2015. Wang Y, Stokes A, Duan Z, Hui J, Xu Y, Chen YP, Chen HW, Lam K, Zhou CJ. Lrp6 modulates Ret signaling in renal development and cystic dysplasia. Journal of the American Society of Nephrology (accepted)
2014. Lassiter RN, Stark MR, Zhao T, Zhou CJ. Signaling mechanisms controlling cranial placode neurogenesis and delamination. Developmental Biology 389:39-49. DOI: 10.1016/j.ydbio.2013.11.025
A core signaling program exists throughout the neurogenic placodes.
Attenuated Notch signaling initiates placode neurogenesis and delamination.
Active FGF signaling also triggers placode neurogenesis and delamination.
Wnt and BMP pathways exert context-dependent roles in neurogenic placodes.
Abstract. The neurogenic cranial placodes are a unique transient epithelial niche of neural progenitor cells that give rise to multiple derivatives of the peripheral nervous system, particularly, the sensory neurons. Placode neurogenesis occurs throughout an extended period of time with epithelial cells continually recruited as neural progenitor cells. Sensory neuron development in the trigeminal, epibranchial, otic, and olfactory placodes coincides with detachment of these neuroblasts from the encompassing epithelial sheet, leading to delamination and ingression into the mesenchyme where they continue to differentiate as neurons. Multiple signaling pathways are known to direct placodal development. This review defines the signaling pathways working at the finite spatiotemporal period when neuronal selection within the placodes occurs, and neuroblasts concomitantly delaminate from the epithelium. Examining neurogenesis and delamination after initial placodal patterning and specification has revealed a common trend throughout the neurogenic placodes, which suggests that both activated FGF and attenuated Notch signaling activities are required for neurogenesis and changes in epithelial cell adhesion leading to delamination. We also address the varying roles of other pathways such as the Wnt and BMP signaling families during sensory neurogenesis and neuroblast delamination in the differing placodes.
2014. Zhao T*, Gan Q*, Stokes A, Lassiter RN, Wang YP, Chan J, Han J, Pleasure DE, Epstein JA, Zhou CJ. Beta-catenin regulates Pax3 and Cdx2 for caudal neural tube closure and elongation. Development 141:148-157. DOI: 10.1242/dev.101550
Abstract. Non-canonical Wnt/planar cell polarity (PCP) signaling plays a primary role in the convergent extension that drives neural tube closure and body axis elongation. PCP signaling gene mutations cause severe neural tube defects (NTDs). However, the role of canonical Wnt/β-catenin signaling in neural tube closure and NTDs remains poorly understood. This study shows that conditional gene targeting of β-catenin in the dorsal neural folds of mouse embryos represses the expression of the homeobox-containing genes Pax3 and Cdx2 at the dorsal posterior neuropore (PNP), and subsequently diminishes the expression of the Wnt/β-catenin signaling target genes T, Tbx6 and Fgf8 at the tail bud, leading to spina bifida aperta, caudal axis bending and tail truncation. We demonstrate that Pax3 and Cdx2 are novel downstream targets of Wnt/β-catenin signaling. Transgenic activation of Pax3 cDNA can rescue the closure defect in the β-catenin mutants, suggesting that Pax3 is a key downstream effector of β-catenin signaling in the PNP closure process. Cdx2 is known to be crucial in posterior axis elongation and in neural tube closure. We found that Cdx2 expression is also repressed in the dorsal PNPs of Pax3-null embryos. However, the ectopically activated Pax3 in the β-catenin mutants cannot restore Cdx2 mRNA in the dorsal PNP, suggesting that the presence of both β-catenin and Pax3 is required for regional Cdx2 expression. Thus, β-catenin signaling is required for caudal neural tube closure and elongation, acting through the transcriptional regulation of key target genes in the PNP.
