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The molecular and cellular logic of vertebrate neural development

Periodic Reporting for period 2 - LogNeuroDev (The molecular and cellular logic of vertebrate neural development)

Reporting period: 2019-07-01 to 2020-12-31

How are the right types of cells produced in the right place, at the right time and in the right amounts in an embryo? We study these questions in the central nervous system (CNS). Despite its complexity, the CNS is assembled in a remarkably precise and reliable manner. This precision is necessary for the wiring of nerves into the functional neural circuits that gives the CNS its function. Our research focuses on the spinal cord, which is the part of the CNS that contains the nerves that allow us to sense our environment and respond to it by moving muscles. Our goal is to identify the genes involved in spinal cord development and determine how they work to produce and organize the different types of nerve cells found in this part of the CNS. This will contribute to understanding the development of the spinal cord as well as shed light on diseased and damaged nervous systems.

A central problem in biology and key to realising the potential of regenerative medicine is understanding the mechanisms that produce and organize cells in the complex tissues of an embryo. In broad terms, initially uncommitted cells acquire their identity in response to signals that control gene activity programmes. How these programmes are established and cell fate decisions implemented is poorly understood. An example of this is the formation of different neuron types in the vertebrate spinal cord. We take an interdisciplinary approach that combines our in vivo expertise in vertebrate embryology with three recent advances in our group. First, we have developed in vitro differentiation systems that use embryonic stem cells to recapitulate development processes. Second, we have embraced new technologies that provide unprecedented ability to manipulate and assay single cells. Finally, we have established interdisciplinary collaborations to develop computational tools and construct data driven mathematical models. Using these approaches, alongside established embryological methods, we are establishing a platform for manipulating and analysing mechanisms by which the cells that form the spinal cord acquire specific identities. We aim to identify the rules by which cells make decisions and to define the logic that leads to distinct cell fate choices. Together these approaches will provide the knowledge and technical foundations for rational, predictive tissue engineering of the spinal cord.
We have previous developed mathematical models of the process of neural tube patterning. To test predictions of these models we have used genome engineering to introduce targeted alterations to a regulatory element controlling a key component of on the genes encoding a component of the neural tube regulatory network. We tested the effect of this in ESCs and in mutant mice. These results revealed a major and surprising role for the regulatory network in determining the precision of tissue patterning. This is leading to new mathematical modelling work and further predictions that we are now pursuing. A report of this work is available (BioRxiv doi.org/10.1101/721043).

We have taken an unbiased approach to extend our knowledge of the gene regulatory programmes involved in neural tube development. Using single cell mRNA sequencing technology we have profiled the transcriptome of cells in the developing mouse neural tube between embryonic days 9.5-13.5. We confirmed that the data accurately recapitulates neural tube development and identified new components of the transcriptional network. In addition, the analysis highlighted a previously under-appreciated temporal component to the mechanisms that generate neuronal diversity. This is offering new insight into the mechanisms that are responsible for neuronal specification and a catalogue of gene expression for classifying spinal cord cell types that will support our future studies of neural tube development. This work is published (Development DOI: 10.1242/dev.173807).

We have also described a tissue scale model of cell behaviour in the neural tube (Development DOI: 10.1242/dev.176297). We used experimental measurements to develop a model based on cell mechanics of the apical surface of the neuroepithelium that incorporates inter-kinetic nuclear movement and spatially varying rates of neuronal differentiation. Simulations predicted that tissue growth and the shape of lineage-related clones of cells differ with the rate of differentiation. These predictions were consistent with experimental observations. The absence of directional signalling in the simulations indicates that global mechanical constraints are sufficient to explain the observed differences in anisotropy. This provides insight into how the tissue growth rate affects cell dynamics and growth anisotropy and opens up possibilities to study the coupling between mechanics, pattern formation and growth in the neural tube. We are now building on this model to incorporate models of molecular signalling and gene regulation.

We have also continued to develop mathematical tools to help analyse the dynamics of gene regulatory networks, in particular extending some techniques from statistical physics for use for the type of problems we study (arXiv:2005.04751). We have also recently uncovered evidence that properties such as how stable proteins are affects the tempo of embryonic development and may explain species specific pace of neural development (BioRxiv: doi.org/10.1101/2019.12.29.889543).
Our goals remain to understand how the gene regulatory network in the spinal cord controls neuronal subtype identity and how these mechanisms affect the dynamics and formation of reproducible pattern. We wish to decipher how cells in developing tissue decide between alternative fates and select appropriate identities to result in well patterned and proportioned tissues. We will continue to develop in silico and in vitro models that accurately recapitulate in vivo development in order to facilitate tissue engineering of the muscloskeletal system. In the next period we anticipate making progress on imaging pattern formation and signaling in our in vitro systems. We will use these, together with our existing methods, to examine the changes in gene expression in cells as cells transition between states. Combining gene induction assays with perturbations of specific signaling pathways will allow us to establish epistasis relationships between the factors controlling the induction of spinal cord identity and we are working with methods that allow us to reconstructed the changes in the genomic landscape as cells transition between states. We will also compare and contrast the dynamics of mouse and human spinal cord patterning and test whether changes in the dynamics of morphogen signaling; changes in genomic regulatory element sensitivity; changes in the stability or expression rates of genes accounts for the observed species differences.

The control of cell fate in developing embryos is fundamentally a dynamic process that depends on the architecture and operation of transcriptional networks. Conventional developmental genetics has been successful at identifying components of these networks The approaches we are taking will provide the quantitative and conceptual understanding of how these networks operate and offer insight into pathway architecture, predict responses to diverse perturbations, and ultimately allow re-engineering for applications in tissue engineering and regenerative medicine.
Microscopy imaging of the developing neural tube