A large scale screen for neural stem cell markers in Xenopus retina

Neural stem cell research suffers from a lack of molecular markers to specifically assess stem or progenitor cell properties. The organization of the Xenopus ciliary marginal zone (CMZ) in the retina allows the spatial distinction of these two cell types: stem cells are confined to the most peripheral region, while progenitors are more central. Despite this clear advantage, very few genes specifically expressed in retinal stem cells have been discovered so far in this model. To gain insight into the molecular signature of these cells, we performed a large-scale expression screen in the Xenopus CMZ, establishing it as a model system for stem cell gene profiling. Eighteen genes expressed specifically in the CMZ stem cell compartment were retrieved and are discussed here. These encode various types of proteins, including factors associated with proliferation, mitotic spindle organization, DNA/RNA processing, and cell adhesion. In addition, the publication of this work in a special issue on Xenopus prompted us to give a more general illustration of the value of large-scale screens in this model species. Thus, beyond neural stem cell specific genes, we give a broader highlight of our screen outcome, describing in particular other retinal cell markers that we found. Finally, we present how these can all be easily retrieved through a novel module we developed in the web-based annotation tool XenMARK, and illustrate the potential of this powerful searchable database in the context of the retina.     

Genetic dissection of the zebrafish retinal stem-cell compartment

In a large-scale forward-genetic screen, we discovered that a limited number of genes are required for the regulation of retinal stem cells after embryogenesis in zebrafish. In 18 mutants out of almost 2000 F2 families screened, the eye undergoes normal embryonic development, but fails to continue growth from the ciliary marginal zone (CMZ), the post-embryonic stem-cell niche. Class I-A mutants (5 loci) display lower amounts of proliferation in the CMZ, while nearly all cells in the retina appear differentiated. Class I-B mutants (2 loci) have a reduced CMZ with a concomitant expansion in the retinal pigmented epithelium (RPE), suggesting a common post-embryonic stem cell is the source for these neighboring cell types. Class II encompasses three distinct types of mutants (11 loci) with expanded CMZ, in which the progenitor population is arrested in the cell cycle. We also show that in at least one combination, the reduced CMZ phenotype is genetically epistatic to the expanded CMZ phenotype, suggesting that Class I genes are more likely to affect the stem cells and Class II the progenitor cells. Finally, a comparative mapping analysis demonstrates that the new genes isolated do not correspond to genes previously implicated in stem-cell regulation. Our study suggests that embryonic and post-embryonic stem cells utilize separable genetic programs in the zebrafish retina.     

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.     

Adult neural stem cells: response to stroke injury and potential for therapeutic applications

The plasticity of neural stem/progenitor cells allows a variety of different responses to many environmental cues. In the past decade, significant research has gone into understanding the regulation of neural stem/progenitor cell properties, because of their promise for cell replacement therapies in adult neurological diseases. Both endogenous and grafted neural stem/progenitor cells are known to have the ability to migrate long distances to lesioned sites after brain injury and differentiate into new neurons. Several chemokines and growth factors, including stromal cell-derived factor-1 and vascular endothelial growth factor, have been shown to stimulate the proliferation, differentiation, and migration of neural stem/progenitor cells, and investigators have now begun to identify the critical downstream effectors and signaling mechanisms that regulate these processes. Both our own lab and others have shown that the extracellular matrix and matrix remodeling factors play a critical role in directing cell differentiation and migration of adult neural stem/progenitor cells within injured sites. Identification of these and other molecular pathways involved in stem cell homing into ischemic areas is vital for the development of new treatments. To ensure the best functional recovery, regenerative therapy may require the application of a combination approach that includes cell replacement, trophic support, and neural protection. Here we review the current state of our knowledge about endogenous adult and exogenous neural stem/progenitor cells as potential therapeutic agents for central nervous system injuries.     

