How do mechanics and signalling interact during neural tube inflation?

Context and challenge: The neural tube is the precursor to the brain and spinal cord. Congenital neural tube defects are prevalent in humans (>1/1000 births). Designing prevention strategies is complicated by the many different mechanisms that must occur at a subcellular level to control this tissue-scale event. Adding to this complexity, the neural tube forms via different mechanisms along its length. For example, most of the human neural tube forms via folding a sheet of cells and subsequent inflation (primary neurulation). One important subcellular mechanism behind this process is mechanical contractility at the belt of cell-cell junctions that connect cells. This bends the tissue into shape. Contractility is driven by a protein complex called actomyosin. However, the base of the spinal neural tube forms via inflation of a fluid-filled cavity within an initially solid tissue, from the inside out (hollowing neurulation). Although this mechanism of cavity inflation has relevance to the development of many structures (kidney, gut, early embryo cavities), the complex interrelationship between cell mechanics and chemical signalling has prevented a full understanding of the cell biology behind this process. Using zebrafish embryos as a tractable model of hollowing neurulation, we recently discovered that actomyosin contractility is required for neural tube inflation. However, instead of acting at cell-cell junctions (as in primary neurulation), actomyosin is active at the base of small structures called primary cilia, where it appears to modulate their function. Acting like ‘antennae’ for the cell, cilia project into the fluid-filled cavity and integrate chemical and mechanical information, translating this into signals within the cell. Interestingly, we found that both zebrafish with defective cilia and zebrafish with defective actomyosin contractility had similar inflation defects. Together, this suggests that actomyosin is playing a role in regulating the pressure within the inflating neural tube, via a role in cilium function. Aims: We aim to uncover the mechanisms linking cell mechanics and cell signalling during inflation of the vertebrate neural tube. Objectives: To unpick the interrelationship between actomyosin contractility, cilium signalling and lumen inflation, we propose an optogenetic approach developed in the lab, which will allow us to specifically manipulate actomyosin contractility at cilia in response to light. We will also use light-activated approaches to manipulate cilia signalling with precise temporal control and will modulate the pressure within the neural tube cavity via microinjection. Using fluorescently labelled sensors for cilia and for organ structure and signalling components, we will carry out high resolution imaging within live embryos. This will allow us to assess the effects of these manipulations from the subcellular to the tissue scale in real time. Using this approach, we will determine how A) actomyosin regulates cilium structure and function, B) ciliary signalling regulates tube inflation, C) cilia transduce mechanical signals. Applications and benefits: Together, this research will 1) further understanding of how early organs form, 2) uncover links between cell mechanics, cell signalling and the shaping of animals, 3) uncover new roles for actomyosin and cilia in cell biology and signalling. These applications and benefits have relevance to both animal development and disease. Relevance to BBSRC: As well as furthering our understanding of the important and disease-relevant developmental process of neurulation (BBSRC objective 3.2 ‘understanding the rules of life’), this proposal will further develop cutting-edge optogenetic approaches within a live vertebrate (BBSRC objective 5.1 transformative technologies).