the neuroectoderm, triggering the loss of posterior neural fates. Ectopic Meis3 protein expression is sufficient to rescue this phenotype. Moreover, Wnt3a induction of the posterior nervous system requires functional Meis3 in the neural plate. Using ChIP and promoter analysis, this study shows that Meis3 is a direct target of Wnt/beta-catenin signaling. This suggests a new model for neural anteroposterior patterning, in which Wnt3a from the paraxial mesoderm induces posterior cell fates via direct activation of a crucial transcription factor in the overlying neural plate (Elkouby, 2010).

Is canonical Wnt activation of Meis gene expression a conserved phenomenon? In C. elegans, the PSA-3/Meis protein is required for daughter cell fates after asymmetric cell division, and it is a direct target of the Wnt pathway. POP-1/TCF protein binds the psa-3/Meis promoter and its binding site is required for expression. In Drosophila, the wingless (Wg/Wnt) protein activates expression of the sole Meis protein homolog, the homothorax (hth) gene, which is required for wing hinge development. In both beetles and spiders, hth expression seems to be regulated by Wg during appendage development. Thus, in diverged invertebrate systems, Wnt signaling controls Meis/hth family gene expression. This study shows the first evidence for Wnt regulation of Meis gene expression in vertebrates (Elkouby, 2010).

The canonical Wnt, FGF and RA signaling pathways are required for posterior CNS formation in Xenopus. In a unifying model, it is suggested that Wnt3a signaling from the Dorsal-lateral marginal zone (DLMZ) triggers Meis3 expression in the overlying neural plate. Meis3 protein directly activates FGF3 and FGF8 gene expression, and Meis3 protein cannot induce posterior cell fates in the absence of downstream FGF signaling. Supporting these data, in caudalized Xenopus explants, FGF acts downstream of canonical Wnt signaling. FGF3 and FGF8 expression in Wnt-caudalized explants is dependent on Meis3 protein activity. RA signaling also interacts with Meis3 protein to optimize Hox gene expression in the early neural plate to fine-tune the hindbrain pattern. Thus, in a regulatory network controlling posterior neural cell fates, Meis3 acts downstream to canonical Wnt, upstream to FGF, and in concert with RA signaling to activate gene expression (Elkouby, 2010).

What is the organizer's role in neural patterning? Clearly the DMZ is indispensable for neural induction, which is a prerequisite for neural patterning. In explants, BMP antagonism does not induce Meis3 expression but, in combination with Wnt signaling, BMP antagonism optimally induces Meis3 expression. BMP antagonism induces competent neuroectoderm that is responsive to caudalizing signals from the DLMZ. Optimal Meis3 expression in the embryo depends on Zic1 protein activity, and Zic gene family expression requires BMP antagonism. While the organizer does not caudalize neuroectoderm, it provides the competence for the Wnt pathway to activate Meis3 gene transcription (Elkouby, 2010).

There are still many open questions. In the presumptive hindbrain region of gastrula embryos, Meis3-expressing cells act as a hindbrain-inducing center by inducing posterior neural cell fates non-cell autonomously. At gastrula stages, when this center is active, the target cells are clustered in close proximity. How does Meis3 induce different cell fates in such proximal, but distinct, embryonic regions? How are the different posterior neural cell types, such as hindbrain, primary neuron and neural crest specified to such exact regions by similar signaling pathways and transcription factors during overlapping time windows? The future challenge is to determine how Meis3 protein acts with these signaling pathways and other transcription factors to generate multiple nervous system cell fates (Elkouby, 2010).

Wnt signaling determines ventral spinal cord cell fates in a time-dependent manner

The identity of distinct cell types in the ventral neural tube is generally believed to be specified by sonic hedgehog (Shh) in a concentration-dependent manner. However, recent studies have questioned whether Shh is the sole signaling molecule determining ventral neuronal cell fates. This study provides evidence that canonical Wnt signaling is involved in the generation of different cell types in the ventral spinal cord. Wnt signaling is active in the mouse ventral spinal cord at the time when ventral cell types are specified. Furthermore, using an approach that stabilizes β-catenin protein in small patches of ventral spinal cord cells at different stages, this study shows that Wnt signaling activates different subsets of target genes depending on the time when Wnt signaling is amplified. Moreover, disruption of Wnt signaling results in the expansion of ventrally located progenitors. Finally, this study shows genetically that Wnt signaling interacts with Hh signaling at least in part through regulating the transcription of Gli3. These results reveal a novel mechanism by which ventral patterning is achieved through a coordination of Wnt and Shh signaling (Yu, 2008).

At least three different mechanisms could account for the switching of ventral progenitor cell fates in response to Wnt signaling. First, the switching of cell fate is dependent on Wnt signal strength. In this scenario, a strong Wnt signal induces dorsal cell types, whereas a weak signal induces ventral cell types. However, because different cell types were induced by stabilized β-catenin, Wnt signal strength, although possible, is unlikely to play a crucial role in cell fate determination. The second possibility is that cell fate switching is dependent on the duration of the Wnt signal. For example, the longer that Wnt signaling is maintained in a cell, the more likely it is that the cell will adopt a dorsal cell fate, similar to a mechanism that has been proposed for Shh action. In this scenario, the dorsal-most cell type, d1, is specified because these cells receive the longest exposure to Wnt signaling (from E8.5 to E10.5), whereas other cells adopt more-ventral cell fates because they receive shorter exposure to Wnt signaling. However, extending the length of Wnt signaling does not appear to alter the expression of dorsal markers. Lastly, it is possible that different cell fates are specified depending on when Wnt signaling is active. In this scenario, Wnt signaling is capable of activating different genes at different time points, based on the changing competence of the cells. Indeed, it was found that early Wnt signaling (induced with TM at E7.5) activated the expression of several dorsal markers, Pax7, Gsh1/2 and Msx1/2. By contrast, Wnt signaling induced with TM at E8.5 could only induce the expression of Msx1/2, and Wnt signaling induced subsequently did not activate dorsal markers. The results therefore strongly support the time-dependent mechanism of Wnt signaling in the ventral spinal cord (Yu, 2008).

Only ~70%-90% of ectopic Msx1/2+ or Pax7+ cells coexpressed TCF/LEF-lacZ. However, this is likely to be an underestimate because the TCF/LEF-lacZ transgenic reporter was not expressed in all E10.5 progenitors that expressed stabilized β-catenin. The action of Wnt signaling on cell fate changes is likely to be cell-autonomous. However, non-autonomous effects cannot be completely excluded, particularly in light of the reduction in the Irx3 ventral expression domain, which lies outside of the Olig1-Cre expression domain, in β-catenin mutant embryos. Nevertheless, no upregulation of phosphorylated Smad1/5/8 was observed in embryos expressing stabilized β-catenin, suggesting that BMP pathways were not activated in response to stabilized β-catenin (Yu, 2008).

Although the removal of β-catenin using Olig1-Cre affects the morphology of the floor plate at E10.5, the action of Wnt signaling on cell type switching appears to be direct, based on the following observations. First, there was no significant change in the level of Shh protein, the number of Shh-expressing cells or in the response to Shh at E9.5, when cell fate is being specified. Even at E10.5, when most of the cells have been specified, no significant changes in the expression of Gli2, Shh, Ptch1 or Gli1 were observed, although the floor plate appeared to be less compact. In fact, deletion of floor plate does not affect the generation of most ventral neurons, except for V3 cells, as has been demonstrated in Gli2 mutants. Lastly, activation of Wnt signaling in small patches of cells using Gli1-CreER reveals that activation of Wnt signaling directly affects cell fate (Yu, 2008).

