Download embor7400751-sup

Supplemental data
Temporal and spatial delineation of mouse Otx2 functions
Nicolas Fossat, Gilles Chatelain, Gilbert Brun and Thomas Lamonerie
Figure S1. Generation of the Otx2flox allele, a conditional null allele. (A) The structure of
Otx2 wild type locus (first line), targeting vector (second line), floxN allele after homologous
recombination (third line), flox allele after removal of the Neo cassette (fourth line) and ∆e2
allele after excision of the Otx2 exon 2 (fifth line) is presented. Purple boxes are Otx2 coding
regions, white box (Neo) is PGK-Neo selection cassette. Red and grey triangles are loxP and
FRT sites respectively. Translation start (ATG) and STOP codons are indicated. A colour
code for Southern blot analysis, PCR products and size indication is used. Thin lines show
Nhe I-BamH I (blue) and Hind III-BamH I (purple) fragments detected by Southern blot
1
analysis using probes A and B (thick lines) respectively, and PCR products using primers
(arrows) a and b (cyan) and primers c, d and e (pink). The position of exon2 probe (orange
thick line) used for in situ hybridization analysis, the size of restriction fragments and PCR
products are indicated. Scale bar is indicated. B, BamH I; D, Nde I; H, Hind III; N, Nhe I; S,
Spe I; X, Xba I. (B) PCR analysis of NeoR ES clones using primers a and b showing a nonhomologous (NR) and an floxN homologous recombinant (R). (C) Southern blot analysis
using Nhe I-BamH I digested genomic DNA of floxN recombinant and wild type clones using
probe A. Genotypes are indicated. (D) Allele identification of wild type and recombinant ES
cells with floxN, flox and ∆e2 loci by Southern blot analysis using probe B, showing
diagnostic 5.4 kb, 4.2 kb, 3 kb and 4.7kb Nhe I-BamH I fragments respectively.
Corresponding genotypes are indicated. (E, F) PCR genotyping of mice and embryos using c,
d and e primers. Wild type, flox and ∆e2 loci direct amplification of 500bp, 543bp and 451bp
fragments respectively. Deduced genotypes are indicated. (G) E7.5 embryo of the indicated
genotype are shown. Otx2∆e2/∆e2 embryos have been obtained by breeding Otx2flox/flox mice
with the Sycp1-Cre transgenic animals which promote deletion upon fertilization (Vidal et al.,
1998). Arrow indicates the constriction at the embryonic-extraembryonic junction,
characteristic of Otx2 null embryos. Scale bar: 100 µm.
2
Figure S2. Generation of the knock-in Otx2CreERT2 allele. (A) The structure of Otx2 wild
type locus (first line), targeting vector (second line), CreER allele after homologous
recombination (third line) and fragments detected by Southern blot analysis using probe A
and PCR products using primers a and b is presented as in Fig. S1A. Yellow box represents
the CreERT2 gene. Thick coloured bars B and Cre are probes used for northern blot and in situ
hybridization analysis. Fragment size and scale bar are indicated. H, HindIII; N, NheI; X,
XbaI. (B) PCR analysis of NeoR clones using primers a and b, showing a non-homologous
(NR) and an CreERT2 homologous recombinant (R). (C) Southern blot analysis using Nhe I
digested genomic DNA of wild type and CreERT2 recombinant clones using probe A.
Genotypes are indicated. (D) Detection of Otx2 and CreERT2 mRNA by northern blot analysis
using Cre or Otx2 B probe in ES cells of the indicated genotypes.
Figure S3. Short term analysis of E10.5 and E12.5 injected animals.
A-N. In situ
hybridization with Gbx2 (A-D), En1 (E-H) and Math1 (I-N) of sagittal sections of
Otx2flox/CreERT2 embryos injected with tamoxifen (Tam) or solvent (Mock) and collected at
3
indicated times. M-N. Detailed view of the caudal part of mesencephalon. Rostral limit of
Gbx2 and En1 expression are marked by black arrows. Ectopic Math1 expression is
delineated by red brackets. O-P. KI67 immunocytochemistry of sagittal sections of the
mesencephalon of Otx2flox/CreERT2 embryos injected with tamoxifen (Tam) or solvent (Mock) at
E10.5 and collected at E12.5. Scale bars: 500 µm.
