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.     

Epigenetic activation of Sox2 gene in the developing vertebrate neural plate

One of the earliest manifestations of neural induction is onset of expression of the neural marker Sox2, mediated by the activation of the enhancers N1 and N2. By using loss and gain of function, we find that Sox2 expression requires the activity of JmjD2A and the Msk1 kinase, which can respectively demethylate the repressive H3K9me3 mark and phosphorylate the activating H3S10 (H3S10ph) mark. Bimolecular fluorescence complementation reveals that the adaptor protein 14-3-3, known to bind to H3S10ph, interacts with JMJD2A and may be involved in its recruitment to regulatory regions of the Sox2 gene. Chromatin immunoprecipitation reveals dynamic binding of JMJD2A to the Sox2 promoter and N-1 enhancer at the time of neural plate induction. Finally, we show a clear temporal antagonism on the occupancy of H3K9me3 and H3S10ph modifications at the promoter of the Sox2 locus before and after the neural plate induction. Taken together, our results propose a series of epigenetic events necessary for the early activation of the Sox2 gene in neural progenitor cells.     

Early expression of Tubulin Beta-III in avian cranial neural crest cells

Neural crest cells are a transient stem-like cell population that forms in the dorsal neural tube of vertebrate embryos and then migrates to various locations to differentiate into diverse derivatives such as craniofacial bone, cartilage, and the enteric and peripheral nervous systems. The current dogma of neural crest cell development suggests that there is a specific hierarchical gene regulatory network (GRN) that controls the induction, specification, and differentiation of these cells at specific developmental times. Our lab has identified that a marker of differentiated neurons, Tubulin Beta-III (TUBB3), is expressed in premigratory neural crest cells. TUBB3 has previously been identified as a major constituent of microtubules and is required for the proper guidance and maintenance of axons during development. Using the model organism, Gallus gallus, we have characterized the spatiotemporal localization of TUBB3 in early stages of development. Here we show TUBB3 is expressed in the developing neural plate, is upregulated in the pre-migratory cranial neural crest prior to cell delamination and migration, and it is maintained or upregulated in neurons in later developmental stages. We believe that TUBB3 likely has a role in early neural crest formation and migration separate from its role in neurogenesis.     

In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation

Trichostatin A (TSA) inhibits all histone deacetylases (HDACs) of both class I and II, whereas trapoxin (TPX) cannot inhibit HDAC6, a cytoplasmic member of class II HDACs. We took advantage of this differential sensitivity of HDAC6 to TSA and TPX to identify its substrates. Using this approach, alpha-tubulin was identified as an HDAC6 substrate. HDAC6 deacetylated alpha-tubulin both in vivo and in vitro. Our investigations suggest that HDAC6 controls the stability of a dynamic pool of microtubules. Indeed, we found that highly acetylated microtubules observed after TSA treatment exhibited delayed drug-induced depolymerization and that HDAC6 overexpression prompted their induced depolymerization. Depolymerized tubulin was rapidly deacetylated in vivo, whereas tubulin acetylation occurred only after polymerization. We therefore suggest that acetylation and deacetylation are coupled to the microtubule turnover and that HDAC6 plays a key regulatory role in the stability of the dynamic microtubules.     

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.     

Misexpression of BRE gene in the developing chick neural tube affects neurulation and somitogenesis

The brain and reproductive expression (BRE) gene is expressed in numerous adult tissues and especially in the nervous and reproductive systems. However, little is known about BRE expression in the developing embryo or about its role in embryonic development. In this study, we used in situ hybridization to reveal the spatiotemporal expression pattern for BRE in chick embryo during development. To determine the importance of BRE in neurogenesis, we overexpressed BRE and also silenced BRE expression specifically in the neural tube. We established that overexpressing BRE in the neural tube indirectly accelerated Pax7⁺ somite development and directly increased HNK-1⁺ neural crest cell (NCC) migration and TuJ-1⁺ neurite outgrowth. These altered morphogenetic processes were associated with changes in the cell cycle of NCCs and neural tube cells. The inverse effect was obtained when BRE expression was silenced in the neural tube. We also determined that BMP4 and Shh expression in the neural tube was affected by misexpression of BRE. This provides a possible mechanism for how altering BRE expression was able to affect somitogenesis, neurogenesis, and NCC migration. In summary, our results demonstrate that BRE plays an important role in regulating neurogenesis and indirectly somite differentiation during early chick embryo development.     

Neural crest development is regulated by the transcription factor Sox9

The neural crest is a transient migratory population of stem cells derived from the dorsal neural folds at the border between neural and non-neural ectoderm. Following induction, prospective neural crest cells are segregated within the neuroepithelium and then delaminate from the neural tube and migrate into the periphery, where they generate multiple differentiated cell types. The intrinsic determinants that direct this process are not well defined. Group E Sox genes (Sox8, Sox9 and Sox10) are expressed in the prospective neural crest and Sox9 expression precedes expression of premigratory neural crest markers. Here, we show that group E Sox genes act at two distinct steps in neural crest differentiation. Forced expression of Sox9 promotes neural-crest-like properties in neural tube progenitors at the expense of central nervous system neuronal differentiation. Subsequently, in migratory neural crest cells, SoxE gene expression biases cells towards glial cell and melanocyte fate, and away from neuronal lineages. Although SoxE genes are sufficient to initiate neural crest development they do not efficiently induce the delamination of ectopic neural crest cells from the neural tube consistent with the idea that this event is independently controlled. Together, these data identify a role for group E Sox genes in the initiation of neural crest development and later SoxE genes influence the differentiation pathway adopted by migrating neural crest cells.     

The early migration of neural crest cells in the trunk region of the avian embryo: an electron microscopic study

Four phases of neural crest migration characteristic of early avian trunk regions are described: (a) appearance, during which crest cells reside in the dorsal neural tube, but are separated from each other dorsally by large spaces; (b) condensation, during which large spaces between the crest cells become reduced, the cells elongate, flatten upon the surface of the neural tube, and become oriented tangentially (i.e., with their long axes perpendicular to the longitudinal axes of the neural tube); (c) early migration, during which the crest population expands uniformly to meet the dorsal apex of the somites; and (d) advanced migration, during which crest cells appear in the extracellular space dorsal to the somites. At the most advanced phases, the crest population at the dorsal midline decreased in number, with a concomitant loss of tangential orientation and the appearance of spaces between the cells. Extracellular components of the acellular spaces through which crest cells migrate are also described. The observations are discussed in terms of (1) those morphological changes undergone by crest cells during migration, and (2) possible factors that might delimit crest pathways. It is suggested that the operation of contact inhibition of movement within the crest population is sufficient to determine the direction of crest migration.