Wnt2b controls retinal cell differentiation at the ciliary marginal zone

The ciliary marginal zone of the vertebrate retina contains undifferentiated progenitor cells that continue to proliferate and add new neurons and glia peripherally during the embryonic stages - even after the formation of a functional retina. To understand the molecular mechanism that controls the prolonged progenitor cell proliferation in the ciliary marginal zone, we employed a candidate molecule approach, focusing on Wnt2b (formerly know as Wnt13), which is expressed in the marginal most tip of the retina. Frizzled 4 and 5, seven-pass transmembrane Wnt receptors, were expressed in the peripheral and central part of the retina, respectively. LEF1, a downstream Wnt signaling component, was expressed at high levels in the ciliary marginal zone with expression gradually decreasing towards the central retina. The LEF1-expressing region, which is where Wnt signaling is supposedly activated, expressed a set of molecular markers that are characteristic of the progenitor cells in the ciliary marginal zone. Overexpression of Wnt2b by use of in ovo electroporation in the central retina inhibited neuronal differentiation and induced the progenitor cell markers. Blocking of the Wnt downstream signaling pathway by a dominant-negative LEF1 inhibited proliferation of the cells in the marginal area, which resulted in their premature neuronal differentiation. The progenitor cells in the ciliary marginal zone differentiated into all the neuronal and glial cell types when cultured in vitro, and they proliferated for a longer period than did centrally located progenitor cells that underwent a limited number of cell divisions. In addition, the proliferation of these progenitor cells was promoted in the presence of Wnt2b. These results suggest that Wnt2b functions to maintain undifferentiated progenitor cells in the ciliary marginal zone, and thus serves as a putative stem cell factor in the retina.     

Inducible expression of Wnt genes during adult hepatic stem/progenitor cell response

In injured livers where hepatocyte growth is severely limited, facultative hepatic stem/progenitor cells, termed oval cells in rodents, are known to emerge and contribute to the regeneration process. Here, we investigated a possible involvement of Wnt signaling during mouse oval cell response and found significant upregulation of several Wnt genes including Wnt7a, Wnt7b, and Wnt10a. Accordingly, increase of β-catenin protein was observed in oval cell compartments. Pharmacological activation of the canonical Wnt/β-catenin signaling induced proliferation of cultured hepatic stem/progenitor cell lines. These results together implicate the role of Wnt/β-catenin signaling in adult hepatic stem/progenitor cell response.     

NFκB signaling regulates embryonic and adult neurogenesis

Both embryonic and adult neurogenesis involves the self-renewal/proliferation,survival,migration and lineage differentiation of neural stem/progenitor cells.Such dynamic process is tightly regulated by...     

Wnt/BMP signal integration regulates the balance between proliferation and differentiation of neuroepithelial cells in the dorsal spinal cord

Multiple signaling pathways regulate proliferation and differentiation of neural progenitor cells during early development of the central nervous system (CNS). In the spinal cord, dorsal signaling by bone morphogenic protein (BMP) acts primarily as a patterning signal, while canonical Wnt signaling promotes cell cycle progression in stem and progenitor cells. However, overexpression of Wnt factors or, as shown here, stabilization of the Wnt signaling component beta-catenin has a more prominent effect in the ventral than in the dorsal spinal cord, revealing local differences in signal interpretation. Intriguingly, Wnt signaling is associated with BMP signal activation in the dorsal spinal cord. This points to a spatially restricted interaction between these pathways. Indeed, BMP counteracts proliferation promoted by Wnt in spinal cord neuroepithelial cells. Conversely, Wnt antagonizes BMP-dependent neuronal differentiation. Thus, a mutually inhibitory crosstalk between Wnt and BMP signaling controls the balance between proliferation and differentiation. A model emerges in which dorsal Wnt/BMP signal integration links growth and patterning, thereby maintaining undifferentiated and slow-cycling neural progenitors that form the dorsal confines of the developing spinal cord.     

mRNA-Seq and MicroRNA-Seq Whole-Transcriptome Analyses of Rhesus Monkey Embryonic Stem Cell Neural Differentiation Revealed the Potential Regulators of Rosette Neural Stem Cells

