Formation of Proximal and Anterior Limb Skeleton Requires Early Function of Irx3 and Irx5 and Is Negatively Regulated by Shh Signaling

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1 Developmental Cell, Volume 29 Supplemental Information Formation of Proximal and Anterior Limb Skeleton Requires Early Function of Irx3 and Irx5 and Is Negatively Regulated by Shh Signaling Danyi Li, Rui Sakuma, Niki A. Vakili, Rong Mo, Vijitha Puviindran, Steven Deimling, Xiaoyun Zhang, Sevan Hopyan, and Chi-chung Hui

2 Figure S1. Expression patterns of Irx3 and Irx5 during limb bud development and skeletal phenotypes of single and compound mutants of Irx3 and Irx5, related to Figure 1 (A-L) Whole-mount in situ hybridization of Irx3 and Irx5 in wild-type forelimb and hindlimb buds at indicated stages. The expression patterns are similar between Irx3 and Irx5. (M-AA) Skeletal staining reveals E18.5 forelimb (including scapula) and hindlimb (including pelvis) structures of indicated genotypes (red staining bone, blue staining cartilage). Red arrows in P, V and Y indicate hypoplastic scapula. The insets in N, Q, T, W and Z show a dorsal view of hindlimb autopod structures.

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4 Figure S2. Irx3 protein levels in Cre-ER TM ;Irx3/5-CKO embryos post tamoxifen administration, morphological phenotype of Irx3/5 DKO hindlimb buds, cell proliferation and cell death in Irx3/5 DKO hindlimb buds, related to Figure 2 (A) Irx3 and Actin protein levels detected by Western blot in the hindlimb bud lysates of Irx3 flox 5 EGFP /3-5 EGFP (flox/-) and Cre-ER TM ;Irx3/5-CKO (MT1 and MT2) embryos 24hr, 36hr, and 48hr post tamoxifen treatment. Irx3 protein level was quantified and normalized to Actin using densitometry. Numbers below western blot images indicate relative level of Irx3 comparing to controls (flox/-). (B) The curve of relative Irx3 protein levels in mutant lysate (comparing to controls) post tamoxifen treatment. Error bars represent standard deviations. (C) Morphology of wild-type (WT) and Irx3/5 DKO hindlimb buds at 29, 32, 34 and 36-somite stages. Irx3/5 DKO hindlimb buds seem initiate normally (29-somite stage), but the anterior aspect of mutant buds become flat (black arrowheads) during outgrowth. (D) Quantification of the frontal plane area of control and mutant hindlimb buds at 36-somite stage (n=6, p<0.001, two-tailed Student s t-test). Mutant hindlimb buds are about 25% smaller than that of controls. Error bars represent standard deviations. (E) Section immunofluorescence of phosphohistone H3 (ph3) and DAPI staining in 29-somite stage hindlimb bud section of control and Irx3/5 DKO embryos, white broken lines indicate anterior border of limb buds. (F) Quantification of ph3-positive cell in the anterior hindlimb field of control and Irx3/5 DKO embryos at E from multiple sections of several limb buds. No obvious difference was observed. Error bars represent standard deviations. (G) Section immunofluorescence of ph3, cleaved Caspase-3 (Caspase 3) and DAPI staining in 37-somite stage hindlimb buds of control and mutants. Caspase 3-positive cells are increased in the anterior region of the base of mutant hindlimb buds. (H) Lysotracker staining in 37-somite stage hindlimb buds of control and mutants. White arrowheads indicate the region with increased cell death in mutant bud. White broken lines indicate limb bud outlines.

5 Figure S3. Hindlimb phenotype of Irx3/5 DKO;Gli3 P1-4/+ mutant, Alx4 expression and ph3-positive cell quantification in the forelimb buds of control, Irx3/5 DKO, Kif7 -/- and Irx3/5;Kif7 TKO embryos, and measurement of frontal plane area of control and Irx3/5 DKO forelimb buds, related to Figure 3 (A-D) Cartilage staining of E14.5 hindlimbs of indicated genotypes. The anterior preaxial polydactytly in Gli3 P1-4/+ hindlimb is lost when Irx3/5 are removed. Interestingly, we noted that Irx3/5 DKO;Gli3 P1-4/+ forelimbs display more severe polydactyly than Gli3 P1-4/+, which is different from the forelimb phenotype in Irx3/5;Kif7 TKO embryos. One possible explanation is that Shh pathway activity in forelimb buds of Irx3/5 DKO;Gli3 P1-4/+ is not as elevated as that of Irx3/5;Kif7 TKO. This is consistent with the less severe polydactyly phenotype of Gli3 P1-4/+ limbs than that of Kif7 -/- (data not shown). (E-J) Alx4 expression (marked by black lines) in E10.5 (33 to 35-somite stages) forelimb buds is decreased in Irx3/5 DKO and Kif7 -/- and almost lost in Irx3/5;Kif7 TKO mutants. (I and J) Reduction of Alx4 expression in Irx3/5 DKO forelimb buds during limb initiation at E9.25 (22 to 23- somite stage) (red asterisk in F). D for dorsal view, L for lateral view. (K) Quantification of ph3-positive cells in the anterior hindlimb field of indicated genotypes at E9.75. No obvious difference among all the genotypes. Error bars represent standard deviations. (L) Quantification of the frontal plane area of control and mutant forelimb buds at 29-somite stage. Irx3/5 DKO forelimb buds are about 20% smaller than that of controls (p<0.05, two-tailed Student s t-test). Error bars represent standard deviations.

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7 Figure S4. Quantification of Shh position, relative size of Gli1-expressing population in developing limb buds, and phenotypes of Irx3/5 DKO;Shh +/- hindlimbs, related to Figure 4 (A-W ) Whole-mount in situ hybridization of Shh (A-J) and Gli1 (K-W ) in wild-type forelimb and hindlimb buds at indicated somite stages (at bottom right corner of each panel). Black lines in A-J indicate the lengths of limb buds along the AP axis. The posterior end is set as 0 and the anterior end is set as 100. Red arrowheads mark the center of Shh domain along the AP limb axis. Red numbers indicate the relative positions of the center of Shh domain from the posterior boundary of limb buds. (K and K ) An example of quantification of the area of Gli1 domain relative to the limb bud using a threshold value to mark the expression domain based on saturated staining of RNA in situ hybridization. Dorsal view images of Gli1 in situ hybridization in wild-type limb buds were carefully taken. Limb bud outline was manually selected based on the morphology (red circled region). Images were converted to greyscale mode, and the Gli1 domain was selected automatically with K value 30% using the Color Range command (black circled region). (L-W ) Examples of ratios of Gli1 domain to limb bud area (%) in forelimb and hindlimb buds at multiple comparable stages. Black and red circled regions mark the limb regions and Gli1 domains respectively. (n 4 for each stage) (X) Quantification of frontal plane areas of hindlimb buds of indicated genotypes at 40 somite stage. Difference between control and Irx3/5 DKO and control and Irx3/5 DKO;Shh +/- were significant (p<0.01 and p< respectively, two-tailed Student s t-test). No obvious difference between Irx3/5 DKO and Irx3/5 DKO;Shh +/- was observed. Error bars represent standard deviations. (Y-AA ) Cartilage structures of hindlimbs in control, Irx3/5 DKO and Irx3/5 DKO;Shh +/- with rescued hindlimb phenotype to different extent.