Rest-Mediated Regulation of Extracellular Matrix Is Crucial for Neural Development

et al. (2008) Rest-Mediated Regulation of Extracellular Matrix Is Crucial for Neural Development. PLoS ONE 3(11): e3656. doi:10.1371/journal.pone.0003656 Rest-Mediated Regulation of Extracellular Matrix Is Crucial for Neural Development Yuh-Man Sun 0 Megan Cooper 0 Sophie Finch 0 Hsuan-Hwai Lin 0 Zhou-Feng Chen 0 Brenda P. Williams 0 Noel J. Buckley 0 Patrick Callaerts, Katholieke Universiteit Leuven, Belgium 0 1 Centre for the Cellular Basis of Behaviour (CCBB), The James Black Centre, Institute of Psychiatry, King's College London , London , United Kingdom , 2 Departments of Anesthesiology, Psychiatry, and Developmental Biology, Washington University School of Medicine Pain Center , Saint Louis, Missouri , United States of America Neural development from blastocysts is strictly controlled by intricate transcriptional programmes that initiate the downregulation of pluripotent genes, Oct4, Nanog and Rex1 in blastocysts followed by up-regulation of lineage-specific genes as neural development proceeds. Here, we demonstrate that the expression pattern of the transcription factor Rest mirrors those of pluripotent genes during neural development from embryonic stem (ES) cells and an early abrogation of Rest in ES cells using a combination of gene targeting and RNAi approaches causes defects in this process. Specifically, Rest ablation does not alter ES cell pluripotency, but impedes the production of Nestin+ neural stem cells, neural progenitor cells and neurons, and results in defective adhesion, decrease in cell proliferation, increase in cell death and neuronal phenotypic defects typified by a reduction in migration and neurite elaboration. We also show that these Rest-null phenotypes are due to the dysregulation of its direct or indirect target genes, Lama1, Lamb1, Lamc1 and Lama2 and that these aberrant phenotypes can be rescued by laminins. - . These authors contributed equally to this work. During mouse embryo development, the blastocyst differentiates into pluripotent primitive ectoderm and gives rise to a structure known as the epiblast [1]. The epiblast responds to extrinsic signals and generates three primary germ layers (ectoderm, mesoderm and endoderm) [2]. During neurulation, the ectoderm gives rise to the neuroectoderm in the form of a neural plate, which subsequently folds to generate the neural tube, composed of a single layer of neuroepithelial cells or neural stem cells (NSCs), where a series of ring-like constrictions mark the boundaries between the primordia of the major brain regions [34]. This process of neural development is orchestrated and accompanied by wholesale changes in transcriptional programmes and patterns of gene expression. However, due to the difficulties in accessing and manipulating early embryos, the transcriptional network that regulates neural development is poorly understood, especially in mammals. Embryonic stem (ES) cells derived from blastocysts retain the ability to recapitulate neural development in vitro, and offer an invaluable model to study early events in embryogenesis. The RE1 Silencing Transcription Factor / Neuron Restrictive Silencer Factor (Rest/Nrsf) is a zinc finger transcription repressor that has been postulated to act as a master regulator of neuronal gene expression in both the developing and mature nervous systems [56]. We and others have shown that Rest is highly expressed in blastocysts and ES cells, but that expression decreases as neural development proceeds [78]. In fact, downregulation of Rest has been proposed to be obligate for differentiation of neural progenitors [8] and more recently, it has been proposed that Rest haplodeficiency results in loss of pluripotency markers and a reciprocal gain in differentiation markers [9]. Taken together, these observations suggest that Rest may play a crucial role at several stages of neural development. Here, we determine the function of Rest during neural development from ES cells through NSCs and neural progenitor cells (NPCs) to mature neurons using an in vitro ES cell-derived neural differentiation model. Rest exerts its function by binding to both canonical and noncanonical RE1-sites identified at over 2000 loci in the mammalian genome [1011] and is implicated in the regulation of both coding and non-coding genes [10,12], many of which represent neuronspecific transcriptional units. The observation that many of these target genes are expressed by differentiated neurons, including ion channels, neurotransmitter receptors, neurotrophins, synaptic vesicle associated proteins, cell adhesion molecules, growth-associated and cytoskeletal proteins, gave rise to the initial perception that Rest acted as a silencer of neuron-specific genes in NPCs and non-neural cells to prevent precocious expression of neuronal characteristics. However, recent studies emerge that Rest has more versatile roles and can regulate its target genes either by activation, repression or silencing, depending upon the developmental stage and cell type [7,13]. Rest recruits multiple cofactors, histone modifying and chromatin remodelling activities, all of which underwrite the complexity of Rest activity [1416]. The diverse roles of Rest have been shown in both neural and non-neural pathologies including Huntingtons disease, cardiac hypertrophy, medulloblastoma, malignant rhabdoid tumor, small cell lung cancer, ovarian cancer, and ischemia (see review for references [13]). Despite the wealth of knowledge in identifying target genes [10 12] and in delineating the mechanistic actions of Rest [1416], the biological function of Rest during neural development remains unclear. Rest2/2 mice die around embryonic day (E)11.5, with embryo degeneration, neural tube malformations and widespread apoptosis evident from E9.5 [6]. Constitutive expression of Rest in chick spinal cord does not cause defects in neurogenesis but does result in axon pathfinding errors [17]. However, in Xenopus, disruption of Rest function disturbs ectoderm patterning and expands the neural plate [18], suggesting that Rest is indeed required for normal neural plate formation and neurogenesis. Collectively, these studies paint a somewhat ambiguous picture of the role of Rest in the development of NSCs and neurons. We have sought to address this issue by using a combination of gene targeting and RNAi to create ES lines expressing a range of Rest concentrations, which we have used to investigate the effect of Rest deficiency during ES cell-derived neural development. Importantly, in contrast to a recent study [9], we find that deletion of a single Rest allele does not result in any change in neural differentiation. Instead, we find that Rest levels have to be decreased by more than 92% to precipitate any phenotype. Rest ablation impairs the extracellular matrix (ECM) components and impedes the production of Nestin+ NSCs, NPCs and neurons. Furthermore, neurons derived from REST-null ES cells are devoid of elaborate processes, (...truncated)