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Blood, Vol. 113, Issue 7, 1444-1454, February 12, 2009
Impaired function of primitive hematopoietic cells in mice lacking the Mixed-Lineage-Leukemia homolog Mll5
Blood Madan et al.
113: 1444
Supplemental materials for: Madan et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 205 KB)
- Figure S1. Characterization of the murine Mll5 gene (JPG, 86.3 KB)
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(A) Comparison of exon/intron structures highlighting the very similar organization of human MLL5 and mouse Mll5. (B) Detection of Mll5 transcripts on a “Multiple Tissue RNA Array.” (C) Analysis of Mll5 expression by RT-PCR with primers annealing to exon 23 and 25-encoded sequences (ethidium bromide-stained agarose gel). (D) Expression of Mll5 in day 14.5 murine embryonic fibroblasts (MEFs) as shown by semi-quantitative RT-PCR. RNA from muscle and lung was included as positive control (ethidium bromide-stained agarose gel). (E) Detection of Mll5 message in the Lin−, Sca-1+, c-kit+ (LSK) bone marrow population. RNA was extracted from 25,000 sorted LSK cells pooled from three C57BL/6 mice and reverse transcribed. cDNA equivalent to 1800, 600 and 200 cells was amplified with Mll5-specific primers as in (C). RNA from an equal number of thymocytes was included as positive control. PCR products were blotted on nylon membrane and hybridized with a radiolabelled Mll5-specific oligonucleotide probe.
- Figure S2. Schematic structure of the expected Mll5 mRNA in wild-type (WT) and Mll5−∕− mice (KO) (JPG, 63.8 KB)
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Arrows on top of the transcript indicate primers used for RT-PCR amplification of total RNA, the stippled lines below indicate the predicted size of the PCR bands, with or without NcoI digestion. The size of the alternatively spliced exon 16 is also shown. Numbers within the rectangles indicate from which Mll5 exon the respective mRNA segment is derived. Note that splicing of exon 15 to 17 maintains the reading frame.
- Figure S3. Post-natal growth defects, but no obvious skeletal malformations in Mll5-deficient mice (JPG, 69.8 KB)
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(A) Comparison of total body weight of homozygous Mll5-deficient mice with their respective heterozygous or wild-type littermates six weeks after birth. Ø = average weight in grams. (B) No differences in total body weights of day 18.5 live embryos (obtained in timed matings with >30 male and >100 female Mll5+∕− parents). Average body weights (grams) are: WT/WT 1.10 ± 0.17 (n=55); KO/WT 1.13 ± 0.17; KO/KO 1.13 ± 0.18 (n=53). (C) Alizarin red/Alcian blue staining of a representative skeleton from an Mll5−∕− embryo at embryonic day E18.5. The bent tail-tip is a handling artifact.
- Figure S4. Mll5-deficiency causes male sterility by abrogating nuclear elongation during spermiogenesis (JPG, 176 KB)
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(A) Representative Hematoxylin/Eosin-stained sections of testes and epididymides from an 8-week-old Mll5-deficient male (KO/KO) and a wild-type littermate control (WT/WT). Note the lack of mature sperm in testis and epididymus of the knock-out male. Magnification, ×200 for epididymides and ×400 for testes. (B) Increased numbers of apoptotic cells in testis of Mll5-deficient males. The upper panels show the result of an in situ TUNEL-assay to detect apoptotic cells on testis sections. The bar graphs below show quantitative evaluation of TUNEL-positive cells in testis sections. Each bar represents the average number of labelled cells (LC) or tubules containing apoptotic cells (LT) from 3 males of the indicated age and genotype. LT/TT = labelled tubules per total number of tubules; LC/LT = labelled cells per labelled tubule. (C, D) Defective nuclear elongation in haploid spermatids of Mll5-deficient mice. Sections of epididymus (C) and testis (D) of 12-week-old wild-type and Mll5−∕− males, stained with haematoxylin and periodic acid Schiff reagent. (C) The number of spermatozoa is greatly reduced in the caput epididymidis of Mll5−∕− males. Mll5−∕− spermatozoa are greatly malformed, being round, instead of hook-shaped. (D) Wild-type haploid spermatids undergo nuclear elongation, starting at stage VIII and lasting until stage XII. Mll5−∕− spermatids initiate nuclear elongation at stage VIII, but then undergo progressive nuclear compaction without elongation (e.g. displayed at stage XII). At later stages, all late spermatids are hypercondensed. Arrows: elongating and elongated spermatids; stars: spermatocytes at metaphase I, characteristic of stage XII.
- Figure S5. The over-all architecture of lymphoid organs is preserved in the absence of Mll5 (JPG, 104 KB)
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Frozen sections of spleen, thymus and lymph node isolated from a 14-week-old Mll5−∕− mouse (KO/KO) and a wild-type littermate (WT/WT) were stained with antibodies against CD3 (T cells), B220 (B cells) or CD4 and CD8 (DP and SP thymocytes), as indicated. Representative examples from 3 independent experiments (3 different Mll5−∕− and Mll5+∕+ mice) are shown. M = medulla; C = cortex; BF = B-cell follicles; PC = paracortex.
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