Wnts and the patterning of the forebrain

The expression patterns of four genes that are potential regulators of development were examined in the CNS of the embryonic day 12.5 mouse embryo. Three of the genes, Dlx-1, Dlx-2 (Tes-1), and Gbx-2, encode homeodomain-containing proteins, and one gene, Wnt-3, encodes a putative secreted differentiation factor. These genes are expressed in spatially restricted transverse and longitudinal domains in the embryonic neural tube, and are also differentially expressed within the wall of the neural tube. Dlx-1 and Dlx-2 are expressed in two separate regions of the forebrain in an identical pattern. The Gbx-2 gene is expressed in four domains, two of which share sharp boundaries with the domains of the Dlx genes. One boundary is in the basal telecephalon between deep and superficial strata of the medial ganglionic eminence; the other boundary is in the diencephalon at the zona limitans intrathalamica. The Wnt-3 gene is expressed in a dorsal longitudinal zone extending from the hindbrain into the diencephalon, where its expression terminates at the zona limitans intrathalamica. Reciprocal patterns of expression are found within the dorsal thalamus for the Gbx-2 and Wnt-3 genes. These findings are consistent with neuromeric theories of forebrain development (Bulfone, 1993).

Wnt signaling regulates a wide range of developmental processes such as proliferation, cell migration, axon guidance, and cell fate determination. The expression of secreted frizzled related protein-2 (SFRP-2), which codes for a putative Wnt inhibitor, in the developing nervous system, has been studied. SFRP-2 is expressed in several discrete neuroepithelial domains, including the diencephalon, the insertion of the eminentia thalami into the caudal telencephalon, and the pallial-subpallial boundary (PSB). Wnt-7b expression is similar to SFRP-2 expression. Because many of these structures are disrupted in Pax-6 mutant mice, SFRP-2 and Wnt-7b expression was examined in the forebrains of Pax-6 Sey/Sey mice. Pax-6 mutants were found to lack SFRP-2 expression in the PSB and diencephalon. Interestingly, Pax-6 mutants also lack Wnt-7b expression in the PSB, but Wnt-7b expression in the diencephalon is preserved. Furthermore, in the spinal cord of Pax-6 mutants, SFRP-2 and Wnt-7b expression is greatly reduced. These results suggest that by virtue of its apposition to Wnt-7b expression, SFRP-2 may modulate Wnt-7b function, particularly at boundaries such as the PSB, and that changes in Wnt signaling contribute to the phenotype of Pax-6 mutants (Kim, 2001).

One of the earliest manifestations of anteroposterior pattering in the developing brain is the restricted expression of Six3 and Irx3 in the anterior and posterior forebrain, respectively. Consistent with the role of Wnts as posteriorizing agents in neural tissue, Wnt signaling was found to be sufficient to induce Irx3 and repress Six3 expression in forebrain explants. The position of the zona limitans intrathalamica (zli), a boundary-cell population that develops between the ventral (vT) and dorsal thalamus (dT), is predicted by the apposition of Six3 and Irx3 expression domains. The expression patterns of several inductive molecules are limited by the zli, including Wnt3, which is expressed posterior to the zli in the dT. Wnt3 and Wnt3a were sufficient to induce the dT marker Gbx2 exclusively in explants isolated posterior to the presumptive zli. Blocking the Wnt response allows the induction of the vT-specific marker Dlx2 in prospective dT tissue. Misexpression of Six3 in the dT induces Dlx2 expression and inhibits the expression of both Gbx2 and Wnt3. These results demonstrate a dual role for Wnt signaling in forebrain development. First, Wnts direct the initial expression of Irx3 and repression of Six3 in the forebrain, delineating posterior and anterior forebrain domains. Later, continued Wnt signaling results in the induction of dT specific markers, but only in tissues that expressed Irx3 (Braun, 2003).

Recent findings implicate embryonic signaling centers in patterning the mammalian cerebral cortex. Mouse in utero electroporation and mutant analysis was used to test whether cortical signaling sources interact to regulate one another. Interactions were identified between the cortical hem (part of the dorsomedial edge of each cerebral cortical hemisphere), rich in Wingless-Int (WNT) proteins and bone morphogenetic proteins (BMPs), and an anterior telencephalic source of fibroblast growth factors (FGFs). Expanding the FGF8 domain suppressed Wnt2b, Wnt3a and Wnt5a expression in the hem. Next to the hem, the hippocampus was shrunken, consistent with its dependence for growth on a hem-derived WNT signal. Maintenance of hem WNT signaling and hippocampal development thus require a constraint on the FGF8 source, which is likely to be supplied by BMP activity. When endogenous BMP signaling is inhibited by noggin, robust Fgf8 expression appears ectopically in the cortical primordium. Abnormal signaling centers were further investigated in mice lacking the transcription factor EMX2, in which FGF8 activity is increased, WNT expression is reduced, and the hippocampus is defective. Suggesting that these defects are causally related, sequestering FGF8 in Emx2 homozygous mutants substantially recovered WNT expression in the hem and partially rescued hippocampal development. Because noggin can induce Fgf8 expression, noggin and BMP signaling were examined in the Emx2 mutant. As the telencephalic vesicle closed, Nog expression expanded and BMP activity reduced, potentially leading to FGF8 upregulation. These findings point to a cross-regulation of BMP, FGF, and WNT signaling in the early telencephalon, integrated by EMX2, and required for normal cortical development (Shimogori, 2004).

The Emx2 mutant mouse line provides an informative illustration of the consequences of signaling center defects. Homozygous mutants display an expanded FGF8 domain, and predictably, given the present findings, a partial loss of WNT gene expression in the hem. Evidence has been provided that shifts in region-specific gene expression in the Emx2 mutant neocortex are in part caused by excess FGF8. Findings from the present study indicate that the expanded FGF8 source in the mutant reduces WNT signaling from the cortical hem, which in turn could contribute to defective development of the hippocampus (Shimogori, 2004).

Emx2 is expressed broadly in the cortical primordium, but its loss does not lead to a broad expansion of Fgf8 expression. Instead, the normally medial and anterior FGF8 domain is enlarged laterally and posteriorly, but retains clear boundaries. Findings from the present study suggest a partial explanation. A likely cause of the expanded FGF8 domain in the Emx2 mutant is early overexpression of noggin at the telencephalic midline, decreasing local BMP activity. BMP inhibition of Fgf8 expression is thereby relieved close to the midline, but not at a distance. Remaining BMP activity may contain further lateral spread of Fgf8 expression (Shimogori, 2004).

It is suggested that cortically expressed EMX2 influences signaling centers by direct gene regulation in the cortical primordium. However, an indirect influence by EMX2 function outside the cortical primordium remains a formal possibility. Emx2 expression appears at E8-8.5 in rostral brain, and continues in both the cortical and subcortical forebrain, where EMX2 has diverse roles in development. These complexities challenge easy interpretation of specific defects in the Emx2 mutant. For example, a misrouting of thalamocortical axons, first ascribed to the absence of EMX2 in the neocortex, may be partially due to loss of gene function in the ventral telencephalon where the thalamocortical pathway begins (Shimogori, 2004).

Ultimately, the timing and sites of Emx2 expression that are crucial to particular aspects of development will be resolved by appropriate conditional deletions, or regional misexpression, of the gene. A recent advance has been the generation of a mouse that overexpresses Emx2 under the control of the nestin promotor. FGF8 levels appear unaffected, perhaps because Emx2 is overexpressed too late, yet area boundaries are shifted. These findings, together with the current ones, indicate a primary effect of EMX2 on cortical patterning, and a secondary effect via two signaling sources (Shimogori, 2004).

It is proposed that early in telencephalic development, EMX2 acts directly or indirectly on noggin to derepress BMP activity. BMP activity constrains expansion of the anterior FGF8 source, and keeps the cortical hem clear of FGF8, protecting local WNT gene expression. Meanwhile, normal levels of midline noggin allow the FGF8 source to be established and maintained. Effectively completing a negative feedback loop, FGF8 downregulates Emx2 expression. These interactions help to ensure FGF and WNT/BMP sources of appropriate size, position and duration to regulate cortical patterning and growth (Shimogori, 2004).

