microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. We use the sea urchin embryo to achieve a comprehensive understanding the function of miR-1 in a developing embryo. Results indicate that miR-1 regulates gut contractions, specification, and positioning of serotonergic neurons, as well as mesodermally-derived muscles, pigment cells, and skeletogenic cells. Gain-of-function of miR-1 generally leads to more severe developmental defects than its loss-of-function. We identified that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Notch, Nodal, and Wnt1 signaling components. We found that miR-1-mediated direct suppression of Nodal may indirectly regulate FoxQ2 to impact serotonergic neurons. Excess miR-1 may lead to decreased Nodal and Notch that result in decreased circumpharnygeal muscle fibers and the number of pigment cells. The striking ectopic skeletal branching induced by miR-1 mimic injections may be explained by miR-1-mediated direct suppression of Nodal that leads to expression changes of Vegf3, and Fgfa that mediate skeletogenesis. This work demonstrates that miR-1 plays a diverse regulatory role that impacts tissues derived from all germ layers.