Rosette neural stem cells (R-NSCs) represent early stage of neural development and possess full neural differentiation and regionalization capacities. R-NSCs are considered as stem cells of neural lineage and have important implications in the study of neurogenesis and cell replacement therapy. However, the molecules regulating their functional properties remain largely unknown. Rhesus monkey is an ideal model to study human neural degenerative diseases and plays intermediate translational roles as therapeutic strategies evolved from rodent systems to human clinical applications. In this study, we derived R-NSCs from rhesus monkey embryonic stem cells (ESCs) and systematically investigated the unique expressions of mRNAs, microRNAs (miRNAs), and signalling pathways by genome-wide comparison of the mRNA and miRNA profilings of ESCs, R-NSCs at early (R-NSCP1) and late (R-NSCP6) passages, and neural progenitor cells. Apart from the R-NSCP1-specific protein-coding genes and miRNAs, we identified several pathways including Hedgehog and Wnt highly activated in R-NSCP1. The possible regulatory interactions among the miRNAs, protein-coding genes, and signalling pathways were proposed. Besides, many genes with alternative splicing switch were identified at R-NSCP1. These data provided valuable resource to understand the regulation of early neurogenesis and to better manipulate the R-NSCs for cell replacement therapy.     

Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes

For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.     

Histone deacetylase 1 regulates retinal neurogenesis in zebrafish by suppressing Wnt and Notch signaling pathways

In the developing vertebrate retina, progenitor cells initially proliferate but begin to produce postmitotic neurons when neuronal differentiation occurs. However, the mechanism that determines whether retinal progenitor cells continue to proliferate or exit from the cell cycle and differentiate is largely unknown. Here, we report that histone deacetylase 1 (Hdac1) is required for the switch from proliferation to differentiation in the zebrafish retina. We isolated a zebrafish mutant, ascending and descending (add), in which retinal cells fail to differentiate into neurons and glial cells but instead continue to proliferate. The cloning of the add gene revealed that it encodes Hdac1. Furthermore, the ratio of the number of differentiating cells to that of proliferating cells increases in proportion to Hdac activity, suggesting that Hdac proteins regulate a crucial step of retinal neurogenesis in zebrafish. Canonical Wnt signaling promotes the proliferation of retinal cells in zebrafish, and Notch signaling inhibits neuronal differentiation through the activation of a neurogenic inhibitor, Hairy/Enhancer-of-split (Hes). We found that both the Wnt and Notch/Hes pathways are activated in the add mutant retina. The cell-cycle progression and the upregulation of Hes expression in the add mutant retina can be inhibited by the blockade of Wnt and Notch signaling, respectively. These data suggest that Hdac1 antagonizes these pathways to promote cell-cycle exit and the subsequent neurogenesis in zebrafish retina. Taken together, these data suggest that Hdac1 functions as a dual switch that suppresses both cell-cycle progression and inhibition of neurogenesis in the zebrafish retina.     

Retinal stem/progenitor properties of iris pigment epithelial cells

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.     

Neural stem cells in the mammalian eye: types and regulation

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina. Recently, such cells have been reported to be present, in a mitotically quiescent state, in the ciliary epithelium of the adult mammalian eye. The retinal and ciliary epithelium stem cells/progenitors appear to share similar signaling pathways that are emerging as important regulators of stem cells in general. Yet, they are different in certain respects, such as in the potential to self-renew. These two neural stem cell/progenitor populations not only will serve as models for investigating stem cell biology but also will help explain the relationships between embryonic and adult neural stem cells/progenitors.     

Expression patterns of chick Musashi-1 in the developing nervous system

Vertebrate homologues of musashi have recently been referred to as neural stem cell markers because of their expression patterns and RNA-binding interactions. In the context of the notch signaling pathway, Musashi-1 (Msi-1) is a regulator of neural cell generation, cooperating with notch to maintain mitosis. In an effort to identify definitive stem cell markers of the neural retina, a portion of the Msi-1 cDNA was cloned, and the expression of Msi-1 in the chick eye was analyzed. Using an Msi-1-specific antibody and RNA probe, we show that expression of Msi-1 in the early neural tube is consistent with neural stem identity. In the neural retina, expression starts shortly before embryonic day 3 (E3) and continues up to and including E18. A BrdU incorporation assay shows Msi-1 to be found in both proliferating and differentiating cells of E5 neural retina. At E8 (when proliferation is complete in the fundus of the retina) and E18 (mature retina) Msi-1 expression was found in the ciliary marginal zone (CMZ) as well as in a subpopulation of differentiated cells, including photoreceptors and ganglion cells.     

