Ulrika Marklund

The enteric nervous system (ENS) contains an extensive range of neural subtypes that collectively control essential gut functions largely independent of the central nervous system (CNS). Enteric neuronal diversity is critical for maintaining normal gut function, and a selective dysfunction or local neuronal loss leads to gastrointestinal (GI) disorders. As current treatments of ENS-related disorders are suboptimal, development of novel regenerative approaches has the potential to significantly improve GI health for millions worldwide. As a first milestone towards cell-based regenerative therapies, the Marklund lab recently established a molecular classification of intestinal enteric neurons and discovered that they diversify through a conceptually new stepwise principle during development. The proposed project, DeSTENy, will significantly expand on these discoveries, and investigate how time and spatial context of developing GI tract controls the developing ENS to acquire the appropriate neuron identities at the right time and region within the mouse gut. DeSTENy will integrate cutting-edge regular and spatial single-cell transcriptomics to identify potentially important spatiotemporal driver genes in the ENS of the intestine versus stomach and oesophagus (upper GI-tract). While studies of the developing ENS has been challenged by its inaccessible location within the gut wall, DeSTENy will overcome this limitation by the utilization a precise, rapid and large-scale gene manipulation strategy based on ultrasound-guided transduction of ENS precursor cells followed by temporally controlled gene expression. Apart from transforming our understanding of the largest division of the peripheral nervous system and fundamental developmental processes, DeSTENy will open avenues for regenerative medicine in treating neurological gut disorders, in particular the many affecting the upper GI-tract.

Inflammatory Bowel Disease (IBD) encompasses two gastrointestinal conditions - Crohn’s disease and ulcerative colitis - which currently lack satisfactory treatment. IBD is associated with alterations to the enteric nervous system (ENS) of the gut, including the formation of new neurons (‘neurogenesis’) and changes in the properties of existing neurons (‘neuroplasticity’). Growing evidence suggests an interplay between acute inflammation and ENS pathology. Despite this, detailed characterisation of ENS alterations, their underlying molecular mechanisms, and how inflammation may induce them during IBD, is not well understood. The Marklund laboratory has recently uncovered a stepwise diversification model of enteric neurogenesis, suggesting that during embryonic development, subtype identities are specified through identity conversions in post-mitotic neurons. This work leads to the intriguing possibility that the identity of terminally-differentiated enteric neurons may, in fact, be flexible. However, whether stepwise diversification is present during adult inflammation-induced neurogenesis, and whether neuronal identity can be altered by extrinsic stimuli such as inflammation, remains to be assessed. Using a colitis model of IBD, single cell RNA sequencing of ENS and immune cells and spatial transcriptomics, NeurogENSity will characterise the composition of the inflamed ENS, revealing the cellular changes occurring as a result of acute inflammation. Pseudotime trajectory analysis, in combination with lineage tracing experiments, will address the nature of adult enteric neuronal identity specification. ENS-immune interactions which may underlie neurogenic and neuroplastic alterations will be identified, and their relevance assessed using cell depletion assays. Together, NeurogENSity will identify mechanisms underlying inflammation-induced alterations to the ENS, paving the way for targeted treatments of ENS dysfunction during IBD.

The enteric nervous system (ENS) provides the gut autonomous control of its physiology. An even broader significance of the ENS is apparent from its recently demonstrated communication with the immune system, microbiota, brain and accessory organs. The vast and heterogenous structure of the ENS has however hampered studies of its specific functions and contribution to disease. My lab recently overcame these challenges by transcriptome analysis of single cells and accomplished a molecular definition of enteric neuron classes. In this consolidating project we will utilize the unique neuron signatures to make a comprehensive mapping of the intrinsic and inter-organ ENS connectome. We will predict disease vulnerability and assess the functional roles of neuron classes at homeostatic and inflamed conditions. This will be achieved by viral-based targeting approaches enabling visualisation of specific neuron classes, their proximity to executive cells of the gut, synaptic interactions and selective manipulation of their activity or gene expression. Changes in neuro-immune units will be addressed in resected tissue from patients with inflammatory bowel disease and a complementary mouse model. At sight, our dissection of functional and anatomical features of enteric neuron classes could be instrumental in the development of better diagnostic and therapeutic principles for the many patients suffering from gut ailments with unclear etiology.

How stem cells decide to develop into specific cell types, such as nerve or liver cells, is not known today. Nor how mature cells retain their identity through life, but in some cases they are transformed into other cell types in response to bodily injury or illness. Researchers at Karolinska Institutet will now investigate this. The group has developed a new technology to investigate stem cell development and ability to change. It will be used to understand developmental principles in two structures in mouse embryos. In the neural plate, which develops to the brain and the peripheral nervous system, and in the liver, which develops to the liver and immune system. By comparing different organs, it is possible to draw conclusions about the development of cell types with a fixed identity in the adult state, such as nerve cells, and the development of more plastic cells, such as liver cells. Creates the family tree of the cells The technology is based on labeling stem cells with fluorescent signals and biological barcodes that give each cell a unique ID number. With them, the stem cells can be traced during development. With RNA sequencing, it is possible to read the barcodes and cell IDs of individual cells, and in this way define the family trees of the mature cell types. The project group combines expertise in developmental biology, neuroscience, liver biology, immunology and physiology. - By connecting fields that are traditionally studied individually, we create a basis for unexpected discoveries about the principles that govern the identity and plasticity of the cells in our bodies, says Emma Andersson, researcher at the Department of Cell and Molecular Biology, Karolinska Institutet. The knowledge can be used in regenerative medicine, which is about repairing damaged tissue, and leading to new ways of attacking cells with abnormal developmental potential in cancer. Text Sara Nilsson Photo by Stefan Zimmerman Photo stem cells with fluorescent signals Katrin Mangold