Fig. 2. Partial rescue of the Tbx6 mutant phenotype with Tbx6 transgenes. Embryos were dissected at E10.5 (A-C,E), E13.5 embryos (D), and E15.5 (F-M). Embryos were derived from the following crosses: Tbx6^tm1Pa/+ Tg46/+ x Tbx6^tm1Pa/+ (A-I), Tbx6^tm1Pa/+ Tg130/+ x Tbx6^tm1Pa/+ (J,K), and Tbx6^tm1Pa/+ Tg46/+ x Tbx6^tm1Pa/+ Tg130/+ (L,M). Genotypes are indicated in the panels with Tbx6^+/– or Tbx6^–/– denoting hetero- or homozygosity for the Tbx6^tm1Pa allele, respectively and Tg46/+ or Tg130/+ denoting hemizygosity for the Tg46 or Tg130 transgene, respectively. (A-C) X-Gal staining reveals predominant expression of the Tg46 transgene in the tailbud, with perdurance of ß-galactosidase in the formed somites of both control and Tg46 rescued embryos. (B,C) A Tg46 partially rescued Tbx6 mutant, in which posterior somites are clearly seen in the high magnification of the tail region (C). X-gal staining is present in the caudal-most somites and predominantly in the dorsal region of the expanded tailbud. (D) Branching morphology of an E13.5 tail from a Tg46 rescued embryo. (E) Whole-mount in situ hybridization of Tbx6 expression in the tails of Tbx6^tm1Pa/+ and Tbx6^tm1Pa/Tbx6^tm1Pa Tg46/+ embryos, showing that although Tbx6 is expressed in both the primitive streak and the PSM, transcripts are downregulated in the Tg46 rescued embryos compared with the heterozygotes. (F,G) Gross morphological analysis reveals the short stature and short tail (red arrow indicates the tip of the tail in G) of the Tbx6^tm1Pa/Tbx6^tm1Pa Tg46/+ compared with the Tbx6^tm1Pa/+ Tg46/+ embryo. (H,I) Alcian Blue and Alizarin Red stained skeletons of the embryos pictured in F,G reveal fused vertebrae and fused ribs along the entire AP axis of the Tbx6^tm1Pa/Tbx6^tm1Pa Tg46/+ (I). Fusions of the ribs are detected in both proximal and more distal regions of the ribs. (J,K) Alcian Blue and Alizarin Red skeletal staining of embryos derived from a Tbx6^tm1Pa/+ Tg130/+ x Tbx6^tm1Pa/+ cross. Tbx6+/+ or Tbx6^tm1Pa/+ embryos with or without the Tg130 transgene were indistinguishable (J), while Tbx6^tm1Pa/Tbx6^tm1Pa Tg130/+ embryos showed almost complete absence of vertebrae and ribs (K). (L,M) Side (L) and dorsal (M) views of a Tbx6^tmPa1/Tbx6^tmPa1 Tg46/+ Tg130/+ embryonic skeleton showing more complete rescue of the mutant phenotype than with either transgene alone (compare L,M with I,K). Fusions were predominantly found in the cervical (not visible in these panels) and thoracic regions (red arrow in L and red asterisks in M), while the lumbar or sacral regions appeared normal.

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