Agrin is a critical protein for neuromuscular synaptogenesis, as shown in agrin-deficient mutant mice. These mice exhibit severe defects in neuromuscular differentiation, including reduced, smaller, and less dense AChR clusters in muscle fibers. Despite this, some postsynaptic differentiation occurs, suggesting the presence of a second nerve-derived signal. Additionally, intramuscular nerve branching and presynaptic differentiation are abnormal, possibly due to agrin's role or impaired retrograde signaling. Agrin, a proteoglycan synthesized by motoneurons, is essential for clustering AChRs on myotubes. It is neurally derived and plays a key role in synaptic organization. Agrin-deficient mice lack z-agrin, a neuron-specific isoform, and show reduced agrin protein levels. Despite this, recombinant z-agrin can induce AChR clustering in myotubes, indicating that agrin's role is not solely dependent on z-agrin. Presynaptic defects in agrin mutants include abnormal axonal branching and arborization, with axons extending parallel to myotubes and lacking synaptic specialization. Postsynaptic defects include abnormal AChR clustering, reduced synaptic components, and impaired transcriptional specialization of synaptic nuclei. These defects suggest that agrin is crucial for both pre- and postsynaptic differentiation. The study highlights agrin's role in synaptic organization, but also suggests that other factors may contribute to postsynaptic differentiation. Agrin deficiency leads to aneural AChR clusters, possibly due to impaired nerve-muscle interactions. The results support the agrin hypothesis, indicating that agrin is essential for proper synaptic development. However, the exact mechanisms by which agrin influences synaptic differentiation remain to be fully elucidated.Agrin is a critical protein for neuromuscular synaptogenesis, as shown in agrin-deficient mutant mice. These mice exhibit severe defects in neuromuscular differentiation, including reduced, smaller, and less dense AChR clusters in muscle fibers. Despite this, some postsynaptic differentiation occurs, suggesting the presence of a second nerve-derived signal. Additionally, intramuscular nerve branching and presynaptic differentiation are abnormal, possibly due to agrin's role or impaired retrograde signaling. Agrin, a proteoglycan synthesized by motoneurons, is essential for clustering AChRs on myotubes. It is neurally derived and plays a key role in synaptic organization. Agrin-deficient mice lack z-agrin, a neuron-specific isoform, and show reduced agrin protein levels. Despite this, recombinant z-agrin can induce AChR clustering in myotubes, indicating that agrin's role is not solely dependent on z-agrin. Presynaptic defects in agrin mutants include abnormal axonal branching and arborization, with axons extending parallel to myotubes and lacking synaptic specialization. Postsynaptic defects include abnormal AChR clustering, reduced synaptic components, and impaired transcriptional specialization of synaptic nuclei. These defects suggest that agrin is crucial for both pre- and postsynaptic differentiation. The study highlights agrin's role in synaptic organization, but also suggests that other factors may contribute to postsynaptic differentiation. Agrin deficiency leads to aneural AChR clusters, possibly due to impaired nerve-muscle interactions. The results support the agrin hypothesis, indicating that agrin is essential for proper synaptic development. However, the exact mechanisms by which agrin influences synaptic differentiation remain to be fully elucidated.