Figure 4

Novel Gdf11 deletion alleles recapitulate skeletal defects observed in Gdf11 knockout mice. (A) Schematic of Gdf11 deletion alleles recovered from Rounds #3 and #4 of CRISPR injections. Dotted lines denote deleted region of the DNA sequence. Size of deletion noted above dotted line. (B) Schematic of exogenous amino acids added (underlined) to the C-terminal domain of GDF11 in the four Gdf11 deletion alleles. Native amino acids are not underlined. *Stop codon. (C) Images of E17.5 or E18.5 embryos from lines 4A, 4B and 11 depicting loss of the tail in homozygous animals. (D) Skeletal preparation analysis of Gdf11^+/+, Gdf11^4B/+ and Gdf11^4B/4B embryos stained with Alcian Blue (to mark cartilage) and Alizarin Red (to mark bone). Numbers indicate thoracic vertebrae. Black arrow denotes tail, which is visibly shortened in the homozygote. hl: hindlimb. (E) Top: Skeletal preparations of skulls from Gdf11^+/+, Gdf11^4B/+ and Gdf11^4B/4B embryos stained with Alcian Blue and Alizarin Red. oc: otic capsule. White arrow denotes oc. Insets depict isolated oc. Bottom: Isolated oc from Gdf11^+/− and Gdf11^−/− and embryos. Scale bar: 1 mm. (F,G) Quantification of total number of vertebrae within embryos from F, each individual deletion allele, and (G), all deletion alleles pooled together. (H) Schematic of vertebrae depicting quantification of cervical (C), thoracic (T) and lumbar (L) vertebrae. (I) Quantification of the number of cervical, thoracic and lumbar vertebrae among the genotypes indicated.