Cells of the developing and regenerating Central Nervous System (CNS) acquire the correct identity according to their position. The floorplate plays a central role in this patterning process by the production of the sonic hedgehog (SHH) morphogen. Neural stem cells respond to SHH in a time- and concentration- dependent manner resulting in an orderly array of specific progenitors along the dorsoventral (DV) axis. Naturally, correct placement and size of the floorplate are crucial for the subsequent generation of correct CNS patterning.

The notochord has, traditionally, been considered as the key floorplate inducing factor. The Tanaka group at the Institute of Molecular Pathology in Vienna, however, has managed to identify two biological contexts in which the floorplate is generated without the notochord. With this in mind, we ask what is the molecular mechanism and temporal dynamics of the floorplate self-organisation? We are also interested in examining how the surrounding neural stem cells respond to the nascent floorplate with time. This work has important implications for directing patterning in neural organoids, which currently exhibit relatively uncontrolled patterning.

To address our questions, we plan to use an organoid system derived from a clonal murine neural tube that undergoes spontaneous floorplate formation in response to global retinoic acid (RA) addition. FOXA2 shows an initial “salt-and-pepper” style expression which, with time, resolves into a cluster that turns on SHH to pattern the rest of the organoid.

We will use live imaging and single cell RNAseq to identify the signaling pathways required to gain stable floor plate expression. We will investigate to what extent Turing-like activator/inhibitor systems are involved in floorplate organization and address the role of self-adhesion among the “winner” cells.

We are also interested in determining the kinetics of how surrounding cells respond to the floor plate during its genesis. To achieve this, we will work together with the Kicheva group, to generate live reporter mouse ES cells for visualizing cellular responses to different signals. We will then join forces with the von Haeseler group single cell RNASeq analysis to characterize the temporal dynamics of cell patterning and cell proliferation effects of these morphogen pathways in the neural organoid system.

As a different approach we will use the axolotl as a model of regeneration to study the role of self-organization events in floorplate regeneration. Using FoxA2 and SHH transgenic reporter axolotls we will track how floor plate regenerates with time. In conjunction with the Hippenmeyer group, we hope to develop the MADM system in axolotl in order to test for the role of retinoic acid in this phenomenon.

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