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 forebrain is patterned along the dorsoventral (DV) axis by Sonic Hedgehog (Shh). However, previous studies have suggested the presence of an Shh-independent mechanism. Our study identifies Wnt/beta-catenin-activated from the telencephalic roof-as an Shh-independent pathway that is essential for telencephalic pallial (dorsal) specification during neurulation. We demonstrate that the transcription factor Foxg1 coordinates the activity of two signaling centers: Foxg1 is a key downstream effector of the Shh pathway during induction of subpallial (ventral) identity, and it inhibits Wnt/beta-catenin signaling through direct transcriptional repression of Wnt ligands. This inhibition restricts the dorsal Wnt signaling center to the roof plate and consequently limits pallial identities. Concomitantly to these roles, Foxg1 controls the formation of the compartment boundary between telencephalon and basal diencephalon. Altogether, these findings identify a key direct target of Foxg1, and uncover a simple molecular mechanism by which Foxg1 integrates two opposing signaling centers.

The Hox paralogous group 1 (PG1) genes are the first and initially most anterior Hox genes expressed in the embryo. In Xenopus, the three PG1 genes, Hoxa1, Hoxb1 and Hoxd1, are expressed in a widely overlapping domain, which includes the region of the future hindbrain and its associated neural crest. We used morpholinos to achieve a complete knockdown of PG1 function. When Hoxa1, Hoxb1 and Hoxd1 are knocked down in combination, the hindbrain patterning phenotype is more severe than in the single or double knockdowns, indicating a degree of redundancy for these genes. In the triple PG1 knockdown embryos the hindbrain is reduced and lacks segmentation. The patterning of rhombomeres 2 to 7 is lost, with a concurrent posterior expansion of the rhombomere 1 marker, Gbx2. This effect could be via the downregulation of other Hox genes, as we show that PG1 function is necessary for the hindbrain expression of Hox genes from paralogous groups 2 to 4. Furthermore, in the absence of PG1 function, the cranial neural crest is correctly specified but does not migrate into the pharyngeal arches. Embryos with no active PG1 genes have defects in derivatives of the pharyngeal arches and, most strikingly, the gill cartilages are completely missing. These results show that the complete abrogation of PG1 function in Xenopus has a much wider scope of effect than would be predicted from the single and double PG1 knockouts in other organisms.

The telencephalon is the most complex brain region, controlling communication, emotion, movement and memory. Its adult derivatives develop from the dorsal pallium and ventral subpallium. Despite knowledge of genes required in these territories, we do not understand how evolution has shaped telencephalon diversity. Here, using rock-and sand-dwelling cichlid fishes from Lake Malawi, we demonstrate that differences in strength and timing of opposing Hedgehog and Wingless signals establish evolutionary divergence in dorsal-ventral telencephalon patterning. Rock dwellers exhibit early, extensive Hedgehog activity in the ventral forebrain resulting in expression of foxg1 before dorsal Wingless signals, and a larger subpallium. Sand dwellers show rapid deployment of Wingless, later foxg1 expression and a larger pallium. Manipulation of the Hedgehog and Wingless pathways in cichlid and zebrafish embryos is sufficient to mimic differences between rock-versus sand-dweller brains. Our data suggest that competing ventral Hedgehog and dorsal Wingless signals mediate evolutionary diversification of the telencephalon.