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Characterisation of cis-acting sequences reveals a biphasic, axon-dependent regulation of Krox20 during Schwann cell development

¹ Unité 368 de l’Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 46 rue d’Ulm, 75230 Paris Cedex 05, France
² Department of Cell Biology and Genetics, Erasmus University Rotterdam, P.O. Box 1738, 3000DR Rotterdam, The Netherlands

View larger version (39K): [in a new window]   Fig. 1. Construction of transgenic lines. (A) Krox20 gene and extragenic regions extending from –31 kb to +40 kb relative to the start site of transcription (upper). Cosmid inserts extending from –31 kb to –2.3 kb and +3 kb to +40 kb as well as the –4.5 kb to +7 kb Krox20/lacZ construct used to generate the –31/+7 Krox20/lacZ and –4.5/+40 Krox20/lacZ transgenic lines (lower). Restriction enzymes used to prepare these fragments for transgenesis and for Southern analysis are shown. Krox20 exons are indicated as solid black rectangles. Solid squares identify vector sequences. (B) Southern analysis of genomic DNA. A lacZ SstI/NdeI fragment was used as a probe. Enzymes used to diagnose recombination are indicated. Four –31/+7 Krox20/lacZ founders that were positive in PCR for both lacZ and cosmid vector sequences were analysed (left panel). DNA from Krox20lacZ/+ mice was included to identify the size of the recombinant band as the lacZ sequence was inserted at the BglII site in both the knock-in and transgenic mice (Schneider-Maunoury et al., 1993). Three –4.5/+40 Krox20/lacZ transgenic founders positive in PCR for both fragments injected were analysed (right panel). The size control consisted of a Krox20/lacZ plasmid with genomic sequences extending from –4.5 kb to +10.5 kb. The arrowheads indicate the size of bands consistent with recombination, which are 11.7 kb and 8.5 kb for the –31/+7 Krox20/lacZ and –4.5/+40 Krox20/lacZ transgenes, respectively. In both panels DNA from the B6D2 genetic background was included as a negative control.

View larger version (72K): [in a new window]   Fig. 2. Expression profile of the –31/+7 Krox20/lacZ transgenic line. (A,B) Transgenic embryos stained for ß-galactosidase activity at 10.5 and 15.5 dpc, respectively. (C) Transverse section through the lumbar region of the embryo shown in B (dorsal to the right). The scale bar represents 200 µm. (D) Hindlimb view of a 18.5 dpc transgenic embryo stained for ß-galactosidase. (E,F) Transverse cryosection and teased preparation of the distal sciatic nerve of transgenic mice at P3 and P30, respectively, stained for ß-galactosidase. The scale bars represent 25 µm. The preparations shown in C, E and F were counterstained with Nuclear Fast Red. V, trigeminal nerve; VII/VIII, facial/acoustic nerve; XI, accessory nerve; VNR, ventral nerve root; DNR, dorsal nerve root; ScN, sciatic nerve; CN, cutaneous nerve.

View larger version (67K): [in a new window]   Fig. 3. Expression profile of the –4.5/+40 Krox20/lacZ transgenic line. (A-D) Transgenic embryos stained for ß-galactosidase activity at 10.5 (A), 15.5 (B) and 18.5 (C,D) dpc (hindlimb, C; thorax, D). (E) Transverse section from the distal sciatic nerve at P3 prepared by cryosectioning and subsequent staining for ß-galactosidase. (F) Teased preparation of the sciatic nerve from a P30 mouse stained for ß-galactosidase and immunolabelled with an antibody against GFAP (red-brown deposit). Preparations in E and F were counterstained with Nuclear Fast Red. The scale bars represent 25 µm.

View larger version (69K): [in a new window]   Fig. 4. Axon-dependent regulation of the –31/+7 and –4.5/+40 Krox20/lacZ transgenes during nerve regeneration. (A-O) The sciatic nerves from adult –31/+7 and –4.5/+40 Krox20/lacZ transgenic and Krox20lacZ/+ mice, either untreated (A-C), or cryolesioned (D-O). –31/+7 Krox20/lacZ transgenic and Krox20lacZ/+ nerves after development for 8 days (D,G,F,I), 16 days (J,L) and 32 days (M,O) post-cryolesion. (E,H,K,N) –4.5/+40 Krox20/lacZ transgenic nerves after development for 12 days (E,H), 16 days (K) and 32 days (N). Teased nerves of the distal region of the sciatic nerve femural branch (A-F, J-O; distal) and proximal to the lesion on the distal side (G-I; proximal). (P-R) Nerves were also transected and teased from the proximal region of the distal stump (proximal) at 8 days for –31/+7 Krox20/lacZ transgenic (P) and Krox20lacZ/+ (R) and 12 days for –4.5/+40 Krox20/lacZ transgenic (Q). Cryolesioned nerves from both the –31/+7 and –4.5/+40 Krox20/lacZ lines were analysed by immunohistochemistry for GFAP at 8 and 12 days, respectively (S,T; red-brown deposit). MBP from –31/+7 Krox20/lacZ lines was analysed at 8 (U) and 12 (W) days and at 12 days (V,X) from –4.5/+40 Krox20/lacZ lines. ß-galactosidase activity was analysed in teased nerves originating from the most distal side of the lesion (S-V) where significant activity was detected, or adjacent to the lesion (W,X). All preparations were stained for ß-galactosidase activity and counterstained with Nuclear Fast Red. The scale bar represents 25 µm.