A systems biology approach to model neural stem cell regulation by notch, shh, wnt, and EGF signaling pathways

The Notch, Sonic Hedgehog (Shh), Wnt, and EGF pathways have long been known to influence cell fate specification in the developing nervous system. Here we attempted to evaluate the contemporary knowledge about neural stem cell differentiation promoted by various drug-based regulations through a systems biology approach. Our model showed the phenomenon of DAPT-mediated antagonism of Enhancer of split [E(spl)] genes and enhancement of Shh target genes by a SAG agonist that were effectively demonstrated computationally and were consistent with experimental studies. However, in the case of model simulation of Wnt and EGF pathways, the model network did not supply any concurrent results with experimental data despite the fact that drugs were added at the appropriate positions. This paves insight into the potential of crosstalks between pathways considered in our study. Therefore, we manually developed a map of signaling crosstalk, which included the species connected by representatives from Notch, Shh, Wnt, and EGF pathways and highlighted the regulation of a single target gene, Hes-1, based on drug-induced simulations. These simulations provided results that matched with experimental studies. Therefore, these signaling crosstalk models complement as a tool toward the discovery of novel regulatory processes involved in neural stem cell maintenance, proliferation, and differentiation during mammalian central nervous system development. To our knowledge, this is the first report of a simple crosstalk map that highlights the differential regulation of neural stem cell differentiation and underscores the flow of positive and negative regulatory signals modulated by drugs.     

Wnt2b inhibits differentiation of retinal progenitor cells in the absence of Notch activity by downregulating the expression of proneural genes

During the development of the central nervous system, cell proliferation and differentiation are precisely regulated. In the vertebrate eye, progenitor cells located in the marginal-most region of the neural retina continue to proliferate for a much longer period compared to the ones in the central retina, thus showing stem-cell-like properties. Wnt2b is expressed in the anterior rim of the optic vesicles, and has been shown to control differentiation of the progenitor cells in the marginal retina. In this paper, we show that stable overexpression of Wnt2b in retinal explants inhibited cellular differentiation and induced continuous growth of the tissue. Notably, Wnt2b maintained the undifferentiated progenitor cells in the explants even under the conditions where Notch signaling was blocked. Wnt2b downregulated the expression of multiple proneural bHLH genes as well as Notch. In addition, expression of Cath5 under the control of an exogenous promoter suppressed the negative effect of Wnt2b on neuronal differentiation. Importantly, Wnt2b inhibited neuronal differentiation independently of cell cycle progression. We propose that Wnt2b maintains the naive state of marginal progenitor cells by attenuating the expression of both proneural and neurogenic genes, thus preventing those cells from launching out into the differentiation cascade regulated by proneural genes and Notch.     

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.     

Wnt and Shh signals regulate neural stem cell proliferation and differentiation in the optic tectum of adult zebrafish

Adult neurogenesis occurs more commonly in teleosts, represented by zebrafish, than in mammals. Zebrafish is therefore considered a suitable model to study adult neurogenesis, for which the regulatory molecular mechanisms remain little known. Our previous study revealed that neuroepithelial‐like neural stem cells (NSCs) are located at the edge of the dorsomedial region. We also showed that Notch signaling inhibits NSC proliferation in this region. In the present study, we reported the expression of Wnt and Shh signaling components in this region of the optic tectum. Moreover, inhibitors of Wnt and Shh signaling suppressed NSC proliferation, suggesting that these pathways promote NSC proliferation. Shh is particularly required for maintaining Sox2‐positive NSCs. Our experimental data also indicate the involvement of these signaling pathways in neural differentiation from NSCs. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1206–1220, 2017