SummaryThe GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

The tremendous complexity of the adult forebrain makes it a challenging task to elucidate how this structure forms during embryonic development. Nevertheless, we are beginning to understand how a simple epithelial sheet of ectoderm gives rise to the labyrinthine network of cells that constitutes the functional forebrain. Here, we discuss early events in forebrain development--those that lead to the establishment of the anterior neural plate and the regional subdivision of this territory into the different domains of the prospective forebrain.

Cells at the anterior boundary of the neural plate (ANB) can induce telencephalic gene expression when transplanted to more posterior regions. Here, we identify a secreted Frizzled-related Wnt antagonist, Tlc, that is expressed in ANB cells and can cell nonautonomously promote telencephalic gene expression in a concentration-dependent manner. Moreover, abrogation of Tlc function compromises telencephalic development. We also identify Wnt8b as a locally acting modulator of regional fate in the anterior neural plate and a likely target for antagonism by Tlc. Finally, we show that tlc expression is regulated by signals that establish early antero-posterior and dorso-ventral ectodermal pattern. From these studies, we propose that local antagonism of Wnt activity within the anterior ectoderm is required to establish the telencephalon.

Zebrafish embryos homozygous for the masterblind (mbl) mutation exhibit a striking phenotype in which the eyes and telencephalon are reduced or absent and diencephalic fates expand to the front of the brain. Here we show that mbl −/− embryos carry an amino-acid change at a conserved site in the Wnt pathway scaffolding protein, Axin1. The amino-acid substitution present in the mbl allele abolishes the binding of Axin to Gsk3 and affects Tcf-dependent transcription. Therefore, Gsk3 activity may be decreased in mbl −/− embryos and in support of this possibility, overexpression of either wild-type Axin1 or Gsk3␤ can restore eye and telencephalic fates to mbl −/− embryos. Our data reveal a crucial role for Axin1-dependent inhibition of the Wnt pathway in the early regional subdivision of the anterior neural plate into telencephalic, diencephalic, and eye-forming territories.

During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. We show that two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/beta-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/beta-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results allow us to postulate a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells.