Expression and functional analysis of musashi-like genes in planarian CNS regeneration

The remarkable regenerative ability of planarians is made possible by a system of pluripotent stem cells. Recent molecular biological and ultrastructural studies have revealed that planarian stem cells consist of heterogeneous populations, which can be classified into several subsets according to their differential expression of RNA binding protein genes. In this study, we focused on planarian musashi family genes. Musashi encodes an evolutionarily conserved RNA binding protein known to be expressed in neural lineage cells, including neural stem cells, in many animals. Here, we investigated whether planarian musashi-like genes can be used as markers for detecting neural fate-restricted cells. Three musashi family genes, DjmlgA, DjmlgB and DjmlgC (Dugesia japonica musashi-like gene A, B, C), and Djdmlg (Dugesia japonica DAZAP-like/musashi-like gene) were obtained by searching a planarian EST database and 5′ RACE, and each was found to have two RNA recognition motifs. We analyzed the types of cells expressing DjmlgA, DjmlgB, DjmlgC and Djdmlg by in situ hybridization, RT-PCR and single-cell RT-PCR analysis. Although Djdmlg was expressed in X-ray-sensitive stem cells and various types of differentiated cells, expression of the other three musashi-like genes was restricted to neural cells, as we expected. Further detailed analyses yielded the unexpected finding that these three planarian musashi family genes were predominantly expressed in X-ray-resistant differentiated neurons, but not in X-ray-sensitive stem cells. RNAi experiments suggested that these planarian musashi family genes might be involved in neural cell differentiation after neural cell-fate commitment.     

Zebrafish arl6ip1 is required for neural crest development during embryogenesis

Background

Although the embryonic expression pattern of ADP ribosylation factor-like 6 interacting protein 1 (Arl6ip1) has been reported, its function in neural crest development is unclear.

Methods/Principal Findings

We found that knockdown of Arl6ip1 caused defective embryonic neural crest derivatives that were particularly severe in craniofacial cartilages. Expressions of the ectodermal patterning factors msxb, dlx3b, and pax3 were normal, but the expressions of the neural crest specifier genes foxd3, snai1b, and sox10 were greatly reduced. These findings suggest that arl6ip1 is essential for specification of neural crest derivatives, but not neural crest induction. Furthermore, we revealed that the streams of crestin- and sox10-expressing neural crest cells, which migrate ventrally from neural tube into trunk, were disrupted in arl6ip1 morphants. This migration defect was not only in the trunk neural crest, but also in the enteric tract where the vagal-derived neural crest cells failed to populate the enteric nervous system. We found that this migration defect was induced by dampened Shh signaling, which may have resulted from defective cilia. These data further suggested that arl6ip1 is required for neural crest migration. Finally, by double-staining of TUNEL and crestin, we confirmed that the loss of neural crest cells could not be attributed to apoptosis.

Conclusions/Significance

Therefore, we concluded that arl6ip1 is required for neural crest migration and sublineage specification.     

PlexinA1 interacts with PTK7 and is required for neural crest migration

Members of the plexin protein family are known regulators of axon guidance, but recent data indicate that they have broader functions in the regulation of embryonic morphogenesis. Here we provide further evidence of this by showing that PlexinA1 is expressed in Xenopus neural crest cells and is required for their migration. PlexinA1 expression is detected in migrating cranial neural crest cells and knockdown of PlexinA1 expression using Morpholino oligonucleotides inhibits neural crest migration. PlexinA1 likely affects neural crest migration by interaction with PTK7, a regulator of planar cell polarity that is required for neural crest migration. PlexinA1 and PTK7 interact in immunoprecipitation assays and show phenotypic interaction in co-injection experiments. Considering that plexins and PTK7 have been shown to genetically interact in Drosophila axon guidance and chick cardiac morphogenesis, our data suggest that this interaction is evolutionary conserved and may be relevant for a broad range of morphogenetic events including the migration of neural crest cells in Xenopus laevis.     

A new role for the Endothelin-1/Endothelin-A receptor signaling during early neural crest specification

The neural crest is induced at the border of the neural plate in a multistep process by signals emanated from the epidermis, neural plate and mesoderm. In this work we show for the first time the existence of a neural crest maintenance step which is dependent on signals released from the mesoderm. We identified Endothelin-1 (Edn1) and its receptor (Ednra) as key players of this signal and we show that Edn1/Ednra signaling is required for maintenance of the neural crest by a dual mechanism of cell specification and cell survival. We show that: (i) Ednra is expressed in prospective neural crest; (ii) loss-of-function experiments with antisense morpholino or with specific chemical inhibitor suppress the expression of early neural crest markers; (iii) gain-of-function experiments expand the neural crest territory; (iv) epistatic experiments show that Ednra/Edn1 is downstream of the early neural crest gene Msx1 and upstream of the late genes Sox9 and Sox10; and (v) Edn1/Ednra signaling inhibits apoptosis and controls cell specification of the neural crest. Together, our results provide insight on a new role of Edn1/Ednra cell signaling pathway during early neural crest development.     