View larger version (79K): [in a new window]   Fig. 5. The POU domain transcription factor Oct6 acts upstream of the MSE to regulate Krox20. (A) Analysis of the –31/+7 and –4.5/+40 Krox20/lacZ transgenes in a Krox20 null-mutant background. (Left) 15.5 dpc Krox20cre/cre embryo carrying the –31/+7 Krox20/lacZ transgene stained for ß-galactosidase activity. The arrowhead identifies the sciatic nerve. (Right) Section of a ß-galactosidase-stained sciatic nerve from a 18.5 dpc Krox20cre/cre embryo carrying the –4.5/+40 Krox20/lacZ transgene. The section was counterstained with Nuclear Fast Red. (B upper) Immunofluorescent analysis of sections of P0 wild type (left) and Krox20lacZ/lacZ mutant (right) nerves using an antibody to Oct6 (-Oct6). (B lower) Immunofluorescent analysis of sections of P8 wild-type (left) and Oct6ß–geo/ß–geo mutant (right) nerves using an antibody to Krox20 (-Krox20). The scale bars in A and B represent 25 µm. (C) Semiquantitative RT-PCR analysis of endogenous Krox20 transcripts (447 bp) and transcripts from the –4.5/+40 Krox20/lacZ transgene (607 bp) from 18.5 dpc sciatic nerves from Oct6ß–geo/+ and Oct6ß–geo/ß–geo embryos without transgene (left) or carrying the –4.5/+40 Krox20/lacZ transgene (right). Analysis of the 18S rRNA (389 bp) was included as a control. The results are representative of two independent experiments.

View larger version (39K): [in a new window]   Fig. 6. Isolation of the MSE on a conserved 1.3 kb element. (A) Murine Krox20 gene and extragenic sequences extending from –4.5 kb to +40 kb (upper). Restriction enzymes used to generate overlapping subfragments are shown. These fragments were fused to a ß-globin minimal promoter/lacZ reporter and tested in transgenesis (lower). The number of mice that showed ß-galactosidase activity in sciatic nerve biopsies at P2/3 and P30 and the total number of transgenic mice analysed (n) is indicated. For fragments #4 and #5 the mice that expressed ß-galactosidase at P30 were among those that expressed at P2/3. (B) Homology plot generated using the VISTA algorithm (Mayor et al., 2000) between murine fragment #4 (horizontal axis; numbering in kb is relative to the Krox20 gene) and corresponding human sequences. The vertical axis indicates percent homology in a window of 100 bp with a resolution of 7 bp. Homology >80% is highlighted in gray. Note the base homology shown is 50%. Mice transgenic for construct #5 were tested for ß-galactosidase activity in the sciatic nerve at P3 (C) and P30 (D) as described in Fig. 3. Nonmyelinating Schwann cells were detected using an antibody to GFAP (D; red-brown deposit). The scale bars represent 25 µm.

View larger version (45K): [in a new window]   Fig. 7. Conserved sequences within the 1.3 kb MSE contain multiple candidate Oct6 binding sites. Two regions of highest sequence conservation between human and mouse fragment #5 (Fig. 6B) were analysed for candidate Oct6 binding sites. These consisted of 478 nt (domain A) and 436 nt (domain B) showing 84% and 90% sequence identity, respectively. Nucleotide numbering corresponds to fragment #5. Conserved residues are indicated with a dash in the human sequence. Nucleotide sequences corresponding to candidate Oct6 binding sites were identified by searching for sequences satisfying the Brn2 binding site consensus, C/AATnT/AAAT, where n=0,2,3 nt (Li et al., 1993). Brn2 and Oct6 are closely related class III POU proteins that are thought to exhibit similar binding characteristics (Schreiber et al., 1997). Conserved and non-conserved sites are overlined with a solid and hatched bar, respectively.