| Bone morphogenetic proteins(BMPs) originally identified as an inducing factor for the formation of bone and cartilage are known to regulate tooth, kidney, skin, hair, muscle, haematopoietic and neuronal development, and maintain iron metabolism and vascular homeostasis. Bmp2 a as a member of Bmps that binds to type I and type II receptors transduces signals through Smad signaling. Previous studies indicated that bmp2 a is expressed in the zebrafish pineal gland, implicating that Bmp2 a may contribute to circadian regulation. However, little is known about Bmp2a’s role in the circadian clock. Here we show that bmp2 a is rhythmically expressed in zebrafish larvae; down-regulated in the clock1a-/- mutant fish but up-regulated in the per2-/- mutant fish. Luciferase reporter assays show that bmp2 a promoter activities are activated by co-transfection of Clock1 a and Bmal1 b but repressed by Cry1aa; and Ch IP assays show that Bmal1 b binds to the E-box in the bmp2 a promoter; indicating that bmp2 a is regulated directly by the circadian clock. Light impulses can induce bmp2 a expression. Also, we find that bmp2 a is a light-inducible gene which can be activated by Tefa. We employed CRISPR-Cas9, a versatile genome-editing tool, to generate a zebrafish bmp2 a mutant. Behavioral assays showed that the period of locomotor activities of bmp2a-/- mutant fish is significantly lenghtened under constant dark(DD) condition, consistent with our previous observation of per2-/- mutant fish. Deep-sequencing transcriptome analysis revealed that per2 is significantly down-regulated in bmp2a-/-mutant fish, reconfirmed by quantitative RT-PCR. Luciferase assays showed that Bmp2 a enhances transcriptional activities of per2 in a dosage-dependent manner, independent of the Clock1a-Bmal1 b complex, suggesting direct involvement of Bmp2 a in circadian regulation. Together, our results highlight a novel regulatory role of Bmp2 a signaling in the zebrafish circadian clock. |
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