Fig 1.
Sall4 conditional expression transgene.
(A) Targeting vector used to insert the conditional expression construct for Sall4 into the Rosa26 locus in ES cells. (B) Schematic of the targeting strategy. The targeting vector also contains a diphtheria toxin negative selection cassette (PGK-DTA-bGH-PA) with diphtheria toxin expressed from the PGK promoter and followed by a bovine growth hormone poly-adenylation site. Homologous recombination into the Rosa26 locus removed PGK-DTA-bGH-PA and produced the single copy transgene CAG-Stop-Sall4-IRES-EGFP. Cre-mediated recombination deletes the “floxed” stop cassette to generate CAG-Sall4-IRES-EGFP. (C) Correct homologous recombination of the 5’ homology arm of the targeting vector into Rosa26 was confirmed by PCR of ES cell genomic DNA using primers F1 and R1 (indicated in panel B), which amplify between exon 1 of Rosa26 and the CAG promoter. No amplification product was detected in wild-type ES cells, but the predicted 1248 bp amplicon was detected in a targeted ES cell clone (#4). Control DNA was prepared from the tail clip of conditional Dmrt1 expression mouse that was generated using the same knockin system (33). (D) Homologous recombination of the 3’ vector arm was confirmed by Southern blotting using the 3’ probe indicated in panel B. Wild-type ES cell DNA generated in detection of the predicted 11 kb fragment, whereas targeted clones (two right lanes) generated both the 11 kb fragment and a 9 kb fragment corresponding to the targeted allele.
Fig 2.
TCre-dependent overexpression of Sall4.
(A-C) Endogenous expression pattern of Sall4 at E9.5. A lateral view of a whole embryo (A), a dorsal view at the forelimb level (B) and a dorsal view of the caudal part of the embryo (C) are shown. (D-F) Sall4 expression in the TCre+/tg; R26-Sall4+/tg embryo. A lateral view shows strong expression of Sall4 in the trunk (D). A dorsal view at the forelimb level shows strong expression in the somite and forelimb bud (E). A dorsal view of the caudal part of the embryo shows strong expression in this region (F). (G, H) Lateral views of the TCre+/tg; R26-Sall4+/tg embryo. A bright field image (G) and an EGFP image (H) are shown. The EGFP signal is detected in the trunk, similar to strong Sall4 expression in D. (I) Western blotting of lysate from TCre+/+; R26-Sall4+/tg embryos (control) and TCre+/tg; R26-Sall4+/tg embryos (overexp.). Stronger bands for SALL4A and SALL4B are detected with extracts from the TCre+/tg; R26-Sall4+/tg embryo. Abbreviations. f: forelimb bud, h: head, n: neural tube, t: tail bud.
Fig 3.
Analysis of neonatal skeletal development after overexpression of Sall4 in the T lineage.
Skeleton of wild-type (A-D, TCre+/+; R26-Sall4+/+) and Sall4-overexpression (E-H, TCre+/tg; R26-Sall4+/tg) neonates. Lateral views of the entire body (A, E), dorsal views of the lumber region (B, F), forelimbs (C, G) and hindlimbs (D, H) are shown. Scale bar, 5 mm in A and E, 1 mm in B-D, F-H. (I, J) Box plot of the length of the humerus (I) and femur (J) of neonates with the indicated genotypes. WT and Tg indicate wild type and the +/Tg genotype, respectively. Sample numbers are indicated in the panel. Abbreviations. fe: femur, fi: fibula, h: humerus, pg: pelvic girdle, r: radius, sg: shoulder girdle, t: tibia, u: ulna.
Fig 4.
Sall4 expression at the digit tip.
(A) Whole-mount LacZ stained Sall4+/LacZ embryo at E14.5. Blue and red arrowheads point to signals at the tail tip and genital organ, respectively. Arrows point to signals at the digit tip. Scale bar, 1 mm. (B, C) Ventral views of the wild-type digit tip of the hindlimbs after lacZ staining at indicated stages. (D, E) Ventral views of the Sall4+/LacZ digit tip of the hindlimbs after lacZ staining at indicated stages. Arrows point to the LacZ signals. Scale bar, 1 mm in A, 0.2 mm in B-E.
Fig 5.
Sall4 overexpression does not alter the regenerative ability of the digit tip.
(A) Western blotting of lysate from TCre+/+; R26-Sall4+/Tg and TCre+/tg; R26-Sall4+/tg neonatal digits. Arrows point to SALL4A and SALL4B bands. (B, C) Alcian blue/Alizarin red-stained digits from 21 day-old control (TCre+/tg; R26-Sall4+/+) and Sall4-overexpression (TCre+/tg; R26-Sall4+/tg) mice, amputated at the tip of the terminal phalange level at the P0 stage. (D, E) Alcian blue/Alizarin red-stained digits from 21 day-old control (TCre+/tg; R26-Sall4+/+) and Sall4-overexpression (TCre+/tg; R26-Sall4+/tg) mice, amputated at the second phalange level at the neonatal stage. Scale bar: 500 μm in B-E. P2 and P3 in panels B-E indicate the second phalange and the terminal phalange, respectively.