Abstract
Secondary neurulation (SN) is the process by which the neural tube (NT) extends caudally in the vertebrate embryo, forming the developmental precursor to the caudal spinal cord. Defective SN is implicated in closed neural tube defects, and the process of SN is thought to proceed in a similar fashion to spinal cord regeneration in amphibians. Despite its importance, the mechanisms by which SN is controlled genetically are poorly understood. This research aimed to uncover the genetic mechanisms underpinning SN by investigating four candidate genes: crabp2, fgf20, inhba.L, and st8sia6.S. CRISPR gene editing was used to knock down these genes in Xenopus laevis embryos, and HCR RNA-FISH was used to analyze their expression patterns. CRISPR knockdown was inconclusive in determining the role of fgf20.L/S in SN, as the knockdown of this gene resulted in an early defect in gastrulation. CRISPR knockdown of crabp2.L/S caused truncation of the tail, suggesting this gene may be involved in SN, a conclusion supported by the expression of crabp2.L/S in NT precursor tissues. Knockdown of inhba.L did not cause NT defects, however inhba.L was found to be expressed in the NT and its precursor tissues, suggesting that this gene may be involved in, but not required for, SN. Additionally, inhba.L knockdown resulted in a delay in tail regeneration, suggesting inhba.L may be required for NT regeneration. Knockdown of st8sia6.S did not cause NT defects, suggesting this gene is not required for SN, a conclusion supported by HCR RNA-FISH which did not find st8sia6.S expression in the NT. However, st8sia6.S knockdown resulted in dorsally biased tail regeneration, possibly due to delayed regeneration of the NT, suggesting st8sia6.S may be involved in NT regeneration. Overall, these results suggest that SN may involve the genes crabp2.L/S and inhba.L, and does not involve st8sia6.S, while no conclusions could be drawn regarding the involvement of fgf20.L/S. These findings inform on the molecular mechanisms underpinning SN, potentially relevant to developing human spinal cord regenerative therapies and enhancing our understanding of closed neural tube disorders.