The forebrain is patterned along the dorsoventral (DV) axis by Sonic Hedgehog (Shh). However, previous studies have suggested the presence of an Shh-independent mechanism. This study identifies Wnt/β-catenin (activated from the telencephalic roof) as an Shh-independent pathway that is essential for telencephalic pallial (dorsal) specification during neurulation. The transcription factor Foxg1 coordinates the activity of two signaling centers: Foxg1 is a key downstream effector of the Shh pathway during induction of subpallial (ventral) identity, and it inhibits Wnt/β-catenin signaling through direct transcriptional repression of Wnt ligands. This inhibition restricts the dorsal Wnt signaling center to the roof plate and consequently limits pallial identities. Concomitantly to these roles, Foxg1 controls the formation of the compartment boundary between telencephalon and basal diencephalon. Altogether, these findings identify a key direct target of Foxg1, and uncover a simple molecular mechanism by which Foxg1 integrates two opposing signaling centers (Danesin, 2009).

The cortical hem, a source of Wingless-related (WNT) and bone morphogenetic protein (BMP) signaling in the dorsomedial telencephalon, is the embryonic organizer for the hippocampus. Whether the hem is a major regulator of cortical patterning outside the hippocampus has not been investigated. This study examined regional organization across the entire cerebral cortex in mice genetically engineered to lack the hem. Indicating that the hem regulates dorsoventral patterning in the cortical hemisphere, the neocortex, particularly dorsomedial neocortex, was reduced in size in late-stage hem-ablated embryos, whereas cortex ventrolateral to the neocortex expanded dorsally. Unexpectedly, hem ablation also perturbed regional patterning along the rostrocaudal axis of neocortex. Rostral neocortical domains identified by characteristic gene expression were expanded, and caudal domains diminished. A similar shift occurs when fibroblast growth factor (FGF) 8 is increased at the rostral telencephalic organizer, yet the FGF8 source was unchanged in hem-ablated brains. Rather hem WNT or BMP signals, or both, were found to have opposite effects to those of FGF8 in regulating transcription factors that control the size and position of neocortical areas. When the hem is ablated a necessary balance is perturbed, and cerebral cortex is rostralized. These findings reveal a much broader role for the hem in cortical development than previously recognized, and emphasize that two major signaling centers interact antagonistically to pattern cerebral cortex (Caronia-Brown, 2014).

Wnt targets in the telencephalon

Pattern formation of the dorsal telencephalon is governed by a regionalization process that leads to the formation of distinct domains, including the future hippocampus and neocortex. Recent studies have implicated signaling proteins of the Wnt and Bmp gene families as well as several transcription factors, including Gli3 and the Emx homeobox genes, in the molecular control of this process. The regulatory relationships between these genes, however, remain largely unknown. Transgenic analysis was used to investigate the upstream mechanisms for regulation of Emx2 in the dorsal telencephalon. An enhancer from the mouse Emx2 gene has been identified that drives specific expression of a lacZ reporter gene in the dorsal telencephalon. This element contains binding sites for Tcf and Smad proteins, transcriptional mediators of the Wnt and Bmp signaling pathway, respectively. Mutations of these binding sites abolish telencephalic enhancer activity, while ectopic expression of these signaling pathways leads to ectopic activation of the enhancer. These results establish Emx2 as a direct transcriptional target of Wnt and Bmp signaling and provide insights into a genetic hierarchy involving Gli3, Emx2 and Bmp and Wnt genes in the control of dorsal telencephalic development (Theil, 2002).

The analysis presented here has revealed several aspects of the complexity of Emx2 regulation. Although the 4.6 kb fragment mediates reporter gene expression in the dorsal telencephalon indistinguishable from the expression pattern of the endogenous gene, enhancer activity was not observed in the early developing dorsal forebrain. This difference suggests that the spatial and temporal control of Emx2 expression might involve the use of distinct regulatory modules. A similar conclusion was obtained for the control of the segmental expression of the Epha4 gene and of the Hox genes in the hindbrain (Theil, 2002).

Several observations of this study indicate a cooperative interaction between Wnt and Bmp signaling to regulate Emx2 expression in the telencephalon. While mutations of the Tcf- and Smad-binding sites abolish Emx2 enhancer activity in the telencephalon, the single site mutations only affect specific aspects of reporter gene expression. Furthermore, in vitro binding of the Tcf/Smad factors is enhanced in the presence of both factors. Similarly, ectopic expression experiments show an increased induction of the telencephalic enhancer through both Wnt and Bmp signaling. Synergy between Tgfß and Wnt signaling to regulate developmental events has been observed in various cases and may involve direct interactions between Lef1 and Smad proteins. Since expression of Bmp family members is confined to the dorsomedial telencephalon, a cooperative effect between Wnt and Bmp signaling would mainly be restricted to development of the hippocampus and adjacent medial neocortex. Interaction between these signaling pathways therefore provides a molecular mechanism to specify the gradient of Emx2 expression along the medial/lateral axis of the telencephalon (Theil, 2002).

Within the neocortical neuroepithelium, control of regional Emx2 expression requires a functional Tcf-binding site. The similarities between the Wnt7b expression and the ß-galactosidase staining pattern of the Emx2 enhancer construct just containing the functional Tcf-binding site make this Wnt family member a good candidate for being an upstream regulator of Emx2 expression in the telencephalon. This idea is further supported by recent findings showing that Wnt7b can induce the formation of a free cytoplasmic pool of ß-catenin and can stimulate the expression of the Tcf target gene Cdx1. In addition, enhancer activity in the ventral diencephalon coincides with another prominent Wnt7b expression domain. Alternatively, control of Emx2 expression could involve other yet to be identified Wnt genes with expression in the cortical neuroepithelium. The Tcf-binding site alone, however, only confers weak lacZ expression in the telencephalon, suggesting a requirement for additional factors. Although Bmp expression and signaling is mainly confined to the dorsomedial telencephalon, mutational analysis suggests an important role for the Smad-binding site in this regulation (Theil, 2002).

While the data establish Bmps and Wnts as essential components of the molecular mechanisms governing regional Emx2 expression, several lines of evidence suggest that activation of Bmp and Wnt signaling is not sufficient for the induction of Emx2 expression during normal development. (1) Even within the neural tube, co-expression of several Wnt and Bmp genes is widespread while Emx2 transcription as well as Emx2 enhancer activity are confined to the forebrain. (2) A second regulatory element, DT2, was defined that is required for reporter gene expression in the dorsal telencephalon. While DT1 on its own is not sufficient to mediate this activity, a fusion construct consisting of just DT1 and DT2 drives lacZ expression in a pattern indistinguishable from the original enhancer construct. This data indicates that DT2 does not solely act to inhibit potential repressive elements within the Emx2 enhancer but functions as a positive regulator and synergises with DT1 in the tissue-specific regulation of Emx2. Region specific expression of the yet unknown factor(s) binding to the DT2 element might therefore be responsible for conferring forebrain specific activation of the Emx2 enhancer (Theil, 2002).

The identification of Wnts/Bmps as regulators of Emx2 expression places this homeobox gene downstream of these signaling pathways in the genetic hierarchy controlling telencephalic development. Consistent with this idea, hippocampal development is affected by both the Wnt3a and the Emx2 mutation, though to different extents. Similar to the Gli3 mutation, loss of Wnt3a function leads to a loss of the hippocampus, while it is reduced in size in the Emx2 mutant. This difference suggests the involvement of Wnt target genes other than Emx2 in the control of this developmental process, such as the Lhx5 homeobox gene. In addition, a role for Bmps in Emx2 regulation could be demonstrated by the finding that ectopic expression of Bmp4 throughout the dorsal telencephalon, as observed in Bf1 mutant mice, coincides with an expansion of the Emx2 expression domain into the ventral telencephalon. Furthermore, the unaltered expression patterns of Gli3 and Wnt genes in the Emx2 mutant telencephalon show that these genes are not regulated by Emx2 (Theil, 2002).

