Apical accumulation of MARCKS in neural plate cells during neurulation in the chick embryo

BACKGROUND: The neural tube is formed by morphogenetic movements largely dependent on cytoskeletal dynamics. Actin and many of its associated proteins have been proposed as important mediators of neurulation. For instance, mice deficient in MARCKS, an actin cross-linking membrane-associated protein that is regulated by PKC and other kinases, present severe developmental defects, including failure of cranial neural tube closure. RESULTS: To determine the distribution of MARCKS, and its possible relationships with actin during neurulation, chick embryos were transversely sectioned and double labeled with an anti-MARCKS polyclonal antibody and phalloidin. In the neural plate, MARCKS was found ubiquitously distributed at the periphery of the cells, being conspicuously accumulated in the apical cell region, in close proximity to the apical actin meshwork. This asymmetric distribution was particularly noticeable during the bending process. After the closure of the neural tube, the apically accumulated MARCKS disappeared, and this cell region became analogous to the other peripheral cell zones in its MARCKS content. Actin did not display analogous variations, remaining highly concentrated at the cell subapical territory. The transient apical accumulation of MARCKS was found throughout the neural tube axis. The analysis of another epithelial bending movement, during the formation of the lens vesicle, revealed an identical phenomenon. CONCLUSIONS: MARCKS is transiently accumulated at the apical region of neural plate and lens placode cells during processes of bending. This asymmetric subcellular distribution of MARCKS starts before the onset of neural plate bending. These results suggest possible upstream regulatory actions of MARCKS on some functions of the actin subapical meshwork

Neuron's little helper: the role of primary cilia in neurogenesis

The generation of new neurons involves a great variety of cell-extrinsic and cell-intrinsic signals. The primary cilium, long regarded as an "evolutionary vestige," has emerged as an essential signaling hub in many cells, including neural progenitors and differentiating neurons. Most progenitors harbor an apically-localized primary cilium, which is assembled and disassembled following the cell cycle, while the presence, position and length of this organelle appears to be even more variable in differentiating neurons. One of the main extracellular cues acting through the cilium is Sonic Hedgehog, which modulates spatial patterning, the progression of the cell cycle and the timing of neurogenesis. Other extracellular signals appear to bind to cilia-localized receptors and affect processes such as dendritogenesis. All the observed dynamics, as well as the many signaling pathways depending on cilia, indicate this organelle as an important structure involved in neurogenesis.Agencia Nacional de Investigación e InnovaciónPEDECIBAInstitut Pasteur de Montevideo–FOCEM Mercosu

Neuron’s little helper: the role of primary cilia in neurogenesis

The generation of new neurons involves a great variety of cell-extrinsic and cell-intrinsic signals. The primary cilium, long regarded as an “evolutionary vestige,” has emerged as an essential signaling hub in many cells, including neural progenitors and differentiating neurons. Most progenitors harbor an apically-localized primary cilium, which is assembled and disassembled following the cell cycle, while the presence, position and length of this organelle appears to be even more variable in differentiating neurons. One of the main extracellular cues acting through the cilium is Sonic Hedgehog, which modulates spatial patterning, the progression of the cell cycle and the timing of neurogenesis. Other extracellular signals appear to bind to cilia-localized receptors and affect processes such as dendritogenesis. All the observed dynamics, as well as the many signaling pathways depending on cilia, indicate this organelle as an important structure involved in neurogenesis

Characterization of primary cilia during the differentiation of retinal ganglion cells in the zebrafish

