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Fig. S1. Strategy to generate epiblast-specific Acvr1 conditional knockout in mouse. (A) Schematic illustration of mating strategy to obtain epiblast-specific Acvr1 conditional knockout mouse embryos. (B) Mox2-Cre recombination was analyzed using ROSA26 reporter mice (R26R). The upper panel shows a frontal view of mouse embryos and the lower panel shows a view from the ventral surface of node at E8.0. A, anterior; P, posterior; L, left; R, right. (C) Msx1 expression was examined by whole-mount in situ hybridization at E9.5 and E10.5. In Acvr1 cKO embryos, expression level of Msx1 was significantly downregulated in the forelimb (E9.5) and craniofacial region (E10.5) (red arrows). fl, forelimb; lnp, lateral nasal process; man, mandibular process; max, maxillary process. (D) The level of phospho-SMAD1/5/8 was examined by western blotting. To delete the Acvr1 flox alleles, adenovirus-Cre (Ad-Cre) was used (middle lane). In Acvr1-deficient MEF cells (Ad-Cre), around 70% phospho-SMAD1/5/8 (P-SMAD1/5/8) reduction was observed compared with control MEF cells, which was transduced with adenovirus-GFP (Ad-GFP; left lane). As a positive control for P-SMAD1/5/8 in western blotting, we established MEF cells from transgenic mice, which conditionally express the constitutively active form of Acvr1 (ca-Acvr1) (Fukuda et al., 2006). After Ad-Cre transduction with ca-Acvr1 MEF cells, strong induction of P-SMAD1/5/8 was seen (right lane). The relative intensity of P-SMAD1/5/8 is shown in the bottom of a lane. Signal intensity of P-SMAD1/5/8 in control MEF was set as 1.0. An asterisk shows a non-specific band. β-Actin was used as a loading control.
Additional reference
Fukuda, T., Scott, G., Komatsu, Y., Araya, R., Kawano, M., Ray, M. K., Yamada, M. and Mishina, Y. (2006). Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2. Genesis 44, 159-167.
Fig. S2. Abnormal limb bud development in Acvr1 cKO embryos. (A,B) Fgf8 expression was examined by whole-mount in situ hybridization. Fgf8 expression was expanded in AER (A; yellow arrow) and ectopic Fgf8 expression in posterior side of forelimb was observed in Acvr1 cKO embryos (B; yellow arrows). A, anterior; P, posterior.
Fig. S3. Establishment of Acvr1-deficient mouse embryonic fibroblasts (MEFs). (A) MEFs harvested from Acvr1flox/flox embryos that carried ROSA26 reporter transgene (Acvr1flox/flox:R26R) were transduced with either recombinant lacZ adenovirus (Ad-lacZ) or recombinant Cre adenovirus (Ad-Cre). After transduction, MEFs were stained by fluorescein di-β-galactoside (FDG) and sorted by flow cytometry. Fluorescence from Ad-lacZ-transduced cells was derived from the recombinant adenovirus, whereas fluorescence from Ad-Cre-transduced cells was derived from Cre-recombined ROSA26 reporter. Cells incubated with FDG without Ad-Cre transduction were used as a negative control. (B) Efficient recombination of Acvr1 targeted exon was confirmed by RT-PCR. RT-PCR using primer specific for sequences in Acvr1 exons 6 and 8 (upper panel) showed that the floxed exon 7 was completely deleted in Ad-Cre-transduced MEFs (Acvr1 KO, 192 bp) compared with Ad-lacZ-transduced MEFs (Acvr1 WT, 469 bp) (lower panel). β-Actin was used as an internal control. Reverse transcription-positive (RT+) and -negative (RT−) samples were used for β-actin (245 bp).
Fig. S4. Cell growth is normal in Acvr1-deficient MEF cells. (A) MEF cells (1×105) were seeded into each well of a six-well tissue culture plate. The total cell number per well was counted on day 2 and day 5. Cell growth was comparable in control (Ad-lacZ) and Acvr1-deficient (Ad-Cre) MEF cells. N.S., not significant. Error bars indicate s.e.m. (B) Apoptosis was examined by TUNEL assay. No cell death was observed during cell growth in control (Ad-lacZ) and Acvr1-deficient (Ad-Cre) MEF cells (left column). Cells treated with DNase I (middle column) and cells without terminal transferase (right column) were used as positive and negative control, respectively.
Fig. S5. Localization of p27Kip1 during primary cilia development. (A,B) Control (Ad-lacZ) and Acvr1-deficient (Ad-Cre) MEF cells were stained with acetylated tubulin (green) and p27 (red). Note that p27Kip1 was localized in the nucleus in control MEF cells (A) but no production of p27Kip1 was seen in Acvr1-deficient MEF cells (B). Cells were counterstained with DAPI (blue).
Fig. S6. Both p27Kip1 and phospho-p27Kip1 are exclusively produced at the node in wild-type mouse embryos. (A,B) Double staining with either Ki-67 (red)/p27Kip1 (green) (A) or Ki-67 (red)/P-p27Kip1 (green) (B) is shown. Sagittal sections of a wild-type mouse embryo at E8.0 were examined and counterstained with DAPI (blue). Both p27Kip1 (A) and p27Kip1 phospho-Ser10 (P-p27) (B) were exclusively produced in the ventral surface of node where Ki-67-stained cells were absent. A, anterior; P, posterior.
Fig. S7. p21Cip1 and p57Kip2 are not produced at the node in wild-type mouse embryos. (A,B) Double staining with either Ki-67 (red)/p21Cip1 (green) (A) or Ki-67 (red)/p57Kip2 (green) (B) of sagittal sections of a wild-type mouse embryo at E8.0. Neither p21Cip1 nor p57Kip2 was produced in the node at E8.0. (C,D) At E14.5, p21Cip1 was detectable in the dental epithelium (left column) (Jernvall et al., 2000) and p57Kip2 was specifically produced in glomerulus (left column) (Nagahama et al., 2001). These specimens were used as positive controls to validate p21Cip1 and p57Kip2 antibodies. Right columns in C and D show negative controls (without primary antibody). White asterisks illustrate non-specific auto-fluorescence signals from erythrocytes (C,D). A, anterior; P, posterior.
Additional references
Jernvall, J., Keranen, S. V. and Thesleff, I. (2000). Evolutionary modification of development in mammalian teeth: quantifying gene expression patterns and topography. Proc. Natl. Acad. Sci. USA 97, 14444-14448.
Nagahama, H., Hatakeyama, S., Nakayama, K., Nagata, M. and Tomita, K. (2001). Spatial and temporal expression patterns of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse development. Anat. Embryol. 203, 77-87.
Movie 1. Leftward fluid flow in the node was visualized by adding 1 µm beads into the node of a control mouse embryo. Movie is twice as fast as original speed.
Movie 2. Leftward fluid flow in the node was visualized by adding 1 µm beads into the node of an Acvr1 cKO embryo. Movie is twice as fast as original speed.
