T-type Calcium Channel Regulation of Neural Tube Closure and EphrinA/EPHA Expression

Abstract

A major class of human birth defects arise from aberrations during neural tube closure (NTC). We report on a NTC signaling pathway requiring T-type calcium channels (TTCC) that is conserved between primitive chordates (Ciona) and Xenopus. With loss of TTCCs there is a failure to seal the anterior neural folds. Accompanying loss of TTCCs is an upregulation of EphrinA effectors. Ephrin signaling is known to be important in NTC, and ephrins can affect both cell adhesion and repulsion. In Ciona, ephrinA-d expression is down regulated at the end of neurulation, while with loss of TTCC ephrinA-d remains elevated. Accordingly, overexpression of ephrinA-d phenocopied TTCC loss of function, while overexpression of a dominant negative Ephrin receptor was able to rescue NTC in a Ciona TTCC mutant. We hypothesize that signaling through TTCCs is necessary for proper anterior NTC through down regulation of ephrins, and possibly elimination of a repulsive signal.

Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona

“…Large deletions of up to 23 kb of intervening DNA resulting from NHEJ between two CRISPR/Cas9-induced DSBs have been reported in Ciona (Abdul-Wajid et al 2015). For functional analyses of protein-coding genes, such deletions would more likely produce null mutations than small deletions resulting from the action of lone sgRNAs.…”

“…By using developmentally regulated cis -regulatory elements to drive Cas9 expression in specific cell lineages or tissue types, we were thus able to control the disruption of Ebf function with spatiotemporal precision. Following this proof-of-principle, tissue-specific CRISPR/Cas9 has rapidly propagated as a simple yet powerful tool to elucidate gene function in the Ciona embryo (Abdul-Wajid et al 2015; Cota and Davidson 2015; Segade et al 2016; Tolkin and Christiaen 2016). …”

ACAM, a novel member of the neural IgCAM family, mediates anterior neural tube closure in a primitive chordate

“…The OTX dominant negative construct (OTX∷EnR) (Haeussler et al, 2010) was amplified and cloned into pCR8/GW/TOPO and recombined into ETR>RfA (Abdul-Wajid et al, 2015). …”

CRISPR Knockouts in Ciona Embryos

“…This increases the odds of generating at least one out-of-frame indel, and the large deletions spanning multiple targets have been consistently observed in Ciona embryos ( Gandhi et al 2016), the largest deletion reported being ~13 kb ( Abdul-Wajid et al 2015). …”

“…However, a chimeric “single-guide RNA” (sgRNA) is sufficient to mimic the roles of these two components ( Jinek et al 2012). This small but profound improvement has helped launch CRISPR/Cas9 as a cheap, simple, and efficient system for targeted mutagenesis in a remarkably wide variety of organisms ( Perry and Henry 2015; Iaffaldano et al 2016; Long et al 2016; Nomura et al 2016; Nymark et al 2016; Tian et al 2016), as well as in tunicates ( Sasaki et al 2014; Stolfi et al 2014; Abdul-Wajid et al 2015; Cota and Davidson 2015; Gandhi et al 2016; Segade et al 2016; Tolkin and Christiaen 2016). …”