Clock1a affects mesoderm development and primitive hematopoiesis by regulating Nodal-Smad3 signaling in the zebrafish embryo

Abstract

Circadian clock and Smad2/3/4-mediated Nodal signaling regulate multiple physiological and pathological processes. However, it remains unknown whether Clock directly cross-talks with Nodal signaling and how this would regulate embryonic development. Here we show that Clock1a coordinated mesoderm development and primitive hematopoiesis in zebrafish embryos by directly up-regulating Nodal-Smad3 signaling. We found that Clock1a is expressed both maternally and zygotically throughout early zebrafish development. We also noted that Clock1a alterations produce embryonic defects with shortened body length, lack of the ventral tail fin, or partial defect of the eyes. Clock1a regulates the expression of the mesodermal markers ntl, gsc, and eve1 and of the hematopoietic markers scl, lmo2, and fli1a Biochemical analyses revealed that Clock1a stimulates Nodal signaling by increasing expression of Smad2/3/4. Mechanistically, Clock1a activates the smad3a promoter via its E-box1 element (CAGATG). Taken together, these findings provide mechanistic insight into the role of Clock1a in the regulation of mesoderm development and primitive hematopoiesis via modulation of Nodal-Smad3 signaling and indicate that Smad3a is directly controlled by the circadian clock in zebrafish.

Keywords: SMAD transcription factor; clock gene; hematopoiesis; mesoderm; nodal; transcription promoter; zebrafish.

Figure 1.

Clock1a spatiotemporal expression patterns. A, spatiotemporal expression pattern of clock1a mRNA in zebrafish embryo, detected by whole-mount in situ hybridization at indicated stages. Embryos before segmentation period were lateral views with the animal pole oriented to the top, whereas the others were oriented with anterior to the left. B, distribution of endogenous Clock1a protein in zebrafish embryo at the indicated stage, visualized by immunofluorescence (red); the nuclei (blue) were stained with DAPI.

Figure 2.

Alterations in Clock1a resulted in defective embryonic development. A, knockdown efficiency of clcock1a morpholino was tested using the corresponding Clock1a-GFP expression constructs. Embryos were photographed at the 70% epiboly. B, the specificity of Clock1a antibody and the validity of clock1a-mRNA on Clock1a protein expression were verified by Western blotting (WB) analysis. clock1a-mRNA was injected with a concentration gradient (0 to 100 to 200 pg per embryo) at the one-cell stage. and the proteins were harvested at shield stage. C, lateral views of live embryos at 24 hpf and 72 hpf are shown. The arrowheads show the tail fins and tails, and the asterisk show fusion of the eyes. D, Western blotting analysis of Clock1a expression in embryos injected with 1 ng of clock1a-MO, 1 ng of clock1a-cMO, 150 pg of clock1a-mRNA, and 1 ng of clock1a-MO+150 pg of clock1a-mRNA .

Figure 3.

Effects of Clock1a knockdown and overexpression on mesoderm and primitive hematopoiesis. Embryos were examined for the expression of the indicated markers at the indicated stage by in situ hybridization, altered marker expression patterns in Clock1a knockdown, and overexpression of embryos. Statistical data were indicated around the corresponding image. A, the mesodermal markers were examined at shield stage. B, the mesodermal markers were examined at the six-somite stage. C, expression patterns of hematopoietic marker genes at the six-somite stage. D, embryos stained by O-dianisidine at 36 hpf.

Figure 4.

Genetic interactions between Clock1a and nodal signals. A, cyc and lefty1 expression in embryos injected with clock1a-MO or clock1a-cMO or clock1a-mRNA. B, overexpression effects of sqt were antagonized by clock1a-MO and facilitated by clock1a-mRNA. A group of embryos was shown for gsc and lmo2. Statistical data are indicated below the corresponding image. C, effects of Clock1a on nodal signaling at the level of promoted nodal of Smad2/3/4 in zebrafish embryos at shield stage. D, Clock1a-regulated Smad2/3/4 expression at mRNA levels in zebrafish embryos at shield stage, 6-somites stage, and 24 hpf. Quantitative real-time PCR assay was performed, and -fold change was normalized by control to an arbitrary value of one. Data are presented as the mean ± S.D. *, p < 0.05.

Figure 5.

Clock1a regulated expression levels of Smad3 through the E-box1 element of smad3a promoter. A, quantitative real-time PCR analysis of circadian rhythm of zebrafish clock1a and smad2/3/4 expression under LD conditions. Standard loading was indicated by β-actin expression. The results were presented as the average ± S.D. B and C, sequence analysis of zebrafish smad3a promoter regions that contain E-boxes and a schematic diagram showing various E-box-included fragments of the zebrafish smad3a promoter region. Data are the average luciferase activity ± S.D. *, p < 0.05 in 293T cells transfected with various E-box-included fragments of the zebrafish smad3a promoter-luciferase. NS, not significant. D and E, average luciferase activity ± S.D. *, p < 0.05) in zebrafish embryos injected with various E-box-included fragments of the zebrafish smad3a promoter-luciferase. F, ChIP assay of Clock1a binds to E-box 1 of the zebrafish smad3a promoter. NC, negative control. G, changes in Clock1a binding to E-box 1 by quantitative ChIP and Smad3 expression by quantitative real-time-PCR in clock1a-MO-, clock1a-cMO-, and clock1a-mRNA-treated zebrafish embryos was measured by the average ± S.D. *, p < 0.05.

Figure 6.

Smad3a overexpression antagonized the effects of clock-MO and cooperatives clock1a-mRNA on mesendoderm development and primitive hematopoiesis. Embryos at the one-cell stage were injected with 100 pg of smad3a-mRNA in combination with clock-MO, clock-cMO, or clock-mRNA and examined for the expression of ntl, gsc, and eve1 at the shield stage and scl stage at the 6-somite stage by in situ hybridization. Statistical data were shown below the corresponding image.

Figure 7.

hCLOCK was involved in regulation of hSMAD3. A, immunofluorescent staining of hSmad3 in transfected cells with hClock expression constructs or shRNA expression vectors. Red, hSmad3; blue, DAPI. B, Western blotting of hSmad3 expression at protein level under the basal or TGF-β stimulated expression. shNC, control of shCLOCK.

Figure 8.

Model of Clock1a affects mesoderm development and primitive hematopoiesis by regulating Nodal-Smad3 signals in zebrafish embryos.