2014. Gan Q*, Lee A*, Suzuki R*, Yamagami T, Stokes A, Nguyen B, Pleasure D, Wang J, Chen HW, Zhou CJ. Pax6 mediates ß-catenin signaling for self-renewal and neurogenesis by neocortical radial glial stem cells. Stem Cells 32:45-58. DOI: 10.1002/stem.1561
Abstract. The Wnt/ß-catenin pathway is a critical stem cell regulator and plays important roles in neuroepithelial cells during early gestation. However, the role of Wnt/ß-catenin signaling in radial glia, a major neural stem cell population expanded by mid-gestation, remains poorly understood. This study shows that genetic ablation of ß-catenin with hGFAP-Cre mice inhibits neocortical formation by disrupting radial glial development. Reduced radial glia and intermediate progenitors are found in the ß-catenin-deficient neocortex during late gestation. Increased apoptosis and divergent localization of radial glia in the subventricular zone are also observed in the mutant neocortex.
In vivo and in vitro proliferation and neurogenesis as well as oligodendrogenesis by cortical radial glia or by dissociated neural stem cells are significantly defective in the mutants. Neocortical layer patterning is not apparently altered, while astrogliogenesis is ectopically increased in the mutants. At the molecular level, the expression of the transcription factor Pax6 is dramatically diminished in the cortical radial glia and the sphere-forming neural stem cells of ß-catenin-deficient mutants. Chromatin immunoprecipitation and luciferase assays demonstrate that ß-catenin/Tcf complex binds to Pax6 promoter and induces its transcriptional activities. The forced expression of Pax6 through lentiviral transduction partially rescues the defective proliferation and neurogenesis by ß-catenin-deficient neural stem cells. Thus, Pax6 is a novel downstream target of the Wnt/ß-catenin pathway, and ß-catenin/Pax6 signaling plays critical roles in self-renewal and neurogenesis of radial glia/neural stem cells during neocortical development.
2011. Guo F, Maeda Y, Ma J, Delgado M, Sohn J, Miers L, Ko EM, Bannerman, Xu J, Wang Y, Zhou CJ, Takebayashi H, Pleasure D. Macroglial plasticity and the origins of reactive astroglia in experimental autoimmune encephalomyelitis. Journal of Neuroscience 31:11914-11928.
Abstract. Accumulations of hypertrophic, intensely glial fibrillary acidic protein-positive (GFAP(+)) astroglia, which also express immunoreactive nestin and vimentin, are prominent features of multiple sclerosis lesions. The issues of the cellular origin of hypertrophic GFAP(+)/vimentin(+)/nestin(+) "reactive" astroglia and also the plasticities and lineage relationships among three macroglial progenitor populations-oligodendrocyte progenitor cells (OPCs), astrocytes and ependymal cells-during multiple sclerosis and other CNS diseases remain controversial. We used genetic fate-mappings with a battery of inducible Cre drivers (Olig2-Cre-ER(T2), GFAP-Cre-ER(T2), FoxJ1-Cre-ER(T2) and Nestin-Cre-ER(T2)) to explore these issues in adult mice with myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis (EAE). The proliferative rate of spinal cord OPCs rose fivefold above control levels during EAE, and numbers of oligodendroglia increased as well, but astrogenesis from OPCs was rare. Spinal cord ependymal cells, previously reported to be multipotent, did not augment their low proliferative rate, nor give rise to astroglia or OPCs. Instead, the hypertrophic, vimentin(+)/nestin(+), reactive astroglia that accumulated in spinal cord in this multiple sclerosis model were derived by proliferation and phenotypic transformation of fibrous astroglia in white matter, and solely by phenotypic transformation of protoplasmic astroglia in gray matter. This comprehensive analysis of macroglial plasticity in EAE helps to clarify the origins of astrogliosis in CNS inflammatory demyelinative disorders.
2011. Wang YZ, Plane JM, Jiang P, Zhou CJ, Deng W. Concise Review: Quiescent and active states of endogenous adult neural stem cells: identification and characterization. Stem Cells 29:907-912.
