PMC

Skeletal development of Dmrt4 mutant embryos. Skeletal preparations of 1 day postnatal (P1) wild-type (WT) and Dmrt4 mutants are shown.

Polyovular follicles in Dmrt4 mutant ovaries. Shown are examples of polyovular follicles from Dmrt4 mutant adult female ovaries. (A) Penta-ovular follicle. (B and C) Biovular follicles. (D) Pair of biovular follicles.

Olfactory neurons in Dmrt4 mutant embryos. Whole-mount immunohistochemistry with NCAM antibody staining of E11.5 embryos is shown. An arrow indicates the position of nasal placode olfactory epithelium. Both embryos are at the ∼35-somite developmental stage.

Histology of wild-type and Dmrt4^Δ/Δ tissues. Left column shows hematoxylin-eosin-stained sections of wild-type (WT) organs, and right column shows Dmrt4^Δ/^Δ tissues. Tissues are ovary (A and B), testis (C and D), epididymis (E and F), salivary gland (G and H), and VNO (I and J).

Dmrt4 adult mRNA expression. RT-PCR analysis of cDNA from the adult tissues indicated, performed as described in Materials Methods. Equal amounts of cDNA were amplified with primers directed against Dmrt4 and Hprt. m. eminence, median eminence; neg. cont., negative control (no cDNA template).

Expression of Dmrt4 RNA from an alternative upstream promoter. RT-PCR of cDNA from indicated wild-type and Dmrt4^Δ/Δ adult tissues, amplified with primers specific for the alternative Dmrt4 transcript (altDmrt4), as described in Materials and Methods, and Hprt primers as a positive control. sem. ves., seminal vesicle.

Disruption of Dmrt4 by homologous recombination. (A) Targeting strategy. Homologous recombination of targeting vector pJB15 inserts a loxP site (recognition site for Cre DNA recombinase; black triangles) upstream of the Dmrt4 transcriptional start site and a second loxP site into intron 1. A neomycin positive selection cassette is inserted into intron 1 and is flanked by frt sites (gray ovals), which are recognized by Flpe DNA recombinase. Homologous recombination generates the targeted allele Dmrt4^loxP and introduces a new BamHI site, as indicated. Breeding to Flpe-expressing mice can delete the neomycin cassette (not shown). Breeding to Cre-expressing mice deletes sequences between the loxP sites, generating the putative null allele Dmrt4^Δ. SP1 and SP2 denote probes used for Southern analysis of neomycin resistant clones. P1, P2, P3, and P4 are PCR primers used to genotype animals. (B) Homologously targeted ES cell clone. Southern blots of ES cell DNA digested with BamH1 and probed with SP1 (left panel) and SP2 (right panel) probes. Both probes detect the 15.5-kb wild-type BamHI fragment from the untargeted Dmrt4 allele. In each panel the first lane contains DNA from a correctly targeted clone, as indicated by the presence of a novel 10.2-kb BamHI fragment detected by SP1 and a novel 6.2-kb BamHI fragment detected by SP2. The second lane contains DNA from a control ES cell clone. WT, wild type.