Background: Retinal ganglion cell (RGC) differentiation in vivo is a highly stereotyped process, likely resulting from the interaction of cell type-specific transcription factors and tissue-derived signaling factors. The primary cilium, as a signaling hub in the cell, may have a role during this process but its presence and localization during RGC generation, and its contribution to the process of cell differentiation, have not been previously assessed in vivo. Methods: In this work we analyzed the distribution of primary cilia in vivo using laser scanning confocal microscopy, as well as their main ultrastructural features by transmission electron microscopy, in the early stages of retinal histogenesis in the zebrafish, around the time of RGC generation and initial differentiation. In addition, we knocked-down ift88 and elipsa, two genes with an essential role in cilia generation and maintenance, a treatment that caused a general reduction in organelle size. The effect on retinal development and RGC differentiation was assessed by confocal microscopy of transgenic or immunolabeled embryos. Results: Our results show that retinal neuroepithelial cells have an apically-localized primary cilium usually protruding from the apical membrane. We also found a small proportion of sub-apical cilia, before and during the neurogenic period. This organelle was also present in an apical position in neuroblasts during apical process retraction and dendritogenesis, although between these stages cilia appeared highly dynamic regarding both presence and position. Disruption of cilia caused a decrease in the proliferation of retinal progenitors and a reduction of neural retina volume. In addition, retinal histogenesis was globally delayed albeit RGC layer formation was preferentially reduced with respect to the amacrine and photoreceptor cell layers. Conclusions: These results indicate that primary cilia exhibit a highly dynamic behavior during early retinal differentiation, and that they are required for the proliferation and survival of retinal progenitors, as well as for neuronal generation, particularly of RGCs

Application of the DNA-Specific stain methyl green in the fluorescent labeling of embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophoreconjugated phalloidi

Polarization and orientation of retinal ganglion cells in vivo.

In the absence of external cues, neurons in vitro polarize by using intrinsic mechanisms. For example, cultured hippocampal neurons extend arbitrarily oriented neurites and then one of these, usually the one nearest the centrosome, begins to grow more quickly than the others. This neurite becomes the axon as it accumulates molecular components of the apical junctional complex. All the other neurites become dendrites. It is unclear, however, whether neurons in vivo, which differentiate within a polarized epithelium, break symmetry by using similar intrinsic mechanisms. To investigate this, we use four-dimensional microscopy of developing retinal ganglion cells (RGCs) in live zebrafish embryos. We find that the situation is indeed very different in vivo, where axons emerge directly from uniformly polarized cells in the absence of other neurites. In vivo, moreover, components of the apical complex do not localize to the emerging axon, nor does the centrosome predict the site of axon emergence. Mosaic analysis in four dimensions, using mutants in which neuroepithelial polarity is disrupted, indicates that extrinsic factors such as access to the basal lamina are critical for normal axon emergence from RGCs in vivo.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Cell extrusion in development and cancer, what MARCKS the difference for epithelial integrity?

Cell extrusion is an active mechanism to eliminate non-viable or supernumerary cells in healthy epithelia. It also plays a role in carcinogenesis, both in tumor growth (apical extrusion) and metastasis (basal extrusion). Embryonic tissues like the neuroepithelium, on the other hand, present rates of proliferation comparable to that of carcinomas, without the occurrence of cell extrusion. However, the downregulation or phosphorylation of actin modulating proteins like MARCKS, causes extensive neuroepithelial apical cell extrusion. As changes in MARCKS proteins phosphorylation and expression have also been correlated to carcinogenesis, we propose here an integrated model for their functions in epithelial integrity.CSIC: 125-34

Asymmetric inheritance of the apical domain and self-renewal of retinal ganglion cell progenitors depend on Anillin function.

Divisions that generate one neuronal lineage-committed and one self-renewing cell maintain the balance of proliferation and differentiation for the generation of neuronal diversity. The asymmetric inheritance of apical domains and components of the cell division machinery has been implicated in this process, and might involve interactions with cell fate determinants in regulatory feedback loops of an as yet unknown nature. Here, we report the dynamics of Anillin - an essential F-actin regulator and furrow component - and its contribution to progenitor cell divisions in the developing zebrafish retina. We find that asymmetrically dividing retinal ganglion cell progenitors position the Anillin-rich midbody at the apical domain of the differentiating daughter. anillin hypomorphic conditions disrupt asymmetric apical domain inheritance and affect daughter cell fate. Consequently, the retinal cell type composition is profoundly affected, such that the ganglion cell layer is dramatically expanded. This study provides the first in vivo evidence for the requirement of Anillin during asymmetric neurogenic divisions. It also provides insights into a reciprocal regulation between Anillin and the ganglion cell fate determinant Ath5, suggesting a mechanism whereby the balance of proliferation and differentiation is accomplished during progenitor cell divisions in vivo.journal articleresearch support, non-u.s. gov't2015 Mar 012015 02 05importe