Wnt signaling and its downstream target N-myc regulate basal progenitors in the developing neocortex

Basal progenitors (also called non-surface dividing or intermediate progenitors) have been proposed to regulate the number of neurons during neocortical development through expanding cells committed to a neuronal fate, although the signals that govern this population have remained largely unknown. This study shows that N-myc mediates the functions of Wnt signaling in promoting neuronal fate commitment and proliferation of neural precursor cells in vitro. Wnt signaling and N-myc also contribute to the production of basal progenitors in vivo. Expression of a stabilized form of beta-catenin, a component of the Wnt signaling pathway, or of N-myc increased the numbers of neocortical basal progenitors, whereas conditional deletion of the N-myc gene reduced these and, as a likely consequence, the number of neocortical neurons. These results reveal that Wnt signaling via N-myc is crucial for the control of neuron number in the developing neocortex (Kuwahara, 2010).

Wnt signaling and its downstream target N-Myc play a key role in the production of basal progenitors. Expression of N-myc or stabilized β-catenin increases, while conditional gene deletion of N-myc decreases the numbers of basal progenitors found in the developing neocortex, as determined by the numbers of Tbr2-positive cells and non-surface dividing cells. The increase in basal progenitors by the Wnt-N-myc axis can be ascribed to either: (1) differentiation of apical progenitors into basal progenitors; or (2) proliferation (and survival) of basal progenitors, or both. The observation that retroviral expression of stabilized β-catenin or N-myc in the neocortex reduced the number of apical progenitors while increasing that of the basal progenitors supports a role for the former mechanism (Kuwahara, 2010).

Members of the Myc family have been reported to be involved in differentiation processes in other cell types, including epithelial, neural crest and hematopoietic stem cells, although previous reports have not directly demonstrated that Myc is involved in fate commitment by a lineage-tracing analysis. In this study, the clonal analysis suggests that N-myc instructs commitment of NPC fate into the neuronal lineage at the expense of the glial lineage and reduces multipotent neurosphere-forming NPCs. This function of N-myc is similar to the reported function of Wnt signaling (Kuwahara, 2010).

It is not known what transcriptional targets of N-myc are involved in instructing neurogenesis. Possible candidates include the proneural gene Ngn1, as deletion of N-myc was observed to cause a decrease in the level of Ngn1 mRNA in the developing neocortex. As Ngn1 is also a direct target of the β-catenin/Tcf transcription complex, it would be interesting to examine the interaction between N-myc and these transcription factors on the Ngn1 promoter. The Myc family has also been shown to function in the regulation of the global chromatin state, in addition to its function as a classical transcription factor; thus it is possible that mechanisms other than direct target gene activation are also involved in N-myc regulation of neurogenesis and proliferation of NPCs (Kuwahara, 2010).

This study also provides evidence that N-myc is directly regulated by the β-catenin/Tcf transcription complex and mediates the functions of Wnt signaling to stimulate neocortical NPC proliferation and differentiation: (1) Wnt3a treatment and stabilized β-catenin expression induced N-myc expression, whereas expression of a dominant-negative form of Tcf3 reduced N-myc expression in NPC cultures; (2) misexpression of stabilized β-catenin in the ventral telencephalon induced ectopic N-myc expression in vivo; (3) N-myc is expressed in the developing neocortex in a pattern similar to that of a Tcf reporter transgene; (4) Tcf3 directly binds to a Tcf-consensus site 1.6 kb upstream of the N-myc gene; (5) Wnt stimulation of proliferation and differentiation in NPC cultures was abrogated by deletion of the N-myc gene. These results provide evidence that N-myc is a key downstream mediator of Wnt-β-catenin signaling in the developing neocortex. It is of note that N-myc is not the only downstream target responsible for the functions of Wnt signaling in the neocortex (Kuwahara, 2010).

The Wnt-β-catenin pathway exerts multiple functions in a context-dependent manner. For instance, persistent expression of stabilized β-catenin in NPCs results in overproliferation of apical progenitors and horizontal/tangential expansion of the cortex in addition to the reduction of Tbr2-positive basal progenitors. However, when the same stabilized β-catenin was expressed by retroviral infection in a small proportion of NPCs located at the VZ, it had the opposite effect: increasing the numbers of basal progenitors and decreasing the number of apical progenitors. This difference does not appear to be due to the differential requirement of N-myc, as N-myc gene deletion rescued both proliferative and differentiating effects of activation of β-catenin. This difference might be rather due to the aberrant brain architecture generated in the β-catenin-δEx3 mice (mutant for β-catenin), to other non-cell autonomous effects of β-catenin or to differences in the levels or timing of active β-catenin expression. Indeed, different levels of active β-catenin expression result in different outcomes in hair follicle stem cells (Kuwahara, 2010).

Although it has previously been postulated that β-catenin exerts its different functions via distinct targets, this study observed that both the proliferating and neurogenic functions of Wnt-β-catenin signaling in the developing neocortex are mediated in common by N-myc. It is noteworthy that c-Myc can also exert distinct functions depending on its expression levels, such as in epithelial stem cells, raising the possibility that the levels of N-myc might determine the cellular output. Importantly, heterozygous mutation of N-MYC (MYCN) in humans causes Feingold syndrome, comprising several defects including microcephaly, supporting the notion that the levels of N-myc in the nervous system are crucial for determining the neuronal number and brain size. It is also possible that N-myc alters its function in a developmental-stage-dependent manner. This possibility is consistent with a previous finding that canonical Wnt signaling promotes proliferation of neocortical neural precursor cells at a relatively early stage (E10.5) but promotes their differentiation at a relatively late stage (E13.5) (Kuwahara, 2010).

Which Wnt ligands are responsible for the activation of N-myc and consequent regulation of basal progenitors in the developing brain? Wnt7a is expressed in NPCs at the VZ and might be important for increase in cells localized in the SVZ. Wnt7b, which is expressed in the deep-layer neurons (neurons at the layer VI), might elicit a feed-forward signal to increase the number of basal progenitors that in turn contribute to the generation of the upper-layer neurons. It is plausible that extracellular signals other than Wnt ligands are also involved in the activation of N-myc and regulation of basal progenitors. N-myc is induced by Shh signaling in cerebellar granule cells, and a recent report shows that Shh protein is localized in the IMZ of the neocortex and contributes to the production of basal progenitors. Growth factors expressed in NPCs such as Fgf2 and epidermal growth factor (Egf) might also participate in the activation of N-myc. Growth factor receptors activate the PI3K (Pik3r1 - Mouse Genome Informatics) pathway, which induces phosphorylation and stabilization of N-myc protein. In addition, Egfr as well as Frs2, an adaptor of Fgfr/Egfr, have been shown to regulate the production of basal progenitors. The RNA-binding protein HuC/D is another candidate that could regulate N-myc function in basal progenitors, as it binds to and stabilizes N-myc mRNA and is localized in the SVZ (Kuwahara, 2010).

As a mechanism of neocortical expansion during animal evolution, the increase of basal progenitors is considered to be a key event, given that basal progenitors increase the number of neurons from a given number of apical progenitors through extra cell division and that the number of basal progenitors dramatically increases during animal evolution. The observation in this study that N-myc deletion decreases Tbr2-positive cells and non-surface dividing cells without marked reduction of Pax6-positive cells supports the notion that Wnt signaling, via N-myc, promotes differentiation from apical progenitors to basal progenitors and promotes indirect neurogenesis. It would be interesting to investigate possible roles of this signaling pathway in the neocortical expansion during animal evolution in future studies (Kuwahara, 2010).