Abstract. The adult mammalian central nervous system (CNS) lacks the capacity for
regeneration, making it a highly sought-after topic for researchers. The
identification of neural stem cells (NSCs) in the adult CNS wiped out a
long-held dogma that the adult brain contains a set number of neurons
and is incapable of replacing them. The discovery of adult NSCs (aNSCs)
stoked the fire for researchers who dream of brain self-repair.
Unfortunately, the quiescent nature and limited plasticity of aNSCs
diminish their regenerative potential. Recent studies evaluating aNSC
plasticity under pathological conditions indicate that a switch from
quiescent to active aNSCs in neurogenic regions plays an important role
in both repairing the damaged tissue and preserving progenitor pools.
Here, we summarize the most recent findings and present questions about
characterizing the active and quiescent aNSCs in major neurogenic
regions, and factors for maintaining their active and quiescent states,
hoping to outline an emerging view for promoting the endogenous
2011. Wang YZ, Yamagami T, Gan Q, Wang Y, Zhao T, Hamad S, Lott P,
Schnittke N, Schwob JE, Zhou CJ. Canonical Wnt signaling promotes
proliferation and neurogenesis of peripheral olfactory stem
cells during postnatal development and adult regeneration.
Journal of Cell Science 124:1553-1563. (journal cover)
Abstract. The mammalian olfactory epithelium (OE) has a unique stem cell or progenitor niche, which is responsible for the constant peripheral neurogenesis throughout the lifespan of the animal. However, neither the signals that regulate the behavior of these cells nor the lineage properties of the OE stem cells are well understood. Multiple Wnt signaling components exhibit dynamic expression patterns in the developing OE. We generated Wnt signaling reporter TOPeGFP transgenic mice and found TOPeGFP activation predominantly in proliferating Sox2(+) OE basal cells during early postnatal development. FACS-isolated TOPeGFP(+) OE basal cells are required, but are not sufficient, for formation of spheres. Wnt3a significantly promotes the proliferation of the Sox2(+) OE sphere cells. Wnt-stimulated OE sphere cells maintain their multipotency and can differentiate into most types of neuronal and non-neuronal epithelial cells. Also, Wnt activators shift the production of differentiated cells toward olfactory sensory neurons. Moreover, TOPeGFP(+) cells are robustly increased in the adult OE after injury. In vivo administration of Wnt modulators significantly alters the regeneration potential. This study demonstrates the role of the canonical Wnt signaling pathway in the regulation of OE stem cells or progenitors during development and regeneration.
Cover caption: The newly constructed Wnt signalling reporter TOPeGFP (green) is predominantly activated in the olfactory epithelial basal stem cells in early postnatal transgenic mice. Olfactory marker protein immunolabelling (red) shows the bipolar olfactory receptor neurons with extended dendrites towards the lumenal surface and axon bundles distributed in the lamina propria. See article by Wang et al. (pp. 1553-1563).
2011. He F, Xiong W, Wang Y, Li L, Liu C, Yamagami T, Taketo MM, Zhou
CJ, Chen Y. Epithelial Wnt/ß-catenin signaling regulates palatal shelf
fusion through regulation of Tgfß3 expression. Developmental Biology 350:511-519. (Collaborative contribution to the Chen Lab's revision experiment)
Abstract. The canonical Wnt/β-catenin
signaling plays essential role in development and diseases. Previous
studies have implicated the canonical Wnt/β-catenin signaling in the
regulation of normal palate development, but functional Wnt/β-catenin
signaling and its tissue-specific activities remain to be accurately
elucidated. In this study, we show that functional Wnt/β-catenin
signaling operates primarily in the palate epithelium, particularly in
the medial edge epithelium (MEE) of the developing mouse palatal
shelves, consistent with the expression patterns of β-catenin and
several Wnt ligands and receptors. Epithelial specific inactivation of
β-catenin by the K14-Cre transgenic allele abolishes the canonical Wnt
signaling activity in the palatal epithelium and leads to an abnormal
persistence of the medial edge seam (MES), ultimately causing a cleft
palate formation, a phenotype resembling that in Tgfβ3 mutant mice.
