The role of actomyosin contractility in zebrafish neural tube formation through hollowing.

The neural tube is an epithelial tube that forms the embryonic brain and spinal cord through the process of neurulation. In amniotes, it forms in two different ways: in the anterior region, primary neurulation occurs through the folding of an already polarised epithelial sheet; and posteriorly, secondary neurulation occurs through de novo apical-basal cell polarisation in the centre of a solid rod and the tube opens through hollowing. The zebrafish hindbrain forms via a secondary-like mechanism, making it an ideal in vivo model to study hollowing lumen formation. While the role of actomyosin contractility is well understood in primary neurulation, for example through constriction of apical-lateral junctions to mediate neural plate bending, its role in secondary neurulation is less clear. In this thesis, I investigated the role of actomyosin contractility over the course of zebrafish neurulation to understand its role in lumen inflation. To explore where forces are active during zebrafish neural tube lumen opening, I conducted live imaging and collaborated with the Cambridge Advanced Imaging Centre to generate 3D segmentations of the lumen and surrounding tissue over time. These quantifications were then used to build a 2D vertex model of a cross section of the lumen in collaboration with the Richards Lab at the University of Exeter to investigate the force balance that could lead to proper lumen opening. The live imaging showed that an increase in lumen occupancy over time is accompanied by an increase in apical surface area, tissue volume, and apical-basal tissue width. The model predicted that the observed lumen growth and triangular-shaped lumen could be explained by tissue and lumen expansion, and the presence of higher apical tension compared to basal tension in the cells. This suggests that a balance of hydrostatic pressure and apical tension is important during lumen opening. I next investigated the role of actomyosin contractility in generating this force balance by inhibiting non-muscle myosin II (NMYII) contractility with the drug Blebbistatin over the course of cell polarisation, apical surface generation, and lumen inflation. This resulted in a narrower lumen, collapsed tissue shape, and buckled apical surface. The apical surface buckled at hinge points which suggests a conservation of passive hinge point formation in different modes of neurulation. Testing the consequences of changing the balance of the lumen volume (to represent hydrostatic pressure) and apical surface area (to represent apical tension) in the 2D vertex model led to a buckled apical surface, suggesting that NMYII is involved in regulating the balance between hydrostatic pressure and apical surface tension. I then compared the size, shape, and barrier function of apical-lateral junctions in control and Blebbistatin-treated embryos to determine how NMYII regulates hydrostatic pressure or apical surface tension. The geometry and size of apical rings can reflect epithelial tension. However, my measurements showed that the apical rings in Blebbistatin-treated embryos were very slightly smaller than controls, with no significant shape differences. This suggests that there is no biologically relevant difference in the apical surface tension even though the lumen size is significantly reduced. I tested the junctional barrier function through a neuroepithelial permeability assay where fluorescent dye was injected into the neural tube lumen and the amount of leakage through the tissue was measured. I found that Blebbistatin treatment significantly increased the amount of dye leakage from the neural tube lumen, suggesting that the barrier function is impaired, which could lead to a reduction in hydrostatic pressure inside the lumen. This demonstrates that NMYII contractility is necessary to regulate hydrostatic pressure via a role in barrier function. However, quantifying Zona Occludens 1 fluorescence and continuity showed no overt physical defects in tight junctions, suggesting an alternative mechanism for NMYII-dependent hydrostatic pressure modulation. Therefore, I next investigated the localisation of active NMYII on the apical surface. I stained for phosphorylated NMYII, which in neural tubes that form through primary neurulation is mostly found at the apical-lateral junctions facing the neural tube lumen. Surprisingly, I found that the predominant localisation was in bright puncta within the middle of the apical rings, associated with basal bodies of primary cilia. This unexpected localisation of active NMYII then led me to investigate whether cilia could be involved in the lumen inflation defect seen when NMYII contractility is inhibited. Through immunofluorescence of cilia proteins, I observed a fragmentation of basal bodies when NMYII contractility was inhibited. As well as this, independent of NMYII contractility perturbation, a zebrafish mutant for IFT88 (a gene involved in cilium biogenesis) had significantly smaller neural tube lumens than wildtype. Together, this suggests a novel, NMYII-dependent role for cilia in regulating lumen inflation. Whilst there is some literature linking primary cilia function to the mechanosensation of fluid flow and regulation of barrier function in endothelial vessels, the role of primary cilia as mechanosensors has been controversial. Cilium-mediated mechanotransduction within the neural tube is so far unexplored and could represent an additional mechanosensory system within inflating tubes, alongside mechanotransduction at the apical junctions. This work therefore lays the groundwork for exciting new research into the interrelationship between actomyosin contractility, cilia mechanotransduction, and epithelial tube lumen inflation.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Race, Amelia

Advisors dc:contributor.advisor

• Buckley, Clare
• Franze, Kristian

• zebrafish
• neurulation
• cilia
• lumenogenesis
• mechanotransduction
• blebbistatin
• actomyosin contractility

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/950cb1b4-933d-4240-93ef-e3803b1f7679/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.117332

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/382573