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Saller, Maximilian (2016): Molecular mechanisms of phrenic nerve outgrowth and innervation of the diaphragm. Dissertation, LMU München: Medizinische Fakultät
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Abstract

The highly structured process of breathing is controlled by automatic respiratory centers in the brainstem, which signal to a specialized group of motor neurons in the cervical spinal cord that constitute the phrenic nerves. In mammals, the thoracic diaphragm that separates the thorax from the abdomen adopts the function of the primary respiratory musculature. Faithful innervation by the phrenic nerves is therefore a prerequisite for the functionality of this highly specialized musculature and thus, ultimately, the viability of the entire organism. Here, we employed a genetic approach to investigate the involvement of the Sema3-Npn-1 signaling pathway during phrenic nerve targeting and fasciculation, as well as during establishment of the diaphragm. We utilized mouse lines in which either the binding site of Npn-1 for class 3 Semaphorins was mutated systemically (Npn-1Sema-), or in which Npn-1 was removed selectively from somatic motor neurons (Npn-1cond;Olig2-Cre) by tissue-specific activity of Cre-recombinase. Whole-mount immunohistochemistry revealed initial defasciculation of phrenic motor axons within the brachial plexus at developmental stage E10.5. Interestingly, the axons refasciculated and formed one distinct nerve bundle before reaching the primordial diaphragm, the pleuroperitoneal fold, at E11.5 in both mutant mouse lines. During the development of the costal muscles of the diaphragm, Sholl analysis revealed increased defasciculation of the phrenic nerve branches which persisted until the end of primary myogenesis at E16.5 and beyond. Additionally, significantly more axons extended into the central tendon region (CTR) of the diaphragm at all investigated embryonic stages when compared to littermate controls. Intriguingly, we observed formation of ectopic muscles patches within the CTR that were innervated by misprojecting axons in both mutant mouse lines. We therefore asked whether ectopic muscle development was a direct result of manipulating Sema3A-Npn-1 signaling, or a secondary effect regarding interaction of ectopically migrating phrenic axons and muscle progenitor cells (MPCs). To elucidate the underlying mechanisms of ectopic recruitment of MPCs to the CTR, we focused on the Slit-Robo signaling pathway, which is employed by motor neurons during axon targeting and bundling, and was shown to be involved in targeted MPC migration at later developmental stages in Drosophila. We showed that Slit2 and it’s corresponding Robo receptors are expressed in phrenic motor neurons and migrating MPCs during diaphragm development and innervation, respectively. In vitro chemotaxis experiments revealed an attractive effect of Slit1 and Slit2 onto primary MPCs, while Sema3A acts strongly repulsive. Taken together, our data indicate that Sema3A-Npn-1 signaling cell-autonomously influences phrenic nerve growth and targeting, while aberrant muscle formation in the CTR of the diaphragm may be triggered by ectopically invading axons, independently of the Sema3A-Npn-1 signaling pathway. Thus, we postulate an influence of factors released by motor neuron growth cones on the migration properties of myoblasts during establishment of the diaphragm. Conditional genetic approaches may prove the interdependency of motor axon growth cones and myoblast progenitor cells for faithful establishment of neuromuscular connectivity during embryonic development.