Wnts and the patterning of the midbrain

In the mouse, Engrailed-1 is a target of Wnt-1 signaling in the midbrain. In Wnt-1 knockout mice, En1 and En2 are first expressed normally, but subsequently domains of En expression are lost, concomitant with a failure of midbrain and anterior hindbrain (cerebellum) development. Although neither single En mutant has a severe a phenotype, compound mutants have a similar midbrain and anterior hindbrain phenotype to that of Wnt-1 knockouts. An Engrailed-1 transgene can completely or partially rescue Wnt-1 mutants morphologically, and the expression domains of Pax-5, Fgf-8, En, and in a few cases, Wnt-1 are only slightly reduced relative to wild-type littermates. Nevertheless, two cranial motor nerves, III (oculomotor) and IV (trochlear), which normally develop adjacent to Wnt-1-expressing cells, are not present in Wnt-1 knockouts rescued with Engrailed-1. Thus there may be additional functions of Wnt-1 signaling that cannot be replaced by En-1 (Danielian, 1996).

Gain-of-function assays in Xenopus have demonstrated that Xwnt-3a can pattern neural tissue by reducing the expression of anterior neural genes, and elevating the expression of posterior neural genes. To date, no loss-of-function studies have been conducted in Xenopus to show a requirement of endogenous Wnt signaling for patterning of the neural ectoderm along the anteroposterior axis. Expression of a dominant negative Wnt in Xenopus embryos causes a reduction in the expression of posterior neural genes, and an elevation in the expression of anterior neural genes, thereby confirming the involvement of endogenous Wnt signaling in patterning the neural axis. The ability of Xwnt-3a to decrease expression of anterior neural genes in noggin-treated explants (noggin is a neural inducer) is dependent on a functional FGF signaling pathway, while the elevation of expression of posterior neural genes does not require FGF signaling. In Xenopus, eGFG, FGF3 and XFGF-9 are expressed in the posterior dorsal mesoderm during gastrulation, consistent with potential roles in neural patterning. The previously reported ability of FGF to elevate the expression of posterior neural genes in noggin-treated explants is found to be dependent on endogenous Wnt signaling. It is concluded that neural induction occurs initially in a Wnt-independent manner, but that generation of complete anteroposterior neural pattern requires the cooperative actions of Wnt and FGF pathways. Noggin induces the anterior markers Xanf-1 and Otx-2 in animal cap explants but in the presence of Xwnt-3a, expression of both markers is reduced. At the same time there is an elevation in expression of the posterior neural markers En-2 and Krox-2, although not the spinal cord marker Hox B9. In the presence of FGF, noggin (in contrast) does not reduce the expression of Xanf-1 or Otx-2, while there is a concurrent induction of posterior genes, including Hox B9. Thus Wnts and FGF can both pattern neural tissues but these factors exhibit differences in their neural patterning activities. Xwnt-3a cannot suppress anterior neural genes in the absence of FGF signaling, indicating that the two pathways work together in neural patterning (McGrew, 1997).

Genes encoding fibroblast growth factors (FGFs) are expressed in early Xenopus neurulae in the prospective midbrain--hindbrain boundary (MHB) region of the neural plate. These expression domains overlap those of XWnt-1 and XEn-2, raising the question of the role of FGF signaling in the regulation of these genes, and more generally about the function of FGF during Xenopus midbrain development. Explants from the prospective MHB grafted into the anterior neural plate in midneurula stage embryos induce XWnt-1 expression and, at a lower frequency, XEn-2 expression in the vicinity of the graft. Such a process is likely to involve FGF signaling. Implantation of FGF4- or FGF8-soaked beads in the prospective forebrain at neurula and tailbud stages causes the up-regulation of XWnt-1 and XEn-2 in the dorsal and lateral region of the anterior midbrain. This effect is not relayed by endogenous FGF genes since exogenous FGFs inhibit the expression of endogenous XFGF3 or XFGF8. However, consequences of grafting MHB or implanting FGF4 or FGF8 beads on tadpole brain development are different. MHB grafts induce ectopic mesencephalic structures, strongly suggesting that a region homologous to the isthmic organizer of amniotes is specified as early as the midneurula stage. In contrast, exogenous FGFs do not cause the formation of ectopic mesencephalic structures but an overgrowth of mesencephalon and diencephalon. It is proposed that FGF signals from the prospective MHB play a crucial role in the spatial regulation of XWnt-1 and XEn-2 expression in the posterior midbrain, but that the full organizing activity of the MHB involves other factors in combination with FGF (Riou, 1998).

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence that is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, a 110 base pair regulatory sequence has been identifed within the 5.5 kilobase enhancer that is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1(-/- )embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain, although later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and the mutation of two putative low affinity sites does not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. This analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction (Rowitch, 1998).

The patterns of the Gbx2, Pax2, Wnt1, and Fgf8 gene expression were analyzed in the chick with respect to the caudal limit of the Otx2 anterior domain, taken as a landmark of the midbrain/hindbrain (MH) boundary. The Gbx2 anterior boundary is always concomitant with the Otx2 posterior boundary. The ring of Wnt1 expression is included within the Otx2 domain and Fgf8 transcripts included within the Gbx2 neuroepithelium. Pax2 expression is centered on the MH boundary with a double decreasing gradient. A new nomenclature is proposed to differentiate the vesicles and constrictions observed in the avian MH domain at stage HH10 and HH20, based on the localization of the Gbx2/Otx2 common boundary (Hidalgo-Snachez, 1999).

The isthmus is the organizing center for the tectum and cerebellum. Fgf8 and Wnt1 are secreted molecules expressed around the isthmus. The function of Fgf8 has been well analyzed, and is now accepted as the most important organizing signal. Involvement of Wnt1 in the isthmic organizing activity was suggested by analysis of Wnt1 knockout mice. But its role in isthmic organizing activity is still obscure. Lmx1b is expressed in the isthmic region and it may occupy higher hierarchical position in the gene expression cascade in the isthmus. Specifically, Lmx1b is expressed in connection with Wnt1. Misexpression by the retrovirus vector has shown that Lmx1b can induce Wnt1 expression. Misexpression experiments of Lmx1b and Wnt1 were carried out, and their role in the isthmic organizing activity has been considered. Lmx1b or Wnt1 misexpression causes expansion of the tectum and cerebellum. Fgf8 is repressed in cells that misexpress Lmx1b, but Fgf8 expression is induced around Lmx1b-misexpressing cells. Since Lmx1b induces Wnt1 and Wnt1 induces Fgf8 expression in turn, Wnt1 may be involved in non cell-autonomous induction of Fgf8 expression by Lmx1b. Wnt1 can not induce Lmx1b expression so that Lmx1b may be put at the higher hierarchical position than Wnt1 in the gene expression cascade in the isthmus (Matsunaga, 2002).

Whether Gbx2 (Drosophila homolog: unplugged) is required after embryonic day 9 (E9) to repress Otx2 in the cerebellar anlage and position the midbrain/hindbrain organizer was examined. In contrast to Gbx2 null mutants, mice lacking Gbx2 in rhombomere 1 (r1) after E9 (Gbx2-CKO) are viable and develop a cerebellum. A Gbx2-independent pathway can repress Otx2 in r1 after E9. Mid/hindbrain organizer gene expression, however, continues to be dependent on Gbx2. Fgf8 expression normally correlates with the isthmus where cells undergo low proliferation and in Gbx2-CKO mutants this domain is expanded. It is proposed that Fgf8 permits lateral cerebellar development through repression of Otx2 and also suppresses medial cerebellar growth in Gbx2-CKO embryos. This work has uncovered distinct requirements for Gbx2 during cerebellum formation and provides a model for how a transcription factor can play multiple roles during development (Li, 2002).