A Systematic Survey of Expression and Function of Zebrafish frizzled Genes

Wnt signaling is crucial for the regulation of numerous processes in development. Consistent with this, the gene families for both the ligands (Wnts) and receptors (Frizzleds) are very large. Surprisingly, while we have a reasonable understanding of the Wnt ligands likely to mediate specific Wnt-dependent processes, the corresponding receptors usually remain to be elucidated. Taking advantage of the zebrafish model''s excellent genomic and genetic properties, we undertook a comprehensive analysis of the expression patterns of frizzled (fzd) genes in zebrafish. To explore their functions, we focused on testing their requirement in several developmental events known to be regulated by Wnt signaling, convergent extension movements of gastrulation, neural crest induction, and melanocyte specification. We found fourteen distinct fzd genes in the zebrafish genome. Systematic analysis of their expression patterns between 1-somite and 30 hours post-fertilization revealed complex, dynamic and overlapping expression patterns. This analysis demonstrated that only fzd3a, fzd9b, and fzd10 are expressed in the dorsal neural tube at stages corresponding to the timing of melanocyte specification. Surprisingly, however, morpholino knockdown of these, alone or in combination, gave no indication of reduction of melanocytes, suggesting the important involvement of untested fzds or another type of Wnt receptor in this process. Likewise, we found only fzd7b and fzd10 expressed at the border of the neural plate at stages appropriate for neural crest induction. However, neural crest markers were not reduced by knockdown of these receptors. Instead, these morpholino knockdown studies showed that fzd7a and fzd7b work co-operatively to regulate convergent extension movement during gastrulation. Furthermore, we show that the two fzd7 genes function together with fzd10 to regulate epiboly movements and mesoderm differentiation.     

An FGF3-BMP Signaling Axis Regulates Caudal Neural Tube Closure,Neural Crest Specification and Anterior-Posterior Axis Extension

During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is a major advance toward understanding how these tissue layers interact during axis extension with important implications in human disease.     

Mammalian Merkel cells are descended from the epidermal lineage

Merkel cells are specialized cells in the skin that are important for proper neural encoding of light touch stimuli. Conflicting evidence suggests that these cells are lineally descended from either the skin or the neural crest. To address this question, we used epidermal (Krt14^Cre) and neural crest (Wnt1^Cre) Cre-driver lines to conditionally delete Atoh1 specifically from the skin or neural crest lineages, respectively, of mice. Deletion of Atoh1 from the skin lineage resulted in loss of Merkel cells from all regions of the skin, while deletion from the neural crest lineage had no effect on this cell population. Thus, mammalian Merkel cells are derived from the skin lineage.     

Regulation of cadherin expression in the chicken neural crest by the Wnt/β-catenin signaling pathway

In neural crest cell development, the expression of the cell adhesion proteins cadherin-7 and cadherin-11 commences after delamination of the neural crest cells from the neuroepithelium. The canonical Wnt signaling pathway is known to drive this delamination step and is a candidate for inducing expression of these cadherins at this time. This project was initiated to investigate the role of canonical Wnt signaling in the expression of cadherin-7 and cadherin-11 by treating neural crest cells with Wnt3a ligand. Expression of cadherin-11 was first confirmed in the neural crest cells for the chicken embryo. The changes in the expression level of cadherin-7 and -11 following the treatment with Wnt3a were studied using real-time RT-PCR and immunostaining. Statistically significant upregulation in the mRNA expression of cadherin-7 and cadherin-11 and in the amount of cadherin-7 and cadherin-11 protein found in cell-cell interfaces between neural crest cells was observed in response to Wnt, demonstrating that cadherin-7 and cadherin-11 expressed by the migrating neural crest cells can be regulated by the canonical Wnt pathway.Key words: neural crest, Wnt, cadherin-7, cadherin-11     

Conditional deletion of Hand2 reveals critical functions in neurogenesis and cell type-specific gene expression for development of neural crest-derived noradrenergic sympathetic ganglion neurons

Neural crest-derived structures that depend critically upon expression of the basic helix-loop-helix DNA binding protein Hand2 for normal development include craniofacial cartilage and bone, the outflow tract of the heart, cardiac cushion, and noradrenergic sympathetic ganglion neurons. Loss of Hand2 is embryonic lethal by E9.5, obviating a genetic analysis of its in-vivo function. We have overcome this difficulty by specific deletion of Hand2 in neural crest-derived cells by crossing our line of floxed Hand2 mice with Wnt1-Cre transgenic mice. Our analysis of Hand2 knock-out in neural crest-derived cells reveals effects on development in all neural crest-derived structures where Hand2 is expressed. In the autonomic nervous system, conditional disruption of Hand2 results in a significant and progressive loss of neurons as well as a significant loss of TH expression. Hand2 affects generation of the neural precursor pool of cells by affecting both the proliferative capacity of the progenitors as well as affecting expression of Phox2a and Gata3, DNA binding proteins important for the cell autonomous development of noradrenergic neurons. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting differentiation and cell type-specific gene expression in neural crest-derived noradrenergic sympathetic ganglion neurons. Hand2 has a pivotal function in a non-linear cross-regulatory network of DNA binding proteins that affect cell autonomous control of differentiation and cell type-specific gene expression.     

