doi: 10.1242/10.1242/jcs.00121
Conditional ablation of the Mat1 subunit of TFIIH in Schwann cells provides evidence that Mat1 is not required for general transcription
Nina Korsisaari1,*,
Derrick J. Rossi1,*,
Anders Paetau2,
Patrick Charnay3,
Mark Henkemeyer4 and
Tomi P. Mäkelä1,
1 Haartman Institute and Helsinki University Central Hospital, Biomedicum Helsinki, PO Box 63, Haartmaninkatu 8, 00014 University of Helsinki, Finland
2 Department of Pathology, Haartman Institute and Helsinki University Central Hospital, P.O. Box 21, 00014 University of Helsinki, Finland
3 Unité 368 de l'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d'Ulm, F-75230 Paris Cedex 05, France
4 Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9133, USA
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Fig. 1. Generation of Mat1 conditional (flox) mice. (A) Partial genomic structure of the murine Mat1 gene and a target vector used to generate the conditional targeted allele Mat1flox. (B) Southern blotting analysis of targeted ES cell lines. SacI-digested genomic DNA from wild-type (wt) and targeted ES cell lines flox1 and flox2 yielding the predicted 9.3 kb band in addition to the 10.5 kb wild-type band. (C) PCR genotyping of the litter resulting from crossing Mat1flox/+ males to Mat1+/- females. A 385 bp wild-type band, a 477 bp conditional band, and a 310 bp null allele band are amplified with M10-M12 and M10-N4 primer pairs indicated in A. (D) PCR genotyping of control and experimental animals generated by crossing Mat1flox/+;KCN males with Mat1flox/flox females, with the Krox-20-Cre allele denoted KCN. The asterisk marks an introduced SacI site.
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Fig. 2. Mat1 is essential for mitotic germ cells. (A) Hematoxylin eosin (H&E) stained sections of control and mutant testis at 3 weeks (P21) and at 6 weeks (P42) of age. The asterisk marks an example of an abnormal seminiferous tubule where spermatogonia and spermatocytes are absent. The arrow marks pycnotic spermatogonia. (B) Macroscopic view (top panel) and H&E stained histological sections (middle panel 10x, lower panel 400x) of male reproductive organs from control and mutant animals at 3 months of age (P100). Abbreviations: Se, Sertoli cells; sp, spermatids; sv, seminal vesicle; bl, urinary bladder; de, ductus epididymis; ce, cauda epididymis; te, testis.
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Fig. 3. Targeted disruption of Mat1 in myelinated Schwann cells. (A) Indirect immunofluorescence detection of Krox-20 and Cre recombinase in longitudinal sections of sciatic nerves from control (Mat1flox/-) and mutant (Mat1flox/-;KCN) animals. Counterstaining of mutant sections was performed with Hoechst (H). Arrows point to non-targeted, Krox-20/Cre-negative cells. (B) Staining of control (Mat1flox/+;Z/AP) and mutant (Mat1flox/-;KCN;Z/AP) sciatic nerve sections for ß-galactosidase (lacZ) and alkaline phosphatase (AP). (C) Indirect immunofluorescence detection of Mat1 in control (Mat1flox/-) and mutant (Mat1flox/-;KCN) animals. Counterstaining was performed with Hoechst.
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Fig. 4. Mat1-deficient Schwann cells attain a mature myelinated phenotype. (A) Toluidine blue staining of osmicated, plastic embedded sciatic nerve cross sections (1 µm) from control and mutant animals at approximately 1 month (P37) and 2 months (P67) of age. (B) Delayed onset demyelination of Mat1-deficient myelinated Schwann cells. Histopathology of mutant animals at 3 months (P97) and 5 months (P157) of age. Arrowheads point to examples of remyelinating Schwann cell/axon units; arrows point to examples of denuded large diameter axons; and the asterisk points to myelin debris.
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Fig. 5. Electron microscopy of the sciatic nerves of mutant animals. (A) Overview of P157 mutant sciatic nerve showing multiple remyelinating Schwann cell/axon units, a reactive non-myelinated Schwann cell (nm), and a macrophage (mp). (B) An arrow points to a dying myelinated Schwann cell with blebbing myelin sheath. Note that the denuded axon (a) is ensheathed by a reactive pro-myelinating Schwann cell (pm). (C) An early stage of remyelination with five myelin lamellae produced by a reactive myelinating Schwann cell (m). (D) Non-myelinated Schwann cell with a transcriptionally active nucleus (nm) associated with multiple small diameter axons. (E-F) Higher magnification of myelin sheaths of representative thickness surrounding medium (3.0-3.5 µm) diameter axons from control (48 lamellae) and mutant (13 lamellae) animals showing normal lamellae compaction. Scale bars: 2 µm in A-D, 200 nm in E-F.
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Fig. 6. Increased proliferation of the Schwann cell lineage in the mutant sciatic nerves. (A) Merged images of indirect immunofluorescence detection of BrdU (green) and Krox-20 (red) in longitudinal sections of sciatic nerves from control and mutant animals at P97. Krox-20/BrdU double-positive cells are shown in yellow. Counterstaining was performed with Hoechst. (B) Quantitation of BrdU/Krox-20 double-positive cells from control and mutant animals at P37, P97, and P157.
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Fig. 7. Biochemical properties of the mutant sciatic nerves. Western blot analysis of Mat1, Cdk7, p44, CNPase, and 18.5, 17 and 14 kDa myelin basic protein (MBP) isoforms from whole sciatic nerve protein lysates from control (control 1; Mat1flox/-, control 2; Mat1flox/+;KCN) and mutant (Mat1flox/-;KCN) animals at P70.
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© The Company of Biologists Ltd 2002