Consistent with this phenotype is the down-regulation of Tgfβ3 and
suppression of apoptosis in the MEE of the β-catenin mutant palatal
shelves. Application of exogenous Tgfβ3 to the mutant palatal shelves in
organ culture rescues the midline seam phenotype. On the other hand,
expression of stabilized β-catenin in the palatal epithelium also
disrupts normal palatogenesis by activating ectopic Tgfβ3 expression in
the palatal epithelium and causing an aberrant fusion between the palate
shelf and mandible in addition to severely deformed palatal shelves.
Collectively, our results demonstrate an essential role for
Wnt/β-catenin signaling in the epithelial component at the step of
palate fusion during palate development by controlling the expression of
Tgfβ3 in the MEE.
2011. Wang Y, Song L, Zhou CJ. The canonical Wnt/beta-catenin
signaling pathway regulates Fgf signaling for early facial development.
Developmental Biology 349:250-260.
Abstract. The canonical Wnt/β-catenin signaling pathway has implications in early facial development; yet, its function and signaling mechanism remain poorly understood. We report here that the frontonasal and upper jaw primordia cannot be formed after conditional ablation of β-catenin with Foxg1-Cre mice in the facial ectoderm and the adjacent telencephalic neuroepithelium. Gene expression of several cell-survival and patterning factors, including Fgf8, Fgf3, and Fgf17, is dramatically diminished in the anterior neural ridge (ANR, a rostral signaling center) and/or the adjacent frontonasal ectoderm of the β-catenin conditional mutant mice. In addition, Shh expression is diminished in the ventral telencephalon of the mutants, while Tcfap2a expression is less affected in the facial primordia. Apoptosis occurs robustly in the rostral head tissues following inactivation of Fgf signaling in the conditional mutants. Consequently, the upper jaw, nasal, ocular and telencephalic structures are absent, but the tongue and mandible are relatively developed in the conditional mutants at birth. Using molecular biological approaches, we demonstrate that the Fgf8 gene is transcriptionally targeted by Wnt/β-catenin signaling during early facial and forebrain development. Furthermore, we show that conditional gain-of-function of β-catenin signaling causes drastic upregulation of Fgf8 mRNA in the ANR and the entire facial ectoderm, which also arrests facial and forebrain development. Taken together, our results suggest that canonical Wnt/β-catenin signaling is required for early development of the mammalian face and related head structures, which mainly or partly acts through the initiation and modulation of balanced Fgf signaling activity.
2010. Zhou CJ, Wang YZ, Yamagami T, Zhao T, Song L, Wang K.
Generation of Lrp6 conditional gene-targeting mouse line for modeling
and dissecting multiple birth defects/congenital anomalies. Developmental Dynamics 239:318-326.
Abstract. Lrp6 is a key coreceptor in
the canonical Wnt pathway that is widely involved in tissue/organ
morphogenesis. We generated a loxP-floxed Lrp6 mouse line. Crossing with
a general Cre deleter, we obtained the Lrp6-floxdel mice, in which the
loxP-floxed exon 2 of Lrp6 gene has been deleted ubiquitously. The
homozygotes of Lrp6-floxdel mice reproduced typical defects as seen in
the conventional Lrp6-deficient mice, such as defects in eye, limb, and
neural tube, and die around birth. We also found new phenotypes
including cleft palate and agenesis of external genitalia in the
Lrp6-floxdel mice. In addition, the Lrp6-deficient embryos are known to
be defective in other systems and internal organs including the heart
and brain. Thus, by selectively crossing with a lineage-specific or
inducible Cre mouse line, the Lrp6 conditional gene-targeting mice will
allow us to model specific types of birth defects for mechanism and
2010. Song L, Li Y, Wang K, Zhou CJ. Cardiac neural crest and outflow tract defects in Lrp6 mutant mice. Developmental Dynamics 239:200-210.
