Fig 2: Hand2 interacts with Hoxd13 and is part of the chromatin complexes bound to the ZRS in limb buds.(A) Scheme of the ∼1.7 kb mouse ZRS cis-regulatory and the flanking genomic regions. The ZRS is located within an intron of the mouse Lmbr1 gene (indicated on the left) and located ∼800 kb upstream of the Shh proximal promoter and coding exons (indicated on the right, see also Figure S5). The evolutionary conserved ZRS region drives expression of a LacZ reporter gene in Shh-like pattern in mouse limb buds [28], while deletion of the MFCS1 core region (indicated in white) disrupts Shh activation in limb buds [29]. Six Ebox sequences in the ZRS, which could potentially interact with Hand2 proteins are numbered “1” to “6”. Black lines indicate the approximate positions and sizes of the PCR amplicons for ChIP analysis. Note that amplicons “b” to “d” reside within the mouse ZRS, while amplicons “a” and “e” are located ∼2 kb upstream and ∼6 kb downstream of the ZRS and serve as non–ZRS controls. (B) Luciferase transactivation assay in NIH3T3 fibroblasts. Cells were co-transfected with ZRS-Luc and the expression plasmids indicated. Bars represent standard deviations. P<0.0001 for all samples except Gli3R alone: P = 0,0519. (C) Co-immunoprecipitation of Hand2 and Hoxd13 from wild-type limb buds (E10.5) using anti-Hoxd13 antibodies (α-Hoxd13) or IgGs (control). Hand2 proteins associated to Hoxd13 protein complexes were detected by Western blotting. (D,E) ChIP from wild-type limb buds (E11.0) to detect Hand2-containing chromatin complexes bound to the ZRS. (D) Analysis of amplicon “c” by conventional PCR (186 bp). Input: DNA isolated from cross-linked chromatin of E11.0 limb buds prior to ChIP was used as a positive control for PCR amplification. α-H2: ChIP using Hand2 antibodies. α-IgG: ChIP using non-specific goat IgGs as a control. Un: undiluted sample; dilutions as indicated. (E) Q–PCR analysis of three completely independent ChIP experiments using freshly cross-linked chromatin and α-Hand2 antibodies. The average values ± standard error are shown. Values obtained by amplifying a particular region from ChIP experiments using non-specific goat IgGs were arbitrarily set at 1 and used to calculate the values for the α-Hand2 ChIP experiments. Statistical evaluation by the Mann-Whitney test shows that the amplicons within the ZRS (“b” to “d”) are enriched in a statistically highly significant manner in comparison to the adjacent non-ZRS amplicons (“a” and “e”; p = 0.0018).

Fig 3: Forelimb buds lacking Hand2 and Gli3 lack AP polarity along the entire PD axis.(A) Skeletal preparations of H2 ?/?c Gli3 Xt/Xt, H2 ?/?c, and Gli3 Xt/Xt single mutant and control (H2 ?./) forelimbs at E14.5. The black arrowheads point to the duplicated elbow-like structure while the white arrowheads point to the symmetrical zeugopodal skeletal elements in H2 ?/?c Gli3 Xt/Xt limbs. Note the shortening of the stylopod in double mutant limbs. (B) Expression of Sox9 in limb buds at E10.75 (38 somites) and E11.5. Black brackets indicate the non-expressing distal mesenchyme that is reduced in H2 ?/?c Gli3 Xt/Xt limb buds. (C) Runx2 expression in wild-type limb buds marks the presumptive stylopod (s) and zeugopodal domains (r/u) at E12.0. Note that establishment of anterior expression domain is delayed in Gli3 Xt/Xt mutant limbs as it becomes visible by E12.5 (data not shown). Black arrowheads point to the apolar proximal expression of Runx2 in H2 ?/?c Gli3 Xt/Xt mutant limb buds. In wild-type limb buds, the presumptive expression domains for Sox9 and Runx2 are indicated as previously defined [42],[63]. sc: scapula; s: stylopod; z: zeugopod; a: autopod; u: ulna; r: radius. All limb buds are oriented with the anterior to the top and the posterior to the bottom.

Fig 4: Delayed inactivation of Hand2 in hindlimb buds results in rather normal Shh expression and development.(A) Hindlimb buds skeletons at E14.5, stained with alcian blue (cartilage) and alizarin red (bone). Prx1-Cre mediated inactivation of Hand2 (H2 ?/?c) in hindlimb buds results in all cases in fusion of the tarsals (arrowheads) and formation of 5 (n = 11/24) or 4 (n = 13/24) digits. Please note that in latter case the formation of digit 2 and/or 3 (not shown), which depend mostly on long-range SHH signaling [7] is always affected. (B) Shh expression in wild-type and Hand2 deficient hindlimb buds at E10.75 (37 somites). Note that the expression domain is correctly positioned in H2 ?/?c hindlimb buds, but expression levels are reduced.

Fig 5: Alk2 is required during the decidualization of human endometrial stromal cells.Expression of decidual markers, IGFBP1 (A) and PRL (B) in human endometrial stromal cells (hESC) cultured in growth medium (blue columns), treated with a hormonal cocktail containing estradiol, medroxyprogesterone acetate (MPA) and cAMP (EPC, green columns), treated with EPC after transfection of non targeting siRNA (red columns) or siRNA targeted to ALK2 (purple columns). Day 0 and day 3 refer to EPC treatment; the siRNA transfections were performed at day -1 and -2. (* p<0.05, ** p<0.01). N = 3. C–D) Expression of CEBPB (C) and PGR (D) at day 0 and after 3 days of EPC treatment in cells treated with non targeting (NT) siRNA (black column) and siRNA targeted to ALK2 (ALK2 siRNA, red column) (* p<0.05). N = 3. E) CEBPB and PGR (isoform A and B) protein levels in NT siRNA-treated and ALK2 siRNA-treated hESC, quantified by western blotting at day 0 and day 3 of EPC treatment. ß-ACTIN was used as loading control. F–H) Expression of HAND2 (F), WNT4 (G) and FOXO1 (H) at day 0 and day 3 of EPC treatment in NT siRNA-treated (black column) and ALK2 siRNA-treated (red column) hESC (* p<0.05). N = 3. Experiments were performed on cells from 3 patients. Data are means ± SEM.