Role of neuronal positioning in jaw motor circuit function in zebrafish

Development of the vertebrate nervous system involves substantial cell migration, where immature neurons move to specific locations to generate functional circuits. Precise neuronal migration and positioning are essential for proper brain architecture and function. Abnormal neuronal migration can contribute to neurological disorders such as lissencephaly, autism and schizophrenia. However, the consequences of abnormal neuronal migration for circuit organization and functional output are poorly understood. To provide some insight, I used the facial branchiomotor (FBM) neurons in zebrafish as a model system to analyze the effects of aberrant neuronal migration on circuit function. The FBM neurons are a subset of the branchiomotor neurons, which are generated in the vertebrate hindbrain and innervate facial and jaw muscles. During development in zebrafish and mice, FBM neurons migrate caudally from rhombomere 4 (r4) to r6 to form the facial motor nucleus and innervate jaw and gill muscles (in fish). In order to examine the consequences of aberrant neuronal migration, one must first characterize the normal functional output of the FBM circuit that drives jaw movements. In collaboration with colleagues in the MU Department of Computer Science, we developed an automated image analysis system to extract motion features from video recordings of jaw movement, enabling rapid and accurate high-throughput analysis. We used this software to examine the emergence of jaw movement in zebrafish larvae between 3-9 days post fertilization (dpf). While gape, the displacement of the lower jaw to form the mouth opening, was minimal at 3 dpf, gape frequency increased sharply by 5 dpf, and stabilized by 7 dpf. A detailed analysis of branchiomotor axons and neuromuscular junctions (NMJs) on jaw muscles suggest that this "maturation" of branchiomotor circuit output may be driven by changes in presynaptic structures at the jaw NMJs. To evaluate the consequences of defective neuronal migration on circuit output, I examined whether jaw movement was affected in the zebrafish off-limits (olt) mutant in which FBM neurons fail to migrate out of r4. In olt mutants, the increase in gape frequency occurred normally between 3-5 dpf. However, the average gape frequency was [approximately] 50 [percent] lower than wildtype siblings from 5-9 dpf while gape amplitude was unaffected. Given the jaw movement defect in olt mutants, I evaluated food intake, an independent measure of jaw movement and another functional output of the branchiomotor circuit. Olt mutants ate poorly compared to their wildtype siblings, consistent with their reduced jaw movement. I then tested several potential mechanisms that could generate the functional deficits in olt mutants. While fzd3a, the gene inactivated in olt mutants, is ubiquitously expressed in neural and non-neural tissues, jaw cartilage and muscle developed normally in olt mutants, and muscle function also appeared to be unaffected. Although FBM neurons were mispositioned in olt mutants, axon pathfinding to jaw muscles were unaffected. Moreover, neuromuscular junctions established by FBM neurons on jaw muscles were similar between wildtype siblings and olt mutants. Interestingly, FBM axons innervating the interhyoideus jaw muscle were frequently defasciculated in olt mutants. Furthermore, GCaMP imaging revealed that mutant FBM neurons were less active than their wildtype counterparts. These data suggest that aberrant positioning of FBM neurons in olt mutants results in subtle defects in fasciculation and neuronal activity, potentially generating defective functional outputs. In the future, we will examine modulatory inputs from other brain regions to the branchiomotor neurons and examine their roles in impacting circuit output in olt mutants.