Randall T. Moon

Zebrafish Prickle, a Modulator of Noncanonical Wnt/Fz Signaling, Regulates Gastrulation Movements

In addition to the canonical Wnt/beta-catenin signaling pathway, at least two noncanonical Wnt/Fz pathways have been described: the planar cell polarity (PCP) pathway in Drosophila [1] and the Wnt/calcium pathway in vertebrate embryos [2]. Recent work suggests that a vertebrate pathway homologous to the PCP pathway acts to regulate the convergent extension movements of gastrulation [3-7]. To further test this hypothesis, we have identified two zebrafish homologs of the Drosophila PCP gene prickle (pk) [8], both of which show discrete and dynamic expression patterns during gastrulation. Both gain and loss of pk1 function cause defects in convergent extension. Pk1 localizes to both the cytoplasm and the cell membrane, and its normal localization is partially dependent on its C-terminal prenylation motif. At the cell membrane, Pk1 is frequently localized asymmetrically around the cell and can colocalize with the signaling molecule Dishevelled (Dsh). In overexpression assays, Pk1 is able to activate AP-1-mediated transcription and inhibit activation of Wnt/beta-catenin signaling. Like noncanonical Wnts [9-10], overexpression of Pk1 increases the frequency of calcium transients in zebrafish blastulae. Our results support the idea that a vertebrate PCP pathway regulates gastrulation movements and suggest that there is overlap between the PCP and Wnt/calcium pathways.

Disruptive CHD8 mutations define a subtype of autism early in development.

Autism spectrum disorder (ASD) is a heterogeneous disease in which efforts to define subtypes behaviorally have met with limited success. Hypothesizing that genetically based subtype identification may prove more productive, we resequenced the ASD-associated gene CHD8 in 3,730 children with developmental delay or ASD. We identified a total of 15 independent mutations; no truncating events were identified in 8,792 controls, including 2,289 unaffected siblings. In addition to a high likelihood of an ASD diagnosis among patients bearing CHD8 mutations, characteristics enriched in this group included macrocephaly, distinct faces, and gastrointestinal complaints. chd8 disruption in zebrafish recapitulates features of the human phenotype, including increased head size as a result of expansion of the forebrain/midbrain and impairment of gastrointestinal motility due to a reduction in postmitotic enteric neurons. Our findings indicate that CHD8 disruptions define a distinct ASD subtype and reveal unexpected comorbidities between brain development and enteric innervation.

Dapper, a Dishevelled-Associated Antagonist of β-Catenin and JNK Signaling, Is Required for Notochord Formation

Dapper was isolated in a screen for proteins interacting with Dishevelled, a key factor in Wnt signaling. Dapper and Dishevelled colocalize intracellularly and form a complex with Axin, GSK-3, CKI, and beta-catenin. Overexpression of Dapper increases Axin and GSK-3 in this complex, resulting in decreased soluble beta-catenin and decreased activation of beta-catenin-responsive genes. Dapper also inhibits activation by Dishevelled of c-Jun N-terminal kinase (JNK), a component of beta-catenin-independent Frizzled signaling. Inhibition of Dapper activates both beta-catenin-responsive genes and an AP1-responsive promoter, demonstrating that Dapper is a general Dishevelled antagonist. Depletion of maternal Dapper RNA from Xenopus embryos results in loss of notochord and head structures, demonstrating that Dapper is required for normal vertebrate development.

Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-β-catenin signaling.

