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Elkouby YM, Frank D. Wnt/β-Catenin Signaling in Vertebrate Posterior Neural Development. San Rafael (CA): Morgan & Claypool Life Sciences; 2010.

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Wnt/β-Catenin Signaling in Vertebrate Posterior Neural Development.

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Chapter 2Making the Neural Rear

In 1987, the Nusse group identified the mouse mammary oncogene int-1 as the homolog of the Drosophila segment polarity gene wingless (Rijsewijk et al., 1987). The convergence of two independent disciplines, of cancer research and embryology, had opened the door for great discoveries in cell and developmental biology. In the following two decades, the canonical Wnt signaling pathway has been established as a key regulator of cell fate specification, differentiation, and growth in multiple developing systems throughout the animal kingdom. This may be best demonstrated by the development of the posterior nervous system of the vertebrate embryo. During this complex process, Wnt/β-catenin signaling is involved in multisteps, including the initial specification of the posterior regions of the early nervous system, the fine-tuned patterning of each region, the determination of different cell fates within it, and the coordination of proliferation and differentiation of a given cell type population. In the caudal nervous system, mid-hindbrain border (MHB), hindbrain, spinal cord, primary neuron, and neural crest (NC) cell fates are all induced during late gastrula stages in intimately close regions of the neural plate, and primarily by the same signals of Wnt, RA, and FGF. We will generically refer to these cell fates collectively as “posterior neural cell fates.”

Several Wnt ligands, such as Wnt-1, Wnt-3, Wnt-3a, Wnt-4, Wnt-8, Wnt-8b and Wnt-10, are expressed in the early developing nervous system of vertebrate embryos (Hollyday et al., 1995; Hume and Dodd, 1993; Kelly et al., 1993; McGrew et al., 1995; McGrew et al., 1992; Molven et al., 1991; Roelink and Nusse, 1991; Wolda et al., 1993). Their role in posterior neural development was first implied in Wnt1- and Wnt3a-deficient mouse embryos, where midbrain, hindbrain, and spinal cord structures failed to develop properly (Augustine et al., 1993; Augustine et al., 1995; McMahon et al., 1992). Wnt3 null mouse embryos showed a similar phenotype of posterior truncation, with a dramatic expansion of the forebrain marker Otx2 and a loss of that of the hindbrain marker HoxB1 (Liu et al., 1999b). A similar scenario was observed in HH stage 4 chick embryos implanted with beads soaked with a soluble form of the Frz receptor (mFrz8-CRD) that sequesters and antagonizes Wnt ligands signaling (see Chapter 9, “Anti-Wnt Anterior Determinants”). Such treated embryos exhibited the same typical expansion of forebrain markers (Otx2 and Pax6) and down-regulation of mid-hindbrain junction (En1) and hindbrain (Gbx2) markers (Nordstrom et al., 2002). Ex vivo cultured chick neural plate explants support these findings. Neural plate explants taken from A–P level of the hindbrain and spinal cord expressed CdxB and CdxC in an equivalent of HH stage 8 (three somites), and Krox20, HoxB4, HoxB8, and HoxC9 in the HH 17 (30 somites) equivalent, while explants taken from the level of the rostral hindbrain expressed Gbx2 and Krox20 at HH stage 4. Addition of mFrz8-CRD to the culture medium eliminated the expression of all of these posterior markers (Nordstrom et al., 2002; Nordstrom et al., 2006). This requirement for Wnt for posterior neural development was also confirmed in Xenopus embryos, where expression of a dominant-negative Wnt protein (inhibiting Wnt1, Wnt3a, and Wnt8 activity) or the Wnt antagonist Dkk-1 protein yielded an anteriorized phenotype (Figure 2.1A). Like Wnt3–/– mouse embryos, these embryos also exhibited neural tube closure defects (McGrew et al., 1997). In addition, expression of the XAnf1 and Otx2 forebrain markers was posteriorly expanded, while expression of the MHB En2 and hindbrain Krox20 markers was depleted (McGrew et al., 1997). Specific Wnt3a- and Wnt8- MO targeting also suggested a role in hindbrain and spinal cord development in zebrafish embryos (Figure 2.1B) (Erter et al., 2001; Lekven et al., 2001). Knockdown of Wnt3a seemed to specifically affect the neural plate, whereas knockdown of Wnt8 activity may reflect a more upstream mesodermal and thus indirect neural inhibition phenotype (see Chapter 12, “The Role of Mesoderm and Specific Wnt Ligands in Neural Patterning”). Indeed, specific Wnt3a knockdown by MO targeting in Xenopus, revealed a strict requirement for this ligand in hindbrain formation (Elkouby et al., 2010). Wnt3a morphant embryos exhibited neural convergent and extension defects, having the typical caudal expansion of forebrain markers (XAnf1, Otx2), with depletion of hindbrain markers (Krox20, HoxB3) at neurula stages. Moreover, during gastrula stages, when hindbrain induction initially takes place, expression of the early posteriorizing homeoproteins Meis3 and HoxD1 was lost (Elkouby et al., 2010). Wnt3a MO phenocopied the neural phenotypes of the general Wnt/β-catenin inhibitor Dkk1 (see Chapter 9, “Anti-Wnt Anterior Determinants”), without altering neither Wnt8 expression nor activity (Elkouby et al., 2010), further supporting a role for Wnt3a as the primary posterior neural inducer in vertebrates.

