Dlk1 Membrane-to-Nuclear Signalling During Motor Neuron Functional Diversification
by Nidhi Subhashini
Date of Examination:2016-11-21
Date of issue:2017-11-20
Advisor:Prof. Dr. Till Marquardt
Referee:Prof. Dr. André Fischer
Referee:Dr. Judith Stegmüller
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EnglishMotor neurons of the spinal cord and hindbrain are responsible for translating neural commands into movements. For the execution of smooth movements, it is necessary that motor neuronal impulses are in turn translated into the gradual build-up of muscle force. This task is achieved by the diversification of motor neurons into a range of ‘slow’ to ‘fast’ types that are recruited by presynaptic activity according to the amount and manner of force they generate in the muscle. During embryonic development motor neurons are generated from neural progenitor cells residing in the ventral neural tube. A lot of information has become available in recent years about how the motor neuron progenitors are specified and how motor neurons differentiate into different ‘positional identities’ according to the specific muscles or muscle groups they supply. However, very little is known about how the motor neurons diversify into the different slow and fast types required for the automated gradation and task-dependent application of muscle force. Dlk1, a type1 transmembrane protein, was recently identified as a determinant of motor neuron functional diversification by promoting fast motor neuron properties. Howbeit, the signalling mechanisms that allow Dlk1 to alter gene expression and promote fast motor neuron properties still remained unresolved. The present thesis attempted to dissect the signalling mechanism in a stepwise fashion starting from the plasma membrane, where the Dlk1 protein resides, to the nucleus where it affects gene expression. As a starting point, the supervisor’s laboratory had identified the Notch and NFATc4 as interacting partners of Dlk1. Knowing that Notch is a principal player in generation of cellular diversity throughout development while NFATc4 is a transcription factor that shuttles between cytosol and nucleus, I divided my study into two parts. In the first part of the thesis, I used biochemical and immunohistochemical assays to resolve the physical interaction between the Dlk1 extracellular segment with Notch, its impact on Notch activation by canonical ligands and its influence on motor-neurogenesis. In the second part of my study, I show that Dlk1 is cleaved intracellularly and that the resulting Dlk1 intracellular segment interacts with NFATc4. My study also indicates the transcriptional regulation of Dlk1 target genes by NFAT. With the help of electrophysiological recordings in genetically manipulated chick spinal cord, I further found that suppressing NFAT activity alters the physiological properties of motor neurons. The result of my studies thus give rise to a model according to which Dlk1, by physically connecting Notch and NFAT signalling pathways, directly connects neurogenesis to motor neuron functional diversification.
Keywords: Motor Neuron, Functional Diversification, Signalling Pathway, Dlk1