Fig 1.
Srg3 is essential for anteroposterior limb skeletal patterning.
(A−D) Skeletal preparations of control and Srg3 CKO limbs at P0. The inset in (B) shows another scapula phenotype. Red arrowheads denote hypoplastic scapulae and black arrowheads indicate the loss of clavicle, deltoid tuberosity, and radius in the Srg3 CKO forelimb. The insets in (C) and (D) show a dorsal view of a hindlimb autopod marked with digit numbers. Red arrows point to the fused digits with soft tissues. cv, clavicle; dt, deltoid tuberosity; fe, femur; fi, fibula; hu, humerus; pg, pelvic girdle; r, radius; sc, scapula; ti, tibia; u, ulna; 1−5, digit identity. Scale bars: 1mm. (E) Percentages of digit number in Srg3 CKO forelimbs and hindlimbs. Upper panels show various types of cartilage structures in Srg3 CKO forelimb digits compared with control digits. Asterisks indicate hypoplastic digits.
Fig 2.
Srg3 CKO forelimb buds establish distinct Hh pathways in the anterior and posterior mesenchyme.
(A, B) Spatiotemporal distribution of Gli1 and Ptch1 transcripts in control and Srg3 CKO forelimb buds at indicated stages. Arrowheads denote the ectopic activation of Shh target genes in the anterior mesenchyme. Scale bars: 100 μm. (C−F) Spatial distribution of Shh, Grem1, Bmp4, and Fgf4 in E10.5 control and Srg3 CKO forelimb buds. Brackets in (C) mark the spatial extent of the Shh expression domain. The black arrow in (D) indicates the anterior expansion of Grem1 expression. Black arrowheads in (F) indicate the anterior and posterior end of the AER. Scale bars: 100 μm. (G−K) Spatial distribution of Hand2, Gli3, Hoxd13, and Alx4 in control and Srg3 CKO forelimb buds at indicated stages. Arrows in (I) and (J) indicate the anterior expansion of Hand2 and Hoxd13. Scale bars: 50 μm in (G−H); 100 μm in (I−K).
Fig 3.
Mesenchymal Srg3 deficiency induces ectopic Shh expression and distalizes epithelial-mesenchymal signaling at late stages.
(A) Expression pattern of Shh in control and Srg3 CKO forelimb buds at indicated stages. Arrowheads and arrow denote the anterior ectopic expression of Shh and its anterior expansion, respectively. (B) Western blot analysis of Gli3FL and Gli3R in lysates from the anterior (Ant) and posterior (Post) halves of E11 control and Srg3 CKO forelimb buds. Values represent the relative levels of Gli3R protein. α-tubulin served as a loading control. (C−E) Spatial distribution of Grem1, Fgf8, and Hoxd13 in control and Srg3 CKO forelimb buds. Red arrowheads denote the anterior ectopic expression. In (C), the black bracket indicates the dissociation of Grem1 expression domains and red brackets mark the distance between the distal expression of Grem1 and the AER. Black arrowheads in (D) point to the posterior end of Fgf8 expression. Asterisks in (E) indicate the distalized expression domain of Hoxd13. Scale bars in (A) and (C−E): 200 μm.
Fig 4.
Srg3 is required for the transcriptional activation and repression of Gli1 and Ptch1 in developing limbs.
(A, B) Quantitative PCR analysis of Gli1 and Ptch1 mRNA in Srg3f/f MEF infected with empty vector (Empty) or Cre-expressing viral vector (Cre). Each of infected MEFs was treated with an ethanol vehicle (EtOH) or cyclopamine (Cyc) (A), or incubated in Shh-conditioned media (+) or control media (−) (B). Error bars represent SD from six independent experiments. (*)P < 0.05; (**)P < 0.01. (C) Reciprocal immunoprecipitation of Srg3 with Gli2 and Gli3 proteins in E11.5 control limb buds. (D) Schematic representation of the relative positions of primer sets in the Gli1 locus (g1, g2) and Ptch1 locus (p1−p5) around the putative Gli-binding sites (red boxes) is shown on the top. Direction of transcription (arrows) and exons (numbers above boxes) are indicated. ChIP−qPCR analyses of DNA fragments precipitated with anti-Brg1 (b), anti-Srg3 (s), and IgG (i) in E11.5 control and Srg3 CKO limb buds. (E, F) ChIP−qPCR analyses of DNA fragments precipitated with anti-Gli2 (left) or anti-Gli3 (right) in MEFs (E) and in E11.5 limb buds (F). (G, H) ChIP−qPCR analyses of DNA fragments precipitated with anti-H3K27me3 from MEFs cultured in a basal condition (G) or in Shh-conditioned media (H). All data in (D−H) are represented as the percentage of input DNA, normalized to the value of nonspecific binding to the Gapdh promoter. Error bars represent SD from three independent experiments.
Fig 5.
Loss of mesenchymal Srg3 disrupted BMP signaling and caused defective chondrogenesis in forelimb buds.
(A, B) Spatiotemporal distribution of Msx2 and Bmp4 expression domains in control and Srg3 CKO forelimb buds at indicated stages. Red and black arrowheads indicate the anterior and posterior domains, respectively. (C) Spatiotemporal distribution of Sox9 in control and Srg3 CKO forelimb buds at indicated stages. Red and black arrowheads indicate the primordia corresponding to scapula and zeugopod, respectively. a, autopod; h, humerus; r, radius; sc, scapula; u, ulna; asterisks, digit rays. (D) Expression of Hoxa13 in E11.75 control and Srg3 CKO forelimb buds. Red and black arrowheads point to anterior expansion and posterior decrease, respectively. Scale bars in (A−D): 200 μm.
Fig 6.
Bifunctional action of the SWI/SNF complex in the Hh pathway regulates the spatiotemporal expression of Grem1.
(A, B) Distribution of Grem1 and Msx2 transcripts in control and Srg3 CKO forelimb autopods at indicated stages. Black bracket points to the distance between two domains of Grem1. Spatial restriction to the interdigital mesenchyme (arrowheads), combined region of separate domains (arrow), and enhanced region (red bracket) of Grem1 are indicated. (C) Expression pattern of Sox9 and Col2a1 in E12.5 control and Srg3 CKO forelimb autopods. Brackets indicate the delayed region of chondrogenic differentiation. Numbers 1 to 5 indicate the primordia of digit rays 1 to 5. Asterisks indicate the preceding chondrogenesis of shortened digit primordia in the posterior. (D) Distribution of apoptotic cells by Lysotracker Red staining in control and Srg3 CKO forelimb autopods at E12.5 and E13.5. Arrow points to increased cell death. Arrowheads indicate the reduction of interdigital cell death. Scale bars in (A−D): 200 μm.