DEV019950 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Insertional mutagenesis of Jaws. (A) Schematic illustrating insertion of the secretory gene trap into the third intron of the Jaws gene, containing 5 exons and located on chromosome 4. The gene trap comprises a type II transmembrane domain (TM) fused to a βgeo reporter, followed by an internal ribosome entry site (IRES) upstream of a human placental alkaline phosphatase (PLAP) reporter, all flanked by a strong splice acceptor (SA) and a polyadenylation signal (pA). Integration as shown yields a fusion protein incorporating the first 213 amino acids of JAWS upstream of the TM domain and βgeo reporter. Arrowheads indicate forward and reverse primers used for RT-PCR analysis. (B) Widespread expression of Jaws, shown by northern blotting of tissue samples from 10-day-old mice or whole E9.5 and E14.5 embryos. Note the absence of the 8.0 kb and 6.0 kb bands in the Jaws^−/− E14.5 embryo. +/+, wild type; −/−, Jaws^−/−; EtBr, ethidium bromide. (C) Quantitative RT-PCR analysis of E10.5 embryos, confirming undetectable expression of wild-type Jaws mRNA in Jaws^−/− mice. (D) Immunoblot of E13.5 embryo lysates with JAWS (top) or β-tubulin (bottom) antibodies. Blocking peptide was preincubated with JAWS antiserum for 2 hours at room temperature (right panels). The arrowhead indicates a non-specific band migrating slightly below the JAWS protein.

• Supplemental Figure S2 -

  Fig. S2. Cleft secondary palate in Jaws^-/- mice. Coronal sections from E18.5 embryos stained with Hematoxylin and Eosin. Arrows indicate the position of the primary (top) and secondary (bottom) palate, the latter of which is absent in the Jaws^−/− embryo.

• Supplemental Figure S3 -

  Fig. S3. Defective chondrogenesis in Jaws^−/− embryos. (A) Cartilage preps of E12.5 embryos, showing reduced Alcian Blue staining throughout the Jaws^−/− skeleton. (B) By E14.5, ectopic joint formation is evident in Jaws^−/− digits (asterisks), the deltoid tuberosity is absent (arrow), and both Meckel’s cartilage (arrowhead) and the long bones of the forelimb are mis-shapen.

• Supplemental Figure S4 -

  Fig. S4. Comparable expression of chondrogenic markers at E12.5. Whole-mount in situ hybridization for Sox9 and its target genes Sox5 and Col2a1 in E12.5 forelimbs. Similar results were obtained for E11.5 limb buds.

• Supplemental Figure S5 -

  Fig. S5. Failure of knee joint formation and tibial development in Jaws^−/− mice. Alcian Blue/von Kossa staining (top) and in situ hybridization for Col2a1 (bottom) on histological sections of E18.5 hindlimbs. Arrows indicate the region of the presumptive knee joint in the Jaws^−/− hindlimb. The Jaws^−/− tibia retains a homogeneous population of small rounded Col2a1-expressing chondrocytes.

• Supplemental Figure S6 -

  Fig. S6. Gdf5 expression in the developing forelimb. Whole-mount in situ hybridization for Gdf5 in E12.5 (top) and E13.5 (bottom) forelimbs. The domain of Gdf5 expression outlining wild-type and Jaws^−/− digits is similar at E12.5, but by E13.5 an expansion of this domain is seen in the forming metacarpophalangeal joints of Jaws^−/− digits. At both stages, aberrant Gdf5 expression is evident in the prospective elbow joint (arrows).

• Supplemental Figure S7 -

  Fig. S7. Comparable levels of apoptosis in wild-type and Jaws^-/- digits. TUNEL analysis of E14.5 (top) and E15.5 (bottom) digits, showing similar numbers of labeled cells in wild-type and ectopic Jaws^−/− joints (arrows). Extensive labeling can also be seen in interdigital regions. Similar results were obtained for E17.5 digits.

• Supplemental Figure S8 -

  Fig. S8. Normal polarity of developmental axes in Jaws^−/− limbs. (A) Dorsoventral patterning, assayed by expression of the dorsal ectoderm marker Lmx1b and the ventral apical ectodermal ridge (AER) marker En1. (B) Proximodistal patterning, assayed by expression of Fgf8 in the AER of E11.5 limb buds. The bottom panels show lateral views, with anterior upwards and proximal towards the left. (C) Anterior/posterior patterning, assayed by expression of Shh (E11.5), Hoxd11 and Hoxd13 (E12.5). The Shh panels show lateral views, with anterior upwards and proximal towards the left.

• Supplemental Figure S9 -

  Fig. S9. Expression of key regulators of chondroitin sulfate biosynthesis and transport. Quantitative RT-PCR analyses showing the relative expression levels of C4st1 (Chst11 − Mouse Genome Informatics), Slc26a2 and Papss2 in E14.5 limbs (n=5). Expression was normalized to that of β-tubulin. n.s., not significant.

• Supplemental Figure S10 -

  Fig. S10. Unchanged expression of collagen 2 and link protein in Jaws^−/− cartilage. Immunostaining for collagen 2 (A) and link protein (B) demonstrating robust, uninterrupted staining in the Jaws^-/- humerus (E14.5).