Tyrosine kinase inhibitors (TKIs) are highly effective in treatment of chronic myeloid leukemia (CML) but do not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSCs, suggesting that additional kinase-independent mechanisms contribute to LSC preservation. We investigated whether signals from the bone marrow (BM) microenvironment protect CML LSCs from TKI treatment. Coculture with human BM mesenchymal stromal cells (MSCs) significantly inhibited apoptosis and preserved CML stem/progenitor cells following TKI exposure, maintaining colony-forming ability and engraftment potential in immunodeficient mice. We found that the N-cadherin receptor plays an important role in MSC-mediated protection of CML progenitors from TKI. N-cadherin-mediated adhesion to MSCs was associated with increased cytoplasmic N-cadherin-β-catenin complex formation as well as enhanced β-catenin nuclear translocation and transcriptional activity. Increased exogenous Wnt-mediated β-catenin signaling played an important role in MSC-mediated protection of CML progenitors from TKI treatment. Our results reveal a close interplay between N-cadherin and the Wnt-β-catenin pathway in protecting CML LSCs during TKI treatment. Importantly, these results reveal novel mechanisms of resistance of CML LSCs to TKI treatment and suggest new targets for treatment designed to eradicate residual LSCs in CML patients.

Structurally Related Receptors and Antagonists Compete for Secreted Wnt Ligands

Since Frzb can block the signaling activity of ectopic Xwnt-8, this raises the question of whether Frzb and Wnts are ever expressed in a manner consistent with their normally functioning in an antagonistic manner. In mouse embryos, Frzb is expressed in the primitive streak during gastrulation, and in mouse and human adults Frzb is expressed in a variety of adult organs, including the heart, brain, and skeletal muscle, pancreas, and kidney (Leyns et al. 1997xLeyns, L., Bouwmeester, T., Kim, S.-H., Piccolo, S., and DeRobertis, E.M. Cell. 1997; 88Abstract | Full Text | Full Text PDF | PubMed | Scopus (505)See all ReferencesLeyns et al. 1997). In Xenopus embryos, Frzb is also expressed during gastrulation, in a remarkable region of the embryo known as the gastrula organizer, or Spemanns organizer (Figure 1Figure 1) (8xLeyns, L., Bouwmeester, T., Kim, S.-H., Piccolo, S., and DeRobertis, E.M. Cell. 1997; 88Abstract | Full Text | Full Text PDF | PubMed | Scopus (505)See all References, 16xWang, S., Krinks, M., Lin, K., Luyten, F.P., and Moos, M. Jr. Cell. 1997; 88Abstract | Full Text | Full Text PDF | Scopus (371)See all References). This dorsal region, when transplanted to the ventral side of a host embryo, is able to induce the formation of a new body axis, complete with notochord, neural structures, and muscle (Spemann 1938xSee all ReferencesSpemann 1938; reviewed byLemaire and Kodjabachian 1996xLemaire, P. and Kodjabachian, L. Trends Genet. 1996; 12: 525–531Abstract | Full Text PDF | PubMed | Scopus (138)See all ReferencesLemaire and Kodjabachian 1996). Like Frzb, Wnts are also expressed in vertebrate embryos and adult tissues, including some of the tissues that express Frzb (9xMcMahon, A.P. Trends Genet. 1992; 8: 236–242Abstract | Full Text PDF | Scopus (89)See all References, 10xNusse, R. and Varmus, H.E. Cell. 1992; 69: 1073–1087Abstract | Full Text PDF | PubMed | Scopus (656)See all References). While careful mapping of the spatial patterns of expression of Frzb, Frizzled homologs, and Wnts has not yet been conducted, the complementary patterns of Frzb and Xwnt-8 in Xenopus (Figure 1Figure 1) are certainly consistent with these secreted factors working in an antagonistic manner in the future mesoderm.The ability of ectopic Frzb to block embryonic responses to ectopic Xwnt-8 does not establish that Frzb normally works to counteract the functions of endogenous Xwnt-8; however, further experiments by Leyns et al. 1997xLeyns, L., Bouwmeester, T., Kim, S.-H., Piccolo, S., and DeRobertis, E.M. Cell. 1997; 88Abstract | Full Text | Full Text PDF | PubMed | Scopus (505)See all ReferencesLeyns et al. 1997 and Wang et al. 1997xWang, S., Krinks, M., Lin, K., Luyten, F.P., and Moos, M. Jr. Cell. 1997; 88Abstract | Full Text | Full Text PDF | Scopus (371)See all ReferencesWang et al. 1997 support this likelihood. As noted above, endogenous Xwnt-8 is expressed in prospective ventro-lateral mesoderm of the gastrula, and its overexpression in regions of the embryo in which it is not normally expressed can divert the fate of those cells to a ventro-lateral mesodermal fate (Christian and Moon 1993xChristian, J.L. and Moon, R.T. Genes Dev. 1993; 7: 13–28CrossRef | PubMedSee all ReferencesChristian and Moon 1993). If a normal function of Xwnt-8 were to participate in forming ventro-lateral mesoderm, then its loss-of-function should perturb the somites and skeletal muscle, which are derived from this region (Figure 1Figure 1). Supporting this role, a dominant negative Xwnt-8 blocks induction of MyoD in the embryo, and inhibits formation of skeletal muscle (Hoppler et al. 1996xHoppler, S., Brown, J.D., and Moon, R.T. Genes Dev. 1996; 10: 2805–2817CrossRef | PubMedSee all ReferencesHoppler et al. 1996). Consistent with Frzb functioning to antagonize endogenous Xwnt-8, ectopic expression of Frzb throughout early Xenopus embryos also blocks induction of MyoD, and inhibits formation of skeletal muscle (8xLeyns, L., Bouwmeester, T., Kim, S.