Homeotic transformations of the axial skeleton that accompany a targeted deletion of E2f6

Abstract

E2F transcription factors play an important role in regulating mammalian cell proliferation. E2F6, the most recently identified E2F family member, is a transcriptional repressor. In an effort to ascertain the in vivo biological function of E2F6, we have generated an E2f6 mutant mouse strain. Mice lacking E2F6 are viable and healthy. Surprisingly, E2f6-/- embryonic fibroblasts proliferate normally. However, E2f6-/- animals display overt homeotic transformations of the axial skeleton that are strikingly similar to the skeletal transformations observed in polycomb mutant mice. This observation is compatible with the recent finding that endogenous E2F6 and one or more mammalian polycomb proteins are components of the same multiprotein complex. The accumulated evidence suggests that, during development, E2F6 participates in the recruitment of polycomb proteins to specific target promoters.

Fig. 1. Targeting strategy and germline transmission. (A) Top: genomic organization of the E2f6 gene. Middle: targeting vector. Bottom: targeted allele. Exons are shown by boxes and introns by lines. The open box represents the non-coding region of exon 1. (B) Southern blot of genomic DNA isolated from different ES cell lines digested with XbaI and hybridized with probe B [see (A)]. Correct targeting of the left flank was confirmed by hybridizing EcoRI-digested genomic ES cell DNA with probe D (not shown). Genotypes are indicated. (C) Northern blot analysis to confirm the absence of E2F6 mRNA in MEFs derived from E2f6–/– animals. Genotypes are indicated. (D) Western blot analysis of lysates from E2f6+/+ and E2f6–/– MEFs to confirm the absence of the E2F6 protein in animals.

Fig. 2. Testicular abnormalities in E2f6–/– animals. Histological sections (hematoxylin and eosin) of testes (A and B) and ductus epididymidis (C and D) of wt (A and C) and E2f6–/– (B and D) animals. An increase of interstitial Leydig cells (asterisk) and of eosinophilic material (residual bodies) in the seminiferous tubuli (arrow) is evident in E2f6–/– testes (B). Incomplete filling of the ductus epididymidis with spermatocytes in E2f6–/– animals (D). In (C), a wt ductus epididymidis is shown at the same magnification as (D).

Fig. 3. Skeletal transformations of E2f6–/– animals. (A–C) In situ hybridization of E2F6 in 14.5-day-old mouse embryos: (A) Bright field; (B) E2F6 antisense probe; (C) E2F6 sense probe (control). (D–M) Alcian Blue (cartilage) and Alizarin Red (bone) staining of cleared newborn animals. Overview of the skeletons of wt (D) and E2f6–/– (E) skeletons. Ventral view of the thoracic region (F) and lateral view of the cervical region (G) of E2f6–/– animals. Note the normal number and attachment of the ribs, and normal size and appearance of the C1 and C2 vertebrae. Dorsal view of the thoraco-lumbo-sacral region of wt (H) and E2f6–/– (I and J) animals. In (J), the thirteenth ribs are degenerated (arrows). Moreover, the sixth lumbar vertebra (L6) has been transformed into the first sacral vertebra (S1) on one side (I and L) or two sides (J and M) (white arrowheads). Black arrowheads point to normal L6 vertebral elements. The skeletal transformations of E2f6–/– animals can also be seen in the ventral view of the lumbo-sacral region (L and M). The formations of sacro-iliac joints at L6 are evident in (L) and (M). Compare with the lumbo-sacral region of a wt animal (K).

Fig. 4. Properties of E2f6 null fibroblasts. (A) Proliferation of wt and E2f6–/– MEFs in medium with 10% FCS. Growth values represent the average of three independently isolated wt MEF cultures and three different E2f6–/– MEF cultures isolated from littermate embryos. Error bars represent the standard deviation. (B) Growth of E2f6–/– and E2f6+/+ MEFs under low serum conditions [for details see (A)]. (C) Cell cycle distribution of independently isolated asynchronously growing E2f6–/– MEF lines. (D) Serum starvation and re-entry into the cell cycle of E2f6+/+ and E2f6–/– MEFs was analyzed by BrdU incorporation. (E) Wild-type and E2f6–/– MEFs were passaged according to a standard 3T3 protocol. Cell numbers were determined at each passage. (F) Premature senescence of E2f6+/+ and E2f6–/– MEFs: E2f6+/+ and E2f6–/– MEFs were infected with either a control retrovirus or a RasV12-expressing retrovirus together with a puromycin resistance gene. Dishes were stained with Crystal Violet following 10 days of selection with puromycin.