FIGURE 2.1. Wnt/β-catenin activity is required for the formation of posterior structures.


Wnt/β-catenin activity is required for the formation of posterior structures. Anterior forebrain structures are enlarged and extend more caudally, and posterior hindbrain and spinal cord are reduced when Wnt activity is compromised. (A) Xenopus (more...)

Complementing these loss-of-function studies, various Wnt gain-of-function assays revealed its ability to sufficiently induce posterior neural cell fates and repress anterior ones (Figure 2.2). The Xenopus animal cap (AC) explant system has provided a great tool for examining the role of Wnt signaling in neural patterning. Overexpression of BMP4 antagonist or BMP4 dominant-negative receptor proteins in AC explants induces neural tissue. Such neuralized explants mimic the initial state of the newly induced embryonic neural plate. These AC explants express pan-neural and anterior neural markers, and will develop as anterior forebrain/cement gland in the absence of additional caudalizing signals. Neuralized AC explants overexpressing different Wnt ligands or downstream effectors, such as β-catenin or inducible constitutively active Tcf, robustly induced expression of mid-hindbrain junction, hindbrain, primary neuron, and neural crest markers, while strongly suppressing anterior neural marker expression (Domingos et al., 2001; Elkouby et al., 2010; McGrew et al., 1995; Monsoro-Burq et al., 2005; Wu et al., 2005). In addition, expression of the earliest hindbrain specifying homeoproteins, Meis3, HoxD1, HoxA2, and Gbx2, along with caudalizing FGF3 and FGF8 genes, were also induced in gastrula-stage neuralized AC explants overexpressing either Wnt3a or β-catenin protein (Elkouby et al., 2010; Li et al., 2009). In a similar fashion, chick early, HH stage 4, rostral forebrain explants were caudally transformed by Wnt3a added to the culture medium (supplemented with FGF) expressing Pax6, En1, Gbx2, and Krox20, instead of Otx2 (Nordstrom et al., 2002). Wnt/β-catenin induction of posterior neural cell fates was shown in both frogs and zebrafish to occur specifically during mid-late gastrula stages (Domingos et al., 2001; Elkouby et al., 2010; Erter et al., 2001; Shimizu et al., 2005). Thus, Wnt/β-catenin signaling can induce the caudalizing transformation step required for proper A–P neural patterning.

Overactivation of Wnt/β-catenin in vivo by overexpression of either different ligands or downstream effectors also supports this conclusion (Figure 2.2). Mouse embryos ubiquitously expressing Wnt8c, driven by a β-actin promoter, had severe anterior truncations with depleted Hesx and anteriorly expanded Wnt1 expression (Popperl et al., 1997). Zebrafish embryos overexpressing a Hsp-Wnt8 driver plasmid that were heat-shocked at gastrula stages exhibited an anterior shift in expression of MHB and hindbrain markers, with forebrain markers pushed to the anterior extremity (Erter et al., 2001). In addition, the zebrafish headless mutant lacked structures of the eye, forebrain, and part of the midbrain (Figure 2.2A), and hardly expressed Anf1, Six3, and Rx3 forebrain markers. Concomitantly, the more posterior Pax2, En2, and Krox20 were expanded anteriorly (Kim et al., 2000). headless was identified as a point mutation in the Wnt downstream negative-effector Tcf 3 gene, mutant TCF3 protein was unable to translocate to the nucleus or to bind DNA, thus causing a loss-of-function phenotype (Kim et al., 2000). In cells where the Wnt/β-catenin pathway is not activated, together with other co-repressor proteins, Tcf3 represses expression of pathway target genes. The loss of Tcf3-mediated repression in the headless mutants reflects an overactive Wnt/β-catenin pathway in the embryos and is therefore responsible for the caudalized phenotype (Kim et al., 2000). Xenopus embryos overexpressing either an inducible β-catenin protein activated at gastrula stages, or a CMV-promoter driven Wnt3a, which is transcribed post-MBT, also induced caudalized embryos (Figure 2.2B) with ectopic expansion of MHB and hindbrain markers anteriorly, and down-regulation of anterior markers (Domingos et al., 2001; Elkouby et al., 2010). This anterior transformation in morphology and gene expression pattern to more caudal fates was also evident in Dkk1 null mouse embryos (Figure 2.2C), in which the Wnt/β-catenin pathway is overactivated because of the loss of this pathway antagonist (see Chapter 9, “Anti-Wnt Anterior Determinants”) (MacDonald et al., 2004), and in 14-somite-stage chick embryos, implanted with Wnt3a soaked beads during HH stage 4 (Nordstrom et al., 2002) (Figure 2.2D).

FIGURE 2.2. Wnt/β-catenin activity caudalizes the embryonic nervous system.


Wnt/β-catenin activity caudalizes the embryonic nervous system. Anterior forebrain structures fail to form and posterior hindbrain and spinal cord are expanded ectopically to the anterior end in Wnt-activated embryos. (A) Zebrafish headless mutant, (more...)

Copyright © 2010 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK53467
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