-H., Piccolo, S., and DeRobertis, E.M. Cell. 1997; 88Abstract | Full Text | Full Text PDF | PubMed | Scopus (505)See all References, 16xWang, S., Krinks, M., Lin, K., Luyten, F.P., and Moos, M. Jr. Cell. 1997; 88Abstract | Full Text | Full Text PDF | Scopus (371)See all References). Local ectopic expression of Frzb in ventral blastomeres at the 4-cell stage results in partial dorsalized phenotypes, also likely by antagonizing the ventral signal of endogenous Xwnt-8 (Wang et al. 1997xWang, S., Krinks, M., Lin, K., Luyten, F.P., and Moos, M. Jr. Cell. 1997; 88Abstract | Full Text | Full Text PDF | Scopus (371)See all ReferencesWang et al. 1997). These experiments support the conclusion that endogenous Frzb is a novel antagonist of the functions of some endogenous Wnts, including Xwnt-8.The presence of Frzb in the gastrula organizer may serve several functions, depending upon the largely unknown extent of diffusion of both Frzb and Xwnts. Within the extracellular space of the gastrula organizer, Frzb may locally block the instructive signal of Xwnt-8 which promotes ventro-lateral mesoderm formation, thereby allowing the development of dorsal cell fates. Second, there may be a region at the boundary of expression of Xwnt-8 and Frzb where the extracellular environment contains both proteins, which could yield a graded response to Xwnt-8 in the prospective lateral mesoderm. Third, since signals from the organizer induce and pattern the nervous system (Hemmati-Brivanlou and Melton 1997xHemmati-Brivanlou, A. and Melton, D. Cell. 1997; 88: 13–17Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesHemmati-Brivanlou and Melton 1997), it is possible that Frzb is involved in modulating neural cell fate, or endodermal cell fate, in addition to its likely roles in the mesoderm. Further testing of the functions of Frzb requires both a better understanding of the range of action of Frzb and Wnt signals, as well as genetic loss-of-function data. The loss-of-function studies would presumably need to come from another organism, since methods for interfering with gene expression in Xenopus embryos are not very precise, and the approach of interfering with protein function through design of a dominant negative protein is probably not suitable when the protein in question is essentially a dominant negative protein to begin with.While showing that the gastrula organizer is the source of a Wnt antagonist was completely unexpected, the new work on Frzb also adds to the growing evidence that the gastrula organizer is the surprising source of multiple secreted antagonists. Previously, the gastrula organizer has been shown to be a source of noggin, follistatin, and chordin that can bind to bone morphogenetic proteins (BMPs), thereby preventing their activating receptor-mediated signaling pathways (reviewed by7xLemaire, P. and Kodjabachian, L. Trends Genet. 1996; 12: 525–531Abstract | Full Text PDF | PubMed | Scopus (138)See all References, 3xHemmati-Brivanlou, A. and Melton, D. Cell. 1997; 88: 13–17Abstract | Full Text | Full Text PDF | PubMedSee all References). The consequence of antagonizing BMPs is that some of the dorsal ectodermal tissue then follows a default developmental pathway and becomes the nervous system. These examples underscore the important role of inhibitory signals, along with receptor-activating signals, in establishing regions of distinct cell fate and function in the embryo.In developmental biology one is constantly reminded that modern cell and molecular studies are often filling the gaps in processes identified or suspected many years ago by the embryologists and philosophers who asked how eggs exploit adults to make more eggs. The idea that different regions of embryos can be the sources of different signals that work in gradients, or in synergistic or antagonistic combinations, was well developed from the work of Boveri, Child, Holtfreter, and others (reviewed bySpemann 1938xSee all ReferencesSpemann 1938). Indeed, “the inducing effect of the organizer was, from the beginning, characterized as a releasing one” (Spemann 1938xSee all ReferencesSpemann 1938). One suspects that these embryologists would have been satisfied to learn of the complementary patterns of expression of Xwnt-8 and Frzb and to learn of their antagonistic functions in the Xenopus embryo.sc

