Apical accumulation of MARCKS in neural plate cells during neurulation in the chick embryo
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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.
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.
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.
KeywordsActin Filament Neural Tube Neural Plate Neuroepithelial Cell Neural Tube Closure
Major tissue movements during neurulation include neural plate bending as well as neural folds elevation and its convergence to fuse and close the neural tube. These movements of the neural plate result from the actions of extrinsic and intrinsic forces , and the latter are believed to be mainly driven by the actin cytoskeleton [2,3,4,5]. Neural plate cells are polarized cells; actin and myosin are mainly restricted to regions of cell narrowing, especially to the apical border of the epithelium [2,6]. In the apical region, cells are joined together by extensive actin-associated zonula adherens cell junctions, which are thought to be important in invagination processes [7,8]. Knockout analyses in mice have shown that some actin binding or adherens junction proteins are important for neural tube formation. Examples of these proteins are: vinculin , shroom , and the two closely related actin cross-linking proteins MARCKS (Myristoylated Alanine-Rich C Kinase Substrate)  and MacMARCKS (also called F52 and MRP) .
MARCKS is a ubiquitous protein substrate for different PKC family kinases and proline directed kinases such as MAPK and Cdks [13,14,15,16,17]. Its PKC-phosphorylation domain or PSD (Phosphorylation Site Domain) is highly conserved and it is also the site for interaction with other molecules, such as calcium-calmodulin, negatively charged membrane phospholipids and F-actin [13,14]. Binding to calcium-calmodulin and plasma membrane, as well as actin filament cross-linking activity, are antagonized by PSD phosphorylation [13,14,18]. Conversely, calcium-calmodulin binding inhibits PSD phosphorylation and actin crosslinking. In addition to neural tube closure, MARCKS and MacMARCKS have been implicated in several other events related to actin cytoskeleton, such as cell motility, cell spreading, membrane ruffling, phagocytosis, exocytosis and neurite outgrowth [13,19,20,21,22,23,24].
To examine possible anatomical relationships between MARCKS and actin during bending movements, we double labelled chick embryo cryosections at levels showing cranial and spinal neurulation. To compare with other invaginating epithelia we also analyzed the localization of these proteins in the lens placode finding that, in both cases, MARCKS is transiently accumulated in the apical border of the bending epithelia, in a position very close to the apical actin belt. In our knowledge, this is the first report showing a polarized distribution of MARCKS towards an apical cell border, as well as its association with the progression of an essential morphogenetic movement.
Results and Discussion
We performed all our fluorescence microscopy analysis by double labeling serial chick embryo transverse sections (H-H stages 6-15 ) with a polyclonal anti-carboxy terminal chicken MARCKS antibody  and with rhodamine-conjugated phalloidin (for F-actin labeling).
MARCKS in the open neural plate
Neural tube closure at the presumptive prosencephalic region
Neural tube closure at the presumptive rhombencephalic and spinal regions
Subcellular localization of MARCKS and F-actin during neural tube bending
Formation of the lens vesicle
Although evidence is lacking, the changes in the subcellular localization of MARCKS described here could be explained, in principle, by two mechanisms: MARCKS could be either re-distributed from a formerly homogeneous peripheral distribution, or newly synthesized protein could be directly driven to the apical region of the epithelial cells. At the opposite, the change observed after neural tube or lens vesicle closure could be explained by a redistribution of the protein, or by a specific degradation in the apical region of the epithelia. Northern-blot and reporter gene expression in neurulating mice embryos showed an important rise in MARCKS expression in the closing cranial and caudal neural plate .
In polarized, confluent MDCK cells, MARCKS localization at the lateral membranes depended on PKC activity and was correlated with the subcellular localization of other membrane and actin interacting protein, fodrin . For MacMARCKS, a basolateral membrane targeting determinant has been found into the PSD , this domain is almost identical to MARCKS PSD  therefore, it is tempting to speculate that a similar mechanism could determine the subcellular localization of MARCKS in some polarized cells. In the case of the neural plate described here, apical localization could be explained by a singular signal, perhaps related to other phosphorylation site(s). Defects in neural tube closure, from the truncal to the cranial region, and in eye formation were reported after incubating neurulating rat embryos in the absence of methionine . In these experiments, some cytoskeletal proteins, including actin, were hypomethylated, and a change of the actin and tubulin distribution was observed in the neural plate.
Another question remains: is the maintenance of apical MARCKS dependent on an interaction with the plasma membrane, the actin cytoskeleton, or both? MARCKS association to the plasma membrane is known to be reversible , and a permanent pool of cytosolic MARCKS has been described . Our observations consistently showed a peripheral distribution of MARCKS in the closing neural plate and lens placode. Similar results were obtained by Blackshear's group in neurulating mice . Altogether, these observations suggest that MARCKS is mainly associated to the plasma membrane during neural plate and lens placode bending. MARCKS can bind to membranes by at least two different sites: a, the myristoyl group at its amino-terminus, that inserts into the lipid bilayer, and b, the positively charged PSD, that, while unphosphorylated, electrostatically interacts with acidic head groups of phospholipids . However, the transgenic expression of modified forms of the protein in MARCKS null mice suggest that membrane association is not necessary for neural tube closure promoting activity. Either the expression of a nonmyristoylatable  or a nonmyristoylatable and pseudophosphorylated (where serines in the PSD were substituted by glutamic acid residues) forms of MARCKS in knockout mice resulted in the complete rescue of the neural tube closure phenotype.
Our results show that MARCKS protein is transiently accumulated to the apical border of neural plate and lens placode cells, in close apposition to the apical actin meshwork, during the processes of neural tube and lens vesicle formation. These observations provide additional structural counterparts to the knockout and transgenic mice analyses, although they also generate new problems, as respect to the role of MARCKS in spinal neural plate bending. In addition, these new results concerning an apical concentration of MARCKS open new questions about the mechanisms able to generate and transiently maintain its asymmetric distribution.
Materials and methods
Fertilized hen eggs were kindly supplied by Prodhin (Uruguay) and incubated in our laboratory until the desired stages. Whole embryos were fixed by immersion in 3.7% paraformaldehyde in PBS for 12-36 hrs and cryoprotected in 5% and 20% sucrose in PBS. They were then gelatin embedded  and quickly frozen in liquid N2. Transverse cryosections (4-5 μm) were made on a Reichert-Jung Cryocut E cryostat and adhered to gelatin-subbed slides. At least two embryos were analyzed at each stage. Anti carboxy-terminal chick MARCKS antibody (a kind gift of Dr. Pico Caroni, Friedrich Miescher Institute, Basel, Switzerland ) was diluted 1:2000-1:3000 in blocking solution (PBS/1% BSA). Secondary antibody: FITC-conjugated goat anti mouse IgG (Gibco BRL, UK), 1:200. TRITC-conjugated phalloidin (Molecular Probes Inc., USA) was diluted 1:4000. Labeled sections were observed and photographed using a Nikon Microphot FXA microscope equipped with epifluorescence.
Supported in part by the programs PEDECIBA and Investigación Científica (CSIC), Universidad de la República. Uruguay. We greatly aknowledge Drs. P. Caroni and S. McLoon for kindly gifting of antibodies. We are indebted to Prof. H.Trenchi for continuous support supplying us fertilized avian eggs.
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