Abstract. The role of a key Wnt
coreceptor Lrp6 during heart development remains unclear. Here we show
that ablation of Lrp6 in mice causes conotruncal anomalies including
double-outlet right ventricle (DORV), outflow tract (OFT) cushion
hypoplasia, and ventricular septal defect (VSD). Cardiac neural crest
cells are specifically lost in the dorsal neural tube and caudal
pharyngeal arches of the mutant embryos. We also demonstrate that Lrp6
is required for proliferation and survival of cardiac progenitors and
for the expression of Isl1 in the secondary heart field. Other known
cardiogenic regulators such as Msx1, Msx2, and Fgf8 are also
significantly diminished in the mutant pharyngeal arches and/or OFT.
Unexpectedly, the myocardium differentiation factors Mef2c and Myocardin
are upregulated in the mutant OFT. Our results indicate that Lrp6 is
essential for cardiac neural crest and OFT development upstream of
multiple important cardiogenic genes in different cardiac lineage cells
during early cardiogenesis.
2009. Yamagami T, Molotkov A, Zhou CJ. Canonical Wnt signaling activity during synovial joint development. Journal of Molecular Histology 40:311-316.
signaling plays important roles in skeletal development. However, the
activation and function of canonical Wnt signaling in joint development
remains unclear. We analyzed the lineage identity and developmental
changes of the Wnt-responsive cells during synovial joint formation as
well as adulthood in the Wnt signaling reporter TOPgal transgenic mice.
At embryonic day (E) 12.5, we found that the TOPgal was inactivated in
the presumptive joint forming interzone, but it was intensively
activated in the cartilage anlage of developing long bones and digits.
At E14.5, the TOPgal activity was found in a subgroup of the articular
chondrocyte lineage cells, which were co-immunolabeled with Doublecortin
intensively and with Vinculin weakly. At E18.5, the TOPgal/Doublecortin
co-immunolabeled cells were found in the superficial layer of the
developing articular cartilage. During postnatal development, the
TOPgal(+) articular chondrocytes were abundant at P7 and decreased from
P10. A small number of TOPgal(+) articular chondrocytes were also found
in adult joints. Our study suggests an age- and lineage-specific role of
canonical Wnt signaling in joint development and maintenance.
2009. Song L, Li Y, Wang K, Wang YZ, Molotkov A, Gao L, Zhao T,
Yamagami T, Wang Y, Gan Q, Pleasure DE, Zhou CJ. Lrp6-mediated
canonical Wnt signaling is required for lip formation and fusion. Development 136:3161-3171.
the mechanisms that govern lip morphogenesis nor the cause of cleft lip
are well understood. We report that genetic inactivation of Lrp6, a
co-receptor of the Wnt/beta-catenin signaling pathway, leads to cleft
lip with cleft palate. The activity of a Wnt signaling reporter is
blocked in the orofacial primordia by Lrp6 deletion in mice. The
morphological dynamic that is required for normal lip formation and
fusion is disrupted in these mutants. The expression of the homeobox
genes Msx1 and Msx2 is dramatically reduced in the mutants, which
prevents the outgrowth of orofacial primordia, especially in the fusion
site. We further demonstrate that Msx1 and Msx2 (but not their potential
regulator Bmp4) are the downstream targets of the Wnt/beta-catenin
signaling pathway during lip formation and fusion. By contrast, a
;fusion-resistant' gene, Raldh3 (also known as Aldh1a3), that encodes a
retinoic acid-synthesizing enzyme is ectopically expressed in the upper
lip primordia of Lrp6-deficient embryos, indicating a region-specific
role of the Wnt/beta-catenin signaling pathway in repressing retinoic
acid signaling. Thus, the Lrp6-mediated Wnt signaling pathway is
required for lip development by orchestrating two distinctively
different morphogenetic movements.
