Neurulation is a complex and multi-step process that gives rise to the chordate central nervous system. Problems in neurulation are the source of some of the most common human birth defects. In this renewal application we are proposing to build on and extend findings from the current funding period that bring to light previously unknown gene functions in neural plate induction and neural tube closure. These discoveries were made using a novel forward genetic screen in the primitive chordate Ciona. The mutant line frimousse (frm) revealed a requirement for connexins (the subunit components of gap junctions) in maintenance of neural induction. In the absence of Ciona connexin-11, anterior neural plate derivatives are initially induced but then lose specification and take on an epidermal fate. A different mutant line, bugeye (bug), revealed a requirement for T-type Ca2+ channels in neural tube closure (NTC). In the absence of the T-type Ca2+ channel CAV3, neural induction and posterior NTC proceed normally, but the anterior neuropore fails to close completely and ultimately reopens, exposing the anterior brain. Knockdown of the orthologous gene product in Xenopus (CAV3.2) gave a nearly identical phenotype, indicating that the CAV3 requirement in NTC is ancient in the chordates. Other findings on bug point to a failure to properly down-regulate ephrinA signaling as a primary cause of the bug phenotype. Not only is ephrinA-d upregulated in the bug mutant, inhibition of EphrinA signaling with a dominant ephrin receptor (Eph3) rescues the bug phenotype. Proposed experiments will investigate the mechanism of CAV3 in NTC. We hypothesize that CAV3 plays a role in monitoring the morphogenetic events of NTC. Our present results also indicate the CAV3 operates by regulating levels of specific transcripts.
In Specific Aim 1, we will use RNA profiling to identify differentially expressed (DE) transcripts in bug versus wild type embryos. These DE transcripts will form the basis of investigation of transcriptional and post-transcriptional regulatory mechanisms. To test whether CAV3 signaling is linked to the events of NTC, rather than operating by autonomous program, we will disrupt closure and test for effects on Ca2+ transients in the neural tube and on target transcripts, including ephrinA-d.
In Specific Aim 2 we will continue to investigate the role of CAV3.2 in vertebrate NTC, and as an initial step we will work with the National Xenopus Resource to make a stable CAV3.2 knockout line. This knockout line will provide a tool for exploring, among other things, changes in cell adhesion in the closing anterior neuropore.
In Specific Aim 3, we will identify connexin genes expressed in the Xenopus neural plate and systematically knock them down to determine if the connexin requirement revealed by the frm mutant is conserved between higher and lower chordates. Finally we will continue to screen for new and informative mutants (Specific Aim 4).
Neural tube closure defects, such as spina bifida, are among the most common human birth defects. This proposal will use animal models to investigate two newly discovered molecular regulators of central nervous system neural development and closure.
Showing the most recent 10 out of 26 publications