In Gbx2-CKO embryos, the juxtaposition of the Wnt1 and Fgf8 expression domains is present at the 8 somite stage, but, consistent with previous studies showing that an interaction between Otx2 and Gbx2 positions the mid/hindbrain organizer, the border is shifted posteriorly to the new Otx2/Gbx2 border. In contrast, at E9.5 when Gbx2 transcripts are no longer detected in r1, Wnt1 and Fgf8 were broadly coexpressed in the alar plate of r1. The derepression of Wnt1 in the alar plate of r1 where Gbx2 is normally expressed demonstrates a cell-autonomous requirement for Gbx2 in repression of Wnt1 expression after E9.5, in agreement with previous studies. Since ectopic expression of Wnt1 in r1 can induce Fgf8 in chick embryos, derepression of Wnt1 in r1 cells in Gbx2-CKO embryos could contribute to the expansion of Fgf8 expression in this region. Furthermore, the expression domain of Pax2 in the isthmus is expanded posteriorly in Gbx2-CKO embryos from E9.5 and largely overlaps with that of Fgf8, consistent with the observation that Pax2 is essential for induction of Fgf8. These experiments show that Gbx2 is required from E8.5 onward to repress Wnt1 expression in r1 and maintain the normal relative expression domains of Wnt1 and Fgf8 (Li, 2002).

Development of the CNS involves highly combinatorial actions of transcription factors. Gbx2 is initially required to repress Otx2 before E8.5 to allow specification of the cerebellar primordium. After E8.5, Gbx2 is not essential for the repression of Otx2 because a second pathway is induced that can repress Otx2. Gbx2 is nevertheless still required for maintenance of normal expression of Wnt1 and Fgf8. The temporal changing requirement for Gbx2 during cerebellar development demonstrated in this work provides a different paradigm for how the same transcription factor can control sequential events during a single developmental process (Li, 2002).

Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b. This linkage relationship may reflect the existence of an ancient cluster of three wnt paralogs (wnt1, wnt6, and wnt10) present before the divergence of protostomes and deuterostomes. The function of wnt10b during embryogenesis has not been explored. In zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. A deficiency allele has been generated for these closely linked loci and morpholino antisense oligo knockdown was performed to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain-hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1-wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB (Lekven, 2003).

In zebrafish, mutagenesis screens have identified three loci required for the formation of the MHB. acerebellar (ace) mutants, which carry mutations in the fgf8 gene, fail to develop a MHB and cerebellum and lose the expression of MHB markers during early somite stages. no isthmus (noi) mutants, which carry mutations in the pax2.1 gene, also fail to form the MHB, but also lack a portion of the midbrain, the optic tectum. In both of these mutants, the expression of several markers for the prospective MHB is initiated properly but quickly disappears, indicating that these genes are required for the early maintenance of gene expression at the MHB. spiel ohne gretzen (spg) mutants also fail to form the MHB, and similar to ace mutants, have enlarged optic tecta. The spg locus encodes zebrafish Pou2, and analysis of early markers of the MHB domain show that spg is also required for the early maintenance of gene expression. The function of spg may be to mediate regional competence in the neurectoderm to Fgf8 signals. Thus, a complex genetic network exists that includes at least Pax2.1, Fgf8, Pou2, and Wnt1, to perform several aspects of MHB formation: initiation of the MHB program, maintenance of gene expression, and morphogenesis (Lekven, 2003).

During two large-scale mutagenesis screens in the zebrafish, only the ace, noi, and spg loci were identified as being required for MHB formation; no wnt1 mutants were recovered. However, wnt1 has been isolated in zebrafish and is expressed in a conserved pattern similar to other vertebrate wnt1 homologs. The conservation of the spatial arrangement of wnt1 and wnt10b raises the possibility that their expression may be regulated in a coordinated fashion. If zebrafish wnt10b and wnt1 are expressed in similar patterns and are at least partially functionally redundant, this could explain the lack of an identified wnt1 point-mutant recovered from two large-scale mutagenesis screens (Lekven, 2003).

The MHB arises at the point in the neural tube where cells expressing otx2 form an interface with cells expressing gbx2 (or gbx1 in zebrafish), and this interface is where pax2, fgf8, and wnt1 expression arises. These three genes form a crossregulatory network to maintain each other's expression. The importance of Fgf8 for the organizing function of the MHB is illustrated by the fact that beads soaked in Fgf8 protein can mimic the polarizing properties of the MHB when implanted into avian embryonic midbrain tissue, and expression of fgf8 isoforms under the control of a wnt1 enhancer results in patterning defects of the midbrain that can result in the induction of wnt1, en, and other MHB genes. In the zebrafish, a suite of genes have been identified that are required not for the positioning or initiation of gene expression at the prospective MHB, but rather for the maintenance of their expression. In noi mutants, expression of wnt1, her5, and en2 is initiated but very quickly disappears, and en3 expression is not properly initiated, possibly indicating a direct role for pax2.1 in its regulation. Pou2, the product of the spg locus, also appears to be required for early steps in maintenance of the MBH regional identity. Similar to noi mutants, the expression of MHB markers (like wnt1, pax2.1, and en2) in spg mutants is initiated but very quickly disappears. Pou2 appears to play a permissive role in patterning the MHB; however, since overexpression does not lead to ectopic expression of either wnt1 or pax2.1, leading to the conclusion that the role of Pou2 is perhaps to provide regional competence in the neurectoderm for reception of Fgf signals, in this case Fgf8. These mutants also point to the establishment of an Fgf8 gradient as being the ultimate output of the MHB. ace mutants lose gene expression later than noi mutants, and they fail to form a cerebellum but do form the optic tectum. Thus, a complex genetic network must exist which involves cross-regulatory interactions, but few studies have been reported to date regarding cis-regulatory sequences for any of these genes. One exception to this is the finding that several pax2/5/8 binding sites are present in the mouse en2 promoter, perhaps indicating that pax2.1 functions through regulation of en2. Consistent with this, simultaneous morpholino knockdown of zebrafish en2 and en3 results in a reduction of the optic tectum and the lack of MHB formation, a phenotype very similar to that seen in noi mutants (Lekven, 2003 and references therein).

Where do wnt1 and wnt10b fit into the MHB genetic hierarchy? In the mouse, the primary function of wnt1 may be to maintain the expression of engrailed homologs, since mutant embryos can be rescued by the expression of en1 under the control of the wnt1 enhancer. However, the expression of fgf8 is also lost in mouse wnt1 mutants, and fgf8 expression can also induce en2. In the case of zebrafish, the results indicate that a portion, but not all, of engrailed and pax2.1 expression is dependent on the activity of wnt1 and wnt10b. This does not rule out the possibility of engrailed and pax2.1 expression in all of the MHB being dependent on Wnt activity, as at least two other Wnts, wnt8b and wnt3a, are still expressed in Df w5 embryos. Further, the result that Dfw5 embryos are sensitized to alterations in the levels of pax2.1 and fgf8 suggests that at least part of the function of wnt1 and wnt10b is to participate in a gene regulatory network that maintains threshold levels of these products at the MHB. Thus, in the absence of wnt1 and wnt10b, threshold levels of en2, pax2.1, fgf8, and other gene products are expressed in at least a portion of the MHB, and the presence of an adequate regulatory network is sufficient for the morphogenetic changes involved in the formation of the MHB. In contrast, when the levels of Pax2.1 or Fgf8 are reduced in the absence of wnt1 and wnt10b, the network is not maintained sufficiently to support the morphogenesis of the MHB. It is concluded that zebrafish wnt1 and wnt10b may be components of a regulative mechanism that protects the embryo against abnormal phenotypes that would result from variability in gene activity (Lekven, 2003).

After the primary anterior-posterior patterning of the neural plate, a subset of wnt signaling molecules including Xwnt-1, Xwnt-2b, Xwnt-3A, Xwnt-8b are still expressed in the developing brain in a region spanning from the posterior part of the diencephalon to the mesencephalon/metencephalon boundary. In this expression field, they are colocalized with the HMG-box transcription factor XTcf-4. Using antisense morpholino loss-of-function strategies, it was demonstrated that the expression of this transcription factor depends on Xwnt-2b, which itself is under the control of XTcf-4. Marker gene analyses reveal that this autoregulatory loop is important for proper development of the midbrain and the isthmus. Staining for NCAM reveals a lack of dorsal neural tissue in this area. This reduction is caused by a reduced proliferation rate as shown by staining for PhosphoH3 positive nuclei. In rescue experiments, it was demonstrated that individual isoforms of XTcf-4 control the development of different parts of the brain. XTcf-4A restores the expression of the mesencephalon marker genes pax-6 and wnt-2b but not the isthmus marker gene en-2. XTcf-4C, in contrast, restores en-2, but has only weak effects on pax-6 and wnt-2b. Thus, autoregulation of canonical Wnt signaling and alternative expression of different isoforms of XTcf-4 is essential for specifying the developing CNS (Kunz, 2004).

Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yields differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain–hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, a full length cDNA was isolated and its expression and function were examined via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development (Buckles, 2004).

Studies in mouse, Xenopus and chicken have shown that Otx2 and Gbx2 expression domains are fundamental for positioning the midbrain-hindbrain boundary (MHB) organizer. Of the two zebrafish gbx genes, gbx1 is a likely candidate to participate in this event because its early expression is similar to that reported for Gbx2 in other species. Zebrafish gbx2, in contrast, acts relatively late at the MHB. To investigate the function of zebrafish gbx1 within the early neural plate, a combination of gain- and loss-of-function experiments was used. Ectopic gbx1 expression in the anterior neural plate reduces forebrain and midbrain, represses otx2 expression and repositions the MHB to a more anterior position at the new gbx1/otx2 border. In the case of gbx1 loss-of-function, the initially robust otx2 domain shifts slightly posterior at a given stage (70% epiboly), as does MHB marker expression. Ectopic juxtaposition of otx2 and gbx1 leads to ectopic activation of MHB markers fgf8, pax2.1 and eng2. This indicates that, in zebrafish, an interaction between otx2 and gbx1 determines the site of MHB development. This work also highlights a novel requirement for gbx1 in hindbrain development. Using cell-tracing experiments, gbx1 was found to cell-autonomously transform anterior neural tissue into posterior. Previous studies have shown that gbx1 is a target of Wnt8 graded activity in the early neural plate. Consistent with this, it was shown that gbx1 can partially restore hindbrain patterning in cases of Wnt8 loss-of-function. It is proposed that in addition to its role at the MHB, gbx1 acts at the transcriptional level to mediate Wnt8 posteriorizing signals that pattern the developing hindbrain. These results provide evidence that zebrafish gbx1 is involved in positioning the MHB in the early neural plate by refining the otx2 expression domain. In addition to its role in MHB formation, gbx1 is a novel mediator of Wnt8 signaling during hindbrain patterning (Rhinn, 2009).

Temporally controlled modulation of FGF/ERK signaling directs midbrain dopaminergic neural progenitor fate in mouse and human pluripotent stem cells

Effective induction of midbrain-specific dopamine (mDA) neurons from stem cells is fundamental for realizing their potential in biomedical applications relevant to Parkinson's disease. During early development, the Otx2-positive neural tissues are patterned anterior-posteriorly to form the forebrain and midbrain under the influence of extracellular signaling such as FGF and Wnt. In the mesencephalon, sonic hedgehog (Shh) specifies a ventral progenitor fate in the floor plate region that later gives rise to mDA neurons. This study systematically investigated the temporal actions of FGF signaling in mDA neuron fate specification of mouse and human pluripotent stem cells and mouse induced pluripotent stem cells. A brief blockade of FGF signaling on exit of the lineage-primed epiblast pluripotent state initiates an early induction of Lmx1a and Foxa2 in nascent neural progenitors. In addition to inducing ventral midbrain characteristics, the FGF signaling blockade during neural induction also directs a midbrain fate in the anterior-posterior axis by suppressing caudalization as well as forebrain induction, leading to the maintenance of midbrain Otx2. Following a period of endogenous FGF signaling, subsequent enhancement of FGF signaling by Fgf8, in combination with Shh, promotes mDA neurogenesis and restricts alternative fates. Thus, a stepwise control of FGF signaling during distinct stages of stem cell neural fate conversion is crucial for reliable and highly efficient production of functional, authentic midbrain-specific dopaminergic neurons. Importantly, evidence is provided that this novel, small-molecule-based strategy applies to both mouse and human pluripotent stem cells (Jaeger, 2011).

This study demonstrates a functional impact of the FGF/ERK signaling level on the course of mDA neuron differentiation of mouse and human pluripotent stem cells. Pharmacological inactivation of FGF/ERK activity upon exit of the lineage-primed epiblast pluripotent state initiates transcription activities that govern early mesencephalic patterning of both the anterior-posterior and dorsal-ventral axes, leading to the induction of mDA neural progenitor characteristics and maintenance of dopaminergic competence. The consolidation of these characteristics, however, requires a period of autocrine/paracrine FGF/ERK signaling immediately after neural induction. Either continued FGF/ERK blockade in newly derived neural progenitors, or enhancing FGF signaling activity by exogenous FGF8 in these cells, abolishes the effects of FGF receptor inhibitor PD173074. These findings demonstrate a previously unrecognized inhibitory role of FGF/ERK in the induction of ventral midbrain neural progenitors and offer a novel strategy for mDA neuron production from mouse and human pluripotent stem cells and iPSCs. Furthermore, the current method represents a simple, small-molecule-based paradigm for significantly improved efficiency and high reproducibility compared with previously reported transgene-free protocols. Importantly, this strategy directs a midbrain regional identity in the derived dopamine neurons, a property that is essential for functional integration of transplanted dopamine neurons in the Parkinsonian brain (Jaeger, 2011).

Stimulation of embryonic stem cell-derived neural progenitors with Shh and FGF8 is used by almost all dopamine differentiation protocols. However, unless combined with genetic manipulation of mDA transcription factors, such as Pitx3 or Lmx1a, the midbrain regional identity of the dopamine neurons generated has remained uncertain. Furthermore, the yield of Th+ neurons has often proved unreliable between experiments and even highly variable between different microscopic fields within a single culture. A major limiting factor is the temporal and spatial heterogeneity of embryonic stem cell-derived neural progenitors. The current findings demonstrate that the above issues can be addressed using epiblast stem cells (EpiSCs). in the absence of FGF/ERK signaling manipulation, nearly 40% of Th+ neurons generated by EpiSCs already co-expressed Pitx3. This represents a significant improvement over ESC-derived monolayer cultures, where Pitx3+ neurons are rarely observed. This improvement is likely to be due to the more synchronous conversion of EpiSCs to the neuroepithelial fate, which would allow for the effective capture of mDA-competent progenitors (Jaeger, 2011).

However, without additional FGF/ERK inhibitor treatment at the neural induction phase, the total numbers of Th+ Pitx3+ cells remained low due to the overall poor efficiency in producing Th+ cells. The early induction of both Lmx1a and Foxa2 by inhibiting FGF receptor or ERK is likely to be a key factor in the observed high efficiency in these experiments. This hypothesis is based on the following observations: (1) d5 PD-treated (EpiSC) MD cultures are highly enriched for Foxa2+ Lmx1a+ neural progenitors compared with untreated controls; (2) although Shh treatment in d5-9 MD results in comparable numbers of Foxa2+ Lmx1a+ cells in PD-primed and no-PD cultures, mDA neuron production was not enhanced in the manner observed with PD treatment; (3) replacing PD with Shh, which turned out to be a slower and less effective inducer of Lmx1a and Foxa2, also led to poor mDA production; and (4) previous reports have credited the dopaminergic-promoting activity of Lmx1a to its early transgene expression in ESC-derived neural progenitors and indicated that Lmx1a functions by cooperating with Foxa2 in specifying mDA fate during midbrain development (Jaeger, 2011).