Editorial Highlight. Cleft lip, with or without cleft palate, occurs in around 1 in 700 human
newborns, but little is known about the mechanisms involved. Now, on
p. 3161, Chengji Zhou
and colleagues identify the Wnt co-receptor Lrp6 as being crucial for normal
lip morphogenesis in mice. All mice in which Lrp6 is deleted develop a cleft
lip with cleft palate. These defects correlate with blocked Wnt/β-catenin
signalling and with reduced cell proliferation in the primordia of the lip and
palate (known as the orofacial primordia) earlier in development.
Concomitantly with reduced Wnt/β-catenin signalling, the expression of
the homeobox genes Msx1 and Msx2, which are important for
mesenchyme proliferation, is decreased in the orofacial primordia. Conversely,
the expression of Raldh3, which encodes a retinoic acid-synthesising
enzyme that counteracts tissue fusion, expands. The authors demonstrate that
Msx1 and Msx2 are direct targets of Wnt/β-catenin
signalling and conclude that Lrp6-mediated Wnt/β-catenin signalling
regulates lip formation and fusion by balancing the activities of Msx1 and
Msx2 with the opposing activity of Raldh3.
2008. Zhou CJ, Molotkov A, Song L, Li Y, Pleasure DE, Pleasure SJ,
Wang YZ. Ocular coloboma and dorsoventral neuroretinal patterning
defects in Lrp6 mutant eyes. Developmental Dynamics 237:3681-3689.
Abstract. Coloboma, an ocular birth
defect seen in humans and other species, is caused by incomplete closure
of the optic fissure. Here, we demonstrate that genetic deletion of
Lrp6, a bottleneck coreceptor in the canonical Wnt signaling pathway,
results in ocular coloboma and neuroretinal patterning defects in mice.
The expression of ventral neuroretinal patterning gene Vax2 was
conserved but with dorsally shifted expression domains; however, the
dorsal neuroretinal patterning gene Tbx5 was lost in the Lrp6-mutant
eyes at embryonic day 10.5. Both Bmp4 and phosphorylated Smad 1/5/8 were
also significantly attenuated in the dorsal neuroretina. In addition,
the retinoic acid synthesizing enzymes Raldh1 and Raldh3 were
significantly changed in the mutant eyes. Our findings suggest that
defective retinal patterning causes coloboma in the Lrp6-deficient mice,
and that canonical Wnt signaling plays a primary role in dorsal
neuroretinal patterning and related morphogenetic movements by
regulation of both Bmp and retinoic acid signaling pathways.
2008. Wang YZ, Molotkov A, Song L, Li Y, Pleasure DE, Zhou CJ. Activation of the Wnt/beta-catenin signaling reporter in developing mouse olfactory nerve layer marks a specialized subgroup of olfactory ensheathing cells. Developmental Dynamics 237:3157-3168.
Abstract. Wnt reporter TOPgal mice carry a beta-galactosidase (betagal) gene under the control of the Wnt/beta-catenin signaling responsive elements. We found that the intensely immunolabeled betagal+ cells were co-immunolabeled with Nestin and formed a tangentially oriented single-cell layer in the "connecting or docking zone" where the olfactory sensory axons attached to the brain surface during mid-gestation. During early postnatal development, betagal+ cells were located in the inner olfactory nerve layer (ONLi) and co-labeled with olfactory ensheathing cell (OEC) markers S100beta and NPY but not with lineage-specific markers for neurons, oligodendrocytes, astrocytes, and microglia, demonstrating that the TOPgal marked a subpopulation of OECs. By confocal microscopy, we found that TOPgal activated processes extended along the developing glomerulus and formed multiple tunnel-like structures that ensheathe and bridge olfactory sensory axonal bundles from ONLi to the glomerulus, which may play a key role in glomerulus formation and convergent sorting of the peripheral olfactory axons.