Characterization of Agrin Function in Chicken and Zebrafish Embryogenesis.

Abstract

Agrin is an extracellular matrix heparan sulfate proteoglycan that plays a key role in the development of the neuromuscular junction (NMJ) by inducing the clustering of acetylcholine receptors at synaptic sites of the NMJ. Although recent studies have extended our understanding of agrin's function in the nervous system, its function in the CNS is not clearly understood.

The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the basic fibroblast growth factor (FGF-2), using both PC12 cells, and chick retina neuronal cultures. Agrin increases the efficacy of FGF-2 stimulation of neurite outgrowth, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF-2. Agrin augments and sustains a transient early phosphorylation of ERK (extracellular signal-regulated protein kinase) in the presence of FGF-2. Neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF-2.

To overcome the lethality of agrin gene disruption and the difficulty of embryonic manipulation of agrin function in mice, a gene encoding zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS, the NMJ, and non-neural structures such as the pronephric duct, and endodermal tissues. A morpholino-based gene targeting against agrin significantly impair development of tail and the NMJ, and cause severe defects in motor neuron axon outgrowth and formation of the midbrain-hindbrain boundary, eye, and otic vesicles. Morphants subsequently develop paralysis, and die at larvae stages.

Knockdown of agrin in zebrafish strikingly resembles phenotypes of zebrafish FGF-related mutants, such as disruption of the MHB, optic and otic vesicles during zebrafish development. Inhibition of FGFR synergizes defects from agrin knockdown resulting in MHB disruption, a shortened tail, small eyes and otic vesicles, which suggest that agrin modulates the activity of FGF signaling pathways.

In conclusion, my studies show that agrin is essential for NMJ formation as well as sensory and motor neuron axonal growth and pathway formation in zebrafish development. Importantly, the HSPG agrin may be involved in regulation of early CNS development via maintenance and regulation of FGF signaling.