Expression of hindbrain boundary markers is regulated by FGF3

Summary Compartment boundaries act as organizing centers that segregate adjacent areas into domains of gene expression and regulation, and control their distinct fates via the secretion of signalling factors. During hindbrain development, a specialized cell-population forms boundaries between rhombomeres. These boundary cells demonstrate unique morphological properties and express multiple genes that differs them from intra-rhombomeric cells. Yet, little is known regarding the mechanisms that controls the expression or function of these boundary markers. Multiple components of the FGF signaling system, including ligands, receptors, downstream effectors as well as proteoglycans are shown to localize to boundary cells in the chick hindbrain. These patterns raise the possibility that FGF signaling plays a role in regulating boundary properties. We provide evidence to the role of FGF signaling, particularly the boundary-derived FGF3, in regulating the expression of multiple markers at hindbrain boundaries. These findings enable further characterization of the unique boundary-cell population, and expose a new function for FGFs as regulators of boundary-gene expression in the chick hindbrain

A new role of hindbrain boundaries as pools of neural stem/progenitor cells regulated by Sox2

Background: Compartment boundaries are an essential developmental mechanism throughout evolution, designated to act as organizing centers and to regulate and localize differently fated cells. The hindbrain serves as a fascinating example for this phenomenon as its early development is devoted to the formation of repetitive rhombomeres and their well-defined boundaries in all vertebrates. Yet, the actual role of hindbrain boundaries remains unresolved, especially in amniotes. Results: Here, we report that hindbrain boundaries in the chick embryo consist of a subset of cells expressing the key neural stem cell (NSC) gene Sox2. These cells co-express other neural progenitor markers such as Transitin (the avian Nestin), GFAP, Pax6 and chondroitin sulfate proteoglycan. The majority of the Sox2+ cells that reside within the boundary core are slow-dividing, whereas nearer to and within rhombomeres Sox2+ cells are largely proliferating. In vivo analyses and cell tracing experiments revealed the contribution of boundary Sox2+ cells to neurons in a ventricular-to-mantle manner within the boundaries, as well as their lateral contribution to proliferating Sox2+ cells in rhombomeres. The generation of boundary-derived neurospheres from hindbrain cultures confirmed the typical NSC behavior of boundary cells as a multipotent and self-renewing Sox2+ cell population. Inhibition of Sox2 in boundaries led to enhanced and aberrant neural differentiation together with inhibition in cell-proliferation, whereas Sox2 mis-expression attenuated neurogenesis, confirming its significant function in hindbrain neuronal organization. Conclusions: Data obtained in this study deciphers a novel role of hindbrain boundaries as repetitive pools of neural stem/progenitor cells, which provide proliferating progenitors and differentiating neurons in a Sox2-dependent regulation. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0277-y) contains supplementary material, which is available to authorized users

Calponin 2 Acts As an Effector of Noncanonical Wnt-Mediated Cell Polarization during Neural Crest Cell Migration

Neural crest cells (NCCs) migrate throughout the embryo to differentiate into cell types of all germ layers. Initial directed NCC emigration relies on planar cell polarity (PCP), which through the activity of the small GTPases RhoA and Rac governs the actin-driven formation of polarized cell protrusions. We found that the actin binding protein calponin 2 (Cnn2) was expressed in protrusions at the leading edge of migratory NCCs in chicks and frogs. Cnn2 knockdown resulted in NCC migration defects in frogs and chicks and randomized outgrowth of cell protrusions in NCC explants. Morphant cells showed central stress fibers at the expense of the peripheral actin network. Cnn2 acted downstream of Wnt/PCP, as migration defects induced by dominant-negative Wnt11 or inhibition of RhoA function were rescued by Cnn2 knockdown. These results suggest that Cnn2 modulates actin dynamics during NCC migration as an effector of noncanonical Wnt/PCP signaling

Matrix metalloproteinase 9/gelatinase B is required for neural crest cell migration

