Northern blot analysis of MTB expression during embryogenesis, in adult tissues, and in hematopoietic cell lines. (A) MTB expression can be detected as early as E7.0, as revealed by hybridization of a Clontech embryonic tissue blot with the C-terminal AD4 domain of MTB. Two primary forms of MTB, of approximately 6.8 and 4.4 kb, exist (arrows). These two transcripts differ in the 5′ and 3′ untranslated regions (data not shown). (B) Hybridization of a Clontech multiple tissue blot with the MTB probe revealed that MTB expression is restricted to the spleen, lung, and testis of adult mice. (C) MTB is expressed in megakaryocytic cells (H2), myeloid cells (416B), EML cells, and a variety of pre- and pro-B-cell lines (18.8, 3.1, and 70Z/3), as well as in multiple T-cell lines (R1-1, RML11, and BW5147). The first two lanes contain poly(A)⁺ RNA, while the other lanes contain total RNA. Hybridization of all three blots with actin is shown below the MTB blots. MTB is also expressed in the erythroid cell lines MEL and G1E (data not shown).

Targeted disruption of Mtb. (A) Targeting strategy for the Mtb locus, replacing approximately 1.3 kb of coding sequence in the middle of the locus with the neomycin cassette. Open boxes indicate known coding exons, and shaded boxes indicate the probe sequence used for Southern hybridization. S, SacI; X, XbaI; B, BamHI. (B) Southern blot analysis of genomic DNA isolated from tail clippings and digested with SacI. The wild-type (WT) allele gives rise to 9- and 7-kb fragments, while digestion of the mutant allele generates 15- and 7-kb fragments. (C) Single-reaction PCR genotyping analysis with no template DNA, genomic DNA (for wild-type and heterozygous reactions), or the targeting construct in the case of the null. The wild-type band is 434 bp, and the mutant band is 616 bp.

Increased incidence of apoptosis in Mtb^−/− blastocysts. Blastocysts were isolated at E3.5, fixed, and subjected to TUNEL. (A) Quantitation of TUNEL positivity was performed by fluorescence microscopy (see Materials and Methods). Mtb^−/− blastocysts have a significantly higher number of TUNEL-positive nuclei than their wild-type (P < 0.001) or Mtb^+/− (P < 0.05) littermates, as determined by Student's t test assuming unequal variances. There was not a significant difference in the number of apoptotic cells between wild-type and Mtb^+/− blastocysts. (B and C) Differential interference contrast (B) and fluorescein isothiocyanate (C) images of a single Mtb^−/− blastocyst, collapsed because of manipulation.

Amino acid sequence of murine MTB. The region with homology to a leucine zipper domain is shaded.

MTB expression in early- and mid-gestation embryos. (A) Whole-mount in situ hybridization, using the AD4 probe, of a wild-type embryo at E8.5 highlights the broad expression pattern of MTB at this early stage of development. (B) In situ hybridization of a mid-sagittal section of a wild-type embryo at E12.5 with the AD4 probe. Note the intense staining of the fetal liver, forebrain, and midbrain. (C, D, and E) In situ hybridization of an E15.5 mid-sagittal embryo section with the MTB probe. MTB expression is abundant in the fetal liver (C), thymus (D), and olfactory epithelium (E).

Mtb^−/− embryos fail to undergo a proliferative burst in vivo and in vitro. (A and B) Hematoxylin- and eosin-stained sections of mouse uterus containing presumed wild-type (A) and Mtb^−/− (B) embryos at E5.5. (C and D) Blastocyst outgrowths of wild-type (C) and Mtb^−/− (D) blastocysts isolated from a uterine flush, cultured for 6 days on gelatinized dishes in embryonic stem cell medium, and genotyped by nested PCR. The ICM of Mtb^−/− embryos does not expand in the uterus or in culture.