Wnt signaling: why is everything so negative?

The Wnt proteins constitute a family of secreted glycoproteins the members of which have essential signaling roles during embryogenesis. The recent identification of several new regulators of this signal transduction pathway have revealed unexpectedly intricate levels of constraint on Wnt-dependent gene activation, and studies in developing embryos and in cell culture systems have allowed a more complete understanding of the functional and biochemical interactions between components of this evolutionarily conserved pathway.

β-Catenin Signaling Increases in Proliferating NG2+ Progenitors and Astrocytes during Post-Traumatic Gliogenesis in the Adult Brain

Wnt/β‐catenin signaling can influence the proliferation and differentiation of progenitor populations in the hippocampus and subventricular zone, known germinal centers in the adult mouse brain. It is not known whether β‐catenin signaling occurs in quiescent glial progenitors in cortex or spinal cord, nor is it known whether β‐catenin is involved in the activation of glial progenitor populations after injury. Using a β‐catenin reporter mouse (BATGAL mouse), we show that β‐catenin signaling occurs in NG2 chondroitin sulfate proteoglycan+ (NG2) progenitors in the cortex, in subcallosal zone (SCZ) progenitors, and in subependymal cells surrounding the central canal. Interestingly, cells with β‐catenin signaling increased in the cortex and SCZ following traumatic brain injury (TBI) but did not following spinal cord injury. Initially after TBI, β‐catenin signaling was predominantly increased in a subset of NG2+ progenitors in the cortex. One week following injury, the majority of β‐catenin signaling appeared in reactive astrocytes but not oligodendrocytes. Bromodeoxyuridine (BrdU) paradigms and Ki‐67 staining showed that the increase in β‐catenin signaling occurred in newly born cells and was sustained after cell division. Dividing cells with β‐catenin signaling were initially NG2+; however, by four days after a single injection of BrdU, they were predominantly astrocytes. Infusing animals with the mitotic inhibitor cytosine arabinoside prevented the increase of β‐catenin signaling in the cortex, confirming that the majority of β‐catenin signaling after TBI occurs in newly born cells. These data argue for manipulating the Wnt/β‐catenin pathway after TBI as a way to modify post‐traumatic gliogenesis. STEM CELLS 2010;28:297–307

Regulation of ribosomal S6 protein kinase-p90(rsk), glycogen synthase kinase 3, and beta-catenin in early Xenopus development.