AbstractThis study determined the role of MMP9/gelatinase B during the migration onset of Neural Crest Cells (NCC) in avian embryos. NCC are neuroepithelial progenitors that convert into mesenchyme and migrate along defined paths throughout the embryo. To engage in migration, NCC loose cell contacts, detach from the neural tube and invade the surrounding environment. Multiple signals and transcription factors that regulate these events have been identified. Nevertheless, little is known regarding effectors that act downstream to execute the actual NCC migration. Matrix metalloproteinases (MMPs) compose a large family of enzymes whose principal substrates are basement membranes, adhesion proteins and the extracellular matrix (ECM) components. A major subgroup of MMPs, the gelatinases (MMP9 and 2) are central to many adult physiological and pathological processes, such as tumor metastasis and angiogenesis, in which cell–cell and cell–matrix contacts are degraded to allow migration. As NCC undergo similar processes during development, we hypothesized that MMP9 may also promote the migration of NCC.MMP9 was found to be expressed in delaminating and migrating NCC of both cranial and trunk axial levels. Blocking MMP9 resulted in a dramatic inhibition of NCC delamination and migration, without perturbing specification or survival. This inhibition occurred at regions containing both premigratory and migrating cells, indicative for the central role of MMP9 in executing the detachment of NCC from the neural tube as well as their migration. Conversely, excess MMP9 enhanced mesenchymalization and delamination of NCC and accelerated progenitors to undergo precocious migration. Examination of the mechanistic activity of MMP9 revealed its capability to degrade the adhesion molecule N-cadherin as well as the basement-membrane protein laminin within or around NCC, respectively. Altogether, our study reveals MMP9 as a novel effector which is required for NCC delamination and migration

Hindbrain boundaries as niches of neural progenitor and stem cells regulated by the extracellular matrix proteoglycan chondroitin sulphate.

Peer reviewed: TrueAcknowledgements: We thank Einat Zelinger and Daniel Waiger from the Center for Scientific Imaging, Core Facility, Faculty of Agriculture, Food and Environmental Sciences, The Hebrew University of Jerusalem, for their help with the SEM and IMARIS analyses. We thank Adi Turjeman from The Center for Genomic Technologies, The Hebrew University of Jerusalem, for assisting with the RNA-sequencing. We thank Efrat Hagai from the Life Sciences Core Facilities, Weizmann Institute of Science, for assisting with the FACS procedure.Publication status: PublishedFunder: The Hebrew University of Jerusalem; doi: http://dx.doi.org/10.13039/501100003483The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development

Additional file 7: of A new role of hindbrain boundaries as pools of neural stem/progenitor cells regulated by Sox2

Expression of neural-differentiation markers in the hindbrain. A. (a) Representative flow cytometry plot from 18 HH hindbrain stained with Sox2 and Tuj1. Quantification of relative abundance of Sox2/Tuj1-expressing cells is shown. (b) Graphic representation of Sox2/Tuj1 distribution as percentage of total stained cells. B. Representative flat-mounted views of 15HH and 18HH hindbrains in situ hybridized with RNA probes against NeuroD, NSCL1 and Brn3a, or immunostained for HuC/D (n = 10/marker) (e-h). Expression of NSCL1 and HuC/D shifts from punctuated rhombomeric expression in 15HH to boundary-enhanced expression at 18HH. C. (a-c) Representative flat-mounted views of 18HH hindbrains stained for Sox2 and HuC/D (n = 10). Merged image is shown in (c). (d-g) Sequential Z-stack analysis from 0 to –20 μm of a boundary area. Arrows indicate site of neural differentiation. Scale bars = 100 μm. D. Scheme of the clonal-analysis of HB cell-labeling experiment using injection of AFP plasmid into single cells and harvesting at two time points. (TIF 4496 kb

Additional file 6: of A new role of hindbrain boundaries as pools of neural stem/progenitor cells regulated by Sox2

CM-DiI labeling of boundary and rhombomere cells. Representative flat-mount confocal views of CM-DiI labelled rhombomere (a,b) or boundary (c,d) (n = 5 hindbrains). Arrows indicate injection site, yellow lines indicate boundaries. Outlined areas in (b,d) show dye expansion. (TIF 11336 kb

Additional file 9: of A new role of hindbrain boundaries as pools of neural stem/progenitor cells regulated by Sox2

Detailed description of clones and plasmids used in this study. (DOCX 13 kb