The robust induction of Wnt1 and its targets in naïve neural progenitors is likely to be a key downstream mediator that confers the observed early induction of Lmx1a, in light of the recent finding that it can be directly regulated by Wnt1/β-catenin signaling. The same study also showed that, although Otx2 itself had no effect in promoting the expression of terminal mDA neuronal marker genes such as Th, Pitx3 and Nurr1, it significantly enhanced the regulatory effect of Lmx1a and Foxa2 on the expression of these genes. Thus, Otx2 plays a permissive role in Lmx1a/Foxa2-mediated mDA neuronal production. It is worth noting that a significant effect of FGF/ERK blockade is the maintenance of Otx2 in derived neural progenitors (Jaeger, 2011).

This study also shows that, in addition to inducing a regulatory cascade for ventralizing nascent neural progenitors, FGF/ERK inhibition suppresses forebrain specification while promoting anterior neural induction, as demonstrated by the strong and consistent repression of the forebrain regulator genes Six3 and Foxg1 and the hindbrain marker Gbx2. Thus, blocking FGF/ERK at the onset of neural induction leads to a direct and early induction of the midbrain fate at the expense of forebrain and caudal neural fates. This finding is consistent with the developmental role of FGF signaling in regionalization of the forebrain (Jaeger, 2011).

Furthermore, this study demonstrated the importance of precise temporal control of cell signaling and its cross-regulation with other signaling pathways in mDA neuronal fate specification. During development, Fgf8-mediated signaling can induce the patterned expression of many midbrain/rostral hindbrain genes and is required for normal development of the midbrain and cerebellum. Fgf8-induced Wnt1 and engrailed are key regulators of midbrain and cerebellum patterning, as well as of the differentiation and survival of dopamine neurons. In EpiSC-derived neural cultures, after an initial burst of upregulation induced by PD exposure, Wnt1 expression was subsequently reduced to a level below the no-PD control by unknown factors in the newly generated neural progenitors in d3-5 MD. This is the period when Shh, Lmx1a and Foxa2 expression levels continued to rise. Given that Shh and Wnt1 play opposing roles with regard to mDA neurogenesis, these findings suggest that the delay in FGF reactivation, which suppresses Wnt1 levels, might be crucial for achieving high numbers of Th+ neurons by consolidating Lmx1a and Foxa2 expression via Shh signaling (Jaeger, 2011).

From a technological standpoint, this study describes a novel method of mDA neuron differentiation that employs temporally controlled exposure of human and mouse pluripotent stem cells to an FGF/ERK-deficient environment. The highly reliable nature of this method was demonstrated using five independent mouse EpiSC lines, a mouse iPS cell line and two human ESC lines. This protocol offers several advantages over current methods of generating midbrain-specific DA neurons in that it is adherent culture-based and free from genetic manipulation and thus could be readily applied to other cell lines of interest. Furthermore, because it is fully chemically defined, this paradigm could be readily adapted for use in a clinical setting or scaled up for toxicity and drug screening relevant to developing new therapeutics for Parkinson's disease (Jaeger, 2011).

Wnts and axon guidance

Guidance cues along the longitudinal axis of the CNS are poorly understood. Wnt proteins attract ascending somatosensory axons to project from the spinal cord to the brain. Wnt proteins repel corticospinal tract (CST) axons in the opposite direction. Several Wnt genes were found to be expressed in the mouse spinal cord gray matter, cupping the dorsal funiculus, in an anterior-to-posterior decreasing gradient along the cervical and thoracic cord. Wnts repel CST axons in collagen gel assays through a conserved high-affinity receptor, Ryk, which is expressed in CST axons. Neonatal spinal cord secretes diffusible repellent(s) in an anterior-posterior graded fashion, with anterior cord being stronger, and the repulsive activity is blocked by antibodies to Ryk (anti-Ryk). Intrathecal injection of anti-Ryk blocks the posterior growth of CST axons. Therefore, Wnt proteins may have a general role in anterior-posterior guidance of multiple classes of axons (Liu, 2005).

Ryk is novel Wnt receptor in both Caenorhabditis elegans and Drosophila melanogaster. Ryk-Wnt interactions have been shown to guide corticospinal axons down the embryonic mouse spinal cord. In Ryk-deficient mice, cortical axons project aberrantly across the major forebrain commissure, the corpus callosum. Many mouse mutants have been described in which loss-of-function mutations result in the inability of callosal axons to cross the midline, thereby forming Probst bundles on the ipsilateral side. In contrast, loss of Ryk does not interfere with the ability of callosal axons to cross the midline but impedes their escape from the midline into the contralateral side. Therefore, Ryk-/- mice display a novel callosal guidance phenotype. Wnt5a acts as a chemorepulsive ligand for Ryk, driving callosal axons toward the contralateral hemisphere after crossing the midline. In addition, whereas callosal axons do cross the midline in Ryk-/- embryos, they are defasciculated on the ipsilateral side, indicating that Ryk also promotes fasciculation of axons before midline crossing. In summary, this study expands the emerging role for Wnts in axon guidance and identifies Ryk as a key guidance receptor in the establishment of the corpus callosum. This analysis of Ryk function further advances understanding of the molecular mechanisms underlying the formation of this important commissure (Keeble, 2006).

Computational modelling has suggested that at least two counteracting forces are required for establishing topographic maps. Ephrin-family proteins are required for both anterior-posterior and medial-lateral topographic mapping, but the opposing forces have not been well characterized. Wnt-family proteins are recently discovered axon guidance cues. Wnt3 is expressed in a medial-lateral decreasing gradient in chick optic tectum and mouse superior colliculus. Retinal ganglion cell (RGC) axons from different dorsal-ventral positions show graded and biphasic response to Wnt3 in a concentration-dependent manner. Wnt3 repulsion is mediated by Ryk, expressed in a ventral-to-dorsal decreasing gradient, whereas attraction of dorsal axons at lower Wnt3 concentrations is mediated by Frizzled(s). Overexpression of Wnt3 in the lateral tectum repels the termination zones of dorsal RGC axons in vivo. Expression of a dominant-negative Ryk in dorsal RGC axons causes a medial shift of the termination zones, promoting medially directed interstitial branches and eliminating laterally directed branches. Therefore, a classical morphogen, Wnt3, acting as an axon guidance molecule, plays a role in retinotectal mapping along the medial-lateral axis, counterbalancing the medial-directed EphrinB1-EphB activity (Schmitt, 2006).

Wnts and synaptogenesis

Wnt proteins play a crucial role in several aspects of neuronal circuit formation. Wnts can signal through different receptors including Frizzled, Ryk and Ror2. In the hippocampus, Wnt7a stimulates the formation of synapses; however, its receptor remains poorly characterized. This study demonstrates that Frizzled-5 (Fz5) is expressed during the peak of synaptogenesis in the mouse hippocampus. Fz5 is present in synaptosomes and colocalizes with the pre- and postsynaptic markers vGlut1 and PSD-95. Expression of Fz5 during early stages of synaptogenesis increases the number of presynaptic sites in hippocampal neurons. Conversely, Fz5 knockdown or the soluble Fz5-CRD domain (Fz5CRD), which binds to Wnt7a, block the ability of Wnt7a to stimulate synaptogenesis. Increased neuronal activity induced by K+ depolarization or by high-frequency stimulation (HFS), known to induce synapse formation, raises the levels of Fz5 at the cell surface. Importantly, both stimuli increase the localization of Fz5 at synapses, an effect that is blocked by Wnt antagonists or Fz5CRD. Conversely, low-frequency stimulation, which reduces the number of synapses, decreases the levels of surface Fz5 and the percentage of synapses containing the receptor. Interestingly, Fz5CRD abolishes HFS-induced synapse formation. These results indicate that Fz5 mediates the synaptogenic effect of Wnt7a and that its localization to synapses is regulated by neuronal activity, a process that depends on endogenous Wnts. These findings support a model where neuronal activity and Wnts increase the responsiveness of neurons to Wnt signalling by recruiting Fz5 receptor at synaptic sites (Sahores, 2010).

Table of contents

wingless continued: Biological Overview | Transcriptional regulation |Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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