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First published online 15 March 2006
doi: 10.1242/dev.02302

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Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development

Stefan Glaser¹, Julia Schaft^1,*, Sandra Lubitz¹, Kristina Vintersten, Frank van der Hoeven, Katharina R. Tufteland², Rein Aasland², Konstantinos Anastassiadis¹, Siew-Lan Ang³ and A. Francis Stewart^1,

¹ Genomics, BioInnovationsZentrum, Dresden University of Technology, Am Tatzberg 47, Dresden 01307, Germany.
² Department of Molecular Biology and Computational Biology Unit at BCCS, University of Bergen, HiB, Bergen N5020, Norway.
³ National Institute for Medical Research, The Ridgeway Mill Hill, London NW7 1AA, UK.

View larger version (40K): [in a new window]   Fig. 1. Classification of murine SET domain proteins. An exhaustive search for SET domains in the mouse genome revealed 50 proteins that were grouped into 11 subclasses using a tree based on a structure-based multiple sequence alignment (see Materials and methods). The tree is shown on the left with protein Accession Number and names. The most likely target specificity for each group is indicated. The most prominent SET domain co-domains are listed in the right-most column. Accession Numbers are for UniProt except those indicated in blue italic, which are from Ensembl (release 30.33f; the full Accession Number is of the form ENSMUSP000000xxxxx, and only the last five digits are shown), and for HYPB, which is a NCBI RefSeq entry. For the PRDM group, the protein names for the human orthologs are given. For the SMYD group, only 3 out of 5 members were included in the alignment, and for the PRDM group, only 7 of 15 members were included. Mll2, which resides on mouse chromosome 7, has also been called MLL4 and Wbp7 (FitzGerald and Diaz, 1999; Huntsman et al., 1999; Bedford et al., 1997).

View larger version (35K): [in a new window]   Fig. 2. Targeted disruption of the mouse Mll2 gene. (A) The mouse Mll2 cDNA sequence is illustrated at the top to indicate the positions of domains and motifs (see Fig. S1 in the supplementary material for more details). Below is a diagram of the gene with exons shown as numbered boxes connected by known splicing events. The exact start site of transcription is not known but arises in the indicated CpG island. Polyadenylation signals are indicated by red circles. The RT-PCR primers and in situ probe are indicated below, as are the alleles after targeting, FLPe and Cre recombination. (B) RT-PCR analysis of E8.5 embryos. An Mll2-specific primer pair amplified a reaction product from Mll2+/– and Mll2+/+ embryos, but not from a Mll2–/– embryo. (C) Western blot analysis of wild-type and Mll2–/– ES cells probed with an antibody raised against Mll2 amino acids 864-980. The strong band detected in wild-type extract probably corresponds to the predicted proteolytic 225 kDa Mll2 fragment processed by Taspase. The weaker band in wild-type extract could correspond to the 284 kDa full-length protein. No bands were detected in extracts from Mll2–/– cells and the blot was controlled using antibodies against CBP, which is also very large. (D) Expression of Mll2 at E8.5. The expression is ubiquitous in the wild-type embryo and absent in the homozygous embryo. (E,F) Mll2–/– embryos and Mll2FC/FC embryos have an identical embryonic lethal phenotype, as illustrated here for E9.5.

View larger version (88K): [in a new window]   Fig. 3. Phenotype of Mll2–/– embryos up to the last observable stage. Litters were taken from E6.5 to E11.5, as indicated, and stained (A-D) or not (E,F) for ß-galactosidase (E6.5-E9.5). Intensely staining embryos were homozygous and lighter staining embryos were heterozygous for the null allele. Staining seems absent from the extra-embryonic tissues (A,B).

View larger version (88K): [in a new window]   Fig. 4. Mll2 is cell autonomously required. Chimeras made with wild type +/+ (A,E,I), Mll2+/– (B,F,J) or Mll2–/– (C,D,G,H,K,L) ES cells were harvested at the times indicated and stained for ß-galactosidase expression after sectioning (E18.5) or not (E8.5, E9.5). Embryos with Mll2–/– cells were divided into low (<50%) or high (>50%) percentage according to the intensity of staining. Blue stained cells were broadly distributed in all Mll2+/– and low percentage Mll2–/– embryos, except for E18.5–/– (K), where only a few blue staining cells could be observed in the condensing oral cartilage (L, a magnification of the boxed region in K), as well as the endogenous ß-galactosidase activity in the gut (I,J,K).