Requirement for Twist1 in frontonasal and skull vault development in the mouse embryo

Using a Cre-mediated conditional deletion approach, we have dissected the function of Twist1 in the morphogenesis of the craniofacial skeleton. Loss of Twist1 in neural crest cells and their derivatives impairs skeletogenic differentiation and leads to the loss of bones of the snout, upper face and skull vault. While no anatomically recognizable maxilla is formed, a malformed mandible is present. Since Twist1 is expressed in the tissues of the maxillary eminence and the mandibular arch, this finding suggests that the requirement for Twist1 is not the same in all neural crest derivatives. The effect of the loss of Twist1 function is not restricted to neural crest-derived bones, since the predominantly mesoderm-derived parietal and interparietal bones are also affected, presumably as a consequence of lost interactions with neural crest-derived tissues. In contrast, the formation of other mesodermal skeletal derivatives such as the occipital bones and most of the chondrocranium are not affected by the loss of Twist1 in the neural crest cells.     

Dll1 and Dll4 function sequentially in the retina and pV2 domain of the spinal cord to regulate neurogenesis and create cell diversity

Signalling mediated by Notch receptors is known to have multiple functions during vertebrate neural development, regulating processes like progenitor differentiation and cell type diversification. Various Notch ligands are expressed in the developing nervous system and their activities might contribute to this multiplicity of functions. Here, we show that two Delta-like genes, Dll1 and Dll4, are sequentially expressed in differentiating neurons of the embryonic mouse retina and spinal cord's pV2 domain, with Dll1 starting to be expressed before Dll4. Analysis of Dll1 mutants reveals this gene is necessary and sufficient to maintain a pool of progenitors in the embryonic neuroepithelium. Accordingly, in the spinal cord domains where Dll1 is the only expressed Notch ligand, its inactivation leads to an increased rate of neurogenesis and premature differentiation of neural progenitors. In contrast, in the pV2 domain and retina where Dll1 is co-expressed with Dll4, progenitors are not exhausted and cell diversity is maintained. Together, our results support a model where Dll1 and Dll4 are part of a unique genetic circuitry that regulates subsequent steps of neurogenesis in the retina and pV2 domain: while Dll1 serves to prevent the untimely differentiation of neural progenitors, Dll4 might function to generate diversity within the population of differentiating neurons.     

Cell communication with the neural plate is required for induction of neural markers by BMP inhibition: evidence for homeogenetic induction and implications for Xenopus animal cap and chick explant assays

In Xenopus, the animal cap is very sensitive to BMP antagonists, which result in neuralization. In chick, however, only cells at the border of the neural plate can be neuralized by BMP inhibition. Here we compare the two systems. BMP antagonists can induce neural plate border markers in both ventral Xenopus epidermis and non-neural chick epiblast. However, BMP antagonism can only neuralize ectodermal cells when the BMP-inhibited cells form a continuous trail connecting them to the neural plate or its border, suggesting that homeogenetic neuralizing factors can only travel between BMP-inhibited cells. Xenopus animal cap explants contain cells fated to contribute to the neural plate border and even to the anterior neural plate, explaining why they are so easily neuralized by BMP-inhibition. Furthermore, chick explants isolated from embryonic epiblast behave like Xenopus animal caps and express border markers. We propose that the animal cap assay in Xenopus and explant assays in the chick are unsuitable for studying instructive signals in neural induction.     

Class III β-tubulin,a novel biomarker in the human melanocyte lineage

It is generally thought that class III β-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III β-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III β-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.     

Epigenetic Regulation of miR-302 by JMJD1C Inhibits Neural Differentiation of Human Embryonic Stem Cells

It has been recently reported that the regulatory circuitry formed by OCT4, miR-302, and NR2F2 controls both pluripotency and neural differentiation of human embryonic stem cells (hESCs). We show here that JMJD1C, a histone 3 lysine 9 (H3K9) demethylase expressed in hESCs, directly interacts with this circuitry. hESCs with stable knockdown of JMJD1C remain pluripotent while having reduced miR-302 expression, decreased BMP signaling, and enhanced TGFβ signaling. JMJD1C binds to the miR-302 promoter and reduces H3K9 methylation. Withdrawal of basic fibroblast growth factor (bFGF) from the culture induces neural differentiation of the knockdown, but not the control, cells within 3 days, accompanied by elevated NR2F2 expression. This can be attenuated with miR-302 mimics or an H3K9 methytransferase inhibitor. Together, our findings suggest that JMJD1C represses neural differentiation of hESCs at least partially by epigenetically sustaining miR-302 expression and that JMJD1C knockdown is sufficient to trigger neural differentiation upon withdrawal of exogenous bFGF.