The role of AKAP12 in axon generation during motor neuron development of zebrafish

Abstract

AKAP12 is a scaffolding protein which interacts with multiple molecules, such as PKA, PKC, and Calmodulin. Tumor suppressor activity, mediation of cell migration, and formation of blood-brain barrier are some of the various functions that are carried out by AKAP12. During the development of zebrafish, AKAP12 is known to be expressed in the slow muscle cells and their precursors, the adaxial cells. It also plays a key role in the movement of adaxial cells from the notochord to the lateral surface. However, it is believed that the interaction between motor neurons and muscle cells is crucial for proper generation of motor neurons in zebrafish. Therefore, this study was conducted to identify the role of AKAP12 during motor neuron generation in zebrafish. We observed an uncontrolled, branch-like patterned motor neuron generation in AKAP12 morphants generated by microinjection of AKAP12 splice-blocking morpholinos. However, AKAP12 is expressed in muscle cells, not in neuron cells. Therefore, it regulates motor neuron generation in a cell non-autonomous manner. Thus, we hypothesized that the AKAP12 affects the motor neuron by controlling the secretion of a downstream molecule. We screened for the molecule and observed that the amount of HSPG around the somite cell is upregulated in AKAP12 morphants. HSPG is a proteoglycan that exists in ECM and promotes generation of axons by controlling many receptor-ligand interactions. AKAP12 spatiotemporally regulates the expression of HSPG. Therefore, AKAP12 morphants which express less AKAP12 could not inhibit HSPG, so HSPG expression was disregulated. However, it has been reported that several genes have different phenotypes between morphants and knockouts. Therefore, we constructed AKAP12 knockout zebrafish using TALEN and confirmed whether they show similar phenotypes compared to the morphants. In selecting the AKAP12 knockout, we used T7E1 and melting curve of qPCR, and these selected knockouts also showed uncontrolled motor neuron sprouting as the morphants. In conclusion, we found that AKAP12, which is expressed in the muscle precursor cells, controls the HSPG, and when this muscle precursor cell migrates, the remaining HSPG regulates motor neuron generation near the notochord.