View larger version (52K): [in a new window]   Fig. 5. Widespread apoptosis in E9.5 Mll2–/– embryos. Paraffin sections were taken from an Mll2–/– embryo (A) and a Mll2+/+ littermate (B), stained with TUNEL and then with Hematoxylin and Eosin. For greater contrast, A shows a section after TUNEL but before Hematoxylin and Eosin staining. (C) The average ratio of apoptotic cells to total cells from three embryos each yields the apoptotic index.

View larger version (113K): [in a new window]   Fig. 6. In situ hybridization analysis of the paraxial mesoderm marker Mox1 indicates a defect in expression maintenance in Mll2–/– mutant embryos. Whole-mount embryos (A-D) and transverse sections (E-G) are depicted. (A) Expression of Mox1 in the presomitic mesoderm and somites of an E8.4 wild-type embryo (eight somites). (B) Expression of Mox1 in an E9.0 Mll2–/– embryo with eight somites. (C) In an E9.5 Mll2–/– embryo with 10 somites, Mox1 expression was lost in the anterior somites. (D) In an E9.75 Mll2–/– embryo, almost no Mox1 expression was detected. Defective longitudinal extension of paraxial mesoderm is the most likely cause of the uneven and compressed shape of the neural tube in mutant embryos at this stage. (E) Transverse section of the E8.4 wild-type embryo displayed in A shows mox1 expression in paraxial mesoderm. (F) TUNEL stained transverse section of the E9.5 Trx2–/– embryo displayed in C. Only a few apoptotic nuclei appear throughout the section; therefore, the Mox1 signal (arrow) is not decreasing because of apoptosis of the entire paraxial mesoderm at this stage. (G) TUNEL-stained transverse section of an E10.5 Mll–/– embryo. At this stage, the entire embryo section is positively stained, with the highest amount of apoptotic cells in the paraxial mesoderm (arrow).

View larger version (85K): [in a new window]   Fig. 7. In situ hybridization analysis of Wnt1, Otx2 and T. The expression of these markers was relatively unaffected in Mll–/– embryos (B,D-F). Expression of Wnt1 in wild-type E9.5 embryo (A) compared with Mll–/– E10.5 (B). Buckling of the neural tube is caused by the loss of somites. Expression of Otx2 in wild-type E9.5 embryo (C) compared with Mll–/– E10.5 embryo (D). Expression of brachyury (T) in Mll–/– embryos at E8.5 (E) and E9.5 (F) shows that it was correctly established and maintained.

View larger version (91K): [in a new window]   Fig. 8 In situ hybridization analysis of Hoxb1 indicates a defect in expression maintenance in Mll2–/– mutant embryos. Wild-type embryos from E8.75 (A) and E9.5 (B), and Mll2–/– embryos representing approximately E7.75 (C), E8.25 (D), E8.5 (E) and E8.75 (F) were hybridized with a Hoxb1 probe.

View larger version (50K): [in a new window]   Fig. 9. Quantitative PCR analysis of Hox complex genes during embryoid body differentiation. The E14 cells used for targeting (wild type), the double-targeted Mll–/– cells and their FLPe rescued derivatives were differentiated in suspension in the absence of LIF or retinoic acid and harvested for RT-PCR analysis every second day. Each data point is the average from three parallel experiments. That is, three culture plates were differentiated, mRNA was harvested, cDNA was produced and Q-PCR analysis took place for each data point in parallel. Results are plotted as Ct values (one Ct value corresponds to a doubling of signal and all values less than 31 were assumed to be zero, i.e. 31). Pbx1 served as a control for RNA input. In our experience, the earlier activation of Hoxb2 and Hoxb5 seen in the rescued cells compared with wild type represents an implicit degree of variability involved in ES cell differentiation experiments, owing to either clone-to-clone or slight onset of differentiation differences.