ABSTRACT β-Catenin is a multifunctional protein that binds cadherins at the plasma membrane, HMG box transcription factors in the nucleus, and several cytoplasmic proteins that are involved in regulating its stability. In developing embryos and in some human cancers, the accumulation of β-catenin in the cytoplasm and subsequently the nuclei of cells may be regulated by the Wnt-1 signaling cascade and by glycogen synthase kinase 3 (GSK-3). This has increased interest in regulators of both GSK-3 and β-catenin. Searching for kinase activities able to phosphorylate the conserved, inhibitory-regulatory GSK-3 residue serine 9, we found p90 rsk to be a potential upstream regulator of GSK-3. Overexpression of p90 rsk in Xenopus embryos leads to increased steady-state levels of total β-catenin but not of the free soluble protein. Instead, p90 rsk overexpression increases the levels of β-catenin in a cell fraction containing membrane-associated cadherins. Consistent with the lack of elevation of free β-catenin levels, ectopic p90 rsk was unable to rescue dorsal cell fate in embryos ventralized by UV irradiation. We show that p90 rsk is a downstream target of fibroblast growth factor (FGF) signaling during early Xenopus development, since ectopic FGF signaling activates both endogenous and overexpressed p90 rsk . Moreover, overexpression of a dominant negative FGF receptor, which blocks endogenous FGF signaling, leads to decreased p90 rsk kinase activity. Finally, we report that FGF inhibits endogenous GSK-3 activity inXenopus embryos. We hypothesize that FGF and p90 rsk play heretofore unsuspected roles in modulating GSK-3 and β-catenin.

Altered splicing of ATP6AP2 causes X-linked parkinsonism with spasticity (XPDS)

We report a novel gene for a parkinsonian disorder. X-linked parkinsonism with spasticity (XPDS) presents either as typical adult onset Parkinsons disease or earlier onset spasticity followed by parkinsonism. We previously mapped the XPDS gene to a 28 Mb region on Xp11.2-X13.3. Exome sequencing of one affected individual identified five rare variants in this region, of which none was missense, nonsense or frame shift. Using patient-derived cells, we tested the effect of these variants on expression/splicing of the relevant genes. A synonymous variant in ATP6AP2, c.345C>T (p.S115S), markedly increased exon 4 skipping, resulting in the overexpression of a minor splice isoform that produces a protein with internal deletion of 32 amino acids in up to 50% of the total pool, with concomitant reduction of isoforms containing exon 4. ATP6AP2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy, a pathway frequently affected in Parkinsons disease. Reduction of the full-size ATP6AP2 transcript in XPDS cells and decreased level of ATP6AP2 protein in XPDS brain may compromise V-ATPase function, as seen with siRNA knockdown in HEK293 cells, and may ultimately be responsible for the pathology. Another synonymous mutation in the same exon, c.321C>T (p.D107D), has a similar molecular defect of exon inclusion and causes X-linked mental retardation Hedera type (MRXSH). Mutations in XPDS and MRXSH alter binding sites for different splicing factors, which may explain the marked differences in age of onset and manifestations.

Requirement of Wnt/β-catenin signaling in pronephric kidney development

The pronephric kidney controls water and electrolyte balance during early fish and amphibian embryogenesis. Many Wnt signaling components have been implicated in kidney development. Specifically, in Xenopus pronephric development as well as the murine metanephroi, the secreted glycoprotein Wnt-4 has been shown to be essential for renal tubule formation. Despite the importance of Wnt signals in kidney organogenesis, little is known of the definitive downstream signaling pathway(s) that mediate their effects. Here we report that inhibition of Wnt/beta-catenin signaling within the pronephric field of Xenopus results in significant losses to kidney epithelial tubulogenesis with little or no effect on adjoining axis or somite development. We find that the requirement for Wnt/beta-catenin signaling extends throughout the pronephric primordium and is essential for the development of proximal and distal tubules of the pronephros as well as for the development of the duct and glomus. Although less pronounced than effects upon later pronephric tubule differentiation, inhibition of the Wnt/beta-catenin pathway decreased expression of early pronephric mesenchymal markers indicating it is also needed in early pronephric patterning. We find that upstream inhibition of Wnt/beta-catenin signals in zebrafish likewise reduces pronephric epithelial tubulogenesis. We also find that exogenous activation of Wnt/beta-catenin signaling within the Xenopus pronephric field results in significant tubulogenic losses. Together, we propose Wnt/beta-catenin signaling is required for pronephric tubule, duct and glomus formation in Xenopus laevis, and this requirement is conserved in zebrafish pronephric tubule formation.