Research Article: Deficiency of mDia, an Actin Nucleator, Disrupts Integrity of Neuroepithelium and Causes Periventricular Dysplasia

Date Published: September 28, 2011

Publisher: Public Library of Science

Author(s): Dean Thumkeo, Ryota Shinohara, Keisuke Watanabe, Hirohide Takebayashi, Yosuke Toyoda, Kiyoshi Tohyama, Toshimasa Ishizaki, Tomoyuki Furuyashiki, Shuh Narumiya, Cara Gottardi.

Abstract: During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that mDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both mDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells.

Partial Text: Neuroepithelial cells function as neural stem cells, which proliferate for self-renewal and to produce progenitor cells for lineages of cells in the central nervous system (CNS). After several rounds of cell division, the progenitor cells become differentiated to precursors of neurons or glial cells, which start to migrate towards the destination of final differentiation [1]. Neural stem cell division and differentiation thus need to be tightly controlled spatiotemporally. Neuroepithelial cells have the apical and the basal processes, and, most significantly, the cell-cell adhesion structure called the apical adherens junction which exists at the end feet of the apical process [2]. Apical adherens junctions are indispensable for the formation and maintenance of the apical-basal polarity of neuroepithelium [3]. Many studies in mouse and human genetics have identified importance of this polarity in homeostatic control of neural stem cells [4], [5], which functions together with differentiation machineries such as Notch and Sonic-Hedgehog signaling [5], [6]. Adherens junction is composed of the cadherin-catenin complex. Gene deletion of components of the adherens junction of neuroepithelial cells such as αE-catenin [6] and N-cadherin [7] perturbs their apical-basal polarity, releases neural stem cells from tight control to proliferate and differentiate autonomously, and induces formation of abnormal cellular mass. Since αE-catenin and N-cadherin function not only in apical adherens junctions but also in adherens junctions at other sites, mass can grow in any direction and invariably protrudes into the ventricle. Occurrence of such periventricular dysplastic mass demonstrates the importance of the apical adherens junction in maintenance of the neuroepithelial integrity. Histological analysis previously identified an actin belt linking apical adherens junctions and lining the apical surface of neuroepithelial cells. However, whether and how this apical actin belt is formed, maintained and supports the integrity of the apical adherens junctions of neuroepithelial cells remain poorly understood.

In the present study, we have generated mice lacking both mDia1 and mDia3, the two isoforms of mDia expressed in the neuroepithelial cells, and have found formation of aberrant periventricular dysplastic mass associated with the hydrocephalus in these mice. The apical actin belt and adherens junction of neuroepithelial cells in the region of periventricular dysplastic mass were disorganized, and the associated apical-basal polarity was lost. In addition, distortion of apical adherens junction, bulging of apical membrane into ventricular space and shrinkage of neuroepithelial cells were widely observed in the remaining region of the ventricular wall throughout the developing brain. Adherens junction components such as N-cadherin and αE-catenin concentrated on the apical surface were previously reported to be involved in the regulation of neuroepithelium structural integrity and apical-basal polarity, and their deletion was found to cause ectopic mass in ventricular space similar to the periventricular dysplastic mass observed in this study [6], [7]. It is known that the filamentous actin belt is associated with such apical adherens junctions in neuroepithelial cells. However, little is known as to how this actin belt is formed, maintained and linked to them. Presumably, the actin belt is organized by the actions of several classes of molecules involved in regulation of actin filament structure, including actin nucleators, actin cross-linking proteins and actin severing proteins [23]. Notably, mutations of filamin-A, an actin cross-linking protein, cause periventricular heterotopia (PVH) in humans [24]. However, what molecules are responsible for de novo actin filament formation in neuroepithelial cells is not known. mDia is one of the two major actin nucleators in mammals and known to promote formation of straight filaments [14]. Previous studies utilizing cultured epithelial cells in vitro have suggested the possible involvement of mDia1-dependent actin polymerization in the formation and maintenance of the adherens junctions [25]–[27]. However, the role of mDia in adherens junction regulation in the intact body of mammals has not been shown. The present study has utilized mice deficient in mDia and revealed a critical role of mDia in the maintenance of the adherens junction in vivo. This study has further shown that this mDia mechanism functions in the specialized structure of the neuroepithelium, the apical adherens junction and the apical actin belt. Consistently, mDia deficiency induces an ectopic mass into the ventricle but the overall cortical structure in mDia-DKO brain is apparently normal in contrast to the N-cadherin and αE-catenin knockout mice that show a severely disorganized cortex [6], [7]. Thus, loss of mDia impairs specifically the apical surface of the neuroepithelium.