Materials and Methods
Generation of the targeting vectors, mouse lines production and genotyping. For Otx2flox
targeting molecule, a BamH I -tagged loxP site was inserted in the Spe I site of a 2.9 kb Xba
I-HindIII genomic fragment encompassing Otx2 exon 2 and part of exon 3. This fragment and
the 4.3 kb Xba I 5’ adjacent genomic fragment were cloned into pBSK+ (Stratagene, La Jolla
CA, USA). Both loxP and FRT oligonucleotides were inserted upstream and one FRT
downstream the neo gene of pPNT (Tybulewicz et al., 1991) to create a selection cassette
which was inserted into the Nde I restriction site of Otx2 intron 2. The Not I linearized
targeting plasmid was electroporated into ENS ES cells (Gauthier et al., 1999), homologous
recombinant clones were identified by PCR using primers a and b and confirmed by
hybridization of Nhe I and BamH I digested genomic DNA with probe A (Fig. S1). One
positive clone was transfected with a plasmid (pFlp-puro) expressing the flp recombinase to
remove the neo cassette (Otx2flox allele). Mice were produced by standard blastocyst injection
procedure. Genotypes were assessed by PCR using the primers c and d (Fig. 2a).
The Otx2CreERT2 targeting molecule was generated by creating a Nhe I site 10 bp
upstream the Otx2 translation start site and ligating together the resulting 4.1 kb 5’-Xba I-Nhe
I and a 1.4 kb 3’-Nhe I-Hind III genomic fragments into pBSK+. The coding sequence from
pCRERT2 (Feil et al., 1997) and the neo cassette from pPNT were ligated in opposite
orientation into the Nhe I site of the above plasmid. After Not I linearization, the targeting
4
plasmid was transfected into ENS ES, cells homologous recombinant clones were identified
by PCR and confirmed by hybridization of Nhe I digested genomic DNA as above (Fig. S2).
Genotypes were determined by PCR using primers a and b. Conditions are available upon
request.
Nucleic acid analyses. PCR, Southern blot and Northern blot analyses were performed
according to standard protocols using DNA form ES cells, mouse tail, whole embryos,
embryo head, brain or yolk sac, PN10 brain and RNA from ES cells.
Primers: a: 5’-CCTACCCGGTAGAATTGAC; b: 5’-TCCAAGCAATCAGTGGTTGA; c:
5’-GAACAAACGTCCCTGTGGTG;
d:
5’-AGAGCTTCCAGAACGTCGAG;
e:
5’-
ACCAAATGAGGTATGGCCTTG.
Probes: A: 0.6 kb EcoR I-BstX I genomic fragment; B: 0.4 kb Sac I-Hind III fragment from
exon 3; Cre: Cre coding sequence purified from plasmid pMCCre (Gu et al., 1994).
Proliferation analysis. 1:100 Rabbit KI67 Clone SP6 (LabVision, CA, USA)
immunocytochemistry was done as described (Vernay et al., 2005).
References
Feil, R., Wagner, J., Metzger, D. and Chambon, P. (1997) Regulation of Cre recombinase
activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res
Commun, 237, 752-757.
Gauthier, K., Chassande, O., Plateroti, M., Roux, J.P., Legrand, C., Pain, B., Rousset, B.,
Weiss, R., Trouillas, J. and Samarut, J. (1999) Different functions for the thyroid
hormone receptors TRalpha and TRbeta in the control of thyroid hormone production
and post-natal development. Embo J, 18, 623-631.
Gu, H., Marth, J.D., Orban, P.C., Mossmann, H. and Rajewsky, K. (1994) Deletion of a DNA
polymerase beta gene segment in T cells using cell type-specific gene targeting [see
comments]. Science, 265, 103-106.
Tybulewicz, V.L., Crawford, C.E., Jackson, P.K., Bronson, R.T. and Mulligan, R.C. (1991)
Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl
proto-oncogene. Cell, 65, 1153-1163.
Vernay, B., Koch, M., Vaccarino, F., Briscoe, J., Simeone, A., Kageyama, R. and Ang, S.L.
(2005) Otx2 regulates subtype specification and neurogenesis in the midbrain. J
Neurosci, 25, 4856-4867.
5
Vidal, F., Sage, J., Cuzin, F. and Rassoulzadegan, M. (1998) Cre expression in primary
spermatocytes: a tool for genetic engineering of the germ line. Mol Reprod Dev, 51,
274-280.
6