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doi: 10.1242/10.1242/dev.00431

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Mammary gland, limb and yolk sac defects in mice lacking Tbx3, the gene mutated in human ulnar mammary syndrome

Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, 701 W 168th Street, New York, NY 10032, USA

View larger version (33K): [in a new window]   Fig. 1. Targeted disruption of mouse Tbx3 to produce the Tbx3tm1Pa allele by homologous recombination in embryonic stem (ES) cells, followed by in vitro Cre-mediated excision. (A) The Tbx3 cDNA and exons 1-6 of the endogenous locus are shown. The T-box is shaded, coding exons are shaded or open boxes, the 5' and 3' UTR are shown as black boxes, and loxP sites are black arrowheads. The targeting construct, containing 7.2 kb of total homology, replaced three coding exons of the T-box with a loxP-flanked hsv-tk PGK-neomycin resistance cassette. A ß-actin-diptheria toxin (ß-actin DT) cassette (Maxwell et al., 1987) was used for negative selection. (B) Southern analysis of BamHI-digested genomic DNA from wild-type and targeted ES cell clones using the 5' external (ext) and 3' internal (int) probes indicated in A to distinguish a 15 kb endogenous band (+) from a 6.1 or 9.3 kb targeted band (–t) with the 5' or 3' probes, respectively. (C) PCR genotyping of mice using primers indicated in A to distinguish a 330 bp wild-type and a 516 bp mutant band from the Cre-excised allele, Tbx3tm1Pa (–e).

View larger version (111K): [in a new window]   Fig. 2. Mammary gland development in wild type (+/+) and heterozygous Tbx3tm1Pa (+/–) adult females and in wild-type (either +/+ or +/–) and Tbx3tm1Pa/Tbx3tm1Pa (–/–) embryos. (A,B) Wholemounts of the right inguinal mammary glands of 6-week-old virgin females showing a similar extent of duct growth within the fat pad (in, inguinal lymph node). (C,D) Mammary glands from pregnant and lactating heterozygous females, respectively, showing normal ductal growth and lactation that was indistinguishable from controls. (E-H) Wnt10b expression in 11.5 and 12.5 dpc embryos. No mammary buds are evident in the mutant embryos (F,H), whereas buds are evident in the wild-type embryos (E,G; arrows). (I-L) Lef1 expression in 12.5 and 13.5 dpc embryos. No mammary buds are evident in the mutant embryos (J,L), whereas they are clearly visible in the wild-type embryos (I,K; arrows). The development of the forelimb and vibrissae in the 13.5 dpc mutant embryo (L) indicates that it is at a similar developmental stage as the 12.5 dpc control (I). (M-P) Sagittal and transverse sections of 12.5 and 13.5 dpc embryos, respectively. No mammary buds were seen in the 12.5 dpc mutant embryos (N), although three buds out of an expected 20 were seen in two mutant embryos at 13.5 dpc (P). Insets are higher magnification images of the boxed area showing details of the mammary bud region.

View larger version (126K): [in a new window]   Fig. 3. Yolk sac defects in Tbx3tm1Pa mutant mice. (A) 12.5 dpc wild-type (+/+) and homozygous Tbx3tm1Pa mutant (–/–) embryos within the yolk sacs, with the placentas (p) still attached. Vasculature is present in the mutant, but the vessels are smaller. (B-D) PECAM immunostaining on dissected yolk sacs at 10.5 dpc showing extensive endothelial cell organization into vessels in the wild-type (C) yolk sac but a variable degree of endothelial development in the homozygous mutants (B,D). (E,F) Histological sections of yolk sacs from heterozygous and homozygous mutant embryos at 10.5 dpc showing normal development of endoderm and mesoderm layers of the yolk sac, and endothelial cell-lined blood islands or blood vessels. (G,H) Histological sections of yolk sacs from heterozygous and homozygous mutant embryos at 12.5 dpc, showing cell death and degeneration of the endoderm layer and blood islands in the homozygous mutant (H). The endoderm cells in the mutant are pyknotic (arrow); asterisk indicates the remains of a vessel or blood island with degenerating blood cells. There were no obvious differences between +/+ and +/– embryos. en, endoderm; et, endothelium; m, mesoderm; p, placenta.

View larger version (48K): [in a new window]   Fig. 4. Limb abnormalities in Tbx3tm1Pa homozygous mutant (–/–) mice. (A-C) Limb morphology of 12.5 dpc embryos. The hindlimb of Tbx3 mutant mice (B,C) is more severely affected than the forelimb, although the phenotype is variable between mice and between contralateral sides of the same mouse. (D) Histology of the forelimb and hindlimb (inset) of a homozygous mutant embryo showing abnormal development of the hand plate and severe truncation of the hindlimb. (E,F) Left and right sides of a homozygous mutant embryo recovered dead near term. (G) Skeletal preparation of limbs from embryo in E,F. The forelimbs exhibit variable abnormalities of the posterior elements, including the ulna and digits, whereas the hindlimb shows severe truncations with a posterior deflection of the zeugopod and no development of the autopod. (H) Right hindlimb of a late term homozygous mutant fetus at higher magnification, showing the single digit attached to the single zeugopod element. (I) Skeletal preparation of the limbs from a normal age-matched control, at the same magnification as G. AER, apical ectodermal ridge.

View larger version (41K): [in a new window]   Fig. 5. In situ hybridization showing expression of differentiation and patterning markers in normal (+/+ or +/–) and Tbx3tm1Pa homozygous mutant (–/–) embryos. (A) Fgf8 expression in 10.5 dpc wild-type and homozygous mutant embryos showing the thinner and anteroposteriorly less extensive AER. (B) Tbx2 expression is absent from the posterior margins of the mutant limbs (arrows indicate posterior margins) but is present at the anterior margin at 11.5 dpc. (C,D) Tbx4 and Tbx5 expression in the hind- and forelimbs, respectively, is normal in mutant embryos at 11.5 and 10.5 dpc, respectively. (E,F) Shh expression at 10.5 and 11.5 dpc, respectively. Expression is absent or reduced in homozygous mutants compared with wild-type controls. (G) Hand2 (dHand) expression is reduced in the forelimb and absent from the hindlimb of mutant embryos at 10.5 dpc.

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