Neural convergence (NC) is the evolutionary conserved mechanism that narrows the neural plate (NP) enabling neural tube (NT) closure. Failure of this critical developmental process contributes to neural tube defects (NTDs), which occur in 1/1000 live births. Among the many candidate genes identified in model organisms that mediate NC, only those implicated in the planar cell polarity (PCP) pathway have so far been implicated in the etiology of human NTDs.
This study investigates the role of the Repulsive Guidance Molecule A (RGMa) and Neogenin signaling as regulators of NC in the zebrafish NP. RGMa is chemorepulsive to emerging retinal ganglion cell axons and prohibitive to the regeneration of damaged nerves. However, evidence from mouse, frog, and zebrafish suggest that RGMa/Neogenin signaling is required for NT formation. Although this latter function is poorly understood, the Cooper lab determined that RGMa signaling directly affected NT closure, the latest stage of tube formation. Absent from the literature is an understanding of the role of RGMa/Neogenin signaling in the earliest, most conserved stage of anterior NT development, NC, without which adequate NT closure is improbable due increased NP tissue width. We found that insufficient ligand or receptor similarly disrupted NC. While control embryos underwent adequate NP narrowing, RGMa and Neogenin-deficient embryos had widened NPs at the same somitic stage. To determine the cellular behaviors contributing to this NC defect, we examined movies of hindbrain NP cells in vivo from control and loss-of-function (LOF) embryos. Our quantitative data show that control embryos extend membranous protrusions toward the midline and lateral surface of the tissue. However, both RGMa and Neogenin LOF randomized the protrusiveness of NP cell membranes. Cells deficient in this pathway were also rounded instead of polarized perpendicular to the midline. The lack of polarization in these embryos is reminiscent of defects observed when microtubule (MT) stability/organization is disrupted. Therefore, we investigated the requirement of RGMa and Neogenin for effective MT organization. As we have reported previously, NC in the zebrafish involves rearrangement of the MT cytoskeleton into a progressively linear array perpendicular or oblique to the midline. In this study, control NP cells were found to have archetypical MT organization. However, RGMa and Neogenin LOF severely shortened the length of MT arrays. These observations suggest that MTs may be downstream targets of RGMa/Neogenin signaling to promote convergence of the NP and thus proper NT formation. Ongoing work investigates the connection between RGMa/Neogenin and cadherin-based cell adhesion to mediate cell behavior essential to NC. .