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The Roof plate-specific spondin 3 (RSPO3) plays crucial roles during embryogenesis by promoting Wnt/β-catenin signaling pathway in amphibian and mammals.However, the role of Rspo3 in other vertebrate was poorly understood.Here, we show that Rspo3 is required for dorsoanterior development by negatively regulating Wnt/β-catenin signaling pathway in zebrafish.